Purification and biological properties of a cell growth-stimulating factor from Clostridium perfringens FERM P-14028.

The active component that stimulates fibroblast growth was purified from cultured Clostridium perfringens FERM P-14028, and a quantitative assay method for the biological activity was established. The active component was named cell growth-stimulating factor (CGSF), and the molecular weight of this factor was estimated to be 420 kDa on gel permeation chromatography. CGSF(50-100 ng/ml) stimulated the growth rate of BHK-21 (C-13) cells in the logarithmic growth phase, and shortened the doubling time by 16-18%, but had no effect on confluent cells. These actions were indicated only with medium containing fetal bovine serum. These results suggest that CGSF is a novel protein that regulates cell growth via a mechanism of action different from that of other growth factors.

Structural elucidation of a variety of GalNAc-containing N-linked oligosaccharides from human urinary kallidinogenase.

Fifteen different structures of terminal GalNAc-containing N-linked oligosaccharides from human urinary kallidinogenase have been identified. These N-linked oligosaccharides were mostly neutral, because sialic acid content was lower than 0.13 mol of sialic acid/mol of sugar chain, and sulfate was not detected. The oligosaccharides were released from pepsin-digested protein by glycoamidase A (from almond) digestion. The reducing ends of the oligosaccharide chains were aminated with a fluorescent reagent, 2-aminopyridine. The resulting mixture of pyridylamino derivatives of the oligosaccharides were separated by high performance liquid chromatography on an ODS-silica column, and 15 oligosaccharides were isolated. The structure of each oligosaccharide fraction was analyzed by two-dimensional sugar mapping, component sugar analysis, high resolution proton nuclear magnetic resonance and methylation analysis. It was found that each N-linked oligosaccharide associated with human urinary kallidinogenase contains unsubstituted GalNAc residues at the nonreducing terminal. These 15 oligosaccharides include 5 biantennary, 7 triantennary, and 3 tetraantennary oligosaccharides.

Two-dimensional elution map of GalNAc-containing N-linked oligosaccharides.

We have previously published a two-dimensional (2-D) mapping technique for N-linked oligosaccharides using pyridylaminated derivatives (PA-oligosaccharides) (N. Tomiya et al. Anal. Biochem. 171, 73-90, 1988). We now report an extension of this method to GalNAc-containing N-linked oligosaccharides. The new 2-D map was prepared from the elution data of 40 different GalNAc-containing oligosaccharides, 16 of which were obtained directly from human urinary kallidinogenase by digestion with glycopeptidase A. The other 24 oligosaccharides were derived by subsequent digestion of the 16 original oligosaccharides with beta-galactosidase or alpha-fucosidase. Each of the 40 oligosaccharide derivatives was separated by high-performance liquid chromatography using ODS-silica and amide-silica columns. The 2-D map constructed by plotting elution position of each oligosaccharide (expressed in terms of glucose units) can be useful as such in delineating the structure of an unknown oligosaccharide by direct placement of its elution positions in the 2-D map. Multiple regression analysis of the data as performed previously yielded parameters related to the contribution of each component monosaccharide unit to the elution profile. The best results were obtained when the GalNAc-containing PA-oligosaccharides were classified into an F-series (those containing Fuc alpha 6GlcNAc-PA) and a Z-series (all others), based on our previous classification method. These calculated values are useful in predicting oligosaccharide structure from known elution values as well as to predict elution volumn from a known structure. The structure of a minor GalNAc-containing oligosaccharide in human urinary kallidinogenase was elucidated using these newly calculated values.

Determination of monosaccharides and sugar alcohols in tissues from diabetic rats by high-performance liquid chromatography with pulsed amperometric detection.

