PubMed Health⌕ Search

Biomedical subjects

S Mohan

Publications and source records attributed to S Mohan.

At least 235 records · Page 13Linked to original sources
PubMed1991

Vitamin D deficiency causes a selective reduction in deposition of transforming growth factor beta in rat bone: possible mechanism for impaired osteoinduction.

We demonstrated previously that implants of bone matrix prepared from vitamin D-deficient (-D) rats were less osteoinductive and contained less extractable mitogenic activity compared with control implants prepared from vitamin D-replete (+D) rats and proposed that bone from -D rats is deficient in one or more specific growth factors. To test this hypothesis, bones from rats that were fed either +D or -D diets and kept in the dark for 8 wk were extracted and assayed for insulin-like growth factors I and II (IGF-I and IGF-II) and transforming growth factor beta (TGF-beta), the three most abundant growth factors in rat bone, and osteocalcin. Serum calcium, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were determined at sacrifice. In -D rats, there were significant reductions in serum calcium, 25-hydroxyvitamin D3, and 1,25(OH)2D3 and skeletal TGF-beta but no differences in extractable skeletal protein, IGF-I, IGF-II, or osteocalcin compared with +D rats. To determine whether 1,25(OH)2D3 increased TGF-beta production by bone cells, we treated mouse calvaria for 6 days and mouse osteoblasts for 2 days with 10 nM 1,25(OH)2D3. Production of TGF-beta was increased almost 100% by 1,25(OH)2D3. We conclude that vitamin D deficiency reduces deposition of TGF-beta in rat bone and that diminished skeletal TGF-beta could contribute to the previously observed decrease in osteoinduction in implants from -D rat bone. The findings support the possibility that vitamin D and bone-derived TGF-beta are required for normal repair of the skeleton.

Animals↗
PubMed1991

Isolation of a novel insulin-like growth factor (IGF) binding protein from human bone: a potential candidate for fixing IGF-II in human bone.

Insulin-like growth factor-II (IGF-II) is the most abundant growth factor stored in human bone. Upon release from this storage depot, IGF-II could act in bone repair and in the coupling of bone formation to bone resorption, a process inherent to bone which is a key regulatory process for maintenance of bone tissue. In this study, we report the isolation and characterization of a novel IGF binding protein (IGFBP) from human bone and describe how this IGFBP may be involved in the fixation of IGF-II in human bone. This new IGFBP has an apparent molecular weight of 29 kDa and has several fold higher affinity for IGF-II than IGF-I which could explain the much greater abundance of IGF-II than IGF-I in human bone. In terms of biological activity, this IGFBP was found to potentiate the proliferative actions of IGF-II on bone cells. This work raises the possibility that this IGFBP may participate in mediating some of the actions of IGF-II.

Amino Acid Sequence↗
PubMed1991

Evidence that tartrate-resistant acid phosphatases from osteoclastomas and hairy cell leukemia spleen are members of a multigene family.

1. Osteoclasts and hairy cell leukemia spleen both contain large amounts of a band 5-tartrate-resistant acid phosphatase (TrACP). 2. We have recently purified to homogeneity a band 5 TrACP from human osteoclastomas and two isoforms of band 5 TrACP (5a and 5b) from the spleen of a patient with hairy cell leukemia. 3. Although the N-terminal amino acid sequences and the apparent molecular weights of the osteoclastoma, hairy cell leukemia spleen TrACPs were identical, there were several differences in the physical and biochemical properties between the three isoenzymes. 4. Based on these findings, it is concluded that these isoenzymes are different enzymes, but that they could have originated from a similar ancestral gene. 5. It is proposed that the osteoclastoma and hairy cell leukemia band 5 TrACPs are members of a multigene family.

Acid Phosphatase↗
PubMed1991

Quantitation of growth factors in ossein-mineral-compound.

Study was undertaken to identify polypeptide factors in the commercially available ossein-mineral-compound and to see if they are present in a biologically relevant quantity. Using the guanidine-EDTA extraction, 35.7 +/- 0.1 mg proteins were obtained from 1 g of the ossein-mineral-compound. At concentration 1 micrograms/ml, guanidine-EDTA-extractable proteins stimulated the incorporation of thymidine into DNA by human bone cells to 581 +/- 122% (p less than 0.001) of that by bovine serum albumin-treated control cells, decreasing thereafter. Similarly, it stimulated the activity of alkaline phosphatase in the human bone cells. Growth factors IGF-I, IGF-II, and TGF-beta were identified in the ossein-mineral-compound. This leads to speculation regarding possible role of growth factors in explaining the beneficial effects of the compound in retarding bone loss in patients with osteoporosis.

