Biomedical subjects

C D Boyd

Publications and source records attributed to C D Boyd.

At least 73 records · Page 4Linked to original sources

PubMed1991

Changes in aortic levels of tropoelastin mRNA following treatment of rats with the antihypertensive drugs captopril and hydralazine.

This manuscript describes changes in the steady state levels of aortic tropoelastin mRNA in spontaneously hypertensive rats (SHR) and normotensive controls (WKY) following treatment with two antihypertensive drugs. Three-week-old WKY and SHR rats were treated with hydralazine (15 mg/kg/day) or captopril (25 mg/kg/day). Tail artery blood pressure was monitored twice weekly. Both drugs prevented the development of hypertension in the SHR rat. At 6 weeks of age, total aortic RNA was extracted and the steady state levels of mRNAs coding for tropoelastin and pro alpha 1 (III) collagen were determined by slot blot hybridization analysis using radiolabeled tropoelastin and pro alpha 1 (III) collagen cDNA clones. Hydralazine treatment resulted in a threefold increase in tropoelastin mRNA levels in both the SHR and the WKY animals (P less than 0.01). Captopril-treated SHR animals demonstrated a similar significant increase. In contrast, no differences in pro alpha 1 (III) collagen mRNA levels were observed in the aorta of SHR or WKY rats following treatment with either captopril or hydralazine. These data suggest that antihypertensive agents can act specifically to directly induce tropoelastin mRNA levels in large arteries and thus may induce vascular remodeling independent of an increase in blood pressure.

Aging↗

PubMed1991

Alternative splicing of rat tropoelastin mRNA is tissue-specific and developmentally regulated.

Sequence analysis of cDNA clones coding for rat tropoelastin previously has identified two variants that potentially corresponded to alternatively spliced tropoelastin mRNAs (Pierce et al., 1990). We have now used S1 nuclease protection analysis of total RNA from aorta, skin and lungs of 10-day and 6-week old rats to localize all sites of alternative splicing in the tropoelastin mRNA and to examine tissue-specific and developmental regulation of the use of these sites. This analysis revealed multiple sites of alternative splicing involving rat tropoelastin coding sequences corresponding to exons 12 through 15 of the bovine tropoelastin gene and a single site of alternative splicing at sequences corresponding to exon 33. Messenger RNAs from all three tissues at both developmental stages were alternatively spliced at the same sites; there was no evidence for the use of an alternative splice site unique to a particular tissue or developmental stage. However, both tissue-specific and developmentally regulated differences were apparent in the proportion of rat tropoelastin mRNA alternatively spliced at exon 33. Tropoelastin mRNA from the aorta and lungs of neonatal rats was alternatively spliced at exon 33 ten time more frequently than tropoelastin mRNA from skin. Between 10 days and 6 weeks of development, the use of this site of alternative splicing decreased by twenty-fold in RNA from skin, ten-fold in RNA from lungs and two-fold in RNA from aorta. In contrast, alternative splicing at exons 12 through 15 occurred in a small percentage of the mRNA and use of these sites exhibited minimal tissue-specific differences or developmental regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

PubMed1991

Mammalian tropoelastin: multiple domains of the protein define an evolutionarily divergent amino acid sequence.

We have recently derived the complete amino acid sequence of rat tropoelastin from a series of overlapping cDNA clones. Comparison of this protein sequence to bovine and human tropoelastin has revealed significant differences in the rates of evolutionary divergence of the various domains of tropoelastin. The overall rate of divergence of the hydrophobic domains of tropoelastin was twice as fast as the cross-link domains of the protein. Certain hydrophobic domains, however, are as conserved as cross-link regions, particularly the hydrophobic sequence coded for by exon 33, the only exon subject to alternate usage in all three mammalian species and the most conserved domain in rat, bovine and human tropoelastin. This conservation of sequence strongly suggests a more complex function of the hydrophobic region encoded by exon 33, beyond the elastic recoil characteristic of all hydrophobic domains of tropoelastin. A comparison of average rates of divergence of hydrophobic and cross-link domains of tropoelastin to functionally-defined domains of other structural proteins, such as collagen, has also revealed that overall, tropoelastin is a highly divergent amino acid sequence, comparable to proteins such as globin and the fibrino-peptides.

