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We found that daidzein stimulated bone resorption in the pit formation assay at the concentration of 10(-8)-10(-10) M. On the other hand, genistein and ipriflavone at these concentrations did not have any effect on pit formation. We speculated that daidzein had the ability to induce osteoclasts directly or indirectly from their progenitors and might be a tool by study osteoclast differentiation.

We searched hop extract for active component(s) that inhibited bone resorption in the pit formation assay, and isolated xanthohumol and humulone as active ingredients. Especially humulone had extraordinarily strong inhibitory activity and the IC50 (concentration of 50% inhibition) value was 5.9 x 10(-9)M.

Factors affecting the production of recombinant human protein C were investigated. When recombinant cells producing human protein C were cultured with microcarrier in two different scales, we found that (1) as cells grew to confluence, specific productivity of the protein was decreased and that (2) the efficiency of gamma-carboxylation of the generated protein C was lower in a larger culture than in a smaller culture. Higher cell density was shown to influence the specific productivity unfavorably. On the other hand, the amount of oxygen supply as demonstrated by oxygen consumption rate in the Opticell culture system correlated well with the efficiency of gamma-carboxylation, suggesting that oxygen metabolism is somehow implicated in the post-translational modification of recombinant protein C.

For the development of a perfusion culture producing recombinant human protein C, the effects of fetal calf serum and growth factors on cell growth and recombinant protein production were investigated. Although the growth of recombinant cells was stimulated by serum in a dose-dependent manner, a lower concentration of serum (2%) could support both synthesis and post-translational modification of protein C as efficiently as 10% serum. Among the growth factors tested, transferrin enhanced protein C production to the level comparable with 10% serum, while insulin was effective in maintaining cellular metabolism. Based on these results, a perfusion culture for a scale-up production of recombinant protein C was done using an Opticell culture system. A good productivity of the recombinant protein was obtained in low serum or serum-free medium for more than one month.

Thirty-three bacterial strains were isolated from soil, utilizing optically asymmetric degradation of dl-2-hydroxy-4-methylpentanoic acid (dl-HMPA) as the screening probe. Those strains were distributed in the following group and genera: Coryneform and Bacillus, Pseudomonas, and Streptomyces. Among them, the most potent strains, Bacillus freudenreichii NRS-137KH20B and Brevibacterium albidum NRS-130KH20B, could perform the resolution of more than 30 g of dl-HMPA per liter within 4 to 5 days of fermentation. Optically pure l- and d-HMPA enantiomers were obtained in more than 80% theoretical yield, whereas the transformed enantiomer was almost quantitatively recovered as 2-oxo-4-methyl-pentanoic acid in the culture broth. The enantiospecific dehydrogenation responsible for this resolution reaction had a rather wide substrate specificity on straight or branched aliphatic C(4) to C(16) 2-hydroxy acids, exhibiting the optima at chain lengths of either C(7) or C(5), although the enantiospecificity was not changed by chain length. The process was thus successfully extended to the preparation of optically pure C(5) to C(9) 2-hydroxy acids.

The mutagenicity of 6 marketed non-steroidal anti-inflammatory drugs (aspirin, flufenamic acid, diclofenac sodium, indomethacin, naproxen and chloroquine) as well as 2 new anti-inflammatory drugs (tenoxicam and carprofen) was examined by using in vitro bacterial systems (repair test and reversion test). None of them was mutagenic on Ames' reversion test. However, they differed in their responses to repair tests. Tenoxicam, carprofen, aspirin, flufenamic acid and naproxen were not mutagenic in either rec- or pol-assays, whereas chloroquine only showed positive results in the pol-assay system. Indomethacin and diclofenac sodium exhibited a slightly stronger inhibitory activity against B. subtilis rec- mutant than against its rec+ counterpart in rec-assay, which was much weaker than AF-2. Thus their mutagenicity was questionable. These results confirm the usefulness of DNA-repair assays as a complementary endpoint to gene mutation in assessing the genotoxic potential of environmental compounds.

