Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast.

Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide-containing, mild base-resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI-anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI-anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI-anchored proteins.     

Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium

Although factor VII/factor VIIa (FVII/FVIIa) is known to interact with many non-vascular cells, activated monocytes, and endothelial cells via its binding to tissue factor (TF), the interaction of FVII/FVIIa with unperturbed endothelium and the role of this interaction in clearing FVII/FVIIa from the circulation are unknown. To investigate this, in the present study we examined the binding of radiolabeled FVIIa to endothelial cells and its subsequent internalization. (125)I-FVIIa bound to non-stimulated human umbilical vein endothelial cells (HUVEC) in time- and dose-dependent manner. The binding is specific and independent of TF and negatively charged phospholipids. Protein C and monoclonal antibodies to endothelial cell protein C receptor (EPCR) blocked effectively (125)I-FVIIa binding to HUVEC. FVIIa binding to EPCR is confirmed by demonstrating a marked increase in (125)I-FVIIa binding to CHO cells that had been stably transfected with EPCR compared with the wild-type. Binding analysis revealed that FVII, FVIIa, protein C, and activated protein C (APC) bound to EPCR with similar affinity. FVIIa binding to EPCR failed to accelerate FVIIa activation of factor X or protease-activated receptors. FVIIa binding to EPCR was shown to facilitate FVIIa endocytosis. Pharmacological concentrations of FVIIa were found to impair partly the EPCR-dependent protein C activation and APC-mediated cell signaling. Overall, the present data provide convincing evidence that EPCR serves as a cellular binding site for FVII/FVIIa. Further studies are needed to evaluate the pathophysiological consequences and relevance of FVIIa binding to EPCR.     

Biochemical,machine learning and molecular approaches for the differential diagnosis of Mucopolysaccharidoses

This study was aimed to construct classification and regression tree (CART) model of glycosaminoglycans (GAGs) for the differential diagnosis of Mucopolysaccharidoses (MPS). Two-dimensional electrophoresis and liquid chromatography–tandem mass spectrometry (LC–MS/MS) were used for the qualitative and quantitative analysis of GAGs. Specific enzyme assays and targeted gene sequencing were performed to confirm the diagnosis. Machine learning tools were used to develop CART model based on GAG profile. Qualitative and quantitative CART models showed 96.3% and 98.3% accuracy, respectively, in the differential diagnosis of MPS. The thresholds of different GAGs diagnostic of specific MPS types were established. In 60 MPS positive cases, 46 different mutations were identified in six specific genes. Among 31 different mutations identified in IDUA, nine were nonsense mutations and two were gross deletions while the remaining were missense mutations. In IDS gene, four missense, two frameshift, and one deletion were identified. In NAGLU gene, c.1693C?>?T and c.1914_1914insT were the most common mutations. Two ARSB, one case each of SGSH and GALNS mutations were observed. LC–MS/MS-based GAG pattern showed higher accuracy in the differential diagnosis of MPS. The mutation spectrum of MPS, specifically in IDUA and IDS genes, is highly heterogeneous among the cases studied.

     

Tid1-S attenuates LPS-induced cardiac hypertrophy and apoptosis through ER-a mediated modulation of p-PI3K/p-Akt signaling cascade

Myocardial dysfunction is clinically relevant? repercussion that follows sepsis. Tid 1 protein has been implicated in many biological process. However, the role of Tid 1 in lipopolysaccharide (LPS)-induced cardiomyocyte hypertrophy and apoptosis remains elusive. In the current research endeavor, we have elucidated the role of Tid1-S on LPS-induced cardiac hypertrophy and apoptosis. Interestingly, we found that overexpression of Tid1-S suppressed TLR-4, NFATc3, and BNP protein expression which eventually led to inhibition of LPS-induced cardiac hypertrophy. Moreover, Tid1-S overexpression attenuated cellular apoptosis and activated survival proteins p-PI3K and p^ser473Akt. Besides this, Tid1-S overexpression enhanced ER-a protein expression. Collectively, our data suggest that Tid1-S plausibly enhance ER-a protein and further activate p-PI3K and p ^ser473Akt survival protein expression; which thereby led to attenuation of LPS-induced apoptosis in cardiomyoblast cells. Interestingly, our data suggest that Tid1-S is involved in attenuation of cardiomyoblast cells damages induced by LPS.     

