Systematic search for single nucleotide polymorphisms in the insulin gene: evidence for a high frequency of -23T-->A in Japanese subjects

It has recently been shown that the A/A genotype at g.-23 of the insulin gene correlates with impaired insulin secretion in response to body weight gain in subjects of European descent. To examine whether there are single nucleotide polymorphisms (SNPs) in the insulin gene associated with type 2 diabetes, all exons with their flanking sequences for 113 Japanese type 2 diabetic patients and 99 nondiabetic control subjects were analyzed using PCR direct sequencing. We have only found g.-23T --> A, 806G --> C, 1128T --> C, and 1141A --> C, which have previously been reported in alpha (A-C-C-C) and beta (T-G-T-A) alleles. The allele frequency of -23T --> A in control Japanese subjects was 97.4%, whereas that in Europeans is about 30%. The A/A genotype was found in 94 of 99 Japanese subjects (94.9%) and the allele frequencies of 806G --> C, 1128T --> C, and 1141A --> C were all 96.5%. The estimated haplotype frequencies were (A-C-C-C) (96.0%), (T-G-T-A) (2.0%), (A-G-T-A) (1.5%), and (T-C-C-C) (0.5%). No association of these SNPs or haplotypes with type 2 diabetes was evident. Thus, the A/A genotype at the g.-23 of insulin gene was generally high in Japanese subjects, which could account for the fact that they typically secrete lower levels of insulin.     

Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress

In the present study, we find that cyclopentenone prostaglandins (PGs) of the J(2) series, naturally occurring derivatives of PGD(2), are potential inducers of intracellular oxidative stress that mediates cell degeneration. Based on an extensive screening of diverse chemical agents on induction of intracellular production of reactive oxygen species (ROS), we found that the cyclopentenone PGs, such as PGA(2), PGJ(2), Delta(12)-PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2), showed the most potent pro-oxidant effect on SH-SY5Y human neuroblastoma cells. As the intracellular events that mediate the PG cytotoxicity, we observed (i) the cellular redox alteration represented by depletion of antioxidant defenses, such as glutathione and glutathione peroxidase; (ii) a transient decrease in the mitochondrial membrane potential (Deltapsi); (iii) the production of protein-bound lipid peroxidation products, such as acrolein and 4-hydroxy-2-nonenal; and (iv) the accumulation of ubiquitinated proteins. These events correlated well with the reduction in cell viability. In addition, the thiol compound, N-acetylcysteine, could significantly inhibit the PG-induced ROS production, thereby preventing cytotoxicity, suggesting that the redox alteration is closely related to the pro-oxidant effect of cyclopentenone PGs. More strikingly, the lipid peroxidation end products, acrolein and 4-hydroxy-2-nonenal, detected in the PG-treated cells potently induced the ROS production, which was accompanied by the accumulation of ubiquitinated proteins and cell death, suggesting that the membrane lipid peroxidation products may represent one of the causative factors that potentiate the cytotoxic effect of cyclopentenone PGs by accelerating intracellular oxidative stress. These data suggest that the intracellular oxidative stress, represented by ROS production/lipid peroxidation and redox alteration, may underlie the well documented biological effects, such as antiproliferative and antitumor activities, of cyclopentenone PGs.     

Molecular cloning and characterization of mouse Tspan-3, a novel member of the tetraspanin superfamily, expressed on resting dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells and play an essential role for triggering T-cell-mediated immune responses. In search for novel cell surface molecules expressed on DCs involved in T cell priming by representational differential analysis, we identified a mouse homologue of Tspan-3 (mTspan-3), a novel member of the tetraspanin superfamily. The mTspan-3 consists of four hydrophobic, putative transmembrane regions, forming a small and a large extracellular loop, with short intracellular amino and carboxil tails. Although the mTspan-3 is expressed on a variety of immune cell types including resting DCs, its expression on DCs is downregulated during activation induced by cross-linking CD40 with anti-CD40 monoclonal antibody. These results suggest that mTspan-3 may be involved in the function of DCs in association with T cell stimulation.     

