Biomedical subjects

Y C Chao

Publications and source records attributed to Y C Chao.

At least 55 records · Page 3Linked to original sources

PubMed1994

Novel gene expression system for plant cells based on induction of alpha-amylase promoter by carbohydrate starvation.

The 5' regulatory region and putative signal sequence of a rice alpha-amylase gene, alpha Amy8, was fused to a bacterial gene encoding beta-glucuronidase (GUS) and introduced into rice, tobacco, and potato via Agrobacterium-mediated transformation systems. Expression of this chimeric gene in suspension cells of transgenic plants was suppressed by the presence of sucrose in the medium and induced by its absence. Induction or suppression of GUS expression in transgenic rice could be reversed by the deprivation or replenishment, respectively, of sucrose in the medium. The expressed GUS fusion protein was translocated to the endoplasmic reticulum, modified by glycosylation, and secreted into the culture medium of transgenic cells. In the presence of a glycosylation inhibitor, tunicamycin, the enzymatically active form of GUS was assembled in the endoplasmic reticulum. The yield of GUS secreted by transgenic cells was estimated to be as high as 40% of total secreted proteins. The reversible induction of the alpha-amylase promoter in culture cells by sugar level in the medium provides an excellent inducible expression system for basic research in plant science. Combination of the alpha-amylase promoter and signal sequence also offers a novel approach for large scale production of low cost, easily purified, secreted recombinant proteins.

Amino Acid Sequence↗

PubMed1994

Evolution rate of hepatitis delta virus RNA isolated in Taiwan.

The complete RNA sequences of hepatitis delta viruses (HDV) isolated at 3 years apart from a chronic delta hepatitis patient in Taiwan were determined. The sequence analysis showed an overall evolution rate of 3.18 x 10(-3) substitutions/nucleotide/year. The evolution rates in different parts of HDV RNA varied. The hypervariable region evolved faster (4.55 x 10(-3) substitutions/nucleotide/year) than the hepatitis delta antigen (HDAg)-coding region (2.60 x 10(-3) substitutions/nucleotide/year) and the autocatalytic region (1.11 x 10(-3) substitutions/nucleotide/year). These data are compatible with the previous finding that the hypervariable region is more divergent than the HDAg-coding region and the autocatalytic regions among the HDV isolates from different geographic areas. No substitution was found in the four previously identified conserved domains of HDV RNA, further confirming their functional importance in viral replication. The evolution rate of this HDV RNA is higher than that determined from the partial RNA sequences of two Japanese HDV isolates and similar to that found in a Lebanon isolate. Further, it was found that this HDV RNA retained the same microheterogeneities at 15 nucleotide positions detected in the RNA 3 years earlier. It is concluded that HDV RNA in patients' serum is extremely heterogeneous, and that the nucleotide substitutions in certain nucleotide positions likely have conferred evolutionary advantages for HDV. Viral sequence evolution is a possible mechanism for chronic HDV infection.

Adult↗

PubMed1994

Genome characterization and identification of viral-associated dsDNA component of banana bunchy top virus.

The single-stranded (ss) and double-stranded (ds) viral-associated DNAs (VADs) of banana bunchy top virus (BBTV) were characterized. The ssVADs were shown to be the viral genomic DNA and the dsVADs are the likely replication forms. Both dsVADs and ssVADs contain DNA species that cross-hybridize to each other. Under the electron microscope, both circular and linear DNAs were observed from these VAD species. Libraries were constructed from VADs to further characterize them. Two overlapping VAD clones were isolated and their combined sequence indicated that their corresponding VAD, named as BBTV component 2, is circular with a size of 1095 nt. The largest ORF spans from nucleotides 82 to 390, which is sandwiched by a putative TATA box, 54 nt upstream from the first ATG codon, and a AATAAA motif, 7 nt downstream from the UAA codon. Although the sequence of this VAD is different from the previously reported BBTV component 1 (Harding et al., J. Gen. Virol. 74, 323-328, 1993) and the genome of coconut foliar decay virus (Rohde et al., Virology 176, 648-651, 1990), stretches of highly homologous sequences were found between BBTV component 2 and the other two viral genomes by computer analysis. The sequence of BBTV component 1 is highly homologous to one of the clones, pBTs26, in the VAD library. Nevertheless, our libraries still contain many clones which hybridized with ss viral genomic DNAs but not with pBTs26 and BBTV component 2, indicating that the viral genome consists of more than two components.

