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L Chen

Publications and source records attributed to L Chen.

At least 1,171 records · Page 65Linked to original sources
PubMed1994

Fluorescent spot test method for specific detection of beta-lactamases.

A simple, sensitive fluorescent spot test method for specific detection of microbial beta-lactamases has been developed, based on the modification of a previously disclosed method (K. C. S. Chen, October 23, 1990, U.S. Patent 4,965,193). The new fluorescence developer used in the present study consisted of 0.5 mM HgCl2, in 0.5 M sodium citrate buffer, pH 4.5, prepared in 0.5% formaldehyde aqueous solution. A beta-lactam substrate solution consisting of a beta-lactam antibiotic with an acyl side chain containing an alpha-amino group and an alpha-phenyl group, or its derivatives, was incubated with a beta-lactamase-producing organism. One volume of the fluorescence developer was added to 4 vol of the incubated beta-lactam substrate solution, followed by heating the mixture at 45 degrees C for 10 min. The mixture was spotted on filter paper. Production of fluorophore indicated beta-lactamase activity. Each fluorophore was analyzed by TLC and its chemical identity was determined. Using ampicillin as the penicillinase substrate and cephalexin as the cephalosporinase substrate, the new method can be conveniently carried out by using dropping bottles for storing and dispensing the substrate solutions and the fluorescence developer. This modified method also provided more favorable conditions for the penicillinases to remain active during fluorescence development. Therefore, the sensitivity of the test was increased.

Ampicillin↗
PubMed1994

Crystallization and preliminary crystallographic analysis of a DNA (cytosine-5)-methyltransferase from Haemophilus aegyptius bound covalently to DNA.

A DNA (cytosine)-5-methyltransferase from Haemophilus aegyptius (M.Hae III), which catalyzes methyl transfer from S-adenosyl-L-methionine to DNA, has been crystallized as a covalent complex with a suicide oligonucleotide substrate. Crystals of the co-complex were grown by vapor diffusion with hanging droplets, using polyethylene glycol 3500 as the precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1); the unit cell parameters are a = 57.6 A, b = 108.0 A, c = 155.8 A with two protein-DNA complexes in the asymmetric unit. Complete sets of native and derivative data have been collected to 2.7 A using a laboratory source.

Base Sequence↗
PubMed1994

Structure of an electron transfer complex: methylamine dehydrogenase, amicyanin, and cytochrome c551i.

The crystal structure of a ternary protein complex has been determined at 2.4 angstrom resolution. The complex is composed of three electron transfer proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase, the blue copper protein amicyanin, and the cytochrome c551i. The central region of the c551i is folded similarly to several small bacterial c-type cytochromes; there is a 45-residue extension at the amino terminus and a 25-residue extension at the carboxyl terminus. The methylamine dehydrogenase-amicyanin interface is largely hydrophobic, whereas the amicyanin-cytochrome interface is more polar, with several charged groups present on each surface. Analysis of the simplest electron transfer pathways between the redox partners points out the importance of other factors such as energetics in determining the electron transfer rates.

Bacterial Proteins↗
PubMed1994

Three-dimensional structures of two plant beta-glucan endohydrolases with distinct substrate specificities.

The three-dimensional structures of (1-->3)-beta-glucanase (EC 3.2.1.39) isoenzyme GII and (1-->3,1-->4)-beta-glucanase (EC 3.2.1.73) isoenzyme EII from barley have been determined by x-ray crystallography at 2.2- to 2.3-A resolution. The two classes of polysaccharide endohydrolase differ in their substrate specificity and function. Thus, the (1-->3)-beta-glucanases, which are classified amongst the plant "pathogenesis-related proteins," can hydrolyze (1-->3)- and (1-->3,1-->6)-beta-glucans of fungal cell walls and may therefore contribute to plant defense strategies, while the (1-->3,1-->4)-beta-glucanases function in plant cell wall hydrolysis during mobilization of the endosperm in germinating grain or during the growth of vegetative tissues. Both enzymes are alpha/beta-barrel structures. The catalytic amino acid residues are located within deep grooves which extend across the enzymes and which probably bind the substrates. Because the polypeptide backbones of the two enzymes are structurally very similar, the differences in their substrate specificities, and hence their widely divergent functions, have been acquired primarily by amino acid substitutions within the groove.

Base Sequence↗
PubMed1994

Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization.

Comparative genomic hybridization was applied to 5 breast cancer cell lines and 33 primary tumors to discover and map regions of the genome with increased DNA-sequence copy-number. Two-thirds of primary tumors and almost all cell lines showed increased DNA-sequence copy-number affecting a total of 26 chromosomal subregions. Most of these loci were distinct from those of currently known amplified genes in breast cancer, with sequences originating from 17q22-q24 and 20q13 showing the highest frequency of amplification. The results indicate that these chromosomal regions may contain previously unknown genes whose increased expression contributes to breast cancer progression. Chromosomal regions with increased copy-number often spanned tens of Mb, suggesting involvement of more than one gene in each region.

Breast Neoplasms↗
PubMed1994

An RNA-binding peptide from bovine immunodeficiency virus Tat protein recognizes an unusual RNA structure.

