Inhibition of rat liver rhodanese by di-, tricarboxylic, and alpha-keto acids.

Rat liver rhodanese [EC 2.8.1.1] purified by ammonium sulfate fractionation, CM-cellulose and Sephadex G-200 chromatography yielded two active fractions (I & II). Their molecular weights were estimated to be 1.75 X 10(4) (I) and 1.26 X 10(4) (II) by the gel filtration method. Kinetic studies revealed that Fraction I rat liver rhodanese catalyzes thiocyanate formation from thiosulfate and cyanide by a double displacement mechanism. Carboxylic acids such as DL-isocitric, citric malic, pyruvic, and oxaloacetic acid were competitive inhibitors with respect to thiosulfate, whereas fumaric, succinic, and alpha-ketoglutaric acids were noncompetitive inhibitors with respect ot thiosulfate. Incubation of mitochondria with sulfate and alpha-ketoglutaric acid caused a significant decrease in rhodanese activity.     

Cultured fetal rat myoblasts release peptide growth factors which are immunologically and biologically similar to somatomedin

The production of immunologically and biologically active somatomedin activity from isolated myoblasts and fibroblasts from fetal rats of 21 days gestational age was investigated. Myoblast-rich cell populations were derived from primary cultures of dispersed muscle cells by the tendency of myoblasts to become detached from the culture dish in the presence of cytochalasin B. Fibroblasts were obtained from fetal muscle. Culture medium conditioned by exposure to myoblasts for 48 hours produced an increased incorporation of both [35S]sulphate and [3H]thymidine by explants of fetal rat costal cartilage in vitro compared to fresh medium. Myoblast-conditioned medium also contained somatomedin-C-like immunoreactivity which diluted in parallel with partially purified human somatomedin-C (3,271 +/- 446 mU/mg cell protein; mean +/- SEM, seven experiments). Medium conditioned by exposure to fetal rat fibroblasts did not promote isotope uptake by fetal rat cartilage above control values, and contained only low levels of somatomedin-C-like immunoreactivity (343 +/- 89 mU/mg cell protein, three experiments). The release of both somatomedin bioactivity and immunoreactivity into conditioned medium was significantly reduced by the incubation of myoblasts in the presence of rat growth hormone (100 ng/ml and 500 ng/ml). We conclude that fetal rat myoblasts released growth factor activity during culture which exhibited biological and immunologic characteristics of somatomedin. Since the bioactivity was demonstrated on skeletal tissues from rat fetuses of the same gestational age as those that yielded myoblasts such growth factor release may be physiological.     

枯草芽孢杆菌C-D6对辣椒炭疽菌附着胞形成的抑制作用研究

【目的】研究枯草芽孢杆菌(Bacillus subtilis) C-D6菌株对辣椒炭疽菌(Colletotrichum capsici)附着胞形成的抑制作用,探索炭疽病生物防治的新途径。【方法】通过对峙培养测定C-D6菌株的抗菌活性,应用摇瓶培养结合生物测定筛选产生抗菌活性成分的最适培养基,采用硫酸铵分级沉淀、Sephadex G-75凝胶柱层析和阴离子交换层析对抗菌蛋白进行分离纯化,应用聚丙烯酰胺凝胶电泳测定蛋白分子量。【结果】C-D6菌株在PDA平板上对辣椒炭疽菌显示明显的抑制作用,其YPD培养液能完全抑制该菌的附着胞形成。摇瓶培养的结果显示C-D6菌株产生抗菌活性物质的最适培养基为YPD培养基。C-D6菌株在该培养基中培养14 h后,所形成的活性物质可完全抑制辣椒炭疽菌的附着胞形成。从该菌的YPD培养液中分离获得一个分子量为32 kD,能明显抑制辣椒炭疽菌附着胞形成的抗菌蛋白。【结论】C-D6菌株的生防特征显示该菌株对防治辣椒炭疽菌引起的炭疽病具有潜在的应用价值。     

A muscle-derived substrate-bound factor that promotes neurite outgrowth from neurons of the central and peripheral nervous systems