Animals↗
PubMed1991

Contribution of marrow stromal cells to the regulation of osteoblast proliferation in rats: evidence for the involvement of insulin-like growth factors.

Fibroblast-like marrow stromal cells are believed to play a role in the maintenance of osteoblast populations at the marrow-bone interface. We now report that this interaction may be very specific. Stromal cell conditioned medium (SC-CM) stimulated DNA synthesis and proliferation in culture of neonatal rat calvarial osteoblasts at low concentrations (1.25-5%), but was inhibitory at 10%. At growth promoting effective concentrations, the activity of osteoblast alkaline phosphatase was decreased. This action was selective since SC-CM failed to promote the growth of rat calvarial fibroblasts. Characterization of the SC-CM indicated the cells produced IGF-I and -II and a wide range of molecular weight fractions with putative stimulatory action (FPLC analysis using Superose 12 and 6 gel permeation columns). HPAE-PAD analysis showed that some elements were glycosylated, and the composition suggested the presence of N- and O-linked oligosaccharide chains. Because rat marrow stromal fibroblast-like cells produce a number of osteotropic factors which affect calvarial osteoblast growth, these interactions may be important to considerations about the etiology of the osteoporoses.

Alkaline Phosphatase↗
PubMed1991

Studies on the cross stand hydrogen bonds in DNA double helices.

A systematic analysis has been carried out to examine all the stereochemically possible bifurcated hydrogen bonds including those of cross strand type between propeller twisted base pairs in DNA double helices by stereochemical considerations involving base pairs alone and by molecular mechanics studies on dimer and trimer duplexes. The results show that there are limited number of combinations of adjacent base pairs that would facilitate bifurcated cross-strand hydrogen bond (CSH). B-type helices concomitant with negative propeller twist seem to be more favored for the occurrence of CSH than canonical A-type helices because of slide in the latter. The results also demonstrate that helices with appropriate sequences may possess continuous run of these propeller twist driven cross strand hydrogen bonds indicating that they may in fact be considered as yet another general structural feature of DNA helices.

Base Composition↗
PubMed1991

1,25-Dihydroxyvitamin D3 differentially regulates the production of insulin-like growth factor I (IGF-I) and IGF-binding protein-4 in mouse osteoblasts.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation and inhibits proliferation in many cell types including bone cells. These effects may be mediated by the modulation of the insulin-like growth factor (IGF) regulatory system. Therefore we investigated the effects of 1,25-(OH)2D3 on transcript and protein levels of both IGF-I and IGF binding proteins (IGFBPs) in clonal mouse osteoblasts. Subconfluent cultures were treated in serum-free medium with 1,25-(OH)2D3. Secreted IGF-I was measured using a RIA under conditions eliminating the interference of IGFBPs. 1,25-(OH)2D3 (10(-11)-10(-8) M) inhibited IGF-I release in a dose dependent manner at 24 h (maximally to 30 +/- 5% of control, mean +/- SEM of seven independent experiments). In a time course study IGF-I increased in the media of control cultures over a 48-h period, while IGF-I secretion was completely prevented from 6 h onward in 1,25-(OH)2D3 treated cultures. Northern blot analysis revealed four IGF-I transcripts of 0.9, 1.8, 4.4, and 7.5 kilobases (kb). 1,25-(OH)2D3 decreased levels of the 7.5 kb IGF-I transcript from 4-48 h, with maximal inhibition occurring at 24 h (25% of control). Western ligand blots of the culture medium demonstrated secretion of a 25-kilodalton IGFBP, which comprised greater than or equal to 90% of the secreted IGFBPs. The 25-kilodalton IGFBP had previously been shown to have sequence similarity with IGFBP-4, a binding protein which inhibits the action of IGFs on bone cells. 1,25-(OH)2D3 treatment increased secretion of IGFBP-4 up to 14-fold over 24 h. 1,25-(OH)2D3 also increased IGFBP-4 (2.2 kb) transcript levels within 30 min, with the maximal stimulation of 8-fold occurring after 8 h. [3H]Thymidine incorporation into cells was inhibited by 1,25-(OH)2D3 both under basal and serum-stimulated conditions. Our results are consistent with the hypothesis that the effects of 1,25-(OH)2D3 on osteoblast proliferation may be mediated in part by decreased levels of IGF-I and increased concentrations of inhibitory IGFBP-4. It is proposed that this alteration in the IGF system may be an important functional autocrine or paracrine switch in the transition of osteoblasts from states of proliferation to differentiation.

