Transfer of Actinomadura spadix Nonomura and Ohara 1971 to Actinoallomurus spadix gen. nov., comb. nov., and description of Actinoallomurus amamiensis sp. nov., Actinoallomurus caesius sp. nov., Actinoallomurus coprocola sp. nov., Actinoallomurus fulvus sp. nov., Actinoallomurus iriomotensis sp. nov., Actinoallomurus luridus sp. nov., Actinoallomurus purpureus sp. nov. and Actinoallomurus yoronensis sp. nov.

Ten actinomycete strains that form chains of spiral or looped spores were isolated from soil and dung samples in Japan. They contained D- and L-lysine, meso-diaminopimelic acid (A2pm), D-glutamic acid and D- and L-alanine in the cell-wall peptidoglycan, madurose as a characteristic whole-cell sugar, MK-9(H6) and MK-9(H8) as the major isoprenoid quinones and iso-C16:0 as the major cellular fatty acid and showed genomic DNA G+C contents of 69-74 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that the isolated actinomycete strains consistently formed a monophyletic cluster with Actinomadura spadix NBRC 14099T and a separate line of descent in the phylogenetic cluster of the family Thermomonosporaceae. Actinomadura spadix NBRC 14099T also contained D- and L-lysine in addition to meso-A2pm. This genetic and phenotypic evidence revealed that the actinomycete strains could be clearly differentiated from the other members of the family Thermomonosporaceae and that they warranted separate genus status. We conclude that Actinomadura spadix should be assigned the status of the type species of a new genus as Actinoallomurus spadix gen. nov., comb. nov. (type strain NBRC 14099T=ATCC 27298T=BCRC 13386T=CBS 261.72T=CIP 105479T=DSM 43459T=JCM 3146T=KCTC 9252T=NCIMB 11118T=NRRL B-16128T). Further, we conclude that the ten new isolates should be assigned to the novel species Actinoallomurus amamiensis sp. nov. (type strain TT00-28T=NBRC 103682T=KCTC 19537T), Actinoallomurus caesius sp. nov. (type strain A3015T=NBRC 103678T=KCTC 19535T), Actinoallomurus coprocola sp. nov. (type strain TT04-09T=NBRC 103688T=KCTC 19542T), Actinoallomurus fulvus sp. nov. (type strain TT99-66T=NBRC 103680T=KCTC 19536T), Actinoallomurus iriomotensis sp. nov. (type strain TT02-47T=NBRC 103685T=KCTC 19539T), Actinoallomurus luridus sp. nov. (type strain TT02-15T=NBRC 103683T=KCTC 19538T), Actinoallomurus purpureus sp. nov. (type strain TTN02-30T=NBRC 103687T=KCTC 19541T) and Actinoallomurus yoronensis sp. nov. (type strain TTN02-22T=NBRC 103686T=KCTC 19540T).

Total synthesis and determination of the absolute configuration of FD-838, a naturally occurring azaspirobicyclic product.

The first asymmetric total synthesis of FD-838, a naturally occurring azaspirobicyclic product, has been accomplished allowing determination of its absolute stereochemistry.

Application of log D for the prediction of hydrophobicity in the advanced Marfey's method.

In the advanced Marfey's method, the resolution between the diastereomers derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA) and 1-fluoro-2,4-dinitrophenyl-5-D-leucinamide (D-FDLA) is reflected by the difference of hydrophobicity of the two functional groups at the asymmetric carbon. However, no effective method has been developed for the estimation of hydrophobicity so far. For this purpose, we introduced log D from the ACD Labs LogD and applied it to relatively simple primary amines, amino acids and secondary alcohols in the present study. It was found that the difference of the retention times (Delta t(R)) correlated with that of log D (Delta log D) for both diastereomers based on the obtained experimental results. Based on these results, the following procedure was proposed for the non-empirical determination of the absolute configuration of primary amines including amino acids and secondary alcohols: (1) estimate the hydrophobicity by the calculation of log D for the two substituent groups at the asymmetric carbon, (2) locate the trans-type arrangement of the two more hydrophobic substituents in the L-DLA derivative and judge the asymmetric carbon to be R or S in the trans-type that is eluted first, (3) derivatize the desired compound with L- or D-FDLA and analyze by LC/MS, and (4) compare the elution order with the prospective one and determine the absolute configuration at the asymmetric carbon. Furthermore, log D could also be used to predict the retention times of unavailable amino acids and small peptides, indicating that the combination of the advanced Marfey's method with log D would provide more reliable structural information on a mixture composed of amino acids and small peptides. The developed method is being applied to more complicated compounds.

Further investigation of microbial degradation of microcystin using the advanced Marfey method.

