Comparative analysis of the secretory proteome of human adipose stromal vascular fraction cells during adipogenesis

Adipogenesis is a complex process that is accompanied by a number of molecular events. In this study, a proteomic approach was adopted to identify secretory factors associated with adipogenesis. A label‐free shotgun proteomic strategy was implemented to analyze proteins secreted by human adipose stromal vascular fraction cells and differentiated adipocytes. A total of 474 proteins were finally identified and classified according to quantitative changes and statistical significances. Briefly, 177 proteins were significantly upregulated during adipogenesis (Class I), whereas 60 proteins were significantly downregulated (Class II). Changes in the expressions of several proteins were confirmed by quantitative RT‐PCR and immunoblotting. One obvious finding based on proteomic data was that the amounts of several extracellular modulators of Wnt and transforming growth factor‐β (TGF‐β) signaling changed during adipogenesis. The expressions of secreted frizzled‐related proteins, dickkopf‐related proteins, and latent TGF‐β‐binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF‐β signaling. This study provides useful tools and important clues regarding the roles of secretory factors during adipogenic differentiation, and provides information related to obesity and obesity‐related metabolic diseases.     

The novel adipocytokine visfatin exerts direct cardioprotective effects

Visfatin is an adipocytokine capable of mimicking the glucose-lowering effects of insulin and activating the pro-survival kinases phosphatidylinositol-3-OH kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinase kinase 1 and 2 (MEK1/2)-extracellular signal-regulated kinase 1 and 2 (Erk 1/2). Experimental studies have demonstrated that the activation of these kinases confers cardioprotection through the inhibition of the mitochondrial permeability transition pore (mPTP). Whether visfatin is capable of exerting direct cardioprotective effects through these mechanisms is unknown and is the subject of the current study. Anaesthetized C57BL/6 male mice were subjected to in situ 30 min. of regional myocardial ischaemia and 120 min. of reperfusion. The administration of an intravenous bolus of visfatin (5 x 10(-6) micromol) at the time of myocardial reperfusion reduced the myocardial infarct size from 46.1+/-4.1% in control hearts to 27.3+/-4.0% (n>or= 6/group, P<0.05), an effect that was blocked by the PI3K inhibitor, wortmannin, and the MEK1/2 inhibitor, U0126 (48.8+/-5.5% and 45.9+/-8.4%, respectively, versus 27.3+/-4.0% with visfatin; n>or= 6/group, P<0.05). In murine ventricular cardiomyocytes subjected to 30 min. of hypoxia followed by 30 min. of reoxygenation, visfatin (100 ng/ml), administered at the time of reoxygenation, reduced the cell death from 65.2+/-4.6% in control to 49.2+/-3.7%(n>200 cells/group, P<0.05), an effect that was abrogated by wortmannin and U0126 (68.1+/-5.2% and 59.7+/-6.2%, respectively; n>200 cells/group, P>0.05). Finally, the treatment of murine ventricular cardiomyocytes with visfatin (100 ng/ml) delayed the opening of the mPTP induced by oxidative stress from 81.2+/-4 sec. in control to 120+/-7 sec. (n>20 cells/group, P<0.05) in a PI3K- and MEK1/2-dependent manner. We report that the adipocytokine, visfatin, is capable of reducing myocardial injury when administered at the time of myocardial reperfusion in both the in situ murine heart and the isolated murine cardiomyocytes. The mechanism appears to involve the PI3K and MEK1/2 pathways and the mPTP.     

Piezoelectric effect stimulates the rearrangement of chondrogenic cells and alters ciliary orientation via atypical PKCζ

Therapeutic ultrasound was administered to patients suffering from bone fracture with FDA approval. Bone and cartilage are piezoelectric materials. To investigate the effects of piezoelectricity on the cells of chondrogenic lineage, we applied ultrasound stimulation on an AT-cut quartz coverslip to generate electric field fluctuations. The bone-marrow-derived mesenchymal stem cells (BMMSC) and primary chondrocytes were cultured on either glass or quartz coverslips for ultrasound stimulation. The cells were immunofluorescent-labeled for the assessment of cell arrangement and ciliary orientation. Ultrasound and piezoelectricity both stimulate cell migration and disrupt ciliary orientation induced by directional migration. In particular, piezoelectric effects on cell rearrangement can be abolished by the inhibitor specifically targeting atypical Protein kinase C zeta (PKCζ). Our findings shed light on the possibility of cellular modulation by using piezoelectric manipulation.     

