Incorporation of radioactive precursors into glycosaminoglycans by rat muscle fibroblasts exposed to a solubilized rat bone matrix fraction

Confluent cultures of rat muscle fibroblastic cells respond by increased glycosaminoglycan (GAG) synthesis when cultured in medium containing a solubilized bone matrix fraction (SBM) at a concentration of 100 micrograms/ml. The metabolism of the GAG associated with the cell pellet, the cell surface and the tissue culture medium fractions was studied, in the presence and absence of SBM, by measuring the incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the isolated GAG. Net synthesis of hyaluronic acid and of chondroitin sulfate in the medium fraction increased more rapidly in cultures containing SBM compared to controls, and the accumulation of labelled GAG in the medium of the treated cultures was approximately linear with respect to the length of incubation. The addition of SBM also resulted in increased incorporation of 3H and of 35S into the GAG of the cell surface and cell pellet fractions. In these fractions, stimulation of incorporation of radioactivity occurred in two waves: an early, relatively minor increase and a later relatively major increase. The relatively major stimulation of radioactivity into the GAG of the cell surface fraction occurred between 24 and 48 h and was independent of any apparent effect of serum.     

Isolation and purification of a low-molecular-weight skeletal growth factor from human bones

A low-molecular-weight potent bone cell mitogen termed human skeletal growth factor (human SGF) was purified to homogeneity from human bone matrix. Extraction and initial purification steps were done under dissociative conditions to separate human SGF from high-molecular-weight complexes of bone matrix proteins. SGF activity was extracted from human femoral heads by demineralization with 10% EDTA in the presence of 4 M guanidine-HCl and proteinase inhibitors and was purified by hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was homogeneous by HPLC reverse-phase chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of human SGF purified under dissociative conditions was 11,000. Human SGF stimulated bone cell proliferation ([3H]thymidine incorporation and cell number) at picomolar concentrations, with half maximum activity at 2-3 ng/ml (180-270 pM). Human SGF constitutes 0.00024% of organic bone matrix by weight.     

"Matrigenin" activity from bovine bone--I. Partial purification of activity

1. Bovine bone contains an extractable activity which stimulated the synthesis of glycosaminoglycans by bovine synovial, human synovial and mouse 3T3 fibroblastic cells in culture. Human cells were used to develop an assay for purification of the stimulatory activity ("matrigenin" activity) from bovine bone. 2. Partial purification of "matrigenin" activity was achieved by precipitation of the EDTA extract at pH 3.5 and Sepharose CL-6B chromatography in 4 M guanidinium HCl. Dissociative conditions were necessary to prevent aggregation. 3. On SDS-polyacrylamide gel electrophoresis the activity ran with a mobility equivalent to a Mr = 27,500 and could be recovered from the SDS gels.     

"Matrigenin" activity from bovine bone--III. Effects on glycosaminoglycans and proteoglycans of human synovial cells in culture

1. Human synovial fibroblastic cells were cultured in the presence and absence of an extract from bovine bone containing "matrigenin" activity. The rate of incorporation of radioactivity into the glycosaminoglycans of the medium of "matrigenin"-treated cultures increased after 24 hr of incubation, compared to "controls". 2. Higher serum concentrations had a greater effect on the incorporation of radioactivity into hyaluronic acid synthesized by "matrigenin"-treated cultures, than by "controls". 3. Incorporation of radioactive precursors into the proteoglycans isolated from the medium was greater in the "matrigenin"-treated cultures than in "controls". The synthesis of a large mol. wt proteoglycan was specifically stimulated.     

Newly synthesized glycoconjugates from two cell lines derived from rat osteogenic sarcoma: effects of Matrigenin activity from bone

An activity isolated from bovine bone was previously shown to stimulate proteoglycan synthesis by several connective tissue cell lines from normal tissues (Matrigenin activity). The effect of this activity on glycoconjugate synthesis by two osteoblastic cell lines, ROS 17/2 and UMR-106, derived from rat osteogenic sarcoma, was examined after labelling of the cells with [3H]glucosamine and [35S]sulfate. The glycoconjugates from the cell layers and the media were separated by DEAE-Sephacel chromatography and the anionic glycoconjugates of the media were further analyzed by chromatography on Sepharose CL-2B and enzymatic digestion of the papain-released glycosaminoglycans. The ROS 17/2 cells secreted at least two distinct species of proteoglycan (one heparan sulfate rich and the other chondroitin sulfate rich), whereas the UMR-106 secreted primarily an anionic glycoprotein. The addition of Matrigenin activity to the ROS 17/2 cells resulted in stimulation of incorporation of radioactivity into the proteoglycan and hyaluronic acid, but in UMR-106 cultures it resulted in decreased incorporation into the anionic glycoprotein. The decrease in incorporation into the anionic glycoprotein from the medium was shown, by alkaline beta-elimination, to have occurred mainly in the oligosaccharide fraction, relative to control cultures.     

