Genetically Compromising Phospholipid Metabolism Limits Candida albicans’ Virulence

Mycopathologia - Perturbing ergosterol synthesis has been previously shown to reduce the virulence of Candida albicans. We tested the hypothesis that further altering cell membrane composition by...     

p38-mediated regulation of an Fas-associated death domain protein-independent pathway leading to caspase-8 activation during TGFbeta-induced apoptosis in human Burkitt lymphoma B cells BL41

On binding to its receptor, transforming growth factor beta (TGFbeta) induces apoptosis in a variety of cells, including human B lymphocytes. We have previously reported that TGFbeta-mediated apoptosis is caspase-dependent and associated with activation of caspase-3. We show here that caspase-8 inhibitors strongly decrease TGFbeta-mediated apoptosis in BL41 Burkitt's lymphoma cells. These inhibitors act upstream of the mitochondria because they inhibited the loss of mitochondrial membrane potential observed in TGFbeta-treated cells. TGFbeta induced caspase-8 activation in these cells as shown by the cleavage of specific substrates, including Bid, and the appearance of cleaved fragments of caspase-8. Our data show that TGFbeta induces an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and caspase-9 and -3 activation. Caspase-8 activation was Fas-associated death domain protein (FADD)-independent because cells expressing a dominant negative mutant of FADD were still sensitive to TGFbeta-induced caspase-8 activation and apoptosis. This FADD-independent pathway of caspase-8 activation is regulated by p38. Indeed, TGFbeta-induced activation of p38 and two different inhibitors specific for this mitogen-activated protein kinase pathway (SB203580 and PD169316) prevented TGFbeta-mediated caspase-8 activation as well as the loss of mitochondrial membrane potential and apoptosis. Overall, our data show that p38 activation by TGFbeta induced an apoptotic pathway via FADD-independent activation of caspase-8.     

Inhibition of Dengue Virus Polymerase by Blocking of the RNA Tunnel

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)₂₀]. Chemical modification of the initial “hit” improved the compound potency to an IC₅₀ (that is, a concentration that inhibits 50% RdRp activity) of 0.7 μM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC₅₀ of 5 μM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC₅₀s of >100 μM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.The family Flaviviridae consists of three genera: Flavivirus, Pestivirus, and Hepacivirus. The genus Flavivirus contains about 73 viruses, many of which are arthropod-borne and pose major public health threats worldwide (15). The four serotypes of dengue virus infect 50 to 100 million people each year, with approximately 500,000 cases developing into life-threatening dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS), leading to about 20,000 deaths. In addition to DENV, West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV) also cause significant human diseases. No antiviral therapy is currently available for treatment of flavivirus infections. Human vaccines are only available for YFV, JEV, and TBEV (15). Development of antiviral therapy and new vaccines is urgently needed for flaviviruses.The flavivirus genome is a single-stranded RNA of plus-sense polarity. The genomic RNA contains a 5′ untranslated region (UTR), a single open reading frame, and a 3′ UTR. The single open reading frame encodes a long polyprotein that is processed by viral and host proteases into 10 mature viral proteins. Three structural proteins (Capsid [C], premembrane [prM], and envelope [E]) are components of virus particles. Seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are responsible for viral replication (40), virion assembly (19, 21, 24, 33), and innate immunity antagonism (4, 16, 23, 29, 30). Two viral proteins encode enzymatic activities that have been targeted for antiviral development. NS3 functions as a protease (with NS2B as a cofactor), helicase, 5′-RNA triphosphatase, and nucleoside triphosphatase (7, 14, 42). The N-terminal part of NS5 is a methyltransferase that methylates the N7 and 2′-O positions of the viral RNA cap structure (13, 18, 37); the C-terminal part of NS5 has an RNA-dependent RNA polymerase (RdRp) activity (1, 39). The RdRp activity is unique to RNA viruses and therefore represents an attractive antiviral target.Two types of inhibitors could be developed to suppress viral polymerases. Type 1 inhibitors are nucleoside/nucleotide analogs that function as RNA or DNA chain terminators; about half of the current antiviral drugs are nucleotide analogs (10). For flaviviruses, a nucleoside analog (7-deaza-2′-C-methyl-adenosine), originally developed for hepatitis C virus (HCV) RdRp, showed anti-DENV activity (32, 38). We recently reported a similar adenosine analog (7-deaza-2′-C-acetylene-adenosine) that potently inhibited DENV both in cell culture and in mice; unfortunately, this compound showed side effects during a 2-week in vivo toxicity study (44). Nevertheless, these studies have proved the concept that nucleoside analogs could potentially be developed for flavivirus therapy. Type 2 inhibitors are non-nucleoside inhibitors (NNI) which bind to allosteric pockets of protein to block enzymatic activities; the mechanism of action of NNI includes structural alteration of polymerase to an inactive conformation, blocking the conformational switch from polymerase initiation to elongation, or impeding the processivity of polymerase elongation (11). A broad range of chemical classes have been identified as NNI, including inhibitors of HIV (9, 35) and HCV (3, 5, 11, 25).In the present study, we performed high-throughput screening (HTS) to search for NNI of DENV RdRp. The HTS and chemistry synthesis led to the identification of N-sulfonylanthranilic acid derivatives as inhibitors of DENV RdRp. The compounds specifically inhibit DENV RdRp. UV cross-linking experiments mapped the compound binding site to the RdRp domain of DENV NS5. Amino acid Met343, located at the entrance of RNA template tunnel of the DENV RdRp, was cross-linked to the compound. These results, together with biochemistry experiments, suggest that the compound blocks the RdRp activity through binding to the RNA template tunnel of the polymerase.     