Potentiating activity of alafosfalin was examined in detail with 8 cephalosporins and mecillinam against 164 urinary bacteria representing 8 genera. Alafosfalin was generally comparable in activity to cefamandole and mecillinam but superior to other cephalosporins tested. When the minimal fractional inhibitory concentration indices were compared, synergism was observed with all of the beta-lactams tested for all species except Streptococcus faecalis. Marked synergism was observed with Escherichia coli, Citrobacter, and enterobacter and, among the drugs tested, with cefamandole and mecillinam. Although alafosfalin is inactive against Pseudomonas aeruginosa, potentiation was seen with cefsulodin. The most favorable combination ratio of alafosfalin/cephalosporin to elicit potentiation was determined for each species and occurred in a rather narrow range of combinations (1:4 to 4:1 in general).

Representative polyethers of each group in Westley's classification and related ionophore antibiotics were examined for in vitro activity against 25 anaerobic strains. Lasalocid (X-537A) and nigericin were found to have activity comparable in potency to some leading antianaerobe antibiotics, newer cephalosporins and nitroimidazoles, with a different spectrum as revealed by a spectral comparison. A strict relationship between their antianaerobe activity and either their structural features or cation transporting properties was not apparent.

Taxonomic characteristics of strains NR-320-OM7HB and NR-320-OM7HBS producing a homologous series of new alpha-amylase inhibitors named trestatins are described, together with the comparison of these strains with similar Streptomyces species by DNA-DNA hybridization. A new species, Streptomyces dimorphogenes sp. nov. Watanabe and Maruyama is proposed as a result of the studies. The type strain of this species is strain NR-320-OM7HB (ATCC 31484), and the morphovar is strain NR-320-OM7HBS (ATCC 31485). The productivity of trestatin A, major and most active component, using these two strains in flask culture is also presented.

Of some 350 microorganisms screened, four strains of Pithomyces species were found to carry out regio-selective hydroxylation of patchoulol, a sesquiterpene, to 10-hydroxypatchoulol: Pithomyces sp. NRJ201, P. chartarum NRJ210, and, to a lesser extent, P. cynodontis ATCC 26150 and P. atro-olivaceus IFO 6651 were found to catalyze this reaction. A method has been developed by which 10-hydroxypatchoulol was obtained in 25 to 45% yields in 1- to 5-liter fermentation jars at 2 to 4 g of patchoulol per liter and isolated as pure material in 30% yields.

Trestatin complex which exhibited a potent inhibitory activity on various alpha-amylases has been isolated from the culture filtrate of Streptomyces dimorphogenes nov. sp. NR-320-OM7HB. Three major components, trestatins A, B and C, have been purified by adsorption and ion-exchange chromatography. Their spectral and chemical properties suggested that trestatins were novel basic oligosaccharide homologues each characterized by the possession of a trehalose moiety at the non-reducing end of the molecule.

Two cell surface mutants of Escherichia coli isolated through supersensitivity to sodium dodecyl sulfate were found to be agglutinated by concanavalin A in the intact rod form. By using these strains, the assay method for the concanavalin A-dependent agglutination was greatly simplified since neither spheroplast formation nor protease treatment is needed. This rod cell agglutination was characterized and applied for a semiquantitative determination of the amount of bound concanavalin A.

A novel antibiotic, penitricin, Ro 09-0804, has been produced in the culture filtrate of Penicillium aculeatum NR 5165. This antibiotic was purified by repeated extraction of culture filtrate with 1-butanol, and passage of the crude extracts through Sephadex G-10, followed by HPLC (Shodex Ionpak S-801). Physico-chemical characterization was made on penitricin, while two open-ringed penitricins B and C co-produced by the producer strain were also identified.

5'-Deoxy-5-fluorouridine (5'-DFUR) was parenterally and orally effective on various transplantable tumors and its activity was better than that of other fluorinated pyrimidines. However, like 5-fluorouracil and 2'-deoxy-5-fluorouridine (FUdR), 5'-DFUR was ineffective on L1210 leukemia resistant to 5-fluorouracil, suggesting that it would exert its antitumor activity through converted 5-fluorouracil. In tissue culture, 5'-DFUR inhibited the growth of various tumor cells similarly to ther fluorinated pyrimidines. However, 5'-DFUR was unique in that uridine completely reversed its inhibitory effect. Enzymological study clarified that uridine inhibited the conversion of 5'-DFUR to 5-fluorouracil by a uridine phosphorylase, in parallel to its reverse effect on cell growth inhibition by 5'-DFUR. Furthermore, a subline of L1210 leukemia resistant to 5'-DFUR but not to 5-fluorouracil was found to lack the uridine phosphorylase. These results indicate that 5'-DFUR is a depot form of 5-fluorouracil which can be promptly activated by uridine phosphorylase. In addition, the uridine phosphorylase was found to be abundant in sarcoma-180 solid tumor, leading to a significantly higher concentration of converted 5-fluorouracil in this tumor than in other normal tissues. This provides a good explanation for the high chemotherapeutic index of 5'-DFUR against this tumor, which may be applicable also for other tumors.