A sensitive and simple high-performance liquid chromatographic method has been developed to determine the concentration of monosaccharides and sugar alcohols in animal tissues. Five neutral monosaccharides (D-glucose, D-galactose, D-mannose, D-fructose, and D-ribose) and three neutral sugar alcohols (myo-inositol, glycerol, and D-sorbitol) predominate in the renal cortices and sciatic nerves of rats. These monosaccharides and sugar alcohols were extracted with distilled water, purified by deproteinization with ethanol, a Sep-Pak C18 cartridge, and columns of Dowex 50W-X8 and Amberlite CG-400, then separated on Ca2+ and Pb2+ cation-exchange columns, eluted with deionized distilled water at 80 degrees C, and detected using integrated pulsed amperometry. About 10 pmol of each sugar was detectable with a signal-to-noise ratio of 10:1. D-Glucose, D-fructose, D-sorbitol, and D-mannose were higher in both the renal and sciatic tissues of diabetic rats than in those of normal animals. D-Ribose and glycerol were higher in the renal cortex of diabetic animals.

Calculated two-dimensional sugar map of pyridylaminated oligosaccharides: elucidation of the jack bean alpha-mannosidase digestion pathway of Man9GlcNAc2.

We have developed a two-dimensional (2-D) mapping of pyridylaminated oligosaccharides as an aid to structural determination of glycoprotein-derived oligosaccharides. Using the available data of reverse-phase HPLC of pyridylamino-oligosaccharides, this was further extended to parameterization of unit contribution by each sugar component, which allows the prediction of possible structures from the elution volume. We have extended this approach to the data obtained with amide-silica HPLC column to obtain a calculated 2-D mapping technique for the oligomannose-type oligosaccharides (M-series). In this method, the elution volumes of all possible pyridylamino-oligosaccharides up to the size of Glc1Man9GlcNAc2 (50 in total) are calculated from the established UC values to construct a 2-D map. To test the validity of the calculated 2-D map, the structures of intermediate PA-oligosaccharides generated during the alpha-mannosidase (jack bean) digestion of Man9GlcNAc2 (porcine thyroglobulin) were analyzed to establish the digestion pathway. The validity of this approach is substantiated by an independent deduction of the intermediate structures based on structural relationships and the coincidence of elution volumes. Our results agree well with the recently published digestion pathway of Man5GlcNAc2 by the same enzyme and that of Man9GlcNAc2 by lysosomal alpha-mannosidase.

Structural analyses of asparagine-linked oligosaccharides of porcine pancreatic kallikrein.

The structures of asparagine-linked oligosaccharides of porcine pancreatic beta-kallikrein are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The mixture of pyridylamino oligosaccharides was separated by reverse-phase and amide-adsorption high-performance liquid chromatography. The pyridylamino oligosaccharides were separated into more than 50 kinds of oligosaccharides. The structures of 5 kinds of triantennary and 12 kinds of tetraantennary oligosaccharides were determined by the use of high-resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Furthermore, the structures of five kinds of oligomannose-type oligosaccharides were elucidated by a combination of exoglycosidase digestion and high-performance liquid chromatography. 1H NMR data for 14 out of the 17 kinds of N-acetyllactosamine-type oligosaccharides reported here have not previously been described in the literature. (1) It has been shown that fucose containing tri- and tetraantennary oligosaccharides is predominant in porcine pancreatic beta-kallikrein B. (2) It has also been shown that the heterogeneity of the structure in these types of oligosaccharides is derived from the variety of the positions of galactose residues linked to outer N-acetylglucosamine residues. (3) The distribution of oligosaccharides into two glycosylation sites, asparagine-95 and asparagine-239, of beta-kallikrein B was determined. It has been found that oligomannose-type oligosaccharides are exclusively present at asparagine-239, although N-acetyllactosamine-type oligosaccharides occur at both glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Analyses of N-linked oligosaccharides using a two-dimensional mapping technique.

We propose a two-dimensional sugar map method for the simple, reproducible, and sensitive analysis of the structures of N-linked oligosaccharides. The structure of an unknown oligosaccharide can be characterized from its position on the map. The data base for the sugar map is prepared by the use of 113 kinds of standard oligosaccharides, 58 of whose structures have been confirmed by 1H NMR spectroscopy. The present method involves six steps, (i) preparation of oligosaccharides from glycopeptides by N-oligosaccharide glycopeptidase (almond) digestion, (ii) derivatization of the reducing ends of oligosaccharides with a fluorescent reagent, 2-amino-pyridine, by using sodium cyanoborohydride, (iii) separation of oligosaccharide derivatives by high-performance liquid chromatography with an ODS-silica column, (iv) analysis of the size of each separated oligosaccharide on an amide-silica column, (v) plotting of the elution position of a sample on the two-dimensional sugar map obtained for the standard oligosaccharides, and (vi) structural analysis of the oligosaccharides by a combination of sequential exoglycosidase digestion and the steps (iii-v). The present method was applied to the identification of the structures of oligosaccharides in hen ovalbumin. It was found that two unusual oligosaccharides that have not yet been reported exist in ovalbumin.

