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Biomedical subjects

D E Humphries

Publications and source records attributed to D E Humphries.

At least 19 recordsLinked to original sources
PubMed1999

Synthesis of 8-epi-prostaglandin F2alpha by human endothelial cells: role of prostaglandin H2 synthase.

The experiments described in this paper were designed to determine the mechanism underlying the increase in 8-isoprostaglandin F(2alpha) (8-epi-PGF(2alpha)) production by cultured human endothelial cells during reoxygenation following hypoxia. Human umbilical artery endothelial cells were grown on microcarrier beads and exposed to sequential periods of normoxia, hypoxia, and reoxygenation. The amount of 8-epi-PGF(2alpha) in the medium was determined by ELISA. The production of 8-epi-PGF(2alpha) decreased by greater than 90% during hypoxia. Upon reoxygenation 8-epi-PGF(2alpha) production increased linearly for 90 min reaching nearly 3 times normoxic levels. When added to the medium during reoxygenation, neither superoxide dismutase nor Tiron, a cell-permeable superoxide scavenger, inhibited 8-epi-PGF(2alpha) production. However, 8-epi-PGF(2alpha) production was inhibited by catalase. The production of 8-epi-PGF(2alpha) was also inhibited by indomethacin and aspirin. Exogenous hydrogen peroxide stimulated 8-epi-PGF(2alpha) production by normoxic cells, and aspirin inhibited the hydrogen peroxide-mediated increase in 8-epi-PGF(2alpha) production. These results indicate that the reactive oxygen species responsible for 8-epi-PGF(2alpha) synthesis during reoxygenation is hydrogen peroxide and that in endothelial cells 8-epi-PGF(2alpha) synthesis is mediated by prostaglandin H(2) synthase (PGHS). To verify the role of PGHS in 8-epi-PGF(2alpha) synthesis, human PGHS-1 was expressed in COS-7 cells, a PGHS negative cell line that does not synthesize 8-epi-PGF(2alpha). In the presence of exogenous arachidonic acid the COS-7 cells expressing human PGHS-1 produced substantial amounts of PGE(2) and 8-epi-PGF(2alpha). These data indicate that human PGHS-1 can support the synthesis of 8-epi-PGF(2alpha) and that 8-epi-PGF(2alpha) synthesis by cultured human endothelial cells during reoxygenation is dependent on the activity of PGHS-1.

Animals↗
PubMed1999

Heparin is essential for the storage of specific granule proteases in mast cells.

All mammals produce heparin, a negatively charged glycosaminoglycan that is a major constituent of the secretory granules of mast cells which are found in the peritoneal cavity and most connective tissues. Although heparin is one of the most studied molecules in the body, its physiological function has yet to be determined. Here we describe transgenic mice, generated by disrupting the N-deacetylase/N-sulphotransferase-2 gene, that cannot express fully sulphated heparin. The mast cells in the skeletal muscle that normally contain heparin lacked metachromatic granules and failed to store appreciable amounts of mouse mast-cell protease (mMCP)-4, mMCP-5 and carboxypeptidase A (mMC-CPA), even though they contained substantial amounts of mMCP-7. We developed mast cells from the bone marrow of the transgenic mice. Although these cultured cells contained high levels of various protease transcripts and had substantial amounts of mMCP-6 protein in their granules, they also failed to express mMCP-5 and mMC-CPA. Our data show that heparin controls, through a post-translational mechanism, the levels of specific cassettes of positively charged proteases inside mast cells.

3T3 Cells↗
PubMed1998

cDNA cloning, genomic organization and chromosomal localization of human heparan glucosaminyl N-deacetylase/N-sulphotransferase-2.

The cDNA and gene encoding human heparan glucosaminyl N-deacetylase/N-sulphotransferase-2 have been cloned. The cDNA encoded a protein of 883 amino acids that was 94% similar to heparan N-sulphotransferase-2 from mouse mast cells. Comparison of the deduced amino acid sequences of human heparan N-sulphotransferase-1 and -2 showed that the enzymes were 70% similar; greater than 90% of the amino acids between residues 418 and 543 were identical. The least conserved amino acids were found in the N-terminus/putative transmembrane regions of the two enzymes. The human heparan N-sulphotransferase-2 gene was localized to chromosome arm 10q (band 10q22) by in situ fluorescent hybridization. The gene contains 13 exons spanning 6.5 kb, ranging in size from 88 bp (exon 2) to >1 kb (exon 1), and 12 introns, which were found to occur at similar sites within the coding sequence of the human heparan N-sulphotransferase-1 gene. The structure of the two genes differed in that the heparan N-sulphotransferase-1 gene contained one additional intron. The similarity of the heparan N-sulphotransferase-1 and -2 proteins and their similar exon-intron organization suggest that they derive from a common ancestral gene.

