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Biomedical subjects

C M Chiang

Publications and source records attributed to C M Chiang.

11 recordsLinked to original sources
PubMed1992

Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins.

We have shown that E1 and E2 proteins of human papillomavirus type 11 (HPV-11) were essential to support the replication of the homologous viral origin (ori) in a transient replication assay, similar to reports on bovine papillomavirus type 1 (BPV-1). Unexpectedly, matched or even mixed combinations of E1 and E2 proteins from HPV-11 or BPV-1 replicated either ori in human, monkey, and rodent cell lines of epithelial or fibroblastic lineage, albeit with varied efficiencies. Either set of viral proteins was also able to initiate replication of ori-containing plasmids from many other human and animal papillomaviruses. Thus the interactions among the cis elements and trans factors of papillomaviruses are more conserved than expected from the other members of the papovavirus family, simian virus 40 and polyomavirus, for which large tumor antigen does not replicate a heterologous ori in either permissive or nonpermissive cells. We infer that the stringent species and tissue specificities observed for papillomaviruses in vivo are not entirely due to direct restrictions on viral DNA replication. Rather, transcriptional control of viral gene expression must play a dominant role.

3T3 Cells↗
PubMed1992

Properties of bovine papillomavirus E1 mutants.

Ostensibly comparable mutants of bovine papillomavirus type 1 (BPV-1) affecting the E1 open reading frame that were constructed in several laboratories have been reported to exhibit either reduced or increased transformation efficiencies in established mouse cell lines relative to wild-type BPV-1 DNA. To resolve these discrepancies, we have reexamined many of the mutants in mouse C127 cells by using focus formation assays. Our primary conclusions are that all E1 mutants tested consistently generated reduced numbers of transformants and that the reduced transformation was not due to cell toxicity associated with E1 mutations, as had been proposed. Our results can best be explained by the inability of the E1 mutants to replicate extrachromosomally, therefore leading to a rapid loss of the BPV-1 DNA and consequently, reduced transformation. In support of this hypothesis, we demonstrated that the human papillomavirus type 11 E1 protein was able to suppress BPV-1 transformation, probably because of interference with BPV-1 replication. Therefore, we attribute the phenotypic disparities reported by the various laboratories to still undefined differences in assay conditions.

Animals↗
PubMed1992

Ventricular tachycardia detection using bipolar electrogram analysis is site specific.

While algorithms for bipolar intraventricular electrogram analysis have potential use in complementing rate criteria for ventricular tachycardia (VT) detection by implantable antitachycardia devices, the sensitivity of such algorithms to the intracavitary site of electrogram detection has not been determined. In this study, unfiltered (1-500 Hz) electrograms were recorded from a bipolar electrode catheter initially positioned at the right ventricular (RV) apex (site 1) of 12 patients during sinus rhythm (SR1) and during induced monomorphic VT (VT1). Sinus rhythm (SR2) and the identical VT (VT2) were recorded a second time after repositioning the same electrode catheter within the RV apex (site 2) 7-44 mm (mean +/- SD = 15 +/- 10) from its original site. The data were digitized at 1,000 Hz. Templates from SR1 and SR2, respectively, were compared subsequently with individual intraventricular electrograms from 15-25 sec passages of SR1 and VT1 and SR2 and VT2, respectively, using correlation waveform analysis. At site 1, the mean patient correlation coefficient ranged from 0.982-0.998 during SR1 and 0.062-0.975 during VT1. At site 2, the mean patient correlation coefficient ranged from 0.995-0.998 during SR2 and 0.113-0.983 during VT2. Using a correlation threshold of 0.9, VT was differentiated from SR in 11/12 patients (91%) overall: 8/12 patients (67%) at site 1, 9/12 patients (75%) at site 2, and 6/12 patients (50%) at both sites. Thus, while discrimination of VT from SR is feasible with morphological analysis of bipolar right ventricular intracavitary electrograms, the accuracy of bipolar intraventricular electrogram analysis may depend upon intracavitary electrode location in selected patients.

Algorithms↗
PubMed1992

Control of human papillomavirus type 11 origin of replication by the E2 family of transcription regulatory proteins.

