PA residues in the 2009 H1N1 pandemic influenza virus enhance avian influenza virus polymerase activity in mammalian cells

The 2009 pandemic influenza virus (pH1N1) is a swine-origin reassortant containing human, avian, and swine influenza genes. We have previously shown that the polymerase complex of the pH1N1 strain A/California/04/2009 (Cal) is highly active in mammalian 293T cells, despite the avian origin of both its PA and PB2. In this study, we analyzed the polymerase residues that are responsible for high pH1N1 polymerase activity in the mammalian host. Characterization of polymerase complexes containing various combinations of Cal and avian influenza virus A/chicken/Nanchang/3-120/01 (H3N2) (Nan) by reporter gene assay indicates that Cal PA, but not PB2, is a major contributing factor to high Cal polymerase activity in 293T cells. In particular, Cal PA significantly activates the otherwise inactive Nan polymerase at 37 and 39°C but not at the lower temperature of 34°C. Further analysis using site-directed mutagenesis showed that the Cal PA residues 85I, 186S, and 336M contribute to enhanced activity of the Cal polymerase. Recombinant A/WSN/33 (H1N1) (WSN) viruses containing Nan NP and polymerase (PA, PB1, PB2) genes with individual mutations in PA at residues 85, 186, and 336 produced higher levels of viral protein than the virus containing wild-type (WT) Nan PA. Interestingly, compared to the WT, the virus containing the 85I mutation grew faster in human A549 cells and the 336M mutation most significantly enhanced pathogenicity in a mouse model, among the three PA mutations tested. Our results suggest that multiple mutations in PA, which were rarely present in previous influenza isolates, are involved in mammalian adaptation and pathogenicity of the 2009 pH1N1.     

Yeast killer toxin: site-directed mutations implicate the precursor protein as the immunity component

Yeast killer toxin and a component giving immunity to it are both encoded by a gene specifying a single 35 kd precursor polypeptide. This precursor is composed of a leader peptide, the alpha and beta subunits of the secreted toxin, and a glycosylated gamma peptide separating the latter. The toxin subunits are proteolytically processed from the precursor during toxin secretion. Using site-directed mutagenesis, we have identified a region of the precursor gene necessary for expression of the immunity phenotype. This immunity-coding region extends through the C-terminal half of the alpha subunit into the N-terminal part of the gamma glycopeptide. Mutations in other parts of the gene allow full immunity but produce precursors that fail to be processed. The precursor can therefore confer immunity, and we propose that it does so in the wild type by competing with mature toxin for binding to a membrane receptor.     

Yeast KEX1 gene encodes a putative protease with a carboxypeptidase B-like function involved in killer toxin and alpha-factor precursor processing

The yeast KEX1 gene product has homology to yeast carboxypeptidase Y. A mutant replacing serine at the putative active site of the KEX1 protein abolished activity in vivo. A probable site of processing by the KEX1 product is the C-terminus of the alpha-subunit of killer toxin, where toxin is followed in the precursor by 2 basic residues. Processing involves endoproteolysis following these basic residues and trimming of their C-terminal by a carboxypeptidase. Consistent with the KEX1 product being this carboxypeptidase is its role in alpha-factor pheromone production. In wild-type yeast, KEX1 is not essential for alpha-factor production, as the final pheromone repeat needs no C-terminal processing. However, in a mutant in which alpha-factor production requires a carboxypeptidase, pheromone production is KEX1-dependent.     

Phylogeography of the marine macroalga Sargassum hemiphyllum (Phaeophyceae,Heterokontophyta) in northwestern Pacific