Amidohydrolases↗
PubMed1998

Hypothesis: link between endogenous retroviruses and cardiovascular disease.

A two-order-magnitude increase in the expression of an endogenous retrovirus was found in myocytes from spontaneously hypertensive rats (SHR) relative to Wistar-Kyoto rats (WKY). It has now been demonstrated that the increase in proviral transcripts is not secondary to the effects of pressure overload, as WKY hearts with hypertrophy due to aortic banding do not express the retrovirus. Crossing SHR with WKY results in intermediate levels of blood pressure, hypertrophy and retroviral gene expression, suggesting co-segregation of genotype and phenotype. Findings suggest a possible linkage between an endogenous retrovirus and genetic hypertension in the SHR. A brief overview of endogenous retroviruses is presented and a discussion of their potential for inducing cardiovascular pathology is considered.

Animals↗
PubMed1997

Localization of human heparan glucosaminyl N-deacetylase/N-sulphotransferase to the trans-Golgi network.

In order to determine the intracellular location of heparan N-deacetylase/N-sulphotransferase, cDNAs encoding human heparan glucosaminyl N-deacetylase/N-sulphotransferase were cloned from human umbilical vein endothelial cells. The deduced amino acid sequence was identical to that of the human heparan N-sulphotransferase cloned previously [Dixon, Loftus, Gladwin, Scambler, Wasmuth and Dixon (1995) Genomics 26, 239-244]. RNA blot analysis indicated that two heparan N-sulphotransferase transcripts of approx. 8.5 and 4 kb were produced in all tissues. Expression was most abundant in heart, liver and pancreas. A cDNA encoding a Flag-tagged human heparan N-sulphotransferase (where Flag is an epitope with the sequence DYKDDDDK) was transfected into mouse LTA cells. Immunofluorescence detection using anti-Flag monoclonal antibodies demonstrated that the enzyme was localized to the trans-Golgi network. A truncated Flag-tagged heparan N-sulphotransferase was also retained in the Golgi, indicating that, as for many other Golgi enzymes, the N-terminal region of heparan N-sulphotransferase is sufficient for retention in the Golgi apparatus.

Amidohydrolases↗
PubMed1997

Glucocorticoids inhibit the cytokine-induced proliferation of mast cells, the high affinity IgE receptor-mediated expression of TNF-alpha, and the IL-10-induced expression of chymases.

Mast cells are a heterogeneous family of immune cells that, when activated through their high affinity IgE receptors (Fc epsilonRI), release various granule mediators (e.g., neutral proteases and serglycin proteoglycans) and proinflammatory cytokines (e.g., IL-6 and TNF-alpha). We and others have shown that the growth and differentiation of immature, nontransformed mouse bone marrow-derived mast cells (mBMMC) can be regulated in vitro by IL-3, IL-10, and c-kit ligand. We now report that glucocorticoids inhibit the c-kit ligand- and IL-3-induced proliferation of mBMMC, the Fc epsilonRI-mediated expression of TNF-alpha, and the IL-10-mediated expression of the two chymases designated mouse mast cell protease (mMCP)-1 and mMCP-2. In contrast, glucocorticoids induce mBMMC to increase their expression of serglycin proteoglycan and carboxypeptidase A. As assessed by nuclear run-on and RNA blot analyses, dexamethasone inhibited the IL-10-mediated expression of mMCP-1 and mMCP-2, primarily by inducing rapid degradation of their transcripts. The stimulative effect on serglycin proteoglycan expression and the inhibitory effect on chymase expression were dose and time dependent and glucocorticoid specific. These findings indicate that glucocorticoids exert profound and diverse effects on the growth, cytokine expression, and granule differentiation of mouse mast cells, and that at least some of this regulation occurs through a post-transcriptional mechanism.

Animals↗
PubMed1997

Localization of alpha1(I) collagen mRNA in myocardium from the spontaneously hypertensive rat during the transition from compensated hypertrophy to failure.

