Fasting-Induced Lipolysis and Hypothalamic Insulin Signaling Are Regulated by Neuronal Glucosylceramide Synthase.

Central nervous regulation of body weight and adipose tissue function is mainly conducted by hypothalamic neurons. Neuronal function depends on the integrity of the membrane lipid microenvironment. Lipid microdomains contain large quantities of cholesterol and glycosphingolipids, including glucosylceramide synthase (GCS) (gene Ugcg)-derived gangliosides. The current study demonstrates that Ugcgf/f//CamKCreERT2 mice with genetic GCS deletion in forebrain neurons, dominantly targeting mediobasal hypothalamus (MBH), display impaired fasting-induced lipolysis accompanied by a decreased norepinephrine content in white adipose tissue (WAT). MBH insulin receptor (IR) levels and signaling are increased in Ugcgf/f//CamKCreERT2 mice. These results are in concordance with reports stating that MBH insulin signaling restrains sympathetic nervous outflow to WAT in fasted mice. In line with the in vivo data, pharmacological GCS inhibition by Genz123346 also increases IR levels as well as IR phosphorylation in insulin-stimulated hypothalamic cells. In addition to studies suggesting that simple gangliosides like GM3 regulate peripheral IR signaling, this work suggests that complex neuronal gangliosides also modulate hypothalamic IR signaling and protein levels. For example, the complex ganglioside GD1a interacts dynamically with the IRs on adult hypothalamic neurons. In summary, our results suggest that neuronal GCS expression modulates MBH insulin signaling and WAT function in fasted mice.

Inhibitory effects of the phenolic fraction from the pomace of Vitis coignetiae on biofilm formation by Streptococcus mutans.

OBJECTIVES: The anti-cariogenic properties of the phenolic fraction from the pomace of Vitis coignetiae (VcPP) were evaluated by in vitro assays and compared with fruit juices from V. coignetiae and common grapes and with other phenolic fractions. The effects of VcPP against the biofilm of Streptococcus mutans were investigated. DESIGN: Sucrose-dependent biofilm formation by S. mutans cultured in the presence of VcPP was measured by crystal violet dye uptake. Inhibition of adhesion to the saliva-coated hydroxyapatite (sHA) beads was quantified using fluorescent-labelled cells. The MIC for S. mutans was determined by colony counting on agar plates containing VcPP. The ability of VcPP to inhibit glucan synthesis by three distinct recombinant glucosyltransferases (Gtfs) was assessed by quantifying the production of water-soluble and -insoluble polysaccharides in bacterial cultures. In addition, the buffering effect of VcPP in cultures of S. mutans was evaluated. RESULTS: VcPP reduced adhesion of S. mutans to sHA and biofilm formation in a dose-dependent manner. The MIC of VcPP was 7.50mg/ml. VcPP inhibited GtfB activity associated with the synthesis of water-insoluble glucans. It also inhibited GtfD activity associated with the synthesis of water-soluble glucans at a concentration which was lower than that used for inhibition of GtfB. VcPP had no effect on acidification associated with glucose utilization by S. mutans. CONCLUSIONS: The current study supports the potential of VcPP as a food additive for reducing caries by inhibiting adhesion to the tooth surface and GtfD-mediated soluble glucan synthesis.

In vitro activity of echinocandins against non-Candida albicans: is echinocandin antifungal activity the same?

The echinocandins anidulafungin, caspofungin, and micafungin have a broad and similar spectrum of in vitro and in vivo activity against most Candida spp. Minimal inhibitory concentrations (MICs) for Candida spp. are usually below 1 µg/mL for most isolates. The exceptions are Candidaparapsilosis and C. guilliermondii. Species-specific clinical breakpoints (CBPs) and epidemiologic cutoff values (ECVs) have been proposed by the Clinical and Laboratory Standards Institute (CLSI) for the eight most common Candida spp. versus each echinocandin; these values are useful to detect in vitro antifungal resistance (CBPs) and to identify isolates harboring fks mutations or having reduced susceptibility (ECVs). This paper presents a review of the literature (2006-2010) regarding the in vitro activity similarities or differences among the three echinocandins against Candida spp.; different parameters or measurements of in vitro potency were evaluated. The focus of the review is the non-Candida albicans species.