The development and survival of spinal motor neurons depends upon muscle-derived trophic factors. Some circumstantial evidence suggested to us that the regulatory subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase (cAMP-dPK)-type II might be involved in neuritic outgrowth from spinal neurons. In the present study, we tested a commercial preparation of cAMP-dPK for neurite-promoting activity. Commercial cAMP-dPK-type II from skeletal and cardiac muscles elicited a significant neurite outgrowth from cultured embryonic chicken neurons when the enzyme preparation was bound to polylysine-coated substrata; type I cAMP-dPK from skeletal muscle was ineffective. Neither cAMP-dPK-type I nor -type II had a significant effect on the survival of spinal neurons in culture. Type II cAMP-dPK also stimulated neurite outgrowth from chicken cerebral hemisphere neurons, dorsal root ganglionic neurons, ciliary ganglionic neurons, and rat sympathetic ganglionic neurons in culture. The neurite-promoting activity appears to reside in a contaminant of the preparation since neither the purified regulatory nor catalytic subunits of cAMP-dPK-type II had an effect on neurite outgrowth per se from cultured neurons and since neurite-promoting activity did not correlate with [3H]cAMP binding or cAMP-dependent kinase activity. The neurite-promoting protein was then partially purified from commercial cAMP-dPK-type II by gel filtration on Sephadex G-200 followed by ion-exchange chromatography on DE-52 cellulose. Sodium dodecyl sulfate gel electrophoresis of the active protein peak revealed a major protein band (MW 50 kDa) and several minor bands (e.g., MW 200 kDa, 52 kDa, 45 kDa). Also, immunoblot analysis and immunoprecipitation revealed that the partially purified neurite-promoting protein was distinct from laminin, heparan sulfate proteoglycan, nerve growth factor, neural cell adhesion molecule, and fibronectin. Furthermore, the neurite-promoting activity was not diminished by treatment with heparinase nor was it bound to heparin conjugated to Sepharose. Our results demonstrate that a protein unrelated to laminin or its associated macromolecules and which copurifies with the type II cAMP-dPK of striated muscle stimulates neurite outgrowth from neurons of the central and peripheral nervous systems.     

Catabolism of newly synthesized decorin by explant cultures of bovine ligament.

The catabolism of newly synthesized decorin by explant cultures of bovine collateral ligament was investigated. The tissue was placed in explant culture for 6 days then incubated with radiolabeled sulfate for 6 h and replaced in culture for 5 days to allow for the loss of the radiolabeled large proteoglycan. The metabolic fate of the remaining radiolabeled decorin present in the matrix of the tissue over the next 9-day period was determined. It was shown that this pool of decorin was lost from ligament explant cultures either directly into the culture medium or taken up and degraded within the cells of the tissue. The intracellular degradation of the radiolabeled pool of decorin by ligament explant cultures was shown to result in the generation of [35S]sulfate. This process required metabolically active cells and involved the lysosomal system since sulfate generation was inhibited when cultures were maintained at 4 degrees C or in the presence of either 10 mM ammonium chloride or 0. 05 mM chloroquine. The inhibition of intracellular processing of decorin resulted in an increase in the rate of loss of this proteoglycan into the medium of the cultures. The inhibition of intracellular degradation of decorin was reversible on incubation of the explant cultures at 37 degrees C or removal of ammonium chloride from the culture medium. After removal of the ammonium chloride from the culture medium the rate of intracellular catabolism was greater than that observed in cultures maintained in medium alone, which suggested that there was an intracellular accumulation of native and/or partially degraded material within the cells.     

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.     

Purification and characterization of lymphocytic clonal growth factor (LCGF) derived from human-human hybridoma SH-76 cells

Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma.     

Growth control in cultured 3T3 fibroblasts. Assays of cell proliferation and demonstration of a growth inhibitory activity

Treatment of sparse, proliferating cultures of 3T3 cells (target cells) with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium (UCM). This differential effect of conditioned medium (CM) and UCM on target cells was demonstrated using three assay systems: (a) assessment of total cell number; (b) measurement of [3H]thymidine incorporated into acid-precipitable DNA; and (c) determination of the percentage of radioactively labeled nuclei in individual cells after incorporation of [3H]thymidine. The difference in the total incorporation of [3H]thymidine in CM-treated and UCM-treated cells was reflected by a difference in the percent of labeled cells. There was no differences in the average number of grains per labeled cell in the two cultures. Moreover, the inhibitory effect of the CM on target cell proliferation was reversible. Finally, this growth inhibitory activity can be collected in serum-free medium, precipitated by ammonium sulfate, and fractionated by gel filtration. In these purification procedures, the inhibitory activity was consistently found to be associated with the protein-containing fractions of the CM. No activity was found upon similar treatment with UCM. These results suggest that a system has been developed for the purification and molecular analysis of growth inhibitory factors that may mediate growth control in culture fibroblasts.     