Modification of acyl-plasmin-streptokinase complex with polyethylene glycol. Reduction of sensitivity to neutralizing antibody.

Acyl-plasmin-streptokinase complex has advantages as a 'site' directed fibrinolytic agent with the active site protected from the plasma protease inhibitors. But, in clinical use, the fibrinolytic potential of this acyl-enzyme complex is modified or abolished by the presence of streptokinase antibodies in the patients. Therefore, better therapeutic agents are required. In this work, chemical modification of the acyl-plasmin-streptokinase complex with polyethylene glycol was found to result in marked resistance to neutralization with streptokinase antibodies.

Effects of purified hog pancreatic kallikrein on the kinin-prostaglandin system and renin-angiotensin-aldosterone system.

In the present study, hog pancreatic kallikrein (HPK, 2,000 KU/kg body weight) was intramuscularly injected into male rabbits and several plasma hormones [kinin, prostaglandin E (PGE), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), thromboxane B2 (TXB2), plasma renin activity (PRA), aldosterone, and ACTH] were measured before and after the HPK administration, in order to clarify the role of glandular kallikrein in the blood. The plasma kinin concentrations were significantly (p less than 0.001) increased from 1 +/- 1 (mean +/- SE), the baseline level, to 230 +/- 22, 288 +/- 36 and 130 +/- 9 pg/ml at 30, 60, and 120 min, respectively, after HPK administration. The plasma level of PGE were slightly increased after HPK administration, but the change was not significant compared to the mean baseline level. The plasma levels of 6-keto-PGF1 alpha were significantly increased from 229 +/- 38, the baseline level, to 594 +/- 131 (p less than 0.05) at 30 min after the administration of HPK and tended to increase (378 +/- 67) at 60 min after the HPK administration, but fell to 278 +/- 37 pg/ml at 120 min after the administration of HPK. On the other hand, the changes in plasma TXB2, aldosterone, ACTH, and PRA before and after HPK administration were not significant. The present results showed that exogenous intramuscular administration of HPK increased the plasma levels of kinin and prostacyclin, but caused no elevation in the plasma levels of other hormones including PRA. It is concluded, therefore, that in this acute experiment, there was a close relationship between the kallikrein-kinin system and prostaglandins.

Saturated fatty acid-starved cells of Saccharomyces cerevisiae grown in the presence of cerulenin and oleic acid.

Cell growth of Saccharomyces cerevisiae ATCC 12341 inhibited by the antibiotic cerulenin, a specific inhibitor of fatty acid synthesis, was restored by oleic acid (18 : 1) to give saturated fatty acid-starved cells, which could not grow when again transferred into a fresh synthetic medium containing the antibiotic and oleic acid. The growth of the saturated fatty acid-starved cells was restored when they were transferred into a medium supplemented with myristic acid (14 : 0), pentadecanoic acid (15 : 0), and palmitic acid (16 : 0) in the presence of cerulenin and oleic acid. Cellular saturated fatty acid content in the growth-restored cells was also restored to about two-thirds of that of the normal yeast cells. The DNA, RNA, and cell wall synthetic capabilities of the saturated fatty acid-starved cells were almost normal, but the L-leucine uptake and cytochrome pattern were severely impaired. These impairments were reversed on supplying palmitic acid. The decrease of L-leucine uptake of the yeasts was also caused by the addition of cerulenin alone. However, since the decrease occurred later than the inhibition of fatty acid synthesis, it was considered to be a secondary effect. These results, obtained by using the saturated fatty acid-starved cells, indicate that the membranes of S. cerevisiae require certain amounts of saturated fatty acid and that the membrane functions (energy metabolism, transport, and so on) are impaired by starvation of saturated fatty acids.