Base Sequence↗

PubMed1994

Sequence determination of hepatitis C virus genome isolated from Taiwan.

The partial genome sequence of the hepatitis C virus (HCV) was determined in the serum of a Taiwanese patient with chronic community-acquired type C hepatitis. The cDNA fragments synthesized with the HCV RNA as a template were amplified by polymerase chain reaction using specific oligonucleotide primers. The amplified fragments represented the regions coding for the putative core, matrix and envelope proteins as well as the N-terminal amino acid sequence of the nonstructural protein NS1, the partial nonstructural NS3 and NS4 proteins and the region of the partial 5'-end noncoding sequence. The cDNA fragments were cloned and sequenced. Sequence analysis of these clones showed that they share 83.7%, 93.2% and 93.6% similarity at the nucleotide level, and 86.6%, 94.1% and 92.9% homology at the amino acid level, with the previously published American, Japanese and Taiwanese isolates, respectively. Accordingly, the RNA genome we obtained is HCV type II, probably, the predominant subtype in Taiwan.

Adult↗

PubMed1994

Polymorphism of alcohol and aldehyde dehydrogenase genes and alcoholic cirrhosis in Chinese patients.

Liver alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), the principal enzymes responsible for the oxidation of ethanol, are polymorphic at the ADH2, ADH3 and ALDH2 loci in human beings. Our previous studies have shown that, compared with nonalcoholic individuals, Chinese alcoholic patients without liver disease had significantly lower frequencies of the ADH2*2 and ADH3*1 alleles, which encode high maximum velocity beta 2- and gamma 1-ADH subunits, respectively, as well as a lower frequency of the ALDH2*2 allele, which encodes an enzymatically inactive subunit. The data strongly suggest that genetic variation in both ADH and ALDH may influence drinking behavior and the risk of alcoholism developing through acetaldehyde formation. To further investigate the possible role of acetaldehyde in the pathogenesis of alcoholic liver disease, we determined the ADH and ALDH genotype frequencies in patients with alcohol-related cirrhosis (n = 27), viral hepatitis-related cirrhosis (n = 29) and gastric and duodenal ulcer without relevance to alcohol (n = 30). We developed a new restriction fragment length polymorphism method to genotype the mutant and normal ALDH2 alleles by using polymerase chain reaction-directed mutagenesis, which proved to be simpler and faster than the conventional detection methods that use hybridization with allele-specific oligonucleotide probes. We found that the frequencies of the alleles ADH2*2 (57%), ADH3*1 (78%) and ALDH2*2 (9%) in the alcoholic cirrhotic patients were significantly lower than those in the healthy controls and in the patients with cirrhosis from viral hepatitis and with gastric and duodenal ulcer.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗

PubMed1994

Genotyping of alcohol dehydrogenase at the ADH2 and ADH3 loci by using a polymerase chain reaction and restriction-fragment-length polymorphism in Chinese alcoholic cirrhotics and non-alcoholics.

We investigated the genotype of ADH2 and ADH3 in Chinese alcoholic cirrhotics and non-alcoholics by using a polymerase chain reaction and restriction-fragment-length polymorphism. This method is non-radioactive, easy to implement with good reproducibility. In the Chinese population, the frequencies of the ADH2*1 and ADH3*2 alleles were significantly higher in the alcoholic cirrhotic patients (53%; 23%) than in the viral hepatitis cirrhotics (32%; 8%) and the gastric and/or duodenal ulcer control patients (25%; 6%). On the other hand, the gastric and/or duodenal ulcer control patients and the viral hepatitis cirrhotic patients showed similar allele frequencies for the polymorphic ADH2 and ADH3 genes. These findings suggest that the alleles ADH2*2 and ADH3*1, coding for the high-Vmax beta 2-ADH and gamma 1-ADH, respectively, may play a protective role against alcoholism in Chinese patients.

