The pro-peptide of Streptomyces mobaraensis transglutaminase functions in cis and in trans to mediate efficient secretion of active enzyme from methylotrophic yeasts

Transglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation.     

Fungicide activity through activation of a fungal signalling pathway

Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis. Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway. The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose. The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil. The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress. Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth. In the presence of fludioxonil, the wild-type fungus was not able to infect the host plant because of a failure of appressorium-mediated penetration, whereas osc1 mutants successfully infected plants. Analysis using a OSC1-GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1. Furthermore, fludioxonil activates Hog1-type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea. These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.     

Roles and modes of action of nectins in cell-cell adhesion

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules (CAMs), which comprise a family consisting of four members. Each nectin homophilically and heterophilically trans-interacts and causes cell-cell adhesion. Biochemical, cell biological, and knockout mice studies have revealed that nectins play important roles in formation of many types of cell-cell junctions and cell-cell contacts, including cadherin-based adherens junctions (AJs) and synapses. Mode of action of nectins in the formation of AJs has extensively been investigated. Nectins form initial cell-cell adhesion and recruit E-cadherin to the nectin-based cell-cell adhesion sites. In addition, nectins induce activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of cadherin-based AJs through the reorganization of the actin cytoskeleton. Nectins furthermore heterophilically trans-interact with nectin-like molecules (Necls), other Ig-like CAMs, and assist or modify their various functions, such as cell adhesion, migration, and proliferation. We describe here the roles and modes of action of nectins as CAMs.     

In vitro differentiation from naive to mature E-selectin binding CD4 T cells: acquisition of skin-homing properties occurs independently of cutaneous lymphocyte antigen expression

We previously showed that skin-homing CD4 T cells in peripheral blood can be subdivided into three populations on the basis of the expression pattern of the cutaneous lymphocyte Ag (CLA) and fucosyltransferase VII (FucT-VII): FucT-VII(+)CLA(-), FucT-VII(+)CLA(+), and FucT-VII(-)CLA(+). In view of the known late appearance of CLA during T cell differentiation, T cells programmed to attain skin-homing properties may start to generate E-selectin-binding epitopes at early stages of differentiation before induction of CLA expression. To this end, the in vitro differentiation from naive to CLA(+) memory T cells was followed after activation with anti-CD3 mAb. Here we demonstrate that naive skin-homing CD4 T cell precursors undergo a linear differentiation process from the FucT-VII(+)CLA(-) phenotype to the FucT-VII(+)CLA(+) phenotype and eventually to the FucT-VII(-)CLA(+) phenotype. The appearance of the FucT-VII(+)CLA(-) subset coincided with or could be immediately followed by the generation of E-selectin binding epitopes, and even after E-selectin-binding epitopes were no longer detectable, CLA remained expressed for prolonged periods of time, suggesting that induction of functional E-selectin ligands depends primarily on the expression of FucT-VII, but not CLA. Immunofluorescence and confocal microscopy studies of these T cells confirm that most E-selectin ligands were found independently of CLA expression.     

Regulation by nectin of the velocity of the formation of adherens junctions and tight junctions

Cadherins are key Ca(2+)-dependent cell-cell adhesion molecules at adherens junctions (AJs) in fibroblasts and epithelial cells, whereas claudins are key Ca(2+)-independent cell-cell adhesion molecules at tight junctions (TJs) in epithelial cells. The formation and maintenance of TJs are dependent on the formation and maintenance of AJs. Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules which comprise a family of four members, nectin-1, -2, -3, and -4, and are involved in the formation of AJs in cooperation with cadherins, and the subsequent formation of TJs. We show here that the velocity of the formation of the E-cadherin-based AJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in L cells stably expressing E-cadherin and Madin-Darby canine kidney cells. Moreover, the velocity of the formation of the claudin-based TJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in Madin-Darby canine kidney cells. These results indicate that nectins regulate the velocity of the formation of the E-cadherin-based AJs and the subsequent formation of the claudin-based TJs.     

