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Biomedical subjects

D Walls

Publications and source records attributed to D Walls.

At least 19 recordsLinked to original sources
PubMed2006

Optimization of endometrial preparation results in a normal endometrial function test (EFT) and good reproductive outcome in donor ovum recipients.

PURPOSE: Numerous studies have investigated potential markers of endometrial receptivity as predictors of successful implantation. Cyclin E and p27 have recently been studied using the endometrial function test (EFT). Our objective is to determine the correlation between the expression of cyclin E and p27 and the adequacy of uterine preparation of recipients using donor oocytes. METHODS: Twenty recipients undergoing preparatory cycles with leuprolide acetate, estrogen, and progesterone. Endometrial biopsies were obtained 10-12 days after progesterone supplementation following the course of estrogen. The tissue was prepared for histological analysis and immunohistochemical staining for cyclin E assessment. The outcome of their subsequent ovum donation cycle was blinded to the reviewer of the EFT. RESULTS: All recipients showed normal luteal transformation. Nineteen (95%) of the recipients had a normal EFT. This is significantly higher than what we demonstrated, previously, in unexplained infertility patients, where only 40% of such patients had a normal EFT. Thirteen recipients with a normal EFT had a clinical pregnancy, while 6 did not become pregnant in their subsequent transfer cycles. The sole patient with an abnormal EFT did not conceive on 2 subsequent cycles. CONCLUSIONS: While a normal EFT does not guarantee a successful pregnancy, an abnormal EFT appears to be associated with pregnancy failure. This may be useful in identifying women who need adjustments to their stimulation protocols prior to progressing to a physically, emotionally, and financially costly cycle.

Adult↗
PubMed2000

A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions.

We describe a rapid, sensitive and specific polymerase chain reaction (PCR) assay for the detection of BHV1 DNA in a range of routine diagnostic submissions without the need for prior virus isolation. The assay, which is based on the selected amplification of a portion of the viral tk gene, detected both BHV1.1 and BHV1.2 subtypes in a panel of 15 characterised field isolates, and its sensitivity was estimated to be <0.125 TCID(50). BHV2, alcephaline herpesvirus, BHV4, equine herpesvirus 1 (EHV1), EHV4 and pseudorabies virus were not detected confirming the specificity of the assay. One hundred and five diagnostic submissions, including tissues, nasal secretions and nasal swabs were taken from cattle with respiratory disease and tested using the routine methods of virus isolation (VI) and the fluorescent antibody test (FAT), and the results were compared with those obtained by PCR. The PCR assay detected BHV1 DNA in all samples that were positive by VI. BHV1 DNA was also detectable by PCR in raw and extended semen samples at a sensitivity of 1 TCID(50) per 50microl. The assay also detected BHV5, permitting differentiation between it and BHV1 by virtue of the size of the amplified PCR product. The PCR assay is more sensitive and independent of sample quality than either virus isolation or FAT, and it is faster than virus isolation. The sample preparation method is simple with few steps involved. There are no extra post-amplification blotting/hybridisation steps and the assay is not based on a nested PCR strategy that might otherwise exacerbate the problem of oversensitivity/contamination in the routine use of such a test in a diagnostic laboratory. This assay would permit discrimination between those animals naturally infected with wild type BHV1 and those vaccinated with tk-BHV1 strains.

Animals↗
PubMed2000

The bfl-1 gene is transcriptionally upregulated by the Epstein-Barr virus LMP1, and its expression promotes the survival of a Burkitt's lymphoma cell line.

The recently identified bfl-1 gene (also known as A1 or GRS), a homologue of bcl-2, encodes an antiapoptotic protein that suppresses apoptosis induced by the p53 tumor suppressor protein and exhibits proliferative and potent cooperative transforming activities. We show that elevated levels of bfl-1 mRNA are a feature of Epstein-Barr virus (EBV)-immortalized B-cell lines and Burkitt's lymphoma cell lines expressing the full spectrum of EBV latent proteins. Using an EBV-negative Burkitt's lymphoma cell line in which the expression of EBV latent membrane protein 1 (LMP1) is inducibly regulated by tetracycline, we demonstrate that LMP1 expression coincides with a dramatic increase in the level of bfl-1 mRNA. Also in this system, an increase in the level of Bcl-2 protein was seen to occur earlier than that of bcl-2 mRNA, suggesting that both transcriptional and translational mechanisms are involved in the control of Bcl-2 expression by LMP-1. We show that elevated bfl-1 mRNA stability can contribute to this effect of LMP-1, thus providing evidence of a novel mechanism of gene regulation by this EBV protein. Upregulation of bfl-1 by LMP1 was not observed in the T-cell line Jurkat or the epithelial cell line C33A. Ectopic expression of Bfl-1 in an EBV-positive cell line exhibiting a latency type I infection protects against apoptosis induced by growth factor deprivation, thereby providing a functional role for Bfl-1 in this cellular context and adding Bfl-1 to the list of antiapoptotic proteins whose expression is modulated by EBV. This is the first report of the regulation of bfl-1 expression by a viral protein, and this novel finding may thus represent an important link between the EBV oncoprotein LMP1 and its cellular growth-transforming properties.

