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Biomedical subjects

I Davidson

Publications and source records attributed to I Davidson.

At least 91 records · Page 5Linked to original sources
PubMed1993

Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis.

Cadmium-induced metallothioneins from the common sea mussel, Mytilus edulis, were shown to comprise of two groups of isoforms having apparent molecular masses of 10 kDa and 20 kDa. The 10-kDa group was resolved by anion-exchange chromatography into four fractions while the 20-kDa group was resolved into three fractions using this method. After metal removal and S-methylation of the cysteine residues using methyl-p-nitrobenzenesulphonate the complete amino acid sequences were determined. Five isoforms of the 20-kDa group were shown to possess monomeric units consisting of 71 amino acids. These proteins were distinct from the four 72-amino-acid proteins of the 10-kDa group. The FASTA algorithm has been used to compare the degree of similarity between the mussel metallothionein MT-10-IV isoform and other metallothioneins. The mussel MT-10-IV isoform exhibited substantial similarity to other molluscan metallothioneins. Moreover, the mussel metallothionein exhibited more similarity to vertebrate metallothioneins than to those of non-molluscan invertebrates, thus suggesting that the mussel metallothioneins are class I metallothioneins.

Amino Acid Sequence↗
PubMed1993

Sequence-specific transactivators counteract topoisomerase II-mediated inhibition of in vitro transcription by RNA polymerases I and II.

An inhibitor of RNA polymerase II transcription in vitro has been purified from HeLa cell nuclear extracts. Partial amino acid sequences derived from the purified protein revealed that the inhibitor of transcription corresponded to human topoisomerase II. Order of addition experiments provided evidence indicating that topoisomerase II inhibited transcription by binding over the core promoter and blocking preinitiation complex formation. Topoisomerase II-mediated repression could be relieved by sequence-specific transcriptional activators, having different activating and/or DNA binding domains, but antirepression required a transcriptional activation function in addition to a DNA binding domain. Moreover, transcription by RNA polymerase I was also inhibited by topoisomerase II and this inhibition could be relieved by the RNA polymerase I transactivator UBF. These observations suggest that topoisomerase II may participate in a general repression of transcription which can be counteracted by transcriptional activators.

Amino Acid Sequence↗
PubMed1993

Different TBP-associated factors are required for mediating the stimulation of transcription in vitro by the acidic transactivator GAL-VP16 and the two nonacidic activation functions of the estrogen receptor.

The estrogen receptor (ER) contains two nonacidic transcriptional activation functions, AF-1 and AF-2 (formerly TAF-1 and TAF-2). In this study we show that AF-1 and AF-2 are able to stimulate transcription in vitro in a HeLa cell system when fused to the DNA binding domain of the yeast activator GAL4. We also demonstrate that a factor(s) required for the function of the ER AFs is chromatographically separable from a factor(s) necessary for the activity of the acidic activation domain of VP16. Moreover, immunoprecipitation experiments using a monoclonal antibody directed against the TATA box binding protein (TBP) indicate, that these different factors are associated with TBP in distinct TFIID complexes.

Amino Acid Sequence↗
PubMed1993

Distinct TFIID complexes mediate the effect of different transcriptional activators.

Multiple chromatographically separable complexes containing the TATA binding protein (TBP), which exhibit different functional properties, exist in HeLa cells. At least three distinct subpopulations of such complexes can be functionally defined as TFIID since they function with RNA polymerase II. Using a partially reconstituted HeLa cell in vitro transcription system and immunoprecipitation with a monoclonal antibody directed against TBP, we show that stimulation of transcription by the chimeric activators GAL-VP16, GAL-TEF-1 and GAL-ER(EF) requires the presence of factors which are tightly associated with these TFIID complexes. Moreover, the activity of GAL-TEF-1 appears to be mediated by at least two chromatographically distinct populations of TFIID. The factor(s) associated with one of these populations is also required for the activity of GAL-ER (EF) and GAL-VP16, while the factor(s) associated with the other population functions selectively with GAL-TEF-1. These two TFIID populations are composed of both common and unique TBP associated factors (TAFs).

Animals↗
PubMed1993

Characterization of the transcription activation function and the DNA binding domain of transcriptional enhancer factor-1.

