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Biomedical subjects

M Stack

Publications and source records attributed to M Stack.

At least 19 recordsLinked to original sources
PubMed1999

Validation of a western immunoblotting procedure for bovine PrP(Sc) detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE).

In this report we document the results of several independent studies testing the sensitivity, specificity and reliability of the Prionics Western blotting (PWB) procedure to detect bovine and ovine disease-specific, protease-resistant prion protein (PrP(Sc)). Validation of the technique was obtained by blind analysis of samples from cattle affected with bovine spongiform encephalopathy (BSE), clinically normal animals or cattle with neurological diseases unrelated to BSE. Overall, very high sensitivity, specificity and reliability was observed. It became clear that sampling of the correct brain region and the method used for protein extraction are important factors for correct diagnosis. Furthermore, we tested the usefulness of the PWB technique as an instrument for surveillance purposes. We analyzed animals from a culling scheme as well as older animals from abattoirs to determine the number of subclinical BSE cases detectable by histopathological examination, immunohistochemistry for PrP(Sc) and PWB. In both studies, BSE-affected animals with no overt clinical symptoms were detected. These results demonstrate the usefulness of the PWB procedure in surveillance systems serving as a rapid diagnostic tool to identify animals subclinically infected with BSE.

Animals↗
PubMed1999

Group A streptococcal pharyngitis in adults 30 to 65 years of age.

BACKGROUND: Although the frequency of group A streptococcal pharyngitis in adults is assumed to be low, there is little information on frequency other than in military populations. METHODS: A prospective, observational study was done to determine the frequency of group A streptococcal pharyngitis in adults seen in the emergency department. Throat swabs were obtained on adults (30 to 65 years of age) with sore throat and pharyngitis on examination. Swabs were also obtained in a group of control subjects. RESULTS: Of the 148 adults with pharyngitis, 65 (44%) had throat specimens positive for group A streptococci. In the 50 control subjects, all throat cultures were negative for group A streptococci. A significant number of patients with group A streptococcal pharyngitis had school-aged children at home. CONCLUSION: The high rate of detection of group A streptococci in adults outside the military has not been previously reported.

Adult↗
PubMed1998

The human ROX gene: genomic structure and mutation analysis in human breast tumors.

We have recently isolated a human gene, ROX, encoding a new member of the basic helix-loop-helix leucine zipper protein family. ROX is capable of heterodimerizing with Max and acts as a transcriptional repressor in an E-box-driven reporter gene system, while it was found to activate transcription in HeLa cells. ROX expression levels vary during the cell cycle, being down-regulated in proliferating cells. These biological properties of ROX suggest a possible involvement of this gene in cell proliferation and differentiation. The ROX gene maps to chromosome 17p13.3, a region frequently deleted in human malignancies. Here we report the genomic structure of the human ROX gene, which is composed of six exons and spans a genomic region of less than 40 kb. In an attempt to identify possible inactivating mutations in the ROX gene in human breast cancer, we performed a single-strand conformation polymorphism analysis of its coding region in 16 sporadic breast carcinomas showing loss of heterozygosity in the 17p13.3 region. No mutations were found in this analysis. Five nucleotide polymorphisms were identified in the ROX gene, three of which caused an amino acid substitution. These nucleotide changes were present in the peripheral blood DNAs of both the patients and the control individuals. In vitro translated assays did not show a significant decrease in the ability of the ROX mutant proteins to bind DNA or to repress transcription of a driven reporter gene in HEK293 cells. Despite experimental evidence that ROX might act as a tumor suppressor gene, our data suggest that mutations in the coding region of ROX are uncommon in human breast tumorigenesis.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1998

The 30-base-pair deletion in Chinese variants of the Epstein-Barr virus LMP1 gene is not the major effector of functional differences between variant LMP1 genes in human lymphocytes.

One group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-kappaB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-kappaB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.

Amino Acid Sequence↗
PubMed1997

Epstein-Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-kappaB pathway involving TNF receptor-associated factors.

