A methyl viologen-sensitive mutant of the nematode Caenorhabditis elegans

A methyl viologen (paraquat)-sensitive mutant, mev-1 (LG III), in Caenorhabditis elegans was about 4 times more sensitive to methyl viologen than the wild type. This mutant was also hypersensitive to oxygen. The brood size was about 1/4 that of the wild type. The average life span was determined to be 9.3 days as compared to 14.3 days for the wild type. The activity of superoxide dismutase (SOD), a scavenging enzyme for superoxide anion, was about half the wild-type level. We suggest that oxygen radicals may be involved in the normal aging mechanism in C. elegans.     

Characterization of Plasmodium falciparum calcium-dependent protein kinase 4

In Plasmodium berghei, the orthologous gene of P. falciparum calcium-dependent protein kinase 4 (PfCDPK4) was reported to be essential for the exflagellation of male gametocytes. To elucidate the role of PfCDPK4 in P. falciparum gametogenesis, we characterized the biological function of PfCDPK4 in vitro. PfCDPK4 was purified as a fusion protein that was labeled with [γ-³²P]ATP; this labeling was then eliminated by phosphatase. Phosphorylation activity of PfCDPK4 was eliminated when its putative catalytic lysine residue was replaced with alanine. In biochemical analyses, PfCDPK4 was found to have characteristics that were similar to those of homologous proteins from plants. PfCDPK4 phosphorylation was activated when experimental conditions were changed from those characteristic of human blood (37 °C, pH 7.4) to those of the mosquito bloodmeal (at least 5 °C below 37 °C, pH 7.6, with xanthurenic acid (XA)). PfCDPK4 was overexpressed in day 15 gametocytes exposed to XA or human serum. Thus, PfCDPK4 phosphorylation is activated by an increase in Ca²⁺ concentration or pH and by a decrease in temperature, and is associated with the Ca²⁺ signals that facilitate P. falciparum gametogenesis.     

Inhibition of Sphingosine Kinase by Bovine Viral Diarrhea Virus NS3 Is Crucial for Efficient Viral Replication and Cytopathogenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in diverse cellular functions including survival, proliferation, tumorigenesis, inflammation, and immunity. Sphingosine kinase (SphK) contributes to these functions by converting sphingosine to S1P. We report here that the nonstructural protein NS3 from bovine viral diarrhea virus (BVDV), a close relative of hepatitis C virus (HCV), binds to and inhibits the catalytic activity of SphK1 independently of its serine protease activity, whereas HCV NS3 does not affect SphK1 activity. Uncleaved NS2-3 from BVDV was also found to interact with and inhibit SphK1. We suspect that inhibition of SphK1 activity by BVDV NS3 and NS2-3 may benefit viral replication, because SphK1 inhibition by small interfering RNA, chemical inhibitor, or overexpression of catalytically inactive SphK1 results in enhanced viral replication, although the mechanisms by which SphK1 inhibition leads to enhanced viral replication remain unknown. A role of SphK1 inhibition in viral cytopathogenesis is also suggested as overexpression of SphK1 significantly attenuates the induction of apoptosis in cells infected with cytopathogenic BVDV. These findings suggest that SphK is targeted by this virus to regulate its catalytic activity.Bovine viral diarrhea virus (BVDV)² is an enveloped, positive-sense single-stranded RNA virus classified in the genus Pestivirus of the family Flaviviridae. BVDV establishes persistent infections in cattle populations worldwide. Because BVDV shares virological and molecular properties with the Flaviviridae family member hepatitis C virus (HCV), which chronically infects an estimated 200 million patients worldwide (1), BVDV is regarded as a surrogate model for HCV (2). Both HCV and BVDV encode a single large precursor polyprotein that is processed by cellular and viral proteases into mature structural and nonstructural (NS) proteins.BVDV NS3 exhibits serine protease and helicase/ATPase activities that require its cofactor NS4A (3). NS3/4A protease is essential for generating mature NS proteins that are required for viral replication. HCV NS3/4A is well characterized and has been shown to suppress type-I interferons by cleaving the cellular interferon mediators IPS-1 and TRIF (4, 5). However, neither interferon suppression nor cellular targets have been identified for the BVDV NS3/4A protease (6).Lytic and persistent BVDV infections depend on the virus biotype. Cytopathogenic (CP) BVDV causes cytopathic effects via apoptosis, whereas noncytopathogenic (NCP) BVDV does not induce obvious changes in cell morphology and viability. These features are distinguished by NS2-3 processing differences; free NS3 produced by NS2-3 cleavage is generated continuously following CP BVDV infections, whereas NS3 is detected only until ∼9 h postinfection (p.i.) for NCP BVDV due to down-regulation of NS2-3 cleavage by this biotype (7). The CP biotype is characterized by dramatic up-regulation of viral RNA synthesis that could be correlated with the induction of cytopathic effect (7–9). Because free NS3, but not NS2-3, can form an active viral replicase complex with other NS proteins, increased viral RNA synthesis promoted through the release of free NS3 has been suggested to be a determinant of the characteristic lytic phenotype of CP BVDV infections (10). However, little is known about the regulation of cellular signaling by BVDV NS2-3, NS3, and NS3/4A, which is crucial for the control of both viral replication and biotype.Recent studies on the mechanisms of viral replication revealed that HCV RNA synthesis occurs on a lipid raft membrane structure where the active viral replicase complex is found (11, 12). The significance of the lipid raft as a scaffold for viral replication is further demonstrated by the identification of a novel HCV replication inhibitor, NA255, which prevents the biosynthesis of sphingolipids, the major components of lipid rafts (13). Administration of NA255 results in disruption of the HCV replicase complexes from the lipid rafts. This report proposes that the interaction between HCV NS5B and sphingomyelin on lipid rafts plays a crucial role for HCV RNA replication. Cellular sphingolipid metabolism is regulated by a large number of converting enzymes that maintain a homeostasis (14) but viral mechanisms that affect the sphingolipid metabolism to facilitate viral replication have yet to be identified.In a search for potential host proteins that interact with BVDV NS3, we identified sphingosine kinase 1 (SphK1) as a binding partner of NS3 using the yeast two-hybrid system. SphK1 is a lipid kinase that catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), a bioactive sphingolipid implicated in diverse cellular functions, including proliferation, survival, tumorigenesis, development, inflammation, and immunity (14, 15). Here, we analyze the biological significance of the SphK1 interaction with NS3, NS2-3, and NS3/4A. Using purified recombinant SphK1 and NS3, SphK activity was inhibited by NS3 in a dose-dependent manner, independently of its serine protease activity. The inhibition appears to be specific for BVDV NS3 because HCV NS3 had no effect on SphK activity. Using specific chemical inhibitors, small interfering RNA (siRNA), and a catalytically inactive mutant of SphK1, we investigated the significance of SphK inhibition in the viral replication. The present study is the first report demonstrating that SphK1 is targeted by a virus to inhibit its catalytic activity, and this mechanism may contribute to the efficient replication and pathogenesis of BVDV.     

