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L Chen

Publications and source records attributed to L Chen.

At least 1,063 records · Page 59Linked to original sources
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↗
PubMed1995

A tetrad of ionizable amino acids is important for catalysis in barley beta-glucanases.

Determination of the crystal structures of a 1,3-beta-D-glucanase (E.C. 3.2.1.39) and a 1,3-1,4-beta-D-glucanase (E.C. 3.2.1.73) from barley (Hordeum vulgare) (Varghese, J.N, Garrett, T. P. J., Colman, P. M., Chen, L., Høj, P. B., and Fincher, G. B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2785-2789) showed the spatial positions of the catalytic residues in the substrate-binding clefts of the enzymes and also identified highly conserved neighboring amino acid residues. Site-directed mutagenesis of the 1,3-beta-glucanase has now been used to investigate the importance of these residues. Substitution of glutamine for the catalytic nucleophile Glu231 (mutant E231Q) reduced the specific activity about 20,000-fold. In contrast, substitution of glutamine for the catalytic acid Glu288 (mutant E288Q) had less severe consequences, reducing kcat approximately 350-fold with little effect on Km. Substitution of two neighboring and strictly conserved active site-located residues Glu279 (mutant E279Q) and Lys282 (mutant K282M) led to 240- and 2500-fold reductions of Kcat, respectively, with small increases in Km. Thus, a tetrad of ionizable amino acids is required for efficient catalysis in barley beta-glucanases. The active site-directed inhibitor 2,3-epoxypropyl beta-laminaribioside was soaked into native crystals. Crystallographic refinement revealed all four residues (Glu231, Glu279, Lys282, and Glu288) to be in contact with the bound inhibitor, and the orientation of bound substrate in the active site of the glucanase was deduced.

ATP-Binding Cassette Transporters↗
PubMed1995

Ras membrane targeting is essential for glucose signaling but not for viability in yeast.

Ras proteins are small GTP binding proteins that serve as critical relays in a variety of signal transduction pathways in eukaryotic cells. Like most metazoan Ras proteins, yeast Ras is post-translationally modified by addition of a farnesyl and a palmitoyl moiety, and these modifications are required for targeting the protein to the cytoplasmic face of the plasma membrane and for biological activity of the protein. We have constructed mutants of the yeast (Saccharomyces cerevisiae) Ras that are farnesylated in vivo but are not palmitoylated. These mutant proteins are not localized to the plasma membrane but function in the cell as well as the wild-type protein. Such mutants are viable but fail to induce a transient increase in intracellular cAMP concentration in response to glucose addition, although this deficiency does not yield a marked growth phenotype. These results are consistent with the hypothesis that the essential role of the farnesyl moiety on yeast Ras is to enhance productive interaction between Ras and its essential downstream target, adenylyl cyclase, rather than to localize Ras to the plasma membrane.

Alleles↗
PubMed1995

Differential progression of complex and smooth stenoses within the same coronary tree in men with stable coronary artery disease.

OBJECTIVES: We sought to compare the evolution of complex and smooth stenoses within the same coronary tree in patients with stable coronary artery disease. BACKGROUND: Progression of coronary stenosis has prognostic significance and may be influenced by local and systemic factors. Stenosis morphology is a determinant of disease progression, but no previous study has systematically assessed progression of complex and smooth stenoses within the same patient. METHODS: We studied 50 men with stable angina who 1) had one complex coronary stenosis and one smooth stenosis in different noninfarct-related coronary vessels at initial coronary angiography, and 2) had a second angiogram after a median interval of 9 months (range 3 to 24). Patients with lesions > or = 10 mm long, at a major branching point or with > 85% diameter reduction were not included. Coronary lesions were measured quantitatively from comparable end-diastolic frames. Stenosis morphology was determined qualitatively by two independent observers. RESULTS: All patients remained in stable condition during follow-up. Progression, defined as an increase in diameter stenosis by > or = 15% was seen in only eight complex stenosis (16%) but in no smooth lesions (p < 0.01). The severity of complex stenoses changed more than that of corresponding smooth stenoses (mean +/- 1 SD 5.8 +/- 13% vs. -0.06 +/- 6%, p < 0.01). On average, the annual rate of growth was 11.4 +/- 28% and 1.5 +/- 14% for complex and smooth lesions, respectively (p < 0.01). CONCLUSIONS: Few coronary stenoses progress rapidly in stable angina. Complex and smooth coronary stenoses progress at different rates within the same coronary tree. complex stenosis morphology itself is an important determinant of progression of stenosis in patients with apparently clinically stable coronary artery disease.