Binding of arrestin to cytoplasmic loop mutants of bovine rhodopsin

The binding of arrestin to rhodopsin is a multistep process that begins when arrestin interacts with the phosphorylated C terminus of rhodopsin. This interaction appears to induce a conformational change in arrestin that exposes a high-affinity binding site for rhodopsin. Several studies in which synthetic peptides were used have suggested that sites on the rhodopsin cytoplasmic loops are involved in this interaction. However, the precise amino acids on rhodopsin that participate in this interaction are unknown. This study addresses the role of specific amino acids in the cytoplasmic loops of rhodopsin in binding arrestin through the use of site-directed mutagenesis and direct binding assays. A series of alanine mutants within the three cytoplasmic loops of rhodopsin were expressed in HEK-293 cells, reconstituted with 11-cis-retinal, prephosphorylated with rhodopsin kinase, and examined for their ability to bind in vitro-translated, 35S-labeled arrestin. Mutations at Asn-73 in loop I as well as at Pro-142 and Met-143 in loop II resulted in dramatic decreases in the level of arrestin binding, whereas the level of phosphorylation by rhodopsin kinase was similar to that of wild-type rhodopsin. The results indicate that these amino acids play a significant role in arrestin binding.     

Growth of starved Escherichia coli O157 cells in selective and non-selective media.

Escherichia coli O157 strains starved in sterile deionized water (SDW) and filter-sterilized natural river water (SRW) were investigated with specific reference to their culturability in selective and non-selective media. Growth of the strains starved in both SDW and SRW were markedly suppressed with time in selective liquid media such as modified trypticase soy broth supplemented with novobiocin (mTSB+n) and modified E. coli broth supplemented with novobiocin (mEC+n). This suppression was more pronounced when incubated at 42 C than at 37 C, especially with mEC+n. By contrast, such growth suppression was seldom observed when cultured at 37 C in non-selective liquid media such as trypticase soy broth (TSB) and buffered peptone water. In mEC+n at 42 C, the non-starved cells from overnight cultures with an initial density of less than 10(3) colony-forming units (CFU)/ml grew to the density of over 10(7) CFU/ml after 24 hr incubation, whereas those starved for 6 weeks in SRW were only to maintain their initial density or died off after 24 hr incubation under the same culturing conditions. These results indicated that the isolation of starved cells of E. coli O157 from water samples would be most difficult with selective enrichment or direct plating on the selective plate media. It is thus highly recommended that a "resuscitation" of the cells with non-selective enrichment should be performed as a routine practice for maximum recovery of E. coli O157 from water systems.     

Antimicrobial activities of diterpene dialdehydes, constituents from myoga (Zingiber mioga Roscoe), and their quantitative analysis

The antimicrobial activities of the three diterpene dialdehydes, miogadial, galanal A and galanal B, isolated from flower buds of the myoga (Zingiber mioga Roscoe) plant were investigated with some strains of bacteria, yeasts and molds. Among the three compounds, miogadial exhibited relatively greater antimicrobial activity than the others against Gram-positive bacteria and yeasts. Galanals A and B also behaved as antimicrobial agents against Gram-positive bacteria and yeasts. The content of miogadial in the flower buds was much higher than that in the leaves, whereas galanals A and B were contained at high levels in the leaves and rhizomes.     

Formation of N-(hexanoyl)ethanolamine, a novel phosphatidylethanolamine adduct, during the oxidation of erythrocyte membrane and low-density lipoprotein

The primary amino groups of biomolecules such as aminophospholipids, as well as proteins, are the potential targets of covalent modifications by lipid peroxidation products; however, little attention has been paid to the modification of aminophospholipids such as phosphatidylethanolamine (PE). The purpose of this study is to characterize the formation of a novel modified phospholipid, N-(hexanoyl)phosphatidylethanolamine (HEPE), in the reaction of PE with lipid hydroperoxides using mass spectrometric analyses. Upon reaction of egg PE with 13-hydroperoxyoctadecadienoic acid or other oxidized polyunsaturated fatty acids followed by phospholipase D-mediated hydrolysis, the formation of N-(hexanoyl)ethanolamine (HEEA), a head group of HEPE, was confirmed by isotope dilution liquid chromatography/tandem mass spectrometry. Moreover, increasing HEEA was detected in the hydrolysates of oxidized erythrocyte ghosts and low-density lipoprotein with their increasing lipid peroxidation levels. Collectively, these results suggest that the N-hexanoylated product of phospholipid, HEPE, can be generated during lipid peroxidation and may serve as one mechanism for the covalent modification of aminophospholipids in vivo.     

Benzyl isothiocyanate modifies expression of the G2/M arrest-related genes

Naturally occurring isothiocyanates are effective chemoprotective agents against chemical carcinogenesis in experimental animals. In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis. The exposure of HL-60 cells to BITC resulted in the inhibition of the G2/M progression that coincided with the apoptosis induction. We demonstrated that BITC significantly up-regulated expression of the G2/M cell cycle arrest-regulating genes including p21, GADD45, and 14-3-3sigma. Thus, these gathered data further supported that BITC has a potential to induce apoptosis selectively in the proliferating pre-cancerous cells through a cell cycle arrest-dependent mechanism.