Sargassum hemiphyllum is commonly found in Japan and Korea, with a variety, var. chinense, that is found distributed in the southern Chinese coast. We previously reported distinct genetic differentiation between the two taxa based on the PCR‐RFLP data of plastid RubiscoL‐S spacer. The present study aims at elucidating the phylogeographic pattern of S. hemiphyllum based on more markers in the nuclear and extranuclear genomes, with a view to reveal the occurrence of hybridization. The two allopatrically distributed taxa were found to be genetically distinct in nuclear ITS2, plastidial Rubisco (Rbc) and mitochondrial TrnW_I (Trn) spacers. Their divergence was postulated to be attributable to the vicariant event which resulted from the isolation of the Sea of Japan during the late Miocene (6.58–11.25 Mya). Divergence within both S. hemiphyllum and the chinense variety was observed based on Trn spacer, while the divergence in S. hemiphyllum was further confirmed in Rbc spacer. This divergence appears to correspond to the separation of the Japanese populations between the Sea of Japan and the Pacific that occurred around 0.92–2.88 Mya (the early Pleistocene). The presence of an ITS2 clone resembling var. chinense sequences in a Japanese population of S. hemiphyllum (JpNS) raises the possibility of the introgression of var. chinense individuals into S. hemiphyllum population. Compared to that between S. hemiphyllum and the chinense variety, hybridization among the Japanese and Korean populations of S. hemiphyllum is highly probable as all these individuals share a pool of nuclear ITS2 sequences, possibly attributable to incomplete concerted evolution of ITS2.     

Mutual antagonism among killer yeasts: competition between K1 and K2 killers and a novel cDNA-based K1-K2 killer strain of Saccharomyces cerevisiae

Mutually antagonistic K1 and K2 killer strains compete when mixed and serially subcultured. At pH 4.6, where the K1 killer toxin is more stable in vitro, the K1 strain outcompeted the K2 strains at both 18 and 30 degrees C. At pH 4.0, closer to the in vitro pH optimum of the K2 killer toxin, the K1 strain again predominated at 18 degrees C, but at 30 degrees C the K2 strains became the sole cell type on subculture. To show more clearly that these results were dependent upon the respective killer toxins, control experiments were conducted with isogenic, nonkiller strains cured of the dsRNA-based killer virions. Such nonkiller strains were unable to compete with antagonistic killers under conditions where their isogenic killer parents could, strongly suggesting that the killer phenotype was important in these competitions. Double K1-K2 killer strains cannot stably exist, as their dsRNA genomes compete at a replicative level. Using recombinant DNA methodology, a stable K1-K2 killer strain was constructed. This strain outcompeted both K1 and K2 killers when serially subcultured under conditions where either the K1 or the K2 strains would normally predominate in mixed cultures. Such a double killer may be useful in commercial fermentations, where there is a risk of contamination by killer yeasts.     

Acute toxicity and adverse effects of aqueous and ethanol extracts of Parkia biglobosa pods on biochemical parameters of Clarias gariepinus

The acute toxicity of the aqueous and ethanol extracts of Parkia biglobosa pods against Clarias gariepinus was investigated under laboratory conditions. Agitated behaviours and respiratory distress were also observed during the exposure period. The adverse effects on biochemical parameters were assessed using semi-static bioassays for 28 days. The ethanol extract of P. biglobosa pods was found to be more acutely toxic with a 96 h LC₅₀ value of 13.96 mg l^?1 than the aqueous extracts, with a 96 h LC₅₀ value of 19.95 mg l^?1 against C. gariepinus. Both extracts induced agitated behaviours and respiratory distress in exposed organisms. The activities of superoxide dismutase (SOD), catalase (CAT) and the concentration of malondialdehyde (MDA) were significantly lower (p < 0.05) in groups of organisms exposed to extracts of P. biglobosa when compared with the control group after 14 days. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were also significantly (p < 0.05) lower compared with activities of the enzymes in the control group after 28 days. The current study has shown that the introduction of P. biglobosa pods into aquatic ecosystems is acutely toxic to fish and would possibly be to other non-target aquatic organisms especially invertebrates.     

The direct measurement of protein kinase C (PKC) activity in isolated membranes using a selective peptide substrate

A protein kinase C (PKC)-selective peptide substrate was used to develop a method for measuring PKC activity directly and quantitatively in isolated cell membranes without prior detergent extraction and reconstitution of the enzyme with phosphatidylserine and TPA in the presence of excess Ca2+. This simple and rapid method can reliably measure changes in membrane-associated PKC activity induced by various bioactive compounds such as hormones and growth factors. Also, this method, which measures PKC activity in its native membrane-associated state, has the advantage of being able to distinguish between active and inactive PKC associated with cell membranes.     

