Inhibitory activity of shiitake flavor against platelet aggregation

Sulfuric-flavored compounds were extracted from shiitake (Lentinus edodes) and their inhibitory activity against platelet aggregation was investigated. Platelet aggregation induced by arachidonic acid and U-46619, the analog of thromboxane A(2), was inhibited by the essential oil from shiitake that contained lenthionine as a major sulfuric compound. This result indicates that the inhibitory site of the shiitake flavor compounds would be different from that of garlic-flavor compounds because the latter inhibits the passage between arachidonic acid and thromboxane A(2). The effect of the synthesized lenthionine was almost equivalent to that of the essential oil, which indicates that the inhibitory activity of the essential oil from shiitake would be mainly attributed to lenthionine.     

Decreased total nitric oxide production in patients with duchenne muscular dystrophy

Plasma nitric oxide (NO) levels in Duchenne muscular dystrophy (DMD) patients were significantly lower than those observed in both healthy controls and in patients with other neuromuscular disorders. The correlation between NO level and ejection fraction was significant (r=–0.384, p=0.0391) in the DMD group. Disruption of NO systems may contribute to the development of muscular dystrophy and have implications for therapeutic strategies.     

Characterization of Psychrotrophic Bacteria in the Surface and Deep-Sea Waters from the Northwestern Pacific Ocean Based on 16S Ribosomal DNA Analysis

Seventy-eight 4°C-culturable bacteria were isolated using ZoBell 2216E medium from surface (0–200 m) and deep-sea (1000–9671 m) waters in the northwestern Pacific Ocean. Growth studies indicated that all 4°C-culturable bacteria were psychrotrophs. Six phylotypes were observed in the surface water samples and 8 phylotypes in the deep-sea waters. Phylogenetic characterization based on 16S ribosomal DNA sequence analysis of the representative phylotypes revealed that some bacterial genera, Pseudoalteromonas, Photobacterium, and Vibrio, were common to surface and deep-sea waters, and others, Pseudomonas and Halomonas, specifically occurred in surface water. Overall, the members of Vibrionaceae appear to be dominant in both habitats. Received June 13, 2000; accepted March 8, 2001.     

Characterization of the γc chain among 27 unrelated Japanese patients with X-linked severe combined immunodeficiency (X-SCID)

X-linked severe combined immunodeficiency (X-SCID) is a rare fatal disease that is caused by mutations in the gene encoding the gammac chain. In this study, 27 unrelated Japanese patients with X-SCID were examined in terms of their genetic mutations and surface expression of the gammac chain. Among 25 patients examined, excluding two patients with large deletions, 23 different mutations were identified in the IL2RG gene, including 10 novel mutations. One patient bearing an extracellular mutation and all three of the patients bearing intracellular mutations after exon 7 expressed the gammac chain on the cell surface. Overall, 84% of patients lacked surface expression of the gammac chain leading to a diagnosis of X-SCID.     

Development and clinical application of a bioluminescence enzyme immunoassay for oxytocin

The development of a highly sensitive analytical method for oxytocin could be useful in the diagnosis and treatment of autistic spectrum disorder. We previously developed a colorimetric enzyme immunoassay (EIA) for plasma oxytocin measurement. In this study, we developed a method to measure oxytocin concentrations using a higher sensitivity bioluminescent EIA. Biotinylated oxytocin bridged with five lysine residues was used in a competitive format. The standard curve range for oxytocin was 1.0 to 1000 pg/assay. In addition, there was good correlation between the colorimetric and bioluminescent immunoassays in terms of measured oxytocin concentration (r = 0.9665, n = 48). The bioluminescent EIA for plasma oxytocin was more rapid and provided higher sensitivity than the colorimetric immunoassay, making it suitable for clinical application.     

Cultivable butyrate-producing bacteria of elderly Japanese diagnosed with Alzheimer’s disease

The group of butyrate-producing bacteria within the human gut microbiome may be associated with positive effects on memory improvement, according to previous studies on dementia-associated diseases. Here, fecal samples of four elderly Japanese diagnosed with Alzheimer’s disease (AD) were used to isolate butyrate-producing bacteria. 226 isolates were randomly picked, their 16S rRNA genes were sequenced, and assigned into sixty OTUs (operational taxonomic units) based on BLASTn results. Four isolates with less than 97% homology to known sequences were considered as unique OTUs of potentially butyrate-producing bacteria. In addition, 12 potential butyrate-producing isolates were selected from the remaining 56 OTUs based on scan-searching against the PubMed and the ScienceDirect databases. Those belonged to the phylum Bacteroidetes and to the clostridial clusters I, IV, XI, XV, XIVa within the phylum Firmicutes. 15 out of the 16 isolates were indeed able to produce butyrate in culture as determined by high-performance liquid chromatography with UV detection. Furthermore, encoding genes for butyrate formation in these bacteria were identified by sequencing of degenerately primed PCR products and included the genes for butyrate kinase (buk), butyryl-CoA: acetate CoAtransferase (but), CoA-transferase-related, and propionate CoA-transferase. The results showed that eight isolates possessed buk, while five isolates possessed but. The CoA-transfer-related gene was identified as butyryl-CoA:4-hydroxybutyrate CoA transferase (4-hbt) in four strains. No strains contained the propionate CoA-transferase gene. The biochemical and butyrate-producing pathways analyses of butyrate producers presented in this study may help to characterize the butyrate-producing bacterial community in the gut of AD patients.     

