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Biomedical subjects

W M Huang

Publications and source records attributed to W M Huang.

At least 19 recordsLinked to original sources
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↗
PubMed1989

[Effects of berberine on ischemic ventricular arrhythmia].

The effects of berberine on ischemic ventricular arrhythmias induced by ligating the left anterior descending coronary artery (LAD) of canine were reported. The results showed that berberine was able to get 99% suppression (P less than 0.001) on the total ventricular premature beats (VPCs) by 12 hours after ligature of LAD, the paired VPCs, ventricular tachycardias and VPCs with R on T were also significantly suppressed; and the ventricular tachycardia induced by programmed ventricular stimulation was effectively inhibited by berberine. In addition, the results revealed that the decrease of cardiac output caused by ligature of LAD was obviously attenuated by berberine. The mechanisms of the antiarrhythmic effect of berberine on ischemic ventricular tachyarrhythmias were discussed.

Animals↗
PubMed1989

[Research on energy metabolism enzyme in liver of the rabbit during hemorrhagic shock and effect of acupuncture at "renzhong" acupoint by quantitative histochemistry].

The energy crisis has been considered to act an important role during shock recent years. We all know the liver can supply for energy substrate when body is in an emergency condition. According to the theory of Traditional Chinese Medicine, Renzhong acupoint is a very important acupoint to treat "Jue Zheng" (including shock). We divided 36 male rabbits into three groups randomly, they were normal, acupuncture and nonacupuncture. The normal control was anesthetized only with sodium pentobarbital (35mg/kg). The acupuncture and nonacupuncture groups were operated under conscious with method of improving Wiggers(1950). After bleeding 20ml/kg, these animals were in shock. Then the acupuncture group was applied with acupuncture at "Renzhong" and the nonacupuncture group was not during hemorrhagic shock. The samples of liver were excised and stained using enzyme histochemical method. All of them were determined with MPV-2 and Univar-SMSP. Two dehydrogenases (LDH and SDH) in hepatocytes were observed in this work. The results showed that the enzyme's activity in nonacupuncture both LDH and SDH were the weakest among three groups (P less than 0.01 and P less than 0.05). But in acupuncture group, they were stronger than nonacupuncture group and near normal control. These suggest that acupuncture at "Renzhong" acupoint can increase activity of LDH and SDH during hemorrhagic shock. It is useful to resistance shock in a certain degree.

Acupuncture Points↗
PubMed1988

Screening for phenylketonuria mutations by DNA amplification with the polymerase chain reaction.

Single base substitutions have been identified in two mutant phenylalanine hydroxylase (PAH) alleles that cause phenylketonuria (PKU). The two mutant alleles are common among caucasians of northern European ancestry; detection in genomic DNA samples of patients and carriers by hybridisation with oligonucleotides specific for the respective mutant alleles requires fractionation of restriction-enzyme-digested genomic DNA samples by gel electrophoresis. This method is too cumbersome for mass screening of PKU carriers. Identification of carriers of the mutant alleles was achieved by direct analysis of their genomic DNA samples after specific amplification of a sub-genomic DNA fragment containing both mutation sites by polymerase chain reaction. The results suggest that it is technically feasible to develop a programme for carrier detection of the genetic trait in the population for individuals without a family history of PKU.

Alleles↗
PubMed1988

A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60.

A 50-nucleotide untranslated region is shown to be present within the coding sequence of Escherichia coli bacteriophage T4 gene 60, which encodes one of the subunits for its type II DNA topoisomerase. This interruption is part of the transcribed messenger RNA and appears not to be removed before translation. Thus, the usual colinearity between messenger RNA and the encoded protein sequence apparently does not exist in this case. The interruption is bracketed by a direct repeat of five base pairs. A mechanism is proposed in which folding of the untranslated region brings together codons separated by the interruption so that the elongating ribosome may skip the 50 nucleotides during translation. The alternative possibility, that the protein is efficiently translated from a very minor and undetectable form of processed messenger RNA, seems unlikely, but has not been completely ruled out.

Amino Acid Sequence↗
PubMed1988

Translation initiation controls the relative rates of expression of the bacteriophage lambda late genes.

The late operon of bacteriophage lambda contains the genes encoding the morphogenetic proteins of the phage. These genes are transcribed equally from the single late promoter. Although the functional half-lives of the mRNA for the various genes of this operon vary less than 2-fold, their relative rates of expression have been shown to vary by nearly 1000-fold. This variation could result from differing rates of translation initiation, from overlapping upstream translation, or from differential elongation rates due to the presence of codons for which the corresponding tRNAs are rare. To distinguish between these possibilities, we have cloned sequences surrounding the initiator codons of several of these genes and measured their ability to drive synthesis of hybrid lambda-beta-galactosidase proteins. The rates of expression of the hybrid genes thus produced correlate very well with the natural rates of expression of the corresponding phage genes, suggesting that the rate of initiation of translation controls the relative expression rates of these genes.