Animals↗
PubMed1991

Insulin-like growth factor II and transforming growth factor beta 1 regulate insulin-like growth factor I secretion in mouse bone cells.

Bone cells in culture produce and respond to growth factors, suggesting that local as well as systemic factors regulate bone volume. Previous studies have shown that IGF-I is the major mitogen produced by mouse bone cells and that its production is regulated by systemic agents such as PTH and estrogen. Because IGF-II and transforming growth factor beta 1 have been shown, respectively, to increase and decrease MC3T3-E1 cell proliferation, we tested the hypothesis that these two growth factors modulate the production of IGF-I in this cell line. In order to eliminate artifacts owing to IGF binding proteins, conditioned media samples were pretreated with IGF-II before measurement of IGF-I by RIA. After 24 h treatment at a density of 2.5 x 10(4) cells/cm2, IGF-II (10 micrograms/l) induced a 2.2-fold increase compared with untreated control (9.5 +/- 1.5 vs 4.2 +/- 0.44 pg/micrograms protein, p less than 0.001), whereas transforming growth factor beta 1 (1 microgram/l) caused a 66% decrease in IGF-I production (1.5 +/- 0.3 vs 4.2 +/- 0.44 pg/micrograms protein, p less than 0.001). Both IGF-II and transforming growth factor beta 1 regulated IGF-I production in a dose-, time- and cell density-dependent manner. The lowest effective doses for IGF-II and transforming growth factor beta 1 were 1 and 0.01 microgram/l, respectively. These results support a role for IGF-II and transforming growth factor beta 1 as potent modulators of IGF-I secretion in mouse bone cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Evidence that the inhibition of TE85 human bone cell proliferation by agents which stimulate cAMP production may in part be mediated by changes in the IGF-II regulatory system.

Our previous studies have shown that insulin-like growth factor-II (IGF-II) is an important autocrine/paracrine regulator of human bone cell proliferation. In this study, we sought to look at the regulation of IGF-II production by human bone cells since IGF-II synthesis is a key variable that regulates the actions of IGF-II at a local site of bone. For studies of IGF-II regulation, we used TE85 human osteosarcoma cells as a model system since these cells exhibited several characteristics which are similar to that of untransformed normal human bone cells. In this study, we investigated the effects of agents which increase intracellular cAMP (forskolin, isobutylmethyl xanthine and prostaglandin E2) and N6,O2' dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) on the IGF-II regulatory system. It was found that these agents caused an increase in the production of an IGFBP in TE85 cells. Subsequent studies on the purification and characterization of IGFBP from DBcAMP treated TE85 cell conditioned medium revealed that the IGFBP produced by TE85 cells in response to DBcAMP treatment was identical to that of 25 kDa inhibitory IGFBP (now designated as IGFBP-4) purified from TE89 human bone cells based on: 1) N-terminal amino acid sequence, 2) amino acid composition, 3) molecular weight and 4) inhibitory actions on basal and IGF-II induced bone cell proliferation. In addition, forskolin and DBcAMP also caused a dose dependent decrease in the production of IGF-II. Consistent with these results, DBcAMP and agents which increase intracellular cAMP inhibited TE85 cell proliferation in a dose dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1991

Bone growth factors.

Bone volume is determined by the relative rates of bone formation and bone resorption. Recent research in several laboratories suggests that growth factors may act locally to modulate bone formation by stimulating osteoblast proliferation and activity. A number of bone-derived growth factors have been isolated and characterized from bone matrix extracts and from media conditioned by bone cells and bone organs in culture. The growth factors found in bone matrix include insulinlike growth factors I and II, transforming growth factor-beta, acidic and basic fibroblast growth factor, platelet-derived growth factor, and bone morphogenetic proteins. Conditioned medium from bone cells contains several of these growth factors and also hematopoietic factors. These bone matrix-derived growth factors have different biologic activities, including mitogenic, differentiating, chemotactic, and osteolytic activities. Evidence suggests that bone cells produce substantial quantities of growth factors for extracellular storage in bone matrix. Apart from being produced for extracellular storage, it is possible that growth factors secreted by bone cells have acute effects on their neighboring osteoblastic cells, i.e., paracrine action, or on themselves, i.e., autocrine action. The release of matrix-stored growth factors by bone resorption may mean that growth factors act as delayed paracrine agents, e.g., osteoblasts deposit growth factors in bone and later when these growth factors are released from bone via bone resorption, the growth factors stimulate osteoblast precursors to proliferate. The findings that bone is a storehouse for growth factors and that bone cells in culture produce and respond to bone growth factors suggest bone growth factors may act as potential determinants of local bone formation. This review is focused on the structure, regulation, and biologic actions of the known bone growth factors.

