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Biomedical subjects

R Lathe

Publications and source records attributed to R Lathe.

At least 19 recordsLinked to original sources
PubMed1996

Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus.

Mice carrying an ovine beta-lactoglobulin (BLG) transgene secrete BLG protein into their milk. To explore transgene expression stability, we studied expression levels in three BLG transgenic mouse lines. Unexpectedly, two lines exhibited variable levels of transgene expression. Copy number within lines appeared to be stable and there was no evidence of transgene rearrangement. In the most variable line, BLG production levels were stable within individual mice in two successive lactations. Backcrossing demonstrated that genetic background did not contribute significantly to variable expression. Tissue in situ hybridization revealed mosaicism of transgene expression within individual mammary glands from the two variable lines; in low expressors, discrete patches of cells expressing the transgene were observed. Transgene protein concentrations in milk reflected the proportion of epithelial cells expressing BLG mRNA. Furthermore, chromosomal in situ hybridization revealed that transgene arrays in both lines are situated close to the centromere. We propose that mosaicism of transgene expression is a consequence of the chromosomal location and/or the nature of the primary transgene integration event.

Animals↗
PubMed1996

Genetic basis of Creutzfeldt-Jakob disease in the United Kingdom: a systematic analysis of predisposing mutations and allelic variation in the PRNP gene.

Creutzfeldt-Jakob disease (CJD) is a transmissible neurodegenerative disorder characterized by the accumulation of aggregates of a cellular protein, PrP, in the brain. In both human and animals, genetic alterations to the gene encoding PrP (PRNP in human) modulate susceptibility to CJD. The recent epidemic of bovine spongi-form encephalopathy in the UK has raised the possibility of transmission from animal produce to humans. To provide a baseline against which to assess possible risk factors, we have determined the frequencies of predisposing mutations and allelic variants in PRNP and their relative contributions to disease. Systematic PRNP genotype analysis was performed on suspected CJD cases referred to the National Surveillance Unit in the UK over the period 1990-1993. Inspection of 120 candidate cases revealed 67 patients with definite and probable CJD, based on clinical and neuropathological criteria. No PRNP mutations were detected in any of the remaining 53 patients assessed as "non-CJD". A disease-associated mutation in the PRNP gene was identified in nine (13.4%) definite and probable cases of CJD, a reliable estimate of the incidence of PRNP-related inherited CJD based on a prospective epidemiological series. Within the group of sporadic CJD patients (lacking PRNP mutations), we confirmed that the genotype distribution with respect to the common methionine/valine (Met/Val) polymorphism at codon 129 within PRNP was significantly different from the normal Caucasian population. The incidence of Met homozygosity at this site was more than doubled and correlated with increased susceptibility to the development of sporadic CJD. Unlike other recent studies, Val homozygosity was also confirmed to be a significant risk factor in sporadic CJD, with the relative risks for the three genotypes Met/Met: Val/Val:Met/Val being 11:4:1.

Alleles↗
PubMed1995

A novel cytochrome P450 expressed primarily in brain.

hct-1 (hippocampal transcript) was detected in a differential screen of a rat hippocampal cDNA library. Expression of hct-1 was enriched in the formation but was also detected in rat liver and kidney, though at much lower levels; expression was barely detectable in testis, ovary, and adrenal. In liver, unlike brain, expression was sexually dimorphic; hepatic expression was greatly reduced in female rats. In mouse, brain expression was widespread, with the highest levels being detected in corpus callosum; only low levels were detected in liver. Sequence analysis of rat and mouse hct-1 cDNAs revealed extensive homologies with cytochrome P450s (CYPs), a diverse family of heme-binding monooxygenases that metabolize a range of substrates including steroids, fatty acids, and xenobiotics. Among the CYPs, hct-1 is most similar (39% at the amino acid sequence) to cholesterol 7 alpha-hydroxylase (CYP7) and contains a postulated steroidogenic domain present in other steroid-metabolizing CYPs but clearly represents a type of CYP not previously reported. Genomic Southern analysis suggests that a single gene corresponding to hct-1 is present in mouse, rat, and human. hct-1 is unusual in that, unlike all other CYPs described, the primary site of expression is in the brain. Similarity to CYP7 and other steroid-metabolizing CYPs may argue that hct-1 (CYP7B) plays a role in steroid metabolism in brain, notable because of the documented ability of brain-derived steroids (neurosteroids) to modulate cognitive function in vivo.

Amino Acid Sequence↗
PubMed1995

A candidate marsupial PrP gene reveals two domains conserved in mammalian PrP proteins.