Bacteriophage lambda↗
PubMed1987

Initiation of bacteriophage P22 DNA packaging series. Analysis of a mutant that alters the DNA target specificity of the packaging apparatus.

Bacteriophage P22 is thought to package its double-stranded DNA chromosome from concatemeric replicating DNA in a "processive" sequential fashion. According to this model, during the initial packaging event in such a series the packaging apparatus recognizes a nucleotide sequence, called pac, on the DNA, and then condenses DNA within the coat protein shell unidirectionally from that point. DNA ends are generated near the pac site before or during the condensation reaction. The opposite end of the mature chromosome is created by a cut made in the DNA after a complete chromosome is condensed within the phage head. Subsequent packaging events on that concatemeric DNA begin at the end generated by the headful cut of the previous event and proceed in the same direction as the previous event. We report here the identification of a consensus nucleotide sequence for the pac site, and present evidence that supports the idea that the gene 3 protein is a central participant in this recognition event. In addition, we tentatively locate the portion of the gene 3 protein that contacts the pac site during the initiation of packaging.

Base Sequence↗
PubMed1986

Nucleotide sequence of a type II DNA topoisomerase gene. Bacteriophage T4 gene 39.

T4 DNA topoisomerase is a type II enzyme and is thought to be required for normal T4 DNA replication T4 gene 39 codes for the largest of the three subunits of T4 DNA topoisomerase. I have determined the nucleotide sequence of a region of 2568 nucleotides of T4 DNA which includes gene 39. The location of the gene was established by the identification of the first fifteen amino acids in the large open reading frame in the DNA sequence as those found at the amino-terminus of the purified 39-protein. The coding region of gene 39 has 1560 bases, and it is followed by two in-frame stop codons. The gene is preceded by a typical Shine-Dalgarno sequence as well as possible promoter sequences for E. coli RNA polymerase. T4 39-protein consists of 520 amino acids, and it has a calculated molecular weight of 58,478. By comparing the amino acid sequences, T4 39-protein is found to share homology with the gyrB subunit of DNA gyrase. This suggests that these topoisomerase subunits may be equivalent functionally. Some of the characteristics of the 39-protein and its structural features predicted from the DNA sequence data are discussed.

Amino Acid Sequence↗
PubMed1986

The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase.

T4 gene 52 encodes one of the three subunits of T4 DNA topoisomerase. The T4 enzyme is required for normal phage DNA replication. I have cloned the entire gene, and it is expressed in uninfected E. coli cells. The sequence of 1966 nucleotides of T4 deletion delta sa9 surrounding gene 52 has been determined. The reading frame of the gene was established by identifying the first ten amino acids in the large open reading frame derived from the DNA sequence as those at the amino-terminus of the purified 52-protein. Based on the DNA sequence, 52-protein has 441 amino acids and a calculated peptide molecular weight of 50,583 daltons. This T4 topoisomerase subunit shares significant amino acid sequence homology with the gyrA subunit of bacterial gyrases and the carboxyl-half of yeast topoisomerase II in spite of the large differences in their sizes, confirming their functional equivalence in type II enzyme directed DNA topoisomerization. Amino acid sequence homology is highest in the amino-terminal portions of the equivalent peptides. The homology alignment suggests a consensus sequence organization surrounding the reactive tyrosine which is used to form the transient protein-DNA intermediate in the double-stranded DNA passing reaction. The delta sa9 deletion in T4 brings gene 52 to a location 30 nucleotides 3' from the rIIB gene whose C-terminal 167 codons are also reported here.

Amino Acid Sequence↗
PubMed1986

Plasmid vectors useful in the study of translation initiation signals.

The construction and characterization of plasmid vectors useful in the analysis of translation initiation signals in Escherichia coli and in the construction of lacZ gene hybrids are described. Transcription on the vectors proceeds from a cAMP-independent lac promoter through several restriction sites into a truncated lacZ structural gene lacking its first eight codons. Because this gene has no translation initiation signal, its level of expression is extremely low. A DNA fragment containing a translation start signal can be inserted between the promoter and truncated lacZ gene to produce a hybrid protein with functional beta-galactosidase activity. The vectors described here differ in sequence between the EcoRI cloning site and the lacZ gene to allow easy, in-frame joining of DNA containing a translation initiation signal to the lacZ gene. Cells containing plasmids can be screened directly for in-frame inserts by colony color on indicator plates.

Genetic Vectors↗