Detection and partial characterization of a specific plasminogen activator inhibitor in human chondrocyte cultures

Serum-free culture medium collected from primary monolayer cultures of human articular chondrocytes was found to inhibit human urokinase [EC 3.4.21.31] activity. Although chondrocyte culture medium contained a small amount of endothelial-type plasminogen activator inhibitor which could be demonstrated by reverse fibrin autography, most of the urokinase inhibitory activity of chondrocyte culture medium was shown to be due to a different molecule from endothelial-type inhibitor, since it did not react with a specific antibody to this type of inhibitor. The dominant urokinase inhibitor in chondrocyte culture medium was partially purified by concanavalin A-Sepharose affinity chromatography. The partially purified inhibitor inhibited high-Mr urokinase more effectively than low-Mr urokinase, but no obvious inhibition was detected against tissue-type plasminogen activator, plasmin, trypsin, and thrombin. The inhibitor had an apparent Mr of 43,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and it was unstable to sodium dodecyl sulfate, acid, and heat treatments. Inhibition of urokinase by the inhibitor was accompanied with the formation of a sodium dodecyl sulfate-stable high-Mr complex between them. Inhibition and complex formation required the active site of urokinase. The partially purified inhibitor was thought to be immunologically different from the known classes of plasminogen activator inhibitors, including endothelial-type inhibitor, macrophage/monocyte inhibitor, and protease nexin, since it did not react with specific antibodies to these inhibitors.     

COMPARISON OF LEVELS OF ADENOSINE 3'',5''-MONOPHOSPHATE IN HIGHLY PURIFIED AND MIXED PRIMARY CULTURES OF NEURONS AND NON-NEURONAL CELLS FROM EMBRYONIC CHICK SYMPATHETIC GANGLIA

Primary cultures containing ≥99% neurons, ≥99% non-neuronal cells (glia), or both cell types were prepared from the sympathetic ganglia of 12-day chick embryos. Levels of cyclic AMP in the non-neuronal cells (～14 pmol/mg protein) were approximately 3-fold higher than levels in the neurons (～4 pmol/mg protein). Mixed cultures had concentrations of cyclic AMP which fell between the values measured for pure neuronal and pure non-neuronal cultures. The measured cyclic AMP values of mixed cultures were indistinguishable from values predicted by summing the expected contributions of the neurons and non-neuronal cells. Thus, contact between the neurons and non-neuronal cells in these mixed cultures did not appear to alter the level of cyclic AMP in either cell type. Neuronal-glial interactions, such as the specific neuronal stimulation of non-neuronal cell proliferation, occurred independently of any changes in the level of cyclic AMP in the mixed cultures. Cell density was varied in both pure and mixed cultures, and both cyclic AMP concentrations and amounts of [³H]thymidine incorporation into DNA were measured. The cyclic AMP content of the non-neuronal cells varied inversely with cell density. [³H]Thymidine incorporation was independent of cell density in both neuronal and non-neuronal cultures. Parallel density-dependent decreases in cyclic AMP concentration and [³H]thymidine incorporation were observed in mixed cultures as cell density was increased. The data suggest that there is no relationship between changes in rate of non-neuronal cell proliferation and cyclic AMP levels in these cultures.     

Purification and Properties of Adenosine Nucleosidases from Tea Leaves

Three adenosine nucleosidases (adenosine ribohydrolase, EC 3.2.2.7) with high substrate specificity were isolated from the extracts of tea leaves by a procedure including fractionation with ammonium sulfate, column chromatography on DEAE- and CM-cellulose, and gel filtration on Sephadex G-100. They were designated adenosine nucleosidase I, II and III, respectively, and their properties were characterized.

Among the naturally occurring nucleosides only adenosine and 2′-deoxyadenosine were hydrolyzed by these three enzymes and cleavage rate of the N-glycosidic bond in 2′-deoxyadenosine was three or four times greater than that in adenosine.     

