Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (−/−) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (−/−) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.     

Language discrimination by Java sparrows

Java sparrows (Padda oryzivora) were trained to discriminate English from Chinese spoken by a bilingual speaker. They could learn discrimination and showed generalization to new sentences spoken by the same speaker and those spoken by a new speaker. Thus, the birds distinguished between English and Chinese. Although auditory cues for the discrimination were not specified, this is the first evidence that non-mammalian species can discriminate human languages.     

Brucella abortusd-alanyl-D-alanine carboxypeptidase contributes to its intracellular replication and resistance against nitric oxide

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes, and cause abortion in domestic animals and undulant fever in humans. However, the mechanism and factors of virulence are not fully understood. In the present study, a D-alanyl-D-alanine carboxypeptidase (DAP) mutant of Brucella abortus failed to replicate in mouse macrophages and HeLa cells, and showed less virulence than the wild type in mice. Under nitric oxide (NO) stress, the growth of the DAP mutant in vitro decreased and it also had less capability to reduce NO than the wild type. Intracellular replication of the DAP mutant was partially restored by pretreatment of macrophages with the NO synthase inhibitor, 1-phenyl-imidazole, and the level of expression of the NO reductase gene, norB, in the DAP mutant was lower than that in the wild type. These results suggest that DAP contributes to resistance against NO and that it is required for the intracellular growth of the bacterium.     

Occurrence of the Fimbria Gene hifA in clinical isolates of nonencapsulated Haemophilus influenzae

The adherence of Haemophilus influenzae to epithelial cells plays a crucial role in infections. However, little is known about the occurrence of fimbriae. In this study, we examined the distribution of the fimbria gene (hifA) by PCR among 167 H. influenzae strains isolated from patients with respiratory infections. Almost all (163; 98%) of the isolates were nonencapsulated strains. The carriage rate of hifA by the nonencapsulated strains was 18.4%. Electron microscopy showed that fimbriae were abundantly present on the cell surface of hifA-positive strains tested. Only four (2.4%) isolates were encapsulated, all of which were type b and did not possess hifA. The present work suggests that fimbriae may play a considerable role as adhesins in nonencapsulated H. influenzae strains.     

Regeneration of the intestinal epithelia: Regulation of bone marrow-derived epithelial cell differentiation towards secretory lineage cells

The intestinal epithelia consists of four lineages of differentiated cells, all of which arise from stem cells residing in the intestinal crypt. For proper regeneration from epithelial damage, both expansion of the epithelial cell number and appropriate regulation of lineage differentiation from the remaining stem cells are thought to be required. In a series of studies, we have shown that bone-marrow derived cells could promote the regeneration of damaged epithelia in the human intestinal tract. Donor-derived epithelial cells substantially repopulated the gastrointestinal tract of bone-marrow transplant recipients during epithelial regeneration after graft-versus-host disease. Furthermore, precise analysis of epithelial cell lineages revealed that during epithelial regeneration, secretory lineage epithelial cells that originated from bone-marrow significantly increased in number. These findings may lead to a novel therapy to repair damaged intestinal epithelia using bone marrow cells, and provide an alternative therapy for refractory inflammatory bowel diseases.     

Capsinoid is biosynthesized from phenylalanine and valine in a non-pungent pepper, Capsicum annuum L. cv. CH-19 sweet

The biosynthetic pathway of capsinoid in 'CH-19 Sweet' was investigated. [(3)H]Valine and [(14)C]phenylalanine were injected into the fruits of the intact plant. Both of radioactivities were detected in capsinoid fractions. (14)C radioactivity was observed in phenylpropanoid compounds, and in vanillin, vanillylamine, vanillyl alcohol, and vanillic acid. We confirmed that capsinoid is biosynthesized from phenylalanine and valine.     

Non-involvement of the human monocarboxylic acid transporter 1 (MCT1) in the transport of phenolic acid

Phenolic acids such as p-coumaric acid and microbial metabolites of poorly absorbed polyphenols are absorbed by the monocarboxylic acid transporter (MCT)-mediated transport system which is identical to the fluorescein/H(+) cotransport system. We focus here on the physiological impact of MCT-mediated absorption and distribution. We examined whether MCT1, the best-characterized isoform found in almost all tissues, is involved in this MCT-mediated transport system. The induction of MCT1 expression in Caco-2 cells by a treatment with sodium butyrate (NaBut) did not increase the fluorescein permeability. Moreover, the transfection of Caco-2 cells with an expression vector encoding MCT1 caused no increase in either the permeability or uptake of fluorescein. Furthermore, in the MCT1-expressing oocytes, no increase of p-coumaric acid uptake was apparent, whereas the uptake of salicylic acid, a substrate of MCT1, nearly doubled. Our data therefore establish that MCT1 was not involved in the MCT-mediated transport of phenolic acids.     

Purification and characterization of phospholipase B from Candida utilis

Phospholipase B (PLB) from the asporogenous yeast Candida utilis was purified to homogeneity from a culture broth. The apparent molecular mass was 90-110 kDa by SDS-PAGE. The enzyme had two pH optima, one acidic (pH 3.0) and the other alkaline (pH 7.5). At acidic pH the enzyme hydrolyzed all phospholipids tested without metal ions. On the other hand, the PLB showed substrate specificity and required metal ions for alkaline activity.The cDNA sequence of the PLB was analyzed by a combination of several PCR procedures. The PLB encoded a protein consisting of 643 amino acids. The amino acid sequence contained a lipase consensus sequence (GxSxG) and catalytic arginine and aspartic acid motifs which were identified as the catalytic triad in the PLB from Kluyveromyces lactis, suggesting that the catalytic mechanism of the PLB is similar to that of cytosolic phospholipase A(2) (cPLA(2)), found in mammalian tissues.     

Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA

alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.     

Synthesis of gerfelin and related analogous compounds

Gerfelin, an inhibitor of human geranylgeranyl diphosphate (GGPP) synthase that has been isolated from a culture broth of Beauveria felina QN22047, was synthesized in 4 and 3 steps starting from 2,4-dihydroxy-6-methylbenzoic acid and 3,4,5-trihydroxytoluene, respectively. An effective ligand, 2-(di-tert-butylphosphino)biphenyl, was used in the palladium-catalyzed diaryl ether-forming reaction. Five analogous compounds of gerfelin were also synthesized for a study of the structure-activity relationship.