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Biomedical subjects

G T Sheu

Publications and source records attributed to G T Sheu.

9 recordsLinked to original sources
PubMed2001

The association of human papillomavirus 16/18 infection with lung cancer among nonsmoking Taiwanese women.

Lung cancer is the leading cause of cancer death in Taiwanese women since 1982. High lung cancer mortality ratio of male:female in Taiwan (2:1) was observed, although less than 10% of female lung cancer patients are smokers. Until now, the etiological factor remains unknown. We hypothesize that high-risk human papillomavirus (HPV) 16/18 may be associated with lung cancer development based on high prevalence of p53 negative immunostainings in female lung tumors compared with that of male lung tumors. In this study, 141 lung cancer patients and 60 noncancer control subjects were enrolled to examine whether HPV 16/18 DNA existed in lung tumor and normal tissues by nested PCR and in situ hybridization (ISH), respectively. The concordant detection of HPV 16 and 18 DNA between nested PCR and ISH method was 73 and 85.5%, respectively. Our data showed that 77 (54.6%) of 141 lung tumors had HPV 16/18 DNA compared with 16 (26.7%; P = 0.0005) of 60 noncancer control subjects. In addition, ISH data showed that HPV 16/18 DNA was uniformly located in lung tumor cells, but not in the adjacent nontumor cells. When study subjects were stratified by gender, age, and smoking status, nonsmoking female lung cancer patients who were older than 60 years old had significantly high prevalence of HPV 16/18 infection. The odds ratio of HPV 16/18 infection of nonsmoking female lung cancer patients is much higher at 10.12 (95% confidence interval, 3.88-26.38) compared with 1.98 (95% confidence interval, 0.84-4.76) of nonsmoking male lung cancer patients. This result strongly suggests that HPV infection is associated with lung cancer development of nonsmoking female lung cancer patients. The high prevalence of HPV 16/18 infection may explain to a certain extent why Taiwanese women nonsmokers had a higher lung cancer mortality rate.

Female↗
PubMed2000

Recombinant hepatitis delta antigen from E. coli promotes hepatitis delta virus RNA replication only from the genomic strand but not the antigenomic strand.

Hepatitis delta antigen (HDAg) of hepatitis delta virus (HDV) typically consists of two related protein species. The small HDAg (S-HDAg) is a 24-kDa protein of 195 amino acids and the large HDAg (L-HDAg) is a 27-kDa protein with an additional 19 amino acids at its C-terminus. These two proteins have distinct functions in the HDV life cycle. We have developed conditions for expressing S-HDAg and L-HDAg in E. coli as soluble proteins to facilitate large-scale purification. These proteins were purified to homogeneity and shown to be biologically active. Transfection of the purified recombinant S-HDAg together with HDV genomic RNA resulted in viral RNA replication. Surprisingly, the purified S-HDAg could not initiate replication from the antigenomic-sense HDV RNA, even though the latter led to RNA replication when transfected with an mRNA encoding the S-HDAg. These results suggest that initiation of HDV RNA synthesis from the antigenomic RNA may require a form of HDAg that is modified in mammalian cells; in contrast, RNA synthesis from the genomic RNA could be initiated by the recombinant S-HDAg from E. coli. Interestingly, the purified L-HDAg appeared as multiple protein species, including one corresponding to S-HDAg, probably as a result of degradation. The partially proteolyzed L-HDAg also initiated HDV RNA replication under the same conditions. These results add to the mounting evidence that genomic- and antigenomic-strand HDV RNA syntheses are carried out by different mechanisms.

Animals↗
PubMed2000

Template requirement and initiation site selection by hepatitis C virus polymerase on a minimal viral RNA template.

RNA-dependent RNA polymerase, NS5B protein, catalyzes replication of viral genomic RNA, which presumably initiates from the 3'-end. We have previously shown that NS5B can utilize the 3'-end 98-nucleotide (nt) X region of the hepatitis C virus (HCV) genome as a minimal authentic template. In this study, we used this RNA to characterize the mechanism of RNA synthesis by the recombinant NS5B. We first showed that NS5B formed a complex with the 3'-end of HCV RNA by binding to both the poly(U-U/C)-rich and X regions of the 3'-untranslated region as well as part of the NS5B-coding sequences. Within the X region, NS5B bound stem II and the single-stranded region connecting stem-loops I and II. Truncation of 40 nt or more from the 3'-end of the X region abolished its template activity, whereas X RNA lacking 35 nt or less from the 3'-end retained template activity, consistent with the NS5B-binding site mapped. Furthermore, NS5B initiated RNA synthesis from a specific site within the single-stranded loop I. All of the RNA templates that have a double-stranded stem at the 3'-end had the same RNA initiation site. However, the addition of single-stranded nucleotides to the 3'-end of X RNA or removal of double-stranded structure in stem I generated RNA products of template size. These results indicate that HCV NS5B initiates RNA synthesis from a single-stranded region closest to the 3'-end of the X region. These results have implications for the mechanism of HCV RNA replication and the nature of HCV RNA templates in the infected cells.

3' Untranslated Regions↗
PubMed1999

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII.

EF-1alpha binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the betagammadelta complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant beta, gamma, and delta subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the beta and delta subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the beta subunit and reduced the Km, while addition of alpha to beta or the betagammacomplex inhibited phosphorylation by CKII. However, alpha had little effect on phosphorylation of delta. Thus, the beta and delta subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of beta was altered by association with other subunits, while the site on delta was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of(alpha2betagamma2delta)2 is proposed and discussed.

Amino Acid Sequence↗
PubMed1998

Inhibition of Cellular RNA polymerase II transcription by delta antigen of hepatitis delta virus.

