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L Chen

Publications and source records attributed to L Chen.

At least 1,045 records · Page 58Linked to original sources
PubMed1995

Transition state and rate-limiting step of the reaction catalyzed by the human dual-specificity phosphatase, VHR.

The dual-specificity phosphatases are unusual catalysts in that they can utilize protein substrates containing phosphotyrosine as well as phosphoserine/threonine. The dual-specificity phosphatases and the protein-tyrosine phosphatases (PTPases) share the active site motif (H/V)C(X)5R(S/T), but display little amino acid sequence identity outside of the active site. Although the dual-specificity phosphatases and the PTPases appear to bring about phosphate monoester hydrolysis through a similar mechanism, it is not clear what causes the difference in the active-site specificity between the two groups of enzymes. In this paper, we show that the human dual-specificity phosphatase, VHR [for VH1-Related; Ishibashi et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12170-12174], is rather promiscuous toward small phosphate monoesters (including both aryl and alkyl phosphates of primary alcohols) with effectively identical kcat/Km and kcat values while the pKa values of the leaving groups (phenols or alcohols) varied from 7 to 16. Linear free-energy relationship analysis of kcat and kcat/Km of the enzyme-catalyzed hydrolysis reaction suggests that a uniform mechanism is utilized for both the aryl and alkyl substrates. The very small dependency of kcat/Km on the leaving group pKa can be accounted for by the protonation of the leaving group. Pre-steady-state burst kinetic analysis of the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate provides direct kinetic evidence for the involvement of a phosphoenzyme intermediate in the dual specificity phosphatase-catalyzed reaction. The rate-limiting step for the VHR-catalyzed hydrolysis of p-nitrophenyl phosphate corresponds to the decomposition of the phosphoenzyme intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Electrostatic properties of beta-cytidine and cytosine monohydrate from Bragg diffraction.

The charge-density distribution in the crystal structure of the nucleoside beta-cytidine at 123 K has been determined from X-ray diffraction data (Ag K alpha, lambda = 0.5608 A) using all 7233 reflections with sin theta/lambda < or = 1.14 A-1. Maps of electrostatic potential for the cytosine base in cytidine are similar to those derived from previous charge-density studies of cytosine monohydrate and 1-beta-D-arabinosyl-cytosine, after taking chemical differences into account. These results were obtained from pseudoatom multipole refinements including kappa as a variable to describe the expansion or contraction of the spherical valence shell for each atom type. A new structure refinement of this type has also been carried out for cytosine monohydrate. A survey of kappa values for hydrogen indicates that this is a variable which is not well determined experimentally. Variations in kappa H are relayed into the population parameters obtained for other pseudoatoms and can have a small but significant effect on molecular properties, such as the dipole moment. Assuming an average theoretical value of kappa H = 1.24, the molecular dipole moments calculated from the monopole and dipole pseudoatom population parameters are 17 (4) and 8.2 (15) Debye for cytidine nucleoside and cytosine in the monohydrate, respectively. Systems of atomic point charges are presented for cytidine, cytosine and water. These are generally satisfactory in reproducing the experimentally determined molecular electrostatic potentials and dipole moments.

Chemical Phenomena↗
PubMed1995

Multiple lineages of tumors express a common tumor antigen, P1A, but they are not cross-protected.

Whether tumors of different lineages share common Ags is a critical issue for understanding anti-tumor immune responses and for designing Ag-specific tumor immunotherapy. Because of lack of cross-protection among individually derived tumors, it has been proposed that tumor Ags are specific for individual tumors. Here we show that lack of cross-protection is not due to lack of a shared tumor Ag. Thus, a plasmocytoma J558 transfected with the costimulatory molecule B7 activates a cross-reactive CTL response in vivo. The major Ag recognized by the cross-reactive CTL is P1A, which is expressed in mastocytoma P815, plasmocytoma J558, and fibrosarcoma Meth A. Surprisingly, no significant cross-protection can be detected among P1A-expressing tumors after immunization with either P1A-expressing or B7-transfected P815 cells. Our results demonstrate that multiple lineages of tumors are not cross-protected even though they share a tumor Ag that can be recognized by CTL. These results have important implications for tumor immunotherapy.

Amino Acid Sequence↗
PubMed1995

Why is phosphonodifluoromethyl phenylalanine a more potent inhibitory moiety than phosphonomethyl phenylalanine toward protein-tyrosine phosphatases?