Replication of human papillomavirus type 11 (HPV-11) DNA requires the full-length viral E1 and E2 proteins (C.-M. Chiang, M. Ustav, A. Stenlund, T. F. Ho, T. R. Broker, and L. T. Chow, Proc. Natl. Acad. Sci. USA 89:5799-5803, 1992). Using transient transfection of subgenomic HPV DNA into hamster CHO and human 293 cells, we have localized an origin of replication (ori) to an 80-bp segment in the upstream regulatory region spanning nucleotide 1. It overlaps the E6 promoter region and contains a short A + T-rich segment and a sequence which is homologous to the binding site of the bovine papillomavirus type 1 (BPV-1) E1 protein in the BPV-1 ori. However, unlike the BPV-1 ori, for which half an E2-responsive sequence (E2-RS) or binding site suffices, an intact binding site is essential for the HPV-11 ori. Replication was more efficient when additional E2-RSs were present. The intact HPV-11 genome also replicated in both cell lines when supplied with E1 and E2 proteins. Expression vectors of transcription repressor proteins that lack the N-terminal domain essential for E2 transcriptional trans activation did not support replication in collaboration with the E1 expression vector. Rather, cotransfection with the repressor expression vectors inhibited ori replication by the E1 and E2 proteins. These results demonstrate the importance of the N-terminal domain of the E2 protein in DNA replication and indicate that the family of E2 proteins positively and negatively regulates both viral DNA replication and E6 promoter transcription.

Animals↗
PubMed1991

Impact of filtering upon ventricular tachycardia identification by correlation waveform analysis.

Signal analysis of digitized waveforms has been postulated as a method for improving sensitivity and specificity of ventricular tachycardia (VT) detection in implantable antitachycardia devices. Such improvement may alleviate the problem of unwarranted delivery of therapy by adding precision to the identification of the pathological VT. Morphological analysis could also allow distinct therapies to be initialized for multiple VTs in the same patient. Correlation waveform analysis (CWA) has been demonstrated to be effective in separating benign rhythms from VT in wideband recordings (1-500 Hz) but the effect of filtering has not been previously examined. Bipolar (1 cm) intraventricular recordings (1-500 Hz) of sinus rhythm (SR) and 25 distinct VTs in 18 patients were analyzed by CWA using a signal-averaged SR template. Passages contained 65.9 +/- 19.8 VT depolarizations (range 45-108). Digital filtering was performed on all data passages with varying passbands. Results for passages with a bandwidth of 1-250 Hz were equivalent to wideband results, i.e., greater than or equal to 92% paired sets of SR and VT were separable at a 95% confidence level. A bandwidth of 1-100 Hz decreased discrimination to 84%. At a bandwidth of 1-80 Hz, 80% of cases were successfully separated, but at 10-80 Hz these results improved to 88%. Bandwidths of 20-80 and 30-80 Hz reduced reliability of CWA performance to 72% and 60%, respectively. Filtering at typical pacemaker/defibrillator passbands produced morphological analysis results equivalent to those yielded at wideband settings. Differences in the range between SR versus VT decreased in filtered recordings but overall detection of VT was not degraded.

Algorithms↗
PubMed1991

An E1M--E2C fusion protein encoded by human papillomavirus type 11 is asequence-specific transcription repressor.

