Biomedical subjects

L Chen

Publications and source records attributed to L Chen.

At least 19 recordsLinked to original sources

PubMed1995

Regulation of vascular smooth muscle growth by alpha 1-adrenoreceptor subtypes in vitro and in situ.

Rat aorta smooth muscle cells which express all three alpha 1-adrenoreceptors (alpha 1A, alpha 1B and alpha 1D) were used to determine the effect of stimulation of alpha 1-adrenergic receptor subtypes on cell growth. "Combined" alpha 1-adrenoreceptor subtype stimulation with norepinephrine alone caused a concentration-dependent, prazosin-sensitive increase in protein content and synthesis: 48 h of stimulation at 1 microM increased cell protein to 216 +/- 40% of time-matched controls (p = 0.008) and RNA to 140 +/- 13% (p = 0.03); protein synthesis increased to 167 +/- 13% (p < 0.01) after 24 h. Stimulation with norepinephrine plus the selective alpha 1A/alpha 1D antagonist 5-methylurapidil produced greater increases in alpha-actin mRNA (270 +/- 40% at 8 h; p = 0.007), total cell protein (220 +/- 45% at 24 h; p = 0.004), and RNA (135 +/- 8% at 24 h; p = 0.01). These effects were prevented by pretreatment with the selective alpha 1B antagonist chloroethylclonidine. Comparable results were obtained for intact aortae. Stimulation with norepinephrine plus 5-methylurapidil increased (p < 0.05) tissue protein, RNA, dry weight, and alpha-actin mRNA; and as in culture cells, combined stimulation with norepinephrine alone attenuated these responses. By comparison, adventitia (fibroblasts) was unaffected. Removal of endothelial cells had no effect. alpha 1B mRNA decreased by 42 +/- 12% (p = 0.01) in cultured cells during combined alpha 1-adrenoreceptor stimulation and by 23 +/- 8% (p = 0.03) for intact aorta. alpha 1D and beta-actin mRNA were unchanged in cultured cells, aorta media, and adventitia. These findings suggest that prolonged stimulation of chloroethylclonidine-sensitive, possibly alpha 1B-adrenoceptors induces hypertrophy of arterial smooth muscle cells and that stimulation of 5-methylurapidil-sensitive, non-alpha 1B-adrenoreceptors attenuates this growth response.

Actins↗

PubMed1995

Protective immunity induced by B7/CD28-costimulated gamma delta T cells to the EL-4 lymphoma in allogenic athymic mice.

We previously reported that the murine EL-4 lymphoma (H-2b) transduced with a retrovirus containing the murine B7-1 gene (B7+ EL-4) grew transiently for several weeks and subsequently regressed in allogenic BALB/c (nu/nu) athymic mice (H-2d). We now show that, in contrast, B7+ EL-4 cells grow progressively in several combined immunodeficiency mice, including SCID and NIH III mice, which lack T cells expressing either TCR-alpha beta or -gamma delta. Furthermore, depletion of gamma delta T cells with a specific mAb made possible the progressive growth of B7+ EL-4 cells in 90% of athymic mice while depletion of alpha beta T cells allowed tumor growth in 50% of these mice. Immunization of athymic mice with B7+ EL-4 cells prevented the outgrowth of wild-type B7- EL-4 cells. This protective immunity was abrogated by in vivo treatment with an anti-TCR-gamma delta mAb, further indicating that gamma delta T cells play an important role in tumor rejection by athymic mice. A gamma delta T cell line, Tc1, was established from B7+ EL-4-immunized athymic mice by repeated restimulation in vitro with irradiated B7+ EL-4 cells. When tested against a broad spectrum of target cells, Tc1 lysed several murine lymphoma lines, but did not lyse other tumor lines, suggesting that the Ag recognized by Tc1 has a limited distribution. Our data demonstrate that gamma delta T cells, and, to a less extent, extrathymic alpha beta T cells, mediate an immune response against B7+ EL-4 cells in allogeneic athymic mice.

Animals↗

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

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↗

PubMed1995

Only one of the two DNA-bound orientations of AP-1 found in solution cooperates with NFATp.

BACKGROUND: The transcription factor AP-1 activates the expression of numerous genes in response to mitogenic stimuli. AP-1 regulates gene expression both through solitary binding to independent recognition sites and, in cooperation with various heterologous transcription factors, through targeting to composite response elements. The two subunits that make up the AP-1 heterodimer, Fos and Jun, possess identical residues at positions that make sequence-specific contacts to DNA. This degeneracy leaves the protein with no apparent way of orienting itself uniquely on DNA by differentially recognizing its two non-identical half-sites. Here, we have analyzed the orientation of the AP-1 basic-leucine-zipper (bZip) domain on a cognate site, both alone and in the cooperative complex formed together with the 'nuclear factor of activated T cells' (NFATp). RESULTS: The results of affinity cleaving experiments demonstrate that, in solution, the AP-1 bZip binds DNA as a mixture of two orientational isomers. However, in the cooperative complex formed with NFATp on a composite response element, the AP-1 bZip adopts a single orientation, with Jun and Fos bound to the NFATp-proximal and NFATp-distal half-sites, respectively. Protein cross-linking experiments demonstrate that protein-protein contacts are responsible for this 'orientational locking'. CONCLUSIONS: Our results demonstrate that, through protein-protein interactions, one protein can force another to adopt a single DNA-bound orientation. Thus, cooperative interactions between adjacent regulatory proteins can influence not only the energetics of their interactions with DNA, but also their precise geometric and stereochemical arrangement. Because orientational isomers present markedly different structures to the transcriptional apparatus, it seems likely that orientation will exert an effect on the ability to activate transcription.