Expression of the Epstein-Barr virus (EBV) transforming LMP1 in B cells activates the transcription factor NF-kappaB and induces phenotypic changes through two distinct domains in the cytoplasmic C-terminus of the protein. The aa 187-231 domain of LMP1, which is important for growth transformation, binds tumour necrosis factor (TNF) receptor associated factor (TRAF) 1 and TRAF3 and this interaction mediates subsequent signalling events. The TRAFs also associate with CD40, a member of the TNFR family, which upon ligation activates NF-kappaB and induces phenotypic changes similar to those mediated by LMP1. This study demonstrates that LMP1 expression in carcinoma cell lines and SV40-transformed keratinocytes results in induction of the pleiotropic cytokine interleukin 6 (IL6), an effect which is also observed upon CD40 ligation. The mechanism by which either LMP1 expression or CD40 ligation induces IL6 production was found to be NF-kappaB-dependent. Mutational analysis identified domains in the C-terminus of LMP1 which are important for NF-kappaB activation and IL6 secretion. LMP1 and CD40 share a common PxQxT core TRAF binding motif and mutations in or adjacent to this sequence impaired the ability of LMP1 or CD40 to induce NF-kappaB activation and IL6 secretion. The importance of TRAF interactions in mediating these effects was confirmed using dominant negative TRAF2 and TRAF3 mutants which also identified differences in the signalling events mediated by the two NF-kappaB activating domains of LMP1. A20, an anti-apoptotic protein which interacts with TRAF2 and blocks CD40-mediated NF-kappaB activity, also blocked NF-kappaB and IL6 secretion in LMP1-transfected epithelial cells. These results suggest that LMP1 regulates IL6 production in epithelial cells in a manner similar to CD40 ligation and implicate TRAFs as common mediators in the transduction of signals generated via the CD40 and LMP1 pathways. As a role for IL6 in regulating epithelial cell growth has previously been suggested, the control of IL6 secretion via the CD40 and LMP1 pathways may have implications for the growth of both normal and transformed epithelial cells.

Amino Acid Sequence↗
PubMed1996

High levels of loss at the 17p telomere suggest the close proximity of a tumour suppressor.

High levels of loss of distal markers on 17p13.3 in breast cancer suggested the presence within the region of at least one tumour-suppressor gene. Here we describe the derivation of two biallelic polymorphisms from the 17p telomeric yeast artificial chromosome (YAC) TYAC98. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR analysis demonstrated that the high level of allelic imbalance observed in breast tumours represented loss of constitutional heterozygosity (LOH) and that this LOH extended to the telomere. Lung carcinoma (but not Wilms' tumour)-derived DNA again revealed a high level of loss of subtelomeric 17p sequences. Telomeric microsatellite polymorphisms from other chromosome arms did not show such elevated loss in either tumour type. This suggested that the 17p loss observed did not reflect a general telomeric instability and provided further evidence for the presence of a breast cancer tumour-suppressor gene in the distal region of 17p13.3.

Breast Neoplasms↗
PubMed1995

Detailed mapping and loss of heterozygosity analysis suggests a suppressor locus involved in sporadic breast cancer within a distal region of chromosome band 17p13.3.

The chromosome region 17p13.3 is thought to encode a tumour suppressor gene involved in sporadic breast cancer and other malignancies. Physical ordering of markers has been carried out by a series of multicolour fluorescent in situ hybridisation (FISH) experiments, using isolated yeast artificial chromosomes (YACs) and cosmids. Eight polymorphic markers ordered within this new physical map and one external marker were used to investigate the pattern of loss of heterozygosity in a panel of 40 sporadic breast tumour patients. The data revealed a region of high loss (60%) within distal 17p13.3, defined by markers D17S926, D17S695 and D17S849 which mapped close together. A contig of YACs was constructed physically linking these three markers.

Base Sequence↗
PubMed1995

An approach for balancing health and ecological risks at hazardous waste sites.