Evidence in favor of linkage to human chromosomal regions 18q, 5q and 13q for bicuspid aortic valve and associated cardiovascular malformations

The aim of this study was to identify regions of the genome that harbor genes influencing inheritance of bicuspid aortic valve (BAV) and/or associated cardiovascular malformation (CVM). Aortic valve disease is an important clinical problem, which often results in valve replacement, the second most common cardiac surgery in the United States. In every age group, a majority of cases of valve disease involves a BAV. BAV is the most common CVM with a reported prevalence of 1–2%. Heritability studies indicate that BAV determination is almost entirely genetic. We used a family-based genome-wide linkage analysis with microsatellite markers. Parametric and nonparametric analyses were performed with the software GENEHUNTER and SOLAR (Sequential Oligogenic Linkage Analysis Routines). Thirty-eight families (353 subjects) with BAV and/or associated CVM were assessed. Each participant underwent a standardized echocardiographic examination. The highest LOD score, 3.8, occurred on chromosome 18q between markers D18S68 and D18S1161. Two other chromosomal regions, 5q15–21 (between D5S644 and D5S2027) and 13q33-qter (between D13S1265 and 13qter), exhibited suggestive evidence of linkage (LOD > 2.0). Further, two previously reported linkage peaks on 9q34 and 17q24 were replicated in family specific analyses. No significant X chromosome linkage peaks were identified. In this genome-wide scan we demonstrate for the first time, that BAV and/or associated CVM exhibit linkage to chromosomes 18q, 5q and 13q. These regions likely contain genes whose mutation results in BAV and/or associated CVM indicating their important role in valvulogenesis and cardiac development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lisa J. Martin and Vijaya Ramachandran have contributed equally to this work.     

Target of Rapamycin Complex 2 Regulates Actin Polarization and Endocytosis via Multiple Pathways

Target of rapamycin is a Ser/Thr kinase that operates in two conserved multiprotein complexes, TORC1 and TORC2. Unlike TORC1, TORC2 is insensitive to rapamycin, and its functional characterization is less advanced. Previous genetic studies demonstrated that TORC2 depletion leads to loss of actin polarization and loss of endocytosis. To determine how TORC2 regulates these readouts, we engineered a yeast strain in which TORC2 can be specifically and acutely inhibited by the imidazoquinoline NVP-BHS345. Kinetic analyses following inhibition of TORC2, supported with quantitative phosphoproteomics, revealed that TORC2 regulates these readouts via distinct pathways as follows: rapidly through direct protein phosphorylation cascades and slowly through indirect changes in the tensile properties of the plasma membrane. The rapid signaling events are mediated in large part through the phospholipid flippase kinases Fpk1 and Fpk2, whereas the slow signaling pathway involves increased plasma membrane tension resulting from a gradual depletion of sphingolipids. Additional hits in our phosphoproteomic screens highlight the intricate control TORC2 exerts over diverse aspects of eukaryote cell physiology.     

Characterization and Pathogenicity of Colletotrichum truncatum Causing Stem Anthracnose of Red‐Fleshed Dragon Fruit (Hylocereus polyrhizus) in Malaysia

During August 2010 and January 2011, 10 isolates of Colletotrichum were recovered from stem anthracnose lesions of Hylocereus polyrhizus in the states of Kedah and Penang, Malaysia. Based on the morphological characteristics of colony colour and appearance, and shapes of conidia as well as sequences of internal transcribed spacer regions (ITS), β‐tubulin, actin (ACT) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), the fungus was identified as Colletotrichum truncatum. Pathogenicity test showed that C. truncatum isolates were pathogenic to the artificially inoculated H. polyrhizus stem. This is the first report of C. truncatum causing anthracnose on H. polyrhizus stems in Malaysia.