Calpain inhibition mediates autophagy-dependent protection against polyglutamine toxicity

Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington''s disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington''s disease.Huntington''s disease (HD) is a currently incurable, autosomal dominant neurodegenerative disease resulting from the expansion of the trinucleotide (CAG) repeat region of the huntingtin (HTT) IT15 gene, encoding huntingtin protein (Htt). In mutant Htt, the polyglutamine tract encoded by this region contains over 35 glutamines and the length of the tract correlates inversely with the age of disease onset, with longer tracts resulting in earlier onset (reviewed in Imarisio et al.¹). HD is one of the 10 trinucleotide repeat disorders resulting from expansions of polyglutamine tracts in different proteins. These expansions cause disease by conferring toxic gain-of-function properties onto the mutant proteins. Hence, one strategy that has been considered for HD and related diseases is to find ways of decreasing the levels of the mutant protein, for instance by harnessing the cell''s capacity to degrade such aggregate-prone proteins via (macro)autophagy.^{2, 3, 4, 5} Autophagy involves the engulfment of cytoplasmic contents by double-membraned autophagosomes, which then traffic to lysosomes where their contents are degraded. Mutant huntingtin, some other polyglutamine expanded proteins like mutant ataxin 3, and proteins like tau (which mediates toxicity in Alzheimer''s disease and related dementias) are autophagy substrates and their clearance can be enhanced in Drosophila and mouse models by autophagy upregulation, which also reduces their toxicity.^{2, 3, 4,6}Calpains are a family of calcium-activated cysteine proteases (reviewed in Ono and Sorimachi⁷) that inhibit autophagy. Strategies that reduce calpain activity in cell culture increase autophagy and decrease levels of autophagy substrates, like mutant Htt. These effects are likely to be mediated by Gsα, a heterotrimeric G-protein subunit which is activated by calpain cleavage. Similar to calpain inhibition, siRNA knockdown of Gsα, or chemical inhibition by NF449, induces autophagy and decreases the number of aggregates resulting from the overexpression of exon-1 Htt with an expanded polyglutamine repeat region (HttQ74) in cell culture models.⁸ In addition to this mechanism of autophagy upregulation by calpains, the core autophagy protein ATG5 has also been demonstrated to be cleaved and inactivated by calpains,^9,10 suggesting that calpains may act on a number of substrates to negatively regulate autophagy.In mammals, the two most abundantly expressed calpains are μ-calpain and m-calpain, which differ in their affinity for calcium and therefore the calcium concentration required for their activation. As well as being regulated by calcium, they are also controlled by an endogenous inhibitor, calpastatin (CAST). Drosophila have four forms of calpain:¹¹ CalpA and CalpB are the conventional calpains formed by a recent duplication in the Drosophila insect lineage, CalpC is also an evolutionarily recent, but not highly conserved duplication (data not shown) and is thought to be catalytically inactive,¹¹ and CalpD (SOL) is a member of the unconventional family of calpains. Drosophila does not appear to have any obvious orthologs of CAST.A role for calpains in HD has been investigated previously. Following observations that shorter Htt fragments are more toxic than full-length Htt,¹² it was demonstrated that Htt can be cleaved by both caspases¹³ and calpains¹⁴ to generate these toxic, short fragments. Blocking Htt cleavage by calpains by mutating their calpain cleavage sites decreases Htt aggregation and toxicity.¹⁵ In addition, calpain activation has been shown to be increased in HD patients compared with controls.¹⁴In this study, we have investigated a role for calpain activity as a modulator of autophagy in both Drosophila and mouse models of HD. To avoid confounding effects from alterations in cleavage of Htt by calpain, we have used models expressing short fragments of Htt, which do not contain calpain cleavage sites and correspond to the shortest fragments of huntingtin seen in patients.¹⁶ We demonstrate that knockdown of CalpA in Drosophila is sufficient to both reduce the number of Htt aggregates and the toxicity associated with the expression of the mutant protein. Importantly, we show that these effects are autophagy-dependent. Furthermore, we show that overexpression of CAST in mice results in enhanced autophagy and improves locomotor function and delays tremor onset in a mouse model of HD, as well as decreasing the number of Htt aggregates seen in the brain. We extended the analysis of CAST overexpressing mice to investigate the possible adverse effects from long-term calpain inhibition or autophagy upregulation but did not observe any obvious deleterious effects.     

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts.Two-dimensional patterns of ghost proteins were not heavily contaminated (?25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8–10 fold over whole cells.Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.