The regions of transcriptional enhancer factor-1 (TEF-1) required for its activation function and sequence-specific DNA binding have been determined. Deletion analysis of a chimera between TEF-1 and the GAL4 DNA binding domain (DBD) indicated that at least three regions of TEF-1 were involved in transactivation. However, none of these regions functioned as independent activating domains. Moreover, none of the GAL4 chimeras containing individual TEF-1 regions interfered with the activity of endogenous HeLa cell TEF-1, while interference was observed with the GAL4-TEF-1 chimeras which functioned as transactivators. These results indicate that there is a general correlation between the abilities of a given GAL4-TEF-1 chimera to function in transcriptional activation and interference, thus supporting the idea that transactivation by TEF-1 is mediated by a limiting transcriptional intermediary factor. In addition, we show experimentally that the TEA/ATTS domain is a novel class of DBD involved in the sequence-specific DNA binding of TEF-1 and its Drosophila homologue scalloped. Two other regions of TEF-1 are also required for DNA binding. These regions are not part of the minimum DBD, but may function by antagonizing the effect of sequences which negatively regulate DNA binding mediated by both the TEF-1 TEA/ATTS domain and the GAL4 DBD. In addition, analysis of TEF-1 and scalloped derivatives in which their TEA/ATTS domains have been interchanged further indicates that the TEA/ATTS domain is not the only determinant of DNA binding specificity.

Amino Acid Sequence↗
PubMed1993

PCR detection of amplified 132 bp repeats in Marek's disease virus type 1 (MDV-1) DNA can serve as an indicator for critical genomic rearrangement leading to the attenuation of virus virulence.

A radioactive PCR test was developed that amplified the very virulent Marek's disease virus-1 (vvMDV-1) DNA sequence containing the 132 bp repeats. In apathogenic MDV-1 (CVI 988, Rispens), amplified DNA bands containing multiple copies of 132 bp repeats were identified. In the present study this PCR technique was used to monitor the passage level of vvMDV-1 in chicken embryo fibroblasts (CEF) in which the number of tandem 132 bp repeats was increased. It was found that at passage level 32 of vvMDV-1-B isolate, the 132 bp tandem repeat was already markedly amplified and its pattern resembled that of the MDV-1 (CVI 988, Rispens) vaccine virus DNA. In the vvMDV-1Z strain, amplification of the 132 bp repeat was not detectable at a similar passage level. The PCR test demonstrated that the apathogenic MDV-1 Md11/75c virus developed by extensive in vitro passaging has amplified 132 bp DNA repeats similar to those of the commercial vaccine virus (CVI 988, Rispense). It was also found that the pattern of viral RNA from infected cells detectable by Northern blot hybridization was markedly changed from a 2.4 kb RNA species in cells infected with vvMDV-1 viruses, to four RNA species (ranging from 2.2 to 4.4 kb) in cells infected with passage 32 of MDV-1-B strain, to a very large number of undefined RNA species synthesized in cells infected with attenuated MDV-1 viruses (CVI 988, Rispens and Md 11/75c).

Animals↗
PubMed1993

Comparison of epidural methadone with epidural diamorphine for analgesia following caesarean section.

Analgesia provided by either 5 mg diamorphine, or 5 mg methadone administered by the epidural route during elective caesarean section was compared in 40 women. The median time to further analgesia in the methadone group was 395 min, and 720 min in the diamorphine group, P = 0.0003. Linear analogue scores to assess pain were measured 2-hourly for 12 h, then again at 24 h postoperatively. Pain scores were significantly lower in the diamorphine group at 8 and 10 h. The median cumulative i.m. morphine dose administered during the first 24 h was 20 mg in the methadone group and 0 mg in the diamorphine group (P = 0.0005). Nausea and pruritus were common side effects in both groups. Continuous pulse oximetry data were available for 12 h post-operatively in 15 patients receiving methadone, and in 17 patients receiving diamorphine. One or more episodes of significant desaturation (< 90% for 30 s), occurred in three patients receiving methadone, and in nine patients receiving diamorphine. Desaturation to 90-92% occurred in a further three patients given epidural diamorphine, and in one further patient given epidural methadone.

Analgesia, Epidural↗
PubMed1993

Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex.

Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters. Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1). The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple. For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated. It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both. To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions). With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen. A GST fusion containing amino acids 5 to 172 (region T1) efficiently bound TBP. TEF-1 bound neither region T1 nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Polymerase chain reaction for differentiation between pathogenic and non-pathogenic serotype 1 Marek's disease viruses (MDV) and vaccine viruses of MDV-serotypes 2 and 3.

A polymerase chain reaction (PCR) test based on primers flanking the 132 bp tandem repeat in pathogenic MDV-1 DNA was developed. These primers amplify a dimer or a trimer 132 bp repeat in pathogenic MDV-1 DNA from blood and organs of commercial chickens with Marek's disease (MD) symptoms. Using the same primers in a radioactive PCR test, it was possible to distinguish between vvMDV-1 and the non-pathogenic MDV-1 CVI-988 vaccine in which the 132 bp repeats in the DNA were increased up to 9 repeats. The MDV-1 specific primers did not amplify MDV-2 (SB1) and MDV-3 (HVT) DNA. Primers prepared according to the nucleotide sequence of MDV-1 antigen A gene amplified MDV-1 DNA only. Specific primers prepared according to the nucleotide sequence of MDV-3 (HVT) antigen A gene amplified MDV-3 DNA but not MDV-1 nor MDV-2 DNA. The results of the present study show that the PCR tests can be used for the early identification of vvMDV-1 DNA in pathological samples from diseased commercial chickens and to distinguish between the vvMDV-1 and the three types of virus vaccines used to immunize chickens. The tests are accurate and can be performed in the presence of vaccine virus DNA in the sample.

Animals↗
PubMed1992

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Animals↗
PubMed1992

Transcriptional enhancer factor (TEF)-1 and its cell-specific co-activator activate human papillomavirus-16 E6 and E7 oncogene transcription in keratinocytes and cervical carcinoma cells.

The human papillomavirus (HPV)-16 oncogenes, E6 and E7, are transcribed preferentially in keratinocytes and cervical carcinoma cells due to a 5' enhancer. An abundant peptide binding to a 37 nt enhancer element was purified from human keratinocytes by sequence-specific DNA chromatography. This protein was identified as transcriptional enhancer factor (TEF)-1 by complex mobility, binding to wild-type and mutant SV40 and HPV-16 enhansons and antigenic reactivity with two anti-TEF-1 antibodies. TEF-1 is cell-specific, but its transactivation also depends on a limiting, cell-specific TEF-1 'co-activator'. We show that both TEF-1 and the TEF-1 co-activator are active in human keratinocytes and essential for HPV-16 transcription. TEF-1 binding in vivo was necessary for HPV-16 P97 promoter activity. Excess TEF-1 and chimeric GAL4-TEF-1 specifically inhibited the P97 promoter by 'squelching', indicating that HPV-16 transcription also requires a limiting TEF-1 co-activator. TEF-1 and the TEF-1 co-activator functions mirrored HPV-16 transcription by their presence in keratinocytes and cervical carcinoma cells and their absence from lymphoid B-cells, but also functioned in liver cells where the HPV-16 promoter is inactive. TEF-1 and its associated co-activator are thus part of a complex mechanism which determines the restricted cell range of the HPV-16 E6 and E7 oncogene promoter.

Antibodies↗
PubMed1992

Antigen B of the vaccine strains of Marek's disease virus and herpesvirus of turkeys presents heat-labile group and serotype specific epitopes.

Antigen B of Marek's disease virus (MDV) vaccine strains CVI988 and SB1 (serotypes 1 and 2) and herpesvirus of turkeys (HVT) (serotype 3) is formed of oligomeric molecules that are detergent-stable and heat-labile. Immunoblots of native membranal extracts of HVT- and MDV-infected chick embryo fibroblasts (CEF) probed with avian monoserotypic antisera, murine monoclonal antibodies (mAb) to the three serotypes and mAb to antigen B showed two distinct patterns of high molecular weight oligomeric antigens. Serotypes 1 and 3 vaccine viruses formed one set and serotype 2, the other. Avian monotypic sera to serotypes 1 and 3 viruses detected two high molecular weight bands of 230 and > or = 300 kDa in MDV-1 and HVT-infected CEF but only a weak diffuse zone ranging from 130 to 230 kDa in extracts of SB1-infected CEF. No 300 kDa band was discernible in the SB1 extract when blotted with avian monotypic 1 and 3 antisera. MAbs to MDV serotypes 1 and 3 and to antigen B also detected the 230 and > or = 300 kDa antigens, while the mAb to SB1 detected a 50 kDa antigen in the SB1-infected extract only. Furthermore, the antigen B mAb did not reveal high mol. wt. oligomers in SB1-infected CEF extracts. Antigen B oligomers were rapidly destroyed by heating at 95 degrees C and the rate of denaturation of the 230 and > or = 300 kDa oligomers differed for each of the three vaccine viruses. We propose that antigen B of MDV1 and HVT has a complex conformation created by juxtaposition of dimers (230-250 kDa) and trimers (> or = 300 kDa), and is inserted in the infected cell membrane so that conformational, discontinuous epitopes are formed in addition to continuous epitopes. It appears that HVT protects chickens against oncogenic strains of MDV1 by virtue of the cross reactivity of the conformational determinants located on these oligomers. Serotype 2 vaccine shares some of its antigenic determinants with serotypes 1 and 3, while its unique immunogenic features form the basis of the protective synergism achieved when serotypes 2 and 3 vaccines are combined together.