Preferential In Vivo Incorporation of [3H]Arachidonic Acid from Blood into Rat Brain Synaptosomal Fractions Before and After Cholinergic Stimulation

Abstract: Awake adult male rats were infused intravenously with [³H]arachidonic acid for 5 min, with or without prior administration of an M1 cholinergic agonist, arecoline (15 mg/kg i.p.). Methylatropine was also administered (4 mg/kg s.c.) to control and arecoline-treated animals. At 15 min postinfusion, the animals were killed, brains were removed and frozen, and subcellular fractions were obtained from homogenates of whole brain. Total radioactivity and radioactivity in various lipid classes were determined for each fraction following normalization for exposure by use of a unidirectional incorporation coefficient, k ^⋆_brain. In control animals, incorporation was greatest in synaptosomal and microsomal fractions, accounting for 50 and 30% of total label incorporated into membrane lipids, respectively. Arecoline increased incorporation in these two fractions by up to 400% but did not increase incorporation into the myelin, mitochondrial, or cytosolic fractions. Of the incorporated radioactivity, 50–80% was in phospholipid in microsomal and synaptosomal fractions, indicating that phospholipid is the major lipid affected by cholinergic stimulation. These results demonstrate that plasma [³H]arachidonic acid is preferentially incorporated into phospholipids of synaptosomal and microsomal fractions of rat brain. Cholinergic stimulation increases incorporation into these fractions, likely by activation of phospholipase A₂ and/or C in association with acyltransferase activity. Thus, intravenously infused radiolabeled arachidonic acid can be used to examine synapse-mediated changes in brain phospholipid metabolism in vivo.     

The effect of mannose6-phosphate on the turnover of cell surface glycosaminoglycans

Human fibroblasts (SL66) were cultured in medium containing 35SO4(2-) to label the glycosaminoglycans (GAGs). After washing, the labeled cells were chased in the presence or absence of mannose6-phosphate (M6P) and the GAGs were analyzed in terms of three arbitrary fractions: 1, Extracellular (soluble medium), 35S radioactivity higher in cultures without M6P than in cultures with M6P. 2, Pericellular (cell surface-associated), 35S radioactivity lower in cultures without M6P than in cultures with M6P. 3, Intracellular (residue within the intact cell), no difference in 35S radioactivity between the two sets of cultures. In addition, when the 35S-labeled GAGs from corresponding cellular compartments derived from cultures with and without M6P were digested with pronase and chondroitin ABC lyase, and then compared by chromatography on Sepharose CL-6B, distinct molecular differences in both the extracellular and pericellular fractions were observed. Several lines of evidence indicate that the effect of M6P on the turnover of 35S-labeled GAGs in our assay system reflects disruption of cell surface lysosomal enzyme activity. For example, when the experiment was performed with I cells, which lack enzymes carrying the M6P marker, no difference was seen in cultures with or without M6P. The addition of lysosomal enzymes derived from normal human fibroblasts to 35SO4-labeled I cells, however, resulted in the turnover of pericellular GAGs and this effect was inhibited by M6P. These results suggest that one possible function of cell surface receptors recognizing the M6P moiety of lysosomal enzymes is to anchor certain of these enzymes proximate to their substrates at the cell surface.     

Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1)

Human skeletal growth factor (hSGF), an 11-kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3-E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14-hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high-pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid-insoluble protein. HSGF did not increase cell number over a 48-hr period and caused a reversible inhibition of DNA synthesis. Half-maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3-E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.     

Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts

SPARC (secreted protein, acidic and rich in cysteine), also known as osteonectin, is an extracellular Ca⁺²-glycoprotein that inhibits the incorporation of [³H]-and delays the onset of S-phase in synchronized cultures of bovine aortic endothelial (BAE) cells. This effect appears not to be dependent on the functional properties of SPARC associated with changes in cell shape or inhibition of cell spreading. In this study we investigate the conditions under which cell cycle modulation occurs in different types of cells. Human umbilical vein endothelial cells, a transformed fetal BAE cell line, and bovine capillary endothelial cells exhibited a sensitivity to SPARC and a cationic peptide from a non-Ca⁺²-region of SPARC (peptide 2.1, 0.2—0.8 mM) similar to that observed in BAE cells. In contrast, human foreskin fibroblasts and fetal bovine ligament fibroblasts exhibited an increase in the incorporation of [³H]-in the presence of 25 μM—0.2 mM peptide 2.1; inhibition was observed at concentrations in excess of 0.4 mM. This biphasic modulation could be further localized to a sequence of 10 amino acids comprising the N-terminal half of peptide 2.1. A synthetic peptide from another cationic region of SPARC (peptide 2.3) increased [³H]-incorporation by BAE cells and fibroblasts in a dose-dependent manner. In endothelial cells, a stimulation of 50% was observed at a concentration of 0.01 mM; fibroblasts required ～ 100-fold more peptide 2.3 for levels of stimulation comparable to those obtained in endothelial cells. The observation that SPARC and unique SPARC peptides can differentially influence the growth of fibroblasts and endothelial cells in a concentration-dependent manner suggests that SPARC might regulate proliferation of specific cells during wound repair and remodeling. © 1993 Wiley-Liss, Inc.     

The effect of 3-methylindole on the rates of phospholipid and neutral lipid synthesis in cultured fibroblasts

The present study was designed to test the hypothesis that a pneumotoxin, 3-methylindole, alters the basic metabolic pathways involved in phospholipid and neutral lipid synthesis in cultured fibroblasts. Rat skin fibroblasts were obtained from day-old pups. Confluent monolayers were preincubated for up to 24 h with a range of concentrations (0-0.76 mM) of 3-methylindole. Following these treatments, the cell lipids were labelled by incubation for 6 h with [14C]glycerol. The lipids were extracted, separated by thin layer chromatography, and the radioactivity in each fraction was determined. 3-Methylindole had no effect on the total incorporation of [14C]glycerol into lipids, but significantly altered the distribution among lipid fractions. Incubation with 3-methylindole caused a decrease in the incorporation of [14C]glycerol into phosphatidylcholine, while radioactivity accumulated in the neutral lipid fraction. The other lipid fractions responded variably. Similarily, Flow 2000 human diploid lung fibroblasts were incubated for 24 h with 3-methylindole followed by treatment with [14C]glycerol, resulting in a 74% decrease in the incorporation of [14C]glycerol into phosphatidylcholine and a 50% increase in its accumulation in neutral lipid. The results indicate that 3-methylindole inhibits the synthesis of phosphatidylcholine from diacylglycerol precursors on the endoplasmic reticulum in cultured fibroblasts. This is an important observation as it shows that 3-methylindole affects the synthesis of phospholipids required for membrane turnover in cells that are not specialized for the production of phospholipids for surfactant.     

Measurement of [<Superscript>18</Superscript>F]-fluorodeoxyglucose incorporation into human osteoblast–An experimental method

An evaluation of human osteoblast metabolism usually involves measurements of the by-products of bone matrix elaboration. The assessment of glycolytic activity of osteoblasts is not a standard tool in most of the reports, but might be of value by providing a direct indicator of cellular metabolism. Measurement of the incorporation of [¹⁸F]-fluorodeoxyglucose, which is not further degradable following its conversion into glycose-6-phosphate during glycolysis and is trapped in this form within the cells, can be used as an effective research tool for estimation of osteoblast metabolism. In order to estimate the [¹⁸F]-fluorodeoxyglucose incorporation we used cultured human osteoblast-like cells. Following incubation of the culture samples in a glucose free medium with 5 μ Ci [¹⁸F]-fluorodeoxyglucose we measured the radioactivity of the cell fraction, as a percent from the initial dose, and compared to the incorporation values in cells treated by protoporphyrine IX (10⁻⁵ M), an endogenous pro-apoptotic agent. To compare the response of [¹⁸F]-fluorodeoxyglucose incorporation studies, following treatment of cells with the protoporphyrine IX, to other experimental cell metabolism evaluation methods, we performed a parallel comparison of alkaline phospatase activity, which is a standard measurement tool of osteoblast metabolism, in the control and treatment groups. A narrow range of 0.22–1.36% of [¹⁸F]-fluorodeoxyglucose incorporation per million cells was found. Additionally in the protoporphyrine IX treated cells a significant 62% decrease of [¹⁸F]-fluorodeoxyglucose incorporation was observed (p < .05). A parallel significant decrease in alkaline phosphatase activity (p < .001) was found in the cells treated by the protoporphyrine IX. Therefore we suggest that the presented method of [¹⁸F]-fluorodeoxyglucose incorporation measurement can be utilized as an effective research tool for estimation of the cellular glycolitic activity in human osteoblast-like cells in vitro.     