Staphylococcal Hyaluronate Lyase: Purification and Characterization Studies

Staphylococcal hyaluronate lyase (hyaluronidase) derived from a pathogenic strain of staphylococcus was purified by means of salt fractionation with ammonium sulfate and gel filtration through Sephadex G-100. Most of the enzyme activity from concentrated culture supernatant fluids of staphylococci was obtained in a fraction precipitated by 90 to 100% saturation with ammonium sulfate. A small amount of enzyme was also precipitated by 80 to 90% saturation with the salt. The hyaluronidase-rich fractions did not contain other staphylococcal enzymes, such as coagulase, protease, lipase, and staphylokinase. These enzymes were present in the original concentrates. Molecular sieving chromatography of the partially purified enzyme by filtration through Sephadex G-100 resulted in a further increase in specific enzyme activity. However, more than one active peak was obtained after gel filtration, thus suggesting that there may be more than one molecular form of the enzyme. Immunodiffusion in agar gel of the chromatographically purified enzyme fraction, with immune serum from rabbits injected with concentrated staphylococcal culture supernatant fluids, indicated that there was one major antigen. A similar antigen, giving reactions of identity with the purified material, was present in the original culture supernatant fluid.     

Immunochemical and Biochemical Studies of Fungi

Polysaccharides were separated from mycelia and culture filtrates of the filamentous fungi Aspergillus niger and Penicillium chrysogenum, and purified by column chromatography on Sephadex G-50, DEAE- and CM-cellulose, successively. No nitrogen and phosphorus were detected in the polymer, and the sugar components were observed to be galactose and mannose. The polysaccharides were confirmed to be galactomannans which were easily hydrolyzed by weak acid, liberating galactofuranose and oligosugar in dialyzable fractions.     

Acetylcholine Synthesis by Adult Bovine Adrenal Chromaffin Cell Cultures

Adrenal chromaffin cells normally synthesize and release catecholamines. In the present study, [3H]acetylcholine synthesis and another characteristic of cholinergic neurons, [3H]choline uptake, were studied in cultures of adult bovine adrenal chromaffin cells. Chromaffin cell cultures took up [3H]choline from the medium and acetylated the [3H]choline to form [3H]acetylcholine. The rate of [3H]acetylcholine synthesis increased after 19 days in culture and continued to increase up to 28 days in culture. [3H]Acetylcholine synthesis could be increased by stimulating the cells with a depolarizing concentration of K+. The ability for K+ to stimulate synthesis of [3H]acetylcholine developed only after 28 days in culture. [3H]Choline was taken up by the cultures through a single mechanism with a high (to intermediate) affinity for choline. [3H]Choline uptake was enhanced by Na+ omission in day-14 cultures, but was at least partially Na+-dependent in day-29 cultures. Hemicholinium-3 (IC50 less than 10 muM) inhibited [3H]choline uptake into chromaffin cell cultures. It is concluded that bovine adrenal chromaffin cells, maintained in culture, are able to exhibit cholinergic properties and this capacity is retained even by the mature adult cell.     

Isolation,partial purification and preliminary characterization of a bacteriocin from Streptococcus mutans Rm-10

An extracellular bactericidal substance was isolated from the supernatant of Streptococcus mutans Rm-10 culture fluid and partially purified with 60% ammonium sulfate precipitation, differential centrifugation, and gel filtration on Sephadex G-200. There was a good correlation of the sensitivity profiles of indicator strains whether assayed on solid medium or with purified material from cell-free culture fluid, indicating that the same inhibitory substance is produced on solid medium and in broth. Vapor from organic solvents such as chloroform, acetone, ethanol, and ether as well as heat treatment at 100 degrees C for 30 min had little effect on the bactericidal factor. It was sensitive to trypsin and pronase and resistant to deoxyribonuclease, ribonuclease, lysozyme, and phospholipase C. The inhibitor was not infective, and electron microscopic studies failed to reveal phage or phage-like particles in concentrated solutions of the bactericidal material. The results indicate that the extracellular bactericidal substance is indeed a bacteriocin. Activity in broth cultures reached a maximum only after exponential growth had ceased. It was active against other streptococcal strains as well as strains of Actinomyces naeslundii, A. viscosus, Bacillus subtilis and Staphylococcus aureus, but not against strains of Fusobacterium nucleatum and Escherichia coli.     

