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Biomedical subjects

V H Rao

Publications and source records attributed to V H Rao.

At least 37 records · Page 2Linked to original sources
PubMed1989

Decreased thermal denaturation temperature of osteogenesis imperfecta mutant collagen is independent of post-translational overmodifications of lysine and hydroxylysine.

Fibroblasts from many patients with osteogenesis imperfecta (OI) synthesize and secrete Type I collagen which is both overmodified and exhibits a decreased thermal denaturation temperature. We have examined the relationship between overmodification and decreased melting temperature in several favorable OI mutants by selectively inhibiting lysyl hydroxylase activity with the drug Minoxidil and comparing the melting profiles of the resultant undermodified collagen with untreated control. Minoxidil treatment causes an appreciable decrease in hydroxylysine with compensatory increases in lysine content, and the delayed sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobility of the overmodified collagen chains becomes normal. However, the decreased melting temperature was unchanged from untreated OI control. When unhydroxylated collagen produced by normal control and OI fibroblasts incubated with alpha,alpha'-dipyridyl was examined, mutant OI molecules melted at a lower temperature than control. These data indicate that the decreased thermal denaturation temperature of OI mutant collagen is independent of post-translational overmodification of lysine or hydroxylysine. Presumably, substitutions for glycine in the Gly-X-Y structural motif distort the helix and produce lower melting temperatures by presently unknown mechanisms.

Collagen↗
PubMed1987

Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-alpha 2(I) chain.

A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.

Amino Acid Sequence↗
PubMed1986

Identification of a disulfide-bonded 70 Kd type X procollagen in embryonic chick sternum cartilage.

Chondrocytes from the presumptive calcification region of 20 day old embryonic chick sternum were found to synthesize a 70 Kd Type X procollagen precursor in addition to the previously described 59 Kd Type X collagen molecules. The 70 Kd molecules exhibited an additional cyanogen bromide peptide, contained a disulfide-bonded domain, and were converted into the 59 Kd moieties during pulse-chase experiments. The conversion of the 70 Kd to the 59 Kd Type X collagen was prevented upon microtubular transport inhibition with colchicine and resulted in tissue accumulation of the 70 Kd Type X procollagen.

Animals↗
PubMed1984

Cysteine in the triple-helical domain of one allelic product of the alpha 1(I) gene of type I collagen produces a lethal form of osteogenesis imperfecta.

We studied tissue and cultured skin fibroblasts from a newborn with the lethal perinatal form of osteogenesis imperfecta born to a mother with the Marfan syndrome and her unrelated husband. Dermis from the infant was thinner and fibril diameter smaller than control; dermal fibroblastic cells had dilated endoplasmic reticulum. His fibroblasts in culture synthesized two different species of pro alpha 1(I) chains in about equal quantity. One chain was normal, the other contained cysteine within the triple-helical portion of the COOH-terminal cyanogen bromide peptide alpha 1(I)CB6. Molecules which contained two copies of the mutant chain formed alpha 1(I)-dimers linked through interchain disulfide bonds. Molecules which contained either one or two mutant chains were delayed in secretion and underwent excessive lysyl hydroxylation and hydroxylysyl glycosylation of all chains in the molecule, probably as a result of delayed triple-helix formation. Molecules containing either one or two copies of the mutant chain melted at 38 degrees C instead of 41 degrees C. The most likely explanation for these findings is that a cysteine is substituted for a glycine in the triple-helical domain of the products of one of the alpha 1(I) alleles. Such a substitution would interfere with triple-helix formation and stability and thus explain 1) the decreased melting temperature, 2) the increased post-translational modification, 3) the altered rate of secretion and accumulation of intracellular material, 4) the increased intracellular degradation of newly synthesized collagen, and 5) the decreased collagen production. Since neither parental cell strain produced the same mutant chain, the findings are best explained by a new mutation in one of the alpha 1(I) genes. The role of the uncharacterized "Marfan" gene in modifying the phenotype in this patient is unclear.

Alleles↗
PubMed1984

Survival of rabbit embryos after rapid freezing and thawing.