Reinitiation of host DNA synthesis in senescent human diploid cells by infection with simian virus 40

Human diploid fibroblasts, TIG-1, cease to proliferate at about 60-62 population doubling level. In their senescent state used in this study, the percentage of nuclei labeled by [3H]thymidine for 48 h was around 1-2% in fresh medium containing 5-40% fetal bovine serum. The percentage of labelled nuclei increased up to 10-fold after infection with SV40. This increase reflects stimulation of cell DNA synthesis because: 1. The increase also occurred when ts A900 was used for infection at the non-permissive temperature, under these conditions viral DNA synthesis is inhibited; 2, the increase paralleled the stimulation of [3H]thymidine incorporation into DNA in a Hirt-precipitate fraction from SV40-infected cells. UV-irradiated SV40 had reduced ability to induce DNA synthesis. A viable deletion mutant of SV40, d1940, had almost the same activity to induce cell DNA synthesis as did wild-type SV40. Equilibrium density gradient centrifugation analysis of DNA labelled with 5-bromodeoxyuridine (BrdU) supported semiconservative replication rather than repair synthesis. We conclude that a considerable fraction of human diploid cells in a senescent population initiate host DNA replication by infection with SV40, although these cells cannot be stimulated with fetal bovine serum.     

Biosynthesis of a glycosylated keratin by human keratinocytes

Human keratinocytes, cultured in the presence of D-[1-14C]glucosamine, incorporated radioactivity into a cytoskeleton-associated glycoprotein with Mr 53,000. This glycoprotein co-purified with prekeratin when keratinocyte cytoskeletons were extracted with 0.1 M citric acid/0.1 M sodium citrate and subjected to isoelectric precipitation at pH 4.0. Analysis of the prekeratin polypeptides by two-dimensional gel electrophoresis revealed that the radioactivity was restricted to a single polypeptide with an isoelectric point in the pH range 4.5-5.5. Acid hydrolysis of prekeratin followed by paper chromatography of the hydrolysate showed that the radioactivity was incorporated as glucosamine and not by metabolic conversion to amino acids. Control experiments showed that the radioactivity associated with the glycoprotein of Mr 53,000 was not the result of adsorbed glycolipids or non-enzymatic labelling. In contrast to the incorporation of D-[1-14C]glucosamine and D-[6-3H]glucosamine, no appreciable amounts of L-[6-3H]fucose, D-[2-3H]mannose or 32PO4 were incorporated into this glycoprotein. The immunological relationship of the glycoprotein of Mr 53,000 to the keratins was demonstrated by its reactivity with both polyclonal and monoclonal antisera to keratin.     

"Matrigenin" activity from bovine bone--II. Effects on the glycosaminoglycans of bovine articular cartilage in culture

1. Bovine articular cartilage slices were studied in long term culture by periodically pulse-labelling the cultures with radiolabelled precursors of glycosaminoglycans and isolating the glycosaminoglycans from cartilage. 2. Pretreatment of the cartilage slices with bacterial collagenase resulted in stimulation of the incorporation of radioactivity into the glycosaminoglycans. 3. The addition of a fraction from bovine bone, enriched in "matrigenin" activity, to cultures of cartilage pretreated with collagenase resulted in an additional increase in the stimulation of incorporation of radioactivity.     

Microbial metabolism of amino alcohols. Biosynthetic utilization of ethanolamine for lipid synthesis by bacteria.