Alcohol Dehydrogenase↗

PubMed1993

Regulation of prostatic acid phosphatase expression and secretion by androgen in LNCaP human prostate carcinoma cells.

The expression and the secretion of human prostatic acid phosphatase (PAcP), a differentiation antigen which is the major acid phosphatase in prostate epithelial cells, are thought to be regulated by an androgen. We investigated this regulatory mechanism at the post-transcriptional level in LNCaP human prostate carcinoma cells using a cDNA clone for the secretory form of PAcP. 5 alpha-Dihydrotestosterone (DHT, an active form of endogenous androgen) stimulated the secretion of PAcP from cells grown in a steroid-reduced medium and in a defined serum-free medium, respectively. The secreted PAcP activity was increased following a DHT dose in a dose-dependent fashion at concentrations of up to 1 microM. Further, the stimulation of PAcP secretion occurred following the period of exposure to DHT. During a 5-day treatment period, with 10 nM of DHT in the steroid-reduced medium, the secretion of PAcP was stimulated approximately 150% over that from control cells. Nevertheless, PAcP was secreted from cells grown in the absence of added DHT. First, the androgen dependency of PAcP expression was examined. The expression and the secretion of PAcP were observed in cells that were grown in a defined serum-free medium and grown in a steroid-reduced medium, in the absence of DHT. The increased secretion by DHT was further demonstrated to be in part due to an increase in PAcP mRNA level, as evidenced by Northern blot analysis. PAcP mRNA levels were elevated approximately 2-fold and corresponded to an increase of approximately 2.5-fold in the secreted level of newly synthesized 35S-PAcP. Then, the effect of DHT on the secretory process was investigated. Results of pulse-chase labeling experiments indicated that the secretory rate of PAcP was stimulated by about 50% on average by DHT. In conclusion, our data demonstrated that, in LNCaP cells, the expression and the secretion of PAcP regulated by androgen are apparently hormone-responsive processes. Further, DHT stimulation of PAcP secretion operates within at least two levels: increased PAcP mRNA and stimulation of the secretory pathway.

Acid Phosphatase↗

PubMed1993

Superinfection-induced apoptosis and its correlation with the reduction of viral progeny in cells persistently infected with Hz-1 baculovirus.

Differential induction of necrosis or apoptosis was found upon challenge of cells of the insect Spodoptera frugiperda productively or persistently infected with Hz-1 baculovirus, respectively. Unlike parental SF9 cells, which were essentially all killed by virally induced necrosis, persistently infected cells underwent a process of massive cell death by apoptosis; cells which were not killed by apoptosis then reestablished a cell monolayer. Upon viral challenge, the yield of viral progeny was reduced greatly in persistently virus-infected cells but not in parental cells. Immunolabelling of individual cells revealed that upon viral challenge, production of viral progeny was detectable only in necrotic cells and not in apoptotic cells. These results indicated that induction of apoptosis greatly reduces the yield of viral progeny in cells persistently infected with Hz-1 baculovirus. This is the first report of apoptosis induction in persistently infected cells upon viral superinfection.

Animals↗

PubMed1993

Dominance of the mutant ALDH2(2) allele in the expression of human stomach aldehyde dehydrogenase-2 activity.