Crystal structure of serine dehydratase from rat liver

SDH (L-serine dehydratase, EC 4.3.1.17) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent dehydration of L-serine to yield pyruvate and ammonia. Liver SDH plays an important role in gluconeogenesis. Formation of pyruvate by SDH is a two-step reaction in which the hydroxyl group of serine is cleaved to produce aminoacrylate, and then the aminoacrylate is deaminated by nonenzymatic hydrolysis to produce pyruvate. The crystal structure of rat liver apo-SDH was determined by single isomorphous replacement at 2.8 A resolution. The holo-SDH crystallized with O-methylserine (OMS) was also determined at 2.6 A resolution by molecular replacement. SDH is composed of two domains, and each domain has a typical alphabeta-open structure. The active site is located in the cleft between the two domains. The holo-SDH contained PLP-OMS aldimine in the active site, indicating that OMS can form the Schiff base linkage with PLP, but the subsequent dehydration did not occur. Apo-SDH forms a dimer by inserting the small domain into the catalytic cleft of the partner subunit so that the active site is closed. Holo-SDH also forms a dimer by making contacts at the back of the clefts so that the dimerization does not close the catalytic cleft. The phosphate group of PLP is surrounded by a characteristic G-rich sequence ((168)GGGGL(172)) and forms hydrogen bonds with the amide groups of those amino acid residues, suggesting that the phosphate group can be protonated. N(1) of PLP participates in a hydrogen bond with Cys303, and similar hydrogen bonds with N(1) participating are seen in other beta-elimination enzymes. These hydrogen bonding schemes indicate that N(1) is not protonated, and thus, the pyridine ring cannot take a quinone-like structure. These characteristics of the bound PLP suggest that SDH catalysis is not facilitated by forming the resonance-stabilized structure of the PLP-Ser aldimine as seen in aminotransferases. A possible catalytic mechanism involves the phosphate group, surrounded by the characteristic sequence, acting as a general acid to donate a proton to the leaving hydroxyl group of serine.     

Fecal microbial diversity in a strict vegetarian as determined by molecular analysis and cultivation

Fecal microbial diversity in a strictly vegetarian woman was determined by the 16S rDNA library method, terminal restriction fragment length polymorphism (T-RFLP) analysis and a culture-based method. The 16S rDNA library was generated from extracted fecal DNA, using bacteria-specific primers. Randomly selected clones were partially sequenced. T-RFLP analysis was performed using amplified 16S rDNA. The lengths of T-RF were analyzed after digestion by HhaI and MspI. The cultivated bacterial isolates were used for partial sequencing of 16S rDNA. Among 183 clones obtained, approximately 29% of the clones belonged to 13 known species. About 71% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). A total of 55 species or phylotypes were identified among the 16S rDNA library, while the cultivated isolates included 22 species or phylotypes. In addition, many new phylotypes were detected from the 16S rDNA library. The 16S rDNA library and isolates commonly included the Bacteroides group, Bifidobacterium group, and Clostridium rRNA clusters IV, XIVa, XVI and XVIII. T-RFLP analysis revealed the major composition of the vegetarian gut microbiota were Clostridium rRNA subcluster XIVa and Clostridium rRNA cluster XVIII. The dominant feature of this strictly vegetarian gut microbiota was the detection of many Clostridium rRNA subcluster XIVa and C. ramosum (Clostridium rRNA cluster XVIII).     

Stimulation of the secretion of pro-inflammatory cytokines by Bifidobacterium strains

To characterize the ability of bifidobacteria to affect the production of macrophage-derived cytokines, a murine macrophage-like cell line, J774.1, was cultured in the presence of 27 strains of heat-inactivated bifidobacteria. Bifidobacterium adolescentis and B. longum, known as adult-type bifidobacteria, induced significantly more pro-inflammatory cytokine secretion, IL-12 and TNF-alpha, by J774.1 cells, than did the infant-type bifidobacteria, B. bifidum, B. breve, and B. infantis (P<0.01). In contrast, B. adolescentis did not stimulate the production of anti-inflammatory IL-10 from J774.1 cells as the other tested bacteria did. The results suggest that the adult-type bifidobacteria, especially B. adolescentis, may be more potent to amplify but less able to down-regulate the inflammatory response.