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C A Sargent

Publications and source records attributed to C A Sargent.

At least 19 recordsLinked to original sources
PubMed1992

Analysis of chromosome 21 copy number in uncultured amniocytes by fluorescence in situ hybridization using a cosmid contig.

A comparison of the use of chromosome 21-specific libraries, DOP-PCR 21 paints, yeast artificial chromosome (YAC) clones, single cosmids, and a 21q cosmid contig as probes for the detection of the copy number of chromosome 21 in interphase cells by fluorescence in situ hybridization shows that the cosmid contig is a satisfactory probe for interphase analysis of chromosome 21. The contig cCMP21.a, which is 55 kb in length, is highly chromosome 21-specific and produces intense, compact signals in a high proportion of interphase cells. A retrospective blind analysis of coded uncultured amniotic fluid samples correctly detected four trisomy 21 cases out of 49 samples.

Amniocentesis↗
PubMed1992

Quantitation of lysophosphatidylcholine molecular species in rat cardiac tissue.

We have developed a rapid and sensitive procedure for isolation and measurement of 1-acyllysophosphatidylcholine (LPC) species in rat myocardial tissue. Tissues were spiked with heptadecanoyl-LPC internal standard and extracted with chloroform/methanol. The chloroform phase was dried, resuspended in chloroform/propan-2-ol (2/1, v/v), and applied to an aminopropyl-bonded phase (Bond Elut) column. Following stepwise elution with several solvent mixtures, the LPC fraction (ethyl acetate/methanol, 4/6, v/v) was separated by HPLC with direct quantitation of palmitoyl-LPC (P-LPC), oleoyl-LPC (O-LPC), and stearoyl-LPC (S-LPC), using an evaporative light scattering mass detector. Calibration curves were generated for each individual LPC species. Recoveries of added [14C]LPC and of heptadecanoyl-LPC internal standard after extraction and chromatography were 85.8 +/- 1.9% (mean +/- SE, N = 10) and 83.4 +/- 1.8% (N = 15), respectively. This assay showed satisfactory sensitivity, reproducibility, and accuracy for measurement of LPC species in rat myocardial tissue. The major molecular species of LPC in rat myocardium were found to be P-LPC and S-LPC, which were two- to sixfold as abundant as O-LPC. In isolated, crystalloid-perfused rat hearts the time of perfusion was found to significantly influence the content of P-LPC (0 min, 252 +/- 10; 15 min, 178 +/- 10, P less than 0.001, compared with 0 min; 40 min, 131 +/- 4, P less than 0.001; and 70 min, 129 +/- 4, P less than 0.001; nmol/g dry weight), but not the content of O-LPC and S-LPC. The method will be useful for studying the participation of LPC species in physiology, pathophysiology, and therapeutics.

Animals↗
PubMed1992

Effect of calmodulin and protein kinase C inhibitors on globally ischemic rat hearts.

Several calmodulin inhibitors have been reported to be cardioprotective, but the ability of these compounds to inhibit protein kinase C (PKC) suggests that calmodulin inhibition may not be the sole mechanism responsible. To distinguish between the effects, we determined the cardioprotective activity of several calmodulin inhibitors with differing PKC inhibitory potencies in isolated globally ischemic rat hearts. Twenty-five minutes of global ischemia caused significant myocardial dysfunction, contracture formation, and lactate dehydrogenase (LDH) release on reperfusion in vehicle-treated hearts. The calmodulin inhibitors trifluoperazine, W-7, calmidazolium, W-13, and CGS 9343B improved postischemic contractile function and/or reduced LDH release. They also reduced preischemic cardiac function, although cardioprotection did not appear to be correlated with cardiodepression. Calmodulin inhibitors increased preischemic coronary flow (CF) and decreased heart rate (HR), but controlling these parameters did not affect the cardioprotection. Pretreatment of ischemic hearts with trifluoperazine was associated with preservation of myocardial ATP. Pretreatment of ischemic rat hearts with the PKC inhibitors staurosporine, calphostin C, polymyxin B, and H-7 did not result in cardioprotection. Thus, calmodulin inhibition causes cardioprotection that appears to be independent of PKC inhibition.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Effect of the potassium channel opener EMD 56431 on globally ischemic rat hearts.

