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Biomedical subjects

G R Sutherland

Publications and source records attributed to G R Sutherland.

At least 253 records · Page 14Linked to original sources
PubMed1994

Ischemia-induced cellular redistribution of the astrocytic gap junctional protein connexin43 in rat brain.

The distribution and levels of the astrocytic gap junction protein, connexin43 (Cx43) was analyzed in various regions of brain as a function of time after neuronal loss and consequent reactive gliosis induced by bilateral carotid occlusion in rats. In the striatum 2 days after induction of ischemia, immunostaining intensity for Cx43 increased in animals exhibiting mild to moderate striatal damage, whereas areas of reduced staining surrounded by elevated levels of Cx43 immunoreactivity were observed in animals with severe ischemic damage. Immunolabelling of glial cell bodies was evident in ischemic, but not normal, striatum. Similar, though less dramatic, changes were seen at 7 days post-ischemia. Compared with the fine punctate pattern of Cx43 staining seen in normal striatum, ischemic striatal areas contained large aggregates of punctate profiles. In the hippocampus, increased immunostaining was seen at 2 and 7 days post-ischemia and, unlike normal hippocampus, neurons in the CA3 pyramidal cell layer were surrounded by a network of Cx43-immunoreactive puncta at the latter survival time. Immuno-EM analysis of ischemic tissue revealed numerous immunolabelled gap junctions among astrocytic processes in the vicinity of degenerating neurons and elevated levels of intracellular Cx43 immunoreactivity in astrocytic processes and cell bodies. No differences in protein levels or phosphorylation states of Cx43 were detected in either hippocampus or striatum by Western blot analyses of ischemic and control tissue. These results suggest that astrocytes respond to an ischemic insult by reorganizing their gap junctions, that the qualitative nature of their response is dependent on the severity of neuronal damage or loss, and that a pool of Cx43 normally undetectable by immunohistochemistry may contribute to the ischemia-induced elevations of immunolabelling for this protein.

Animals↗
PubMed1994

Haplotype analysis at the FRAXA locus in the Japanese population.

Fragile X syndrome, one of the most common human genetic diseases, is characterized by a unique genetic mechanism which involves dynamic mutation in a heritable unstable DNA sequence, a p(CCG)n repeat, in the FRAXA locus. It has recently been suggested that a few founder chromosomes are responsible for most fragile X mutations in the Caucasian population. In order to investigate the origin of the fragile X mutations in the Japanese population, we analyzed haplotypes of the FRAXA locus in 40 unrelated fragile X chromosomes and 142 normal X chromosomes in Japanese males, by using two polymorphic AC repeats, FRAXAC1 and FRAXAC2, which flank the fragile site. This analysis provided evidence for founder fragile X chromosomes in the Japanese population, similar to that in Caucasians, although different haplotypes are involved. The distribution of normal allele size of the p(CCG)n repeat among the X chromosomes in the Japanese population is very similar to that reported for Caucasians, except that the most frequent copy number (n = 28) is one copy less than that in Caucasians and that there is an additional peak at 35 copies. There is significant correlation between FRAXAC alleles and the p(CCG)n repeat copy number in non-fragile X chromosomes, however, alleles with more than 31 copies of the p(CCG)n repeat do not segregate with either of the fragile X common FRAXAC haplotypes.

Asian People↗
PubMed1994

Implications of FRA16A structure for the mechanism of chromosomal fragile site genesis.

Fragile sites are chemically induced nonstaining gaps in chromosomes. Different fragile sites vary in frequency in the population and in the chemistry of their induction. DNA sequences encompassing and including the rare, autosomal, folate-sensitive fragile site, FRA16A, were isolated by positional cloning. The molecular basis of FRA16A was found to be expansion of a normally polymorphic p(CCG)n repeat. This repeat was adjacent to a CpG island that was methylated in fragile site-expressing individuals. The FRA16A locus in individuals who do not express the fragile site is not a site of DNA methylation (imprinting), which suggests that the methylation associated with fragile sites may be a consequence and not a cause of their genesis.

