Johnson T. Wong, M.D.

Specialty: Allergy

Massachusetts General Hospital

WAC 6-626
15 Parkman Street
Boston, MA 02114


The following is a list of recent publications for which this Partners Asthma Center physician has been cited as an author in PubMed databases. Study abstracts have been provided for your convenience.

Yeung, P. K. and J. T. Wong (2003). "Inhibition of cell proliferation by mechanical agitation involves transient cell cycle arrest at G(1) phase in dinoflagellates." Protoplasma 220(3-4): 173-8.

Cell proliferation of dinoflagellates is negatively affected by mechanical agitation and red tides caused by members of the group have been correlated with periods of calm sea conditions. The mechanism involved in the mechanically transduced inhibition of cell proliferation is thought to involve the disruption of the cell division apparatus. In this study, we used highly synchronized cells and flow cytometry to study the effects of mechanical agitation on cell cycle progression. We observed that mechanical agitation induced transient cell cycle arrest at G(1) phase, in both the heterotrophic dinoflagellate Crypthecodinium cohniiand the photosynthetic dinoflagellate Heteroscapsa triquetra.

Xue, H., K. L. Tong, et al. (2003). "Transfer RNA paralogs: evidence for genetic code-amino acid biosynthesis coevolution and an archaeal root of life." Gene 310: 59-66.

A search has been performed on 2878 tRNA sequences from 60 different genomes in order to detect the existence of closely related 'alloacceptor' tRNAs accepting dissimilar amino acids that could be paralogs generated by gene duplications. This has led to the identification of extremely conserved tRNA(Phe)-tRNA(Tyr) pairs displaying as high as 94% identity between them, and also other potentially paralogous tRNA pairs in archaeal species. These paralogous pairs are enriched for amino acid pairs belonging to the same amino acid biosynthetic family, thus providing evidence for the coevolution of genetic code and amino acid biosynthesis. Overall, the genetic distances between alloacceptor tRNAs yield estimates of how closely clustered in sequence space are the tRNAs in a genome. Among 34 Bacteria, 18 Archaea and 8 Eukarya, Methanopyrus kandleri and Aeropyrum pernix have yielded the lowest alloacceptor distances and largest number of paralogous pairs. Based on a cluster-dispersion model of tRNA evolution, such tight alloacceptor clustering is a measure of primitiveness of tRNA genotypes, and places last universal common ancestor (LUCA) between the branches leading to these two archaea in the tRNA phylogenetic tree.

Wong, J. T., D. C. New, et al. (2003). "Histone-Like Proteins of the Dinoflagellate Crypthecodinium cohnii Have Homologies to Bacterial DNA-Binding Proteins." Eukaryot Cell 2(3): 646-50.

The dinoflagellates have very large genomes encoded in permanently condensed and histoneless chromosomes. Sequence alignment identified significant similarity between the dinoflagellate chromosomal histone-like proteins of Crypthecodinium cohnii (HCCs) and the bacterial DNA-binding and the eukaryotic histone H1 proteins. Phylogenetic analysis also supports the origin of the HCCs from histone-like proteins of bacteria.

Wong, J. T., D. K. Chan, et al. (2003). "Smith-Magenis syndrome and cyanotic congenital heart disease: a case report." Clin Dysmorphol 12(1): 73-4.

We report a male newborn infant of Chinese descent with the Smith-Magenis (SMS) syndrome who presented with a severe cyanotic congenital heart disease. This report adds pulmonary atresia and ventricular septal defect to the spectrum of cardiac defects seen in SMS.

Kwok, A. C. and J. T. Wong (2003). "Cellulose synthesis is coupled to cell cycle progression at G1 in the dinoflagellate Crypthecodinium cohnii." Plant Physiol 131(4): 1681-91.

Cellulosic deposition in alveolar vesicles forms the "internal cell wall" in thecated dinoflagellates. The availability of synchronized single cells, the lack of secondary deposition, and the absence of cellulosic cell plates at division facilitate investigation of the possible roles of cellulose synthesis (CS) in the entire cell cycle. Flow cytograms of cellulosic contents revealed a stepwise process of CS in the dinoflagellate cell cycle, with the highest rate occurring at G(1). A cell cycle delay in G(1), but not G(2)/M, was observed after inhibition of CS. A cell cycle inhibitor of G(1)/S, but not G(2)/M, was able to delay cell cycle progression with a corresponding reduction of CS. The increase of cellulose content in the cell cycle corresponded well to the expected increase of surface area. No differences were observed in the cellulose to surface area ratio between normal and fast-growing G(1) cells, implicating the significance of surface area in linking CS to the coupling of cell growth with cell cycle progression. The coupling of CS to G(1) implicates a novel link between CS and cell cycle control, and we postulate that the coupling mechanism might integrate cell wall integrity to the cell size checkpoint.

Jones, N., D. Agrawal, et al. (2003). "Evaluation of antigen-specific responses using in vitro enriched T cells." J Immunol Methods 274(1-2): 139-47.

Antigen-specific lymphocytes are important in the immune response to viral infection. Peripheral blood mononuclear cells (PBMC) are traditionally used as a source of effector cells in most immunological studies. We described here the use of the bispecific monoclonal antibodies (BSMAB) anti CD3:CD8 (CD3,8) and anti CD3:CD4 (CD3,4B) to expand and selectively enrich CD4+ and CD8+ T cells populations, respectively. The expanded cells demonstrated >90% CD3+CD4+ or CD3+CD8+ by 14 days. We measured HIV- and CMV-specific responses of these subset-enriched T cell and found that sensitivity and specificity is similar or higher when compared to PBMC in various cellular immunology assays (CMI). Vbeta analysis of BSMAB-enriched cells demonstrated comparable repertoire to the parent PBMC. Although both CD45RA(hi) and CD45RO(hi) cell populations were expanded with the BSMAB, selective subset depletion demonstrated that the antigen-specific T cell responses were restricted to the initial CD45RO(hi) memory effector subgroup. In conclusion, BSMAB in vitro enrichment of T cells allows significant expansion of the cell population without loss of specificity. This technique of cell expansion permits studies of T cell subset function in situations where the initial cell source is scarce, and presents an alternative for viable and functional T cells in immunological assays.

Huen, M. S., J. W. Leung, et al. (2003). "5,7-Dihydroxy-6-methoxyflavone, a benzodiazepine site ligand isolated from Scutellaria baicalensis Georgi, with selective antagonistic properties." Biochem Pharmacol 66(1): 125-32.

As part of an effort to identify naturally occurring GABA(A) receptor benzodiazepine binding site (BDS) ligands from traditional medicinal herbs, we previously reported that flavonoid derivatives isolated from Scutellaria baicalensis (S. baicalensis) Georgi exhibited significant affinities for the BDS. The present study describes the characterization of 5,7-dihydroxy-6-methoxyflavone (oroxylin A), one of the major components of the herbal extract. Oroxylin A inhibited [3H]flunitrazepam binding to rat cerebral cortical membrane with a IC(50) value of 1.09+/-0.07 microM. A GABA ratio of 1.09+/-0.04 suggests that oroxylin A interacts as an antagonist at the recognition site. In neuropharmacological studies, oral administration of oroxylin A (3.75-60 mg kg(-1)) did not result in significant changes in animal models routinely employed for benzodiazepine (BD) evaluation. However, oroxylin A selectively abolished the anxiolytic, myorelaxant and motor incoordination, but not the sedative and anticonvulsant effects elicited by diazepam, a BDS agonist. These results add oroxylin A to the list of CNS active flavonoids, and as the first naturally occurring member endowed with selective antagonistic actions via the BDS.

Yeung, P. K., F. T. Wong, et al. (2002). "Mimosine, the Allelochemical from the leguminous tree Leucaena leucocephala, selectively enhances cell proliferation in dinoflagellates." Appl Environ Microbiol 68(10): 5160-3.

Mimosine, the allelochemical from the leguminous tree Leucaena leucocephala, is toxic to most terrestrial animals and plants. We report here that while mimosine inhibits major phytoplankton groups, it enhances cell proliferation in dinoflagellates. On addition to coastal seawater samples, mimosine is able to confer a growth advantage to dinoflagellates. The use of mimosine will promote the isolation and culture of this group of phytoplankton.

Wang, H., K. M. Hui, et al. (2002). "Two flavones from Scutellaria baicalensis Georgi and their binding affinities to the benzodiazepine site of the GABAA receptor complex." Pharmazie 57(12): 857-8.

A new flavone 6,2'-dihydroxy-5,7,8,6'-tetramethoxyflavone (1) together with one known flavone 5,7,2'-trihydroxy-6,8-dimethoxyflavone (2) were isolated from the roots of Scutellaria baicalensis Georgi. Their structures were elucidated on the basis of spectral evidence and their affinities for the benzodiazepine (BDZ) site of the GABAA receptor complex were evaluated with a radioligand receptor binding assay.

Wang, H., K. M. Hui, et al. (2002). "Structure-activity relationships of flavonoids, isolated from Scutellaria baicalensis, binding to benzodiazepine site of GABA(A) receptor complex." Planta Med 68(12): 1059-62.

Twenty-six flavonoids were isolated from Scutellaria baicalensis. Their affinities for the benzodiazepine (BDZ) binding site of GABA A receptor have been studied using [ 3H]flunitrazepam binding to rat cortical membranes in vitro. The structure-activity relationships suggested that 2'-OH flavones exhibited the most potent binding affinity, which could lead to the design and discovery of new BDZ receptor ligands.

Hui, K. M., M. S. Huen, et al. (2002). "Anxiolytic effect of wogonin, a benzodiazepine receptor ligand isolated from Scutellaria baicalensis Georgi." Biochem Pharmacol 64(9): 1415-24.

The search for novel anxiolytics devoid of undesirable side-effects typical of classical benzodiazepines (BDZs) has been intense, and flavonoids, as a relative new class of ligands, have been shown to possess anxiolytic effects in vivo. The present study evaluated the pharmacological properties of a naturally occurring monoflavonoid, 5,7-dihydroxy-8-methoxyflavone or wogonin. The affinity (K(i)) of wogonin for the benzodiazepine site (BZD-S) on the gamma-aminobutyric acid(A) (GABA(A)) receptor complex was 0.92 microM. Using electrophysiological techniques, we showed that wogonin enhanced the GABA-activated current in rat dorsal root ganglion neurons, and in Xenopus laevis oocytes expressing recombinant rat GABA(A) receptors, the enhancement was partially reversed by the co-application of a 1 microM concentration of the BZD-S antagonist anexate (Ro15-1788). Acute toxicity and behavioral effects were examined in mice. Acute lethal activity was low, with an LD(50) of 3.9 g/kg. Oral administration of wogonin (7.5 to 30 mg/kg) elicited an anxiolytic response that was similar to that elicited by diazepam in the elevated plus-maze; a dose-dependent increase in open arm entries and time spent in open arms was observed. More importantly, its anxiolytic effect was blocked by the co-administration of Ro15-1788. In the holeboard test, not only did wogonin-treated mice experience an increased number of head-dips but they also spent more time at it, showing no signs of sedation. Furthermore, wogonin did not cause myorelaxant effects in the horizontal wire test. Taken together, these data suggest that wogonin exerts its anxiolytic effect through positive allosteric modulation of the GABA(A) receptor complex via interaction at the BZD-S. Its anxiolytic effect was not accompanied by sedative and myorelaxant side-effects typical of BDZs.

Hong, S. J., J. T. Wong, et al. (2002). "Reactions to radiocontrast media." Allergy Asthma Proc 23(5): 347-51.

Adverse reactions to radiocontrast media (RCM) occur unexpectedly and may be life-threatening. This article describes an anaphylactoid reaction (AR) in one patient. The term AR refers to a syndrome clinically similar to anaphylaxis, but these reactions are independent of immunoglobulin E antibody-mediated mast cell or basophil degranulation. This article briefly reviews the literature regarding RCMs and types of reactions to RCM. The risk factors for AR to RCM infusions will be discussed along with current concepts of the pathogenesis of RCM-induced ARs. This article also describes the therapeutic management of patients who have had a previous adverse reaction to RCM and provides an approach to patients who have breakthrough reactions despite adequate premedication, but require additional radiographic studies.

Guo, Q., Q. Gong, et al. (2002). "Recognition by tryptophanyl-tRNA synthetases of discriminator base on tRNATrp from three biological domains." J Biol Chem 277(16): 14343-9.

To study the recognition by tryptophanyl-tRNA synthetase (TrpRS) of tRNA(Trp) discriminator base, mutations were introduced into the discriminator base of Bacillus subtilis, Archeoglobus fulgidus, and bovine tRNA(Trp), representing the three biological domains. When B. subtilis, A. fulgidus, and human TrpRS were used to acylate these tRNA(Trp), two distinct preference profiles regarding the discriminator base of different tRNA(Trp) substrates were found: G>A>U>C for B. subtilis TrpRS, and A>C>U>G for A. fulgidus and human TrpRS. The preference for G73 in tRNA(Trp) by bacterial TrpRS is much stronger than the modest preferences for A73 by the archaeal and eukaryotic TrpRS. Cross-species reactivities between TrpRS and tRNA(Trp) from the three domains were in accordance with the view that the evolutionary position of archaea is intermediate between those of eukarya and bacteria. NMR spectroscopy revealed that mutation of A73 to G73 in bovine tRNA(Trp) elicited a conformational alteration in the G1-C72 base pair. Mutation of G1-C72 to A1-U72 or disruption of the G1-C72 base pair also caused reduction of Trp-tRNA(Trp) formation. These observations identify a tRNA(Trp) structural region near the end of acceptor stem comprising A73 and G1-C72 as a crucial domain required for effective recognition by human TrpRS.

Gong, Q., Q. Guo, et al. (2002). "NMR analysis of bovine tRNATrp: conformation dependence of Mg2+ binding." J Biol Chem 277(23): 20694-701.

NMR was used to study the solution structure of bovine tRNA(Trp) hyperexpressed in Escherichia coli. With the use of (15)N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U(71)A(2) by G(2)C(71), U(27)A(43) by G(27)C(43), and G(12)C(23) by U(12)A(23), the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A(73) and the G(12)C(23) base pair on the D stem, two identity elements on bovine tRNA(Trp) that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G(73) and U(12)A(23), respectively, NMR analysis revealed an important conformational change in the U(12)A(23) mutant but not in the G(73) mutant molecule. Thus A(73) appears to be directly recognized by tryptophanyl-tRNA synthetase, and G(12)C(23) represents an important structural determinant. Mg(2+) effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg(2+) binding sites in tRNA(Trp). Strong Mg(2+) binding modes were associated with the residues G(7), s(4)U(8) (where s(4)U is 4-thiouridine), G(12), and U(52). The observations that G(42) was associated with strong Mg(2+) binding in only the U(12)A(23) mutant tRNA(Trp) but not the wild type or G(73) mutant tRNA(Trp) and that the G(7), s(4)U(8), G(24), and G(22) imino protons are associated with a two-site Mg(2+) binding mode in wild type and G(73) mutant but only a one-site mode in the U(12)A(23) mutant established the occurrence of conformational change in the U(12)A(23) mutant tRNA(Trp). These observations also established the dependence of Mg(2+) binding on tRNA conformation and the usefulness of Mg(2+) binding sites as conformational probes. The thermal titration of tRNA(Trp) in the presence and absence of 10 mm Mg(2+) indicated that overall tRNA(Trp) structure stability was increased by more than 15 degrees C by the presence of Mg(2+).

Chan, K. L., D. New, et al. (2002). "Transcript levels of the eukaryotic translation initiation factor 5A gene peak at early G(1) phase of the cell cycle in the dinoflagellate Crypthecodinium cohnii." Appl Environ Microbiol 68(5): 2278-84.

A cDNA encoding a eukaryotic translation initiation factor 5A (eIF-5A) homolog in heterotrophic dinoflagellate Crypthecodinium cohnii (CceIF-5A) was isolated through random sequencing of a cDNA library. The predicted amino acid sequence possesses the 12 strictly conserved amino acids around lysine 52 (equivalent to lysine 50 or 51 in other eukaryotes). A single 1.2-kb band was detected in Northern blot analysis. In synchronized C. cohnii cells, the transcript level peaked at early G(1) and decreased dramatically on the entry to S phase. Although this has not been previously reported, studies of budding yeast (Saccharomyces cerevisiae) and certain mammalian cell types suggest a role for eIF-5A in the G(1)/S transition of the eukaryotic cell cycle. Phylogenetic trees constructed with 26 other published eIF-5A sequences suggest that CceIF-5A, while falling within the eukaryotic branches, forms a lineage separate from those of the plants, animals, and archaebacteria. The posttranslational modification of eIF-5A by a transfer of a 4-aminobutyl moiety from spermidine to conserved lysine 50 or 51, forming amino acid hypusine, is the only demonstrated specific function of polyamines in cell proliferation. It has been suggested that polyamines stimulate population growth of bloom-forming dinoflagellates in the sea. We demonstrate here putrescine-stimulated cell proliferation. Furthermore, ornithine decarboxylase inhibitor D-difluoromethylornithine and the specific hypusination inhibitor N-guanyl-1,7-diaminoheptane exhibited inhibitory effects in two species of dinoflagellates. The possible links of polyamines and saxitoxin synthesis to the arginine cycle are also discussed.

Yip, E. C., Y. H. Wong, et al. (2001). "Bacterial formyl peptide mediated chemotaxis and extracellular acidification in shrimp haemocytes." Dev Comp Immunol 25(4): 269-77.

