Synthesis and characterization of quaternized β-chitin

Water-soluble 2′-O-hydroxypropyltrimethylammoniumchitin chloride (2′-O-HTACCt) was prepared directly from β-chitin and 3-chloro-2-hydroxypropyltrimethylammonium chloride (CTA) in basic medium. The effect of alkali concentration, reaction temperature, reaction time, and dosage of CTA on yield and degree of substitution (DS) of 2′-O-HTACCt were studied. These quaternized chitin derivatives were characterized by FTIR and ¹H NMR spectroscopy, conductometric titration, and elemental analysis methods. Research results indicate that β-chitin can react directly with CTA to produce a water-soluble 2′-O-HTACCt derivative with a high DS. The optimal preparation conditions were determined to be 35-40 wt % (aq NaOH), 40 °C (reaction temperature), 6 h (reaction time), and 4 (molar ratio of CTA to β-chitin unit).     

TFPI or uPA-PAI-1 complex affect cell function through expression variation of type II very low density lipoprotein receptor

Very low density lipoprotein receptors (VLDLR) including type I and type II are known to affect cell functions by binding to its extracellular ligands. However, the effect of these ligands on VLDLR expression remains elusive. Tissue factor pathway inhibitor (TFPI) and urokinase plasminogen activator and plasminogen activator inhibitor 1 (uPA-PAI-1) complex, two ligands of VLDLR, were used to examine their effects on VLDLR expression. TFPI treatment decreased type II VLDLR expression, inhibited cell proliferation and migration, and degradated β-catenin in SGC7901 cells. However, uPA-PAI-1 complex, increased type II VLDLR expression with promoted cell proliferation and migration and stabilization of β-catenin. These results indicated that extracellular ligands can change the expression of type II VLDLR to affect cell proliferation and migration.     

Shear-induced Interleukin-6 Synthesis in Chondrocytes: ROLES OF E PROSTANOID (EP) 2 AND EP3 IN cAMP/PROTEIN KINASE A- AND PI3-K/Akt-DEPENDENT NF-κB ACTIVATION*

Mechanical overloading of cartilage producing hydrostatic stress, tensile strain, and fluid flow can adversely affect chondrocyte function and precipitate osteoarthritis (OA). Application of high fluid shear stress to chondrocytes recapitulates the earmarks of OA, as evidenced by the release of pro-inflammatory mediators, matrix degradation, and chondrocyte apoptosis. Elevated levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E₂, and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated chondrocytes in vitro. Although PGE₂ positively regulates IL-6 synthesis in chondrocytes, the underlying signaling pathway of shear-induced IL-6 expression remains unknown. Using the human T/C-28a2 chondrocyte cell line as a model system, we demonstrate that COX-2-derived PGE₂ signals via up-regulation of E prostanoid (EP) 2 and down-regulation of EP3 receptors to raise intracellular cAMP, and activate protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. PKA and PI3-K/Akt transactivate the NF-κB p65 subunit via phosphorylation at Ser-276 and Ser-536, respectively. Binding of p65 to the IL-6 promoter elicits IL-6 synthesis in sheared chondrocytes. Selective knockdown of EP2 or ectopic expression of EP3 blocks PKA- and PI3-K/Akt-dependent p65 activation and markedly diminishes shear-induced IL-6 expression. Similar inhibitory effects on IL-6 synthesis were observed by inhibiting PKA, PI3-K, or NF-κB using pharmacological and/or genetic interventions. Reconstructing the signaling network regulating shear-induced IL-6 expression in chondrocytes may provide insights for developing therapeutic strategies for arthritic disorders and for culturing artificial cartilage in bioreactors.     

GenePro: a Cytoscape plug-in for advanced visualization and analysis of interaction networks

MOTIVATION: Analyzing the networks of interactions between genes and proteins has become a central theme in systems biology. Versatile software tools for interactively displaying and analyzing these networks are therefore very much in demand. The public-domain open software environment Cytoscape has been developed with the goal of facilitating the design and development of such software tools by the scientific community. RESULTS: We present GenePro, a plugin to Cytoscape featuring a set of versatile tools that greatly facilitates the visualization and analysis of protein networks derived from high-throughput interactions data and the validation of various methods for parsing these networks into meaningful functional modules. AVAILABILITY: The GenePro plugin is available at the website http://genepro.ccb.sickkids.ca.     

