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Biomedical subjects

W M Huang

Publications and source records attributed to W M Huang.

At least 37 records · Page 2Linked to original sources
PubMed1995

Linear chromosomes of Lyme disease agent spirochetes: genetic diversity and conservation of gene order.

We have constructed physical and genetic maps of the chromosomes of 21 Lyme disease agent spirochetes from geographically diverse locations. All have linear chromosomes whose lengths range from 935 to 955 kbp, and all contain multiple linear plasmids in the 16- to 175-kbp size range. The locations of 11 gene clusters on the chromosomes of these different isolates are indistinguishable at the resolution achieved in this study, indicating that the members of this related group of species have highly conserved chromosomal gene orders. However, chromosomal restriction endonuclease cleavage site maps are unique for nearly all isolates. The 22 chromosomal maps currently available define eight classes of Lyme disease agents. Four of these correspond to the previously proposed species Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, and Borrelia japonica. In addition, the North American isolates 21038, DN127 c19-2, 25015, and CA55 typify four additional chromosomal types that are as phylogenetically distinct as the species listed above. These findings support the idea that comparison of restriction maps is currently the most robust and definitive method for determining overall chromosomal relationships among closely related bacteria. In the course of this work, we located on the chromosome the previously unmapped outer surface protein-encoding LA7 gene and genes homologous to the Escherichia coli priA, plsC, parE, and parC genes, and we have substantially refined the locations of the recA, fla, p22A, and flgE genes.

Animals↗
PubMed1995

Gene therapy for hepatocellular carcinoma: chemosensitivity conferred by adenovirus-mediated transfer of the HSV-1 thymidine kinase gene.

We have investigated whether adenovirally mediated gene transfer of the herpes simplex thymidine kinase gene to human hepatocellular carcinoma (HCC) cell lines can sensitize these cells to the prodrug ganciclovir and thereby provide a therapeutic option for this intractable cancer. Two replication-deficient adenoviruses encoding for the herpes simplex virus type-1 (HSV) thymidine kinase (TK) gene were generated in which expression of TK is under the control of either the human cytomegalovirus immediate early promoter (CMV) or the human alpha-fetoprotein (AFP) promoter/enhancer. We demonstrate that the combination of adenovirally mediated TK gene transfer and ganciclovir treatment effectively inhibits proliferation and causes cell death of HCC cells in vitro and that in vivo TK gene transfer and ganciclovir treatment inhibits hepatocellular tumor growth in a mouse model of this cancer. Furthermore, we show that expression of the TK gene can be restricted to those HCCs that express the tumor marker AFP through the incorporation of the AFP enhancer/promoter within an adenoviral vector.

Animals↗
PubMed1995

[Clinical and experimental study on inhibitory effect of sanhuang mixture on platelet aggregation].

Sanhuang mixture (Xinmai capsule) is composed of Astragalus membranaceus, Coptis chinensis and Scutellaria baicalensis. It could inhibit platelet aggregation (PAG) induced by ADP in rats or by ADP and ADR in vivo. Patients with high PAG took Xinmai capsule for 4 weeks, the effective rate was 88.7%, which was same as the efficacy of aspirin 50 mg/day. In Syndrome Differentiation of TCM, the effective rate was the highest (92.3%) in the Syndrome of Qi Deficiency-Blood Stasis, and the lowest (72.7%) in the Syndrome of Stasis Phlegm and combined together.

Adult↗
PubMed1994

Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer.

We have constructed recombinant human adenoviruses that express wild-type human p53 under the control of either the Ad 2 major late promoter (MLP) or the human cytomegalovirus (CMV) immediate early gene promoter. Each construct replaces the Ad 5 E1a and E1b coding sequences necessary for viral replication with the p53 cDNA and MLP or CMV promoter. These p53/Ad recombinants are able to express p53 protein in a dose-dependent manner in infected human cancer cells. Tumor suppressor activity of the expressed p53 protein was assayed by several methods. [3H]Thymidine incorporation assays showed that the recombinant adenoviruses were capable of inhibiting DNA synthesis in a p53-specific, dose-dependent fashion. Ex vivo treatment of Saos-2 tumor cells, followed by injection of the treated cells into nude mice, led to complete tumor suppression using the MLP/p53 recombinant. Following a single injection of CMV/p53 recombinant adenovirus into the peritumoral space surrounding an in vivo established tumor derived from a human small cell lung carcinoma cell line (NIH-H69), we were able to detect p53 mRNA in the tumors at 2 and 7 days post-injection. Continued treatment of established H69 tumors with MLP/p53 recombinant led to reduced tumor growth and increased survival time compared to control treated animals. These results indicate that recombinant adenoviruses expressing wild-type p53 may be useful vectors for gene therapy of human cancer.