Key Amino Acid Residues Involved in the Transitions of L- to R-Type Protofilaments of the Salmonella Flagellar Filament

The flagellar filament enables bacteria to swim by functioning as a helical propeller. The filament is a supercoiled assembly of a single protein, flagellin, and is formed by 11 protofilaments arranged in a circle. Bacterial swimming and tumbling correlate with changes of the various helical structures, called polymorphic transformation, that are determined by the ratios of two distinct forms of protofilaments, the L and R types. The polymorphic transformation is caused by transition of the protofilament between L and R types. Elucidation of this transition mechanism has been addressed by comparing the atomic structures of L- and R-type straight filaments or using massive molecular dynamic simulation. Here, we found amino acid residues required for the transition of the protofilament using fliC-intragenic suppressor analysis. We isolated a number of revertants producing supercoiled filaments from mutants with straight filaments and identified the second-site mutations in all of the revertants. The results suggest that Asp107, Gly426, and Ser448 and Ser106, Ala416, Ala427, and Arg431 are the key residues involved in inducing supercoiled filaments from the R- and the L-type straight filaments, respectively. Considering the structures of the R- and L-type protofilaments and the relationship between the rotation of the flagellar motor and the polymorphic transformation, we propose that Gly426, Ala427, and Arg431 contribute to the first stage of the transition and that Ser106, Asp107, and Ala416 play a role in propagating the transitions along the flagellar filament.     

Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using RecA and a restriction enzyme for enhanced amplification specificity

Our aim was to test and develop the use of loop-mediated isothermal amplification (LAMP) for HLA-DRB1 genotyping. Initially, we found that the conventional LAMP protocols produced non-specific and variable amplification results depending on the sample DNA conditions. Experiments with different concentrations of DNase in the reaction mixture with and without T4 DNA ligase-treated samples suggested that the strand displacement activity of DNA polymerase in LAMP, at least in part, started from randomly existing nicks because T4 DNA ligase treatment of sample DNA resulted in no amplification. Such non-specific amplification due to the randomly existing nicks was improved specifically by the addition of RecA of Escherichia coli and a restriction enzyme, for example, PvuII, to the reaction mixture. We applied the modified LAMP (mLAMP) (1) to detect specific HLA-DRB1 alleles by using only specific primers for amplification or (2) for genotyping in multiple samples with a multi-probe typing system. In the latter case, HLA-DRB1 genotyping was developed by combining the mLAMP with amplicon capture using polymorphic region-specific probes fixed onto the bottom of the wells of a 96-well plate and the captured amplicons visualized as a black spot at the bottom of the well. The multi-probe human leukocyte antigen (HLA) typing method and the specific HLA allele detection method could be applied for point-of-care testing due to no requirement for specific and expensive instruments.     

Unique properties of cluster of differentiation 93 in the umbilical cord blood of neonates

It has previously been reported by these authors that cluster of differentiation (CD) 93 is co‐expressed on naive T‐lymphocytes (CD4⁺CD45RA⁺ cells) in neonatal umbilical cord blood cells (UCBCs) but not on normal adult peripheral blood cells (PBCs). In this study, expression of CD93 on other lymphocyte subsets and the concentration of soluble formed CD93 (sCD93) in serum or culture supernatants from neonatal umbilical cord blood (UCB) was examined. It was found that CD93 is also co‐expressed on CD2⁺, CD16⁺, CD56⁺ or CD25⁺ cells in the lymphocyte population of neonatal UCBCs, but not on normal adult PBCs. The concentrations of sCD93 in serum and culture supernatants from neonatal UCB were significantly greater than those from normal adult peripheral blood. The concentrations of sCD93 in culture supernatants from neonatal UCBCs and normal adult PBCs treated with phorbol 12‐myristate 13‐acetate (PMA) were significantly enhanced compared with those without PMA treatment. The degree of enhancement of sCD93 by PMA in culture supernatants from neonatal UCBCs was significantly greater than that of normal adult PBCs and enhancement of sCD93 by PMA in the culture supernatants from neonatal UCBCs and normal adult PBCs was significantly suppressed by PKC inhibitor. Interestingly, the high concentration of serum sCD93 in neonates was significantly decreased in sera from infants at 1 month after birth. Expression of CD93 on the lymphocyte population of PBCs from infants at 1 month after birth was also significantly decreased, compared with that for neonatal UCBCs. These findings indicate that CD93 in neonatal UCB has unique properties as an immunological biomarker.     

Enantioselective synthesis of derivatives and structure–activity relationship study in the development of NA255 as a novel host-targeting anti-HCV agent

Hepatitis C virus (HCV) infection represents a serious health-care problem. Previously we reported the identification of NA255 from our natural products library using a HCV sub-genomic replicon cell culture system. Herein, we report how the absolute stereochemistry of NA255 was determined and an enantioselective synthetic method for NA255 derivatives was developed. The structure–activity relationship of the NA255 derivatives and rat pharmacokinetic profiles of the representative compounds are disclosed.