Separation and analysis of free ceramides containing 2-hydroxy fatty acids in Sphingobacterium species

Abstract The occurrence of free ceramides was shown in the chloroform-methanol extractable lipids of 16 strains of Sphingobacterium including three species: S. versatilis, S. multivorum and S. mizutae . The predominant long-chain base was identified as a branched-chain, saturated dihydroxy base with a carbon chain consisting of 17 carbon atoms, while the most abundant fatty acid was 2-hydroxy-13-methyltetradecanoic acid. The major molecular species of the intact ceramides were identified as LCB- d - iso -17 : 0-2-OH iso -15 : 0FA, LCB- d - iso -17 : 0- iso -15 : 0FA and LCB- d -n16 : 0- iso -15 : 0FA.     

Inhibitory effects of condiments and herbal drugs on the growth and toxin production of toxigenic fungi

The effects of thirteen kinds of powdered herbal drugs and seven kinds of commercial dry condiments on the growth and toxin production ofAspergillus parasiticus, A. flavus,A. ochraceus, andA. versicolor were observed by introducing these substances into culture media for mycotoxin production.Of the twenty samples tested, cinnamon bark completely inhibited the fungal growth, while the others only inhibited the toxin production.The inhibitors were easily extracted from the samples with solvents such as hot water, chloroform, or ethanol.The extracts from coptis, philodendron bark, mustard, green tea leaves, and zanthoxylum completely inhibited the aflatoxin production ofA. parasiticus, however, they had little or no inhibitory effect againstA. flavus.     

Antagonistic jacalin-related lectins regulate the size of ER body-type beta-glucosidase complexes in Arabidopsis thaliana

PYK10/BGLU23 is a beta-glucosidase that is a major protein of ER bodies, which are endoplasmic reticulum (ER)-derived organelles that may be involved in defense systems. PYK10 has active and inactive forms. Active PYK10 molecules form large complexes with diameters ranging from 0.65 microm to > 70 microm. We identified three beta-glucosidases (PYK10, BGLU21 and BGLU22), five jacalin-related lectins (JALs) and a GDSL lipase-like protein (GLL) in the purified PYK10 complex. Expression levels of JALs and GLLs were lower in the nai1-1 mutant, which has no ER bodies, than in Col-0. The subcellular localization of PYK10 is predicted to be different from the localizations of JALs and GLLs. This suggests that PYK10 interacts with its partners (JALs and GLLs) when the subcellular structure is destroyed by pathogens. The PYK10 complex was found to be larger in the pbp1-1 and jal22-1 mutants than in Col-0, while it was smaller in the jal23-1, jal31-1 and jal31-2 mutants than in Col-0. These results show that two types of JALs having opposite roles regulate the size of the PYK10 complex antagonistically. We define the two types of lectins as a 'polymerizer-type lectin' and an 'inhibitor-type lectin'. Interestingly, the closest homologs of polymerizer-type lectins (JAL31 and JAL23) were inhibitor-type lectins (PBP1/JAL30 and JAL22). The pairs of polymerizer-type and inhibitor-type lectins reported here are good examples of genes that have evolved new functions after gene duplication (neofunctionalization).     

A novel giant gene CSMD3 encoding a protein with CUB and sushi multiple domains: a candidate gene for benign adult familial myoclonic epilepsy on human chromosome 8q23.3-q24.1

We identified a novel giant gene encoding a transmembrane protein with CUB and sushi multiple domains on the human chromosome 8q23.3-q24.1 in which benign adult familial myoclonic epilepsy type 1 (BAFME1/FAME, OMIM:601068) has been mapped. This giant gene consists of 73 exons and spans over 1.2Mb on the genomic DNA region. It showed significant homology to two genes, CSMD1 gene on 8p23 and CSMD2 gene on 1p34, at reduced amino acid sequence level and hence we designated as CSMD3. The CSMD3 gene was expressed mainly in adult and fetal brains. We performed mutation analysis on the CSMD3 gene for seven patients with BAFME1/FAME, but no mutation was found in the coding sequence of the CSMD3 gene. Comparative genomic analysis revealed a conserved family of CSMD genes in the mouse and fugu genomes. Possible functions of the CSMD gene family are discussed.     

Characterization of histidinol phosphate aminotransferase from Escherichia coli

Histidinol phosphate aminotransferase (HPAT) is a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase classified into Subgroup I aminotransferase, in which aspartate aminotransferase (AspAT) is the prototype. In order to expand our knowledge on the reaction mechanism of Subgroup I aminotransferases, HPAT is an enzyme suitable for detailed mechanistic studies because of having low sequence identity with AspAT and a unique substrate recognition mode. Here we investigated the spectroscopic properties of HPAT and the effect of the C4-C4' strain of the PLP-Lys(214) Schiff base on regulating the Schiff base pK(a) in HPAT. Similar to AspAT, the PLP-form HPAT showed pH-dependent absorption spectral change with maxima at 340 nm at high pH and 420 nm at low pH, having a low pK(a) of 6.6. The pK(a) value of the methylamine-reconstituted K214A mutant enzyme was increased from 6.6 to 10.6. Mutation of Asn(157) to Ala increased the pK(a) to 9.2. Replacement of Arg(335) by Leu increased the pK(a) to 8.6. On the other hand, the pK(a) value of the N157A/R335L double mutant enzyme was 10.6. These data indicate that the strain of the Schiff base is the principal factor to decrease the pK(a) in HPAT and is crucial for the subsequent increase in the Schiff base pK(a) during catalysis, although the electrostatic effect of the arginine residue that binds the negatively charged group of the substrate is larger in HPAT than that in AspAT. Our findings also support the idea that the strain mechanism is common to Subgroup I aminotransferases.     

Studies on DNA markers (D4S10 and D4S43/S127) genetically linked to Huntington's disease in Japanese families

Summary This is the first full report on the genetic linkage between Japanese Huntington's disease and the DNA markers D4S10 and D4S43/S127. With use of the HindIII, BglI, and EcoRI polymorphisms detected at D4S10, and the combination of all these polymorphisms to give composite haplotypes, nine Japanese Huntington's disease families were found to be informative. Three recombinants for D4S10 were detected in these families, giving a maximum lod score of 1.662 at a of 0.10. Similarly, when we used the MspI and PvuII polymorphisms detected by D4S43/S127, five families gave informative results. No recombinant was detected in these families, giving a maximum lod score of 3.348 at a of 0.00. These results clearly support the view that the Japanese Huntington's disease gene may be identical with the Western gene, in spite of the lower prevalence rate in Japan.