The effect of the ATP-sensitive potassium channel opener EMD 56431 on coronary vasodilation and cardioprotection in isolated rat hearts was investigated. EMD 56431 caused a significant increase in pre-ischemic coronary flow. Time to contracture and reperfusion function were significantly increased at 3, 10 and 30 microM concentrations. LDH release was significantly reduced at 10 and 30 microM concentrations. 1 microM glyburide completely abolished the protective effects found with 10 microM EMD 56431. When given during reperfusion only, 10 microM EMD 56431 showed no cardioprotection. Thus, EMD 56431 appeared to reduce the severity of ischemia/reperfusion injury. The vasorelaxant versus cardioprotective effects for EMD 56431 are similar to other potassium channel openers, such as cromakalim.

Animals↗
PubMed1992

Effect of the phospholipase A2 inhibitors quinacrine and 7,7-dimethyleicosadienoic acid in isolated globally ischemic rat hearts.

Phospholipase A2 (PLA2) activity results in the formation of lysophospholipids and free fatty acids which may contribute to ischemic myocardial dysfunction. We evaluated the cardioprotective activity of two putative PLA2 inhibitors, quinacrine and 7,7-dimethyleicosadienoic acid (DEDA), in isolated globally ischemic rat hearts. Pretreatment with 1, 5 and 50 microM quinacrine before ischemia did not alter coronary flow but did cause significant cardiodepression. Twenty five minutes of global ischemia and 30 min of reperfusion caused severe myocardial dysfunction and lactate dehydrogenase release. Quinacrine significantly improved reperfusion contractile function and reduced lactate dehydrogenase release, indicative of cardioprotection. In contrast, 30 to 100 microM DEDA produced neither preischemic cardiodepression nor cardioprotective activity. PLA2 inhibition was inferred from measurements of the prostacyclin metabolite, 6-keto-prostaglandin F1 alpha in the coronary effluent and myocardial palmitoyl-lysophosphatidylcholine. Quinacrine and DEDA reduced both 6-keto-prostaglandin F1 alpha and palmitoyl-lysophosphatidylcholine by similar degrees. These results suggest that the cardioprotective activity of quinacrine is independent of PLA2 inhibition. A possible role of calcium inhibition was investigated in rat aortic smooth muscle strips. Norepinephrine-, KCl- and BAY K8644-induced contractions were antagonized in the presence of 5 and 50 microM quinacrine, but were unaffected by 30 to 60 microM DEDA. The ability of quinacrine to inhibit calcium was investigated further in cardiac ventricular myocytes. Measurement of mean whole cell calcium currents showed that quinacrine (5 microM) could inhibit this current up to 70%. Thus, these results suggest that quinacrine-induced cardioprotection may not be due to PLA2 inhibition, but may be related to calcium entry blocking activity.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

An X-linked zinc finger gene mapping to Xq21.1-q21.3 closely related to ZFX and ZFY: possible origins from a common ancestral gene.

We describe a new zinc finger gene sequence (CMPX1 or HGM symbol ZNF6; isolated by cross-hybridization of ZFY to clones in a testis cDNA library) which possesses a zinc finger domain closely related to the transcriptional activator gene ZFX. The putative acidic activation domain is only 11.5% homologous with ZFX, whereas the putative DNA binding domain shares 75% homology and shows the same organisation composed of a basic two fingered repeat unit. ZNF6 has an unusually large 5' untranslated region (UTR) of 1.2 Kb which contains 26 potential ATG initiation codons, only one of which is associated with a long open reading frame. Southern and Northern blot analysis has shown that this 5' UTR is shared with many other sequences in the genome and transcribed associated with a large range of mRNA species. In situ hybridisation, analysis of somatic cell hybrids and male individuals carrying deleted X chromosomes have mapped the gene to Xq21.1-q21.3. The gene is highly conserved amongst the primates, in the mouse and can be detected weakly in the genome of a metatherian mammal (possum). Dosage in male and female mice indicates that it is also X-linked in this species. Possible origins of ZFX, ZFY and CMPX1 from a common ancestral gene are discussed.

Amino Acid Sequence↗
PubMed1991

Effect of potassium channel blockade on the anti-ischemic actions of mechanistically diverse agents.