Animals↗
PubMed1992

Age-associated changes in mesenteric arteries.

Vascular disease increases in incidence with age and is the commonest cause of morbidity and mortality among elderly people. Hypertension is associated with hypertrophy of the arterial media. This study was designed to investigate changes in arterial structure that may occur with age independent of blood pressure. Collapsed sections of human mesenteric arteries (external diameter 2-3 mm) were measured using a semi-automatic image analysis system. There was a nonlinear increase in both the wall/lumen area ratio and the relative intimal area with age. There were no significant relationships between blood pressure and either the wall/lumen ratio or the relative intimal area.

Adult↗
PubMed1991

Cloning, expression, and transcriptional properties of the human enhancer factor TEF-1.

We describe the cDNA encoding the SV40 transcriptional enhancer factor 1 (TEF-1) and show that its translation initiates exclusively at an AUU codon in vivo. Cloned TEF-1, which is unrelated to other known transcription factors, specifically binds the SV40 GT-IIC and Sph enhansons. Cloned TEF-1 does not activate these enhansons in lymphoid MPC11 cells where they are known to be inactive, but represses the endogenous HeLa TEF-1 activity in vivo and in vitro. Repression is also observed with chimeras where the DNA-binding domain of the GAL4 activator replaces that of TEF-1, showing that repression results from interference/squelching. Such chimeras stimulate transcription in HeLa, but not in MPC11, cells in vivo and in HeLa cell extracts in vitro. However, high concentrations result in self-interference/squelching. These results strongly suggest that the trans-activation function of TEF-1 is mediated by a highly limiting, possible cell-specific, titratable transcriptional intermediary factor(s).

Amino Acid Sequence↗
PubMed1991

Identification of viral proteins encoded by two DNA fragments of herpesvirus of turkeys (HVT).

Herpesvirus of turkeys (HVT) vaccine is used worldwide to immunize chickens against Marek's disease (MD). Polyclonal antiserum directed against one virus cross-reacts with proteins of the other, while only 5% homology at the DNA level was demonstrated between the two viruses. A partial library of HVT DNA fragments ranging from 1.5 to 13.5 kbp in size was constructed in pBR 322. Under stringent conditions of hybridization (low salt concentration, 10% dextran sulfate at 68 degrees C), two of the cloned HVT DNA fragments (13.5 and 11.0 kbp) hybridized to three MDV DNA fragments: BamHI-C, D, and G. The 13.5 kbp fragment detected two transcripts of 1.8 and 3.0 kb in RNA extracted from HVT-infected chicken embryo fibroblasts, while the 11.0 kbp fragment detected three transcripts of 1.6, 2.0, and 3.0 kb in the extracted RNA. Using hybrid selection, specific RNA was isolated by hybridization with the two cloned HVT DNA fragments and then was translated in vitro using rabbit reticulocyte lysate, and the resulting proteins were analyzed by NaDodSO4-PAGE. The RNA selected by the two HVT DNA fragments coded for a 30 kD protein and for several smaller proteins 10-20 kD in size, while the RNA selected by the 11.0 kb HVT DNA fragment was translated into a 40 kD protein. Immunoprecipitation of these in vitro synthesized proteins with hyperimmune anti-HVT chicken serum showed that they were of viral origin.

Animals↗