We have isolated a putative, spliced E5 cDNA of human papillomavirus type 11 (HPV-11) by polymerase chain reaction amplification of cDNAs from an experimental condyloma. Using retrovirus-mediated gene transfer, we isolated two novel HPV-11 cDNAs, one of which had a splice linking nucleotides 1272 and 3377. This transcript also existed in experimental condylomata and in cervical carcinoma cells transfected with cloned genomic HPV-11 DNAs. The 5' end of the transcript in transfected cells originated upstream of the initiation codon of the E1 open reading frame (ORF). It could conceptually encode a fusion protein consisting of the amino-terminal 23% of the E1 ORF and the carboxy-terminal 40% of the E2 ORF. This E1M--E2C fusion protein contained both the DNA replication modulator domain E1M, as defined in the bovine papillomavirus system, and the DNA binding domain of the E2 protein, which regulates viral transcriptional activities. Indirect immunofluorescence with polyclonal antibodies raised against the bacterially expressed TrpE-HPV-11 E2 protein demonstrated nuclear localization of the E1M--E2C protein in cells transiently transfected with an expression plasmid. Immunoprecipitation revealed a specific protein with an apparent molecular weight of 42,000 in transfected cells. The chloramphenicol acetyltransferase assay established that the putative E1M--E2C protein was a potent transcriptional repressor of both E2-dependent and E2-independent HPV-11 enhancer/promoter activities. Northern (RNA) blot hybridization indicated the repression was on the transcriptional level. Mutational analysis suggested that the E1M--E2C protein is an E2-binding site-specific repressor. The fusion protein also repressed bovine papillomavirus type 1 (BPV-1) E2 protein-dependent BPV-1 enhancer activity. When constitutively expressed in mouse C127 cells, the E1M--E2C protein inhibited BPV-1 transformation and episomal DNA replication, consistent with a role in the modulation of replication.

Condylomata Acuminata↗
PubMed1989

Characterization of cDNAs of spliced HPV-11 E2 mRNA and other HPV mRNAs recovered via retrovirus-mediated gene transfer.

Human papillomaviruses (HPVs) are associated with hyperproliferations of cutaneous or mucosal epithelium. These viruses cannot be propagated in any cell culture system. Because cloning cDNA copies of HPV mRNAs recovered from human lesions has met with only very limited success, the characterization of HPV mRNAs has been problematic. Using the Moloney murine leukemia virus vector system (C.L. Cepko, B.E. Roberts, and R.C. Mulligan, 1984, Cell 37, 1053-1062), we have recovered cDNAs of spliced E2 mRNAs of human papillomavirus type 11 and additional mRNAs of type 11 and type 18 and determined the utilization of open reading frames (ORFs) in the DNA sequences. The recovery of cDNA copies of messages with splice sites identical to those previously described strongly suggests that the newly characterized splice donors and acceptors are also authentic. The HPV-11 E2 cDNA contains the intact E6 and E7 ORFs and the beginning of the E1 ORF in the first exon, which is then spliced from nt 847 to the second exon at nt 2622, 100 nucleotides upstream from the initiation codon for the E2 ORF. The initiation codon in the E1 ORF is followed by four additional in-frame AUG triplets and an in-frame termination codon positioned 30 nucleotides upstream from the initiation codon for the E2 protein. The authenticity of this putative E2 cDNA was shown by its ability to provide enhancer transactivating activity in chloramphenicol acetyltransferase (CAT) assays in several cell lines. A mutation in the genomic DNA at this splice acceptor site eliminates its activity, demonstrating that the splice is essential for the expression of the E2 protein. We conclude that the translation of the HPV-11 E2 protein requires internal initiation.

Base Sequence↗
PubMed1988

Drug transport from thin applications of topical dosage forms: development of methodology.

There are presently no standards for in vitro research dealing with the release and delivery of drugs from semisolid dosage forms, largely because of inherent experimental difficulties. Among the problems, it has proven difficult to apply dosage forms to membranes mounted in in vitro diffusion cells in facsimile to the manner in which the dosage forms are applied clinically. In the present studies, methodology has been developed which allows films with thicknesses approaching clinical dimensions to be spread evenly over silicone rubber membranes. Using methyl p-aminobenzoate as a test permeant and gelled water and water/propylene glycol solvent systems as test vehicles, it has proven possible to spread films as thin as 75 microns, yielding highly reproducible delivery profiles. Using this application technique, it has been shown how the diffusive clearance of drug from films of fixed composition placed over a resistant membrane is dependent on the thickness of application. For a given medium and thickness of application, when the vehicle composition is enriched in propylene glycol, partitioning into the membrane is suppressed, resulting in a lessening of the absolute rate of delivery and, consequently, a prolongation of the period over which drug is released. Increasing the membrane's resistance, i.e., increasing the membrane's thickness, likewise slows down the absolute delivery rate, extending the effective period of total clearance of drug from the applied film.

4-Aminobenzoic Acid↗