About half of Chinese individuals lack mitochondrial aldehyde dehydrogenase-2 (ALDH2) activity, which is responsible for the oxidation of acetaldehyde produced during ethanol metabolism. The ALDH2 deficiency in Chinese has been implicated in alcohol flush reaction and reported to be a negative risk factor for development of alcohol dependence. To assess the effects of inactive ALDH2 subunits, encoded by the mutant ALDH2(2) allele, on the catalytic activity of tetrameric enzyme molecules, we have phenotyped ALDH2 from 30 gastroendoscopic biopsies by using agarose isoelectric focusing and determined the genotypes from leukocytes of the same individuals by using polymerase-chain-reaction amplification and hybridization with allele-specific oligonucleotide probes. Sixteen subjects were homozygous for the ALDH2(1) allele, one was homozygous for ALDH2(2), and thirteen were the heterozygous genotype. None of the subjects with the mutant homozygotic and the heterozygotic genotypes exhibited the ALDH2 activity band or intermediate bands between ALDH2 and ALDH1 on isoelectric focusing gels. Our results support the notion that the mutant allele is dominant and that the heterotetrameric ALDH2 molecules containing the mutant subunits are enzymatically inactive or far less active.

Aldehyde Dehydrogenase↗

PubMed1992

Evolution of hepatitis delta virus RNA during chronic infection.

The complete RNA sequences of hepatitis delta virus (HDV) isolated at three different time points from a chronic delta hepatitis patient were determined. These time points represented three different periods of clinical flare-ups. The sequence analysis showed that these three different HDV isolates evolved at a rate ranging from 3.0 x 10(-2) to 3.0 x 10(-3) substitutions/nucleotide/year, depending on the period of infection. The evolution rates appeared to correlate with the changes of clinical pictures of hepatitis, i.e., the more drastic the change in the symptom of hepatitis was, the more nucleotide changes were detected. Except during the transition from the acute phase to chronic phase of delta hepatitis, when there was a much larger number of changes in HDV RNA sequence, the overall evolution rate of HDV RNA in the chronic phase appeared to be similar to those of other RNA viruses. Sequence relationship of these HDV RNAs suggested that acute exacerbations in chronic delta hepatitis were associated with the evolution of the persistently infected HDV, rather than resulting from new viral infections. However, some of the mutations were not cumulative, suggesting that HDV isolated at a later time was not directly evolved from the immediately previous one. Thus, HDV at any time point was a mixture of viruses with slight sequence variations, and a specific HDV RNA species was selected from this virus population under different environments. These findings indicate that HDV RNA is heterogeneous and evolves at a fast rate. The evolution rates in different parts of HDV RNA also varied. The evolution rate of HDV RNA determined here was higher than the ones determined previously from partial RNA sequences of two Japanese HDV isolates.

Adult↗

PubMed1992

Differential expression of Hz-1 baculovirus genes during productive and persistent viral infections.

Hz-1 viral RNA transcription was studied during productive and persistent infections. The RNAs were localized to 10- to 30-kb regions within the viral genome, and the timing of their expression was determined. During productive infections, we detected 101 virus-specific transcripts that could be grouped into three categories by time of appearance. At 2 h postinoculation (p.i.), a total of 34 virus-specific transcripts were detected. An additional 51 and 16 virus-specific transcripts appeared between 4 and 6 h p.i. and at 8 h p.i., respectively. After 8 h, no new transcripts were found. Under conditions of persistent infection, we detected only one viral persistency-associated transcript (PAT1). The region of the viral DNA which encodes PAT1 was cloned. During productive infections, three transcripts were derived from this region. Each had the same polarity as PAT1. One of them was of the same size as PAT1 and had similar, if not identical, 3' and 5' ends. This report provides detailed and very useful information concerning sequentially expressed transcripts of the Hz-1 baculovirus.

Animals↗

PubMed1991

Sequence of swallow, a gene required for the localization of bicoid message in Drosophila eggs.

We report the sequence of the Drosophila maternal effect gene swallow, one of the genes whose product is required for the localization of bicoid message during Drosophila oogenesis. The inferred swallow protein contains a domain that is predicted to be an amphipathic alpha-helix similar to those implicated in protein:protein associations in other systems. Another part of the predicted protein appears to be a diverged RNA-binding motif. We discuss these structural features in light of the function of the swallow protein in the bicoid message localization process.

