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Biomedical subjects

T Huang

Publications and source records attributed to T Huang.

At least 163 records · Page 9Linked to original sources
PubMed1992

The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae.

We have constructed a dihydrofolate reductase mutant (dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfr1 mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfr1 mutants. The inability of the mutants to metabolize FA suggests that the DFR1 gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfr1 strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfr1 strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFR1 gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfr1 mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfr1 strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.

Leucovorin↗
PubMed1992

Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed mutagenesis.

1. Juvenile hormone esterase (JHE) is a serine hydrolase selective for hydrolysis of the conjugated methyl esters of insect juvenile hormones. 2. We have investigated the mechanism of catalytic action of this enzyme by site-directed mutagenesis of the cloned enzyme and expression of the mutants in a baculovirus system. 3. A series of individual mutations of JHE were made to residues possibly involved in catalysis of juvenile hormones, and which are highly conserved in both esterases and lipases. 4. Mutation of the serine residue at position 201 to glycine (S201G), or aspartate 173 to asparagine (D173N), or histidine 446 to lysine (H446K), removed all detectable activity and these mutagenized enzymes were determined to be at least 10(6)-fold less active than wild type JHE. 5. Mutation of arginine 47 to histidine (R47H) decreased but did not abolish activity, with Km essentially unchanged at 66 nM for R47H compared to 34 nM for wild type JHE. 6. The kcat for R47H was decreased from 103 min-1 for wild type JHE to 1.9 min-1. 7. In addition, glutamate residue 332 was altered to glutamine (E332Q) and expressed in an Escherichia coli system. 8. This mutation was also found to remove all detectable activity. 9. From the results presented in this study and by comparison of JHE to other serine esterases and lipases, we predict that JHE possesses a Ser201-His446-Glu332 catalytic triad. 10. In addition, aspartate 173 and arginine 47 are essential for the efficient functioning of JHE.

Amino Acid Sequence↗
PubMed1992

Characterization of human herpesvirus-6(U1102) and (GS) gp112 and identification of the Z29-specified homolog.

Monoclonal antibody 2D10 (MAb 2D10) raised toward human herpesvirus-6(U1102) [HHV6(U1102)] immunoprecipitated three glycosylated peptides, M(r) 112,000, 64,000, and 58,000, designated as gp112 from U1102-infected lymphocytes. Pulse-chase experiments suggest that the M(r) 64,000 and 58,000 polypeptides are very likely generated by post-translational cleavage of the M(r) 112,000 polypeptide. MAb 2D10 neutralized virion infectivity in the presence of complement, suggesting that gp112 is located in the virion envelope. MAb 2D10 did not prevent the appearance of HHV6-specific cytopathic effect. MAb 2D10 was reactive with denatured gp112 in immunoblots. HHV6 isolates form two clusters (Schimer, Wyatt, Yamanishi, Rodriguez, and Frenkel, Proc. Natl. Acad. Sci. USA 88, 5922; Ablashi, Balachandran, Josephs, Hung, Krtueger, Kramarsky, Salahuddin, and Gallo, Virology 184, 545). MAb 2D10 reacted by immunofluorescence and immunoprecipitation with the prototypes of each cluster, GS and Z29. Whereas the proteins immunoprecipitated by MAb 2D10 from GS-infected lymphocytes had an electrophoretic pattern very similar to that of U1102 gp112, the homologous glycoprotein immunoprecipitated from Z29-infected lymphocytes consisted of three polypeptides with M(r) 102,000, 59,000, and 50,000. The data suggest a variation among HHV6 isolates as far as this glycoprotein is concerned.

Animals↗
PubMed1992

Overexpression of the influenza virus polymerase can titrate out inhibition by the murine Mx1 protein.

The murine Mx1 protein is an interferon-inducible protein which confers selective resistance to influenza virus infection both in vitro and in vivo. The precise mechanism by which the murine Mx1 specifically inhibits replication of influenza virus is not known. Previously, sensitive replication systems for influenza virus ribonucleoprotein, in which a synthetic influenza virus-like ribonucleoprotein is replicated and transcribed by influenza virus proteins provided in trans, have been developed. With these systems, the antiviral activity of the murine Mx1 protein was examined. It was found that continued expression of influenza polymerase polypeptides via vaccinia virus vectors can titrate out the inhibitory action of the murine Mx1 protein. This titration of inhibitory activity also occurs when the viral PB2 protein alone is overexpressed, suggesting that an antiviral target for the murine Mx1 polypeptide is the viral PB2 protein.

Animals↗
PubMed1991

Rescue of a foreign gene by Sendai virus.

A simple protocol for the rescue of a synthetic genome into a paramyxovirus has been developed. First, a synthetic Sendai virus-like RNA, containing the antisense coding region of the chloramphenicol acetyltransferase gene replacing the coding region of the Sendai virus genome, was transcribed from a cDNA. When introduced into cells that are infected with Sendai virus, this RNA construct was transcribed, replicated, and packaged into infectious virions. The addition of infected cell extract to the RNA prior to transfection markedly enhanced levels of chloramphenicol acetyltransferase expression and rescue. However, this enhancement is not due to encapsidation of the RNA into nucleocapsids as the RNA remains nuclease-sensitive. Uninfected cell extract also enhances expression and rescue efficiency, implying involvement of a cellular factor(s) with the synthetic viral-like RNA construct that allows for enhanced polymerase recognition. This system should allow for the dissection of the various cis-acting RNA signals within the paramyxovirus genome.

Animals↗
PubMed1991

Inhibition of DNA replication of adenovirus type 5 and simian virus 40 by tunicamycin.