A multivariate approach for integrating genome-wide expression data and biological knowledge

MOTIVATION: Several statistical methods that combine analysis of differential gene expression with biological knowledge databases have been proposed for a more rapid interpretation of expression data. However, most such methods are based on a series of univariate statistical tests and do not properly account for the complex structure of gene interactions. RESULTS: We present a simple yet effective multivariate statistical procedure for assessing the correlation between a subspace defined by a group of genes and a binary phenotype. A subspace is deemed significant if the samples corresponding to different phenotypes are well separated in that subspace. The separation is measured using Hotelling's T(2) statistic, which captures the covariance structure of the subspace. When the dimension of the subspace is larger than that of the sample space, we project the original data to a smaller orthonormal subspace. We use this method to search through functional pathway subspaces defined by Reactome, KEGG, BioCarta and Gene Ontology. To demonstrate its performance, we apply this method to the data from two published studies, and visualize the results in the principal component space.     

Ganglioside GD1a regulation of caveolin-1 and Stim1 expression in mouse FBJ cells:Augmented expression of caveolin-1 and Stim1 in cells with increased GD1a content

GD1a was previously shown responsible for regulating cell motility, cellular adhesiveness to vitronectin, phosphorylation of c-Met and metastatic ability of mouse FBJ osteosarcoma cells. To determine the particular molecules regulated by GD1a, FBJ cells were assessed for tumor-related gene expression by semi-quantitative RT-PCR. Caveolin-1 and stromal interaction molecule 1 (Stim1) expression in FBJ-S1 cells, rich in GD1a, were found to be 6 and 4 times as much, respectively, than in FBJ-LL cells devoid of GD1a. Enhanced production of caveolin-1 in protein was confirmed by Western blotting. A low-metastatic FBJ-LL cell variant, having high GD1a expression through β1-4GalNAcT-1 (GM2/GD2 synthase) cDNA transfection (Hyuga S, et al, Int J Cancer 83: 685-91, 1999), showed enhanced production of caveolin-1 and Stim1 in mRNA and protein, compared to mock-transfectant M5. Incubation of FBJ-M5 cells with exogenous GD1a augmented the expression of caveolin-1 in mRNA and protein and Stim1 in mRNA as well. Treatment of FBJ-S1 with fumonisin B1, an inhibitor of N-acylsphinganine synthesis, for 15 days caused the complete depletion of gangliosides and suppressed the expression of caveolin-1 and Stim1. St3gal5 siRNA transfected cells showed decreased expression of caveolin-1 and Stim1 mRNA, as well as St3gal5 mRNA. These findings clearly indicate ganglioside GD1a to be involved in the regulation of the transformation suppressor genes, caveolin-1 and Stim1. Moreover, treatment with GD1a of mouse melanoma B16 cells and human hepatoma HepG2 cells brought about elevated expression of caveolin-1 and Stim1. Li Wang and Shizuka Takaku are equal contributors to the present work     

Outbreak of Trichophyton verrucosum in China Transmitted from Cows to Humans

Twenty percent of 1000 dairy cows were infected with Trichophyton verrucosum in a newly established dairy farm in western China. The disease was transmitted to 30 of 100 animal workers. The source of the outbreak is unknown but it is suspected that naïve cattle which were transported from Holland to China became infected after they arrived in Xinjiang province (West China) where the fungus is endemic in nature, prepuberty school children and various livestock.     