Phosphoprotein enriched in astrocytes-15 kDa expression inhibits astrocyte migration by a protein kinase C delta-dependent mechanism

Phosphoprotein enriched in astrocytes-15 kDa (PEA-15), a phosphoprotein enriched in astrocytes, inhibits both apoptosis and proliferation in normal and cancerous cells. Here, analysis of PEA-15 expression in glioblastoma organotypic cultures revealed low levels of PEA-15 in tumor cells migrating away from the explants, regardless of the expression levels in the originating explants. Because glioblastomas are highly invasive primary brain tumors that can originate from astrocytes, we explored the involvement of PEA-15 in the control of astrocyte migration. PEA-15-/- astrocytes presented an enhanced motility in vitro compared with their wild-type counterparts. Accordingly, NIH-3T3 cells transfected by green fluorescent protein-PEA-15 displayed a reduced migration. Reexpression of PEA-15 restored PEA-15-/- astrocyte motility to wild-type levels. Pharmacological manipulations excluded a participation of extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and calcium/calmodulin-dependent protein kinase II in this effect of PEA-15. In contrast, treatment by bisindolylmaleimide, G?6976, and rottlerin, and chronic application of phorbol 12-myristate 13-acetate and/or bryostatin-1 indicated that PKC delta mediated PEA-15 inhibition of astrocyte migration. PEA-15-/- astrocytes constitutively expressed a 40-kDa form of PKC delta that was down-regulated upon PEA-15 reexpression. Together, these data reveal a new function for PEA-15 in the inhibitory control of astrocyte motility through a PKC delta-dependent pathway involving the constitutive expression of a catalytic fragment of PKC delta.     

Length‐weight analysis of three needlefish species from the Lakshadweep archipelago

Length‐weight relationships (LWRs) of three needlefishes belonging to the family Belonidae viz., Ablennes hians, Tylosurus crocodilus and Tylosurus acus melanotus were estimated based on samples exploited from a gill‐net fishery in Androth, an island in the Lakshadweep archipelago. The estimated allometric co‐efficient b value ranged from 3.047 (T. acus melanotus) to 3.274 (A. hians), and r² value ranged from 0.911 (T. acus melanotus) to 0.973 (A. hians). The first estimate of LWR for these three commercially exploited needlefish species from the Lakshadweep islands indicate local populations to be fairly robust and forms a basis for future management of fishing stock in the region.     