The ATP-sensitive potassium channel opener, cromakalim, protects ischemic hearts and its effect can be reversed by glyburide. It is presently unknown if glyburide can abolish the anti-ischemic effects of mechanistically different agents or if blockers of other potassium channels can abolish the protective effects of cromakalim. Thus, the effect of glyburide on previously reported cardioprotective agents was tested in globally ischemic/reperfused isolated rat hearts. Calcium antagonists, sodium channel blockers and calmodulin antagonists were found to significantly improve postischemic contractile function and reduce lactate-dehydrogenase release after 25 min of global ischemia and 30 min of reperfusion. Glyburide did not reverse their cardioprotective effects. 5-(N,N-dimethyl)amiloride, an inhibitor of Na+/H+ exchange, significantly reduced lactatedehydrogenase release without improving postischemic contractile function, and glyburide did not reverse this. The potassium channel opener, cromakalim, protected ischemic rat hearts (improved recovery of contractile function and reduced enzyme release) and this was abolished by glyburide. Charybdotoxin blocks both calcium-activated potassium channels and voltage-gated potassium channels and E-4031 the delayed rectifier potassium channels. Neither was found to effect the action of the potassium channel opener, cromakalim. These data indicate that glyburide is selective in that it only blocks the anti-ischemic effects of potassium channel openers and not other cardioprotective compounds. In addition, cromakalim is unaffected by blockers of other potassium channels, further indicating selectivity of glyburide for ATP-sensitive potassium channels.

Animals↗
PubMed1990

Human major histocompatibility complex contains a new cluster of genes between the HLA-D and complement C4 loci.

A new cluster of genes has been defined in the human Major Histocompatibility Complex class III region. The seven novel genes, G12 to G18, are localised in a 160 kb segment of DNA extending from the complement gene cluster towards HLA-DR. The genes were identified by isolation of cDNA clones using cosmid genomic inserts as hybridisation probes, and by the detection of the corresponding transcripts in Northern blot analysis. Characterisation of the cosmid genomic DNA inserts, in conjunction with pulsed field gel electrophoresis analysis of uncloned DNA, for the presence of clustered sites for infrequently cutting restriction endonucleases has revealed that at least 5 of the 7 genes are associated with HTF-islands. These unmethylated CpG-rich sequences are frequently found at the 5' ends of ubiquitously expressed genes. Together with previously published data 36 genes have now been defined in a 680kb stretch of DNA within the MHC. With one gene approximately every 20kb of DNA this represents the most densely packed region of the human genome so far characterised, and is of major significance in relation to the mapping and sequence analysis of the rest of the genome.

Blotting, Northern↗
PubMed1990

Characterization of the class III region in different MHC haplotypes by pulsed-field gel electrophoresis.

The class III region of the human major histocompatibility complex (MHC) in seven HLA haplotypes has been analyzed using pulsed-field gel electrophoresis (PFGE), restriction enzymes that cut genomic DNA infrequently, and Southern blotting. In particular, extensive mapping with the enzyme Bss HII, which generates fragments in the size range 8-280 kilobases (kb), has revealed that in the haplotypes studied the DNA content of the class III region does not appear to vary other than as previously observed at the C4 and CYP21 loci.

Blotting, Southern↗
PubMed1990

Polymorphism of the human complement component C4.

The genes encoding the two C4 isotypes, C4A and C4B, lie 10 kb apart in the class III region of the human major histocompatibility complex. The two isotypes exhibit extensive structural polymorphism. Characterisation of a number of C4A and C4B alleles has established the pattern of polymorphism in C4 and this has provided a structural basis for the observed functional and serological differences between the C4 isotypes. An intriguing feature in the genetics of C4 is the unusually high frequency of null alleles forming half null C4A and C4B haplotypes. Duplication of one of the loci has also been recognised. In addition the genes can differ in size due to the presence or absence of a large intron near the 5' end of the genes. These differences in gene size and gene number can be observed directly on different haplotypes using pulsed field gel electrophoresis.

Alleles↗
PubMed1989

Direct observation of the gene organization of the complement C4 and 21-hydroxylase loci by pulsed field gel electrophoresis.

Pulsed field gel electrophoresis and enzymes that cut genomic DNA infrequently have been used to define large RFLPs at the human C4 loci. With the enzymes BssH II or Sac II, and C4 or 21-hydroxylase DNA probes, it has been possible to observe directly the number of C4 genes present on a haplotype, and also whether the C4 genes are long (6-7-kb intron present) or short (6-7-kb intron absent). Haplotypes that have either two long C4 genes or one long and one short C4 gene generate BssH II fragments of approximately 115 or approximately 105 kb, respectively. Haplotypes that have either a single long or a single short C4 gene generate BssH II fragments of approximately 80 or approximately 70 kb, respectively. This technique has been used to analyze the DNA isolated from PBMC and allows the complete definition of the C4 gene organization of an individual without the need for family studies.

Cell Line↗
PubMed1989

Identification of multiple HTF-island associated genes in the human major histocompatibility complex class III region.