Trichoderma songyi sp. nov., a new species associated with the pine mushroom (Tricholoma matsutake)

A new species, Trichoderma songyi, was found to be associated with the pine mushroom (Tricholoma matsutake) in Korea. This species was isolated from three different substrates: Tricholoma matsutake basidiomata, as well as roots of Pinus densiflora and soil in the fairy ring. Based on its molecular and phenotypic characteristics, we demonstrate that Trichoderma songyi is unique and distinguishable from closely related species. We performed phylogenetic analyses based on two molecular markers, the genes for both translation elongation factor 1-alpha and the second largest subunit of RNA polymerase II. Phylogenetic analyses showed that Trichoderma songyi is closely related to Trichoderma koningii aggregate and Trichoderma caerulescens. Morphologically, Trichoderma songyi can be distinguished from these closely related taxa by its growth rates, colony morphology on PDA in darkness, and coconut-like odour. Due to the economic importance of the pine mushroom, the relationship between Trichoderma songyi and Tricholoma matsutake should be studied further.     

Antimicrobial Resistance and Virulence Genes of Escherichia coli Isolates from Swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by ≥8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (≤3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

Design of a description language for generating wrapper to collect biological data

The biological data are scattered in various areas with various formats and they are changing continuously. Therefore, data integration becomes an important issue to provide researcher a dynamic access of data. In the data integration process, the method of extracting heterogeneous data dynamically from the data source is an essential part. Data extraction method using wrapper can provide flexibility and extensibility to an integration system.     

Conservation value of degraded habitats for forest birds in southern Peninsular Malaysia

Clearance of tropical forest for agricultural purposes is generally assumed to seriously threaten the survival of forest species. In this study, we quantified the conservation value, for forest bird species, of three degraded habitat types in Peninsular Malaysia, namely rubber tree plantations, oil palm plantations, and open areas. We surveyed these degraded habitats using point counts to estimate their forest bird species richness and abundance. We assessed whether richness, abundance, and activities of different avian dietary groups (i.e. insectivores and frugivores) varied among the habitats. We identified the critical habitat elements that accounted for the distribution of forest avifauna in these degraded habitats. Our results showed that these habitats harboured a moderate fraction of forest avifauna (approximately 46–76 species) and their functions were complementary (i.e. rubber tree plantations for moving; open habitats for perching; shrubs in oil palm plantations for foraging). In terms of species richness and abundance, rubber tree plantations were more important than oil palm plantations and open habitats. The relatively high species richness of this agricultural landscape was partly due to the contiguity of our study areas with extensive forest areas. Forecasts of forest-species presence under various canopy cover scenarios suggest that leaving isolated trees among non-arboreal crops could greatly attract relatively tolerant species that require tree canopy. The conservation value of degraded habitats in agricultural landscapes seems to depend on factors such as the type of crops planted and distance to primary forest remnants.     

Phylogeny of magpie‐robins and shamas (Aves: Turdidae: Copsychus and Trichixos): implications for island biogeography in Southeast Asia