The phosphonodifluoromethyl phenylalanine (F2Pmp) is superior to phosphonomethyl phenylalanine (Pmp) as a non-hydrolyzable phosphotyrosine (pTyr) mimetic. The difluoromethyl moiety increases the inhibitory potency of a F2Pmp-containing peptide over a Pmp-containing counterpart by 1000-fold toward the protein tyrosine phosphatase (PTPase), PTP1. Fluorine substitution at the methylene carbon have the double effect of lowering the phosphonate pKa2 as well as introducing hydrogen bonding interactions similar to the phosphate ester oxygen in pTyr. The inhibition of PTP1-catalyzed dephosphorylation reaction by both the F2Pmp and Pmp-containing peptides did not vary as a function of pH. The data indicate that both the monoanion and the dianion forms of the phosphonate bind PTP1 with equal efficiency. Thus, the better binding by the F2Pmp-peptide as compared to the Pmp-peptide is not due to the difference in pKa2. Taken together, these results offer an explanation for the increased affinity of F2Pmp for PTP1. The two fluorine atoms in F2Pmp may be able to interact with active site residues in PTP1 in a fashion analogous to that involving the phenolic oxygen and side chains in the active site of PTP1. Ki measurements for a simple phosphonic acid, Pmp- and F2Pmp-containing peptides suggest that although the principal recognition element is F2Pmp itself, the surrounding amino acids are required for high affinity binding. Comparative analysis of the inhibition of PTP1, PTP alpha and LAR by F2Pmp-containing peptides suggests that selective, tight-binding PTPase inhibitors can be developed.

Amino Acid Sequence↗
PubMed1995

Solution structure of a bovine immunodeficiency virus Tat-TAR peptide-RNA complex.

The Tat protein of bovine immunodeficiency virus (BIV) binds to its target RNA, TAR, and activates transcription. A 14-amino acid arginine-rich peptide corresponding to the RNA-binding domain of BIV Tat binds specifically to BIV TAR, and biochemical and in vivo experiments have identified the amino acids and nucleotides required for binding. The solution structure of the RNA-peptide complex has now been determined by nuclear magnetic resonance spectroscopy. TAR forms a virtually continuous A-form helix with two unstacked bulged nucleotides. The peptide adopts a beta-turn conformation and sits in the major groove of the RNA. Specific contacts are apparent between critical amino acids in the peptide and bases and phosphates in the RNA. The structure is consistent with all biochemical data and demonstrates ways in which proteins can recognize the major groove of RNA.

Amino Acid Sequence↗
PubMed1995

Stereochemical course of glucan hydrolysis by barley (1-->3)- and (1-->3, 1-->4)-beta-glucanases.

The stereochemical course of hydrolysis of Laminaria digitata laminarin and barley (1-->3, 1-->4)-beta-glucan by barley (1-->3)-beta-glucanase (E.C. 3.2.1.39) isoenzyme GII and (1-->3, 1-->4)-beta-glucanase (EC 3.2.1.73) isoenzyme EII, respectively, has been determined by 1H-NMR. Both enzymes catalyse hydrolysis with retention of anomeric configuration (e-->e) and may therefore operate via a double displacement mechanism. We predict that all other members of Family 17 of beta-glycosyl hydrolases also follow this stereochemical course of hydrolysis.

Carbohydrate Conformation↗
PubMed1995

Rapid angiographic progression of coronary artery disease in patients with angina pectoris. The role of complex stenosis morphology.

BACKGROUND: Rapid disease progression commonly underlies acute coronary events, and "complex" stenosis morphology may play a role in this phenomenon. METHODS AND RESULTS: We studied the role of complex stenosis morphology in rapid disease progression in 94 consecutive patients awaiting routine coronary angioplasty. Coronary arteriography was repeated at 8 +/- 3 months' follow-up, immediately preceding angioplasty (68 patients) or after an acute coronary event (26 patients). Disease progression of 217 stenoses, of which 79 (36%) were "complex" and 138 (64%) were "smooth," was assessed by computerized angiography. At presentation, 63 patients had stable angina pectoris and 31 had unstable angina that settled rapidly with medical therapy. At follow-up, 23 patients (24%) had progression of preexisting stenoses and 71 (76%) had no progression. Patients with progression were younger (55 +/- 12 years) than those without (58 +/- 9 years) but did not differ with regard to risk factors, previous myocardial infarction, or severity and extent of coronary disease. Twenty-three lesions (11%) progressed, 15 to total occlusion (11 complex and 4 smooth; 65%). Progression occurred in 17 of the 79 complex stenoses (22%) and in 6 of the 138 smooth lesions (4%) (P = .002). Mean stenosis diameter reduction was also significantly greater in complex than in smooth lesions (11.6% versus 3.9% change; P < .001). Acute coronary events occurred in 57% of patients with progression compared with 18% of those without progression (P < .001) and were more frequent in patients who presented with unstable angina (P = .002). CONCLUSIONS: Rapid stenosis progression is not uncommon, and complex stenoses are at risk more than smooth lesions.

Angina Pectoris↗
PubMed1995

In vitro priming of tumor-reactive cytolytic T lymphocytes by combining IL-10 with B7-CD28 costimulation.