Chromosome walking in the major histocompatibility complex (MHC) class III region has resulted in the isolation of 541 kb of genomic DNA in two sets of overlapping cosmid clones. These two sets encompass the 340 kb separating the C2 and tumour necrosis factor (TNF) alpha and beta genes, except for a 22 kb gap 108 kb centromeric to the TNF alpha gene. The genomic DNA inserts have been characterized for the presence of clusters of restriction sites with CpG dinucleotides in their recognition sequence. In conjunction with pulsed field gel electrophoresis the exact sites which cleave in chromosomal DNA have been established and this has suggested the presence of a number of HTF-islands. Genomic probes flanking the HTF-islands have been hybridized to Northern blots of RNA from a number of cell lines. Transcripts ranging in size from 0.6 to 6 kb corresponding to the products of 12 novel, single copy genes have been identified. In addition the human equivalent of the murine B144 gene was mapped approximately 10 kb centromeric of the TNF alpha gene. The location of so many new genes in this region raises the question as to whether they play any role in the observed HLA associations with an individual's susceptibility to develop autoimmune disease.

Base Sequence↗
PubMed1989

An analysis of variation in the long-range genomic organization of the human major histocompatibility complex class II region by pulsed-field gel electrophoresis.

The class II region of the human major histocompatibility complex in seven common HLA haplotypes has been analyzed using pulsed-field gel electrophoresis, restriction enzymes that cut genomic DNA infrequently, and Southern blotting. This analysis has revealed that there are differences in the amount of DNA present in the DQ and DR subregions dependent on the haplotype. The class II region of the DR3 haplotype spans approximately 750 kb and has the same amount of DNA as the class II region of the DR5 and DR6 haplotypes. However, the DR2 haplotype has approximately 30 kb more DNA within the DR subregion. The DR4 haplotype has an additional approximately 110 kb of DNA within the DQ or DR subregions compared to the DR3, DR5, and DR6 haplotypes. These haplotype-specific differences could have some bearing both on the analysis of disease susceptibility and on the ability of chromosomes possessing different HLA haplotypes to recombine within the DQ/DR subregions.

Blotting, Southern↗
PubMed1989

Human major histocompatibility complex contains genes for the major heat shock protein HSP70.

Little is known as to why a large number of human diseases are influenced by the major histocompatibility complex. In some cases, a direct involvement of the products of the polymorphic class I and class II, aas well as the less variable products of the class III, genes has been proposed. During characterization of the class III region for the presence of additional loci, we have located a duplicated locus encoding the major heat shock protein HSP70 between the complement and tumor necrosis factor genes. The HSP70 loci are 12 kilobases apart and lie 92 kilobases telomeric of the C2 gene. As HSP70 proteins have been linked with a protective role during and after cellular stress, and HSP70 analogues are often presented as antigens in bacterial and protozoal infections, this finding may have major implications with regard to the major histo-compatibility complex and associated diseases.

Amino Acid Sequence↗
PubMed1989

Dietary fish oil and ischaemic arrhythmias.

Ventricular fibrillation (VF), a serious arrhythmia, is an important cause of death from ischaemic heart disease. There is substantial evidence that experimental diets with a high polyunsaturated (linoleic acid) to saturated fatty acid ratio offer protection against VF. In this article the effects of large amounts of dietary fish oil (up to 30% cal) are reviewed. It is concluded that in all recently published studies marked increases in the incorporation of n-3 fatty acids in membranes was achieved. Two studies were too small, however, to demonstrate any statistically significant difference in VF. The results from a third, a long term feeding study, show a reduction in VF when compared with a highly saturated fat diet, but not when compared to a polyunsaturated fatty acid (n-6) rich diet. Using a more realistic fish oil supplement (0.4% cal) for 8 weeks, we observed a small but nonsignificant reduction in the incidence of VF during acute myocardial ischaemia in the isolated perfused rat heart. The need for further work on the effect of dose, dietary period and confounding influences of saturated fatty acids and linoleic acid are discussed.

Animals↗
PubMed1988

Molecular mapping of the HLA-linked complement genes and the RCA linkage group.

Phenotypic genetics have established linkage of the genes encoding proteins involved in the activation of the complement component C3. C2, factor B and C4, three of the structural components of the classical and alternative pathway C3 convertases, are encoded by genes which have been mapped to the class III region of the major histocompatibility complex (MHC) on human chromosome 6. The regulatory proteins factor H, C4BP, CR1, CR2 and DAF, which are involved in the control of C3 convertase activity, are encoded by closely linked genes, termed the regulators of complement activation (RCA) linkage group, that have been mapped to human chromosome 1. cDNA clones for all these proteins have been isolated, and this has made it possible to investigate the organization and structure of the MHC class III genes and the genes in the RCA linkage group. This short review summarizes some of the main features which have emerged from recent cloning work.

Animals↗