Alleles↗
PubMed1994

Hydrogen ion compartmentation during and following cerebral ischemia evaluated by 31P NMR spectroscopy.

Two peaks were observed in the inorganic phosphate region in 31P NMR spectra obtained during and shortly after incomplete forebrain ischemia induced in rats under normoglycemic conditions. Pre-ischemia, a single Pi peak was observed at a chemical shift of 2.5 ppm (pH 7.2). This peak shifted upfield to 1.6 ppm (pH 6.5) and, following reperfusion, returned to its pre-ischemia value. During ischemia and for a short time following reperfusion, a second, smaller peak was observed which we assigned to a second pool of inorganic phosphate. The data support the proposal by Kraig and coworkers of pH compartmentation between neurons and glia during and following transient forebrain ischemia.

Animals↗
PubMed1994

Isolation and characterization of transcribed sequences from a chromosome 16 hn-cDNA library and the physical mapping of genes and transcribed sequences using a high-resolution somatic cell panel of human chromosome 16.

A hn-cDNA (heteronuclear complementary DNA) library was constructed from a mouse/human somatic cell hybrid, CY18, which contains chromosome 16 as the only human chromosome. Hexamer primers constructed from consensus 5' intron splice sequences were used to generate cDNA from the immature unspliced mRNA. The resulting cDNA library was screened with a total human DNA probe to identify potential human clones. Rescreening was necessary, and use of a mouse-derived clone with homology to 7SL RNA proved successful in eliminating the majority of mouse clones. Thirteen clones had open reading frames, and of those, five showed homology to human sequences in GenBank. Two clones had homology to random partially sequenced cDNAs, one clone was likely to be a GRP78 pseudogene, one clone mapped the PHKG2 gene to 16p11.2-16p12.1, and one clone had homology to human S13 ribosomal protein. All clones except the latter were mapped to a high-resolution somatic cell panel. Although isolation of human chromosome 16 genes from this library was successful, it was apparent that cDNA synthesis was initiated at sites other than intron splice sites, presumably by mispairing of the hexamers.

Animals↗
PubMed1994

The mouse CD69 gene. Structure, expression, and mapping to the NK gene complex.

CD69 is a rapidly induced T cell activation Ag that is also expressed in an inducible fashion on cells of most, if not all, hematopoietic lineages. Molecular cloning has shown that CD69 is a type II membrane glycoprotein that is a member of the C-type lectin family. In this report we have shown that induction of CD69 mRNA in activated murine thymocytes and T cells is very rapid, peaking between 30 and 60 min poststimulation, and transient, dropping to nearly resting levels by 8 h. An analysis of the mouse CD69 gene structure showed the gene to consist of 5 exons and have a phorbol ester-inducible promoter element within the first 700 bp upstream of the start of transcription. Chromosomal mapping placed the mouse CD69 gene on the long arm of chromosome 6 near the NK gene complex that contains the related NKR-P1 and Ly-49 gene families. The human CD69 gene mapped to chromosome 12p13 near the related NKG2 gene cluster and in a region associated with rearrangements in approximately 10% of cases of childhood acute lymphocytic leukemia.

Amino Acid Sequence↗
PubMed1994

Localization of a human receptor tyrosine kinase (ETK1) to chromosome region 3p11.2.

We have recently described a human receptor tyrosine kinase (hek) that is expressed by some pre-B and thymic T cell lines, but is not detectable on normal adult human tissues. Gene cloning studies established that hek is a new member of the EPH family of receptor tyrosine kinases. The expression of hek may normally be developmentally regulated and inappropriate expression may contribute to oncogenesis. In the present study, we have used Southern blot analysis of somatic cell hybrids and fluorescence in situ hybridization to localize the hek gene to human chromosome region 3p11.2. Karyotype analysis of the cell lines that over-express hek showed no cytogenetically visible abnormality involving the hek locus.