Amino Acid Sequence↗

PubMed1991

Desmoplasia in benign and malignant breast disease is characterized by alterations in level of mRNAs coding for types I and III procollagen.

Desmoplasia, the formation of excessive connective tissue surrounding some neoplasms, is a well documented, but incompletely understood phenomenon. To characterize the fibrotic response in benign breast conditions and malignancy, we examined the steady state levels of mRNA coding for types I and III procollagen in mild fibrocystic changes, in fibroadenoma, and in infiltrating ductal carcinoma. The results indicate that in mild fibrocystic change there is a relative increase in type III procollagen mRNA. In contrast, fibroadenoma and carcinoma are characterized by increased levels of type I procollagen mRNA.

Adult↗

PubMed1991

A restriction fragment length polymorphism in the pigeon pro alpha 2(1) collagen gene: lack of an allelic association with an atherogenic phenotype in pigeons genetically susceptible to the development of spontaneous atherosclerosis.

A high frequency restriction fragment length polymorphism (RFLP) at the 3'-end of the pigeon pro alpha 2(1) collagen gene was detected using the restriction endonuclease EcoR1. The distribution of this allelic variant was analyzed in DNA isolated from White Carneau pigeons genetically susceptible to the development of spontaneous atherosclerosis. The atherogenic phenotype in individual pigeons was measured by the determination of total cholesterol and cholesterol ester levels in the celiac focus of the thoracic aorta of adult White Carneau pigeons. Aortic wall cholesterol levels correlated with an increase in lesion size. No correlation, however, was observed between allelic variants of the pigeon pro alpha 2(1) collagen gene and the atherogenic phenotype in White Carneau pigeons suggesting lack of linkage between this allelic marker and the genetic susceptibility to spontaneous atherogenesis. This is the first study of its kind in this animal model and serves to provide a basis for the further analysis of co-segregation of RFLPS in candidate genes to this polygenic phenotype.

Animals↗

PubMed1991

Glomerular basement membrane expansion in passive Heymann nephritis. Absence of increased synthesis of type IV collagen, laminin, or fibronectin.

The distribution and synthetic rate of glomerular basement membrane components was examined in the Passive Heymann Nephritis model of experimental membranous nephropathy. The extensive tissue injury that developed included subepithelial electron-dense deposits, podocyte foot process effacement, and expansion of the glomerular basement membrane. Levels of mRNA for type IV collagen, laminin, and fibronectin from isolated glomeruli was quantitated by slot-blot analysis and showed no change in experimental animals as compared to controls at either 1 week, 3 weeks, or 3 months after disease induction. Immunoelectron microscopy with gold-labeled anti-laminin IgG revealed no difference in the number of particles bound to the glomerular basement membrane of experimental animals and controls. Immunofluorescence with both type IV collagen antisera and anti-laminin antibody showed no difference in the intensity or pattern of staining. Despite extensive glomerular damage and glomerular basement membrane thickening, no evidence was found for either an increase in the synthetic rate of type IV collagen, laminin, or fibronectin or for an accumulation of basement membrane laminin within the damaged glomeruli. Alternate processes, such as diminished density of matrix components or accumulation of other unmeasured matrix constituents, presumably account for the expansion of the glomerular basement membrane seen in experimental membranous nephropathy.

Animals↗

PubMed1990

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning.

A lambda gt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin cDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA.

Amino Acid Sequence↗

PubMed1990

Changes in vascular extracellular matrix accumulation reflect phenotypic differences between the arterial wall of pigeons resistant and susceptible to the development of spontaneous atherosclerosis.