Spontaneously hypertensive rats (SHR) commonly develop impairment of myocardial function between ages 18-24 months. Isolated muscle studies demonstrate depressed myocardial contractility and increased passive stiffness. Studies of the extracellular matrix in SHR with failure (SHR-F) demonstrate an increased expression of genes encoding extracellular matrix components (ECM), hydroxyproline concentration and fibrosis relative to age-matched non-failing animals. In the present study, tissue sections of hearts from SHR-F, non-failing SHR (SHR-NF) and non-hypertensive Wistar Kyoto rats (WKY) were hybridized with a cDNA probe for alpha1(I) collagen mRNA, which was found by Northern blot analysis to be elevated in SHR-F relative to hearts from control animals. In situ hybridization studies demonstrate increased perivascular and interstitial collagen alpha1(I) gene expression in myocardium from the SHR relative to WKY. In addition, failing hearts from the SHR demonstrate focal alpha1(I) collagen mRNA accumulation in the endocardium and at sites of degenerating single myocardial cells.

Animals↗
PubMed1997

Endogenous retroviral transcripts in myocytes from spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) is a well studied animal model of genetic hypertension and heart disease of unknown cause. With the use of differential display, a transcript was found in SHR myocardium that on sequence analysis was identified as an endogenous retrovirus (ERV). ERV gene expression was greater than an order of magnitude increased in adult SHR hearts relative to age-matched normotensive Wistar-Kyoto rats and was further increased in hearts from SHR with heart failure. In situ hybridization studies demonstrated that increased ERV gene expression was localized to myocardial cells. Increases in ERV transcripts in SHR suggest a possible link between inherited proviral elements and genetic hypertensive heart disease.

Age Factors↗
PubMed1996

Structure and expression of the promoter for the R4/ALK5 human type I transforming growth factor-beta receptor: regulation by TGF-beta.

The type I transforming growth factor-beta (TGF-beta) receptors are serine/threonine kinases that are essential for the action of TGF-beta. In this paper, we describe the molecular cloning and expression of the R4/ALK5 human type I TGF-beta receptor promoter. DNA sequence analysis indicates that the promoter lacks a TATA and CAAT box but is highly GC-rich and contains putative Sp1 binding sites. The transcriptional start site is approx. 232 base pairs upstream of the AUG start codon. In human lung fibroblasts, TGF-beta induced a 3-fold increase in steady-state level for type I receptor mRNA. Exposure of cells transfected with a 618 bp promoter fragment to TGF-beta 1 up-regulated transcriptional activity indicating that a TGF-beta response element is contained within this region.

Activin Receptors, Type I↗
PubMed1996

Enhanced galactosyltransferase expression in the failing hearts of spontaneously hypertensive rats.

To examine differences in cardiac gene expression between spontaneously hypertensive rats with and without heart failure, we have used subtractive hybridization to identify differentially expressed genes. After subtraction, cDNAs were amplified by PCR, cloned and sequenced. One of 36 independent cDNAs was found to be 86% homologous to murine UDP-galactose:N-acetylglucosamine beta-1,4-galactosyltransferase. RNA blot analysis confirmed the approximately 4.0 kb rat galactosyltransferase transcript was increased in failing hearts relative to non-failing hearts. Biochemical assay also showed increased galactosyltransferase activity in the failing hearts.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of the human ryudocan promoter.

The promoter region of human ryudocan was isolated from a human lambda dash genomic library and cloned into pBluescript. The 5'flanking region contained a classical TATA box and GC rich regions that are commonly found in constitutively expressed genes. Two fusion gene constructs, one of 898 bp and the other of 480 bp of the 5' flanking DNA coupled to the luciferase gene, were transiently expressed in a mouse endothelioma cell line and in human umbilical endothelial cell cultures. Both constructs were capable of driving luciferase expression; the 898 bp construct produced greater levels of luciferase activity than the 480 bp construct in both cell types examined. Analysis of the sequence revealed the presence of several potential sites for nuclear transcription factor binding; the relevance of these sites is presently unknown.

Base Sequence↗
PubMed1994

Structure and expression of the promoter for the human type II transforming growth factor-beta receptor.

The type II TGF-beta receptor is a serine/threonine kinase whose expression is essential for the action of TGF-beta. In this paper, we describe the cloning and expression of the human type II TGF-beta promoter. DNA sequence analysis indicates that the region near the transcription initiation site lacks a TATA and CAAT box but contains Sp1 binding sites. A similar promoter type has been identified in the 5' flanking regions of genes that code for certain other growth factor receptors. Transfection of the promoter (888 bp fragment) mediated transcriptional activity in bovine vascular smooth muscle cells and human lung fibroblasts.