Amino Acid Sequence↗

PubMed1991

Stomach and duodenal alcohol and aldehyde dehydrogenase isozymes in Chinese.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozyme phenotypes were determined in surgical and endoscopic biopsies of the stomach and duodenum by agarose isoelectric focusing. gamma-ADH was found to be the predominant form in the mucosal layer whereas beta-ADH was predominant in the muscular layer. Low-Km ALDH1 and ALDH2 were found in the stomach and duodenum. High-Km ALDH3 isozymes occurred only in the stomach but not in the duodenum. The isozyme patterns of gastric mucosal ALDH2 and ALDH3 remained unchanged in the fundus, corpus, and antrum. The stomach ALDH3 isozymes exhibited a Km value for acetaldehyde of 75 mM, and an optimum for acetaldehyde oxidation at pH 8.5. Since the Km value was high, ALDH3 contributed very little, if any, to gastric ethanol metabolism. The activities of ALDH in the gastric mucosa deficient in ALDH2 were 60-70% of that of the ALDH2-active phenotypes. These results indicate that Chinese lacking ALDH2 activity may have a lower acetaldehyde oxidation rate in the stomach during alcohol consumption.

Adult↗

PubMed1991

Molecular cloning and characterization of an isolate of hepatitis delta virus from Taiwan.

The genomic RNA of an Asian isolate of hepatitis delta virus was cloned from a Chinese patient from Taiwan, using the polymerase chain reaction to amplify cDNA for cloning and sequencing. The sequence of this hepatitis delta virus isolate shares an 86% to 88% similarity with the three published hepatitis delta virus RNA sequences, suggesting heterogeneity of hepatitis delta viruses from different geographical areas. Four highly conserved, long stretches of sequence were found. These four regions corresponded to the sequences required for the autocatalytic cleavage activities of the genomic and antigenomic RNAs and the middle and the carboxyl terminal parts of the open reading frame for the delta antigen on the antigenomic strand. The conservation of nucleotide sequence in these four regions was further confirmed by sequencing additional hepatitis delta virus RNAs obtained from three patients with chronic delta hepatitis who lived in Los Angeles. These findings suggest that the conserved sequences are critical for viral replication. These conserved regions offer ideal sites for primer selection to carry out polymerase chain reactions to detect hepatitis delta virus RNA in patients with hepatitis delta virus infection.

Adult↗

PubMed1991

Functional studies of hepatitis delta antigen and delta virus RNA.

We have sequenced an HDV RNA from an acute delta hepatitis patient from the Nauru Islands. By comparison with other HDV sequences previously reported, we have identified three conserved regions: the first one is the sequence around the catalytic cleavage site for genomic-sense RNA; the second is the corresponding site for the antigenomic sense RNA; and the third is the sequence encoding the middle domain of the hepatitis delta antigen. We have shown that the middle domain of the delta antigen can bind specifically to the HDV RNA. This binding was demonstrated both in vitro and in the purified virion. It was suggested that this RNA-protein interaction is important for HDV RNA replication. We also suggest that the conserved sequences provide ideal primers for use in polymerase chain reaction (PCR) in clinical diagnosis of HDV infections. We recommend the use of nucleotides 870-900 and 690-720 (Makino et al., 1987a) as primers for routine screening.

Amino Acid Sequence↗

PubMed1990

Sequence conservation and divergence of hepatitis delta virus RNA.