The bacterial formyl peptide N-formylmethionine-leucine-phenylalanine (fMLP) is a potent chemoattractant for mammalian neutrophils. In this study, we demonstrated the binding of fluorescent dye-conjugated-fMLP to haemocytes of the penaeid shrimp Penaeus penicillatus (Alcock), through the use of flow cytometry. Fluorescence microscopy with rhodamine-fMLP suggested that fMLP receptors are present only in sub-populations of the haemocytes: granulocytes and the semi-granular cells. In addition, fMLP dose-dependently mediated chemotaxis in sub-populations of haemocytes. Microphysiometry experiments demonstrated rapid extracellular acidification upon addition of fMLP, which is in agreement with the observation in neutrophils. t-BOC, the specific fMLP receptor antagonist, was able to block the binding, chemotaxis and extracellular acidification induced by the peptide. The ability of shrimp haemocytes to migrate toward fMLP in vitro suggests that this mechanism may be important for the accumulation of these cells in infected tissues of the shrimps.

Tanay, V. A., M. B. Parent, et al. (2001). "Effects of the antidepressant/antipanic drug phenelzine on alanine and alanine transaminase in rat brain." Cell Mol Neurobiol 21(4): 325-39.

1. Phenelzine (PLZ) is an antidepressant with anxiolytic properties. Acute and chronic PLZ administration increase brain GABA levels, an effect due, at least in part, to an inhibition of the activity of the GABA metabolizing enzyme, GABA transaminase (GABA-T). 2. Previous preliminary reports have indicated that acute PLZ treatment also elevates brain alanine levels. As with GABA, the metabolism of alanine involves a pyridoxal phosphate-dependent transaminase. 3. In the study reported here, the effects of acute PLZ treatment on the levels of various amino acids, some of which are also metabolized by pyridoxal phosphate-dependent transaminases were compared in rat whole brain. Of the 6 amino acids investigated, only GABA and alanine levels were elevated (in a time- and dose-dependent manner). 4. The elevation in brain alanine levels could be explained, at least in part, by a time- and dose-dependent inhibitory effect of PLZ on alanine transaminase (ALA-T), although as with GABA the increases are higher than expected from the degree of enzyme inhibition produced. In addition, we also showed that the elevation in alanine levels and the inhibition of alanine transaminase in the brain are retained after 14 days of PLZ treatment, and that PLZ produces a marked increase in extracellular levels of alanine. 5. These results are discussed in terms of their relevance to synaptic function and to the pharmacological profile of PLZ.

Lam, C. M., C. Chong, et al. (2001). "A dinoflagellate mutant with higher frequency of multiple fission." Protoplasma 216(1-2): 75-9.

The dinoflagellate Crypthecodinium cohnii Biecheler propagates by both binary and multiple fission. By a newly developed mutagenesis protocol based on using ethyl methanesulfonate and a cell size screening method, a cell cycle mutant, mf2, was isolated with giant cells which predominantly divide by multiple fission. The average cell size of the mutant mf2 is larger than the control C. cohnii. Cell cycle synchronization experiments suggest that mutant mf2, when compared with the control strain, has a prolonged G1 phase with a corresponding delay of the G2 + M phase.

Huang, X., T. Liu, et al. (2001). "3D-QSAR model of flavonoids binding at benzodiazepine site in GABAA receptors." J Med Chem 44(12): 1883-91.

With flavone as a structural template, three-dimensional quantitative structure-activity relationship (3D-QSAR) studies and ab initio calculations were performed on a series of flavonoids. A reasonable pharmacophore model was built through CoMFA, CoMSIA, and HQSAR analyses and electrostatic potential calculations. A plausible binding mode for flavonoids with GABA(A) receptors was rationalized. On the basis of the commonly recognized binding site, the specific S1 and S2 subsites relating to substituent positions were proposed. The different binding affinities could be explained according to the frontier orbitals and electrostatic potential (ESP) maps. The ESP could be used as a novel starting point for designing more selective BZ-binding-site ligands.

Yeung, P. K., D. C. New, et al. (2000). "The spindle checkpoint in the dinoflagellate Crypthecodinium cohnii." Exp Cell Res 254(1): 120-9.

Dinoflagellates are a major group of organisms with an extranuclear spindle. As the purpose of the spindle checkpoint is to ensure proper alignment of the chromosomes on the spindle, dinoflagellate cell cycle control may be compromised to accomodate the extranuclear spindle. In the present study, we demonstrated that nocodazole reversibly prolonged the G2 + M phase of the dinoflagellate cell cycle, in both metaphase and anaphase. The regulation of the spindle checkpoint involves the activation and inhibition of the anaphase promoting complex (APC), which in turn degrades specific cell cycle regulators in the metaphase to anaphase transition. In Crypthecodinium cohnii, nocodazole was also able to induce a prolongation of the degradation of mitotic cyclins and a delay in the inactivation of p13(suc1)-associated histone kinase activities. In addition, cell extracts prepared from C. cohnii in G1 phase and G2/M phase (or nocodazole treated) were able to activate and inhibit, respectively, the degradation of exogenous human cyclin B1 in vitro. The present study thus demonstrated the presence of the spindle checkpoint and APC-mediated cyclin degradation in dinoflagellates. This is discussed in relation to a possible role of the nuclear membrane in mitosis in dinoflagellates.

Yan, X., H. Xue, et al. (2000). "NMR studies of Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli. Assignment of imino proton signals and determination of thermal stability." J Biol Chem 275(10): 6712-6.

15N-Labeled Bacillus subtilis tRNA(Trp) wild type and a series of mutants were hyperexpressed in Escherichia coli and purified for NMR studies with the use of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) and three-dimensional NOESY-HSQC techniques. These made possible chemical shift assignments of imino protons and determination of the thermal stability of the tRNA(Trp) molecules. Almost all of the imino protons in the helical regions and the tertiary base pairs were assigned, except three imino protons of the AU base pairs whose peaks were not clearly observed. Several base triplets found in the crystal structure of tRNA were observed in the present study as well. These studies also revealed two components of tRNA(Trp), which could not be separated by high pressure liquid chromatography, corresponding to s(4)U and U at position 8 of the tRNA(Trp), as indicated by two different sets of peaks for the TpsiC and D arms. The modification at position 8 altered the local conformation of the core region of the tRNA. Thermal unfolding experiments showed that the unfolding process is cooperative in the presence of a high concentration of magnesium ions and that the component corresponding to the s(4)U8 is more stable than the U8 component, thus providing evidence that the thiolation of U8 stabilizes the tertiary structure of tRNA.

Wong, J. T., M. Chan, et al. (2000). "Phosphatidylcholine metabolism in human endothelial cells: modulation by phosphocholine." Mol Cell Biochem 207(1-2): 95-100.

Phosphatidylcholine is the principal phospholipid in mammalian tissues, and a major source for the production of arachidonic acid. In this study, the effect of exogenous phosphocholine, a precursor of phosphatidylcholine biosynthesis, on the metabolism of phosphatidylcholine in human umbilical vein endothelial cells was investigated. Incubation of endothelial cells with exogenous phosphocholine at concentrations of 1 to 5 mM was found to inhibit choline uptake and its subsequent incorporation into phosphatidylcholine. Phosphocholine appeared to inhibit choline uptake in a competitive manner. Since phosphatidylcholine is metabolized mainly by the action of phospholipase A2, with the release of arachidonic acid and other fatty acids, the effect of phosphocholine on arachidonic acid release in endothelial cells was also examined. The induction of arachidonic acid release by ATP was enhanced in cells treated with 1 mM phosphocholine. In vitro assays of phospholipase A2 activity in cells incubated with phosphocholine, however, did not produced any significant change in the activity of this enzyme. The results of this study show that phosphocholine modulates the biosynthesis and catabolism of phosphatidylcholine in an indirect manner.

Wong, J. T., C. S. Nagy, et al. (2000). "Rapid oral challenge-desensitization for patients with aspirin-related urticaria-angioedema." J Allergy Clin Immunol 105(5): 997-1001.

BACKGROUND: Acetylsalicylic acid (ASA), commonly known as aspirin, is indicated in the treatment of coronary artery disease (CAD). Many patients are denied treatment with ASA because of a history of ASA or nonsteroidal anti-inflammatory drug (NSAID)-induced urticaria or angioedema. OBJECTIVE: We sought to develop a safe and practical protocol to allow the administration of ASA to patients with a history of ASA- or NSAID-induced urticaria-angioedema. METHODS: Eleven subjects with a history of ASA- or NSAID-induced urticaria-angioedema were challenged-desensitized by oral protocols based on rapidly escalating doses of ASA. Most had CAD, one had a history of pulmonary embolism, and one had refractory chronic sinusitis and asthma. Starting doses ranged from 0.1 to 10 mg and were administered at intervals of 10 to 30 minutes. Dosing was individualized for each patient but followed this general sequence (in milligrams): 0.1, 0.3, 1, 3, 10, 20, 40, 81, 162, 325. RESULTS: Nine patients tolerated the procedure without adverse effects and continued taking ASA for periods ranging from 1 to 24 months, without development of urticaria or angioedema. A patient who had a history of chronic idiopathic urticaria in addition to aspirin-induced urticaria had chest tightness during the protocol. Another patient who had continuing urticaria and angioedema associated with antithyroid antibodies developed angioedema several hours after completing the protocol. CONCLUSION: In patients with historical ASA- or NSAID-induced urticaria-angioedema reactions but who did not have urticaria and angioedema independent of ASA/NSAID, rapid oral challenge-desensitization to ASA was performed safely and permitted patients with CAD and other diseases to receive treatment with ASA.

Tremblay, C. L., F. Giguel, et al. (2000). "Marked differences in quantity of infectious human immunodeficiency virus type 1 detected in persons with controlled plasma viremia by a simple enhanced culture method." J Clin Microbiol 38(11): 4246-8.

Culture of autologous CD4 lymphocytes from peripheral blood mononuclear cells compared favorably with two other methods for the measurement of cell-associated human immunodeficiency virus type 1 (HIV-1). For subjects with undetectable HIV-1 RNA levels in plasma, there was a 10,000-fold range of cell-associated virus detected. This method provides a simple and reproducible means for monitoring cell-associated HIV-1.

Shiu, S. Y., S. C. Xi, et al. (2000). "Inhibition of malignant trophoblastic cell proliferation in vitro and in vivo by melatonin." Life Sci 67(17): 2059-74.

Melatonin inhibited thymidine incorporation into human choriocarcinoma JEG-3 cells at physiological and pharmacological concentrations in the present study. Gene expression of MT2 receptor, but not that of mt1 receptor, was detected in JEG-3 cells by reverse transcription-polymerase chain reaction (RT-PCR). The gene expression profile of the two human melatonin receptor subtypes in JEG-3 cells was identical to that previously reported for JAr cells, whose proliferation had also been shown to be similarly inhibited by physiological and pharmacological concentrations of melatonin. In contrast, melatonin had no effect on thymidine incorporation into 3A-Sub-E cells (a transformed trophoblast cell line), in which gene expression of both receptor subtypes could not be detected. The data suggest that in human placental trophoblasts, a correlation may exist between MT2 receptor gene expression and the direct anti-proliferative action of melatonin. Although melatonin has been reported to induce G1/S delay in cell cycle progression of JAr cells, no significant changes in the percentages of JEG-3 cells in different cell cycle phases upon melatonin treatment was recorded by flow cytometric analysis. This indicates that G1/S transition delay is probably not an important cellular mechanism in the direct anti-proliferative action of melatonin on human JEG-3 cells in vitro. Furthermore, melatonin inhibited the growth of both JAr and JEG-3 xenograft tumors in athymic nude mice, and prolonged the survival of those animals that developed choriocarcinoma. While the number of apoptotic tumor cells was not increased by melatonin, the pineal hormone induced significant decreases in the numbers of JAr and JEG-3 cells expressing proliferating cell nuclear antigen (PCNA) and cyclin A in the tumors. Taking into account both the in vitro and in vivo findings, it is likely that the inhibitory effect of melatonin on choriocarcinoma JAr and JEG-3 cell proliferation in vivo is largely a direct action of the hormone on the tumor cells.

New, D. C., Y. H. Wong, et al. (2000). "Cloning of a novel G-protein-coupled receptor from the sea anemone nervous system." Biochem Biophys Res Commun 271(3): 761-9.

We describe the cloning and analysis of genomic and cDNA copies of a gene from sea anemones that encodes a new member of the G-protein-coupled receptor family. The receptor shows similarity to previously described receptors for biogenic amines such as adrenaline, serotonin, and octopamine, as well as a variety of small molecule agonists and peptides, although we have been unable to determine which ligand is the natural agonist. Antibodies generated against the recombinant receptor protein identify a single protein with a molecular weight of 66 kDa in membrane preparations. Immunofluorescence studies using the same antibody have enabled localization of the receptor in the nervous system. Western blotting and RT-PCR analysis reveal that a homologue of this receptor is expressed in jellyfish and soft coral. We suggest that the receptor plays a role in neurotransmission in the sea anemone and other members of the phylum Cnidaria.

Ahn, J., J. T. Wong, et al. (2000). "The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy." J Biol Chem 275(27): 20399-405.

ABCR is a photoreceptor-specific ATP-binding cassette transporter that has been linked to various retinal diseases, including Stargardt macular dystrophy, and implicated in retinal transport across rod outer segment (ROS) membranes. We have examined the ATPase and GTPase activity of detergent-solubilized and reconstituted ABCR. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized ABCR had ATPase and GTPase activity (K(m) approximately 75 micrometer V(max) approximately 200 nmol/min/mg) that was stimulated 1.5-2-fold by all-trans-retinal and dependent on phospholipid and dithiothreitol. The K(m) for ATP decreased to approximately 25 micrometer after reconstitution, whereas the V(max) was strongly dependent on the lipid used for reconstitution. ABCR reconstituted in ROS phospholipid had a V(max) for basal and retinal activated ATPase activity that was 4-6 times higher than for ABCR in soybean or brain phospholipid. This enhanced activity was mainly due to the high phosphatidylethanolamine (PE) content of ROS membranes. PE was also required for retinoid-stimulated ATPase activity. ATPase activity of ABCR was stimulated by the addition of N-retinylidene-PE but not the reduced derivative, retinyl-PE. ABCR expressed in COS-1 cells also exhibited retinal-stimulated ATPase activity similar to that of the native protein. These results support the view that ABCR is an active retinoid transporter, the nucleotidase activity of which is strongly influenced by its lipid environment.

Wong, J. T., S. T. Wong, et al. (1999). "Ectopic semaphorin-1a functions as an attractive guidance cue for developing peripheral neurons." Nat Neurosci 2(9): 798-803.

Transmembrane and secreted glycoproteins of the semaphorin family are typically classified as inhibitory neuronal guidance molecules. However, although chemorepulsive activity has been demonstrated for several semaphorin family members, little is known about the function of the numerous transmembrane semaphorins identified to date. Here we demonstrated that the extracellular semaphorin domain of a transmembrane semaphorin, semaphorin-1a, could actively perturb axon pathfinding in vivo when presented homogenously as a recombinant freely soluble factor. When ectopic overexpression was limited to defined epithelial regions, semaphorin-1a could directly steer axons by acting as an attractive guidance molecule.

Shiu, S. Y., L. Li, et al. (1999). "Melatonin-induced inhibition of proliferation and G1/S cell cycle transition delay of human choriocarcinoma JAr cells: possible involvement of MT2 (MEL1B) receptor." J Pineal Res 27(3): 183-92.

Melatonin, the pineal neurohormone, is an evolutionarily conserved photoperiodic signaling molecule with diverse functions that include the entrainment of human circadian rhythms. Although evidence supporting a direct inhibitory action of melatonin on human cancer cell proliferation exists in the literature, the molecular and cellular signaling mechanisms involved are largely undefined. In our study, significant inhibition of human choriocarcinoma JAr cell proliferation at physiological and pharmacological concentrations of melatonin was observed. 2-Iodomelatonin, a high affinity melatonin receptor agonist, was more potent than melatonin in inhibiting JAr cell proliferation. In addition, the presence of putative melatonin receptors in choriocarcinoma was suggested by the demonstration of specific 2-[125I]iodomelatonin binding to the tumor. Interestingly, the selective MT2 melatonin receptor ligand, 4-phenyl-2-propionamidotetraline (4-P-PDOT), was found to exert not only concentration-dependent anti-proliferative actions on JAr cells, but also additive effects with melatonin in inhibiting JAr cell proliferation. Furthermore, MT2 melatonin receptor gene expression by JAr cells was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). Taken together, our data suggest that the reported anti-proliferative action of melatonin on human choriocarcinoma JAr cells may be mediated, in part, by MT2 melatonin receptor. Moreover, analysis of melatonin effect on cell cycle kinetics indicated that G1/S transition delay may underlie the observed inhibition of choriocarcinoma cell proliferation by melatonin.

Merrill, D. P., J. Martinez-Picado, et al. (1999). "Improved CD4 lymphocyte outgrowth in response to effective antiretroviral therapy." J Infect Dis 179(2): 345-51.

CD4 lymphocyte regenerative capacity was evaluated by use of an ex vivo outgrowth assay in human immunodeficiency virus (HIV)-1-infected subjects enrolled in a clinical trial (Merck 039). CD4 lymphocytes were selectively expanded in vitro by T cell receptor triggering, which also induces HIV production from latently infected cells. CD4 cell expansion and lack of virus production in cultures correlated well with clinical responses and were best in those receiving an aggressive antiretroviral three-drug regimen. Twelve clinical responders receiving triple-drug therapy monitored for 60 weeks had both excellent ex vivo CD4 cell expansion and lack of HIV replication, often in the absence of added drug in culture. Breakthrough viruses recovered from drug-containing arms of the cultures showed phenotypic resistance to the drugs used in vivo. This CD4 lymphocyte outgrowth assay correlates well with clinical outcome in subjects receiving potent antiretroviral regimens and may predict the emergence of early drug resistance.

