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A series of 3,9 disubstituted [(alkylthio)methyl]- and (alkoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A kinase inhibitory properties approximately 100- to > 500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A, protein kinase C1 protein kinase A, and myosin light chain kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was active in preventing degeneration of cholinergic neurons of the nucleus basalis magnocellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons.

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Hyperthermia has long been known as a radio-sensitizing agent that displays anti-tumor effects, and has been developed as a therapeutic application. The mechanisms of hyperthermia-induced radio-sensitization are highly associated with inhibition of DNA repair. Our investigations aimed to show how hyperthermia inactivate homologous recombination repair in the process of sensitizing cells to ionizing radiation by using a series of DNA repair deficient Chinese Hamster cells. Significant differences in cellular toxicity attributable to hyperthermia at and above 42.5°C were observed. In wild-type and non-homologous end joining repair mutants, cells in late S phase showed double the amount heat-induced radio-sensitization effects of G1-phase cells. Both radiation-induced DNA double strand breaks and chromatin damage resulting from hyperthermia exposure was measured to be approximately two times higher in G2-phase cells than G0/G1 cells. Additionally, G2-phase cells took approximately two times as long to repair DNA damage over time than G0/G1-phase cells. To supplement these findings, radiation-induced Rad51 foci formations at DNA double strand break sites were observed to gradually dissociate in response to the temperature and time of hyperthermia exposure. Dissociated Rad51 proteins subsequently re-formed foci at damage sites with time, and occurred in a trend also related to temperature and time of hyperthermia exposure. These findings suggest Rad51's dissociation and subsequent reformation at DNA double strand break sites in response to varying hyperthermia conditions plays an important role in hyperthermia-induced radio-sensitization.

A novel inhibitor of angiotensin I converting enzyme (ACE), designated K-13, was isolated from the culture broth of Micromonospora halophytica subsp. exilisia K-13. K-13 inhibited ACEnon-competitively whenhippuryl-L-histidyl-L-leucine was used as a substrate. The inhibition constant (Ki) was 0.349 /^m. K-13 hardly inhibited carboxypeptidase A, trypsin, a-chymotrypsin, leucine aminopeptidase, and aminopeptidase B even at a level of 61^m. WhenK-13 was administered intravenously to rats, it inhibited the pressor response to angiotensin I.In previous papers, we reported novel and potent inhibitors of angiotensin I converting enzyme (ACE) from microbial origin, K-261} and K-42). During further screening for ACEinhibitors, we found that an actinomycete strain identified as Micromonospora halophytica subsp. exilisia K-1 3 produced a new ACE inhibitor, K-13 (Fig. 1).In this communication, we report the fermentation, isolation, physico-chemical and biological properties ofK-1 3. The taxonomic studies of the producing organism and structure elucidation of K-138) will be published in separate papers. Materials and Methods MaterialsCarboxypeptidase A (bovine pancreas) was obtained from Sigma ; carboxypeptidase B (porcine pancreas) and leucine aminopeptidase (hog kidney) from Boehringer Mannheim, and a-chymotrypsin (bovine pancreas) and trypsin (bovine pancreas) from Worthington Biochem. Hippuryl-L-histidyl-L-leucine (HHL) and angiotensin I were purchased from Protein Research Foundation, Osaka ; hippuryl-L-phenylalanine, L-arginyl /3-naphtylamide, and L-leucine /J-naphthylamide from Sigma, and benzoyl-L-tyrosine ethyl ester and /j-tosyl-L-arginine methyl ester hydrochloride were from Nakarai Chemicals, Ltd.Microorganisms Micromonospora halophytica subsp. exilisia K-13, isolated from a soil sample was used in this experiment. Mediumand CultureThe seed medium consisted of glucose 1.0 %, soluble starch 1.0 %, beef extract 0.3 %, yeast extract 0.5%, Bacto-tryptone (Difco) 0.5% and CaCO3 0.2%, pH 7.2. The production medium consisted