The complete RNA sequence of the hepatitis delta virus (HDV) obtained from the Nauru Island in the Pacific was determined by cDNA cloning and amplification by polymerase chain reaction (PCR). The sequence showed 14-17% divergence from the two known HDV RNA sequences. There are three highly conserved domains: the region around the autocatalytic cleavage site of the genomic RNA (nucleotides 659 to 772), the region around the autocatalytic cleavage site of the antigenomic-sense RNA (nucleotides 847 to 966), and the region around the middle one-third domain of the open reading frame (ORF) encoding the hepatitis delta antigen on the antigenomic RNA (nucleotides 1267 to 1347). The two autocatalytic activities are required for the cleavage and ligation of HDV RNA during RNA replication. The third conserved domain codes for the RNA-binding domain of HDAg, which specifically interacts with HDV RNA. Three nucleotide changes within the genomic catalytic sequence are present but did not alter the catalytic cleavage activity of the HDV RNA. Microheterogeneity of the RNA sequences was also detected. One of these occurred within the coding region of the delta antigen, creating an amber termination codon in some of the RNA species. Thus, this HDV strain contains two different RNA species, one of which encodes a delta antigen of 214 amino acids and the other 195 amino acids. These two protein species were detected by immunoblotting of the patient's plasma. In contrast to other HDV strains, only three ORFs capable of encoding more than 100 amino acids each are present in this HDV RNA. We recommend that oligonucleotides complementary to the highly conserved sequences should be used as primers for PCR in clinical detection assays of hepatitis delta virus infection.

Adult↗

PubMed1988

Human stomach alcohol and aldehyde dehydrogenases (ALDH): a genetic model proposed for ALDH III isozymes.

Isozyme phenotypes of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) from human gastroendoscopic as well as surgical gastric biopsies were determined by starch gel electrophoresis and agarose isoelectric focusing. gamma gamma ADH isozymes were expressed predominantly in the mucosal layer of the stomach, whereas beta beta isozymes were in the muscular layer. In the 56 gastroendoscopic mucosal biopsies examined, the homozygous ADH3 1-1 phenotype was found in 75% of the samples, and the heterozygous ADH3 2-1 phenotype in 25%. Accordingly, the gene frequencies of the alleles ADH1/3 and ADH2/3 were calculated to be 0.88 and 0.12, respectively. Using a modified agarose isoelectric focusing procedure, gastric ALDH I, ALDH II, and up to five ALDH III forms could be clearly resolved. The ALDH III isozymes accounted for more than 80% of the total ALDH activities in gastric mucosa and exhibited Km values in the millimolar range for propionaldehyde at pH 9.0. Forty-five percent of the 55 gastroendoscopic biopsies studied lacked ALDH I isozyme. The complex gastric ALDH III isozyme phenotypes seen in these biopsies fall into three patterns. They can be interpreted by a genetic hypothesis, based on a dimeric molecule, in which there are two separate genes, ALDH3a and ALDH3b, with the ALDH3b locus exhibiting polymorphism. The homozygous phenotypes ALDH3b 1-1 and ALDH3b 2-2 were found to be 4 and 76%, respectively, and the heterozygous ALDH3b 2-1 phenotype 20%, of the total. Therefore, the allele frequencies for ALDH1/3b and ALDH2/3b were calculated to be 0.14 and 0.86, respectively. Several lines of biochemical evidence consistent with this genetic model are discussed.

Alcohol Dehydrogenase↗

PubMed1988

Molecular analysis of the swallow gene of Drosophila melanogaster.

We report the cloning and molecular characterization of the swallow gene, a maternal-effect gene of Drosophila melanogaster. Maternal-effect genes are transcribed only during oogenesis; one such gene, bicoid, encodes a message that is localized at the anterior end of oocytes, eggs, and early embryos. swallow+ activity is necessary during oogenesis for bicoid message localization. We show here that a fragment of a previously identified clone, introduced into the genome by P-element-mediated transformation, is able to rescue the maternal-effect lethality of swallow mutants, confirming the identity of this clone as swallow. By in situ hybridization, we show that swallow message is synthesized in nurse cells during oogenesis but is not localized along either the anterior-posterior or dorsal-ventral axes. We have confirmed an earlier finding that the localization of bicoid message is defective in swallow- oocytes and eggs.

Animals↗