Liu, Z. and J. T. Wong (1999). "Proliferative and regenerative capacities of CD4(+) T cells upon TCR stimulation." Clin Immunol 93(1): 16-23.

Proliferation of activated CD4(+) T cells effectively amplifies the immune response. Based on a model of selective CD4(+) T cell proliferation using an anti-CD3:anti-CD8 bispecific monoclonal antibody that leads to selective proliferation of CD4(+) T cells, we demonstrated that the peripheral CD4(+) T cells of normal subjects can divide for 17 to 38 generations, expanding 1.7 x 10(5) to 3 x 10(11) fold, following a single short period of anti-TCR stimulation. The proliferating CD4(+) T cells maintained IL2R expression. A large subgroup from each subject maintained the CD45RA(hi)CD45RO(lo) phenotype, demonstrating that activation and proliferation do not automatically convert CD4(+) T cells into CD45RA(lo)CD45RO(hi) phenotype. The enormous number of cells generated demonstrated that the peripheral CD4(+) T cells have remarkable regenerative expansion capacity. The high, but substantially variable, proliferative capacities among subjects have important implications for diseases in which CD4(+) T cells play an important role, such as HIV-infection, graft rejection, and autoimmunity.

Li, L., J. T. Wong, et al. (1999). "Melatonin-induced stimulation of rat corpus epididymal epithelial cell proliferation." Life Sci 65(10): 1067-76.

Stimulation of rat epididymal epithelial cell proliferation by melatonin was demonstrated by thymidine incorporation and flow cytometric analyses. The stimulatory effect of melatonin was dependent on the hormone concentration and the duration of cell exposure to the hormone. Maximal stimulation of [3H]thymidine incorporation into epididymal epithelial cells by melatonin was observed at 1 x 10(-9) M 5alpha-dihydrotestosterone in medium, while lower or higher concentrations of androgen attenuated the stimulatory effect of melatonin. Interestingly, a nuclear melatonin receptor agonist (1-[3-allyl-4-oxothiazolidine-2-ylidene]-4-methyl-thiosemi-carb azone, CGP 52608) induced opposite effect on epithelial cell proliferation to that produced by melatonin. Our data suggest that melatonin-induced stimulation of rat epididymal epithelial cell proliferation is not likely to be mediated by nuclear receptor. Furthermore, sequential changes of cell cycle distribution with melatonin treatment also supports a stimulatory action of melatonin on epididymal epithelial cell proliferation.

Johnson, D. S., J. T. Wong, et al. (1999). "False-positive positron emission tomographic scan for recurrent breast cancer resulting from a bee sting." Clin Nucl Med 24(9): 702-3.

Golfman, L. S., N. J. Haughey, et al. (1999). "Lysophosphatidylcholine induces arachidonic acid release and calcium overload in cardiac myoblastic H9c2 cells." J Lipid Res 40(10): 1818-26.

Lysophosphatidylcholine (lyso-PC) and arachidonate are products of phosphatidylcholine hydrolysis by phospholipase A(2). In this study, the modulation of arachidonate release by exogenous lyso-PC in rat heart myoblastic H9c2 cells was examined. Incubation of H9c2 cells with lyso-PC resulted in an enhanced release of arachidonate in both a time- and dose-dependent fashion. Lyso-PC species containing palmitoyl (C(16:0)) or stearoyl (C(18:0)) groups evoked the highest amount of arachidonate release, while other lysophospholipid species were relatively ineffective. Cells treated with phospholipase A(2) inhibitors resulted in the attenuation of the enhanced arachidonate release in the presence of lyso-PC. Lyso-PC caused the translocation of phospholipase A(2) from the cytosol to the membrane fraction and induced an increase in Ca2+ flux from the medium into the cells. Nimodipine, a specific Ca(2+)-channel blocker, partially attenuated the lyso-PC-induced rise in intracellular Ca2+. Concurrent with Ca2+ influx, lyso-PC caused an enhancement of protein kinase C activity. The lyso-PC-induced arachidonate release was attenuated when cells were pre-incubated with specific protein kinase C and mitogen activated protein kinase kinase inhibitors. Taken together, these results strongly indicate that the lyso-PC-induced increases in levels of intracellular calcium and stimulation of protein kinase C lead to the activation of cytosolic phospholipase A(2) which results in the enhancement of arachidonate release in H9c2 cells.

Wong, J. T., K. Tran, et al. (1998). "Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells." J Biol Chem 273(12): 6830-6.

Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins, and atherosclerotic tissues. It has the ability to alter endothelial functions and is regarded as a causal agent in atherogenesis. In this study, the modulation of arachidonate release by lyso-PC in human umbilical vein endothelial cells was examined. Incubation of endothelial cells with lyso-PC resulted in an enhanced release of arachidonate in a time- and concentration-dependent manner. Maximum arachidonate release was observed at 10 min of incubation with 50 microM lyso-PC. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups elicited the enhancement of arachidonate release, while other lysolipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate were relatively ineffective. Lyso-PC-induced arachidonate release was decreased by treatment of cells with PLA2 inhibitors such as para-bromophenacyl bromide and arachidonoyl trifluoromethyl ketone. Furthermore, arachidonate release was attenuated in cells grown in the presence of antisense oligodeoxynucleotides that specifically bind cytosolic PLA2 mRNA. Treatment of cells with lyso-PC resulted in a translocation of PLA2 activity from the cytosolic to the membrane fractions of cells. Lyso-PC induced a rapid influx of Ca2+ from the medium into the cells, with a simultaneous enhancement of protein kinase C (PKC) activity in the membrane fractions. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific inhibitors of PKC (staurosporine and Ro31-8220) or a specific inhibitor of mitogen-activated protein kinase/extracellular regulated kinase kinase (PD098059). Taken together, the results of this study show that lyso-PC caused the elevation of cellular Ca2+ and the activation of PKC, which stimulated cytosolic PLA2 in an indirect manner and resulted in an enhanced release of arachidonate.

Tsim, S. T., J. T. Wong, et al. (1998). "Regulation of calcium influx and phospholipase C activity by indoleamines in dinoflagellate Crypthecodinium cohnii." J Pineal Res 24(3): 152-61.

Exogenous indoleamines such as melatonin and 5-methoxytryptamine have been shown to induce cyst formation (encystment) in many species of dinoflagellate. Induction of inositol phosphates formation by indoleamine has previously been demonstrated in Crypthecodinium cohnii. In addition, depletion of extracellular Ca2+ blocks the indoleamine-induced encystment. In the present study, 12 indoleamines (including melatonin and related compounds) were examined for their abilities to induce Ca2+ influx, inositol phosphates formation, and encystment in C. cohnii. The results showed that melatonin, 5-methoxytryptamine, and the peptide toxin mastoparan stimulated 45Ca2+ influxes in dose- and time-dependent manners. The EC50 values of 5-methoxytrypramine and mastoparan to stimulate 45Ca2+ uptake were 2 mM and 35 microM, respectively. The 5-methoxytryptamine- and mastoparan-induced 45Ca2+ influx were partially attenuated by the calcium channel blockers, verapamil and ruthenium red. A series of indoleamines were examined for their structure-activity relationship on the induction of encystment and formation of inositol phosphates. Melatonin-induced inositol phosphates formation was completely blocked by U73122, indicating the possible involvement of phospholipase C. Taken together, we conclude that indoleamines may induce encystment of the dinoflagellate C. cohnii via parallel activation of phospholipase C and Ca2+ influx signaling pathways. However, activation of phospholipase C and Ca2+ influx are not always necessary or sufficient for inducing encystment. Also, these data provided the first direct evidence of a Ca2+ influx regulating mechanism in dinoflagellate C. cohnii.

New, D. C. and J. T. Wong (1998). "The evidence for G-protein-coupled receptors and heterotrimeric G proteins in protozoa and ancestral metazoa." Biol Signals Recept 7(2): 98-108.

In higher eukaryotes G-protein-coupled signal transduction pathways are a common mechanism used to detect an extracellular message and transmit a signal, via a membrane-bound receptor and a heterotrimeric G protein, to second messenger producing enzymes and effector proteins. The techniques used to identify components of these pathways are increasingly being applied to protozoa and ancestral metazoa. Many of the organisms studied do seem to express functional homologues of those found in higher eukaryotes and increasingly genes encoding these proteins are being cloned. Sequence analysis of the isolated alpha-subunits of heterotrimeric G proteins shows that these proteins have extensive homology to their mammalian counterparts, and often show absolute sequence identity in functionally significant regions. The receptor clones isolated clearly establish that protozoa and early metazoa express proteins with seven transmembrane spanning domains. Comparisons with mammalian receptors indicate that these proteins are likely to be regulated by phosphorylation and dephosphorylation events, although the pathways which control these are yet to be identified. The postulated regulatory mechanisms and the number of homologous clones isolated from some protozoa suggest that a highly regulated system of transmembrane signalling appeared at a relatively early stage in evolution.

Li, L., J. N. Xu, et al. (1998). "Molecular and cellular analyses of melatonin receptor-mediated cAMP signaling in rat corpus epididymis." J Pineal Res 25(4): 219-28.

By using 2-[125I]iodomelatonin receptor binding studies, we have previously demonstrated high affinity melatonin receptors, the binding activities of which are regulated by testosterone, in the corpus epididymis of rats. In this report, some of the basic molecular and cellular characteristics of these high affinity melatonin receptors in rat corpus epididymis were analyzed. MEL1A and MEL1B receptor mRNAs were expressed by rat corpus epididymal epithelial cells as revealed by in situ hybridization. Functionally, these high affinity melatonin receptors are negatively coupled to adenylyl cyclase via pertussis toxin (PTX) sensitive Gi protein and the inhibitory effects of melatonin on forskolin-stimulated cAMP accumulation were enhanced by 5alpha-dihydrotestosterone (5alpha-DHT). Interestingly, opposing interactions between melatonin and beta-adrenergic receptor signaling in rat epididymal epithelial cells were observed with melatonin inhibiting norepinephrine- and isoproterenol-stimulated cAMP accumulation. In conclusion, our data support a modulatory action of melatonin, mediated via pertussis toxin-sensitive Gi-coupled MEL1A and MEL1B receptors, in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions.

Wong, J. T., W. T. Yu, et al. (1997). "Transmembrane grasshopper Semaphorin I promotes axon outgrowth in vivo." Development 124(18): 3597-607.

Members of the Semaphorin family of glycoproteins play an important role in axonal pathfinding by functioning as inhibitory guidance cues. Here we provide evidence that a transmembrane form of Semaphorin (Semaphorin I), which is expressed by bands of epithelial cells in the developing grasshopper limb bud, functions as an attractive/permissive cue for the growth cones of the subgenual organ. In addition, we demonstrate that Semaphorin I is needed for initial axonal outgrowth from the subgenual organ. These results are consistent with an alternative function for a transmembrane form of Semaphorin and may explain the previously reported arrest of the proximal extension of the subgenual organ growth cones in the absence of the Ti1 pioneer pathway.

Tsim, S. T., J. T. Wong, et al. (1997). "Calcium ion dependency and the role of inositol phosphates in melatonin-induced encystment of dinoflagellates." J Cell Sci 110 ( Pt 12): 1387-93.

The unicellular eukaryotic dinoflagellates shed their flagella and form a new pellicle cyst wall in response to environmental stress. This encystment process can also be induced by indoleamines such as melatonin and 5-methoxytryptamine. To decipher the complex signaling events which lead to encystment, we have investigated the functional roles of Ca2+ and inositol phosphates in indoleamine-induced encystment of the dinoflagellates Alexandrium catenella and Crypthecodinium cohnii. Pretreatment with EGTA, but not with EDTA, effectively blocked the indoleamine-induced encystment of A. catenella in a dose-dependent manner. Conversely, agents that facilitate the influx of Ca2+ (Bay K 8644, A23187 and ionomycin) dose-dependently induced encystment of A. catenella. Endoplasmic Ca2+-ATPase inhibitors such as thapsigargin and the peptide toxin melittin also induced encystment of A. catenella. These results suggest that an elevation of intracellular [Ca2+] may be involved in the encystment response. In terms of the regulation of phospholipase C, melatonin dose- and time-dependently stimulated the formation of inositol phosphates in C. cohnii. The rank order of potency for several indoleamines to stimulate inositol phosphates formation was 2-iodomelatonin > 5-methoxytryptamine > or = melatonin >> N-acetylserotonin > 5-hydroxytryptamine. This rank order was the same as for the indoleamine-induced encystment of C. cohnii as previously reported. Our results indicate that indoleamine-induced activation of phospholipase C and elevation of intracellular [Ca2+] may be proximal steps in the signal transduction pathway leading to encystment in dinoflagellates. Moreover, this is the first demonstration of the possible involvement of Ca2+ and inositol phosphates as second messengers in dinoflagellates.

Shiu, S. Y., L. Li, et al. (1997). "Biology of G protein-coupled melatonin receptors in the epididymis and prostate of mammals." Chin Med J (Engl) 110(8): 648-55.

Leveson, A., F. Wong, et al. (1997). "Cyclins in a dinoflagellate cell cycle." Mol Mar Biol Biotechnol 6(3): 172-9.

The dinoflagellates are distinct eukaryotes in having and extranuclear spindle and permanently condensed chromosomes. These cytologic features implicate special adaptations to the molecular mechanisms of cell cycle control. We have demonstrated the presence of cyclin-box-containing polypeptides in dinoflagellates by immunoblotting using peptide-generated antibodies. We identified four major cyclin-box-containing polypeptides. The cell cycle dynamics of these polypeptides were also investigated in synchronized populations, using a newly developed method. Of the four major cyclin-box-containing polypeptides detected, a triplex with apparent molecular weight of 75 kDa did not change appreciably during the cell cycle. For two other cyclin-box-containing polypeptides with apparent molecular weights of 50 and 65 kDa, we observed an early expression in the cell cycle, with the level accumulating and eventually being degraded on the exit of mitosis. At least on cyclin-box-containing polypeptide (50 kDa) was also observed in a protein complex bound to p13suc1 beads. The bound complex head associated histone kinase activity. Variation of this activity corresponded well with the periodic expression of the 50-kDa cyclin-box-containing polypeptide during the cell cycle of Crypthecodinium cohnii. This demonstrates the presence of cyclins and cyclin-dependent kinases in dinoflagellates.

Hishii, M., D. Andrews, et al. (1997). "In vivo accumulation of the same anti-melanoma T cell clone in two different metastatic sites." Proc Natl Acad Sci U S A 94(4): 1378-83.

In a patient with progressing metastatic melanoma, we showed that the same autologous tumor-cytolytic CD8+ tumor infiltrating lymphocyte (TIL) clone accumulated in two separate metastatic sites. This clone, which represented three of eight independently derived clones from a tumor deposit on the skin of the abdomen, also represented two of eight clones derived from a skin lesion on the shoulder. This clone could be identified by its use of a unique TCRBV2-nD1n-J1S6 sequence, and could also be detected by single-stranded conformational polymorphism (SSCP) as the dominant TCRBV2-expressing clone among CD8+ TILs propagated from both shoulder and abdominal lesions. Using SSCP analysis, we also demonstrated that this clone was dominant in the fresh tumor tissue and in all TILs in which CD8+ were strongly represented, including several separate but parallel cultures. The SSCP pattern for this clone was not apparent among CD4+ TILs or CD8+ peripheral blood mononuclear cells. The SSCP analysis of the tumor tissue prior to in vitro culture is an indication that the selection for this anti-tumor cytotoxic T cell clone was a reflection of its in vivo accumulation. Thus, we provide evidence that melanomas are immunogenic and able to select for cytotoxic antitumor-specific TIL clones that are expanded in vivo and can circulate to accumulate in different tumor sites. However, because these clones were isolated from progressing tumor metastases, the accumulation of these specific cytotoxic T cells was not sufficient to contain tumor growth.

Chen, L., B. Liang, et al. (1997). "Oxidative modification of low density lipoprotein in normal and hyperlipidemic patients: effect of lysophosphatidylcholine composition on vascular relaxation." J Lipid Res 38(3): 546-53.

The elevated level of plasma low density lipoprotein (LDL) in hyperlipidemic patients is an important risk factor for the production of atherosclerosis. Plasma LDL must be modified before it can produce an impairment of endothelium-dependent relaxation in aortic rings or enhancement of uptake by macrophages. The dramatic increase in lysophosphatidylcholine (lysoPC) content in oxidatively modified LDL has been touted as an important biochemical factor for the impairment of endothelium-dependent relaxation. The present study was designed to examine the lysoPC composition of oxidized LDL samples from normal and hyperlipidemic subjects, and their effects on the impairment of endothelium-dependent relaxation. Oxidatively modified LDL from hyperlipidemic patients contained a slightly higher level (17%) of lysoPC, but produced a disproportionately greater impairment of endothelium-dependent relaxation than that from normal subjects. As lysoPC is composed of many molecular species, its composition in oxidized LDL samples was analyzed. In hyperlipidemic patients, lysoPC samples were found to contain a higher proportion of long-chain acyl groups. Subsequent studies revealed that only long-chain lysoPC (C > 16:0) were effective in impairing endothelium-dependent relaxation. Experimental loading of oxidized LDL from normal subjects with long chain lysoPC to mimic levels observed in oxidized LDL from hyperlipidemic patients resulted in further impairment of endothelium-dependent relaxation. We conclude that the greater proportion of long-chain lysoPC found in the oxidized LDL of hyperlipidemic subjects is responsible for the increased impairment of endothelium-dependent vascular relaxation. We propose that the high level of LDL found in the plasma of hyperlipidemic patients, coupled with its enhanced ability to generate long chain species of lysoPC during oxidative modification, are important factors for the development of atherosclerosis in these patients.

