Adrenomedullin is an autocrine/paracrine growth factor for rat vascular smooth muscle cells

Adrenomedullin is a potent vasodilator peptide secreted by vascular endothelial and smooth muscle cells. Adrenomedullin stimulates the proliferation of quiescent rat vascular smooth muscle cells (VSMCs) via p42/p44 ERK/MAP kinase activation. Recently, receptor-activity-modifying proteins (RAMPs) have been shown to transport calcitonin-receptor-like-receptor (CRLR) to the cell surface to present either as CGRP receptor or adrenomedullin receptor. We investigated whether adrenomedullin acts as an autocrine/paracrine growth factor for cultured rat VSMCs and whether coexpressions of RAMP isoform and CRLR may mediate p42/p44 ERK/MAP kinase activation by adrenomedullin. Adrenomedullin dose-dependently stimulated the proliferation of quiescent rat VSMCs, and this effect was inhibited by an adrenomedullin receptor antagonist, a MAP kinase kinase inhibitor and phosphatidylinositol 3-kinase inhibitors. Addition of either CGRP(8-37) or anti-adrenomedullin antibody to exponentially growing rat VSMCs inhibited the serum-induced cell proliferation, suggesting its role as an autocrine/paracrine growth factor. Cotransfection of RAMP2 or RAMP3 with CRLR into rat VSMCs potentiated activation of cAMP activity, but not of p42/p44 ERK/MAP kinase activity in response to adrenomedullin. Our results suggest that adrenomedullin is an autocrine/paracrine growth factor for rat VSMCs via p42/p44 ERK/MAP kinase and phosphatidylinositol 3-kinase pathways and that it is not mediated by human RAMP-CRLR receptors.     

Establishment of hepatic stem-like cell lines from normal adult porcine liver in a poly-D-lysine-coated dish with nair-1 medium

The existence, origin, and bipotency of the hepatic stem cell (HeSC) have been investigated. However, the isolation and culture of HeSCs from adult liver tissue is not yet well established, and the mechanism by which HeSCs differentiate into mature cells remains unclear. On the other hand, the development of HeSC-isolating and -culturing methods and the in vitro clonal analysis of their mechanism of differentiation are required to enable clinical applications of regenerative medicine in the liver. For the purpose of providing HeSCs for these studies, we attempted to establish an HeSC line from a normal adult porcine liver using a unique culture system, a poly-D-lysine-coated culture dish with NAIR-1 medium (the PDL-NAIR-1 culture system). Moreover, we examined the differentiating capacity of HeSCs in vitro. We demonstrated that it was possible in the culture system that immature epithelial cells capable of proliferating grew selectively into aggregates and that two hepatic stem-like cell lines, PHeSC-A1 and PHeSC-A2, were established. The results from our data suggest that these hepatic stem-like cell lines were capable of self-renewing and differentiating into hepatocytes or biliary epithelial cells and show that the PDL-NAIR-1 culture system offers the immense advantage of isolating and culturing HeSCs from a normal adult liver. Furthermore, because of the ability to use a clonal analysis in vitro, these cell lines are useful for the investigation of various mechanisms in which HeSCs seem to participate and their application in the study of regenerative medicine in the liver.     

SOCS-3 regulates onset and maintenance of T(H)2-mediated allergic responses

Members of the suppressor of cytokine signaling (SOCS) family are involved in the pathogenesis of many inflammatory diseases. SOCS-3 is predominantly expressed in T-helper type 2 (T(H)2) cells, but its role in T(H)2-related allergic diseases remains to be investigated. In this study we provide a strong correlation between SOCS-3 expression and the pathology of asthma and atopic dermatitis, as well as serum IgE levels in allergic human patients. SOCS-3 transgenic mice showed increased T(H)2 responses and multiple pathological features characteristic of asthma in an airway hypersensitivity model system. In contrast, dominant-negative mutant SOCS-3 transgenic mice, as well as mice with a heterozygous deletion of Socs3, had decreased T(H)2 development. These data indicate that SOCS-3 has an important role in regulating the onset and maintenance of T(H)2-mediated allergic immune disease, and suggest that SOCS-3 may be a new therapeutic target for the development of antiallergic drugs.     

Crystal structures of bacterial lipoprotein localization factors,LolA and LolB

Lipoproteins having a lipid-modified cysteine at the N-terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram-negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of LolA and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta barrel and an alpha-helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB and from LolB to the outer membrane. Furthermore, the structures of both LolA and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.     