Naive spleen cells from syngeneic mice generated tumor-reactive CTL following three cycles of in vitro culturing with IL-10 and cells from the P815 mouse mastocytoma that expressed the B7-1 or B7-2 costimulator. Unpurified as well as CD8-enriched naive splenocytes could be used for priming. The in vitro primed CTL were CD8+, and their recognition was MHC class I restricted. Both IL-10 and B7-transfected P815 cells were required for the priming. However, a combination of exogenous IL-10 and IL-2 in the presence of B7-negative wild-type P815 cells also induced tumor-reactive CTL. Injection of the neutralizing anti-IL-10 mAb JES-2A5 into mice reduced their ability to mount a primary CTL response after immunization with B7-1+ P815 cells, and inclusion of this mAb in the in vitro cultures inhibited a secondary CTL response. Adoptive transfer of the in vitro primed CTL had a therapeutic effect in mice with P815 established as an ascites tumor. Our results underscore an important role of IL-10 in the induction of a tumor-specific CTL response.

Animals↗
PubMed1995

Solution small-angle X-ray scattering study of the molecular chaperone Hsc70 and its subfragments.

Solution X-ray scattering experiments have been carried out on recombinant bovine Hsc70 (with 650 amino acid residues), a 60 kDa subfragment (residues 1-554) which has ATPase- and peptide-binding activities, a 44kDa subfragment (residues 1-386) which has only ATPase activity, and a peptide-binding fragment (residues 388-554). Modeling based on steady-state values of radii of gyration (Rg's) and P(r) functions shows that the 44 kDa and peptide-binding domains are oblate fragments while Hsc70 and the 60 kDa fragment are prolate and relatively elongated. Rg values decrease significantly in the presence of MgATP relative to their values in the presence of MgADP (delta Rg approximately 4-5 A) for Hsc70 and the 60 kDa fragment; in contrast, they are essentially equal in the presence of either nucleotide for the 44 kDa ATPase fragment. The kinetics of the change of Rg for Hsc70 and the 60 kDa fragment under single-ATPase cycle conditions show that the transition to the ATP-induced Rg occurs significantly more rapidly than ATP hydrolysis while the reverse transition to the larger Rg value does not occur before product release. Altogether, the solution scattering data support a model in which a conformational change in Hsc70 (presumably to the low-peptide-affinity state) is predicated on ATP binding while the reverse transition is predicated on product release.

Adenosine Triphosphatases↗
PubMed1995

Compact and scattered gap junctions in diffusion mediated cell--cell communication.

Gap junctions have been thought of as prime sites for the passive communication of small molecules between two adjacent cells. The sizes of a gap junction plaque may range from a few junctional particles to several thousands particles on the interface of two cells, with little being known about the relative contribution of different patterns of junctional plaques on diffusion mediated cell--cell communication. In this paper, by constructing a simple cubic lattice model of a two-cell system, the effects on communication efficiency of two extreme distribution patterns of individual junctional particles rae investigated. This model indicates that scattered gap junctions are an order of magnitude higher in communication efficiency than compact junctional plaques of equal number of total junctional particles. Thus, it is possible that modulation of cell-cell communication may rely on the interconversion of these two states with compact plaques existing as an inactive communication form. Any correlation of gap junctional membrane with cell-cell communication should therefore also take into account its configuration. It is possible that an increase in number and area of tightly packed gap junctional plaques may actually decrease, instead of increase, cell-cell communication.

Animals↗
PubMed1995

Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions.

The Bacillus subtilis mrgA gene encodes an abundant DNA-binding protein that protects cells against the lethal effects of H2O2. Transcription of mrgA is induced by H2O2 or by entry into stationary phase when manganese and iron levels are low. We have selected for strains derepressed for transcription of mrgA in the presence of Mn(II). The resulting cis-acting mutants define an operator site just upstream of the mrgA promoter. Similar sequences flank the promoters for the catalase gene, katA, and the heme biosynthesis operon, hemAXCDBL. Like mrgA, transcription of the katA and hem genes is repressed by Mn(II), which thereby potentiates the killing action of H2O2. We identified two classes of trans-acting mutants derepressed for mrgA transcription in the presence of Mn(II): some exhibit a coordinate derepression of MrgA, catalase, heme biosynthesis, and alkyl hydroperoxide reductase and are H2O2 resistant, while others have reduced catalase activity and are H2O2 sensitive. These data indicate that the peroxide stress response of B. subtilis is regulated by a repressor that senses both metal ion levels and H2O2.

Bacillus subtilis↗
PubMed1995

In vivo release of interleukin-1 beta into hypothalamic extracellular fluid in rats: effects of repeated sampling.

Interleukin-1 beta (Il-1 beta) concentrations in extracellular fluid (ECF) withdrawn at 10-min intervals through a push-pull cannula (PPC) located in the hypothalamus were studied in freely behaving male rats for 1 h at 24 and 72 h and again at 7 days after PPC implantation. Il-1 beta concentrations in ECF were similar in the latter. However, when ECF was sampled at 3 h and again 7 days after PPC implantation, Il-1 beta concentrations were greatly elevated at 7 days when compared to all other intervals. These results demonstrate how the relationships between Il-1 beta measured in ECF and the conditions of measurement appear to be integral parts of a whole intracerebral system: cytokine concentrations appear to be inextricably bound to intrahypothalamic conditions created by the sampling device presence and frequency of use.

Animals↗