Yeung, P. K., K. F. Kong, et al. (1996). "Sequence data for two large-subunit rRNA genes from an Asian strain of Alexandrium catenella." Appl Environ Microbiol 62(11): 4199-201.

PCR generated two distinct products from a toxic isolate of Alexandrium catenella, which had been taken from Dai Ya Bay (southern China), by using primers for large-subunit rRNA. This pattern is distinct from published data for North American Alexandrium species. Sequences of the two products suggest that the smaller was generated by a deletion event. Single-cell PCR generated the same pattern, confirming that the two products were not the results from different individuals.

Wong, J. T. (1996). "Protozoan cell cycle control." Biol Signals 5(6): 301-8.

Many genes belonging to the cyclin-dependent kinase (cdk) family have been isolated from protozoans. While their role in cell cycle has yet to be proven unequivocally, at least one cdk can complement the cdc2ts/cdc28ts mutants in yeasts. Among the interesting questions relating to cdks in protozoa are: whether one cdk acts throughout the whole cell cycle and whether cyclin partnership is absolutely required. In protozoa, cell cycle control is closely associated with developmental control. Many life cycle differentiation phases can only occur during a specific window in the cell cycle. Because of different DNA biosynthetic pathways, some protozoa among the earliest eukaryotic lineages are unresponsive to common inhibitors of DNA synthesis like hydroxyurea. However, many protozoa do have different checkpoint controls in relation to their response to cell cycle inhibitors.

Wong, J. T., R. Y. Man, et al. (1996). "The effects of lidocaine and hypoxia on phospholipid biosynthesis in the isolated hamster heart." Lipids 31(10): 1059-67.

In this study, the effects of lidocaine and hypoxia on the biosynthesis of phospholipids in the hamster heart were examined. Hamster hearts were perfused with [1,3-3H]glycerol under normal and hypoxic conditions, and in the absence or presence of 0.5 mg/mL lidocaine. After perfusion, the radioactivity incorporated into the various phospholipid fractions was determined. With the exception of phosphatidylcholine, the synthesis of phospholipids was generally stimulated by lidocaine perfusion. In contrast, hypoxia caused a general decrease in phospholipid biosynthesis which was partially restored by lidocaine. ATP and CTP levels were severely reduced under hypoxic conditions, but their levels were not restored by lidocaine treatment. The activities of enzymes for phospholipid synthesis were determined under the various perfusion conditions. The activity of phosphatidic acid phosphatase was elevated by lidocaine and decreased by hypoxic treatment. The activity of CTP:phosphatidic acid cytidylyltransferase was increased under hypoxia, with or without lidocaine. Despite the reduction in phosphatidylcholine biosynthesis, no change in the activity of cytidine diphosphocholine (CDPcholine):diacylglycerol cholinephosphotransferase was detected following lidocaine or hypoxic perfusion. However, enzyme activity was inhibited by the presence of lidocaine in the assay mixture. Our results indicate that the reduction in phospholipid biosynthesis under hypoxic conditions was caused mainly by diminishing high-energy nucleotide levels. The enhancement of phospholipid biosynthesis by lidocaine appeared to be mediated in part by modulation of enzyme activities.

Tslm, S. T., J. T. Wong, et al. (1996). "CGP 52608-induced cyst formation in dinoflagellates: possible involvement of a nuclear receptor for melatonin." J Pineal Res 21(2): 101-7.

Melatonin has been shown to regulate gene transcription through RZR/ROR nuclear receptors in mammalian cells. Thiazolidine dione CGP 52608 is a selective agonist of RZR/ROR receptors with little or no affinity for the cell surface G protein-coupled melatonin receptors. In this study, we addressed whether nuclear signaling may be involved in indoleamine-induced encystment of the unicellular dinoflagellates by examining their responses to CGP 52608. Three species of dinoflagellates (Alexandrium catenella, Amphidinium carterae, and Crypthecodinium cohnii) encysted in the presence of CGP 52608 and the responses were reversible and dose-dependent. Since a previous study has implicated the involvement of G proteins in mediating indoleamine-induced encystment of dinoflagellates, we explored the possibility of cross-talks between G protein-dependent and nuclear signaling pathways. The responses of A. catenella to either mastoparan (a direct activator of mammalian G proteins) or indoleamines were assessed in the presence or absence of CGP 52608. Interestingly, CGP 52608 synergized with either indoleamines or mastoparan to produce a more rapid encystment response. These findings suggest that nuclear signaling may be involved in the indoleamine-induced encystment of dinoflagellates.

Tsim, S. T., J. T. Wong, et al. (1996). "Effects of dibutyryl cAMP and bacterial toxins on indoleamine-induced encystment of dinoflagellates." Biol Signals 5(1): 22-9.

Dinoflagellates are the causative agents of red tides with worldwide occurrence and can be induced to encyst by in doleamines such as melatonin and 5-methoxytryptamine (5-MOT). This biological response may be mediated via indoleamine-binding proteins or receptors. Here we report the initial characterization of the signal transduction mechanisms by which indoleamines induce encystment of dinoflagellates. In particular, we explored the possible involvement of G proteins and cAMP in cyst formation. Both melatonin and 5-MOT promoted the encystment of Gonyaulax tamarensis and Crypthecodinium cohnii. Exposure of dinoflagellates to dibutyryl cAMP, which directly activates cAMP-dependent pathways, did not affect the ability of indoleamines to promote encystment. However, dibutyryl cAMP dose-dependently diminished the indoleamine-induced suppression of cell growth. Exposure of dinoflagellates to the bacterial toxins from Vibrio cholerae and Bordetella pertussis had no effect on the indoleamine-induced encystment response, indicating the lack of involvement of Gs or Gi-like proteins. Moreover, [32P]ADP ribosylation of dinoflagellate membranes by either toxin failed to identify substrate proteins. These results suggest that although the indoleamine-induced encystment of dinoflagellates may involve a G-protein-coupled signal transduction pathway, the identity of the G protein concerned may be distinct from those that regulate adenylyl cyclases in mammalian cells.

Tsai, S. P. and J. T. Wong (1996). "Enhancement of erythrocyte sedimentation rate by polymerized hemoglobin." Artif Cells Blood Substit Immobil Biotechnol 24(5): 513-23.

Development of hemoglobin-based blood substitutes requires the scrutiny of blood rheological parameters that could be influenced by this class of molecules. Accordingly, we have examined the effects of glutaraldehyde-polymerized human hemoglobin on the erythrocyte sedimentation rate (ESR). For this purpose, human hemoglobin (Hb) was polymerized by glutaraldehyde, and its progress was monitored by gel permeation. ESR was measured by addition of hemoglobin or polymerized Hb (Poly-Hb) to citrated rat whole blood. The results indicate that, whereas Hb exerted minimal perturbation of ESR, Poly-Hb obtained under some polymerization conditions induced an over fifty-fold elevation of ESR. When polymerized Hb was fractionated by size, and different fractions were tested for their effects on ESR, a sharp dependence of ESR enhancement on molecular size of polymerized Hb was found. These observations suggest that ESR enhancement is mediated by macromolecular bridging formed by Poly-Hb of an adequate length between the surfaces of two stacking erythrocytes.

Tran, K., J. T. Wong, et al. (1996). "Vitamin E potentiates arachidonate release and phospholipase A2 activity in rat heart myoblastic cells." Biochem J 319 ( Pt 2): 385-91.

Cytosolic phospholipase A2 (cPLA2) selectively catalyses the release of arachidonic acid from the sn-2 position of glycero-phospholipids to produce prostaglandins and leukotrienes. In this study, vitamin E enrichment of rat heart myoblastic H9c2 cells caused an increase in the release of arachidonate during ionophore (A23187) stimulation. PLA2 activity in the cytosolic fraction was also enhanced but enzyme activity in the particulate fraction was not affected by this treatment. Immunoblotting analysis with a polyclonal anti-cPLA2 antibody showed an increased level of the enzyme in vitamin E-treated cells. Direct incorporation of vitamin E into lipid vesicles in the assay mixture resulted in modulation of enzyme activity in a biphasic manner. Pretreatment of cells with phorbol 12-myristate 13-acetate, a known activator of protein kinase C, synergistically potentiated the ionophore-induced arachidonate release in both the control and vitamin E-treated cells. However, vitamin E treatment by itself did not affect the protein kinase C activity, indicating that the vitamin E-induced activation of cPLA2 was independent of the protein kinase C cascade. Collectively, these results suggest that vitamin E potentiates arachidonate release through the direct and/or indirect modulation of cPLA2 activity.

Hogue, C. W., S. Doublie, et al. (1996). "A concerted tryptophanyl-adenylate-dependent conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase revealed by the fluorescence of Trp92." J Mol Biol 260(3): 446-66.

A semi-conserved tryptophan residue of Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was previously asserted to be an essential residue and directly involved in tRNATrp binding and recognition. The crystal structure of the Bacillus stearothermophilus TrpRS tryptophanyl-5'-adenylate complex (Trp-AMP) shows that the corresponding Trp91 is buried and in the dimer interface, contrary to the expectations of the earlier assertation. Here we examine the role of this semi-conserved tryptophan residue using fluorescence spectroscopy. B. subtilis TrpRS has a single tryptophan residue, Trp92. 4-Fluorotryptophan (4FW) is used as a non-fluorescent substrate analog, allowing characterization of Trp92 fluorescence in the 4-fluorotryptophanyl-5'-adenylate (4FW-AMP) TrpRS complex. Complexation causes the Trp92 fluorescence to become quenched by 70%. Titrations, forming this complex under irreversible conditions, show that this quenching is essentially complete after half of the sites are filled. This indicates that a substrate-dependent mechanism exists for the inter-subunit communication of conformational changes. Trp92 fluorescence is not efficiently quenched by small solutes in either the apo- or complexed form. From this we conclude that this tryptophan residue is not solvent exposed and that binding of the Trp92 to tRNATrp is unlikely. Time-resolved fluorescence indicates conformational heterogeneity of B. subtilis Trp92 with the fluorescence decay being best described by three discrete exponential decay times. The decay-associated spectra (DAS) of the apo- and complexed-TrpRS show large variations of the concentration of individual fluorescence decay components. Based on recent correlations of these data with changes in the local secondary structure of the backbone containing the fluorescent tryptophan residue, we conclude that changes observed in Trp92 time-resolved fluorescence originate primarily from large perturbations of its local secondary structure. The quenching of Trp92 in the 4FW-AMP complex is best explained by the crystal structure conformation, in which the tryptophan residue is found in an alpha-helix. The amino acid residue cysteine is observed clearly within the quenching radius (3.6 angstroms) of the conserved tryptophan residue. These tryptophan and cysteine residues are neighbors, one helical turn apart. If this local alpha-helix was disrupted in the apo-TrpRS, this disruption would concomitantly relieve the putative cysteine quenching by separating the two residues. Hence we propose a substrate-dependent local helix-coil transition to explain both the observed time-resolved and steady-state fluorescence of Trp92. A mechanism can be further inferred for the inter-subunit communication involving the substrate ligand Asp132 and a small alpha-helix bridging the substrate tryptophan residue and the conserved tryptophan residue of the opposite subunit. This putative mechanism is also consistent with the observed pH dependence of TrpRS crystal growth and substrate binding. We observe that the mechanism of TrpRS has a dynamic component, and contend that conformational dynamics of aminoacyl-tRNA synthetases must be considered as part of the molecular basis for the recognition of cognate tRNA.

Fong, W. F., W. Lam, et al. (1996). "Partial synergism between dextran-conjugated doxorubicin and cancer drugs on the killing of multidrug resistant KB-V1 cells." Anticancer Res 16(6B): 3773-8.

One of the major problems in cancer chemotherapy is the development of tumor resistance to drug treatment. In in vitro experiments, the stepwise selection of cancer cells resistant to a single antineoplastic agent may lead to resistance to multiple agents (multidrug resistance). One of the well known mechanisms leading to multidrug resistance is the over-expression of the mdr1 gene product, the 170 kDa membrane P-glycoprotein which is an ATP-driven efflux pump of xenobiotics. We studied the effects of dextran-conjugated doxorubicin in combination with colchicine, vinblastine and free doxorubicin respectively on the killing of human KB 3-1 carcinoma cells and its multidrug resistant subclone KB-V-1 cells. Cell survival was quantified by the tetrazolium salt MTT assay Cytotoxicity studies were designed so that data could be analyzed by the medium-effect principle and the calculated Combination Indices at different cell survival levels. When added alone conjugated doxorubicin was not as effective as doxorubicin in cell killing. When conjugated doxorubicin was combined with free doxorubicin or colchicine at high (over 75%) killing rates, a significant degree of synergism was observed in the killing of multidrug resistant KB-V1 cells. This synergism was not observed in non-resistant KB-3-1 cells nor when conjugated doxorubicin was combined with vinblastine.

Dueck, D. A., M. Chan, et al. (1996). "The modulation of choline phosphoglyceride metabolism in human colon cancer." Mol Cell Biochem 162(2): 97-103.

Colorectal cancer has a high incidence of morbidity and mortality in the North American population. Elevated levels of plasmalogens have been reported in some neoplastic tissues including colon tumors, but the mechanism for this increase has not been defined. Since changes in plasmalogen level are usually associated with changes in the other phospholipid subclasses, a general increase in all phospholipid subclasses may also be found in colonic neoplasms. In this study, the levels of the major phospholipids, including their plasmalogen and diacylphospholipid subclasses, were found to be elevated in human malignant colonic tissues. Since phosphatidylcholine is the most prominent type of phospholipid found in both malignant and control tissues, the mechanism for its accumulation during malignancy was investigated. Decreases in phospholipase C and D activities were observed in tumor samples, but an enhancement of the CTP: phosphocholine cytidylyltransferase activity was also detected. Immunoblotting analysis revealed that the elevated cytidylyltransferase activity was caused by a three-fold increase in the level of enzyme protein during tumor development. Based on these enzyme studies, we conclude that the high level of phosphatidylcholine in colon tumors resulted from a decrease in its turnover and an increase in its expression.

Chow, K. C. and J. T. Wong (1996). "Isolation and characterization of the Bacillus subtilis tryptophanyl-tRNA synthetase gene (trpS) conferring 5-fluorotryptophan resistance and temperature sensitivity." Biochim Biophys Acta 1309(1-2): 42-6.

The mutant trpS gene of Bacillus subtilis encoding a thermosensitive tryptophanyl-tRNA synthetase that also confers resistance to growth inhibition by 5-fluorotryptophan at permissive temperature has been isolated and characterized. A point mutation at codon 82 of the gene from a wild-type TCA codon for Ser to a TTA codon for Leu has been identified.

Xue, H. and J. T. Wong (1995). "Interferon induction of human tryptophanyl-tRNA synthetase safeguards the synthesis of tryptophan-rich immune-system proteins: a hypothesis." Gene 165(2): 335-9.

Ever since the discovery that the human tryptophanyl-tRNA synthetase (TrpRS)-encoding gene is induced by interferon (IFN) [J. Fleckner et al., Proc. Natl. Acad. Sci. USA 88 (1991) 11520-11524] and contains IFN-response regulatory elements [Frolova et al., Gene 128 (1993) 237-245], the biological rationale for this induction has remained unresolved. A survey of immune system proteins in this study reveals that the human major histocompatibility complex (MHC) antigens, beta-2-microglobulin (beta MG) and complement factor B, which are known to be induced by IFN, together with immunoglobulins (Ig) are all exceptionally enriched in Trp residues, as compared to human proteins in general. It also reveals the conservation of a sequence motif, CX10-17 WX26-62C, in Ig domains. The conservation of this sequence motif and the utility of Trp residues within antigen-binding sites clearly contribute to the Trp enrichment in Ig. These observations suggest a biological rationale for the induction of TrpRS by IFN in safeguarding Trp incorporation for the IFN-enhanced synthesis of immunological molecules.

Wilson, C. C., J. T. Wong, et al. (1995). "Ex vivo expansion of CD4 lymphocytes from human immunodeficiency virus type 1-infected persons in the presence of combination antiretroviral agents." J Infect Dis 172(1): 88-96.

Expansion of CD4 lymphocytes from human immunodeficiency virus type 1 (HIV-1)-infected persons ex vivo has been limited by enhanced virus replication and cell death. The successful expansion of functional CD4 lymphocytes from HIV-1-infected persons has now been accomplished using a bispecific monoclonal antibody to CD3 and CD8 in combination with three antiretroviral agents. CD4 lymphocytes were polyclonally expanded by a factor of 10(3)-10(7) during 4-8 weeks in culture. Supernatants from most cultures were persistently HIV-1 p24 antigen-negative by day 14 and remained negative despite removal of antiretroviral agents at day 28. In such cultures, HIV-1 could not be recovered by cocultivation, and amounts of HIV-1 DNA declined or remained stable at low levels, eventually becoming undetectable in 2 cases. This approach establishes the feasibility of CD4 lymphocyte expansion in persons with HIV disease and may be useful for immune-based or gene therapies.

MacLean, J. A., Z. Su, et al. (1995). "Anti-CD3:anti-IL-2 receptor-bispecific mAb-mediated immunomodulation. Low systemic toxicity, differential effect on lymphoid tissue, and inhibition of cell-mediated hypersensitivity." J Immunol 155(7): 3674-82.