Hepatitis delta virus (HDV) contains a circular, viroid-like RNA and the hepatitis delta antigen (HDAg) protein. The viral RNA is replicated via RNA-dependent RNA synthesis, which is thought to be mediated by host DNA-dependent RNA polymerase II (pol II). The precise mechanism of HDV RNA replication using RNA as a template remains to be elucidated, though it is clear that HDAg is involved. We demonstrate here that both SP1-activated and basal pol II transcription are inhibited by HDAg. This inhibitory effect of HDAg was observed in vivo in transient cotransfection assays as well as in vitro in HeLa nuclear extracts with purified, recombinant HDAg. The in vitro inhibition of pol II transcription could be reversed with excess HeLa nuclear extracts. Furthermore, HDAg specifically inhibited pol II-mediated transcription but not pol I- or III-mediated transcription. These results provide support for the model in which HDAg participates in a complex with host cell pol II transcription factors to mediate pol II-dependent HDV RNA replication, concomitantly cellular pol II transcription.

Chloramphenicol O-Acetyltransferase↗
PubMed1998

Hepatitis delta antigen mediates the nuclear import of hepatitis delta virus RNA.

Hepatitis delta virus (HDV) RNA replicates in the nuclei of virus-infected cells. The mechanism of nuclear import of HDV RNA is so far unknown. Using a fluorescein-labeled HDV RNA introduced into partially permeabilized HeLa cells, we found that HDV RNA accumulated only in the cytoplasm. However, in the presence of hepatitis delta antigen (HDAg), which is the only protein encoded by HDV RNA, the HDV RNA was translocated into the nucleus, suggesting that nuclear import of HDV RNA is mediated by HDAg. Deletion of the nuclear localization signal (NLS) or RNA-binding motifs of HDAg resulted in the failure of nuclear import of HDV RNA, indicating that both the NLS and an RNA-binding motif of HDAg are required for the RNA-transporting activity of HDAg. Surprisingly, any one of the three previously identified RNA-binding motifs was sufficient to confer the RNA-transporting activity. We have further shown that HDAg, via its NLS, interacts with karyopherin alpha2 in vitro, suggesting that nuclear import of the HDAg-HDV RNA complex is mediated by the karyopherin alpha2beta heterodimer. The nuclear import of HDV RNA may be the first biological function of HDAg in the HDV life cycle.

Binding Sites↗
PubMed1997

Recombinant subunits of mammalian elongation factor 1 expressed in Escherichia coli. Subunit interactions, elongation activity, and phosphorylation by protein kinase CKII.

The first step in elongation requires two different activities; elongation factor (EF)-1alpha transfers aminoacyl-tRNA to the ribosome and is released upon hydrolysis of GTP, EF-1betagammadelta catalyzes exchange of GDP on EF-1alpha with GTP. To analyze the role of the individual subunits of EF-1 in elongation, the cDNAs for the beta, gamma, and delta subunits of EF-1 from rabbit were cloned, and proteins of 225, 437, and 280 amino acids, respectively, were expressed in Escherichia coli. The purified recombinant beta subunit migrates as a dimer and the gamma subunit as a trimer upon gel filtration, whereas the delta subunit forms a large aggregate. Complexes of betagamma, gammadelta and betagammadelta were formed by self-association and eluted with a molecular mass of approximately 160, 530, and 670 kDa, respectively; no interaction was observed between beta and delta. The activity of the recombinant subunits was determined with native EF-1alpha by measuring stimulation of the rate of elongation by poly(U)-directed polyphenylalanine synthesis. Recombinant beta and delta alone stimulated the rate of elongation by 10-fold, with a ratio of 5alpha:2beta or delta. The betagammadelta complex stimulated EF-1alpha activity up to 10-fold with a ratio of 20alpha to 1betagammadelta. Phosphorylation of the beta and delta subunits alone or in betagammadelta by protein kinase CKII had no effect on the rate of elongation.

Amino Acid Sequence↗
PubMed1994

Effects of autophosphorylation on casein kinase II activity: evidence from mutations in the beta subunit.

Casein kinase II is a heterotetramer composed of two catalytic (alpha) and two regulatory (beta) subunits. To examine the effects of autophosphorylation of the beta subunit on enzyme activity, two mutants of the beta subunit from Drosophila were constructed in which either Ser4 or Ser2-4 was changed to alanine residues by oligonucleotide-directed mutagenesis and the proteins were expressed in Escherichia coli. The wild-type alpha and individual beta subunits present in inclusion bodies were renatured, and the biochemical properties of the reconstituted holoenzymes were examined. Analysis of autophosphorylation revealed that phosphate incorporation was about 0.8 mol/mol of beta subunit for the wild type and Ala4 mutant; Ser2 and Ser3 were the major sites of autophosphorylation with some phosphate in Ser4 as shown by Edman degradation. No autophosphorylation was observed with the Ala2-4 mutant. Substitution of alanine for serine residues at positions 4 or 2-4 of the beta subunits did not influence the reassociation of the alpha and beta subunits to form holoenzyme, or the function of the beta subunit in stimulating catalytic activity or in responding to basic compounds. To measure the effects of autophosphorylation on casein kinase II activity, the wild-type and mutant holoenzymes were preincubated in the presence and absence of ATP, and the rate of phosphorylation was measured with various substrates. In the absence of autophosphorylation, the wild-type, Ala4, and Ala2-4 forms of the holoenzyme displayed similar rates of phosphorylation of glycogen synthase. After preincubation with ATP, the rate of phosphorylation of glycogen synthase by the wild-type and Ala4 enzymes was inhibited by 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