The normal function of the pathogenicity-related protein, PrP (or prion protein), is unknown. To shed light on functionally important domains, we have characterized a candidate marsupial PrP gene. The deduced marsupial PrP has an overall identity of about 80% to eutherian PrP at the amino acid (aa) level. This similarity is not equally distributed and two regions (aa 118-142 and 177-223) are particularly highly conserved. In contrast, a repeat region in the N-terminal half of the marsupial PrP shows dipeptide inserts not described in other PrP. Another particular feature of the marsupial gene is the lack of a continuous ORF on the antisense strand, as is found in most eutherian PrP. We propose that antisense ORFs found in other species are artefactual. Comparison with all known PrP argues that the molecule characterised is the true marsupial PrP orthologue.

Amino Acid Sequence↗
PubMed1995

Inherited Creutzfeldt-Jakob disease in a British family associated with a novel 144 base pair insertion of the prion protein gene.

A case of familial Creutzfeldt-Jakob disease associated with a 144 base pair insertion in the open reading frame of the prion protein gene is described. Sequencing of the mutated allele showed an arrangement of six octapeptide repeats, distinct from that of a recently described British family with an insertion of similar size. Thirteen years previously the brother of the proband had died from "Huntington's disease", but re-examination of his neuropathology revealed spongiform encephalopathy and anti-prion protein immunocytochemistry gave a positive result. The independent evolution of at least two distinct pathological 144 base pair insertions in Britain is proposed. The importance of maintaining a high index of suspicion of inherited Creutzfeldt-Jakob disease in cases of familial neurodegenerative disease is stressed.

Base Sequence↗
PubMed1994

Neuropathological phenotype and 'prion protein' genotype correlation in sporadic Creutzfeldt-Jakob disease.

A systematic study of 'prion protein' genotype in cases of sporadic Creutzfeldt-Jakob disease showing amyloid plaques staining with anti-prion protein antibody has been performed. This revealed a relative excess of cases with valine at position 129 of the gene's open reading frame. The observation emphasises the importance of this site of common polymorphism in influencing the neuropathological phenotype in human spongiform encephalopathy.

Aged↗
PubMed1994

Analysing brain function and dysfunction in transgenic animals.

Many neuropsychiatric disorders have a genetic aetiology. In vivo gene modification offers a route to simulating such disorders in transgenic animals, allowing a systematic study of the underlying pathophysiology. However, attempts to mimic diseases such as Alzheimer's disease in transgenic animals have not yet been successful. This principally reflects our lack of knowledge concerning normal brain function, and an understanding of the biochemical mechanisms underlying cognitive processes is a primary objective. We and others have therefore focused on the hippocampus, a brain region involved in learning and memory and an early target for degeneration in Alzheimer's disease. Genetic intervention to date has yielded transgenic animals with apparent functional deficits in the hippocampus, leading the way to a greater understanding of brain function.

Animals↗
PubMed1991

Induction of pS2 and hSP genes as markers of mucosal ulceration of the digestive tract.

The recently discovered pS2 protein is expressed under estrogen control in a subset of estrogen receptor-positive breast cancers and in an estrogen-independent manner in normal stomach mucosa. The pS2 gene belongs to a family of genes encoding peptides that contain a conserved 5-cysteine domain, the P domains. Although the function of the pS2 protein is unknown, it has been suggested that it may have cell growth stimulatory activity. We report here that expression of the pS2 gene in the digestive tract, which is normally restricted to the stomach, is strongly induced by mucosal ulcerations elsewhere in the tract, most notably in Crohn's disease. pS2 gene expression is restricted to the mucosal layers adjacent to the ulcerations, in a region where a novel epidermal growth factor-secreting cell lineage was shown to be induced by mucosal ulceration. The human hSP gene, which contains a tandem duplication of the pS2 gene P domain and is coexpressed with the pS2 gene in normal stomach mucosa but not in breast cancers, is also expressed in Crohn's disease. We suggest that pS2 gene expression may provide a useful marker for mucosal ulcerations of the digestive tract.

Biomarkers↗
PubMed1991

Immunization against human papillomavirus type 16 tumor cells with recombinant vaccinia viruses expressing E6 and E7.

Papillomaviruses are etiological agents of epithelial proliferative disease. In man, neoplastic transformation of the uterine cervix has been linked to infection with specific subtypes of human papillomavirus, particularly types 16 and 18. We previously reported that live vaccinia virus recombinants expressing early transforming proteins of other tumor viruses can immunize against challenge with cognate tumor cells and we have extended this approach to HPV16. Neoplastic transformation by papillomaviruses involves expression of early open reading frames (ORFs) E5, E6, and E7, and we report the construction of vaccinia recombinants separately expressing ORFs E5-E7 of HPV16. Primary rat cell lines cotransformed with HPV16 and an activated ras oncogene were established in order to evaluate the potential of the recombinants to elicit antitumor immunity. We report that inoculation of rats with vaccinia recombinants expressing E6 or E7 retarded or prevented tumor development in a proportion of animals challenged by subcutaneous seeding of tumor cells whereas the recombinant expressing E5 was inactive.

