Inositol hexakisphosphate kinases (IP6Ks) phosphorylate inositol hexakisphosphate (InsP 6 ) to yield 5-diphosphoinositol pentakisphosphate (5-[PP]-InsP 5 or InsP 7 ). In this study, we report the characterization of a selective inhibitor, N 2 -(m-(trifluoromethy)lbenzyl) N 6 -(p-nitrobenzyl)purine (TNP), for these enzymes. TNP dose-dependently and selectively inhibited the activity of IP6K in vitro and inhibited InsP 7 and InsP 8 synthesis in vivo without affecting levels of other inositol phosphates. TNP did not inhibit either human or yeast Vip/PPIP5K, a newly described InsP 6 /InsP 7 1/3-kinase. Overexpression of IP6K1, -2, or -3 in cells rescued TNP inhibition of InsP 7 synthesis. TNP had no effect on the activity of a large number of protein kinases, suggesting that it is selective for IP6Ks. TNP reversibly reduced InsP 7 /InsP 8 levels. TNP in combination with genetic studies was used to implicate the involvement of two pathways for synthesis of InsP 8 in yeast. TNP induced a fragmented vacuole phenotype in yeast, consistent with inhibition of Kcs1, a Saccharomyces cerevisiae IP6K. In addition, it also inhibited insulin release from Min6 cells in a dose-dependent manner further implicating InsP 7 in this process. TNP thus provides a means of selectively and rapidly modulating cellular InsP 7 levels, providing a new and versatile tool to study the biological function and metabolic relationships of inositol pyrophosphates.Inositol(1,4,5)trisphosphate (Ins(1,4,5)P 3 ) 2 is the cytosolic product of inositol phospholipid-specific phospholipase Cs and serves multiple biological functions. In higher eukaryotes, it regulates Ca 2ϩ release from intracellular stores via binding to Ins(1,4,5)P 3 -specific receptors located in the endoplasmic reticulum. In addition, in Saccharomyces cerevisiae and all other eukaryotes that have been studied, Ins(1,4,5)P 3 also undergoes complex metabolism to generate a series of inositol polyphosphates with diverse functions (1, 2). Inositol hexakisphosphate (InsP 6 ) can be synthesized in inositol phospholipid-specific phospholipase C/Ins(1,4,5)P 3 -mediated pathways in most, if not all, eukaryotes. InsP 6 is further metabolized by the inositol hexakisphosphate kinases (IHPKs or IP6Ks), which add a pyrophosphate moiety at the 5-position to generate 5-[PP]-InsP 5 or 5-InsP 7 . Dictyostelium discoideum and S. cerevisiae, each possess one IP6K gene product designated Kcs1 in yeast (3, 4). In mammals, three IP6Ks have been identified (5, 6). In addition, a second InsP 6 /5-InsP 7 kinase, designated Vip/PPIP5K, has been identified in yeast (7) and mammalian cells (8,9). This kinase is distinct from IP6K/Kcs1 in that it phosphorylates the 1/3-position of InsP 6 and 5-[PP]-InsP 5 (10).Insights into the biological functions of inositol pyrophosphates have come from genetic studies in yeast and by manipulating expression of inositol polyphosphate kinases in mammalian cells. More recently, yeast mutants failing to synthesize inositol pyro phosphate molecules have been found to be impaired in s...

The entire genomic RNA of clover yellow mosaic virus was sequenced from cDNA clones and run-off cDNA transcripts.

Introduction HIV‐1‐infected patients develop haematological disorders such as cytopenias. One possible explanation is the inhibition of haematopoiesis at the level of differentiation of CD34+ haematopoietic progenitor stem cells. Based on our previous studies, we hypothesised that there may be viral encoded, or host cellular factors which participate in the process of inhibition of haematopoiesis. Materials and Methods Virus‐depleted media from infected CD4+ T cells was prepared by filtration and added to CD34+ cell differentiation semisolid medium. We have also used the virus‐depleted media to isolate host/viral factors including miRNA. Isolated miRNAs were screened for their haematopoietic inhibitory function using the miRNA mining approach. Results Addition of virus‐depleted media caused a 40% inhibition of differentiation of CD34+ cells into myeloid and erythroid colony formation. Real‐time RT‐PCR showed miR‐15a and miR‐24 from both pIndie‐C1 and pNL4.3 HIV‐1‐infected cells showed a significant differential expression when compared to control media. Conclusion In this study, we have identified two miRNAs, miR‐15a and miR‐24 secreted from purified HIV‐1‐infected CD4+ T cells that inhibited CD34+ haematopoietic progenitor stem cell differentiation into myeloid and erythroid colonies in vitro.

Objective: Japanese encephalitis (JE) is a debilitating disease caused by infection with the JE virus (JEV; family: Flaviviridae), which leaves neurological sequelae in survivors but more often leads to mortality. Neurodegeneration caused by inflammation is the primary pathology behind the clinical manifestation of encephalitis caused by JEV. Bacillus Calmette-Guérin (BCG) has been used in immunoprophylaxis for tuberculosis and in the adjuvant therapy of many malignancies, and has exhibited neuroprotective activities in experimental models of Parkinson and Alzheimer disease. This study aimed at assessing the neuroprotective role of BCG in a murine model of JE. Methods: Suckling mice were inoculated with 10⁶ CFU of BCG and at 18 days postinoculation were challenged with 100 LD₅₀ of JEV. PBS-inoculated mice were used as controls. Mice were sacrificed on days 2, 4, 6, and 8. Brain tissue was homogenized for RNA extraction. One-step real-time RT-PCR was performed to assess the relative gene expressions of TNF-α, IL-6, and iNOS. Results: The BCG-inoculated (BCG+JEV) group exhibited a significant delay in the presentation of neuropathological symptoms, longer survival, and a downregulation in the expression of TNF-α, IL-6, and iNOS on days 2, 4, and 6 post-JEV challenge compared to the JEV group. Conclusion: These findings indicate that the administration of BCG offers neuroprotection in the murine model of JE. BCG should therefore be further investigated as an adjuvant in the management of JE. BCG is an accepted vaccine for tuberculosis in many countries that are endemic for JEV. This approach may have a significant impact on the public health burden in these countries.

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