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From the organic extract of a deep-water Hawaiian sponge Dactylospongia sp., a new potent antioxidant and antimicrobial meroterpenoid, puupehenol (1), was isolated. The structure of 1 was determined using spectroscopic techniques ((1)H and (13)C NMR, MS, IR, UV, [α]D). The known compound puupehenone (2) was also isolated and suggested as a probable artifact of the isolation procedures. Complete unambiguous (1)H and (13)C NMR data are provided for compounds 1 and 2. Bioassays performed with 1 and 2 showed them both to be very effective antioxidants and to have antimicrobial properties.

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.

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.

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.

Sporulation of Saccharomyces cerevisiae G2-2 was inhibited by the antibiotic cerulenin which is known to be a specific inhibitor of fatty acid and sterol synthesis. This inhibition was reversed by various fatty acids, especially by oleic acid (C(18:1)) and pentadecanoic acid (C(15:0)). Ergosterol showed only slight reversibility of this inhibition. When cerulenin was added to the sporulation medium later than 12 h after the start of incubation, the marked inhibition disappeared. When the fatty acid fraction extracted from the sporulated yeasts was added to the cells pretreated with cerulenin for more than 6 h, sporulation became evident 6 h after the fatty acid fraction addition. Therefore, sufficient synthesis of fatty acids required for sporulation was assumed to occur during the first 6 h in phase I of yeast sporulation.

Cerulenin, a specific inhibitor of beta-ketoacyl-(acyl carrier protein) synthetase [EC 2.3.1.41] and 3-hydroxy-3-methylglutaryl-CoA synthetase [EC 4.1.3.5], was studied to determine whether it inhibits 6-methylsalicylic acid synthesis, in which so-called "polyketide" formation, a condensation step similar to that in fatty acid synthesis, is involved. In fact, 100 mug/ml (4.5 X 10(-4) M) of cerulenin inhibited 60% of 6-methylsalicylic acid synthetase activity and 68% of fatty acid synthetase activity of Penicillium urticae.

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).

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.

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.

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.

The structure-activity relationships of derivatives of the antibiotic cerulenin were investigated by chemically modifying dodecanoic acid, its skeletal back-bone. The dimethylamide derivatives were active against both gram-positive and -negative bacteria, and fungi. Among the compounds having modified groups at positions C2 and C4, the most active were those with a carbonyl group at C4 and a double bond at C2. The dimethylamide and pyrrolidine amide derivatives of this structure type were the most active. Activity against bacteria and yeast increased with the number of carbon atoms in the skeleton, with the maximum activity being observed at C=12. No significant differences in activity against fungi were observed with change in chain length.

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.

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)

Two metabolites have been isolated from the fermentation broth of a new species of Kitasatoa, Kitasatoa griseophaeus. These alkaloids have been identified as quinoline-2-methanol and quinoline-2-methanol acetate. The former exhibits hypoglycemic activity in the rat.

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.

Nanomycins are new antibiotics produced by the strain OS-3966 which was designated Streptomyces rosa var. notoensis. Nanomycins A and B were isolated from the culture filtrate by extraction with organic solvent and silica gel chromatography. The physical and chemical properties suggest that nanaomycins A and B are quinone-related compounds having the molecular formulae, C16H14O6 and C16H16O7, respectively. Nanaomycins A and B inhibit mainly mycoplasmas, fungi and Gram-positive bacteria. The acute toxicities (LD50, ip) of nanaomycins A and B in mice are 28.2 and 169 mg/kg, respectively.

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.

Cell growth of Saccharomyces cerevisiae ATCC 12341 inhibited by the antibiotic cerulenin, a specific inhibitor of fatty acid and sterol syntheses, was reversed by various exogenous fatty acids. Myristic acid (14 : 0), pentadecanoic acid (15 : 0), palmitic acid (16 : 0), and oleic acid (18 : 1) reversed effectively the growth inhibition by cerulenin. When these cells were reversed by adding pentadecanoic acid, over 90% of native even-numbered fatty acids was substituted by odd-numbered fatty acids. Those in the cells reversed by adding oleic acid were almost all unsaturated fatty acids. Cerulenin did not inhibit either elongation or desaturation systems in S. cerevisiae.

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.

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.

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.

CM-55 is a synthetic analogue of the antibiotic cerulenin with the chemical structure of 2, 3-dodecenyl-4-oxo-dimethyl amide. This compound inhibited the growth of Saccharomyces cerevisiae ATCC 12341 and inhibited protein and lipid synthesis by 91 and 95%, respectively, at a concentration of 50 mug/ml (2.1 x 10(-4) M). The inhibition of protein synthesis was associated with the partial reduction of ribonucleic acid synthesis and leucine transport. The mechanism of inhibition of lipid synthesis was further investigated in a cell-free extract of the yeast. CM-55 inhibited the incorporation of [(14)C]acetyl Coenzyme A (CoA) into both fatty acid (FAF) and non-saponifiable fractions (NSF). However, it did not inhibit [(14)C]malonyl CoA incorporation into FAF and only slightly inhibited [(14)C]mevalonate incorporation into NSF. The activity of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase was inhibited more strongly than the incorporation of [(14)C]3-hydroxy-3-methylglutaryl CoA into NSF; this could account for the CM-55 inhibition of [(14)C]acetyl CoA incorporation into NSF.

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.