Animals↗
PubMed1991

Vaccinia recombinants expressing secreted and transmembrane forms of breast cancer-associated epithelial tumour antigen (ETA).

Monoclonal antibody H23 identifies a polymorphic epithelial tumour antigen (ETA) that is aberrantly expressed in breast cancer and which may afford a target for active immunotherapy. We recently reported the cloning of H23-ETA genomic and cDNA clones. H23-ETA contains a multiple internal tandem repetition of a 20 amino acid motif and sequence analysis predicted two mRNA species encoding different ETA proteins, one harbouring a C-terminal potentially transmembrane hydrophobic zone (T) and a second form (S) that lacks this zone. We report that both RNA species can be detected in breast cancer cells. To further characterize the encoded proteins we have constructed vaccinia virus recombinants, VV-ETA-S and VV-ETA-T, separately expressing the alternative forms. Despite selective loss of internal tandem repeat elements during propagation of recombinant vaccinia, the encoded polypeptides were efficiently recognized by H23 monoclonal antibody. Immunoprecipitation revealed that ETA encoded by the S recombinant was secreted into the culture medium whereas the T form remained tethered at the cell surface. Both forms were readily detected in infected cells by immunofluorescence. Abnormal mobility of the T polypeptide indicated post-translational cleavage that may permit the extracellular domain of the T-polypeptide to be shed from the cell surface. Further, fluorescence-activated cell sorting analysis shows that the S form of the polypeptide is also partly present at the cell surface. Vaccinia recombinants expressing ETA may be of utility in the active immunotherapy of breast cancer.

Antigens, Neoplasm↗
PubMed1991

Expression of the breast cancer associated gene pS2 and the pancreatic spasmolytic polypeptide gene (hSP) in diffuse type of stomach carcinoma.

Expression of the pancreatic spasmolytic peptide (hSP) gene and pS2 (a gene isolated from oestrogen-induced breast carcinoma cells) were analysed in 36 samples of human stomach carcinoma. 17 tumours were investigated at the RNA level (by northern blots) as well as at the gene product level (by immunochemistry). Since pS2 had been shown to be expressed in normal stomach mucosa its activity in carcinoma samples was expected. Surprisingly, strong pS2 immunoreactivity was noted in the diffuse carcinoma type, whereas the intestinal type displayed weak reactivity. The tumour samples showing strong immunostaining expressed the regular 0.6 kb pS2 RNA band and weak staining was paralleled by aberrant transcripts. Additionally, only in tumour samples with regular pS2 transcription was the typical 0.7 kb hSP RNA band seen; samples with aberrant pS2 bands did not express hSP at all. This is the first demonstration of hSP gene activity in a human tumour.

Blotting, Northern↗
PubMed1991

Precocious mammary gland development and milk protein synthesis in transgenic mice ubiquitously expressing human growth hormone.

A chimeric gene comprising the hydroxymethylglutaryl coenzyme-A reductase promoter and the human GH (hGH) genomic sequences was used to create transgenic mice expressing hGH in all tissues. In transgenic females, morphological development of the mammary gland and milk protein (WAP) expression commences at 3 weeks of age. At 8 weeks of age the mammary gland is morphologically and functionally comparable to that normally reached after 14-15 days of gestation. Precocious development correlated with local expression of hGH in mammary gland. Organ culture in the presence of different lactogenic hormones revealed that insulin and hydrocortisone are sufficient to maintain transcription of the WAP gene in transgenic mammary gland. In contrast, WAP transcription in normal gland required either hGH or PRL in addition to insulin and hydrocortisone. However, the effect of hGH on mammary differentiation does not appear to be solely mediated through an interaction with PRL receptors, since PRL, when added to cultured mammary tissues, did not elicit an equivalent response.

Animals↗
PubMed1991

Abrogation of dominant glucose intolerance in SJL mice by a growth hormone transgene.

Glucose tolerance was studied in transgenic mice (SJL x C57BL/6) expressing human GH under the control of a housekeeping promoter. Parental SJL mice were found to harbour a dominant allele, termed here glid, determining glucose intolerance in pure-bred animals and in F1 hybrids with glucose-tolerant C57BL/6 mice. Blood glucose levels in transgenic SJL x C57BL/6 hybrid mice were well controlled following glucose challenge, whereas non-transgenic hybrids failed to control their glucose adequately. Pancreatic morphology was normal in all animals. In confirmation of a physiological role for GH in glucose regulation. GH-deficient lit/lit mice were pathologically sensitive to glucose.

Alleles↗