Adenoviruses, Human↗
PubMed1994

Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates.

Neisseria gonorrhoeae homologues of gyrA and parC have been identified using hybridization probes generated from conserved regions of diverse gyrA genes. These genes have been tentatively identified as gyrA and parC, based on predicted amino acid sequence homologies to known GyrA homologues from numerous bacterial species and to ParC from Escherichia coli and Salmonella typhimurium. The gyrA gene maps to a physical location distant from the gyrB locus on the gonococcal chromosome, which is similar to the situation found in E. coli. The parC gene is not closely linked (i.e. greater than 9 kb) to an identifiable parE gene in N. gonorrhoeae. The gonococcal GyrA is slightly larger than its E. coli homologue and contains several small insertions near the C-terminus of the predicted open reading frame. A series of ciprofloxacin-resistant mutants were selected by passage of N. gonorrhoeae on increasing concentrations of the antibiotic. Sequential passage resulted in the selection of isolates with minimum inhibitory concentrations approximately 10,000-fold higher than the parental strain. Mutations within gyrA resulted in low to moderate levels of resistance, while strains with high-level resistance acquired analogous mutations in both gyrA and parC. Resistance mutations were readily transferred between N. gonorrhoeae strains by transformation. The frequencies of transformation, resulting in different levels of ciprofloxacin resistance, further support the notion that both gyrA and parC genes are involved in the establishment of extreme levels of ciprofloxacin resistance.

Amino Acid Sequence↗
PubMed1994

Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions of gyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons of gyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = 14) for which the ciprofloxacin MIC was > 2 micrograms/ml. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of 1 in 10(7) to 10(8) at ciprofloxacin concentrations of 2 micrograms/ml, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.

Anti-Infective Agents↗
PubMed1994

Analysis by PCR and direct DNA sequencing of gyrA mutations associated with fluoroquinolone resistance in Enterococcus faecalis.

A region of gyrA, the gene encoding subunit A of DNA gyrase, that is known to be associated with resistance was amplified and sequenced from 16 Enterococcus faecalis and Enterococcus faecium isolates. Six ciprofloxacin-resistant clinical isolates (MICs of ciprofloxacin, 32 to 64 micrograms/ml) and one multistep resistant laboratory mutant of E. faecalis (MIC of ciprofloxacin, 128 micrograms/ml) contained a change from serine to arginine or to isoleucine at codon 83 or a change from glutamic acid to lysine or to glycine at codon 87 (Escherichia coli GyrA coordinates); these changes have been associated with fluoroquinolone resistance in other species. No difference in the region studied was found in two ciprofloxacin-resistant E. faecium isolates (MICs, 32 micrograms/ml) or in four laboratory derived, spontaneous ciprofloxacin-resistant mutants of E. faecalis (MICs, 8 to 16 micrograms/ml), suggesting that other mechanisms may be responsible for fluoroquinolone resistance in some enterococci.

Amino Acid Sequence↗
PubMed1994

gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi.

We have isolated and characterized mutants of Borrelia burgdorferi that are resistant to the antibiotic coumermycin A1, which targets the B subunit of DNA gyrase. Mutants had either 100- or 300-fold higher resistance to coumermycin A1 than wild-type B. burgdorferi. In each case, a single point mutation in the gyrB gene converted Arg-133 to Gly or Ile. Mutations in the homologous Arg residue of Escherichia coli DNA gyrase are also associated with resistance to coumarin antimicrobial agents.

Amino Acid Sequence↗
PubMed1993

Linear chromosomal physical and genetic map of Borrelia burgdorferi, the Lyme disease agent.

A physical map of the 952 kbp chromosome of Borrelia burgdorferi Sh-2-82 has been constructed. Eighty-three intervals on the chromosome, defined by the cleavage sites of 15 restriction enzymes, are delineated. The intervals vary in size from 96 kbp to a few hundred bp, with an average size of 11.5 kbp. A striking feature of the map is its linearity; no other bacterial groups are known to have linear chromosomes. The two ends of the chromosome do not hybridize with one another, indicating that there are no large common terminal regions. The chromosome of this strain was found to be stable in culture; passage 6, 165 and 320 cultures have identical chromosomal restriction maps. We have positioned all previously known Borrelia burgdorferi chromosomal genes and several newly identified ones on this map. These include the gyrA/gyrB/dnaA/dnaN gene cluster, the rRNA gene cluster, fla, flgE, groEL (hsp60), recA, the rho/hip cluster, the dnaK (hsp70)/dnaJ/grpE cluster, the pheT/pheS cluster, and the genes which encode the potent immunogen proteins p22A, p39 and p83. Our electrophoretic analysis detects five linear and at least two circular plasmids in B. burgdorferi Sh-2-82. We have constructed a physical map of the 53 kbp linear plasmid and located the operon that encodes the two major outer surface proteins ospA and ospB on this plasmid. Because of the absence of functional genetic tools for this organism, these maps will serve as a basis for future mapping, cloning and sequencing studies of B. burgdorferi.