Frozen storage of rabbit embryos at the 16-cell stage in 2.0 M dimethylsulfoxide (DMSO) in phosphate-buffered saline (PBS) was achieved by a 2-step procedure. After storage for 10 days at -196 degrees C they were revived by rapidly thawing at 500 degrees C/min. On transfer of these embryos to pseudopregnant foster mothers, 50% survived to term. The difference in in vivo survival between frozen-thawed and frozen-thawed-cultured embryos was not significant.

Animals↗
PubMed1984

Ehlers-Danlos syndrome type IV D: an autosomal recessive disorder.

Two siblings born to consanguineous parents are reported with typical clinical features of the Ehlers-Danlos syndrome type IV. However, their cultured skin fibroblasts synthesize and secrete procollagen type III in normal amounts and proportions. This is probably a new form of the Ehlers-Danlos syndrome with autosomal recessive inheritance classified as Ehlers-Danlos syndrome type IV D.

Biopsy↗
PubMed1984

Xenogenous fertilization of goat ova in the rabbit oviduct.

Eggs from superovulated goats were surgically placed in the oviducts of pseudopregnant rabbits previously inseminated with goat semen. Out of 81 eggs, 30 (37%) exhibited two pronuclei or two blastomeres when recovered 24-36 h later. Transplantation of these embryos into foster nanny goats resulted in the birth of kids.

Animals↗
PubMed1984

Neonatal severe primary hyperparathyroidism and alkaptonuria in a boy born to related parents with familial hypocalciuric hypercalcemia.

We describe a study of a boy with neonatal severe primary hyperparathyroidism (NSPHP) and alkaptonuria born to related parents of Turkish origin. The clinical and chemical courses (e.g. of mineral metabolism, of urinary excretion of amino acids and collagen metabolites) in response to various therapeutic approaches including total parathyroidectomy (PTX) are reported. Urinary excretion of calcium was unusually low before and immediately after PTX, and later during an inadvertent vitamin D intoxication. It corresponded to values typical for patients with familial hypocalciuric hypercalcemia (FHH), an autosomal dominant disorder. Both parents and one sibling had episodes of hypercalcemia with inappropriately high parathormone levels; in the father there was also relative hypocalciuria consistent with FHH. On the basis of the genetic and pathophysiologic data reported here, we speculate that homozygosity for the 'FHH-gene' is the cause of the life-threatening manifestation of NSPHP, whereas heterozygosity for the same gene leads to FHH, by comparison a mild disorder. The association of the two very rare recessively transmitted disorders, alkaptonuria and NSPHP, is unique; close linkage of the two genes, one coding for homogentisic acid oxidase, the other for the unknown gene product defective in NSPHP, can be suspected.

Alkaptonuria↗
PubMed1983

Influence of bleomycin on the metabolism of dermal collagen in albino rats.

The metabolism of collagen in male rats by treatment with bleomycin was studied following the injection of [3H]proline and the determination of specific and total activity of [3H]hydroxyproline in skin collagen fractions and urine. In the case of the bleomycin-treated animals, there was found to be an increase in the neutral salt soluble collagen content with no change in insoluble collagen content as compared to the control group. The specific and total radioactivity of [3H]hydroxyproline in soluble and insoluble collagen fractions was also increased. Examination of [3H]hydroxyproline activity in soluble and insoluble collagen showed that the conversion of soluble to insoluble collagen was improved by the bleomycin-treated group. It was found that this was accompanied by a decrease in urinary excretion of total hydroxyproline and [3H]hydroxyproline during the first 12 hr after the administration of [3H]proline. Therefore, the results of the present investigation clearly indicate that the maturation of soluble to insoluble collagen is promoted and accompanied by a decrease in the catabolism of soluble collagen in the bleomycin-treated animals. In addition, administration of bleomycin increased the synthesis of collagen.

Animals↗
PubMed1983

Effect of protein malnutrition on the cross-linking of gingival and uterine collagen in rats.