White Carneau pigeons have previously been shown to be genetically susceptible to the development of spontaneous atherogenesis. The severity of development of atheromatous lesions is considerably greater than a more resistant breed of Show Racer pigeons. Analysis of levels of total hydroxyproline and isodesmosine in the thoracic aorta and celiac bifurcation of prelesion, six-week-old White Carneau and Show Racer pigeons, revealed an increased accumulation of total collagen and cross-linked elastin in the White Carneau arterial tissue. Using dot blot hybridization, measurements of steady state levels of several mRNAs in total RNA extracted from pigeon aortic tissue were also determined. While the increased deposition of extracellular matrix proteins was paralleled by a significantly greater recovery of mRNAs coding for pro alpha 1(1) collagen and elastin, in RNA extracted from White Carneau aortal tissue, increased recovery of mRNAs coding for an intracellular protein, gamma-actin were also observed in White Carneau aortal tissue. No differences in steady state levels of mRNAs coding for pro alpha 1(1) collagen and elastin were observed in RNA extracted from pigeon liver, suggesting a tissue specific increase in the mRNAs coding for these connective tissue proteins in aorta. A markedly reduced cell population however, was responsible for this overall increase in biosynthetic activity in White Carneau pigeon aortic tissue. This was demonstrated by a reduced cell count and by the recovery of reduced levels of total DNA in the thoracic aorta and celiac bifurcation of the White Carneau pigeon. The cell population in White Carneau aortic tissue exhibits therefore a markedly different phenotype with respect to a capacity for the biosynthesis of extracellular and intracellular proteins.

Actins↗

PubMed1990

Changes in steady-state levels of mRNAs coding for type IV collagen, laminin and fibronectin following capillary basement membrane thickening in human adult onset diabetes.

The development of capillary basement membrane thickening has been linked to microvascular changes known to occur in tissues of patients with type II diabetes. Previous evidence has suggested that capillary basement membrane thickening is due to increased basement membrane synthesis. In this study, skin samples from 8 diabetic patients with confirmed capillary basement membrane thickening and 7 non-diabetic controls were used to assess steady state levels of mRNAs coding for several basement components including pro alpha 1(IV) collagen, laminin and fibronectin. Total RNA was extracted from abdominal skin samples and levels of mRNAs coding for the basement membrane components laminin, fibronectin and pro alpha 1(IV) collagen, a fibrillar collagenous protein, pro alpha 1(I) collagen and an intracellular polypeptide, gamma-actin, were determined by dot blot hybridization analysis. While there were no changes of steady state levels of pro alpha 1(I) collagen mRNA and laminin mRNA, a significant reduction was noted in the quantitative recovery of mRNA levels for pro alpha 1(IV) collagen, gamma-actin and fibronectin in total RNA isolated from the skin of diabetic patients. This reduction in levels of mRNAs coding for basement membrane components contrasts with pathological confirmation of an accumulation of endothelial capillary basement membrane in skin from diabetic patients and suggests that basement membrane thickening arises more as a consequence of reduced basement membrane degradation than elevated synthesis of basement membrane components.

Aged↗

PubMed1989

Characterization of elastin protein and mRNA from salmonid fish (Oncorhynchus kisutch).

1. Elastin was isolated from the bulbus arteriosus of a salmonid fish. Monoclonal and polyclonal antibodies, elicited against a CNBr digest of this protein, immunoprecipitated a polypeptide of Mr 43,000 from fish cell culture medium. 2. Cell-free translation of salmon poly A+ RNA produced a protein of approximately 43 kD that was immunoprecipitated with anti-elastin antibodies. The corresponding mRNA had an approximate Mr of 2 kb. 3. Despite similarities in amino acid composition, the differences in Mr between mammalian and salmon mRNA and protein suggest a divergence of fish and higher vertebrate elastins from an earlier ancestral gene.

Amino Acids↗

PubMed1989

The effect of viable omentum on early bile leakage and healing of liver lacerations.