Human health and ecological risks must be balanced at hazardous waste sites in order to ensure that remedial actions prevent unacceptable risks of either type. Actions that are designed to protect humans may fail to protect nonhuman populations and ecosystems or may damage ecosystems. However, there is no common scale of health and ecological risk that would allow comparisons to be performed. This paper presents an approach to addressing this problem based on classifying all risks (i.e., health and ecological risks due contaminants and remediation) as insignificant (de minimis), highly significant (de manifestis), or intermediate. For health risks the classification is based on standard criteria. However, in the absence of national guidance concerning the acceptability of ecological risks, new ecological criteria are proposed based on an analysis of regulatory precedents. Matrices and flow charts are presented to guide the use of these risk categories in remedial decision making. The assessment of mercury contamination of the East Fork Poplar Creek is presented as an example of the implementation of the approach.

Algorithms↗
PubMed1991

The salmon gene encoding apolipoprotein A-I: cDNA sequence, tissue expression and evolution.

A cDNA encoding an apolipoprotein (Apo) has been isolated from the Atlantic salmon (Salmo salar) and sequenced. It encodes a peptide of 258 amino acids (aa), including a signal peptide of 18 aa, with 5'- and 3'-untranslated regions of the mRNA of 12 and 329 nucleotides, respectively. The protein has structural features in common with other Apo's of human and avian origin, including conserved sequences in the signal peptide and a series of internal repeats of 22 aa. The sequence has been identified as salmon Apo A-I (sApoA-I), and has 23% aa identity with human ApoA-I. Northern-blot analysis using the sApoA-I cDNA probe against total RNA prepared from several salmon tissues detects the expression of this gene in liver, intestine and muscle. A phylogenetic analysis reveals that the mammalian ApoA-I, ApoA-IV and Apo-E aa sequences are more closely related to each other than any of them are to sApoA-I. This suggests that the duplication events, from which A-I, A-IV and E arose, occurred after the divergence of the tetrapod and teleost ancestors.

Amino Acid Sequence↗
PubMed1984

Myelotoxicity and macrophage alteration in mice exposed to ochratoxin A.

Six- to seven-week-old female B6C3F1 mice were administered a total of 0, 20, 40, or 80 mg/kg of ochratoxin A (OCT A) ip on alternate days over an 8-day period. Twenty-four hours following the final dose, histopathology, bone marrow, and macrophage parameters were assayed. There was a dramatic dose related decrease in thymic mass with the mean thymus weight of the high dose animals being only 33% of controls. Histologic evidence of nephrotoxicity was minimal and restricted to the inner cortex. Myelotoxicity was present as evidenced by bone marrow hypocellularity, decreased marrow pluripotent stem cells (CFU-S), granulocyte-macrophage progenitors (CFU-GMs), and decreased 59Fe uptake in marrows and spleens of exposed mice. Peritoneal macrophages from sc as well as ip injected mice demonstrated increased phagocytic capacities and increased capacity to inhibit tumor cell growth. These alterations in bone marrow cells and macrophages suggest myelotoxicity is an additional potential hazard of OCT A exposure.

Animals↗
PubMedDate not supplied

The identification of cDNA clones that include the 3' end of mRNA.

A method is presented that facilitates the identification of cDNA clones corresponding to the polyadenylated 3' end of mRNA. It is based on the use of a poly dT probe that is synthesized by homopolymer extension of commercially available oligo dT. The method is shown to work in Southern blot analysis of plasmid preparations and in situ with colonies.

Blotting, Southern↗
PubMedDate not supplied

Morphological differentiation of Tritrichomonas foetus from other protozoa of the bovine reproductive tract.

Tritrichomonas foetus is a protozoan parasite associated with reproductive failure in the bovine. Its incidence has effectively been controlled in the UK, as in many other countries, through a policy of testing of breeding animals and the widespread use of artificial insemination (AI). As a result there have been only two cases of bovine trichomoniasis in the UK over the past 20 years. Despite the virtual absence of T. foetus in bovine preputial and vaginal samples, routine examinations have occasionally revealed the presence of motile protozoa which require careful identification. In this paper, the morphological characteristics of T. foetus, and other protozoa isolated from the bovine reproductive tract, are presented.

Animals↗