Blotting, Southern↗
PubMed1993

Cloning and nucleotide sequence of the Campylobacter jejuni gyrA gene and characterization of quinolone resistance mutations.

The gyrA gene of Campylobacter jejuni UA580, which encodes the A subunit of DNA gyrase, was cloned and its nucleotide sequence was determined. An open reading frame of 2,589 nucleotides was identified, which could code for a polypeptide of 863 amino acids with a M(r) of 97 kDa. Both the nucleotide sequence and the putative amino acid sequence show ca. 50% identity with those of other gyrA genes from gram-positive and gram-negative bacteria. The locations of the gyrA gene on genome maps of both C. jejuni UA580 and Campylobacter coli UA417 were determined. Six nalidixic acid-resistant isolates of C. jejuni were shown to carry mutations in gyrA. Three clinical isolates had Thr-86-to-Ile substitutions. Three laboratory mutants had substitutions of Thr-86 to Ile, Asp-90 to Ala, and Ala-70 to Thr, respectively. The mutation at Thr-86, which is homologous to Ser-83 in Escherichia coli, was associated with high-level resistance to ciprofloxacin in C. jejuni.

4-Quinolones↗
PubMed1992

Localization of HPV-16 DNA sequence in CaSki cells by electron microscopic hybridocytochemistry.

We investigated the HPV-16 DNA sequence in the CaSki cervical carcinoma cell line by electron microscopic hybridocytochemistry using biotinylated HPV-16/18 probes. At the light microscopic level, reaction product of hybridized HPV-16 DNA sequence was not seen in the cytoplasm but appeared as spots or rods randomly distributed in the nuclei. By electron microscopy, reaction product was seen aggregated in several regions in the nuclei. Most of the stained areas did not reveal particular architecture but showed part of the chromatin structure. In other nuclei, reaction product was observed to be associated with strings of loop-like structure, and some stained loops were seen to be connected directly to the nuclear filamentous chromatin structure. The skeletonized images of hybridized HPV-16 DNA in the nuclei were illustrated by computerized image analysis. In conclusion, we have demonstrated the HPV-16 DNA sequence in the nuclei of CaSki cells by electron microscopy. The identification of stained areas localized only in the chromatin suggests an integrated form of HPV-16 DNA sequence in the cells. This method could be used to identify an integrated or episomal form of viral DNA in the virus-containing cells.

Biotin↗
PubMed1992

Beneficial effects of berberine on hemodynamics during acute ischemic left ventricular failure in dogs.

In 18 dogs ischemic left ventricular failure characterized by a 30 percent reduction in peak rate of rise of left ventricular pressure (+dp/dt) and elevation of left ventricular end-diastolic pressure (LVEDP) to 15 mmHg or more was produced by ligation of the proximal left anterior descending coronary artery followed by serial occlusions of the distal left circumflex coronary artery. In 10 days, administration of berberine in an intravenous bolus injection (1 mg/kg, within 3 minutes) followed by a constant infusion (0.2 mg/kg/min, 30 minutes) increased the cardiac output (CO) from 1.25 +/- 0.12 to 1.61 +/- 0.17 L/min (P < 0.05), and +dp/dt from 810 +/- 85 to 1021 +/- 130 mmHg/s (P < 0.01), and decreased LVEDP from 16.5 +/- 1.3 to 12.0 +/- 1.0 mmHg (P < 0.05), diastolic blood pressure from 94 +/- 6 to 84 +/- 5 mmHg (P < 0.01), systemic vascular resistance from 7303 +/- 278 to 5442 +/- 231 dynes.x/cm5 (P < 0.01), but did not affect the heart rate. Injection of 5% glucose with the same volume did not improve CO and dp/dt (P > 0.05) but increased the LVEDP from 17.1 +/- 1.4 to 17.8 +/- 1.6 mmHg (P < 0.01) in 8 dogs. The levels of plasma concentration of berberine was determined with high-performance liquid chromatography. The changes in plasma drug level were found parallel to hemodynamic effects of berberine. The results of this study showed that berberine was able to improve the impaired left ventricular function by its positive inotropic effect and mild systemic vasodilatation.