When assayed by a newly devised, simple and quantitative method, "motilometry", the motility of E. coli S-26 was found stimulated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) and 8-hydroxy cyclic AMP as well as histamine, catecholamines and inhibited by cyclic guanosine 3':5'-monophosphate (cyclic GMP). The stimulation elicited by cyclic AMP or other biogenic amines was reversed by cyclic GMP. The experimental significance and implicaton of these findings are discussed.

A substance inhibiting Escherichia coli motility was isolated by "motilometry" assay from the culture broth of Streptomyces sp. strain NR9GG8 and was found to be identical with desferrioxamine B. Its inhibitory effect was reversed by cyclic adenosine 3',5'-monophosphate (cAMP), while the motility stimulation by cAMP was diminished by this inhibitor. Its effects on various enzymes involved in cAMP metabolism of function of cAMP were examined.

Several Citrobacter freundii beta-lactamases, discernible by their substrate profiles and sensitivities to inhibition by newer cephalosporins, were inhibited by ceftriaxone in an unusual manner. Inhibition was noncompetitive at low concentrations of cephaloridine but became competitive at concentrations above 600 muM, which is close to the K(m) (570 muM) for cephaloridine.

Long, nonseptate filamentous cells consisting of 5 to 40 single-cell unit lengths were formed from Escherichia coli surface mutant ONT-3 by treatment with a sublethal concentration of sodium dodecyl sylfate. As distinct from several other elongated cells (e.g., thymine-starved filaments), it was found here that stable giant spheroplasts, 5 to 10 micrometers in diameter, were produced by the action of lysozyme in the presence of bovine serum albumin via the gradual fusion of distinct spheroplasting bulbs.

Four pyrazolones in frequent use, i.e. antipyrine (AP), aminopyrine (AMP), sulpyrine (SP) and isopropylantipyrine (IPA), were compared for their reactivity with nitrite and for the in vitro mutagenicity of their reaction products by Ames' reversion tests. In various acidic solutions at 37 degrees C, AP, AMP and SP were found to react easily with nitrite and yield various products including dimethylnitrosamine (DMNA) and 4-nitrosoantipyrine (4-NAP) in the cases of AMP and AP, respectively. When tested with Salmonella typhimurium TA100 and TA98 after lyophilization, the reaction products of AP (AP-N) were found to be mutagenic in both strains, while products of AMP and SP (AMP-N and SP-N) were mutagenic only in TA100. The presence of unknown ultimate mutagens, other than DMNA and 4-NAP, were evidenced in AP-N, AMP-N and SP-N. Incubation with S-9 mixture did not affect the mutagenicity of SP-N and decreased that of AP-N and AMP-N. In clear contrast to AP, AMP and SP, it was found that IPA remained essentially intact upon reaction with nitrite. No mutagenicity was detected with the reaction mixture (IPA-N) in either strain.

The optimal reaction conditions for the determination of DNA-homology in Streptomyces species were established in the presence of formamide using S1 nuclease. The melting temperature of Streptomyces DNA was 90 degrees C in 0.42 M NaCl containing 20% formamide in which the denaturation was completed by boiling for 5 minutes. In the S1 reaction mixture consisting of 5 U of S1 nuclease, 0.168 M NaCl, 1 mM ZnSO4 and 8% formamide at pH 4.8, single-stranded DNA was hydrolyzed by more than 98%, while the hydrolysis of double-stranded DNA was less than 3%. From the analysis of homoduplex formation, the C0t1/2 was found at 20 hours, when a mixture of unlabeled DNA and index DNA was used at a ratio of 500:1.