An anti-CD3:anti-CD25 (CD3,25) bispecific mAb was developed with the objective of combining the advantages of the parent anti-CD3 and anti-CD25 mAbs. The in vivo effects of the CD3,25 were examined in comparison to the parent Abs. The CD3,25 was well tolerated in vivo, in contrast to the parent anti-CD3 mAb, which induced systemic toxicity in recipient animals. Anti-CD3 mAb induced cell death, lymphoblast formation, and T cell activation in peripheral lymphoid organs; these were observed to a lesser extent in CD3,25-treated animals. In the thymus, anti-CD3 caused a progressive depletion of the CD4+ CD8+ "double positive" thymocytes, which was not seen in CD3,25-treated animals. This finding suggests that monovalent CD3 binding is insufficient to induce thymocyte apoptosis. Animals treated with a combination of anti-CD3 and anti-CD25 mAbs demonstrated changes in the lymphoid organs that were similar to anti-CD3-treated mice. This finding demonstrates that the effect of the CD3,25 is different than the sum of the parent Abs and suggests that the bispecific nature of the CD3,25 results in a reagent with unique immunomodulatory properties. The functional efficacy of the CD3,25 was assessed in a murine model of delayed-type hypersensitivity. The CD3,25 was as effective as the anti-CD3 mAb in inhibiting the delayed-type hypersensitivity reaction and was more effective than the parent anti-CD25 mAb. These data demonstrate that appropriately designed bispecific mAbs can be used as effective immunosuppressive agents with low systemic toxicity.

Xue, H. and J. T. Wong (1994). "Preparation of conjugated hemoglobins." Methods Enzymol 231: 308-22.

Wong, J. T., R. E. Ripple, et al. (1994). "Vancomycin hypersensitivity: synergism with narcotics and "desensitization" by a rapid continuous intravenous protocol." J Allergy Clin Immunol 94(2 Pt 1): 189-94.

BACKGROUND: We examined the clinical spectrum of patients with persistent adverse reactions to vancomycin, assessed contributing factors, and evaluated the efficacy and safety of a rapid continuous intravenous "desensitization" protocol in these patients. METHODS: Seven patients with serious staphylococcal infections resistant to beta-lactam antibiotics whose adverse reactions to vancomycin persisted despite slowing of the vancomycin infusion and pretreatment with H1-antihistamine were studied. All seven patients underwent a rapid continuous intravenous desensitization protocol with multiple small increases in vancomycin concentration tightly regulated with a syringe pump. RESULTS: Most of the seven patients safely achieved, during the first day, a vancomycin infusion rate (VIR) sufficient, or close to sufficient, to provide the desired vancomycin dose. In three patients there appeared to be a threshold VIR beyond which adverse reactions were repeatedly elicited; these reactions abated when the VIR was slightly lowered. Narcotic administration was found to adversely affect treatment with vancomycin. After concurrent narcotic administration was discontinued in three patients, they and the other four patients successfully completed the full course of treatment with vancomycin. CONCLUSION: Patients whose adverse reactions to vancomycin did not respond to slowing of the infusion rate and additional H1-antihistamines can be safely treated with a rapid continuous intravenous desensitization protocol and discontinuance of narcotic administration.

Wong, J. T., R. Y. Man, et al. (1994). "The effect of lidocaine on de novo phospholipid biosynthesis in the isolated hamster heart." Lipids 29(6): 391-6.

Lidocaine is used clinically as an antiarrhythmic agent, but its effect on cardiac phospholipid metabolism has not been defined. In this study, hamster hearts were perfused with [1,3-3H]glycerol in the presence of 0.5 mg/mL lidocaine. The incorporation of radioactivities into lysophosphatidic acid, phosphatidic acid, phosphatidylethanolamine, cytidine diphosphate diacylglycerol, phosphatidylinositol, phosphatidylserine, diacylglycerol and triacylglycerol were enhanced by lidocaine treatment, whereas the labelling of phosphatidylcholine was reduced. Analyses of enzyme activities in the heart after perfusion with lidocaine revealed that the activities of phosphatidate phosphatase and acyl-coenzyme A (CoA):1,2-diacylglycerol acyltransferase were enhanced. The presence of lidocaine in the assay did not directly stimulate these enzymes. However, the activity of acyl-CoA:glycerol-3- phosphate acyltransferase was stimulated by lidocaine whereas the activity of cytidine diphosphocholine:1,2-diacylglycerol cholinephosphotransferase was inhibited by lidocaine. We conclude that lidocaine affects the regulation of phospholipid biosynthesis in the heart by both direct and indirect modulation of phospholipid biosynthetic enzymes.

Sherry-McKenna, R. L., J. T. Wong, et al. (1994). "Monoamine oxidase inhibitors: effects on tryptophan concentrations in rat brain." J Neural Transm Suppl 41: 155-63.

It has been suggested that inhibition of tryptophan (Trp) pyrrolase and a subsequent elevation of brain Trp may contribute to the actions of antidepressant drugs. In our laboratories, we have conducted a series of experiments measuring brain Trp levels in the rat after both acute and chronic administration of several monoamine oxidase (MAO) inhibitors. The drugs studied during the course of the long-term (28 day) experiments were phenelzine, N2-acetylphenelzine, tranylcypromine, 4-fluorotranylcypromine, 4-methoxytranylcypromine and (-)-deprenyl. High-pressure liquid chromatography with electrochemical detection was employed to measure Trp levels in brains of both MAO inhibitor- and vehicle-treated animals. No significant increases in brain Trp levels were observed as a consequence of MAO inhibitor treatment. Acute time-response (up to 24 h) and dose-response studies were conducted following the administration of phenelzine and tranylcypromine. Only after administration of high doses of these drugs was an elevation in brain Trp observed and the increase was relatively short-lived. These results suggest that elevation of brain Trp may be an important factor in the actions of MAO inhibitors only at high doses of these drugs.

Logan, C. G., D. G. Lavond, et al. (1994). "Acquisition of classically conditioned eyeblink response following bilateral lesions of flocculus and paraflocculus." Behav Neural Biol 61(1): 102-6.

Stimulation of regions of the cerebellar flocculus can elicit eyeblinks, but the relationship of this floccular eyeblink zone to eyeblink classical conditioning is unknown. In this experiment, New Zealand white rabbits received bilateral lesions of the flocculus and paraflocculus and were subsequently classically conditioned with tone and corneal airpuff on the left and then the right eye. All animals reached training criterion on both eyes, with the exception of one animal whose lesion included the superior cerebellar peduncle and who was unable to learn on the ipsilateral eye. The lesioned group was not significantly different from unlesioned controls in rate of acquisition or conditioned or unconditioned response amplitude. These results indicate that the flocculus and paraflocculus are not by themselves the essential site of plasticity for classical conditioning of the rabbit eyeblink response.

Xue, H., W. Shen, et al. (1993). "Identity elements of tRNA(Trp). Identification and evolutionary conservation." J Biol Chem 268(13): 9316-22.

In this study, the varying reactivities of Bacillus subtilis tryptophanyl-tRNA synthetase toward prokaryotic, eukaryotic, and halophile tRNAs were employed to define the potential identity elements on tRNA(Trp). On this basis mutagenesis was performed to obtain, through in vivo heterologous expression in Escherichia coli and in vitro transcription with T7 RNA polymerase, mutant B. subtilis tRNA(Trp) for comparison with the wild-type. These comparisons served to establish G73 and the anticodon as major identity elements, and A1-U72, G5-C68, and A9 as minor identity elements. While the tryptophanyl-tRNA synthetase from B. subtilis and E. coli require G73 to function, replacement of G73 by A73 favors the enzyme from yeast. This change points to the variation of the identity elements for the same amino acid among different organisms. The similarity in these elements between B. subtilis and E. coli tryptophanyl-tRNA synthetase, however, suggests that identity elements on tRNA, like the active centers on enzymes, undergo evolutionary change at slower rates than less essential portions of the macromolecule.

Xue, H., W. Shen, et al. (1993). "Purification of hyperexpressed Bacillus subtilis tRNA(Trp) cloned in Escherichia coli." J Chromatogr 613(2): 247-55.

To study the effects of evolutionary sequence changes on the molecular interactions of tRNA inside the cell, the Bacillus subtilis tRNA(Trp) gene has been cloned into Escherichia coli JM109 under the control of the lac promotor. Hyperexpression of the gene in minimal medium upon induction yielded 28% of total tRNA in the form of B. subtilis tRNA(Trp). The tRNA(Trp) gene product was purified by the use of a single Vydac C4 high-performance liquid chromatography (HPLC) matrix. This experimental system provided a valuable system for the hyperexpression and purification of a heterologous tRNA for studies in vitro. Moreover, because HPLC fractionation of the heterologous tRNA(Trp) gene product yielded multiple peaks, the system made possible an analysis of the molecular mechanisms for the transcriptional modifications of the tRNA(Trp) gene product in vivo.

MacLean, J. A., Z. Su, et al. (1993). "Anti-CD3:anti-IL-2 receptor bispecific monoclonal antibody. Targeting of activated T cells in vitro." J Immunol 150(4): 1619-28.

T cells are major mediators of graft rejection and many autoimmune diseases. During the Ag recognition process, T cells often become activated. We tested the hypothesis that an anti-CD3:anti-CD25 (CD3,25) bispecific mAb (BSMAB) can effectively and selectively target activated T cells. By flow cytometric analysis, the CD3,25 BSMAB was shown to bind avidly to activated T cells that coexpress CD3 and CD25 (p55 chain of the IL-2R), achieving higher levels than the parent anti-CD3 and anti-CD25 mAb. It bound only weakly to unstimulated T cells. The CD3,25 BSMAB effectively redirected CTL to lyse CD25-bearing PHA-stimulated T lymphoblasts and the IL2-dependent CTLL tumor cell line in chromium release assays. It was highly effective in blocking MLR as shown by inhibition of [3H]TdR incorporation. However, the CD3,25 BSMAB has a low potential to activate resting T cells, as it induced only minimal [3H]TdR incorporation even in the presence of exogenous IL-2. In the absence of exogenous IL-2, the CD3,25 BSMAB was unable to induce [3H]TdR incorporation. In contrast, the parent anti-CD3 mAb induced a high degree of incorporation. In summary, the CD3,25 BSMAB selectively targets activated CD25-expressing T cells and lymphomas although maintaining a low activation potential for unstimulated T cells, potentially advantageous properties that can be exploited for immunotherapy.

Curiel, T. J., J. T. Wong, et al. (1993). "CD4+ human immunodeficiency virus type 1 (HIV-1) envelope-specific cytotoxic T lymphocytes derived from the peripheral blood cells of an HIV-1-infected individual." AIDS Res Hum Retroviruses 9(1): 61-8.

Virus-specific cytotoxic T lymphocytes (CTL) are frequently of the CD8+ surface phenotype, although CTL of the CD4+ surface phenotype have also been described. Published reports of CTL derived from peripheral blood mononuclear cells (PBMC) of individuals infected with human immunodeficiency virus type 1 (HIV-1) have described primarily cells of the CD8+ surface phenotype. However, CD4+ HIV-1 envelope-specific CTL have been reported after in vitro stimulation with HIV-1 envelope protein of peripheral blood cells obtained from HIV-1-seronegative donors, in peripheral blood cells after vaccination of HIV-1-seronegative persons with HIV-1 envelope proteins, and in cerebrospinal fluid cells of HIV-1-infected individuals. Recently, CD4+ HIV-1 gag-specific CTL were also reported. We now report a patient from whom we derived HIV-1 envelope-specific CTL cell lines of the CD4+ surface phenotype. Our cell culture technique did not employ exogenous viral antigenic stimulation, and may therefore yield cells that more closely reflect those in the underlying populations from which they were derived. These CTL did not appear to have the clear human leukocyte antigen (HLA) class II restriction pattern typically seen in CD4-expressing cells and were not functionally inhibited by anti-CD3 antibody. Further work will be required to define the role of CD4+ CTL in the pathogenesis of HIV-1 disease.

Ala, P., H. Xue, et al. (1993). "Crystallization of Bacillus subtilis tryptophanyl-tRNA synthetase." J Mol Biol 230(3): 1089-90.

Tryptophanyl-tRNA synthetase from Bacillus subtilis was overexpressed in Escherichia coli and was purified using column chromatography on DEAE-Sephacel and hydroxyapatite columns. Single crystals of the synthetase were grown by vapor diffusion at 4 degrees C from pH 5.5 solutions of polyethylene glycol 8000 containing magnesium ATP and L-tryptophan. The crystals diffracted to about 4.0 A resolution at -150 degrees C and appeared to belong to the orthorhombic space group P2(1)2(1)2 with unit cell dimensions: a = 143.6 A, b = 111.6 A, c = 50.6 A with one dimer in the asymmetric unit.

Xue, H., X. F. Wu, et al. (1992). "Properties of hemoglobin and dextran-hemoglobin rightshifted by oxidized inositol tetrakisphosphate." Artif Organs 16(4): 427-31.

Phytate was digested by wheat bran phytase to yield inositol tetrakisphosphate. Periodate-oxidized inositol tetrakisphosphate (oxyIP4) was coupled by means of reductive alkylation to hemoglobin and the covalent dextran-hemoglobin conjugate to yield the rightshifted (rs) compounds rsHb and rsDxHb, respectively. The variations of the oxygen dissociation curves of these molecules with pH and temperature were compared to those of hemoglobin. The variations with pH were found to be less pronounced for these rightshifted forms. An extensive decrease in the half-saturation oxygen tension was observed, however, with both rsHb and rsDxHb, as in the case of unmodified Hb. Modification of hemoglobin by oxyIP4 at the polyphosphate site was suggested by the lack of a further rightshifting effect of phytate on rsHb, and by the similarity between the difference spectrum of rs-methemoglobin and the difference spectrum induced by the addition of phytate.

Wong, J. T., J. V. Bonventre, et al. (1992). "Comparative studies of isolated CD3: CD8, CD3: CD3, and monovalent CD3 binding on CD8+ T-cell activation: model of progressive T-cell receptor aggregation synergism." Hum Immunol 35(3): 200-8.

The TCR and CD8 complexes of CD8+ T cells bind to different regions of MHC class I molecules and both play important roles in the response of the CD8+ T cells to Ag/MHC on APCs. In this report, we mimicked common MHC binding with an anti-CD3:anti-CD8 (CD3,8) BSMAB to isolate the effect of CD3: CD8 pairing, compared this with the effect of CD3: CD3 pairing by the parental bivalent anti-CD3 MAB, and with monovalent anti-CD3 binding by an anti-CD3: anti-CD4 (CD3,4) BSMAB. CD3: CD8 pairing induced an increase in cytosolic free [Ca2+] 1.5 to 3.0-fold greater than the increase induced by CD3: CD3 pairing whereas monovalent CD3 binding induced only 20%-30% of the increase. Postbinding receptor migration studies suggested that microaggregation increased from monovalent CD3 binding to CD3: CD3 pairing to CD3: CD8 pairing. Further studies revealed that progressively higher concentrations of antibodies were needed from CD3,8 to CD3,3 to CD3,4 to initiate the same degree of DNA synthesis. These results demonstrated that Ti/CD3 and CD8 can indeed be bridged by a single molecule. A model of direct CD8: CD3 synergism was raised as a possible explanation for the enhanced activation induced by CD3: CD8 pairing. The observed parallel between all three parameters and the number of TCRs that can be directly linked by the Abs raised a nonmutually exclusive model whereby CD3 binding induces activated TCR intermediaries (aTCRi) that progressively synergize with other adjacent aTCRis. In this model, this dominant inter-aTCRi synergism may be enhanced by the di- and multimeric CD8 alpha chains serving as aTCRi-aggregation foci.

Pandolfi, F., L. Trentin, et al. (1992). "T cell heterogeneity in patients with common variable immunodeficiency as assessed by abnormalities of T cell subpopulations and T cell receptor gene analysis." Clin Exp Immunol 89(2): 198-203.

T lymphocyte regulation of immunoglobulin production may be abnormal in some patients with common variable immunodeficiency (CVI). Phenotypic analysis of peripheral blood T lymphocytes from nine patients with CVI was conducted to examine whether an abnormal distribution could be detected in a functionally distinct T lymphocyte subpopulation. The percentage of CD8+ lymphocytes proved to be increased in some patients and decreased in others. In comparison with normal controls, many patients with CVI had reduced percentages of lymphocytes expressing both CD4 and CD45RA, a phenotype associate with naive CD4+ cells. There was no significant difference in CD4+ populations bearing CD29 or leucocyte adhesion molecule-1 (LAM-1) antigens. The pattern of gene rearrangement of the T cell antigen receptor (TCR) was studied using peripheral blood lymphocytes from these patients with CVI. Genomic DNA from freshly isolated lymphocytes as well as from selectively propagated CD4+ or CD8+ populations were examined using Southern blot analysis and a probe for the beta chain of the TCR. A polyclonal pattern of TCR gene rearrangement, without the appearance of dominant non-germline bands, was demonstrated in all patient samples. These data suggest that the T lymphocytes in patients with CVI have a polyclonal pattern of TCR rearrangement despite an abnormal distribution of T cell subpopulations in some patients.

Koziel, M. J., D. Dudley, et al. (1992). "Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis." J Immunol 149(10): 3339-44.