1. Ten bacteria utilizing [2-14C]ethanol-2-amine as the sole or major source of nitrogen for growth on glycerol + salts medium incorporated radioactivity into a variety of bacterial substances. A high proportion was commonly found in lipid fractions, particularly in the case of Erwinia carotovora. 2. Detailed studies of [14C]ethanolamine incorporation into lipids by five bacteria, including E. carotovora, showed that all detectable lipids were labelled. Even where phosphatidylethanolamine was the major lipid labelled, radioactivity was predominantly in the fatty acid rather than the base moiety. The labelled fatty acids were identified in each case. 3. The addition of acetate to growth media decreased the incorporation of radioactivity from ethanolamine into both fatty acid and phosphatidyl-base fragments of lipids from all the bacteria except Mycobacterium smegmatis. Experiments with [3H]ethanolamine and [14C]acetate confirmed that unlabelled acetate decreased the incorporation of both radioactive isotopes into lipids, except in the case of M. smegmatis. 4. Enzyme studies suggested one of two metabolic routes between ethanolamine and acetyl-CoA for each of four bacteria. A role for ethanolamine O-phosphate was not obligatory for the incorporation of [14C]ethanolamine into phospholipids, but correlated with CoA-independent aldehyde dehydrogenase activity.     

人红细胞生成素的分离、纯化及鉴定

 用自制的苯基-琼脂糖CL-4B和羟基邻灰石等层析材料,从再生障碍性贫血病人尿中分离、纯化制得了红细胞生成素(EPO)。用多血小鼠红细胞~(56)Fe参入法测定该制品在体内的生物活力。用小鼠与人骨髓红系祖细胞培养法测其在体外的生物活力。实验结果说明,我们自制的EPO制品,不仅能用于动物,也能用于人骨贿红系祖细胞的培养。用Azocoll法测该制品中蛋白水解酶活力为阴性。     

Stereospecificity of hydrogen transfer by bovine testicular 20 alpha-hydroxysteroid dehydrogenase

The stereospecificity of hydrogen transfer between steroid (17-hydroxyprogesterone) and both natural cofactors by bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) utilizing [17 alpha-3H]estradiol-17 beta and NAD+ or NADP+, respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20 alpha-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20 alpha-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20 alpha-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor. Finally, this work described modifications that represent considerable improvement in the purification and assay of bovine 20 alpha-HSD as originally described.     

Effects of adenosine 3':5'-monophosphate and related agents on ribonucleic acid synthesis and morphological differentiation in mouse neuroblastoma cells in culture.

A variety of compounds were assessed for their ability to induce morphological differentiation and to affect the synthesis of RNA in uncloned mouse neuroblastoma cells in culture. The stimulation of morphological differentiation in uncloned cells after exposure for 48 hours to concentrations of 3 times 10-7 to 3 times 10-4 M papavarine or 10-9 to 10-3 M dibutyryl adenosine 3':5'-monophosphate (dibutyryl-cAMP) was associated, in part, with a concentration-dependent decrease in incorporation of [5-3H]uridine into ribosomal RNA (rRNA) and heterogeneous RNA (HnRNA). The latter effect on cellular RNA produced by papavarine occurred within 1 hour after its addition to the medium and was associated with impaired uptake of radioactive precursor into uridine nucleotides and reduction in the intracellular concentration of uridine 5'-triphosphate (UTP). Dibutytyl-cAMP produced a decreased in the specific radioactivity of UTP without affecting the concentration of UTP in the tumor cells. The effects of papavarine and dibutyryl-cAMP could be distinguished further by the 50% reduction of acetylcholinesterase activity produced by papavarine, but not by dibutyryl-cAMP. Papavarine did not, however, reduce the cellular level of the soluble enzyme, adenine phosphoribosyltransferase. Sodium butyrate, while producing morphological effects similar to those of papavarine and dibutyryl-cAMP at equimolar concentrations, caused no significant changes in the incorporation of [5-3H]uridine into rRNA and HnRNA; however, acetylcholinesterase activity was stimulated 6- to 7-fold above control levels. In contrast to the other differentiating agents examined, addition of 10-9 to 3 times 10-4 M concentrations of cAMP to the tissue culture medium enhanced morphological differentiation of nueroblastoma cells, and caused a 10- to 20-fold stimulation of the incorporation of [5-3H]uridine into rRNA and HnRNA at concentrations of 10-4 M and higher. This effect observed only at high concentrations of cyclic nucleotide was accompanied by an elevation in the specific acitivty of UTP, These studies suggest that the morphological response of neuroblastoma cells is not necessarily associated with concomitant alterations in the synthesis of RNA with agents other than cAMP. Observed changes in incorporation of [5-3H]uridine into RNA appear in most instances to be due to alterations in the uptake of uridine, and in the pool size and specific radioactivity of UTP.