Production of 9-beta-D-arabinofuranosyladenine by a new species of Streptomyces and its herbicidal activity.

9-beta-D-Arabinofuranosyladenine (ara-A) was produced by a new species of Streptomyces designated as S. herbaceus. Ara-A was found to have potent herbicidal activity against Echinochloa crus-galli, Digitaria adscendens and Chenopodium ficifolium by the treatment with ara-A before germination of these plants. However, Oryza sativa had strong resistance to ara-A.

A new antibiotic, setomimycin, produced by a strain of Streptomyces.

A new antibiotic, setomimycin, was isolated from the culture broth of strain AM-2947, which was identified as Streptomyces pseudovenezuelae. The compound is a weakly acidic substance, and has UV-absorption at 228, 268 and 422 nm and a molecular formula of C34H28O9 (MW 580). It is active against Gram-positive bacteria including Mycobacteria, and has antitumor activity against Sarcoma-180 solid tumor in mice.

Isolation and characterization of elasnin, a new human granulocyte elastase inhibitor produced by a strain of Streptomyces.

Elasnin, a new human granulocyte elastase inhibitor, produced by the strain of KM-2753 designated as Streptomyces noboritoensis KM--2753 has been isolated from the fermentation broth by column chromatography on silica gel and neutral alumina. Elasnin is a neutral, colorless, and viscous oil (ND17 = 1.4983, [alpha]18D -0.9 degrees, lambdaEtOHmax 291 nm (epsilon, 7,760) having a molecular formula of C24H40O4 (MW 392) as shown by its elemental analysis and mass spectrum. Elasnin markedly inhibits human granulocyte elastase, but it is almost inactive against pancreatic elastase, chymotrypsin, and trypsin. At 1.3 microgram/ml (3.3 X 10(-6) M), elasnin is 50% inhibitory to human elastase, but it causes 50% inhibition of pancreatic elastase at 30.1 microgram/ml (76.8 X 10(-6) M).

Production of deoxyfrenolicin and a new antibiotic, frenolicin B by Streptomyces roseofulvus strain AM-3867.

Two antibiotics of frenolicin group, antibiotic AM-3867 I and II were isolated from the fermentation broth of Streptomyces roseofulvus strain No. AM-3867, a soil isolate. The former was a new antibiotic designated as frenolicin B and its structure containing gamma-lactone was determined, while the latter was identified as deoxyfrenolicin having been chemically prepared from frenolicin.

Aurantinin, a new antibiotic of bacterial origin.

A new polyene class antibiotic, aurantinin (KM-214), was isolated from the fermentation broth of Bacillus aurantinus MASUMA and OMURA sp. nov. The substance is a conjugated triene with a molecular weight of 618 and molecular formula C35H54O9, and melts at 139 approximately 140 degrees C. The antibiotic is active in vitro against Gram-positive bacteria, but not against yeast and fungi.

New antitumor antibiotics, OS-4742 A1, A2, B1 and B2 produced by a strain of Streptomyces.

New antibiotics, OS-4742 A1, A2, B1 and B2, were isolated from the culture broth of the strain OS-4742, which was designated as Streptomyces matensis subsp. vineus. These compounds have anthracycline chromophores and sugar moieties, but do not contain nitrogen. They possess antimicrobial activities against Gram-positive bacteria and antitumor activities against S-180 solid tumor on mice.

A new alkaloid AM-2282 OF Streptomyces origin. Taxonomy, fermentation, isolation and preliminary characterization.

AM-2282, a new alkaloid has been isolated from cultures of Streptomyces sp. AM-2282 by solvent extraction and silica gel chromatography. The compound exhibits a strong absorption maximum at 292 nm and shows antimicrobial activity against fungi and yeast. The LD50 of its hydrochloride (i.p. in mice) is 6.6 mg/kg. The molecular formula of AM-2282 has been determined as C28H26N4O3. The producing strain, AM-2282 was classified as a new species and the name, Streptomyces staurosporeus AWAYA, TAKAHASHI and OMURA, nov. sp. is proposed.