Animals↗
PubMed1994

Molecular and biological characterization of human 4-1BB and its ligand.

4-1BB was originally described as a cDNA expressed by activated murine T cells and subsequently demonstrated to encode a member of the tumor necrosis factor receptor family of integral membrane proteins. Recently, we identified and cloned a murine ligand for 4-1BB (mu4-1BB-L) and demonstrated it to be a member of an emerging family of ligands with structural homology to tumor necrosis factor. To characterize further the role of 4-1BB in the immune response we undertook to clone the human homologue of 4-1BB-L. However, attempts to isolate a cDNA encoding the human 4-1BB-L by cross-hybridization with the murine cDNA were unsuccessful. Therefore we first utilized cross-species hybridization to isolate a cDNA encoding human 4-1BB (hu4-1BB). A fusion protein consisting of the extracellular portion of hu4-1BB coupled to the Fc region of human immunoglobulin G1 (hu4-1BB.Fc) was then used to identify and clone a gene for human 4-1BB-L from an activated CD4+ T cell clone using a direct expression cloning strategy. Human 4-1BB-L shows 36% amino acid identity with its murine counterpart and maps to chromosome 19p13.3. Scatchard analysis demonstrated high-affinity binding of hu4-1BB.Fc to either native or recombinant human 4-1BB-L. Both monoclonal antibody to hu4-1BB and cells transfected with hu4-1BB-L induced a strong proliferative response in mitogen co-stimulated primary T cells. In contrast, ligation of 4-1BB on T cell clones enhanced activation-induced cell death when triggered by engagement of the TCR/CD3 complex.

Amino Acid Sequence↗
PubMed1994

At least two different regions are involved in allelic imbalance on chromosome arm 16q in breast cancer.

Loss of heterozygosity (LOH) or allelic imbalance, the latter term referring to both loss and gain of an allele, on the long arm of chromosome 16 has been repeatedly found in cancers of, e.g., the breast and prostate. This indicates the presence of one or more tumor suppressor genes on 16q. To locate the gene(s) more precisely, a detailed allelic imbalance map of 20 polymorphic markers on this chromosome arm was made for 79 sporadic breast carcinomas. LOH of one or more markers was found in 63% of the tumors. Some had allelic imbalance on a region of 16q which failed to overlap with the LOH in other tumors. We therefore assigned two separate "smallest regions of overlap" to 16q and suggest that this chromosome arm contains at least two different tumor suppressor genes.

Alleles↗
PubMed1994

Phorbol ester-induced transcription of an immediate-early response gene by human T cells is inhibited by co-treatment with calcium ionophore.

Human T cells require two discrete signals to initiate their proliferation. In Jurkat T cells the first signal can be provided by the phorbol ester TPA and the second by the calcium ionophore A23187. We have isolated a cDNA from Jurkat T cells representing mRNA induced by TPA but inhibited by simultaneous treatment of the cells with antibody, lectin, or A23187. Sequencing revealed identity of the Jurkat clone to a cDNA, termed ETR101, recently isolated from HL60 promyelocytic leukaemia cells and shown to be an immediate early gene expressed upon TPA stimulation of these cells [Shimizu et al.: J Biol Chem 266:12157, 1991]. The gene is also induced very rapidly upon TPA treatment of Jurkat cells and is superinduced by co-treatment with cycloheximide. The predicted amino acid sequence encoded by ETR101 has weak homology to JunB and JunD, therefore it is of some interest that these three genes share the chromosomal localization, 19p13.2. The divergent effects of TPA treatment upon cell proliferation and differentiation in different circumstances allow some speculation about a possible role for the ETR101 gene product upon cellular differentiation.

Base Sequence↗
PubMed1994

1H MRS of high grade astrocytomas: mobile lipid accumulation in necrotic tissue.

