Linkage mapping of the locus responsible for congenital multiple ocular defects in cattle on bovine Chromosome 18

Congenital multiple ocular defects (MOD) in Japanese black cattle is a hereditary ocular disorder with an autosomal recessive manner of inheritance, showing developmental defects of the lens, retina, and iris, persistent embryonic eye vascularization, and microphthalmia. In the present study, we mapped the locus responsible for the disorder by linkage analysis using 240 microsatellite markers covering the entire bovine genome and an inbred pedigree obtained from commercial herds. The linkage analysis demonstrated a significant linkage between the disorder locus and markers on the proximal region of bovine Chromosome (BTA) 18 with the maximum LOD score of 5.1. Homozygosity mapping using the haplotype of the linked markers further refined the critical region. The results revealed the localization of the locus responsible for MOD in an approximately 6.6-cM region of BTA18. Comparison of published linkage and radiation hybrid (RH) maps of BTA18 with its evolutionary ortholog, human Chromosome (HSA) 16, revealed several potential candidate genes for the disorder including the MAF and FOXC2 genes.     

Muscle force per cross-sectional area is inversely related with pennation angle in strength trained athletes

The present study aimed to examine the effect of pennation angle on the force per cross-sectional area for elbow extensor muscles in strength-trained athletes. A total of 52 male bodybuilders (n = 32) and Olympic weightlifters (n = 20) did maximal isometric elbow extension on an isokinetic dynamometer. Muscle cross-sectional area (CSA) and muscle-fiber pennation angle (PA) of the triceps brachii muscles were measured by ultrasonography. Bodybuilders had significantly greater isometric elbow extension force (F), CSA and PA than weightlifters. The ratio of force to CSA (F/CSA) of bodybuilders was significantly lower than that of weightlifters. A significant positive correlation was observed between CSA and PA in both groups (r = 0.832, P < 0.001, and r = 0.682, P < 0.001, for bodybuilders and weightlifters, respectively). The F/CSA was negatively correlated to PA both for bodybuilders (r = -0.408, P < 0.05) and weightlifters (r = -0.465, P < 0.05). Thus present study indicates that the larger pennation angle is associated with the lower force relative to muscle CSA in strength-trained athletes.     

Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin

The serine/threonine protein kinase Akt is involved in a variety of cellular processes including cell proliferation, survival, metabolism and gene expression. It is essential in vascular endothelial growth factor (VEGF)-mediated angiogenesis; however, it is not known how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching and the formation of networks during angiogenesis. Here we report that Akt-mediated phosphorylation of Girdin, an actin-binding protein, promotes VEGF-dependent migration of endothelial cells and tube formation by these cells. We found that exogenously delivered adenovirus harbouring Girdin short interfering RNA in Matrigel embedded in mice, markedly inhibited VEGF-mediated angiogenesis. Targeted disruption of the Girdin gene in mice impaired vessel remodelling in the retina and angiogenesis from aortic rings, whereas Girdin was dispensable for embryonic vasculogenesis. These findings demonstrate that the Akt/Girdin signalling pathway is essential in VEGF-mediated postneonatal angiogenesis.     

Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata

The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of beta-D-xylosidase, beta-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated "pathogenesis-related proteins". These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth.     

Heterotypy in the N-terminal region of growth/differentiation factor 5 (GDF5) mature protein during teleost evolution

Heterotypy is now recognized as a generative force in the formationof new proteins through modification of existing proteins. Wereport that heterotypy in the N-terminal region of the maturegrowth/differentiation factor 5 (GDF5) protein occurred duringevolution of teleosts. N-terminal length variation of GDF5 wasfound among teleost interfamilies and interorders but not withinteleost families or among tetrapods. We further show that increaseof proline and glutamine to the N-terminal region of matureGDF5 occurred in Eurypterygii, the higher lineage of teleosts.Because the basic amino acids, believed to control diffusion,are conserved in this region across all species examined, wesuggest that the N-terminal elongation of the mature GDF5 proteinduring evolution has altered the protein diffusion in Eurypterygii,leading to high concentrations of the protein in the joint ofthe pharyngeal skeleton, the location of cartilage formationduring development.     

Intestinal epithelial cancer cell anoikis resistance: EGFR‐mediated sustained activation of Src overrides Fak‐dependent signaling to MEK/Erk and/or PI3‐K/Akt‐1

Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3‐K/Akt‐1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down‐activation, cancer cells nevertheless exhibit sustained Fak–Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3‐K/Akt‐1 down‐activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3‐K/Akt‐1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak‐mediated signaling to MEK/Erk and PI3‐K/Akt‐1 in T84 cells, as in undifferentiated IECs, whereas PI3‐K/Akt‐1 is Src‐independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak–Src interactions and down‐activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3‐K/Akt‐1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR‐mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak–Src interactions and MEK/Erk activation, thus not only overriding Fak‐mediated signaling to MEK/Erk and/or PI3‐K/Akt‐1, but also the requirement of Fak and/or PI3‐K/Akt‐1 for survival. J. Cell. Biochem. 107: 639–654, 2009. © 2009 Wiley‐Liss, Inc.     

Diphosphorylation of regulatory light chain of myosin IIA is responsible for proper cell spreading

The actomyosin cytoskeleton plays prominent roles in cell spreading and migration. To address the roles of myosin II isoforms and to estimate the region where the myosin IIs are activated in spreading cells, we examined the immunolocalization of myosin II isoforms and phosphorylated RLCs in the spreading MRC-5 cells. We observed the formation of actin ring-like structure at the base of the lamella. Both myosin IIA and IIB were predominantly localized there. Myosin IIA and diphosphorylated RLC were distributed outside of the region where myosin IIB and monophosphoryated RLC were distributed predominantly. Inhibition of Rho-kinase resulted in the disappearance of the diphosphorylation of RLC, moreover, it accelerated the rate of cell spreading and induced an aberrant cell shape at later stage of spreading. These results indicate that diphosphorylation of RLCs of myosin IIA by Rho-kinase in lamella is responsible for the cell to spread properly.     

Solution structure of the SWIRM domain of human histone demethylase LSD1

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.     

Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.