Calyculin and okadaic acid promote perilipin phosphorylation and increase lipolysis in primary rat adipocytes

Lipolysis is primarily regulated by protein kinase A (PKA), which phosphorylates perilipin and hormone-sensitive lipase (HSL), and causes translocation of HSL from cytosol to lipid droplets in adipocytes. Perilipin coats lipid droplet surface and assumes to prevent lipase access to triacylglycerols, thus inhibiting basal lipolysis; phosphorylated perilipin facilitates lipolysis on PKA activation. Here, we induced lipolysis in primary rat adipocytes by inhibiting protein serine/threonine phosphatase with specific inhibitors, okadaic acid and calyculin. The incubation with calyculin promotes incorporation of 32Pi into perilipins, thus, confirming that perilipin is hyperphosphorylated. The lipolysis response to calyculin is gradually accompanied by increased accumulation of phosphorylated perilipin A in a concentration- and time-responsive manner. When perilipin phosphorylation is abrogated by the addition of N-ethylmaleimide, lipolysis ceases. Different from a considerable translocation of HSL upon PKA activation with isoproterenol, calyculin does not alter HSL redistribution in primary or differentiated adipocytes, as confirmed by both immunostaining and immunoblotting. Thus, we suggest that inhibition of the phosphatase by calyculin activates lipolysis via promoting perilipin phosphorylation rather than eliciting HSL translocation in adipocytes. Further, we show that when the endogenous phosphatase is inhibited by calyculin, simultaneous PKA activation with isoproterenol converts most of the perilipin to the hyperphosphorylated species, and induces enhanced lipolysis. Apparently, as PKA phosphorylates perilipin and stimulates lipolysis, the phosphatase acts to dephosphorylate perilipin and attenuate lipolysis. This suggests a two-step strategy governed by a kinase and a phosphatase to modulate the steady state of perilipin phosphorylation and hence the lipolysis response to hormonal stimulation.     

Extremely low penetrance of deafness associated with the mitochondrial 12S rRNA mutation in 16 Chinese families: implication for early detection and prevention of deafness

Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. We report here the clinical, genetic, and molecular characterization of 16 Chinese pedigrees (a total of 246 matrilineal relatives) with aminoglycoside-induced impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects, although these subjects share some common features: being bilateral and sensorineural hearing impairment. Strikingly, these Chinese pedigrees exhibited extremely low penetrance of hearing loss, ranging from 4% to 18%, with an average of 8%. In particular, nineteen of 246 matrilineal relatives in these pedigrees had aminoglycoside-induced hearing loss. Mutational analysis of the mtDNA in these pedigrees showed the presence of homoplasmic 12S rRNA A1555G mutation, which has been associated with hearing impairment in many families worldwide. The extremely low penetrance of hearing loss in these Chinese families carrying the A1555G mutation strongly supports the notion that the A1555G mutation itself is not sufficient to produce the clinical phenotype. Children carrying the A1555G mutation are susceptible to the exposure of aminoglycosides, thereby inducing or worsening hearing impairment, as in the case of these Chinese families. Using those genetic and molecular approaches, we are able to diagnose whether children carry the ototoxic mtDNA mutation. Therefore, these data have been providing valuable information and technology to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside therapy, and eventually to decrease the incidence of deafness.     

A biogeographic delineation of the European Alpine System based on a cluster analysis of Carex curvula-dominated grasslands

Biogeographic delineations within the European temperate mountains remain poorly understood, as there has been little effort to assemble and analyze vegetation relevés covering Pyrenees, Alps, Carpathians and Balkans altogether. Our study tackles this issue by focusing on the widely distributed alpine acidic grasslands dominated by Carex curvula. Cluster analysis of more than 800 vegetation relevés revealed the European-scale spatial patterns of vascular plant diversity in these alpine grasslands. The geographical distribution of floristic clusters was partly congruent with the physiography of European mountains. Southern European ranges (Southern Balkans and Pyrenees) exhibit a high level of endemism and corresponding floristic clusters are well separated from the others. Marked floristic similarities between the Easternmost Alps, the Carpathians, and the Northern Balkans (Stara Planina) supported a major floristic boundary that runs through the Austrian Alps and that is likely the legacy of a shared Quaternary history. Within the Alps, floristic clustering was mainly driven by ecological drivers and not geography. This paper presents the first detailed study of spatial patterns of species distribution within the European Alpine System, based on a comprehensive analysis of within- and between-community species diversity. It shows that the quantitative analysis of large and consistent data sets may question the traditional delineations of biogeographic regions within European mountains.