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A sensitive and simple high-performance liquid chromatographicmethod 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 C 18 cartridge, and columns of Dowex 50W-X8 and Amberlite CG-400, then separated on Ca 2+ and Pb 2+ cation-exchange columns, eluted with deionized distilled water at 80°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.

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.

In the course of our examination for the alkaloid productivities of Streptomyces strains, Streptomyces sp. NA–15 was found to produce a new alkaloid, pyrindicin, in the culture medium. The strain NA–15 was found to be a variant of Streptomyces griseoflavus and was designated as S. griseoflavus var. pyr indie us nov. var.After the culture conditions for pyrindicin production were studied, pyrindicin was obtained as its hydrochloride (mp 145°C, decomp.) from the cultured broth. The compound was shown to possess weak antimicrobial and several pharmacological activities. The LD50 of the hydrochloride (ip, in mice) was 87 mg/kg.

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.

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 μg/ml (2.1 × 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.

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.

The antibiotic cerulenin inhibited the incorporation of 14 C-acetyl-CoA by 67% at a concentration of 9 × 10 −6 M but not that of 14 C-HMG-CoA into the non-saponifiable fraction in a cell-free extract of Saccharomyces cerevisiae . Cerulenin markedly inhibited the activity of partially purified HMG-CoA synthase. No inhibition of acetoacetyl-CoA thiolase activity was observed in the same preparation of HMG-CoA synthase. Therefore, cerulenin inhibition of overall sterol synthesis may be accounted for by the specific inhibition of HMG-CoA synthase activity.

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.

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.

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.

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.

Thr-Thr-Met-Pro-Leu-Trp, the C-terminal hexapeptide of αs1-casein, inhibited the angiotensin I-converting enzyme (I50 = 16 μM). Z-Pro-Leu-Trp also inhibited the enzyme (I50 = 18μM). The Pro-Leu-Trp sequence may be important for the inhibitory activity of this hexapeptide. Z-Pro-Val-Trp, which contains Val in place of Leu, inhibited the enzyme more potently (I50=2.9μM). The antihypertensive activity of this hexapeptide was also investigated. This peptide, when intravenously administered to anesthetized rats at 31.8mg/kg, tended to antagonize the rats' pressor response to angiotensin I.

Thr-Thr-Met-Pro-Leu-Trp, the C-terminal hexapeptide of αsl-casein, inhibited the angiotensin I-converting enzyme (I50 = 16 μM). Z-Pro-Leu-Trp also inhibited the enzyme (I50 = 18 μM). The Pro- Leu-Trp sequence may be important for the inhibitory activity of this hexapeptide. Z-Pro-Val-Trp, which contains Val in place of Leu, inhibited the enzyme more potently (I50 =2.9 μM).The antihypertensive activity of this hexapeptide was also investigated. This peptide, when intravenously administered to anesthetized rats at 31.8 mg/kg, tended to antagonize the rats’ pressor response to angiotensin I.

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.

In the course of our examination for the alkaloid productivities of Streptomyces strains, Streptomyces sp. NA–15 was found to produce a new alkaloid, pyrindicin, in the culture medium. The strain NA–15 was found to be a variant of Streptomyces griseoflavus and was designated as S. griseoflavus var. pyr indie us nov. var.After the culture conditions for pyrindicin production were studied, pyrindicin was obtained as its hydrochloride (mp 145°C, decomp.) from the cultured broth. The compound was shown to possess weak antimicrobial and several pharmacological activities. The LD50 of the hydrochloride (ip, in mice) was 87 mg/kg.

Cerulenin, (2S) (3R)2,3-epoxy-4-oxo-7,10-dodecadienoylamide, an antibiotic isolated from the culture filtrate of Cephalosporium caerulens, inhibits the fatty acid synthetase of Escherichia coli by specifically inhibiting β-ketoacyl-acyl carrier protein synthetase, the enzyme which catalyzes the condensation reaction of fatty acid biosynthesis. Although cerulenin and tetrahydrocerulenin inhibited both β-ketoacyl-acyl carrier protein synthetase and fatty acid synthetase, neither dihydrocerulenin nor hexahydrocerulenin, analogs which lack the epoxide ring, inhibited these enzyme systems. Both β-ketoacyl-acyl carrier protein synthetase and fatty acid synthetase were protected to the same extent against inhibition by cerulenin by prior incubation with acetyl-acyi carrier protein. The acyl group of this thioester is bound at the fatty acyi site of β-ketoacyl-acyl carrier protein synthetase. Detailed studies with homogeneous β-ketoacyl-acyl carrier protein synthetase indicated that cerulenin inhibited model reactions representing both the fatty acyl transacylase and the malonyl-acyl carrier protein decarboxylase components of the β-ketoacyl-acyl carrier protein synthetase reaction. Inhibition of β-ketoacyl-acyl carrier protein synthetase by cerulenin was irreversible, and it was associated with the binding of approximately 1 mole of inhibitor per mole of enzyme when inhibition approached 100 %.

A newly described species of Streptomyces (named Sm. kagawaensis ATCC No. 21811) isolated from soil was found to produce a new antifungal antibiotic, prumycin, which belongs to amino sugar group. Prumycin was isolated from the fermentation broth by ion-exchange adsorption and gel-filtration methods. This antibiotic inhibited specifically the growth of Sclerotinia sp. and Botrytis sp. on flower pot test with kidney bean leaves and also was effective on the field test with various plants.

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.

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.

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.

Porcine pancreatic kallikrein is a serine proteinase which releases vasodepressor peptides, kinins, from kininogens. This enzyme is a glycoprotein and it has been shown that this protein has heterogenity in carbohydrate moiety.

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).

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.

A new antibiotic O-2867 inhibiting the growth of Piricularia oryzae, a plant pathogenic fungus of rice, was isolated from a Streptomyces sp. Antibiotic O-2867 is water-soluble, amphoteric and consists of 2 components, O- 2867-α and β. O-2867-α was obtained as an amorphous powder and O-2867-β as colorless needles. Both antibiotics are active against P. oryzae in vitro but are not effective in pot tests using rice plants infected with P. oryzae.

An alkaloid from Streptomyces sp. NA-337, whose structure was assigned previously as (E, E)-4-methyl-2-pentadienyl-1-pyrroline, is re-investigated and revised as (E, E)-2-pentadienyl-3, 4, 5, 6-tetrahydropyridine.