Studies on Japanese Botryllid Ascidians. III. A new species of the genus Botryllus with a vivid colony color from the vicinity of Shimoda

The morphology and life history of a new species of the genus Botryllus belonging to the family Botryllidae are described in detail. This ascidian was collected from the stony shore in the cove near Shimoda Marine Research Center, University of Tsukuba (Shimoda, Shizuoka Prefecture, Japan). The ascidian colony was easily distinguished from colonies of other botryllids because it was very thin and bright pink in color. The arrangement of ovary and testis in this ascidian was the same as that in other species of the genus Botryllus. This ascidian was prolific, with 1-5 embryos on each side of a zooid, and the embryos of this ascidian developed in the peribranchial cavity without any brooding organs as in Botryllus scalaris. We observed the processes and features of the allorecognition reaction in colony specificity and found that allorejection occurred after fusion of the vascular system between two incompatible colonies. This manner of allorejection is also shown in B. scalaris and Botryllus delicatus; however, the reaction speed of allorejection is faster than that of B. delicatus and similar to that of B. scalaris. These results indicate that this ascidian might be closely related to B. scalaris.     

Geographic variation and diversity of the cytochrome b gene in Japanese wild populations of medaka, Oryzias latipes

We conducted a polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome b gene to elucidate the detailed genetic population structure of Japanese wild populations of medaka, Oryzias latipes. The analysis of 1,225 specimens collected from 303 sites identified 67 mitotypes. Subsequently we determined the nucleotide sequences of the complete cytochrome b gene (1141-bp) to clarify the phylogenetic relationships among mitotypes. The phylogenetic tree based on nucleotide sequences indicated three major clades (A, B and C) that differed by 11.3-11.8%, corresponding to three clusters previously identified by RFLP analysis of entire mitochondrial DNAs. The geographic distribution of mitotypes in clades A and B was fully concordant with the Northern and Southern Populations defined by allozymes. Clade A could be subdivided into three subclades and clade B into eleven, with sequence divergences among subclades of 1.3-5.8%. Each distribution of mitotypes in subclades roughly corresponded to that of mtDNA haplotypes in subclusters previously identified. Mitotypes in clade C were found only in the Kanto district. The phylogenetic relationships and the estimated divergence times suggest that three Japanese clades originated from a common ancestor and were separated during the Pliocene, and that the regional differentiation of subclades was closely connected with the geological history of the Quaternary. This study has also demonstrated the possibility of artificial disturbance of natural distribution especially in the Kanto district and the superior efficacy of PCR-RFLP analysis as a simple method for detecting genetic variation and artificial gene flow of medaka.     

Production of D-arabitol by Metschnikowia reukaufii AJ14787

A potent producer of D-arabitol was isolated by screening of natural sources and identified as Metschnikowia reukaufii AJ14787. Resting cells of this strain can efficiently produce D-arabitol from D-glucose with a weight yield of more than 60%, and can also produce D-arabitol from several other types of sugars such as polyols, ketoses, and aldoses. To improve productivity, various culture conditions such as temperature and the concentrations of D-glucose and nitrogen sources were examined. Under optimal conditions, 206 g/l of D-arabitol was produced from D-glucose with a weight yield of 52% in 100 hours.     

Isolation and characterization of aromatics-degrading microorganisms from the gut of the lower termite Coptotermes formosanus

We isolated aromatics-degrading bacteria from the gut of a lower termite, Coptotermes formosanus, using a mineral salt medium containing various aromatic compounds as the sole carbon source. Two species, Burkholderia sp. strain VE22 and Citrobacter sp. strain VA53, were isolated by aerobic enrichment culture with veratraldehyde and vanillin, respectively. Strain VA53 could also grow and metabolize vanillin anaerobically.     

Identification of volicitin-related compounds from the regurgitant of lepidopteran caterpillars

Volicitin-related compounds were found in the oral secretion of the three noctuid species, Helicoverpa armigera, Mythimna separata and Spodoptera litura, and one sphingid species, Agrius convolvuli. Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine], N-(17-hydroxylinoleoyl)-glutamine, N-linolenoylglutamine and N-linoleoylglutamine were identified in the secretion from the noctuid larvae. In secretions from the sphingid larvae, N-linolenoylglutamine and N-linoleoylglutamine were the main components. Furthermore, there were significant differences in the amounts of the N-acylamino acid conjugates in the secretions from the three noctuid species. These results suggest that the proportion of volicitin-related compounds in the regurgitant was species-specific.