C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.     

Biosynthesis of alkyl lysophosphatidic acid by diacylglycerol kinases

Lysophosphatidic acid (LPA) designates a family of bioactive phosphoglycerides that differ in the length and degree of saturation of their radyl chain. Additional diversity is provided by the linkage of the radyl chain to glycerol: acyl, alkyl, or alk-1-enyl. Acyl-LPAs are the predominate species in tissues and biological fluids. Alkyl-LPAs exhibit distinct pharmacodynamics at LPA receptors, potently drive platelet aggregation, and contribute to ovarian cancer aggressiveness. Multiple biosynthetic pathways exist for alkyl-LPA production. Herein we report that diacylglycerol kinases (DGKs) contribute to cell-associated alkyl-LPA production involving phosphorylation of 1-alkyl-2-acetyl glycerol and document the biosynthesis of alkyl-LPA by DGKs in SKOV-3 ovarian cancer cells, specifically identifying the contribution of DGKα. Concurrently, we discovered that treating SKOV-3 ovarian cancer cell with a sphingosine analog stimulates conversion of exogenous 1-alkyl-2-acetyl glycerol to alkyl-LPA, indicating that DGKα contributes significantly to the production of alkyl-LPA in SKOV-3 cells and identifying cross-talk between the sphingolipid and glycerol lipid pathways.     

Non-conservative surface decoration of hemoglobin: influence of neutralization of positive charges at PEGylation sites on molecular and functional properties of PEGylated hemoglobin

High hydrodynamic volume, high viscosity and high colloidal osmotic pressure (COP) of PEGylated hemoglobin (Hb) have been suggested to neutralize the vasoactivity of acellular Hb. Consequences of non-conservative PEGylation (positive charge of the amino groups at the PEGylation sites is neutralized) using succinimidyl-ester of propionic acid PEG5K on the properties of PEGylated Hb have now been investigated. Non-conservative PEGylation of Hb leads to a much higher increase in the COP and viscosity of Hb than conservative extension arm facilitated (EAF) PEGylation of Hb. Introduction of alphaalpha-fumaryl crosslinking decreased the COP of non-conservative PEGylated Hb by stabilization of interdimeric interactions. Compared to the EAF-PEGylated alphaalpha-fumaryl Hb, non-conservative PEGylated product shows a comparable COP and higher viscosity. Conservative PEGylation of alphaalpha-fumaryl Hb by reductive alkylation chemistry does not increase the COP to this level, but enhanced the molecular volume and viscosity comparable to EAF-PEGylated product. Thus, the molecular properties of PEGylated Hb can be fine tuned using different PEGylation platforms and provide a unique opportunity for the design of second generation PEGylated Hbs.     

Molecular biology and pathogenesis of hepatitis E virus

The hepatitis E virus (HEV) is a small RNA virus and the etiological agent for hepatitis E, a form of acute viral hepatitis. The virus has a feco-oral transmission cycle and is transmitted through environmental contamination, mainly through drinking water. Recent studies on the isolation of HEV-like viruses from animal species also suggest zoonotic transfer of the virus. The absence of small animal models of infection and efficient cell culture systems has precluded virological studies on the replication cycle and pathogenesis of HEV. A vaccine against HEV has undergone successful clinical testing and diagnostic tests are available. This review describes HEV epidemiology, clinical presentation, pathogenesis, molecular virology and the host response to HEV infection. The focus is on published literature in the past decade. Equal contribution     

Interaction of Fusarium Oxysporum f.sp. Cubense with Pseudomonas Fluorescens Precolonized to Banana Roots

Effect of precolonization of banana cv Neeypovan roots with Pseudomonas fluorescens on infection with Fusarium oxysporum f.sp. cubense was studied. Under in vitro conditions Pseudomonas fluorescens clearly inhibited Fusarium oxysporum f.sp. cubense. Fluorescein isothiocyanate-tagged antibodies raised in a rabbit system for Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense separately were used to study the spread of both organisms in banana root. It was observed that precolonization with Pseudomonas fluorescens could reduce Fusarium oxysporum f.sp. cubense colonization by 72%, and also correlated with a number of structural changes in the cortical cells, mainly with densely stained amorphous material and polymorphic wall thickenings as revealed by light and electron microscopic studies. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

Queuine mediated inhibition in phosphorylation of tyrosine phosphoproteins in cancer

Protein phosphorylation or dephosphorylation is the most important regulatory switch of signal transduction contributing to control of cell proliferation. The reversibility of phosphorylation and dephosphorylation is due to the activities of kinases and phosphatase, which determine protein phosphorylation level of cell under different physiological and pathological conditions. Receptor tyrosine kinase (RTK) mediated cellular signaling is precisely coordinated and tightly controlled in normal cells which ensures regulated mitosis. Deregulation of RTK signaling resulting in aberrant activation in RTKs leads to malignant transformation. Queuine is one of the modified base of tRNA which participates in down regulation of tyrosine kinase activity. The guanine analogue queuine is a nutrient factor to eukaryotes and occurs as free base or modified nucleoside queuosine into the first anticodon position of specific tRNAs. The tRNAs are often queuine deficient in cancer and fast proliferating tissues. The present study is aimed to investigate queuine mediated inhibition in phosphorylation of tyrosine phosphorylated proteins in lymphoma bearing mouse. The result shows high level of cytosolic and membrane associated tyrosine phosphoprotein in DLAT cancerous mouse liver compared to normal. Queuine treatments down regulate the level of tyrosine phosphoproteins, which suggests that queuine is involved in regulation of mitotic signaling pathways.