Apoptosis↗
PubMed1994

The Epstein-Barr virus determined nuclear antigens EBNA-3A, -3B, and -3C repress EBNA-2-mediated transactivation of the viral terminal protein 1 gene promoter.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) has been shown to transactivate both cellular and viral gene promoters including the promoter for the viral terminal protein 1 gene (TP-1). We investigated whether three other EBV nuclear antigens EBNA-3A, -3B, and -3C (which themselves share a degree of primary sequence homology) could also play a role in TP-1 gene regulation. The TP-1 promoter sequence was linked to the chloramphenicol acetyltransferase (CAT) gene and used in cotransfection experiments in an EBV negative cell line with various combinations of vectors expressing individual EBNA-3s. In the absence of other EBV proteins, the EBNA-3s did not stimulate TP-1 promoter activity. In the presence of EBNA-2, the EBNA-3s were shown to be capable of reducing the level of TP-1 promoter-driven CAT activity. The EBNA-3s had no effect on a panel of heterologous promoters, indicating that EBNA-2 and/or transcription elements specific to the TP-1 promoter are essential for the observed activity of the EBNA-3s. The functional antagonism between the EBNA-2 and EBNA-3 proteins may be important in the overall viral strategy.

Antigens, Viral↗
PubMed1992

Repression of the viral latent promoter BC-R2 in Epstein-Barr virus negative cell lines.

The regulation of the latent promoter BC-R2 of Epstein-Barr virus (EBV) was examined using the chloramphenicol acetyl transferase (CAT) gene reporter system. A 5' deletion analysis of BC-R2 promoter sequences has been used to characterize a region, described previously as a transcriptional enhancer in EBV positive cell lines, which can repress the BC-R2 transcriptional activity in EBV negative cell lines.

Burkitt Lymphoma↗
PubMed1991

Novel downstream elements upregulate transcription initiated from an Epstein-Barr virus latent promoter.

The modulation of nuclear antigen expression from the Epstein-Barr virus (EBV) genome in virus-infected cells is crucial to the establishment and maintenance of the immortalized state. The EBV nuclear antigen (EBNA) genes are all transcribed from either of two promoters whose alternate usage is not understood. We have studied the EBNA transcriptional domain by using various promoter constructs to transfect a variety of EBV positive cell lines and then measured promoter activity by RNase protection analysis and nuclear run-on transcription assay. These experiments have shown that at least two distinct and novel DNA sequence elements are located in the intronic region situated between both promoters and that their presence results in a 100-fold stimulation of transcription initiated from the upstream promoter. One of these elements is shown to bind nuclear proteins from an EBV-positive cell line. In addition, an important sequence homology is noted between this element and the core sequences of a number of well known viral enhancers. Taken together, the results show that EBNA transcription is controlled by upstream and intronic elements in a regulatory domain that is at least 7 kb long.

Antigens, Viral↗
PubMed1991

The identification of new transcription elements active in Epstein-Barr virus latent gene expression.

The immortalization in vitro of human B cells by the Epstein-Barr virus (EBV) is associated with the expression of a restricted group of nuclear proteins (EBNA). EBNA transcription is driven from either of two mutually exclusive promoters whose modulation is likely to involve an important host/virus interaction. Our experiments on the EBNA transcriptional domain have led us to identify several new transcription elements which are located on the DNA sequence between the two promoters. These elements stimulate the activity of the upstream EBNA promoter by a factor of at least one hundred. One of these elements may also be involved in the in vivo reactivation of EBV in certain cases.

Chromosome Mapping↗
PubMed1990

cDNA cloning and transient expression of the Epstein-Barr virus-determined nuclear antigen EBNA3B in human cells and identification of novel transcripts from its coding region.

Recombinant plasmids containing sequences from the BamHI-E rightward reading frames 2a and 2b (BERF2a and 2b) of the Epstein-Barr virus (EBV) genome were isolated from a library of cDNA clones which had been previously made from the EBV B95-8 lymphoblastoid cell line (M. Bodescot, O. Brison, and M. Perricaudet, Nucleic Acids Res. 14:7103-7114, 1986). The characterization of these clones in combination with RNase mapping experiments led to the identification of one leftward and several rightward transcripts traversing the EBV-determined nuclear antigen EBNA3B coding region. One cDNA (T7) contains a continuous open reading frame generated by the splicing together of BERF2a and BERF2b. The T7 clone was used to reconstruct a complete fused BERF2a/2b open reading frame in an adenovirus-based expression vector. Western immunoblotting and immunofluorescence experiments using human 293 cells showed that the recombinant plasmid is capable of expressing a protein with a size, immunological characteristics, and a subcellular localization indistinguishable from those of native B95-8 EBNA3B.

Amino Acid Sequence↗
PubMed1988

The analysis of EBV proteins which are antigenic in vivo.

We have used small random EBV B95-8 DNA fragments to generate a large genomic bank in a plasmid expression vector. This bank was screened with a pool of sera from individuals with IM thus allowing any EBV antigen which evoked an immune response in man to be identified. The characterization of four immunopositive clones obtained in this way is presented in this study. Three of these clones express viral ORF DNA sequences which are parts of larger ORFs in the BamH1 N(het), V and X regions of the B95-8 viral genome. cDNA cloning has been used to confirm that the cloned sequences from BamH1 N and V are expressed in cell culture and to identify the transcription units involved. The fourth clone expresses an ORF sequence located in the viral BamH1 F fragment in a region not previously recognized as having protein coding potential. The experimental design used here must reflect the situation in vivo and consequently these sequences must be expressed and be antigenic during IM.

Amino Acid Sequence↗
PubMed1988

The expression of novel antigens from the Epstein-Barr virus large internal repeat.

A large Epstein-Barr virus (EBV) B95-8 genomic bank has been prepared in an Escherichia coli expression vector and screened with a pool of sera from human infectious mononucleosis patients. Four immunopositive clones which also contained sequences from the viral large internal repeat were selected. DNA sequence analysis has located them on the repeat sequence and shown that they come from three potential open reading frames and that two of them consist of overlapping reading frames. This must imply extensive intron/exon splicing or that the repeat itself encodes several different proteins. The four expressed epitopes were shown to be present simultaneously in independent cases of infectious mononucleosis. These have not been previously described and based on the experimental design, they must reflect the situation in vivo.

Amino Acid Sequence↗