Hepatitis C virus (HCV) is a major cause of post-transfusion and sporadic hepatitis worldwide, leading to chronic liver disease in at least 50% of infected individuals. The pathogenic mechanisms that result in chronic hepatitis are unknown. Lymphocytes are typically observed within the hepatic parenchyma, but the functional characteristics of these cells have not been defined. In this study, liver-infiltrating lymphocytes from two subjects with chronic HCV hepatitis were cloned at limiting dilution and tested for HCV-specific cytolytic activity using autologous target cells infected with vaccinia viruses expressing recombinant HCV Ag or sensitized with synthetic HCV peptides. In both subjects, HCV-specific, HLA class I-restricted CTL were identified that recognized epitopes in variable regions of either the envelope or nonstructural proteins. These results demonstrate the presence of HCV-specific CTL at the site of tissue damage in persons with chronic HCV hepatitis, and provide a means to evaluate the possible pathogenic role of these cells in HCV infection.

Chow, K. C., H. Xue, et al. (1992). "Mutational identification of an essential tryptophan in tryptophanyl-tRNA synthetase of Bacillus subtilis." J Biol Chem 267(13): 9146-9.

The strongly conserved single tryptophan residue, Trp92, in Bacillus subtilis tryptophanyl-tRNA synthetase has been mutagenized via site direction singly into Gln, Ala, and Phe. All three mutant enzymes were inactive toward the catalysis of tRNA tryptophanylation. The Trp92----Phe mutant has been subcloned into the high expression plasmid pKK223-3 to yield the recombinant plasmid pKSW-F92. Growth of bacteria carrying the latter plasmid made possible the purification of the mutant TrpRS-F92 enzyme to homogeneity. This mutant enzyme was deficient in ultraviolet absorbance and fluorescence relative to the wild type enzyme and inactive in the partial reaction of Trp-activation as well as the overall reaction of tRNA tryptophanylation. Furthermore, unlike the wild type B. subtilis trpS gene, the mutant trpS-F92 gene upon transformation into Escherichia coli trpS 10343 failed to complement the temperature sensitive trpS mutation of the host cells. Trp92 therefore represents an essential residue both in vitro and in vivo for the function of the tryptophanyl-tRNA synthetase.

Wong, J. T. and R. B. Colvin (1991). "Selective reduction and proliferation of the CD4+ and CD8+ T cell subsets with bispecific monoclonal antibodies: evidence for inter-T cell-mediated cytolysis." Clin Immunol Immunopathol 58(2): 236-50.

CD3,4 (anti-CD3:anti-CD4) bispecific monoclonal antibodies (BSMAB) cause a profound decrease in CD4+ T cells and a marked proliferation of CD8+ T cells in peripheral blood mononuclear cells in vitro. CD3,8 (anti-CD3:anti-CD8) BSMAB causes a reciprocal decrease in CD8+ T cells and a proliferation of CD4+ T cells. The major effector of CD4+ T cell cytolysis in the presence of CD3,4 resides in the CD8+ T cell population. In contrast, both the CD4+ and CD8+ T cells are effective mediators of cytolysis of the CD8+ T cells in the presence of the CD3,8. The likely underlying mechanism in each case is bridging of the CD4 and CD8 of the target cells to the CD3 complexes of the effector cells by antibodies, mimicking the natural encounter between a cytolytic T cell and its target. Proliferation studies indicated that CD3,4 and CD3,8 each can induce proliferation of both CD4+ and CD8+ T cells in the presence of accessory cells. These results suggest that the major selection of the BSMABs occurs via selective destruction of one T cell subset with concurrent stimulation of the remaining CD3+ population. Potential applications of the selective destruction and proliferation include study and manipulation of the T cell subsets in HIV infections, tumor infiltrating lymphocytes, autoimmune diseases, and graft rejection.

Wong, J. T. (1991). "Origin of genetically encoded protein synthesis: a model based on selection for RNA peptidation." Orig Life Evol Biosph 21(3): 165-76.

The difficulty in explaining the origin of genetic coding centres on the need to identify selective advantages that could account for the synthesis of peptidyl-tRNA, the essential intermediate in genetically programmed translation. It is resolved by a recognition of the functional advantages derivable from the post-transcriptional addition of peptide cofactors to RNA apo-catalysts. This enables the formulation of a theory for the origin of the genetic encoding of protein synthesis by RNA.

Littaua, R. A., M. B. Oldstone, et al. (1991). "An HLA-C-restricted CD8+ cytotoxic T-lymphocyte clone recognizes a highly conserved epitope on human immunodeficiency virus type 1 gag." J Virol 65(8): 4051-6.

A unique epitope on the gag protein of human immunodeficiency virus type 1 (HIV-1), located at amino acid 145 to 150, has been mapped by using a CD8+ cytotoxic T-lymphocyte (CTL) clone. This epitope is highly conserved among 18 HIV-1 strains. The HIV-1 gag-specific human leukocyte antigen (HLA) class I-restricted CD8+ CTL clone was generated from fresh peripheral blood mononuclear cells of an HIV-seropositive donor by stimulation with gamma-irradiated allogeneic peripheral blood mononuclear cells in the presence of an anti-CD3 monoclonal antibody and recombinant interleukin-2. This gag-specific CTL clone killed autologous target cells infected with a recombinant vaccinia virus containing the gag gene of HIV-1 and target cells pulsed with an authentic p24gag construct expressed in Escherichia coli. Fine specificity was determined by using a panel of overlapping 30-amino-acid-long synthetic peptides and subsequently using smaller peptides to precisely map the CTL domain on p24. The epitope is on a highly conserved region, and it overlaps with a major B-cell epitope of gag. This CD8+ T-cell epitope is restricted by HLA-Cw3, which has not been previously identified as a restricting element for human CTL responses.

Brown, E. E., J. M. Finlay, et al. (1991). "Behavioral and neurochemical interactions between cocaine and buprenorphine: implications for the pharmacotherapy of cocaine abuse." J Pharmacol Exp Ther 256(1): 119-26.

Intravenous self-administration studies in nonhuman primates suggest that the opioid receptor agonist-antagonist buprenorphine may be useful in the pharmacotherapy of cocaine abuse. In the present studies, behavioral and neurochemical interactions between cocaine and buprenorphine were examined using a conditioned place preference (CPP) procedure and in vivo microdialysis. Cocaine-induced CPP was linearly related to the dose administered (0-5.0 mg/kg). Buprenorphine (0-0.9 mg/kg) also elicited CPP in a dose-related manner; an inverted U-shaped function was obtained. Subthreshold doses of cocaine (1.5 mg/kg) and buprenorphine (0.01 mg/kg), themselves incapable of eliciting CPP, produced a significant CPP when given together. Moderate doses of cocaine (5.0 mg/kg) and buprenorphine (0.075 mg/kg), which were individually capable of eliciting CPP, produced a significantly larger CPP when given in combination. In the in vivo microdialysis studies, a low dose of buprenorphine (0.01 mg/kg) produced a progressive increase in extracellular dopamine in the nucleus accumbens, reaching approximately 200% of basal levels after 5 hr. Cocaine (5.0 mg/kg) rapidly increased extracellular dopamine concentrations (180% of basal values within 20 min), which returned to baseline in 2 to 3 hr. This effect of cocaine was significantly potentiated by coadministering buprenorphine (0.01 mg/kg); under this condition the peak increase in extracellular dopamine reached 260% of baseline values. These neurochemical findings are consistent with the CPP results and indicate that buprenorphine can interact with cocaine in a synergistic manner. In contrast to previous speculations, these results suggest that buprenorphine may enhance rather than attenuate the rewarding properties of cocaine.

Baker, G. B., J. T. Wong, et al. (1991). "Effects of the antidepressant phenelzine on brain levels of gamma-aminobutyric acid (GABA)." J Affect Disord 21(3): 207-11.

Time- and dose-response studies were carried out on the effects of the monoamine oxidase-inhibiting antidepressant and antipanic drug phenelzine on GABA levels in rat whole brain. At a dose of 15 mg/kg i.p., phenelzine significantly elevated GABA levels at all time intervals studied (1, 2, 4, 8, 16, and 24 h). A further investigation indicated that this was a dose-dependent effect. The possible importance of GABA in the etiology and pharmacotherapy of depression and panic disorder is discussed.

Wong, J. T., G. B. Baker, et al. (1990). "A rapid, sensitive assay for gamma-aminobutyric acid in brain using electron-capture gas chromatography." Res Commun Chem Pathol Pharmacol 70(1): 115-24.

A rapid, sensitive procedure has been developed for the assay of gamma-aminobutyric acid (GABA) in brain tissue. The method involves sequential reaction with isobutyl chloroformate and pentafluorophenol under aqueous conditions and subsequent separation and quantitation of the derivative on a gas chromatograph equipped with a capillary column and an electron-capture detector. The method produces a derivative with good stability and provides for simultaneous analysis of GABA and five other aliphaticamino acids in very small volumes of supernatant from brain homogenates.

Wong, J. T., A. A. Eylath, et al. (1990). "The mechanism of anti-CD3 monoclonal antibodies. Mediation of cytolysis by inter-T cell bridging." Transplantation 50(4): 683-9.

OKT3, an anti-CD3 MAB, depletes T cells in vivo and is among the most potent inhibitors of acute allograft rejection. The mechanism of this inhibition is unknown. The present studies investigate whether anti-CD3 antibodies have the ability to crosslink CD3 on two different cells and induce TCR-dependent antibody-bridged cell-mediated cytolysis (TCR-ABCMC) between T cells. Two different anti-CD3 antibodies (OKT3 and CD3,3) and OKT3 F(ab')2 were all highly effective in inducing cytolysis of CD8+ and CD4+ T cells by CD8+ T cells, and CD8+ T cells by CD4+ T cells. Monovalent OKT3 Fab was 25-125-fold less potent than OKT3 F(ab')2. Monovalent CD3,X was totally ineffective. The necessity for intercellular bridging was evidenced by the observation that an anti-CD3:anti-CD4 (CD3,4) bispecific MAB (BSMAB) was effective in mediating lysis of CD4+ but not CD8+ T cells by CD8+ T cells. These studies indicate that neither FcR-mediated ADCC nor complement fixation is necessary for bivalent anti-CD3 MAB to lyse T cells. Inter-T cell TCR-ABCMC may be particularly effective in inflammatory tissues, such as rejecting allografts and autoimmune diseases, in which numerous cytolytic T lymphocytes are present in close association with other T cells.

Wong, J. T., G. B. Baker, et al. (1990). "Long-lasting elevation of alanine in brain produced by the antidepressant phenelzine." Brain Res Bull 25(1): 179-81.

Time- and dose-response studies were conducted to determine the effects of the antidepressant and antipanic drug phenelzine (a monoamine oxidase inhibitor) on whole brain levels of the aliphatic amino acid alanine. At a dose of phenelzine of 15 mg/kg IP, there was a significant increase in brain levels of alanine up to 16 hr after drug administration. Dose studies at 4 hr indicated that the alanine elevation after phenelzine was a dose-dependent effect.

Wong, J. T., P. R. Paetsch, et al. (1990). "A rapid procedure for the analysis of phenylalanine in brain tissue utilizing electron-capture gas chromatography." J Neurosci Methods 32(2): 105-9.

A rapid procedure has been developed for the analysis of phenylalanine in brain tissue. Perchloric acid extracts of brain tissue were made basic, and benzoyl chloride was added to derivatize the amine function. The aqueous layer was retained and made slightly acidic. To derivatize the carboxylic acid group, a solution of pentafluorobenzyl alcohol was added in the presence of the coupling agent dicyclohexylcarbodiimide and chloroform. After shaking for 15 min, the organic phase was retained and taken to dryness. The residue was taken up in toluene, washed, and an aliquot used for analysis on a gas chromatograph equipped with an automatic injector, a capillary column and an electron-capture detector. The procedure has been utilized for analysis of phenylalanine in brains of rats treated with vehicle or phenylalanine.

Wong, J. T., E. Schott, et al. (1990). "Immunogenic epitopes of the p55 chain of the IL-2 receptor. Relationships to high-affinity IL-2 binding and modulation of the p55 chain." Transplantation 49(3): 587-96.

Monoclonal antibodies were used to determine the relationships between epitopes on the p55 chain of the IL-2 receptor and high-affinity IL-2 binding. Five monoclonal antibodies to the human P55 chain of the IL-2 receptor were induced by immunizing mice with murine L cells that were transfected with human p55 cDNA. Since the p55 chain is the only human antigen expressed on these cells, all antihuman MABs thus generated were directed against this molecule. These antibodies were used to map epitopes on the p55 chain and determine their relationship to high-affinity IL-2 binding. Extensive flow cytometric studies with these MABs and a large panel of other anti-p55 MABs revealed three major patterns of competition. Type I MABs compete with anti-Tac extensively but not with antibodies of other groups. Type II MABs do not block anti-Tac but do block 7E11. Type III MABs do not block either type I or type II antibodies. 125I-IL2 competition studies under high-affinity conditions revealed that types I and II MABs inhibit IL-2 binding. Type III MABs can be resolved into two subgroups, one that inhibits IL-2 binding and one that does not. Together these data suggest that there are at least four distinct immunogenic epitopes on the human p55 chain, with three epitopes related to IL-2 binding. The competitive component evident by a change in Kd on the Scatchard plots suggests that all three epitopes are close to or part of the IL-2-binding site of the p55 chain. The noncompetitive component, as evidenced by the lower number of high-affinity IL-2 receptors induced by these antibodies, suggests that the same epitopes are also close to the site(s) of interaction between the p55 and p70 chains to form the high-affinity receptor. These studies indicated that the IL-2-binding site and site of interaction between the p55 and p70 chains are close together or identical. Modulation studies revealed that one type II antibody (7E11) modulates the p55 chain in the absence of IL2 and the p70 chain, thus revealing that modulation of the p55 chain can occur by an active process, and not merely passively comodulate by the p70 chain upon IL-2 binding. Modulation of the p55 chain alone has no proliferative effect on IL-2-responsive T lymphoblasts. Potentially this antibody-dependent modulation may be used to deliver toxin to activated lymphocytes.

Shi, W., K. C. Chow, et al. (1990). "High-level expression of Bacillus subtilis tryptophanyl-tRNA synthetase in Escherichia coli." Biochem Cell Biol 68(2): 492-5.

The trpS gene encoding Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was prepared from the pUC8-derived pTSQ2 plasmid, mutagenized to introduce an EcoRI site immediately in front of the ATG start codon, and inserted into the pKK223-3 vector downstream to the tac promoter to yield the pKSW1 plasmid. Upon induction with isopropyl-beta-D-thiogalactopyranoside, Escherichia coli JM109[pKSW1] cells synthesized TrpRS to a level corresponding to 45% of total cell proteins. This high level of gene expression facilitates large scale preparation of TrpRS for physical studies, detection of in vivo degradation of mutant forms of TrpRS, and comparative assays of TrpRS by [3H]Trp-tRNA formation and by Trp-hydroxamate formation for the purpose of mutant characterization. Finally, since pKSW1 could complement the temperature-sensitive TrpRS mutation on E. coli trpS 10343 cells, defective mutations of the trpS gene on pKSW1 would be deductible on the basis of complementation testing.

Bornstein, R. A., G. B. Baker, et al. (1990). "Plasma amino acids in attention deficit disorder." Psychiatry Res 33(3): 301-6.

This study examines plasma amino acids in a group of 28 patients meeting DSM-III criteria for attention deficit disorder (ADD) and 20 control subjects. Compared with controls, the ADD subjects had significantly lower levels of phenylalanine, tyrosine, tryptophan, histidine, and isoleucine. These data suggest a general deficit in amino acid transport, absorption, or both.

Xu, Z. J., M. L. Love, et al. (1989). "Tryptophanyl-tRNA synthetase from Bacillus subtilis. Characterization and role of hydrophobicity in substrate recognition." J Biol Chem 264(8): 4304-11.

The tryptophanyl-tRNA synthetase from Bacillus subtilis was purified to homogeneity and characterized. It has an alpha 2 subunit structure and a molecular weight of 77,000. Tryptophanyl-tRNA synthetase does not catalyze any significant proofreading. It activates tryptophan as well as the three fluorinated analogues, DL-4-fluoro-, DL-5-fluoro-, or DL-6-fluorotryptophan (4F-, 5F-, and 6F-Trp), in the ATP-pyrophosphate exchange reaction. In the aminoacylation reaction, the fluorotryptophans act as competitive inhibitors of Trp. Their relative activities follow the same order in both reactions: Trp greater than 4F-Trp greater than 6F-Trp greater than 5F-Trp. This order is the inverse of the order of relative hydrophobicities of these compounds, pointing to the importance of hydrophobic interactions in the selective recognition by tryptophanyl-tRNA synthetase among this group of substrates. To define the physical basis of the relative hydrophobicities, the crystallographic structure of 4F-Trp was determined and compared to that of trptophan. Charge distributions calculated for tryptophan and its different fluoroanalogues on the basis of molecular structures were supported by their carbon-13 NMR spectra. Correlations between charge distributions and relative hydrophobicities suggest that the polarity of the C-F bond represents an underlying factor determining the hydrophobicities of 4F-, 5F-, and 6F-Trp, thus relating tryptophanyl-tRNA synthetase selectivity toward tryptophan and its fluoroanalogues directly to their electronic configurations.

Wong, J. T., C. E. Pinto, et al. (1989). "Characterization of the CD4+ and CD8+ tumor infiltrating lymphocytes propagated with bispecific monoclonal antibodies." J Immunol 143(10): 3404-11.