Growth control in cultured 3T3 fibroblasts II. Molecular properties of a fraction enriched in growth inhibitory activity

Treatment of sparse, proliferating cultures of 3T3 cells with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium. This growth inhibitory activity was fractionated by ammonium sulfate precipitation and gel filtration, yielding one fraction that was 35-fold enriched in specific activity. Analysis of the chemical and biological properties of this highly active fraction indicated that: (a) it is an endogenous cell product, synthesized by the 3T3 cells and shed into the medium; (b) it is a protein and its activity is sensitive to treatment with pronase; (c) the constituent polypeptide chains have molecular weights of 10,000 and 13,000; and (d) it is not cytotoxic and its effect on target cells are reversible. These results suggest that we have partially purified from conditioned medium an endogenous growth regulatory factor that may play a role in density-dependent inhibition of growth in cultured fibroblasts. We propose the term Fibroblast Growth Regulator to describe this class of molecules.     

Purification of a kappa-carrageenase from marine Cytophaga species

A mixture of extracellular carrageenases was isolated from the cell-free medium of a culture of marine Cytophaga sp. 1k-C783 grown on ZoBell 2216 E broth with 0.1% commercial carrageenan. A single active peak of kappa-carrageenase was separated and purified from the mixture by ammonium sulfate precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration chromatography. Molecular weight of the purified kappa-carrageenase was estimated as 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified kappa-carrageenase had pH optimum 7.6 and temperature optimum 25 C.     

Purification and biologic properties of fibroblast somatomedin

Cultured human fibroblasts produce a peptide growth factor that cross-reacts with antisera to human somatomedin-C (Sm-C). To determine the identity of this species and compare its molecular properties to pure Sm-C, 2 liters of conditioned medium derived from human fibroblast monolayers were concentrated (X10) by ultrafiltration. The concentrated conditioned medium was purified further by CM-Sephadex ion-exchange chromatography. Following elution in 1.0 M NaCl, pH 8.0, the active material was purified by gel filtration on Sephadex G-150. The active fractions which eluted at Kd 0.45 (Mr estimated at 32,000) were further purified by isoelectric focusing. Two peaks of activity electrofocused at pI 5.4 and 7.2, respectively. The pI 5.4 peak contained only binding protein activity. The active fractions from the neutral pool were further purified by reverse-phase high pressure liquid chromatography on a C-18 Bondapak with a linear gradient of acetonitrile (10-60%). The active single peak which eluted at 55% acetonitrile gave a single band when analyzed by polyacrylamide gel electrophoresis. This material stimulated [3H]thymidine incorporation into human fibroblast DNA with approximately 3.2 times the potency of pure Sm-C but was equipotent in stimulating BALB/c 3T3 fibroblasts. It was degraded by fibroblast cultures at a slower rate compared to Sm-C, although it had a similar affinity for Sm-C-binding protein. We conclude that human fibroblasts produce two peptides that react with anti-Sm-C antibody but are chemically distinct from Sm-C. The greater response to fibroblast somatomedin may be due to its affinity for somatomedin-binding protein and slower degradation. These findings may have implications for understanding the regulation of human fibroblast replication.     

Hypoxanthine-DNA glycosylase from Escherichia coli. Partial purification and properties

Hypoxanthine-DNA glycosylase from Escherichia coli was partially purified by ammonium sulfate fractionation and by chromatography on Sephacryl S-200, DEAE-cellulose, and phosphocellulose P-11 columns. Analysis of the enzymatic reaction products was carried out on a minicolumn of DEAE-cellulose and/or by paper chromatography, by following the release of the free base [3H]hypoxanthine from [3H]dIMP-containing phi X174 DNA. In native conditions, the enzyme has a molecular mass of 60 +/- 4 kDa, as determined by gel filtration on Sephadex G-150 and Sephacryl S-200 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a major polypeptide band of an apparent molecular mass of 56 kDa, and glycerol gradient centrifugation indicated a sedimentation coefficient of 4.0 S. Hypoxanthine-DNA glycosylase from E. coli has an obligatory requirement for Mg2+ and is totally inhibited in the presence of EDTA. Co2+ can only partially replace Mg2+. The enzyme is inhibited by hypoxanthine which at 4 mM causes 85% inhibition. The optimal pH range of the enzymatic activity is 5.5-7.8, and the apparent Km value is 2.5 x 10(-7) M.