Sixty-four samples from six grade 4 astrocytomas were investigated ex vivo by 1H MRS at 360 MHz and subsequently by histopathology to obtain percentages of viable and necrotic tumour and grey and white matter. MR-visible lipids were detected in 87% of tumour samples. Necrotic foci were < 3 x 3 x 6 mm3. The means of the intensities/unit weight tissue of the lipid resonances at 5.33, 2.80, 1.29 and 0.89 ppm were significantly higher (p < 0.05) for three sets of comparisons: samples with 85-100% vs 50-75%; with 50-75% vs 10-40% and with 10-40% vs 0-5% necrosis. For the lipid resonance at 2.04 ppm the difference in the means was significant only for samples with 50-75% compared to those with 85-100% necrosis, because for samples with < 50% necrosis resonances from glutamine and possibly small amounts of glutamate, gamma-aminobutyrate and N-acetylaspartate anions contribute significantly to the spectral area at 2.0 ppm. We conclude that necrotic foci below MRI resolution yield the resonances at 1.3 and 0.9 ppm, and contribute to the intense resonance at 2.0 ppm observed in in vivo 1H spectra of some high grade astrocytomas.

Astrocytoma↗
PubMed1994

Mobile lipids and metabolic heterogeneity of brain tumours as detectable by ex vivo 1H MR spectroscopy.

To examine metabolic heterogeneity in primary brain tumour, multiple biopsies (one to five per tumour) from four benign meningiomas, five acoustic schwannomas, eleven glial tumours and five medulloblastomas were each subdivided into one to five samples (total = 194) and investigated using ex vivo 360 MHz 1H MR spectroscopy with histopathological correlation. Low amounts of mobile lipids were detected in meningothelial meningiomas and medulloblastomas, high amounts in some or all samples of all other tumours. The differences in non-lipid metabolites between different tumour types were less marked, with the exception of the medulloblastomas, which had high amounts of N-acetyl aspartate and cholines. The amount of mobile lipid correlated with Antoni type B areas in schwannomas and necrosis in a high grade giant cell tumour. The amount of lipid varied significantly from sample to sample in a grade 2 astrocytoma and grade 4 astrocytoma samples with 0-5% necrosis but did not correlate with the site in the tumour or histopathological characteristics. The metabolic heterogeneity occurred on a spatial scale at least as small as the size of the MR samples, i.e., < or = 60 mg. These results suggest that the detection of metabolic heterogeneity and mobile lipids may have importance for the design of clinical studies.

Astrocytoma↗
PubMed1994

A linkage map of microsatellite markers on the human X chromosome.

The efficiency of mapping and diagnosis of X-linked disorders by linkage depends upon the existence of a high-density genetic map of polymerase chain reaction (PCR)-based markers. DXS1120, DXS1122, DXS1123, DXS1124, DXS1125, DXS1126, and DXS1153 were randomly isolated from a flow-sorted lambda bacteriophage library of the human X chromosome. The CCN (N = A or G) repeat within the androgen receptor was also found to be polymorphic and primers were designed for genotyping the CCN polymorphism in addition to the AGC polymorphism. The above markers, together with microsatellite polymorphisms at DXS237 (GMGX9), 5'DYS-II and 3'DYS MS (within the dystrophin locus), DXS538 (XL27B), PGK1P1, DXS300 (VK29AC), DXS294 (VK17AC), and DXS102 (cX38.1AC), were genotyped in the 40 CEPH reference families. One marker, DXS1153, was found to include cryptic alleles that amplify only in homozygotes and hemizygotes but not heterozygotes. A PCR-based linkage map was constructed using all of the above markers plus PCR-based markers from the CEPH database and those PCR-based markers previously typed in our laboratory: ALAS2, DXS292 (VK14AC), DXS297 (VK23AC), FRAXAC1, and FRAXAC2. The genetic map of the X chromosome incorporates 62 PCR-based marker loci, integrates the Weissenbach markers, and extends from XG near Xpter to DXS52 near Xqter, a distance of 236 cM.

Alleles↗