Animals↗
PubMed1990

Human prostatic cancer cells, PC3, elaborate mitogenic activity which selectively stimulates human bone cells.

Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis (i.e., osteoblast but not stromal fibroblast proliferation). In the present study we sought to test the hypothesis that prostatic cancer cells produce factor(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow fibroblasts. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat fibroblasts. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human fibroblast proliferation [( 3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not fibroblast cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone growth factors, specific assays for these growth factors were performed. PC3 CM contained 10 pg insulin-like growth factor (IGF) I, less than 2 pg IGF II, 54 pg basic fibroblast growth factor, and 16 pg transforming growth factor beta/microgram CM protein. None of these growth factors alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic fibroblast growth factor, IGF I, IGF II, or transforming growth factor beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens. Biochemical characterization supported the hypothesis that the PC3 cell growth factor was unique from other growth factors. The PC3 growth factor did not bind to heparin and was resistant to acid as well as the reducing agent, dithiothreitol. Sephadex G-75 and fast protein liquid chromatography Mono S cation-exchange chromatography revealed the PC3-derived mitogen to be an Mr 26,000-30,000 basic protein. Therefore, we conclude that PC3 cells release a mitogen which exhibits higher specificity for human osteoblasts than human fibroblasts and is unique from other growth factors tested. Production of this mitogen by human prostatic carcinoma cells could play an etiological role in the intense osteoblast-specific stimulation that occurs at sites of bone metastases.

Animals↗
PubMed1990

Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coli K-12 chromosome.

The DNA sequence and derived amino-acid sequence of a 5618-base region in the 74-min area of the Escherichia coli chromosome has been determined in order to locate the structural gene, nirB, for the NADH-dependent nitrite reductase and a gene, cysG, required for the synthesis of the sirohaem prosthetic group. Three additional open reading frames, nirD, nirE and nirC, were found between nirB and cysG. Potential binding sites on the NirB protein for NADH and FAD, as well as conserved central core and interface domains, were deduced by comparing the derived amino-acid sequence with those of database proteins. A directly repeated sequence, which includes the motif -Cys-Xaa-Xaa-Cys-, is suggested as the binding site for either one [4Fe-4S] or two [2Fe-2S] clusters. The nirD gene potentially encodes a soluble, cytoplasmic protein of unknown function. No significant similarities were found between the derived amino-acid sequence of NirD and either NirB or any other protein in the database. If the nirE open reading frame is translated, it would encode a 33-amino-acid peptide of unknown function which includes 8 phenylalanyl residues. The product of the nirC gene is a highly hydrophobic protein with regions of amino-acid sequence similar to cytochrome oxidase polypeptide 1.

Amino Acid Sequence↗
PubMed1990

Purification and N-terminal amino acid sequence of the tartrate-resistant acid phosphatase from human osteoclastoma: evidence for a single structure.

Tartrate-resistant acid phosphatase type-5 was purified to apparent homogeneity from human osteoclastomas by sequential chromatography on CM-Sepharose, Phenyl-Sepharose, concanavalin A-Sepharose, FPLC Superose-12, and FPLC Mono-S. The purification over the original tissue extract was 1167-fold, with a yield of 16%. An identity in the N-terminal amino acid sequence and Mr was found between this enzyme and two type-5 tartrate-resistant acid phosphatases isolated from hairy cell leukemia spleen. However, they appeared to be different as assessed by amino acid composition. In contrast to a previous report, no evidence was found for two subunits of the tartrate-resistant acid phosphatase.

Acid Phosphatase↗
PubMed1990

Skeletal growth factor and other growth factors known to be present in bone matrix stimulate proliferation and protein synthesis in human bone cells.

The purpose of the study was to investigate the effect of skeletal growth factor/insulinlike growth factor II and other growth factors known to be present in bone matrix on the proliferation and differentiation of human bone cells. Cells were isolated by collagenase digestion from femoral heads obtained during hip replacement operations. Cells were cultured in DMEM medium with 10% calf serum. Third to fifth passage cells were plated in multiwell plates and the medium changed to low serum (0.1%) for 2 days. The medium was changed to serum-free medium prior to addition of growth factors. Cell proliferation was measured by the incorporation of [3H]thymidine into DNA and by the percentage of cells that incorporate bromodeoxyuridine. Protein synthesis was measured by the incorporation of [3H]proline into trichloroacetic acid-precipitable material. Skeletal growth factor/insulinlike growth factor II and insulinlike growth factor I stimulated cell proliferation and protein synthesis in a dose-dependent manner. Alkaline phosphatase-specific activity was not increased by these factors. Transforming growth factor beta 1 did not affect cell proliferation but stimulated protein synthesis and increased the specific activity of alkaline phosphatase. Fibroblast growth factor did not affect any of the cell parameters. These studies suggest that skeletal growth factor/insulinlike growth factor II, insulinlike growth factor I, and transforming growth factor beta 1 may play a role in the local control of the proliferation and differentiation of human osteoblasts.