Several chalcone derivatives; e.g. Ro 09-0204, Ro 09-0323 and Ro 09-0501 were found to reduce markedly the revertant increase of Salmonella typhimurium TA100 by benzo(a)pyrene during the incubation with S-9 Mix. The antimutagenic activity was 100 - 700 times stronger than that of L-ascorbic acid. Effect on other mutagens, the structure activity relationship and the possible mechanism of action are briefly discussed.

Several yeast strains belonging to genus Candida were found to selectively hydrogenate l-sorbose with enantiomeric specificity, yielding optically pure l-iditol in the culture broth. The most active strain, isolated from a commercial lemon, was identified as Candida intermedia, which produced 50 g of l-iditol per liter from 150 g of l-sorbose per liter during a 5-day fermentation period (35% yield).

A new antibiotic, fumaramidmycin, has been isolated from a streptomycete NR-7GG1 which was characterized and named Streptomyces kurssanovii. The strain produced the antibiotic only when grown on agar plates but not in the submerged culture broth, where the contact with the vegetative mycelia appears to cause the inactivation of the antibiotic. The antibiotic shows an antimicrobial activity against both Gram-positive and Gram-negative bacteria.

A new phosphonopeptide, alafosfalin, was evaluated for in vitro antibacterial activity and for synergism with beta-lactams, using 475 Japanese clinical isolates from urinary tract infections. Alafosfalin was found to be highly active against Escherichia coli and moderately active against Serratia, Klebsiella, Enterobacter, and Citrobacter, but less active against gram-positive organisms than were beta-lactams such as cephazolin or ampicillin and inactive against indole-positive Proteus, Pseudomonas, and Acinetobacter. Potentiation with the two beta-lactams (fractional inhibitory concentration less than or equal to 0.5) was found in 10 to 40% of susceptible strains in 4:1 and 1:4 combinations, and to a lesser extent in those species or genera that were insensitive to alafosfalin alone. No cross resistance was seen between alafosfalin and the beta-lactams or any other commonly used antibacterial agents tested. Effect on selected ampicillin-resistant strains, differential sensitivity to alafosfalin among resistant strains of various types, and sensitivity of alafosfalin-insensitive E. coli and Klebsiella to other antibiotics are also discussed.

A new antibiotic fumaramidmycin produced by Streptomyces kurssanovii NR-7GG1 was isolated as colorless crystals. The structure was shown to be N-(phenylacetyl) fumaramide. Starting from fumaramic acid, fumaramidmycin has been synthesized in good yield, in which the key stage involves N-acylated imino ether formation followed by mild acid hydrolysis. Five analogues of fumaramidmycin have also been prepared.

The mutagenicity of an anti-epileptic drug, clonazepam, and its metabolites was studied by three repair (rec, pol and uvr) assays and reversion (Ames') tests. No detectable induction of mutation could be found in any tests by them per se or via metabolism in rat and mouse.

When Escherichia coli spheroplasts made by ethylenediaminetetraacetic acid and lysozyme were agglutinated by concanavalin A (con A), the degradation of ribosomal ribonucleic acid (rRNA) was found to occur proportionally to the degree of the agglutination, which was determined by microscopic examination or by a newly devised assay based on the slower settling of aggregates. Methyl-alpha-d-glucoside, low temperature or alkaline pH, all of which reverse the agglutination, also reduced the extent of rRNA degradation. This degradation was not due to the direct action of con A since a similar relationship was found in the case of spontaneous agglutination with concentrated spheroplasts in the absence of con A. The possible importance of a change in the cell membrane associated with the agglutination process is discussed in connection with the initiation of rRNA degradation.

The properties of the abnormal ribonucleoprotein particles produced by Escherichia coli Q-13 starved for glucose were studied. Smaller species of these partially deproteinized particles separable to six distinct sizes contained partially degraded ribonucleic acids. The mode of ribosome degradation under this condition is discussed in terms of differential appearance of these intermediate particles.

Tumor virus RNAs from several mammalian and one reptilian species were purified; their 3'-terminal nucleoside was identified by separation of the trialcohols produced by periodate oxidation followed by reduction and tritiation with NaB(3)H(4). Each virus contained uridine as the predominant terminal nucleoside. Molecular weight estimations based on the tritiation reactions were consistent with a structure consisting of four subunits.