The human immunodeficiency virus (HIV) Tat protein binds specifically to an RNA hairpin, TAR, located at the 5' end of its mRNA. Tat uses a single arginine residue within a short region of basic amino acids to recognize a bulge region in TAR. Here we show that a 17 amino acid arginine-rich peptide from the bovine immunodeficiency virus (BIV) Tat protein also binds to an RNA hairpin at the 5' end of its mRNA (BIV TAR), but recognizes different structural features of the RNA. Mutagenesis, RNase mapping, and chemical interference experiments indicate that bulge and stem regions of BIV TAR are recognized simultaneously by the BIV peptide and that the RNA adopts an unusual structure. BIV Tat binds to its TAR site with high affinity and specificity and, unlike HIV Tat, does not appear to use cellular proteins to stabilize RNA binding in vivo. Thus, two related viral activators have evolved rather distinct ways to recognize their RNA targets.

Amino Acid Sequence↗
PubMed1994

Effects of angiotensin-converting enzyme inhibition on exercise-induced angina and ST segment depression in patients with microvascular angina.

OBJECTIVES: This study was conducted to test the hypothesis that angiotensin-converting enzyme inhibition may lessen myocardial ischemia in patients with microvascular angina. BACKGROUND: Patients with syndrome X (angina pectoris, positive findings on exercise testing and normal coronary arteriogram) have a reduced coronary vasodilator reserve ("microvascular angina") and may show an increased sympathetic drive. Angiotensin-converting enzyme inhibition attenuates sympathetic coronary vasoconstriction in patients with coronary artery disease. METHODS: Ten patients (seven women and three men, mean age [+/- SD] 53 +/- 6 years) with syndrome X and a reduced coronary flow reserve underwent a randomized, single-blind, crossover, placebo-controlled study of the effects of the angiotensin-converting enzyme inhibitor enalapril on angina and exercise-induced ST segment depression. Assessment was by symptom-limited treadmill exercise testing after 2 weeks of treatment with 10 mg/day of enalapril and after 2 weeks of placebo administration. RESULTS: All patients had positive findings on exercise testing (> or = 1 mm ST segment depression and angina) while taking placebo, whereas six patients had a positive test result (four with angina) during enalapril therapy. Total exercise duration and time to 1 mm of ST segment depression were prolonged by enalapril over those obtained with placebo (mean 779 +/- 141 vs. 690 +/- 148 s, p = 0.006 and 690 +/- 204 vs. 485 +/- 241 s, p = 0.007, respectively). The magnitude of ST segment depression was also less with enalapril than with placebo (mean 1.1 +/- 0.4 vs. 1.5 +/- 0.2 mm, p = 0.004). Heart rate and blood pressure at peak exercise and at 1 mm of ST depression were not significantly different during placebo and enalapril treatment. CONCLUSIONS: Angiotensin-converting enzyme inhibition lessens exercise-induced ischemia in patients with syndrome X and microvascular angina, probably by a direct modulation of coronary microvascular tone, which results in an increased myocardial oxygen supply.

Coronary Circulation↗
PubMed1994

Replacement of glutamic acid 29 with glutamine leads to a loss of cooperativity for AMP with porcine fructose-1,6-bisphosphatase.

Mutations in the AMP binding site of porcine fructose-1,6-bisphosphatase were carried out by site-specific mutagenesis based on the crystal structure of the enzyme (Ke, H., Zhang, Y., and Lipscomb, W.L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5243-5247). The mutant and wild-type enzymes were characterized by SDS-polyacrylamide gel electrophoresis, circular dichroism spectrometry, and initial rate kinetics. One of the mutant forms of fructose-1,6-bisphosphatase, Glu-29-->Gln, is ligated to the phosphoryl moiety of AMP, a potent inhibitor of the reaction, whereas the other mutant, Thr-31-->Val, is associated with the purine base of AMP. No discernible alteration in structure as measured by circular dichroism spectrometry was noted for the mutants relative to the wild-type enzyme. As expected, major changes in kinetic parameters between the mutants and the wild-type enzyme were associated with inhibition by AMP. AMP, a competitive inhibitor with respect to Mg2+ in the fructose-1,6-bisphosphatase reaction, exhibits cooperativity in the case of the wild-type and the mutant Thr-31-->Val enzymes with a Hill coefficient of 2.0. On the other hand, cooperativity is completely lost in the case of Glu-29-->Gln fructose-1,6-bisphosphatase.

Adenosine Monophosphate↗
PubMed1994

MgATP-induced conformational changes in the iron protein from Azotobacter vinelandii, as studied by small-angle x-ray scattering.