A study on distribution of natural radionuclide polonium-210 in a pond ecosystem

This paper presents the results of an investigation on the distribution of²¹⁰Po in Mutharasanallur pond ecosystem. It has been demonstrated that²¹⁰Po is non-uniformly distributed within the ecosystem. The results of the study show a dissolved²¹⁰Po concentration in pond water of 1 4mBq 1₋₁. The sediment samplso recorded a²¹⁰Po activity of 59 9 Bq kg₋₁. The aquatic organisms showed differential accumulation of the radionuclide with enhanced bioaccumulation in soft tissues and muscle. The²¹⁰Po activity in the biota fell within the range of 1·2–53 3 Bq kg₋₁ (wet wt). The bivalve mussel,Lamellidens marginalis was identified to accumulate higher concentration of²¹⁰Poin soft tissues, suggesting that these organisms could serve as a bio-monitor of²¹⁰Po radionuclide in a freshwater system. The concentration factors of²¹⁰Po for the biotic components ranged from ∼10²–∼10⁴. Analyses of the results indicate that prawn and fish represent an important source of supply of²¹⁰Po to humans via dietary intake. Results of²¹⁰Po activity in the abiotic and biotic components of the pond ecosystem were higher when compared with those of Cauvery river system, the primary water source of the pond.     

A chemical inhibitor of Apaf-1 exerts mitochondrioprotective functions and interferes with the intra-S-phase DNA damage checkpoint

QM31 represents a new class of cytoprotective agents that inhibit the formation of the apoptosome, the caspase activation complex composed by Apaf-1, cytochrome c, dATP and caspase-9. Here, we analyzed the cellular effects of QM31, as compared to the prototypic caspase inhibitor Z-VAD-fmk. QM31 was as efficient as Z-VAD-fmk in suppressing caspase-3 activation, and conferred a similar cytoprotective effect. In contrast to Z-VAD-fmk, QM31 inhibited the release of cytochrome c from mitochondria, an unforeseen property that may contribute to its pronounced cytoprotective activity. Moreover, QM31 suppressed the Apaf-1-dependent intra-S-phase DNA damage checkpoint. These results suggest that QM31 can interfere with the two known functions of Apaf-1, namely apoptosome assembly/activation and intra-S-phase cell cycle arrest. Moreover, QM31 can inhibit mitochondrial outer membrane permeabilization, an effect that is independent from its action on Apaf-1.     

Metabolic Syndrome and Coronary Artery Disease in Ossabaw Compared with Yucatan Swine