Vitamin D3 enhances ATRA-mediated neurosteroid biosynthesis in human glioma GI-1 cells

Emerging evidence indicates that vitamin D (VD) is an important modulator of brain development and function. To investigate whether VD modulates neurosteroid biosynthesis in neural cells, we investigated the effect of VD(3) on steroidogenic gene expression in human glioma GI-1 cells. We found that VD(3) enhanced CYP11A1 and 3β-hydroxysteroid dehydrogenase gene expression. The induction of CYP11A1 gene expression by VD(3) was dose- and incubation time-dependent. Calcipotriol, a VD(3) receptor (VDR) agonist, also induced CYP11A1 gene expression in GI-1 cells, indicating that VDR is involved in this induction. The induction of progesterone (PROG) de novo synthesis was observed along with the induction of steroidogenic genes by VD(3). Furthermore, VD(3) enhanced all-trans retinoic acid (ATRA)-induced CYP11A1 gene expression and PROG production. This suggests cooperative regulation of steroidogenic gene expression by the two fat-soluble vitamins, A and D. In addition, a mixed culture of neuronal IMR-32 cells and GI-1 cells treated with ATRA and VD(3) resulted in the induction of PROG-responsive gene expression in the IMR-32 cells. This result shows a paracrine action of PROG that is induced in and released by the GI-1 cells. The relationship between neurological dysfunction associated with VD deficiency and neurosteroid induction by VD is discussed.     

Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth

Background

cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.

Methodology/Principal Finding

The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.

Conclusions/Significance

Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.     

Xanthurenic acid is an endogenous substrate for the silkworm cytosolic sulfotransferase, bmST1

Sulfotransferase enzymes are known to regulate physiologically active substances such as steroids and catecholamines in mammals. Although invertebrates also express sulfotransferases, their biological function is mostly unclear. In a previous study, we reported that 4-nitrocatechol and the gallete ester are substrates for the silkworm sulfotransferase bmST1. The K(m) of bmST1 for these substrates is high. However, endogenous substrates of bmST1 have not yet been determined. We therefore investigated endogenous bmST1 substrates and carried out a detailed expression profile analysis of bmST1. We found that xanthurenic acid, a tryptophan metabolite, is a possible endogenous substrate of bmST1. The K(m) of bmST1 for xanthurenic acid is low, in the μM range, which is lower than that for previously reported substrates. Additionally, xanthurenic acid is a tryptophan metabolite that characteristically shows toxicity in vivo. High dose administration of xanthurenic acid resulted in inhibition of cuticular biosynthesis. The expression of the bmST1 gene reached a maximal level in the Malpighian tubule at the 4th molting stage, when amino acid metabolism might be activated. Our results suggest that bmST1 plays a role in detoxification of xanthurenic acid in the silkworm.     

Myelinated Herring bodies in the posterior part of the median eminence of the mouse

Summary The posterior part of the median eminence of the albino mouse (CF # 1-JCL) contains a cluster of myelinated axons beneath the tanycyte layer. Among them, small Herring bodies surrounded by myelin sheaths are revealed by electron microscopy. These structures contain electron-dense bodies, lamellar bodies, autophagic bodies, autophagic vacuoles, and neurofilaments. A few neurosecretory granules and mitochondria are also present. Some myelinated axons contain mostly accumulated neurosecretory granules.     

T cell subpopulations mediating inhibition of feline immunodeficiency virus replication in mucosally infected cats

Feline immunodeficiency virus (FIV) infection induces an increase in two subpopulations (CD8alpha(+)beta(low) and CD8alpha(+)beta(-)) within CD8(+) peripheral blood lymphocytes (PBLs) of cats. It is known that depletion of CD8(+) cells often results in augmentation of FIV proliferation in PBL culture, similarly to the case of human immunodeficiency virus. In this study, we attempted to define PBL subpopulations mediating antiviral activity in five cats intravaginally infected with a molecularly cloned FIV isolate. Several subpopulations (CD8alpha(+)beta(+), CD8alpha(+)beta(-), and CD4(+) cells) were shown to participate in inhibition of the FIV replication, at least in part, in a major histocompatibility complex-unrestricted manner. Moreover, the subpopulations showing anti-FIV activity were different among the individual cats.