Amino Acid Sequence↗

PubMed1995

The crystal structure of HaeIII methyltransferase convalently complexed to DNA: an extrahelical cytosine and rearranged base pairing.

Many organisms expand the information content of their genome through enzymatic methylation of cytosine residues. Here we report the 2.8 A crystal structure of a bacterial DNA (cytosine-5)-methyltransferase (DCMtase), M. HaeIII, bound covalently to DNA. In this complex, the substrate cytosine is extruded from the DNA helix and inserted into the active site of the enzyme, as has been observed for another DCMtase, M. HhaI. The DNA is bound in a cleft between the two domains of the protein and is distorted from the characteristic B-form conformation at its recognition sequence. A comparison of structures shows a variation in the mode of DNA recognition: M. HaeIII differs from M. HhaI in that the remaining bases in its recognition sequence undergo an extensive rearrangement in their pairing. In this process, the bases are unstacked, and a gap 8 A long opens in the DNA.

Amino Acid Sequence↗

PubMed1995

Molecular characterization of two Drosophila guanylate cyclases expressed in the nervous system.

We have isolated, by interspecies hybridization, two classes of Drosophila cDNA each encoding a different guanylate cyclase (GC). One of them encodes an alpha subunit homolog of soluble GC, designated DGC alpha 1, and the other encodes a receptor-type GC, designated DrGC. The dgc alpha 1 cDNA encodes a protein of 676 amino acids and maps to 99B. In situ hybridization to adult tissue sections showed that dgc alpha 1 mRNA is found mainly in the cell bodies of the optic lobe, central brain, and thoracic ganglia. The DGC alpha 1 protein was also localized primarily to the nervous system by immunocytochemical staining, consistent with results of in situ hybridization. However, no detectable expression of this protein was found in the retina. The other class of cDNA, drgc, maps to 76C and encodes a 1525-amino acid protein displaying structural features similar to other known receptor-type guanylate cyclases. However, it has a C-terminal 430 amino acid region that has no homology to any known proteins. drgc RNA is expressed at low levels throughout development and in adult heads and bodies. In situ hybridizations to adult tissue sections showed that drgc mRNA is expressed in a wide range of tissues, including the optic lobe, central brain, thoracic ganglia, digestive tract, and the oocyte.

Amino Acid Sequence↗

PubMed1995

A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.

Amino Acid Sequence↗

PubMed1995

Angiographic stenosis progression and coronary events in patients with 'stabilized' unstable angina.

BACKGROUND: Recent studies suggest that angiographically complex coronary stenoses are associated with an adverse short-term outcome. It is not known, however, if this applies to unstable angina patients who stabilize on medical therapy. METHODS AND RESULTS: We prospectively studied 85 consecutive patients with unstable angina who stabilized on medical therapy but were found to require angioplasty for treatment of obstructive coronary disease. Angiography was carried out at admission, and patients were restudied 8 +/- 4 months (mean +/- SD) after the first angiogram. Ischemia-related stenoses were identified and classified as "complex" (irregular borders, overhanging edges, or thrombus) or "smooth" (absence of complex features). Stenosis progression (> or = 20% diameter reduction or new total occlusion) was assessed by automated edge detection. At initial angiography, there were 198 stenoses (> or = 50%, 102), of which 85 (54 complex and 31 smooth) were ischemia related. At restudy, 21 ischemia-related stenoses and 8 non-ischemia-related stenoses progressed (25% versus 7%, P = .001). Seventeen of the 21 ischemia-related stenoses that progressed developed into total occlusion compared with 3 of the 8 non-ischemia-related stenoses (P = .02). Changes in average stenosis severity and in absolute stenosis diameter were significantly larger in ischemia-related stenoses than in non-ischemia-related stenoses (P = .03). Eighteen (34%) complex stenoses progressed, compared with 3 (10%) smooth lesions (P = .02). During follow-up, 1 patient died (myocardial infarction) and 25 patients had nonfatal coronary events that were associated with progression of ischemia-related stenoses in 14 (56%). CONCLUSIONS: In unstable angina patients who stabilize medically, subsequent short-term stenosis progression and coronary events are common. The unstable coronary lesion (particularly complex stenoses) is often not stabilized and will continue to progress over the ensuing months.

Angina, Unstable↗