The Arabidopsis irregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan, which are essential for secondary cell wall integrity.

The secondary cell wall in higher plants consists mainly of cellulose, lignin, and xylan and is the major component of biomass in many species. The Arabidopsis thaliana irregular xylem8 (irx8) mutant is dwarfed and has a significant reduction in secondary cell wall thickness. IRX8 belongs to a subgroup of glycosyltransferase family 8 called the GAUT1-related gene family, whose members include GAUT1, a homogalacturonan galacturonosyltransferase, and GAUT12 (IRX8). Here, we use comparative cell wall analyses to show that the irx8 mutant contains significantly reduced levels of xylan and homogalacturonan. Immunohistochemical analyses confirmed that the level of xylan was significantly reduced in the mutant. Structural fingerprinting of the cell wall polymers further revealed that irx8 is deficient in glucuronoxylan. To explore the biological function of IRX8, we crossed irx8 with irx1 (affecting cellulose synthase 8). The homozygous irx1 irx8 exhibited severely dwarfed phenotypes, suggesting that IRX8 is essential for cell wall integrity during cellulose deficiency. Taken together, the data presented show that IRX8 affects the level of glucuronoxylan and homogalacturonan in higher plants and that IRX8 provides an important link between the xylan polymer and the secondary cell wall matrix and directly affects secondary cell wall integrity.

Knockout of the AtCESA2 gene affects microtubule orientation and causes abnormal cell expansion in Arabidopsis.

Complete cellulose synthesis is required to form functional cell walls and to facilitate proper cell expansion during plant growth. AtCESA2 is a member of the cellulose synthase A family in Arabidopsis (Arabidopsis thaliana) that participates in cell wall formation. By analysis of transgenic seedlings, we demonstrated that AtCESA2 was expressed in all organs, except root hairs. The atcesa2 mutant was devoid of AtCESA2 expression, leading to the stunted growth of hypocotyls in seedlings and greatly reduced seed production in mature plants. These observations were attributed to alterations in cell size as a result of reduced cellulose synthesis in the mutant. The orientation of microtubules was also altered in the atcesa2 mutant, which was clearly observed in hypocotyls and petioles. Complementary expression of AtCESA2 in atcesa2 could rescue the mutant phenotypes. Together, we conclude that disruption of cellulose synthesis results in altered orientation of microtubules and eventually leads to abnormal plant growth. We also demonstrated that the zinc finger-like domain of AtCESA2 could homodimerize, possibly contributing to rosette assemblies of cellulose synthase A within plasma membranes.

Arabidopsis reversibly glycosylated polypeptides 1 and 2 are essential for pollen development.

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. To date, to our knowledge, no direct evidence exists for the involvement of RGPs in a particular biochemical process. The Arabidopsis (Arabidopsis thaliana) genome contains five RGP genes out of which RGP1 and RGP2 share the highest sequence identity. We characterized the native expression pattern of Arabidopsis RGP1 and RGP2 and used reverse genetics to investigate their respective functions. Although both genes are ubiquitously expressed, the highest levels are observed in actively growing tissues and in mature pollen, in particular. RGPs showed cytoplasmic and transient association with Golgi. In addition, both proteins colocalized in the same compartments and coimmunoprecipitated from plant cell extracts. Single-gene disruptions did not show any obvious morphological defects under greenhouse conditions, whereas the double-insertion mutant could not be recovered. We present evidence that the double mutant is lethal and demonstrate the critical role of RGPs, particularly in pollen development. Detailed analysis demonstrated that mutant pollen development is associated with abnormally enlarged vacuoles and a poorly defined inner cell wall layer, which consequently results in disintegration of the pollen structure during pollen mitosis I. Taken together, our results indicate that RGP1 and RGP2 are required during microspore development and pollen mitosis, either affecting cell division and/or vacuolar integrity.