Aim Magpie‐robins and shamas are forest and woodland birds of south Asia. There are two genera: Trichixos for the monotypic T. pyrrhopygus, and Copsychus for other species. Two species are widespread, whereas the others are restricted to specific islands. Endemicity is highest in the Philippines. Using phylogenetic methods, we examined how this group came to its unusual distribution. Location Mainland Asia from India to southern China, and islands from Madagascar to the Philippines. Particular emphasis is placed on the Greater Sundas and Philippines. Methods The phylogeny was estimated from DNA sequences of 14 ingroup taxa representing all nine currently recognized Copsychus and Trichixos species. The entire mitochondrial ND2 gene and portions of nuclear myoglobin intron 2 (Myo2) and transforming growth factor beta 2 intron 5 (TGFβ2‐5) were sequenced for all but two species. The phylogeny was reconstructed using maximum likelihood and Bayesian methods. The timing of divergence events was estimated using a relaxed molecular clock approach, and ancestral areas were examined using stochastic modelling. Results The group comprises three main clades corresponding to ecological types: Trichixos, a primary‐forest specialist; Copsychus magpie‐robins, open‐woodland and coastal species; and Copsychus shamas, thick‐forest species. Trichixos appears to be sister to the magpie‐robins, rendering Copsychus polyphyletic. The dating of phylogenetic nodes was too ambiguous to provide substantial insight into specific geographical events responsible for divergence within the group. Some patterns are nevertheless clear. Copsychus shamas reached the Philippines, probably in two separate invasions, and split into endemic species. Copsychus malabaricus and C. saularis expanded widely in the Greater Sundas and mainland Southeast Asia without species‐level diversification. Main conclusions Magpie‐robins are excellent dispersers and have diversified into distinct species only on isolated oceanic islands. Trichixos, a poor disperser, is restricted to mature forests of the Malay Peninsula, Sumatra and Borneo. Copsychus shamas are intermediate in habitat preference and dispersal capabilities. Their endemism in the Philippines may be attributed to early colonization and specialization to interior forests. In the Greater Sundas, C. malabaricus and C. saularis populations split and came together on Borneo to form two separate subspecies (of each species), which now hybridize.     

Phosphorylation of focal adhesion kinase at tyrosine 861 is crucial for Ras transformation of fibroblasts

Although elevated expression and increased tyrosine phosphorylation of focal adhesion kinase (FAK) are crucial for tumor progression, the mechanism by which FAK promotes oncogenic transformation is unclear. We have therefore determined the role of FAK phosphorylation at tyrosine 861 in the oncogenic transformation of NIH3T3 fibroblasts. FAK phosphorylation at tyrosine 861 was increased in both constitutively H-Ras-transformed and H-Ras-inducible NIH3T3 cells, in parallel with cell transformation. However, H-Ras-inducible cells transfected with the nonphosphorylatable mutant FAK Y861F showed decreased migration/invasion, focus forming activity and anchorage-independent growth, compared with either wild-type or kinase-defective FAK. In contrast to unaltered FAK/Src activity, the association of FAK and p130(CAS) was decreased in FAK Y861F-transfected cells, and FAK phosphorylation at tyrosine 861 enhanced this association in vitro. Consistently, FAK Y861F-transfected cells were defective in activation of c-Jun NH(2)-terminal kinase and in expression of matrix metalloproteinase-9 during transformation. Taken together, these results strongly suggest that FAK phosphorylation at tyrosine 861 is crucial for H-Ras-induced transformation through regulation of the association of FAK with p130(CAS).     

CD4+CD25hiCD127low Regulatory T Cells Are Increased in Oral Squamous Cell Carcinoma Patients

Regulatory T cells (Tregs), a subset of CD4⁺ T cells plays a pivotal role in regulating the immune system. An increase in Treg numbers enables cancer progression by dampening the immune system and allowing tumor cells to evade immune detection and destruction. An increase in Treg numbers and expression of inhibitory cytokines including TGF-β and IL-10 are mechanisms by which Tregs exert their immune suppressive function. However, the presence of Tregs and inhibitory cytokines in oral cancer patients is still unclear. In this study, the presence of circulating Tregs in 39 oral cancer patients and 24 healthy donors was examined by studying the presence of the CD4⁺CD25^hiCD127^low cell population in their peripheral blood mononuclear cells using flow cytometry. Serum levels of TGF-β and IL-10 were measured by ELISA. T cell subsets of OSCC patients were found to differ significantly from healthy donors where a decrease in CD8⁺ cytotoxic T cells and an increase in Tregs (CD4⁺CD25^hiCD127^low) were observed. Further, the ratio of CD8⁺ T cells/Tregs was also decreased in patients compared to healthy donors. The presence of Tregs was accompanied by a decrease in IL-10 but not TGF-β secretion in OSCC patients when compared to donors; in addition, the analysis also revealed that an increased presence of Tregs was accompanied by better patient survival. Amongst OSCC patients, smokers had significantly higher levels of TGF-β. It is apparent that the immune system is compromised in OSCC patients and the characterization of the Treg subpopulation could form a basis for improving our understanding of the perturbations in the immune system that occur during OSCC tumorigenesis.