It is known that microcystin (MC) is subject to microbial degradation to provide three types of products, linearized MCLR (Adda-Glu-Mdha-Ala-Leu-MeAsp-Arg), tetrapeptide Adda-Glu-Mdha-Ala, and Adda. They can be readily detected by the usual HPLC, because they commonly have an Adda moiety with a diene and an absorption maximum at 238 nm as the chromophore. However, no other degradation products without such a chromophore have been isolated to date. In this study, cell preparation of a bacterium B-9 that can degrade MC and detection of the degradation products were devised. First, we regulated the B-9 hydrolytic activity by washing with sodium chloride solution to obtain a desired cell preparation, which permitted an additional intermediate and the final products of MCLR to be obtained. Second, the resulting products could be firmly identified using the advanced Marfey method with the aid of log D. As a result of these experiments, the following degradation products were further identified: a tetrapeptide, Adda-Glu-Mdha-Ala, tripeptides Adda-Glu-Mdha, Glu-Mdha-Ala, and Arg-MeAsp-Leu, a dipeptide, Glu-Mdha, and amino acids Adda, Arg, and methylamine derived from Mdha. The present study expands the hydrolytic activity of the B-9 strain, which can hydrolyze not only cyanobacterial cyclic peptides but also MC to the intermediates and final products. The established characterization method composed of the advanced Marfey method and log D would be a standard technique for the structural characterization of a mixture of amino acids and peptides.

Reliable and sensitive analysis of amino acids in the peptidoglycan of actinomycetes using the advanced Marfey's method.

For the chemotaxonomic classification of actinomycetes, we developed a new method for the detection of the 2, 6-diaminopimelic acid (A(2)pm) stereoisomers and 3-hydroxy diaminopimelic acid (3-OH A(2)pm) in the cell wall peptidoglycan of actinomycetes using "the advanced Marfey's method", which consists of a chromatographic technique for the separation of amino acids into each of its enantiomers by derivatization with 1-fluoro-2, 4-dinitrophenyl-5-L-luecinamide (L-FDLA) and D-FDLA, and a detection method using liquid chromatography/mass spectrometry (LC/MS). This method was successfully applied to determine the absolute configuration of the A(2)pm and detect the 3-OH A(2)pm included in the cell wall peptidoglycan of the standard strains. Because the procedure can be performed with very small amounts of an amino acid, it was possible to use one colony from an agar plate of the actinomycetes for the acid hydrolysis. In addition, the constituent amino acids and their absolute configurations in the cell wall of the actinomycetes could be simultaneously determined. Thus, a reliable, sensitive and rapid analytical method for amino acids including A(2)pm in the peptidoglycan of the microorganisms was established.

Elution behavior of diaminopimelic acid and related diamino acids using the advanced Marfey's method.

The advanced Marfey's method consists of a chromatography technique for the separation of amino acids into each enantiomer by derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA), and a detection method using liquid chromatography/mass spectrometry (LC/MS) which can determine the non-empirically the absolute configuration of various amino acids including the non-protein ones. However, this method has not been applied to the determination of the absolute configuration of an amino acid with a "meso" configuration such as diaminopimelic acid (A2pm). In the present study, this method was successfully applied to determine the absolute configurations of diaminosuccinic acid (DAS), A2pm, cystine (Cys), selenocystine (SeCys) and homocystine (HomoCys) using a racemization procedure and the DL-FDLA method, and the resulting elution behavior was summarized as follows: (1) the LL- and meso-isomers were eluted prior to the DD-isomer except for one case; (2) the LL- and meso-isomers are closely eluted and the elution was occasionally reversed; (3) the retention time for both the L- and D-derivatives of the meso-isomer was not changed; (4) the complementary use of the two solvent systems using CH3CN and MeOH was effective to obtain a chromatogram with a high resolution; (5) the abnormality, such as the elution order and peak shape, was observed in the elution behavior of DAS.

Increase in secretion of glial cell line-derived neurotrophic factor from glial cell lines by inhibitors of vacuolar ATPase.

Glial cell line-derived neurotrophic factor (GDNF) was reported to be effective for treating subjects with neurodegenerative diseases such as Parkinson's disease. In search of finding a compound which promotes GDNF secretion, we found that concanamycin A (ConA), a vacuolar ATPase (V-type ATPase) inhibitor purified from Streptomyces diastatochromogens, enhanced GDNF secretion from glioma cells. The rat glioma cell line, C6, and the human glioma cell lines, U87MG and T98G, abundantly expressed GDNF mRNA, and secreted GDNF into culture media, and this event was potently enhanced by a Ca(2+) ionophore and by phorbol ester, as noted in other cells. ConA concentration dependently and potently increased GDNF release from C6, U87MG and T98G cells into culture media. In addition, ConA enhanced GDNF secretion from astrocyte primary cultures prepared from the human fetus with the same potency seen in glioma cell lines. Likewise, another V-type ATPase inhibitor, bafilomycinA1 facilitated GDNF release from C6, U87MG and T98G glioma cells, in a concentration-dependent manner. The potencies of these V-type ATPase inhibitors in enhancing GDNF secretion were consistent with those which inhibited V-type ATPase activity. These results suggest that blockade of V-type ATPase potently stimulates the secretion of GDNF from glial cells. The V-type ATPase inhibitors may be beneficial to use for the treatment of diseases in which increase in GDNF could be effective.

A novel neuritogenic compound, NGA0187.

A new neuritogenic compound NGA0187 was isolated from the fermentation broth of Acremonium sp. TF-0356. The structure of NGA0187 was determined by means of spectroscopic analysis and X-Ray diffraction. NGA0187 induced significant neurite outgrowth in PC12 cells. However, survival effect of NGA0187 on the primary culture of cerebral cortical neurons was not observed.