Addition of tunicamycin, a glycosylation inhibitor, to SV40-infected CV-1 cells or Ad5-infected HeLa cells at the beginning of infection was found to inhibit the accumulation of viral DNA at late times after infection. However, tunicamycin did not block viral DNA replication when added to the infected cells at late times after infection. The inhibitory effect of tunicamycin was partially reversible in the presence of acetylglucosamine, suggesting that the effect was due to glycosylation. In spite of diminished amounts of viral DNA accumulated at the late phase of infection in the presence of tunicamycin, the transcription rate of Ad5 late RNA and the amount of adenovirus late proteins and mRNA were not significantly affected by tunicamycin treatment. The inhibitory effect of tunicamycin on Ad5 DNA replication was much reduced in 293 cells which provide E1a gene products in trans. Similar observation was obtained for the replication of SV40 DNA in Cos-7 cells which provide SV40 early gene products in trans. These results suggest that tunicamycin inhibits a glycosylation event induced by the early gene products of Ad5 and SV40 viruses during the early phase of infection.

Adenoviruses, Human↗
PubMed1991

Birth of the first 100 babies in Hawaii after conception in-vitro: experience at the Pacific In-Vitro Fertilization Institute.

This paper reports on the first 100 infants delivered consecutively the result of 63 pregnancies after conception in-vitro at the Pacific In-Vitro Fertilization (IVF) Institute. These pregnancies were established prior to the introduction of embryo-cryopreservation into the program. Ninety-seven percent (97%) of singleton pregnancies delivered after 36 weeks; only 5/31 (16%) required tocolytic therapy. The overall multiple gestation rate was 51%, higher than in many other IVF centers. Maternal complications and neonatal morbidity were associated primarily with these multiple gestations. Forty-one percent (41%) delivered in less than 36 weeks, and tocolytics were administered in 22/27 (81%) of twin and 5/5 (100%) of triplet gestations. Neonatal morbidity was noted in 39 infants (33 from multiple gestations); 28 neonates were admitted to the intensive care unit (27 from multiple gestations). Nine births had or developed minor abnormalities (hernias, pyloric stenosis). One case of Trisomy 21, and one infant death at 11 months occurred in the group. The caesarean section rate for all IVF deliveries was 65%. It is concluded that IVF is clearly established in Hawaii for infertility refractory to other methods of treatment. The risk associated with IVF stem from the possibility of multiple gestations, not whether the pregnancy was initiated in-vitro. In the future, embryo cryopreservation may help to reduce the risk of multiple pregnancy.

Adult↗
PubMed1991

[A study on standard 12-lead ECG QRS scoring system for estimating myocardial infarct size].

Using the standard 12-lead electrocardiographic QRS scoring system modified by Nancy (1985), 99 ECGs of 52 patients with acute myocardial infarction were used for estimating myocardial infarct size (MIS). One point was designed to represent 3% of the left ventricle. Each patient had the first score of ECG at the day ranged from 7 to 12 and the second 25-32 days post acute chest pain except 5 cases died before the second scoring. The results showed that the averaged score was 6.7 +/- 3.1 points accumulated from all 99 ECGs and 11.0 points from 5 cases died. There was no statistic difference between the points value of the first scoring 6.4 +/- 2.9 vs second 6.5 +/- 2.7 points, nor between the points received from Q waves 4.0 +/- 1.5 vs 3.9 +/- 1.4 points in 47 patients. There was nevertheless strong correlation both between the total points of the first and second scoring, r = 0.85 (P less than 0.0005), and those between the points related to Q wave, r = 0.81 (P less than 0.005). Furthermore, early serial serum CPK changes were determined for evaluating MIS in 25 patients, the results were compared to the first and second scores respectively, the correlations were significant, r = 0.58, P less than 0.0025 and r = 0.47, P less than 0.01.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Gossypol-induced alterations of aminophospholipid composition in human sperm.

Gossypol-induced alterations of aminophospholipid composition in human sperm were observed and the effects of some factors on these alterations were investigated. The alterations of aminophospholipid composition of human sperm induced by gossypol included a progressive decrease in the levels of phosphatidylethanolamine (PE) and phosphatidylserine (PS) when gossypol concentrations ranged from 5-500 mumol/L, and a progressive increase in the level of (LPE) at lower gossypol concentrations (5-50 mumol/L). The conversion of PE into lysophosphatidylethanolamine (LPE) was strongly enhanced by Ca2+ and inhibited by 0.5 mmol/L EDTA, while PMSF, NEM, iodoacetamide and Zn2+ had no effect. This conversion was weaker in sperm with stripped surface proteins than in normal sperm. In addition, three surface proteins (MW 80, 60 and 40 kD) were fixed on the plasma membrane by gossypol treatment. The significance of gossypol action and the possibility that sperm PLA2, PE and some surface proteins might play an important role in the physiological acrosome reaction of human sperm, as well as in oocyte penetration, are discussed.

Gossypol↗
PubMed1989

Genetic analysis of olfactory behavior in Drosophila: a new screen yields the ota mutants.

A simple means of measuring Drosophila olfactory response is described, and the behavior which it measures is characterized. The assay was used to screen for X-linked mutants defective in olfactory function. Six ota mutants were isolated and characterized (ota = olfactory trap abnormal). Four of the mutants were found to be abnormal in another chemosensory behavior as well. Two of the mutant phenotypes extend to include another sensory system: they are defective in visual system physiology. All were normal, however, in a test of giant fiber system physiology. Two of the mutations are dominant, and the recessive mutations define two complementation groups. Mutations representing each complementation group, as well as one of the dominant mutations, were mapped. For the mutants with defective visual system physiology, the visual defects were shown to cosegregate with olfactory phenotypes.

Analysis of Variance↗