Alkaline Phosphatase↗
PubMed1990

Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-beta in human dentin.

Human bone matrix is known to contain a battery of polypeptide growth factors. Since dentin is a mineralized tissue similar to bone in composition and perhaps in formation, human dentin was assayed for the presence of similar growth factors. Root dentin proteins were extracted by demineralization in 4 M guanidine hydrochloride (Gu) and 30 mM Tris (pH 7.4) containing 20% EDTA and proteinase inhibitors. Gu-EDTA extracts were desalted and used for the following assays: (1) bone cell proliferation in chick calvarial cell mitogenic assay using the incorporation of [3H]thymidine into TCA-insoluble material; (2) osteocalcin by radioimmunoassay (RIA); (3) insulin-like growth factor I (IGF-I) by RIA; (4) skeletal growth factor/insulinlike growth factor II (SGF/IGF-II) by radioreceptor assay; and (5) transforming growth factor beta (TGF-beta) by bioassay. Gu-EDTA extracts stimulated bone cell proliferation. At 10 micrograms/ml, dentin proteins increased the incorporation of [3H]thymidine by calvarial cells to 320% of that by BSA-treated control cells. Consistent with the presence of mitogenic activity, growth factors were found in dentin in the following concentrations (ng/micrograms Gu-EDTA protein): (1) IGF-I, 0.06; (2) SGF/IGF-II, 0.52; and (3) TGF-beta, 0.017. All three growth factors were present in concentrations lower than that found in human bone. Osteocalcin was detected at a concentration of 3.0 mg/g Gu-EDTA protein, also much lower than that in bone.

Calcium↗
PubMed1990

Insulin-like growth factor-I and insulin-like growth factor-II induce c-fos in mouse osteoblastic cells.

We investigated the expression of c-fos in mouse osteoblast-like cultures treated with insulin-like growth factor (IGF)-I and IGF-II. The IGFs are present in bone, are produced by osteoblast-like cells in culture, and stimulate osteoblast cell proliferation. Quiescent, subconfluent cultures of the clonal osteoblast-like mouse calvarial cell line, MC3T3-E1, were treated with 10 ng/ml of IGF-I or IGF-II. RNA was extracted at 0, 15, 30, 60, 120 and 240 minutes, and c-fos messenger RNA (mRNA) was analyzed on Northern blots. Both IGFs transiently increased c-fos mRNA levels 25-28 fold at 15-30 min. To determine if c-fos induction was unique to the MC3T3-E1 cell line, effects of IGF-1 and IGF-II (3 ng/ml) were also tested in quiescent, serum-free primary mouse calvarial cells. Levels of c-fos mRNA were increased at 15 and 30 minutes (40-fold with IGF-I and 5-fold with IGF-II). These results indicate that IGF-I and IGF-II caused a rapid and transient induction of c-fos mRNA in murine osteoblasts.

Animals↗
PubMed1990

Insulin-like growth factors I and II are present in the skeletal tissues of ten vertebrates.

Previous studies have shown that insulin-like growth factor I and insulin-like growth factor II (IGF I and IGF II) constitute an important family of skeletal growth-regulating peptides. We undertook this study to determine if, as in the case of sera in various animals, IGF I and IGF II are conserved in the skeletal tissues of various vertebrates. Skeletal tissues of ten animals representing five of the six vertebrate classes were studied: monkey, dog, sheep, adult mice, neonatal mice, chicken, lizard, frog, trout, and shark. The skeletal tissues were pulverized and demineralized with 10% ethylenediaminetetraacetic acid (pH 7.0) to extract the soluble matrix proteins. The tissue extracts were concentrated, dialized, and subjected to specific human IGF assays. IGF I and IGF II were measured with a radioimmunoassay and a radioreceptor assay, respectively. We found that 1) there are detectable human IGF I- and II-like substances in all extracts, 2) IGF values obtained in the skeletal extracts were not caused by binding protein artifacts, 3) in general there is more IGF II than IGF I, and 4) the skeletal tissue levels of the IGFs are comparable with their respective serum levels. We conclude that, in the skeletal tissues of vertebrates, the IGFs are conserved and may be important regulators of osteogenesis.

Animals↗