Roles of peptide-peptide charge interaction and lipid phase separation in helix-helix association in lipid bilayer

The roles of peptide-peptide charged interaction and lipid phase separation in helix-helix association in lipid bilayers were investigated using a model peptide, P₂₄, as a transmembrane α-helical peptide, and its four analogues. Fluorescence amino acids, tryptophan (P₂₄W) and pyrenylalanine (P₂₄Pya), were introduced into the sequence of P₂₄, respectively. Association of these peptides permits the resonance excitation energy transfer between tryptophan in P₂₄W and pyrenylalanine in P₂₄Pya or excimer formation between P₂₄Pya themselves. To evaluate the effect of charged interaction on the association between α-helical transmembrane segments in membrane proteins, charged amino acids, glutamic acid (P₂₄EW) and lysine (P₂₄KPya), were introduced into P₂₄W and P₂₄Pya, respectively. Energy transfer experiments indicated that the charged interaction between the positive charge of lysine residue in P₂₄KPya and the negative charge of glutamic acid residue in P₂₄EW did not affect the aggregation of transmembrane peptides in lipid membranes. As the content ratio of sphingomyelin (SM) and cholesterol (Ch) was increased in the egg phosphatidylcholine (PC), the stronger excimer fluorescence spectra of P₂₄Pya were observed, indicating that the co-existence of SM and Ch in PC liposomes, that is, the raft of SM and Ch, promotes the aggregation of the α-helical transmembrane peptides in lipid bilayers. Since the increase in the contents of SM and Ch leads to the decrease in the content of liquid crystalline-order phase, the moving area of transmembrane peptides might be limited in the liposomes, resulting in easy formation of the excimer in the presence of the lipid-raft.     

Chick endogenous lectin enhances chondrogenesis of cultured chick limb bud cells

We have studied the effect of β-d-galactoside-specific lectin purified from 14-day-old chick embryos on the differentiation of the mesenchymal cells dissociated from the limb buds of stage 24 chick embryos, using the micro-mass culture method described previously. When the cells were incubated with the lectin during the initial 12 hr of culture, cell proliferation became slightly activated. The lectin-treated cells formed a greater number of cartilage nodules and incorporated about twice as much as [³⁵S]sulfate per cell than the control cultures. The results of this study show that the chick endogenous lectin promotes cartilage differentiation in vitro and that endogenous lectin may possibly be involved in chondrogenesis in vivo.     

Mapping the human liver/islet glucose transporter (GLUT2) gene within a genetic linkage map of chromosome 3q using a (CA)n dinucleotide repeat polymorphism and characterization of the polymorphism in three racial groups.

The human liver/islet glucose transporter (GLUT2), a candidate gene for diabetes, has been incorporated into a genetic linkage map for chromosome 3q using a (CA)n dinucleotide repeat polymorphism adjacent to the 3'-end of exon 4a. We have found a total of nine alleles ranging in length from 153 to 169 nucleotides in three racial groups and have determined the precise structure of the variable region for four of the alleles by DNA sequencing. Five alleles were found to be common to the American Black, Caucasian, and Pima Indian racial groups studied. One allele (169 bp) was unique to American Blacks, and another rare allele (153 bp) was found only in the Caucasian population studied. Observed heterozygosity of the polymorphism in the Caucasian (CEPH) reference pedigree collection is 60%, for American Blacks 71%, and for Pima Indians 53%. An independent study recently identified the same dinucleotide repeat and found six alleles in a Caucasian population (Froguel et al., 1991), a result that we confirm; however, our sequencing data indicate a different molecular structure for the polymorphism for some of the alleles. We have constructed a new genetic linkage map of chromosome 3q uniquely placing the GLUT2 gene between flanking markers D3S26 and D3S43. The genetic map consists of 23 loci (25 RFLPs and 2 (CA)n dinucleotide repeat markers) with 14 markers uniquely localized with odds of at least 1000:1. Three genes (FTHL4, TF, GLUT2) are integrated into the map, which spans a sex-average distance of 147.3 cM, 103.8 cM in males and 227.0 cM in females.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurochemical effects of prenatal treatment with ochratoxin A on fetal and adult mouse brain

