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Biomedical subjects

V H Rao

Publications and source records attributed to V H Rao.

At least 19 recordsLinked to original sources
PubMed1991

Comparison of morphological and biochemical characteristics of cultured chondrocytes isolated from proliferative and hypertrophic zones of bovine growth plate cartilage.

Primary cultures of chondrocytes isolated from the proliferative and hypertrophic zones of bovine growth plate cartilage were established, and their morphological and biosynthetic characteristics were compared. High-cell-density monolayer cultures maintained a predominantly chondrocytic morphology, preserved their zone-specific collagen phenotype, and produced an abundant proteoglycan matrix. In contrast, monolayer cultures plated at low cell densities contained many cells with fibroblastic morphology and initiated the synthesis of type I collagen within 72 h following seeding. On a per cell basis, the proliferative zone chondrocytes displayed greater biosynthetic activity than chondrocytes from the hypertrophic region. Characterization of newly synthesized collagens showed that type II collagen was the major biosynthetic product of both types of cells. In contrast, type X collagen was synthesized exclusively by hypertrophic chondrocytes and represented about 20% of the total collagen produced by these cells. The type X collagen molecules synthesized by the hypertrophic cells contained disulfide bonds within their triple-helical domains and assembled into a high-Mr aggregate. When polymerase chain reaction (PCR) was employed to amplify type X collagen sequences from cDNA obtained by reverse transcription of mRNA from cultures of both types of chondrocytes, only the hypertrophic chondrocyte mRNA yielded PCR amplification products indicative of the presence of type X collagen transcripts.

Amino Acid Sequence↗
PubMed1991

Characterization of a COL1A1 splicing defect in a case of Ehlers-Danlos syndrome type VII: further evidence of molecular homogeneity.

A child affected by the type VII form of Ehlers-Danlos syndrome (EDS VII) was shown to have a heterozygous structural defect in the amino-terminus of pro-alpha 1(I) collagen. As a result, type I procollagen trimers containing defective subunits are not converted to mature collagen molecules. To identify the cause of the protein abnormality, specifically primed cDNAs and genomic DNA were PCR amplified and sequenced. This analysis disclosed that the protein structural defect is caused by a single base substitution (A for G) at position -1 of the splice donor site of intron 6 of the pro-alpha 1(I) collagen gene (COL1A1). The affected allele produces (a) transcripts lacking exon 6 sequences and (b), in lesser amount, normally spliced transcripts. Furthermore, the rate of exon 6 skipping is temperature dependent, for it appears to decrease substantially when the patient's fibroblasts are incubated at 31 degrees C. These findings are similar to those we previously reported for other unrelated EDS VII cases and, therefore, reemphasize the molecular homogeneity of this rare connective tissue disorder.

Adult↗
PubMed1990

In vivo and in vitro noncovalent association of excised alpha 1 (I) amino-terminal propeptides with mutant pN alpha 2(I) collagen chains in native mutant collagen in a case of Ehlers-Danlos syndrome, type VII.

The cause of the Ehlers-Danlos syndrome Type VII (EDS VII) is considered to be defective removal of the amino-terminal propeptide (N-propeptide) of Type I procollagen due to deficiency of procollagen N-proteinase, the enzyme responsible for the normal proteolytic excision of this precursor-specific domain. Molecules retaining the N-propeptide (pN-collagen molecules) are thought to cause defective fibrillogenesis and cross-linking which eventuate in dramatic joint laxity and joint dislocations, the clinical hallmark of this variety of EDS. Recent studies demonstrate that some EDS VII patients harbor small deletions of either the pro-alpha 1(I) or pro-alpha 2(I) chain of Type I procollagen. We have found an 18-amino acid deletion (due to exon outsplicing) in a mutant pro-alpha 2(I) chain from such a patient. The deleted peptide is the junctional segment (N-telopeptide) linking the alpha 2(I) N-propeptide and major triple helical domains; loss of this short segment results in union of these latter domains and produces a shortened pN alpha 2(I) chain. Directly extracted tissue collagen and pepsin-digested fibroblast collagen contain this mutant pN alpha 2(I) chain and normal alpha 1(I) chains, but not pN alpha 1(I) chains, indicating that the relatively larger alpha 1(I) N-propeptide is excised from the related alpha 1(I) chains. The fate of this alpha 1(I) N-propeptide was unclear and therefore whether or not the intact N-propeptide was, in fact, retained in native mutant collagen was also unclear. In this paper, we describe morphologic, chemical, and immunochemical studies which indicate that the alpha 1(I) N-propeptide is retained in noncovalent association with the mutant pN alpha 2(I) chain in native mutant collagen molecules both in vivo and in vitro. In both instances, the alpha 1(I) N-propeptides are proteolytically cleaved from the related alpha 1(I) chains. These data suggest that retention of a partially cleaved, but essentially intact N-propeptide in mutant collagen may play a role in the pathogenesis of this disease.

Amino Acid Sequence↗
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

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↗
PubMed1979

Synovial fluid and serum hydroxyproline fractions in rheumatoid arthritis.

In rheumatoid arthritis (RA) the synovial fluid lymphocyte count closely parallels the synovial fluid dialysable hydroxyproline, a marker of collagen resorption. This observation stresses the primordial role of lymphocytes in RA joint injury. The actual and eventually potential destruction of any single joint, as expressed by synovial fluid dialysable hydroxyproline levels, seems to reflect the general picture of disease activity as evaluated by the number of active joints (joint count), ESR and rheumatoid factor titres (latex fixation). Although urinary hydroxyproline levels are generally within the normal range in RA, they can be used as index of articular tissue destruction and as a parameter of overall disease activity when groups of patients are studied longitudinally in detail. The present study sheds no further light on the significance of synovial fluid and blood non-dialysable hydroxyproline levels.

Arthritis, Rheumatoid↗
PubMed1979

Serum and synovial fluid hydroxyproline fractions in microcrystalline arthritis and osteoarthritis.

Synovial fluid and serum hydroxyproline fractions were investigated in patients with osteoarthritis and microcrystalline arthritis. Synovial fluid dialysable hydroxyproline levels are higher than serum levels in both conditions. Synovial fluid total and dialysable hydroxyproline are higher in microcrystalline arthritis than in osteoarthritis, while non-dialysable hydroxyproline values are similar in both conditions. In microcrystalline arthritis, synovial fluid dialysable hydroxyproline and polymorphonuclear leukocyte counts closely parallel each other. Irrespective of the type of arthropathy, synovial fluid dialysable hydroxyproline levels correlate with urinary hydroxyproline excretion. While the data suggest overproduction of dialysable hydroxyproline by joints in both conditions, the overproduction appears to be mediated by polymorphonuclear leukocytes in microcrystalline arthritis only. The ratio of serum to synovial total hydroxyproline are further suggestive of a possible differentiation between osteoarthritis and microcrystalline arthritis. In the conditions governing the present study, urinary hydroxyproline may be used as an index of joint tissue collagen resorption. Finally the significance of synovial fluid and serum non-dialysable hydroxyproline is discussed.

Chondrocalcinosis↗