To study the CD4+ and CD8+ tumor infiltrating lymphocytes (TIL) in the antitumor response, we propagated these subsets directly from tumor tissues with anti-CD3:anti-CD8 (CD3,8) and anti-CD3:anti-CD4 (CD3,4) bispecific mAb (BSMAB). CD3,8 BSMAB cause selective cytolysis of CD8+ lymphocytes by bridging the CD8 molecules of target lymphocytes to the CD3 molecular complex of cytolytic T lymphocytes with concurrent activation and proliferation of residual CD3+CD4+ T lymphocytes. Similarly, CD3,4 BSMAB cause selective lysis of CD4+ lymphocytes whereas concurrently activating the residual CD3+CD8+ T cells. Small tumor fragments from four malignant melanoma and three renal cell carcinoma patients were cultured in medium containing CD3,8 + IL-2, CD3,4 + IL-2, or IL-2 alone. CD3,8 led to selective propagation of the CD4+ TIL whereas CD3,4 led to selective propagation of the CD8+ TIL from each of the tumors. The phenotypes of the TIL subset cultures were generally stable when assayed over a 1 to 3 months period and after further expansion with anti-CD3 mAb or lectins. Specific 51Cr release of labeled target cells that were bridged to the CD3 molecular complexes of TIL suggested that both CD4+ and CD8+ TIL cultures have the capacity of mediating cytolysis via their Ti/CD3 TCR complexes. In addition, both CD4+ and CD8+ TIL cultures from most patients caused substantial (greater than 20%) lysis of the NK-sensitive K562 cell line. The majority of CD4+ but not CD8+ TIL cultures also produced substantial lysis of the NK-resistant Daudi cell line. Lysis of the autologous tumor by the TIL subsets was assessed in two patients with malignant melanoma. The CD8+ TIL from one tumor demonstrated cytotoxic activity against the autologous tumor but negligible lysis of allogeneic melanoma targets. In conclusion, immunocompetent CD4+ and CD8+ TIL subsets can be isolated and expanded directly from small tumor fragments of malignant melanoma and renal cell carcinoma using BSMAB. The resultant TIL subsets can be further expanded for detailed studies or for adoptive immunotherapy.

Wong, J. T., G. B. Baker, et al. (1988). "Rapid and simple procedure for the determination of urinary phenylacetic acid using derivatization in aqueous medium followed by electron-capture gas chromatography." J Chromatogr 428(1): 140-6.

Wong, J. T. (1988). "Evolution of the genetic code." Microbiol Sci 5(6): 174-81.

The structure of the genetic code suggests that amino acid biosynthesis and hydrophobicity were important factors in shaping the genetic code, as the primitive code coevolved with new varieties of amino acids generated by the expanding pathways of biosynthesis. The current code is exceptionally stable. Deviant codes nonetheless have been observed in a number of mitochondrial and cellular genomes. Even the membership of encoded amino acids is undergoing expansion to include phosphoserine and selenocysteine. Experimental mutation of the code also has proven feasible, in a replacement of tryptophan by 4-fluorotryptophan as a component constituent of proteins. Such mutations, introducing novel varieties of encoded amino acids, will open up a new dimension in protein engineering and design.

Wong, J. T. (1988). "Rightshifted dextran-hemoglobin as blood substitute." Biomater Artif Cells Artif Organs 16(1-3): 237-45.

Covalent modification of hemoglobin and dextran-hemoglobin by reductive alkylation under aerobic conditions with periodated inositol tetrakisphosphate brought about a rightshifting of their oxygen dissociation curves. The rightshifted dextran-hemoglobin conjugate provided adequate oxygen delivery in macaques exchange-transfused to 90% with respect to their erythrocyte content. The conjugate did not exhibit the nephrotoxicity of free hemoglobin. It was also more resistant to precipitation, and therefore more amenable to viral sterilisation, by ethanol.

Tam, S. C. and J. T. Wong (1988). "Impairment of renal function by stroma-free hemoglobin in rats." J Lab Clin Med 111(2): 189-93.

Hemoglobin infused into rats was excreted mainly by the kidneys. The effects of hemoglobin on renal tubular functions were investigated in this study. During intravenous infusion of 6% stroma-free hemoglobin (SFHB) solution, there was a 30% reduction in glomerular filtration rate (GFR). The reduction in GFR was directly related to the urine hemoglobin concentration. The rate of urinary sodium excretion (8.2 mumol/min) was significantly higher than the control rate (2.8 mumol/min), suggesting proximal tubular damage in the presence of intratubular hemoglobin. This was further substantiated by the demonstration of a 10-fold increase in urinary N-acetyl-beta-D-glucosaminidase (NAG) activity during SFHB infusion. In contrast, dextran-hemoglobin (DXHB) infused into rats was not excreted by the kidneys. During the infusion of 6% stroma-free DXHB solution, there was no significant change in the GFR. Urinary sodium excretion was lower than the control value whereas urinary NAG activity was slightly elevated. The observations suggest that the presence of hemoglobin in the renal tubules gives rise to impairment of tissue function and structure. DXHB, on account of its larger molecular size, does not enter into the renal tubules, and therefore does not cause such impairment.

Chow, K. C. and J. T. Wong (1988). "Cloning and nucleotide sequence of the structural gene coding for Bacillus subtilis tryptophanyl-tRNA synthetase." Gene 73(2): 537-43.

A 1.47-kb DNA fragment that carries the tryptophanyl-tRNA synthetase (TrpRS) gene of Bacillus subtilis has been cloned into the pUC8 plasmid. The recombinant plasmid, pTSQ2, conferred temperature-resistance to the temperature-sensitive trpS ts mutant of B. subtilis through chromosomal transformation, and to that of Escherichia coli through complementation. The pTSQ2 could be stably maintained in E. coli DH5 alpha, causing in the host cell a 200-fold amplification of TrpRS activity. The complete nucleotide sequence of the cloned fragment has been determined. A putative transcriptional promoter, a Shine-Dalgarno sequence, the 990-bp trpS gene proper, as well as a transcriptional terminator have been identified.

Bronskill, P. M. and J. T. Wong (1988). "Suppression of fluorescence of tryptophan residues in proteins by replacement with 4-fluorotryptophan." Biochem J 249(1): 305-8.

The tryptophan-auxotrophic Bacillus subtilis LC33 mutant strain utilizes either tryptophan or 4-fluorotryptophan for growth. Proteins therefore could be isolated from these cells in either tryptophan-containing or 4-fluorotryptophan-containing forms. Since 4-fluorotryptophan is non-fluorescent, tryptophan fluorescence would be suppressed in the 4-fluorotryptophan-containing proteins, facilitating the investigation of other chromophores either on the proteins or interacting with the proteins. This approach, potentially applicable to any protein endogenous to or clonable into B. subtilis, was illustrated by an examination of the fluorescence of B. subtilis ribosomal proteins.

Wong, J. T. and R. B. Colvin (1987). "Bi-specific monoclonal antibodies: selective binding and complement fixation to cells that express two different surface antigens." J Immunol 139(4): 1369-74.

A new dimension of immunotherapeutic selectivity might be achieved if antibodies could distinguish cells that co-express two different surface antigens. Bi-specific monoclonal antibodies (BSMAB) with two different antigen combining sites that share a common Fc region theoretically might have such a potential. Two such BSMAB were produced by hybrid-hybridoma clones prepared by fusion of pre-existing hybridomas and were purified by isoelectric focusing. CD3,4 (IgG2a, IgG2b) recognizes the T cell surface antigens CD3 and CD4, and CD3,8 (IgG2a, IgG2a) recognizes CD3 and CD8. These BSMAB promote complement-mediated lysis of target cells that bear both surface antigens 25 to 3125 times more efficiently than those that express only one of the antigens. This selectivity results from the increased avidity of these antibodies for cells with both antigens, as reflected by the increased surface immunoglobulin concentration detected by flow cytometry. It was also demonstrated that there exists a threshold surface immunoglobulin density necessary for antibody-dependent complement-mediated cytotoxicity microtiter assays for the various IgG antibodies tested in both bivalent and monovalent binding. These results support the associative model of IgG-mediated complement fixation.

Wang, Q. S., P. M. Bronskill, et al. (1987). "Complementary base-pairing properties of cyclized and linear oligonucleotides." Arch Biochem Biophys 255(1): 176-83.

Oligouridylates of varying chain lengths were synthesized by polynucleotide phosphorylase and cyclized by RNA ligase. Over chain lengths from 7 to 15, the bindings of the cyclized and linear oligomers to polyadenylate were measured on the basis of differential migration of bound and free oligomers on a gel exclusion column. Binding of the cyclized oligomers was found to be far weaker than that of their linear counterparts of equal length. Such a general reduction in base-pairing capacity due to the cyclized conformation, by limiting the strength of unintended base-pairing without obstructing the possible development of strong specific base-pairing, may represent an advantage important to the function and evolution of loop structures in tRNA and other RNA molecules.

Baker, G. B., J. T. Wong, et al. (1987). "Simultaneous extraction and quantitation of several bioactive amines in cheese and chocolate." J Chromatogr 392: 317-31.

A method is described for simultaneous extraction and quantitation of the amines 2-phenylethylamine, tele-methylhistamine, histamine, tryptamine, m- and p-tyramine, 3-methoxytyramine, 5-hydroxytryptamine, cadaverine, putrescine, spermidine and spermine. This method is based on extractive derivatization of the amines with a perfluoroacylating agent, pentafluorobenzoyl chloride, under basic aqueous conditions. Analysis was done on a gas chromatograph equipped with an electron-capture detector and a capillary column system. The procedure is relatively rapid and provides derivatives with good chromatographic properties. Its application to analysis of the above amines in cheese and chocolate products is described.

Zhang, S. B., P. M. Bronskill, et al. (1986). "Separation of tRNA by high-performance liquid chromatography at ambient temperature." J Chromatogr 360(1): 282-7.

Wong, J. T. and R. Cedergren (1986). "Natural selection versus primitive gene structure as determinant of codon usage." Eur J Biochem 159(1): 175-80.

Different codons are not utilized equally in known gene sequences. One of the important biases of codon usage is observed in the form of an enrichment of RNY codons, especially within RNN codon families. Such biases could represent the residue of a primitive repeating-RNY gene structure, or the outcome of natural selection, or both. Analyses based on the rates of silent substitutions, the frequencies of base doublets, and synonymous codon ratios for Escherichia coli, yeast, Drosophila and Xenopus proteins have been performed. The results rule out any significant support for a primitive repeating-RNY or repeating-RRY gene structure, and establish the important role of natural selection in determining the choice of codons. With strong intervention by natural selection, the relationship between primitive gene structure and codon usage necessarily becomes minimal.

Wong, J. T. and R. P. Hill (1986). "Biophysical basis of hypoxic radioprotection by deoxygenated dextran-hemoglobin." Int J Radiat Oncol Biol Phys 12(8): 1303-6.

Perfusion with deoxygenated dextran-hemoglobin provides an effective method for inducing hypoxic radioprotection of normal tissues during radiation treatment of tumors. In this study, the dependence of P50, the half-saturation pressure of oxygen binding to dextran-hemoglobin, was analyzed as a function of solution temperature and pH. The variation of attainable radioprotection with P50, and with the amount of collateral blood entering into the perfused region, was calculated. Upon perfusion of canine gracilis muscle with deoxygenated dextran-hemoglobin, a rapid onset of extensive venous hypoxia was observed.

Applegarth, D. A., H. L. Levy, et al. (1986). "Prenatal diagnosis of non-ketotic hyperglycinemia." Prenat Diagn 6(4): 257-63.

We describe successful prenatal diagnosis in four pregnancies at risk for non-ketotic hyperglycinemia, two affected and two unaffected, using the glycine level and the glycine/serine ratio in amniotic fluid obtained at 16 weeks gestational age. Although this method of prenatal diagnosis for non-ketotic hyperglycinemia has been effective in our hands the narrow differences between affected and unaffected pregnancies indicate the need for caution concerning its reliability.

Rivera, J. A., Q. S. Wang, et al. (1984). "A highly thermostable proline:tRNA ligase from Thermus aquaticus. Purification and enzyme-tRNA recognition at elevated temperatures." Can J Biochem Cell Biol 62(6): 507-15.

Proline:tRNA ligase from Thermus aquaticus was purified to homogeneity and characterized. Its molecular weight was found to be 127 000, consisting of two identical subunits. It catalysed the prolylation of tRNAPro from Escherichia coli with a bell-shaped pH dependence peaking at about pH 7 and exhibited extreme thermostability. The Vm/Km ratios of steady-state kinetics for proline and ATP as well as tRNAPro were not extensively diminished even at 85 degrees C, but prolylation became insignificant at 90 degrees C. Since the melting of tRNAPro was in progress, yet incomplete, at 85 degrees C, these findings suggest that some threshold level of conformational integrity of tRNAPro, rather than the entire unmelted conformation of the molecule, is essential to effective recognition by the proline:tRNA ligase.

Hill, R. P., L. S. Porter, et al. (1984). "Initial studies of hypoxic radioprotection by deoxygenated dextran-hemoglobin." Int J Radiat Oncol Biol Phys 10(3): 369-73.

Initial studies were performed to examine the potential of perfused dextran-hemoglobin to protect pig skin or mouse bone marrow cells against radiation damage. Some protection was indicated in both systems. In the pig skin a protection factor of 1.5 was observed for moist desquamation, and 2.0 for necrosis. These results suggest the possibility of using blood substitutes to induce tissue hypoxia for therapeutic purposes.

Wong, J. T. (1983). "Membership mutation of the genetic code: loss of fitness by tryptophan." Proc Natl Acad Sci U S A 80(20): 6303-6.

Bacillus subtilis strain QB928, a tryptophan-auxotroph, was serially mutated to yield strain HR15. For QB928, tryptophan functioned as a competent amino acid and 4-fluorotryptophan as merely an inferior analogue. For HR15, these roles were reversed. The tryptophan/4-fluorotryptophan growth ratio decreased by a factor of 2 X 10(4) in the transition from QB928 to HR15.

Wong, J. T. and L. S. Porter (1983). "Ligand binding to protein measured by gel filtration on a dispersion column." Anal Biochem 130(2): 491-7.

A double conical gel-permeation column is described, which permits an efficient dispersion of solutes in passage. It makes possible a facile determination of ligand binding to protein on the basis of the differential gel permeation of these molecules. The system has been applied, using Sephadex G-25 beads as column packing, to measure [3H]tryptophan binding to bovine serum albumin.

Wang, Q. S. and J. T. Wong (1983). "Purification of isoacceptor transfer RNA by affinity coupling to aldehyde-containing matrix." Anal Biochem 131(2): 360-4.

A method has been developed for the affinity coupling of aminoacyl-tRNA to an aldehyde-containing polymer by means of reduction with sodium cyanoborohydride. Sephadex dialdehyde obtained from periodate oxidation of Sephadex G-50, and Enzacryl polyaldehyde obtained from hydrolysis of Enzacryl polyacetal, were found to provide the requisite polymeric matrix. This coupling reaction permitted the rapid purification of isoacceptor tRNAs of Escherichia coli for the alpha-amino acids glycine, valine, and arginine, as well as the alpha-imino acid proline.

Gu, X. R., K. Nicoghosian, et al. (1983). "Sequences of halobacterial tRNAs and the paucity of U in the first position of their anticodons." Nucleic Acids Res 11(16): 5433-42.

The sequence of three tRNAs from Halobacterium cutirubrum have been determined. The sequences of tRNAValGAC and tRNAValCAC differ by only one nucleotide which is in the 5' terminal anticodon position. These tRNAs as well as that of tRNAAlaCGC are compared to other known halobacterial tRNAs. An observed paucity (or absence) of U in the first anticodon position is unique to archaebacterial tRNAs and may be indicative of unusual decoding properties of these organisms.

Roberts, L., P. Sadowski, et al. (1982). "Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein." Biochemistry 21(23): 6000-5.

Bacteriophage T7 codes for a single-stranded DNA binding protein. This protein is the product of gene 2.5 and has been found previously to stimulate specifically the activity of the phage-coded DNA polymerase. We report here that the T7 DNA binding protein also stimulates the activity of the phage-coded exonuclease. The gene 6 exonuclease is a double-stranded DNA specific 5'-exonuclease that has been implicated in destruction of bacterial DNA, removal of RNA primers during DNA replication, genetic recombination, and DNA maturation. The enzyme is markedly inhibited by physiological concentrations of NaCl. This inhibition, which is due to a marked reduction in the Vmax of the enzyme, can be largely overcome by the phage-coded DNA binding protein. This stimulation is specific since the Escherichia coli DNA binding protein is without effect. The stimulation by the binding protein is apparently not due to its coating of the 3' single-stranded tails generated during the digestion. Kinetic studies show that the stimulation is due to a combined effect on both the Km and Vmax of the exonuclease. These studies are consistent with a loose binding of the binding protein to either the DNA or the exonuclease.

Liu, W., J. Zhou, et al. (1982). "A novel synthesis of 3,5-diiodotyrosine with iodic acid." Anal Biochem 120(1): 204-7.

Lin, S. X., K. C. Chou, et al. (1982). "A thermal-variation method for analysing the rate constants of the Michaelis--Menten mechanism." Biochem J 207(1): 179-81.

By analysing the variations of saturation velocity and Michaelis constant with temperature and invoking the mathematical constraint represented by the Arrhenius equation, it becomes possible to estimate k+2 and indistinguishably k+1 and k-1 for the Michaelis--Menten mechanism of one-substrate enzyme reactions. Distinction between k+1 and k-1 may be obtained through the determination of isotopic rate effects. This procedure thus provides a basis for evaluating all three rate constants of the one-substrate mechanism, and disproves the suggestion that k+1 and k-1 are intrinsically unobtainable from steady-state kinetic measurements.