In order to determine if omental tissue accelerates the healing of liver lacerations, simulated bile leakage and collagen biosynthesis were studied in 53 rabbits. After creating a standardized complex liver laceration, hemostasis was obtained by vessel ligation and electrocoagulation. The wound was either left open (OP) or viable omentum sutured to its base (OM). Simulated bile leakage was noted in all of eight animals (four OM, four OP) studied on day of injury. None of 18 OM and two of 17 OP animals demonstrated extravasation of dye from the wound on the second and third postinjury day (N.S.). The ratio of mRNA for alpha 1(I) procollagen/actin, used as an indicator of wound healing, was 56.3 +/- 7.8 for OM and 50.6 +/- 12.1 for OP at the wound edge, and 63.5 +/- 18.6 and 69.2 +/- 7.5, respectively, for RNA isolated from the scar (N.S.). For alpha 1(III) procollagen mRNA, the ratio was 23.9 +/- 3.5 for OM and 22.4 +/- 8.3 for OP at the wound edge, and 32.4 +/- 6.5 and 31.8 +/- 7.9, respectively, for RNA isolated from the scar (N.S.). There was no difference in the scar hydroxyproline content between the two repair methods. In this model of hepatic injury and repair, bile leakage was minimal by the second postinjury day with both repair methods. Placing the omentum in liver lacerations did not contribute to accelerated wound healing as measured by simulated bile leakage and collagen biosynthesis.

Animals↗

PubMed1989

Pressure-induced connective tissue synthesis in pulmonary artery segments is dependent on intact endothelium.

Physiologic stimuli of connective tissue accumulation in pulmonary vascular remodeling are poorly defined. We postulated that increased pressure within central pulmonary arteries is a stimulus for connective tissue synthesis and the response is dependent on an intact endothelium. Mechanical tension equivalent to 50 mmHg pressure was applied for 4 h to isolated rat main pulmonary arteries (endothelium intact or removed), and incorporation of [14C]proline into collagen, [14C]valine into elastin, [3H]thymidine into DNA and pro alpha 1 (I) collagen mRNA levels were measured. In intact vessels, tension induced synthesis of collagen (3.1 +/- 0.4 vs. 2.3 +/- 0.5 [SEM] dpm X 10(2) [14C]-hydroxyproline/[mg protein.h]) (n = 10) and elastin (6.1 +/- 2.4 vs. 2.9 +/- 0.4 dpm X 10(3) [14C]valine/[mg protein.h]) (n = 5) (both P less than 0.05). Steady state mRNA levels of pro alpha 1 (I) collagen were also increased by tension (46 vs. 30 X 10(2) dpm hybridized/100 ng total RNA). However, the stimulus did not increase [3H]thymidine incorporation into DNA. In denuded vessels, tension had no effect on connective tissue synthesis or mRNA level of pro alpha 1 (I) collagen. Messenger RNA levels for v-sis were induced by tension in intact but not denuded vessels. Our findings establish that induction of vascular connective tissue synthesis by mechanical tension is dependent on an intact endothelium.

Actins↗

PubMed1988

Rat tropoelastin is synthesized from a 3.5-kilobase mRNA.

A lambda gt11 cDNA library was constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats and screened with a human tropoelastin cDNA clone. DNA sequence analysis of several overlapping rat clones confirmed the presence of DNA sequences coding for murine tropoelastin and DNA sequences coding for the 3'-untranslated region of the rat tropoelastin mRNA. Northern blot analysis of total RNA from aortic tissue of neonatal rats using oligonucleotide probes derived from these rat tropoelastin cDNAs demonstrated the presence of a 3.5-kilobase tropoelastin mRNA. The size of this rat tropoelastin mRNA agrees with previous reports for the size of the mRNA coding for tropoelastin in tissue from several vertebrate species but contrasts with several reports suggesting the presence of a higher molecular weight mRNA species responsible for the synthesis of tropoelastin in rodent tissue.

Animals↗

PubMed1988

High recombination between two physically close human basement membrane collagen genes at the distal end of chromosome 13q.

Two basement membrane collagen genes coding for the pro alpha 1 chain and pro alpha 2 chain of type IV collagen map to 13q34 and are linked with a maximum likelihood estimate of recombination of 0.028 at a logarithm of odds (lod) score of 19.98. The single-copy sequence that identifies the locus D13S3 is also closely linked to both collagen genes. Four enzymes reveal polymorphisms with COL4A1, and 10 haplotypes have been observed in Caucasoids. Within COL4A1 a nonrandom association of alleles exists only between alleles defined by Hae III and those defined by the other three enzymes. A random association of alleles of COL4A1 and COL4A2 is observed. Between the two collagen genes were detected three meiotic recombination events that contributed to the estimate of 2.8% recombination. This is higher than expected for two genes that lie within 650 kilobases of each other. The lack of linkage disequilibrium between COL4A1 and COL4A2 is in agreement with the relatively high recombination that is observed.