Small angle x-ray scattering experiments have been carried out on the purified iron proteins of nitrogenase from wild-type Azotobacter vinelandii and from a Nif- mutant strain, A. vinelandii UW91 (which has an A157S mutation). This study was designed to investigate the influence of MgATP and MgADP binding on the protein structure in solution. For the wild-type protein, the binding of MgATP induces a significant conformational change that is observed as a decrease of about 2.0 A in the radius of gyration. In contrast, the binding of MgADP to the wild-type iron protein does not detectably affect the radius of gyration. In the absence of nucleotides, the radius of gyration for the UW91 mutant is indistinguishable from that of the wild-type. However, unlike for the wild-type protein, the radius of gyration of the UW91 iron protein is unaffected by the addition of MgATP. We have previously shown that the UW91 iron protein has a normal [4Fe-4S] cluster and MgATP binding ability but that it is completely blocked for electron transfer and MgATP hydrolysis (Gavini, N., and Burgess, B. K. (1992) J. Biol. Chem. 267, 21179-21186). These x-ray scattering measurements suggest that a conformation different from that of the native state is therefore required for the iron protein to perform electron transfer to the MoFe protein. These results also support the hypothesis that Ala-157 is crucial for the iron protein to establish the electron-transfer-favored conformation induced by MgATP binding.

Adenosine Diphosphate↗
PubMed1994

Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity.

A costimulatory signal through B7 to its counter-receptor CD28 on T cells enhances T cell activation. We have generated recombinant retroviruses containing cDNA for murine B7 and transduced a panel of murine tumor lines with varying immunogenicity to study the effect of B7 costimulation on antitumor immunity. In contrast to the progressive outgrowth of all wild-type (B7-) tumors in unimmunized syngeneic mice, four immunogenic tumors, lymphoma RMA, EL4, mastocytoma P815, and melanoma E6B2, regressed completely when transduced with the B7 gene. In contrast, four nonimmunogenic tumors, sarcomas MCA101, MCA102, and Ag104, and melanoma B16, remained tumorigenic after transduction of the B7 gene. Immunization with B7-transduced immunogenic tumors enhanced protective immunity and increased specific cytotoxic T lymphocyte (CTL) activity against the respective wild-type tumors as compared to immunization with nontransduced or mock-transduced tumors. Moreover, cocultivation of CTL with B7-transduced EL4 cells augmented the specificity of tumor-reactive CTL in long-term cultures. Treatment by injection of B7-transduced tumor cells cured 60% of mice with established wild-type EL4 lymphoma. In contrast, immunization with nonimmunogenic tumors transduced with B7 did not provide protective immunity and did not increase specific CTL activity. Our results show that tumor immunogenicity is critical to the outcome of costimulation of T cell-mediated tumor immunity by B7.

Animals↗
PubMed1994

Elements regulating cell- and stage-specific expression of the C. elegans MyoD family homolog hlh-1.

We investigated the cis-acting sequences regulating expression of the Caenorhabditis elegans gene hlh-1, a homolog of the MyoD family of myogenic regulatory factors. The hlh-1 gene is expressed in mature body wall muscle, in clonal muscle precursors, in a set of early embryonic blastomeres (the MS-granddaughters), and in six glial-like cells called GLRs. The entire natural hlh-1 expression pattern is recapitulated in transgenic animals containing an hlh-1::lacZ fusion with 5.1 kb of hlh-1 sequences beginning upstream of the coding region and extending into the second exon. Deletions and rearrangements in this 5.1-kb sequence were assayed for their effects on reporter gene expression in transgenic animals. Deletions removing a segment 434-550 bp upstream of the coding region resulted in a loss of all aspects of the expression pattern, suggesting that at least one common regulatory factor is required for all expression. Deletions of other regulatory elements affected distinct aspects of the expression pattern; hence, each expression subpattern exhibits a different set of sequence requirements. Interspecies sequence comparison and lacZ expression constructs with the related nematode Caenorhabditis briggsae indicated that the C. briggsae and C. elegans genes contain equivalent sets of control signals. The results from this work implicate the hlh-1 promoter as an integration point for diverse temporal and spatial control signals.

Amino Acid Sequence↗
PubMed1994

Discharge patterns of chicken cochlear ganglion neurons following kanamycin-induced hair cell loss and regeneration.

Hair cells in the basal, high frequency region (> 1100 Hz) of the chicken cochlea were destroyed with kanamycin (400 mg/kg/d x 10 d) and allowed to regenerate. Afterwards, single unit recordings were made from cochlear ganglion neurons at various times post-treatment. During the first few weeks post-treatment, only neurons with low characteristic frequencies (< 1100 Hz) responded to sound. Despite the fact that the low frequency region of the cochlea was not destroyed, neurons with low characteristic frequencies had elevated thresholds, abnormally broad U-shaped or W-shaped tuning curves and low spontaneous discharge rates. At 2 days post-treatment, the spontaneous discharge rates of some acoustically unresponsive units fluctuated in a rhythmical manner. As recovery time increased, thresholds decreased, tuning curves narrowed and developed a symmetrical V-shape, spontaneous rate increased and neurons with higher characteristic frequencies began to respond to sound. In addition, the proportion of interspike interval histograms with regularly spaced peaks increased. These improvements progressed along a low-to-high characteristic frequency gradient. By 10-20 weeks post-treatment, the thresholds and tuning curves of neurons with characteristic frequencies below 2000 Hz were within normal limits; however, the spontaneous discharge rates of the neurons were still significantly lower than those from normal animals.

Animals↗