Metabolic syndrome (MetS), a compilation of associated risk factors, increases the risk of type 2 diabetes and coronary artery disease (CAD, atherosclerosis), which can progress to the point of artery occlusion. Stents are the primary interventional treatment for occlusive CAD, and patients with MetS and hyperinsulinemia have increased restenosis. Because of its thrifty genotype, the Ossabaw pig is a model of MetS. We tested the hypothesis that, when fed high-fat diet, Ossabaw swine develop more features of MetS, greater native CAD, and greater stent-induced CAD than do Yucatan swine. Animals of each breed were divided randomly into 2 groups and fed 2 different calorie-matched diets for 40 wk: control diet (C) and high-fat, high-cholesterol atherogenic diet (H). A bare metal stent was placed in the circumflex artery, and pigs were allowed to recover for 3 wk. Characteristics of MetS, macrovascular and microvascular CAD, in-stent stenosis, and Ca²⁺ signaling in coronary smooth muscle cells were evaluated. MetS characteristics including, obesity, glucose intolerance, hyperinsulinemia, and elevated arterial pressure were elevated in Ossabaw swine compared to Yucatan swine. Ossabaw swine with MetS had more extensive and diffuse native CAD and in-stent stenosis and impaired coronary blood flow regulation compared with Yucatan. In-stent atherosclerotic lesions in Ossabaw coronary arteries were less fibrous and more cellular. Coronary smooth muscle cells from Ossabaw had impaired Ca²⁺ efflux and intracellular sequestration versus cells from Yucatan swine. Therefore, Ossabaw swine are a superior model of MetS, subsequent CAD, and cellular Ca²⁺ signaling defects, whereas Yucatan swine are leaner and relatively resistant to MetS and CAD.Abbreviations: CAD, coronary artery disease; CSM, coronary smooth muscle; IVGTT, intravenous glucose tolerance test; MetS, metabolic syndrome; SERCA, sarco–endoplasmic reticulum Ca²⁺ ATPase; ET1, endothelin 1; SOCE, store-operated Ca²⁺ entryAtherosclerotic coronary artery disease (CAD) is increased at least 2-fold in patients with metabolic syndrome (MetS)²⁷ and is accompanied by marked microvascular dysfunction that further impairs coronary blood flow.¹⁰ MetS generally is diagnosed by the presence of 3 or more of the following conditions: obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension.^17,28 There is strong support for the role of the hyperinsulinemia component of MetS in increased restenosis after percutaneous coronary interventions.^74,75,84,85 Further, our group has shown that severe coronary microvascular dysfunction occurs in MetS.⁵ Because MetS (so-called ‘prediabetes’) affects as much as 27% of the United States population, is increasing dramatically in prevalence,⁹⁴ and can progress to type 2 diabetes, there is great need for basic research using animal models that accurately mimic MetS and the accompanying CAD. Clearly, there is need for study of MetS-induced CAD and in-stent stenosis and the underlying cellular and molecular mechanisms.Mice, rats, and swine are known to recapitulate MetS;^{3,12,36,60,71,72} however, none of these models fully reproduce the combined symptoms of MetS and CAD. Further, transgenic mouse models are simply not adequate for coronary vascular interventions using stents identical to those used in humans,^{18,23,38,55,57,79,83,86} a step that is essential for translation to the clinic. Yucatan and domestic swine are commonly used large animal models for study of cardiovascular disease due to their ability to mimic the neointimal formation and thrombosis observed in humans.⁸⁶ For example, several laboratories have produced severe CAD in swine,^{8,24,51,61,62,68,91} but through toxin-induced pancreatic β-cell ablation and feeding of an atherogenic diet, rather than as a natural development subsequent to MetS or diabetes. Currently, there is a paucity of large animal models that reproduce MetS and CAD.³Research on the obesity-prone Ossabaw miniature swine⁵⁹ clearly indicates that these animals develop MetS and cardiovascular disease when fed a high-calorie atherogenic diet,^{4,5,9,16,19,42,50,52,83,92} Female Ossabaw swine on this type of diet nearly doubled their percentage body fat in only 9 wk, showed insulin resistance, impaired glucose tolerance, dyslipidemia (profound increase in the ratio of low-density to high-density lipoprotein cholesterol, hypertriglyceridemia), hypertension, and early coronary atherosclerosis.¹⁶ These data contrast with those from male Yucatan miniature pigs, which did not develop MetS even after 20 wk on a comparable excess calorie atherogenic diet.^8,68,95 Yucatan swine do not develop MetS through diet manipulation, unlike Ossabaw swine, which consistently recapitulate all MetS characteristics. However, important differences in study design have not allowed direct comparison between Yucatan and Ossabaw swine.Cytosolic Ca²⁺ signaling is involved in ‘phenotypic modulation’ of coronary smooth muscle (CSM), as characterized by proliferation and migration in several in vitro cell culture models^33,35,89,90 and in vivo rodent models of the peripheral circulation (for example, reference 51). The Yucatan swine model of diabetic dyslipidemia shows altered Ca²⁺ extrusion,⁹⁶ Ca²⁺ sequestration by the sarcoplasmic reticulum,^32,34,98 and Ca²⁺ influx through voltage-gated Ca²⁺ channels.⁹⁸ Currently, Ca²⁺ signaling has not been compared directly between MetS Ossabaw and Yucatan swine CSM. Therefore, the purpose of the present study was to test the hypothesis that compared with Yucatan swine on calorie-matched standard chow (for example, Yucatan maintenance diet^8,95) and atherogenic diets, Ossabaw swine have a greater propensity to MetS and CAD with impaired coronary microvascular dysfunction and Ca²⁺ handling in CSM.