The effect of protein malnutrition on gingival and uterine collagen cross-linking was studied in female albino rats. The gingival and uterine samples were taken on the 28th day of the experimental period in both groups; the percent reversibility of neutral salt-soluble collagen, and the susceptibility of insoluble collagen to denaturing agents (to KSCN, urea, or pronase) were increased in gingival and uterine samples of protein-deficient animals. The analysis of gingival and uterine samples showed alpha 1 and alpha 2 subunits of neutral salt-soluble collagen appreciably increased, but beta 11 and beta 12 chains and the aldehyde content were significantly decreased in protein-deficient animals compared to controls. The results indicate that in protein deficiency the cross-linking and maturation of collagen are impaired.

Animals↗
PubMed1983

Relationship of cell growth to collagen synthesis in glucocorticoid treated A/J and C57BL6/J neonatal mouse dermal fibroblasts.

Primary cultures of neonatal dermal fibroblasts from two strains of mice (A/J and C57BL6/J) were utilized as an in vitro system to investigate the effects of glucocorticoids on cell growth and collagen synthesis. Protein and DNA synthesis were lower in untreated (control) A/J fibroblasts than in C57BL6/J fibroblasts. Treatment with glucocorticoids for 4 days resulted in dose-dependent inhibition of [3H]thymidine incorporation into DNA and a reduction in collagen synthesis. Collagen synthesis was differentially more susceptible to glucocorticoids than was total protein synthesis. Neonatal dermal fibroblasts obtained from A/J mice were more sensitive to glucocorticoids than were cells from C57BL6/J mice. Reduction in collagen production by anti-inflammatory steroids in this system may be related to adverse effects observed in vivo following treatment with these steroids.

Animals↗
PubMed1983

Bioflavonoid-mediated stabilization of collagen in adjuvant-induced arthritis.

In rats with adjuvant-induced arthritis, the effect of (+)-catechin (CA) and 0-(beta-hydroxyethyl) rutosides (HR) on the crosslinking of collagen was studied. Compared with controls the arthritic group showed an increase in the reversibility of neutral salt-soluble collagen gel, solubility of acid-insoluble collagen to denaturing agents and the ratio of alpha/beta subunits of neutral salt-soluble collagen. These results suggest an impaired maturation of collagen in arthritic animals. Administration of CA or HR to the arthritic animals caused a decrease in the reversibility of collagen gel and in the solubility of acid-insoluble collagen to denaturing agents and to pronase. In addition, the electrophoretic patterns of neutral salt-soluble collagen on SDS polyacrylamide gels also showed a decrease in the alpha/beta ratio. All these results may collectively indicate that both flavonoids promote the crosslinking of collagen in arthritic animals.

Animals↗
PubMed1982

Effect of protein malnutrition on the crosslinking of dermal collagen in aging.

The effect of protein malnutrition on collagen crosslinking was studied in different age groups of rats. The percent of reversibility of neutral salt-soluble collagen gel and the susceptibility of insoluble collagen to denaturing agents and pronase decreased with aging. The electrophoretic pattern of neutral salt-soluble collagen on sodium dodecyl sulphate polyacrylamide gels showed a decrease of alpha 1 and alpha 2 subunits and an increase of beta-components, resulting in a decrease of alpha/beta ratio in aging. There was a significant decrease in the aldehyde content of neutral salt-soluble collagen during age. Protein deficiency was found to impair the crosslinking and the effect was greater in younger animals than in older ones.

Age Factors↗
PubMed1981

Synovial fluid hydroxyproline fractions before and after osmic acid treatment in rheumatoid arthritis.

Synovial fluid total, dialysable and non-dialysable hydroxyproline were determined in patients with Rheumatoid Arthritis before intra-articular osmic acid injection and on days 2 and 4 after this local treatment. On day 2 the increase in dialysable hydroxyproline paralleled the increase in non-dialysable hydroxyproline. Patients with the highest pre-treatment dialysable hydroxyproline levels also had the highest levels of this fraction on day 2 suggesting that articular damage proceeds after osmic acid injection. However, on day 4 dialysable hydroxyproline levels were consistently lower than before the treatment; the same pattern was observed with non-dialysable hydroxyproline, except for all the joints but one, where effusion recurred 6-12 months after osmic acid treatment. This study suggests that the determination of synovial fluid non-dialysable levels 4 days after osmic acid injection may provide a clue to the prediction of recurrent joint effusion and possibly justify a complementary injection either with osmic acid or with a beta-emitting isotope.

Adult↗