Cunnington, P. G., S. N. Jenkins, et al. (1981). "Oxygen-binding and immunological properties of complexes between dextran and animal haemoglobins." Biochem J 193(1): 261-6.

Complexes of dextran 20 000 with haemoglobins of sheep, rabbit, dog, bovine and human origin were prepared through alkylation of haemoglobin by N-bromoacetylaminoethylamino-dextran. The yields were uniformly high. Complex-formation in each case was accompanied by the disappearance of reactive thiol groups on the haemoglobin, and by an increase in the affinity of the haemoglobin for oxygen. The immunological properties of dog, rabbit and sheep dextran-haemoglobin were investigated in both homologous and heterologous species. The complexes were found to be non-immunogenic in the homologous species. In heterologous species the anti-haemoglobin response induced by each complex was generally of a similar level to that induced by the haemoglobin alone.

Wong, J. T. (1980). "Role of minimization of chemical distances between amino acids in the evolution of the genetic code." Proc Natl Acad Sci U S A 77(2): 1083-6.

The allocation of codons in the genetic code makes possible a moderate minimization of the chemical distances between pairs of neighboring amino acids in the code. However, the code is neither a global nor a local optimum with respect to distance minimization. These findings do not support the physicochemical postulate that distance minimization was a major factor shaping the evolution of the genetic code. They agree with the coevolution theory, which proposes that genetic code evolution was predominantly determined by the concession of codons from precursor to product amino acids in an expansion of the code to accommodate new varieties of amino acids, with distance minimization playing a subsidiary role in deciding the choice of codons to be acquired by the product amino acids from the codon domains of the precursor amino acids.

Tam, S. C. and J. T. Wong (1980). "Modification of hemoglobin upon covalent coupling to dextran: enhanced stability against acid denaturation and reduced affinity for haptoglobin." Can J Biochem 58(9): 732-6.

Alkylation of human hemoglobin by bromoacetylaminoethylamino-substituted dextran gave rise to a covalent dextran-hemoglobin complex with enhanced stability against acid denaturation, and reduced affinity for binding to haptoglobin, compared with free hemoglobin. These effects increased with increasing size of the dextran moiety and were not elicited by either free dextran or alkylation of hemoglobin by iodoacetamide. These observations are consistent with an inhibition by covalently attached dextran of the binding of beta-subunit of hemoglobin to haptoglobin.

Mak, W. W. and J. T. Wong (1980). "Relationship between membrane fluidity and capping of receptors for concanavalin A." Can J Biochem 58(12): 1421-9.

The activities of a range of phenylalaninol-related compounds on capping of concanavalin A and induction of rounding of Chinese hamster ovary tsHl cells, as well as on the fluidity of phosphatidylcholine-cholesterol (1:1) liposomes, have been examined. These compounds include phenylalaninol, histidinol, leucinol, benzyl alcohol, benzylamine, 2-phenylethanol, 2-phenylamine, 3-phenyl-1-propanol, 3-phenyl-1-propylamine, and 3-phenylpropionic acid. The results indicate a strong correlation between the capacities of these compounds to enhance fluidity and their capacities to inhibit capping of concanavalin A. The specificity of this correlation is suggested by the finding that both types of capacities are poorly correlated with the capacities of the various compounds to induce cell rounding.

Mak, W. W., L. Pinteric, et al. (1980). "Enhanced sensitivity of tumorigenic cells to rapid rounding induced by phenylalaninol." Can J Biochem 58(9): 737-44.

This study describes a rounding reaction induced in mammalian cells by the addition of phenylalaninol. In the Chinese hamster ovary tsH1 line the rounding occurred rapidly with a half time of 1 min at 25 mM phenylalaninol. After the removal of phenylalaninol, the rounding was reversed, leading to the reflattening of the cells with a half-time of 3.5 min. Rounding was inhibited by dibutyryl-cAMP and testosterone, and reflattening by cytochalasin B. Either in the case of the tsH1 line and its growth-control revertant GRC+L-73, or in the case of SV40-transformed and untransformed human WI-38 cells, the transformed cells displayed a weakened resistance toward rounding. Likewise rat cells transformed by th highly oncogenic adenovirus-12 were more sensitive to rounding than cells transformed by the poorly oncogenic adenovirus-5, which in turn were more sensitive than untransformed cells. However, drug-resistant cell-surface mutants of the Chinese hamster ovary GAT- line also exhibited an altered sensitivity to rounding. These findings suggest that more than one cellular component determines cellular sensitivity to phenylalaninol-induced rounding. One of these components is specifically altered, giving rise to an enhanced sensitivity, in the course of tumorigenic transformation.

Kwok, Y. and J. T. Wong (1980). "Evolutionary relationship between Halobacterium cutirubrum and eukaryotes determined by use of aminoacyl-tRNA synthetases as phylogenetic probes." Can J Biochem 58(3): 213-8.

The cross-species reactivities between tRNAs and aminoacyl-tRNA synthetases have been employed as a basis to estimate the relatedness of various prokaryotes to the eukaryotes. The tRNA of Halobacterium cutirubrum, unlike that of other prokaryotes tested, including Agrobacterium tumefaciens, Arthrobacter luteus, Bacillus subtilis, Bacillus stearothermophilus, Escherichia coli, Micrococcus luteus, Myxococcus xanthus, Rhodopseudomonas spheroides, and Thermus aquaticus, was found to share with yeast, rat liver, and wheat germ tRNA a distinct preference for aminoacylation by eukaryotic synthetases from yeast as opposed to prokaryotic synthetases from either E. coli or R. spheroides. These results suggest that phylogenetically H. cutirubrum is more closely related to the eukaryotes than to the eubacteria.

Cornish-Bowden, A. and J. T. Wong (1980). "Validity of the jack-knife technique for analysing enzyme kinetic data." Biochem J 185(2): 535-6.

An observation by Duggleby [Biochem. J. (1979) 181, 255-256] that estimates of kinetic parameters by the jack-knife technique [Cornish-Bowden & Wong (1978) Biochem. J. 175, 969--976] are sometimes outside the range of estimates from which they are calculated has been examined. No significant correlation has been found between the occurrence of this behaviour and the actual quality of the estimates.

Wong, J. T. and P. M. Bronskill (1979). "Inadequacy of prebiotic synthesis as origin of proteinous amino acids." J Mol Evol 13(2): 115-25.

The production of some nonproteinous, and lack of production of other proteinous, amino acids in model prebiotic synthesis, along with the instability of glutamine and asparagine, suggest that not all of the 20 present day proteinous amino acids gained entry into proteins directly from the primordial soup. Instead, a process of active co-evolution of the genetic code and its constituent amino acids would have to precede the final selection of these proteinous amono acids.

Wong, J. T. (1978). "Extensor mechanism preventing reduction of finger." Med J Aust 1(2): 101.

Tam, S. C., J. Blumenstein, et al. (1978). "Blood replacement in dogs by dextran-hemoglobin." Can J Biochem 56(10): 981-4.

Exchange transfusions in dogs were performed with a solution of either dextran or a covalent complex between dextran and human hemoglobin. Dogs transfused with dextran alone died when their hematocrit was lowered to 6-10%. Dogs transfused with dextran-hemoglobin complex, however, survived a reduction of their hematocrit to 2% or below. In the latter animals, the dextran-hemoglobin complex disappeared from the circulation with an average half-life of 2.4 days. Correcting for oxidation of the hemoglobin moiety to methemoglobin, the half-life of functional unoxidized dextran-hemoglobin in the circulation was 1.9 days. In compensation for the loss of dextran-hemoglobin, vigorous erythropoiesis was observed at a rate of close to 5% hematocrit per day over the first 2 days following the exchange transfusion. As a result, the total hemoglobin concentration in blood was maintained at 5-6% during this period, and the animals went on to complete recovery in room air without the need for further transfusion with dextran-hemoglobin.

Cornish-Bowden, A. and J. T. Wong (1978). "Evaluation of rate constants for enzyme-catalysed reactions by the jackknife technique. Application to liver alcohol dehydrogenase." Biochem J 175(3): 969-76.

Steady-state measurements of enzyme-catalysed reactions are capable of providing more information about the rate constants of the individual steps than is commonly obtained. We have applied a combination of the jackknife and non-linear regression techniques to measurements of the rate of oxidation of ethanol by NAD+, catalysed by alcohol dehydrogenase from horse liver. This has permitted values and confidence intervals to be assigned to the eight rate constants that characterize the binding of ethanol and NAD+ in random order to the enzyme, and to the net rate constant kcat. for the breakdown of the ternary complex.

Blumenstein, J., S. C. Tam, et al. (1978). "Experimental transfusion of dextran-hemoglobin." Prog Clin Biol Res 19: 205-12.

Chang, J. E. and J. T. Wong (1977). "Synthesis of soluble dextran-hemoglobin complexes of different molecular sizes." Can J Biochem 55(4): 398-403.

Experimental conditions were defined that determined the synthesis of dextran-hemoglobin complexes through the alklation of hemoglobin by N-bromoacetylaminoethylaminodextran. Using appropriate concentrations of the two reactants, over 90% yield of dextran-hemoglobin was obtained for dextrans of average molecular weight of 200 000 110000, 70000, 400000, and 20000. Extensive viscosity increase due to crosslinking could be avoided, and a large molar excess of dextran over hemoglobin made unnecessary, under the optimal conditions.

Wong, J. T. (1976). "The evolution of a universal genetic code." Proc Natl Acad Sci U S A 73(7): 2336-40.

Tarter, M. E., E. O. Rigsbee, et al. (1976). "Interactive editing of biomedical data." Comput Programs Biomed 6(2): 117-23.

Editing options of an interactive graphical-biometry statistical system are described. A method for screening using a highly reduced data file is illustrated. By utilizing properties of sample Fourier coefficients, the problem of component overlap can be resolved. This, in turn, tends to free edited statistical information from the contamination or truncation inherent in the usual screening process.

Tam, S. C., J. Blumenstein, et al. (1976). "Soluble dextran-hemoglobin complex as a potential blood substitute." Proc Natl Acad Sci U S A 73(6): 2128-31.

A complex between soluble dextran and human hemoglobin has been synthesized by two different methods. In the alkylation method, hemoglobin was allowed to react with bromoacetyl groups incorporated into the dextran; the yield of the complex was about 80% in terms of the hemoglobin used. In the dialdehyde method, hemoglobin was allowed to react with dialdehyde groups on the dextran generated by periodate oxidation; the yield of the complex was about 60%. Both soluble dextran-hemoglobin complexes could bind and release oxygen reversibly, but the oxygen-binding curves were shifted to the left relative to that of free hemoglobin. In the rabbit, the complex obtained by the alkylation method was excreted by the kidneys and cleared from the circulation much more slowly than free hemoglobin.

Ganoza, M. C., N. Barraclough, et al. (1976). "Purification and properties of an N-formylmethionyl-tRNA hydrolase." Eur J Biochem 65(2): 613-22.

The isolation and properties of a novel N-formylmethionyl-tRNA hydrolase (hydrolase II) from Escherichia coli are described. This enzyme is difficult to detect in crude extracts; purification, however, unmasks the activity. Sedimentation and gel filtration parameters of this enzyme differ from those of the previously described peptidyl-tRNA hydrolase (hydrolase I), and preparations can be obtained where the two activities are free of each other. A mutant of hydrolase I has wild-type levels of hydrolase II. These data indicate that hydrolase II is a different enzyme, or an altogether different form of hydrolase I. The bulk of the enzymic activity occurs in the ribosome-free cytoplasm; the remainder is found on intact or dissociated 70-S ribosomes. Purified preparations of hydrolase II analyzed by two-dimensional gel electrophoresis contain 2 protein bands. These 2 proteins do not coincide in electrophoretic mobility with any known ribosomal proteins. Analysis after mixing experiments verifies this conclusion. The purified enzyme (hydrolase II) is inhibited by ribosomes bearing bound N-formylmethionyl-tRNA. The inhibition is potentiated by sparsomycin and other antibiotics that block specifically peptide-bond synthesis. The relationship of this enzyme to other hydrolytic activities, including a newly described ribosome-dependent hydrolase, are discussed.

Wong, J. T. (1975). "Radiographic scale grid for supervoltage radiographs." Radiol Technol 47(3): 414-4.

This paper deals with the construction and practical application of a radiographic scale grid incorporated into high energy radiation therapy machines in making port radiographs. The gadet is designed especially for the numerous small radiation therapy departments in community hospitals and private practices that do not have the sophistication provided by a simulator.

Wong, J. T. (1975). "A co-evolution theory of the genetic code." Proc Natl Acad Sci U S A 72(5): 1909-12.

Milne, A. N., W. W. Mak, et al. (1975). "Variation of ribosomal proteins with bacterial growth rate." J Bacteriol 122(1): 89-92.

The composition of ribosomal proteins has been examined as a function of the growth rate of Escherichia coli cells. Seven sets of cultural conditions, utilizing different combinations of carbon and nitrogen sources, were employed to provide a 36-fold spread in growth rate. The cellular content of most of the ribosomal proteins in ribosomes decreased to a similar extent in the very slow-growing cultures. Major exceptions were proteins S6 and L12, which exhibited a much more pronounced decrease , and S21, which exhibited an increase. None of the proteins remained invariant with growth rate.

Beatty, B. G., W. W. Mak, et al. (1975). "Regulation of synthesis of ribosomal proteins during pyrimidine starvation in Escherichia coli." Biochem J 150(3): 463-9.

The synthesis of ribosomal proteins during pyrimidine starvation was investigated. A progressive turn-off of protein synthesis, with a decay half-time of about 5 min, was observed when Escherichia coli cells were starved of uridine. By means of double-labelling, the syntheses of different ribosomal proteins were shown to be turned off unequally during the starvation. Comparison of the turn-off patterns for some proteins and the known polycistronic organization of the structural genes for these proteins suggests that a major cause of the unequal turn-off was the degradation of mRNA molecules for the ribosomal proteins from the 5'-end toward the 3'-end.

Wong, J. T. and C. S. Hanes (1973). "Topological analysis of enzymic mechanisms." Acta Biol Med Ger 31(4): 507-14.

Nazar, R. N. and J. T. Wong (1973). "Azide- or cyanide-induced methionine starvation in Escherichia coli." Biochim Biophys Acta 324(2): 218-25.

Nazar, R. N. and J. T. Wong (1973). "Distinct patterns of inhibitor-induced shift-down responses in mammalian cells." Exp Cell Res 77(1): 271-9.

Gurr, P. A., J. T. Wong, et al. (1973). "Automated determination of enzymatic rate functions by the 'gradient-flow' method." Anal Biochem 51(2): 584-8.

Nazar, R. N., L. A. Tyfield, et al. (1972). "Regulation of ribonucleic acid accumulation in vivo by nucleoside triphosphates." J Biol Chem 247(3): 798-804.

Nazar, R. N. and J. T. Wong (1972). "Nucleotide changes and the regulation of ribonucleic acid accumulation during growth rate shifts in Escherichia coli." J Biol Chem 247(3): 790-7.

Hanes, C. S., P. M. Bronskill, et al. (1972). "Kinetic mechanism for the major isoenzyme of horse liver alcohol dehydrogenase." Can J Biochem 50(12): 1385-413.

Gurr, P. A., P. M. Bronskill, et al. (1972). "Purification of isoenzyme and coenzymes for kinetic study of horse liver alcohol dehydrogenase." Can J Biochem 50(12): 1376-84.

Wong, J. T. and L. Endrenyi (1971). "Interpretation of nonhyperbolic behavior in enzymic systems. I. Differentiation of model mechanisms." Can J Biochem 49(5): 568-80.

Wong, J. T., A. Pincock, et al. (1971). "Site of energy coupling in the carrier mechanism for beta-galactoside transport." Biochim Biophys Acta 233(1): 176-88.

Endrenyi, L., M. S. Chan, et al. (1971). "Interpretation of nonhyperbolic behavior in enzymic systems. II. Quantitative characteristics of rate and binding functions." Can J Biochem 49(5): 581-98.

Bronskill, P. M. and J. T. Wong (1971). "Nature of ribosome-bound beta-galactosidase." J Bacteriol 105(2): 498-503.

Beatty, B. G. and J. T. Wong (1971). "Recognition of early and late biosynthetic classes ribosomal proteins in Escherichia coli." Can J Biochem 49(12): 1276-8.

Wong, J. T. and R. N. Nazar (1970). "Relationship of the MS nucleotides to the regulation of ribonucleic acid synthesis in Escherichia coli." J Biol Chem 245(17): 4591-3.

Nazar, R. N., H. G. Lawford, et al. (1970). "An improved procedure for extraction and analysis of cellular nucleotides." Anal Biochem 35(2): 305-13.

Beatty, B. G. and J. T. Wong (1970). "Synthesis and assembly of ribosomal proteins at the onset of pyrimidine starvation." Biochem Biophys Res Commun 41(1): 99-107.

Wong, J. T. and C. S. Hanes (1969). "A novel criterion for enzymic mechanisms involving three substrates." Arch Biochem Biophys 135(1): 50-9.

Wong, J. T. and M. Mustard (1969). "Regulation of amino acid-specific transfer RNA's in Escherichia coli." Biochim Biophys Acta 174(2): 513-24.

Nazar, R. N. and J. T. Wong (1969). "Inhibitor-induced shift-downs in Escherichia coli." J Bacteriol 100(2): 956-61.

Wong, J. T. and G. R. Williams (1968). "The mechanism of liver alcohol dehydrogenase." Arch Biochem Biophys 124(1): 344-8.

Hastings, P. and J. T. Wong (1968). "A simple procedure for eluting nucleotides from thin-layer plates." Anal Biochem 22(1): 169-71.