Ochratoxin A (OA) is a mycotoxin produced by several storage fungi, such asAspergillus ochraceus and severalPenicillium species. OA (3 mg/kg) was given intraperitoneally to pregnant mice on day 11 of gestation (day 1=day of insemination), and neurochemical changes in brains of their offspring were examined at fetal and adult stages. OA treatment produced retardation of intrauterine growth as well as microencephaly and reductions in total weight and DNA content of fetal brains. Specific activities of lysosomal enzymes in fetal brains began to increase by the 2nd day after treatment and to reach peak activities by the 3rd or 4th day after injection, indicative of cell dealth in the developing brains. Examination of brain regions of offspring three months after birth revealed that both tissue weight and DNA content were reduced to 80% of control in cerebral hemispheres (CHs; cerebral cortex and subjacent white matter, hippocampus, and amygdala) and to 90% of control in remainder of the brain (BGDM; basal ganglia, diencephalon, and mesencephalon). Total content of noradrenaline (NA), dopamine (DA) 5-hydroxytryptamine (5-HT) in treated CH showed about 15% reduction, although, expressed on a tissue weight basis, concentrations of these monoamines were increased by about 15%. Total DA content in BGDM was also reduced to 85% of controls, but total content of NA and 5-HT in BGDM and pons-medulla oblongata did not change. These result suggest that synaptogenesis of monoamine neurons in the cerebrum is imparied by prenatal treatment with OA, and that dopaminergic neurons show a slight selective vulnerability to the toxin.Abbrevations used. Ochratoxin (OA) Ochratoxin A - (CH) cerebral hemisphere - (BGDM) remainder of the brain consisting basal ganglia, diencephalon and mesencephalon - (PM) pons-medulla oblongata - (CE) cerebellum - (NA) noradrenaline - (DA) dopamine - (5-HT) 5-hydroxytryptamine     

Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans

Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated.     

Genome-wide phylogenetic analysis of Gluconobacter, Acetobacter, and Gluconacetobacter

Phylogenetic relationships among three genera, Gluconobacter, Acetobacter, and Gluconacetobacter, of acetic acid bacteria (AAB) are still unclear, although phylogenetic analysis using 16S rRNA gene sequence has shown that Gluconacetobacter diverged first from the ancestor of these three genera. Therefore, the relationships among these three genera were investigated by genome-wide phylogenetic analysis of AAB. Contrary to the results of 16S rRNA gene analysis, phylogenetic analysis of 293 enzymes involved in metabolism clearly showed that Gluconobacter separated first from its common ancestor with Acetobacter and Gluconacetobacter. In addition, we defined 753 unique orthologous proteins among five known complete genomes of AAB, and phylogenetic analysis was carried out using concatenated gene sequences of these 753 proteins. The result also showed that Gluconobacter separated first from its common ancestor with Acetobacter and Gluconacetobacter. Our results strongly suggest that Gluconobacter was the first to diverge from the common ancestor of Gluconobacter, Acetobacter, and Gluconacetobacter, a relationship that is in good agreement with the physiologies and habitats of these genera.     

Global analysis of the genes involved in the thermotolerance mechanism of thermotolerant Acetobacter tropicalis SKU1100

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.     

Accumulation of Metal-Specific T Cells in Inflamed Skin in a Novel Murine Model of Chromium-Induced Allergic Contact Dermatitis

Chromium (Cr) causes delayed-type hypersensitivity reactions possibly mediated by accumulating T cells into allergic inflamed skin, which are called irritants or allergic contact dermatitis. However, accumulating T cells during development of metal allergy are poorly characterized because a suitable animal model is not available. This study aimed to elucidate the skewing of T-cell receptor (TCR) repertoire and cytokine profiles in accumulated T cells in inflamed skin during elucidation of Cr allergy. A novel model of Cr allergy was induced by two sensitizations of Cr plus lipopolysaccharide solution into mouse groin followed by single Cr challenge into the footpad. TCR repertoires and nucleotide sequences of complementary determining region 3 were assessed in accumulated T cells from inflamed skin. Cytokine expression profiles and T-cell phenotypes were determined by qPCR. CD3+CD4+ T cells accumulated in allergic footpads and produced increased T helper 1 (Th1) type cytokines, Fas, and Fas ligand in the footpads after challenge, suggesting CD4+ Th1 cells locally expanded in response to Cr. Accumulated T cells included natural killer (NK) T cells and Cr-specific T cells with VA11-1/VB14-1 usage, suggesting metal-specific T cells driven by invariant NKT cells might contribute to the pathogenesis of Cr allergy.