Animals↗
PubMed1992

The human serglycin gene. Nucleotide sequence and methylation pattern in human promyelocytic leukemia HL-60 cells and T-lymphoblast Molt-4 cells.

The complete nucleotide sequence of the 16.7-kb human gene that encodes the peptide core (serglycin) of a secretory granule proteoglycan was determined, thus representing the first proteoglycan peptide core gene to be sequenced in its entirety. The exons, intron 1, and intron 2 comprised 7, 53, and 40% of the gene, respectively. Nineteen Alu-repetitive DNA sequences were interspersed in the gene, accounting for 28% of the total nucleotides in intron 1 and 40% of the nucleotides in intron 2. The nucleotide sequence was then used in an examination of the methylation pattern of the human serglycin gene in human promyelocytic leukemia HL-60 cells that contain serglycin mRNA and in T-lymphoblast Molt-4 cells that do not. With polymerase chain reaction methodology, 13 DNA probes of 250-880 base pairs in length were generated that corresponded to unique, non-Alu sequences spaced throughout the entire human serglycin gene. When blots containing genomic DNA digested with HpaII or MspI were examined with these genomic probes, it was discovered that the 5'-flanking region and intron 1 of the serglycin gene in HL-60 cells were both substantially less methylated than intron 2. In contrast, the entire serglycin gene in Molt-4 cells was highly methylated. Because hypomethylated genes generally are transcribed more efficiently than hypermethylated genes, the high level of serglycin mRNA in HL-60 cells probably is a consequence of the low level of methylation of intron 1 and the 5'-flanking region of the serglycin gene in these cells.

Base Sequence↗
PubMed1991

Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals.

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of [3H]chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.

Aggrecans↗
PubMed1990

Glycosaminoglycans of bovine aorta endothelial cells: identification and localization by use of a platelet factor 4-fluorescein probe.

We utilized platelet factor 4 (PF4) conjugated to fluorescein to stain the proteoglycans of permeabilized fixed bovine aorta endothelial cells in monolayer culture. Treatment of the monolayers with chondroitin ABC lyase and/or a preparation from Flavobacterium heparinum was used to remove chondroitin sulfate and/or heparan sulfate before staining, with resultant separate identification and partial localization of these glycosaminoglycans. When PF4-fluorescein was utilized with untreated control monolayers, fairly uniform reticular, perinuclear, and cell surface fluorescence was seen. After treatment with chondroitin ABC lyase, fluorescence was retained only on the cell surface. In contrast, treatment with the F. heparinum preparation resulted in the loss of all cell surface fluorescence. Use of both glycosaminoglycan lyases together resulted in loss of essentially all the fluorescence. The cell surface heparan sulfate observed by fluorescence after removal of cell surface chondroitin sulfate appeared to be unevenly distributed, with a heavier accumulation at one pole of each cell. This technique offers a specific method for identification and partial localization of cell surface heparan sulfate.

Animals↗
PubMed1989

Production of [3H]hexosamine-labeled proteoglycans by cultures of normal and diabetic skin fibroblasts: dilution of exogenous [3H]glucosamine by endogenous hexosamine from glucose and other sources.

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine and [35S]-sulfate for varying periods of time. Incorporation of 3H into macromolecules appearing in the medium was linear after approximately 45 min, and incorporation of 35S was linear after approximately 30 min. The amounts of 35S-proteoglycan formed by each of the cultures during 5-h incubations were compared and were found to be fairly similar for the six lines, varying from 0.08 to 0.14 nmol sulfate/microgram DNA. Isolated 3H,35S-glycosaminoglycans were then treated with chondroitin ABC lyase to characterize the location and degree of sulfation. Results indicated a considerable variation in completeness of chondroitin/dermatan sulfation and in proportions of 6-sulfation to 4-sulfation among the various lines. However these variations did not seem to be related to whether the cells were from normals or diabetics. 3H,35S-Labeled disaccharides were isolated and ratios of 3H to 35S determined in order to calculate the [3H]glucosamine dilution by endogenous glucosamine derived from glucose or other sources during the period of incubation. Dilutions varied widely from 160- to 635-fold among the different cell lines, but the variations did not seem to be related to whether the cells were from normals or diabetics.

Adult↗