Basement Membrane↗

PubMed1988

Increased elastin mRNA levels associated with surgically induced intimal injury.

Quantitative levels of mRNAs coding for elastin, types I and III procollagen and gamma-actin were measured in porcine vascular material following balloon catheterization. A balloon catheter was introduced into the thoracic aorta and jugular vein of 3-6 week old pigs; following distention and six days of postoperative recovery, tissue samples were obtained for histopathology, electron microscopy, RNA extraction and mRNA quantitation. Using a series of mammalian cDNA clones and the procedure of slot blot hybridization, we have shown that elastin and types I and III procollagen mRNA levels rose significantly during the postoperative period following vascular distention. The increase correlated with an increase in the cell mass present in both the venous and arterial intimal layers. Changes in gamma-actin mRNA levels were also associated with this rapid proliferative response but in arterial tissue only.

Actins↗

PubMed1988

The genes coding for human pro alpha 1(IV) collagen and pro alpha 2(IV) collagen are both located at the end of the long arm of chromosome 13.

We have isolated and characterized a cDNA clone containing DNA sequences coding for the noncollagenous carboxy-terminal domain of human pro alpha 2(IV) collagen. Using this cDNA clone in both Southern blot analysis of DNA isolated from human-mouse somatic-cell hybrids and in situ hybridization of normal human metaphase chromosomes, we have demonstrated that the gene coding for human pro alpha 2(IV) collagen is located at 13q33----34, in the same position on chromosome 13 as the pro alpha 1(IV) collagen gene.

Animals↗

PubMed1987

Increased procollagen mRNA levels in carbon tetrachloride-induced liver fibrosis in rats.

Carbon tetrachloride-induced liver damage is a well-characterized experimental model for studying liver fibrosis. We used this model to examine alpha 1(I), alpha 1(III), and alpha 1(IV) procollagen mRNA levels during the development of liver fibrosis. Rats were given 0.5 ml of carbon tetrachloride/kg of body weight for 1-6 weeks. The liver tissue was assayed for collagen content by measuring total hydroxyproline content. Specific increases in procollagen mRNAs were assayed by slot blot hybridization. There was a significant increase in hydroxyproline content of liver tissue following 3 weeks of carbon tetrachloride treatment. The increase in tissue collagen content correlated with an increase in alpha 1(I) procollagen mRNA levels. At 5 and 6 weeks of treatment, there was an increase in alpha 1(III) procollagen mRNA levels. alpha 1(IV) procollagen levels increased slightly with five injections of carbon tetrachloride treatment. These results suggest that specific increases in procollagen mRNAs in liver fibrosis parallel, but do not precede, increases in tissue collagen content.

Animals↗

PubMed1987

The gene coding for tropoelastin is represented as a single copy sequence in the haploid sheep genome.

The identity of the primary in vitro translation products of fetal sheep nuchal ligament elastin mRNA was confirmed as two distinct polypeptides of 63 Kdal and 65 Kdal in both rabbit reticulocyte and wheat germ extract cell-free translation systems. Both polypeptides were co-translationally processed by a microsomal membrane signal peptidase, with the removal of 20-25 amino acid residues. A single (3,5 kb) RNA species encodes both tropoelastin polypeptides. Restriction endonuclease mapping of sheep genomic DNA by hydridization with two radiolabelled genomic DNA fragments containing sequences coding for sheep tropoelastin (pSE1-1,3 and pSE1-0.7,) indicated the presence of a single elastin gene. The elastin gene copy number was further quantitated by comparison of hybridisation of pSE1-1.3 and pSE1-0.7 to slot-blots and Southern transfers of sheep genomic DNA and to standard curves constructed with each clone. These results clearly demonstrate that each of these sequences is represented only once per haploid genome, suggesting that the two tropoelastin polypeptides are products of a single elastin gene.

Amino Acid Sequence↗