Aged↗
PubMed1995

Antitumor immunity elicited by tumor cells transfected with B7-2, a second ligand for CD28/CTLA-4 costimulatory molecules.

We have examined the role of the B7-2 costimulatory molecule, a second ligand for CD28/CTLA-4 counter-receptors, in the induction of antitumor immunity. A plasmid containing murine B7-2 cDNA was transfected into the immunogenic mouse mastocytoma P815 of DBA/2 origin. In contrast to the lethal growth of the wild-type (wt) P815 tumor, B7-2-positive (B7-2+) P815 cells inoculated into syngeneic mice regressed, and immunization of mice with such tumor cells protected them against the challenge of wt P815 tumor. Depletion of CD8+, but not of CD4+, lymphocytes in vivo by specific Abs abolished the regression of B7-2+ P815 tumors. CD8+ cytolytic T cells could be generated from mice immunized with B7-2+ P815. They were found to be MHC class I-restricted and specific for the P815 tumor. In contrast, transfection of the B7-2 gene into the nonimmunogenic MCA102 fibrosarcoma of C57BL/6 origin induced neither tumor regression nor protective immunity. Co-expression on MCA102 cells of B7-2 together with the related costimulator B7-1 also failed to induce immunity to MCA102 tumor. Our results indicate that transfection of B7-2 into tumor cells can improve host response to some tumors, and that the effects seen are similar to those previously observed for B7-1.

Abatacept↗
PubMed1995

Recombinant lys-plasminogen, but not glu-plasminogen, improves recombinant tissue-type plasminogen activator-induced coronary thrombolysis in dogs.

OBJECTIVES: This study examined the modification of recombinant tissue-type plasminogen activator (rt-PA)-induced thrombolysis by recombinant lys-plasminogen. BACKGROUND: Recombinant tissue-type plasminogen activator restores flow in the thrombosed coronary artery, but the artery often reoccludes. The rt-PA-induced thrombolysis is a result of activation of plasminogen bound to fibrin in the thrombus and results in generation of the fibrinolytic enzyme plasmin. Small amounts of lys-plasminogen are formed when rt-PA is used. Lys-plasminogen binds to fibrin with a 10-fold greater affinity than the predominant native glu-plasminogen, leading to a loose fibrin structure. METHODS: Dogs with electrically induced occlusive intracoronary thrombus were treated with saline solution (n = 9), glu-plasminogen (2 mg/kg body weight, n = 5) or lys-plasminogen (2 mg/kg, n = 5), followed by infusion of rt-PA (1 mg/kg over 20 min) 10 min later. RESULTS: Reperfusion rates were similar in all groups of dogs, but the time to reflow was lowest in dogs given lys-plasminogen compared with those given saline solution or glu-plasminogen before rt-PA (mean [+/- SE] 14 +/- 2 vs. 22 +/- 2 and 23 +/- 3 min, respectively, p < 0.05). None of the reperfused coronary arteries reoccluded in the lys-plasminogen plus rt-PA group, whereas 75% reoccluded in dogs given saline solution plus rt-PA, and 50% reoccluded in those given glu-plasminogen plus rt-PA. Accordingly, duration of reflow was greater in the lys-plasminogen plus rt-PA group (> 120 vs. 39 +/- 7 and 82 +/- 21 min, respectively, p < 0.05). Plasminogen activator inhibitor-1 activity decreased during rt-PA infusion and thereafter increased in all dogs, but less so in dogs given lys-plasminogen (p < 0.05 vs. those given saline solution before rt-PA). CONCLUSIONS: Treatment with recombinant lys-plasminogen before rt-PA reduces time to reflow and sustains reflow after thrombolysis, whereas glu-plasminogen has no such effect.

Animals↗