Animals↗
PubMed1992

[A study of the antiarrhythmic mechanism of berberine on delayed activation potassium current by voltage clamp].

The effects of berberine (0.1-10 mg/L) on delayed activation potassium current (iK) of canine Purkinje fibers were studied with two-microelectrode voltage clamp technique. Berberine (Ber) significantly reduced the amplitude of iK in concentration-dependent manner. When perfused with 10 mg/L Ber, the amplitude of tail current of the Purkinje fibers decreased immediately and the inhibition rate was 73.6 +/- 4.5% (n = 9, P < 0.001). The inhibition effect of berberine on iK may explain its prolongation effect on action potential duration and its antiarrhythmic action.

Action Potentials↗
PubMed1991

Structure and function of L-glutamate decarboxylase.

Membrane bound L-glutamate decarboxylase (GAD) has been solubilized and partially purified from hog brain. The solubilized GAD appears to exist in two forms, alpha and beta, differing in their size and electrophoretic mobility. The alpha form has similar mobility as that of the soluble GAD in 7.5% and 5-25% gradient polyacrylamide gel electrophoresis suggesting that they are similar in size and charge. In addition, gene encoding for mouse brain GAD has been cloned and characterized. Mouse brain GAD cDNA consists of two DNA fragments with 1.6 and 1.0 Kb. The 1.6 and 1.0 Kb fragments contain 1657 and 974 bP, respectively. The significance of multiple forms of GAD is also discussed.

Animals↗
PubMed1990

A nascent peptide is required for ribosomal bypass of the coding gap in bacteriophage T4 gene 60.

Bacteriophage T4 DNA topoisomerase gene 60 contains a 50 nucleotide untranslated region within the coding sequence of its mRNA. Translational bypass of this sequence by elongating ribosomes has been postulated for the mode of synthesis of an 18 kd polypeptide specified by the split coding segments. Ribosome bypass of the untranslated region also occurs when a segment of gene 60 is fused to lacZ and expressed in E. coli. The efficiency of bypass in these gene 60-lacZ fusions approaches 100%. Here, mutations that delete, insert, or substitute nucleotides from gene 60-lacZ fusions are examined. Essential features necessary for high level gap bypass emerging from this analysis are a cis-acting nascent peptide sequence, a short duplication bordering the gap, and a stop codon contained in a stem-loop structure at the 5' junction of the gap.

Amino Acid Sequence↗
PubMed1990

Study of the effects and mechanisms of berberine on slow-response action potentials.

The effects of berberine on slow-response action potentials (SAP) of guinea pig papillary muscles were studied. SAP was elicited by histamine in a high concentration of potassium solution (27 mmol). The results showed that berberine (24.5 mumol) was able to increase action potential amplitude, maximum rate of depolarization, action potential duration 50, action potential duration 100 (n = 16, p less than 0.01) and effective refractory period (n = 10, p less than 0.01) of SAP by 6.2%, 21.1%, 50.1%, 47.2% and 92.2%, respectively, but did not affect the resting membrane potential (RMP). To the above parameters, except APD 100, berberine was no longer to induce any significant change by pretreating with propranolol. The results suggested that the effects of berberine on slow-response action potentials were mainly related to the facilitating of slow calcium inward current, which might result from stimulating the beta-adrenoceptor.

Action Potentials↗
PubMed1990

Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase.

We used specific polyclonal antibodies against L-glutamate decarboxylase (GAD) to screen a mouse brain cDNA library that was constructed in the expression vector lambda gt11. We obtained 1.5 x 10(6) recombinant DNA clones in the mouse brain cDNA library. One of the clones was positively identified as a GAD clone on the basis of the following results: (i) the clone and its secondary and tertiary clones all reacted strongly with anti-GAD antibodies; (ii) the fusion protein obtained from lambda GAD-Y1089 showed good GAD enzyme activity as determined by both CO2 and gamma-aminobutyric acid methods. The GAD clone thus obtained contains GAD cDNA of approximately 2.6 kilobases that has one internal EcoRI site. After GAD cDNA was cut at the EcoRI site, two DNA fragments of about 1.6 and 1.0 kilobases were obtained at the 5' and 3' ends, respectively. The cDNA insert was found to be composed of 2632 base pairs, the translation initiation site was assigned to the methionine codon ATG, and the termination site was found to be TGA (positions 2216-2218). Furthermore, the coding region in 2169 base pairs was found to consist of 723 amino acids. The protein has a molecular weight of 83,207 and contains 83 strongly basic, 108 strongly acidic, 226 hydrophobic, and 221 polar amino acids with an isoelectric point of 5.355. The relationship of this GAD cDNA to other forms of GAD is discussed.

Amino Acid Sequence↗