Elimination of phosphorylation sites of Semliki Forest virus replicase protein nsP3

nsP3 is one of the four RNA replicase subunits encoded by alphaviruses. The specific essential functions of nsP3 remain unknown, but it is known to be phosphorylated on serine and threonine residues. Here we have completed mapping of the individual phosphorylation sites on Semliki Forest virus nsP3 (482 amino acids) by point mutational analysis of threonine residues. This showed that threonines 344 and 345 represented the major threonine phosphorylation sites in nsP3. Experiments with deletion variants suggested that nsP3 itself had no kinase activity; instead, it was likely to be phosphorylated by multiple cellular kinases. Phosphorylation was not necessary for the peripheral membrane association of nsP3, which was mediated by the N-terminal region preceding the phosphorylation sites. Two deletion variants of nsP3 with either reduced or undetectable phosphorylation were studied in the context of virus infection. Cells infected with mutant viruses produced close to wild type levels of infectious virions; however, the rate of viral RNA synthesis was significantly reduced in the mutants. A virus totally defective in nsP3 phosphorylation and exhibiting a decreased rate of RNA synthesis also exhibited greatly reduced pathogenicity in mice.     

Contribution of HIF-P4H isoenzyme inhibition to metabolism indicates major beneficial effects being conveyed by HIF-P4H-2 antagonism

Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs 1–3) are druggable targets in renal anemia, where pan-HIF-P4H inhibitors induce an erythropoietic response. Preclinical data suggest that HIF-P4Hs could also be therapeutic targets for treating metabolic dysfunction, although the contributions of HIF-P4H isoenzymes in various tissues to the metabolic phenotype are inadequately understood. Here, we used mouse lines that were gene-deficient for HIF-P4Hs 1 to 3 and two preclinical pan-HIF-P4H inhibitors to study the contributions of these isoenzymes to the anthropometric and metabolic outcome and HIF response. We show both inhibitors induced a HIF response in wildtype white adipose tissue (WAT), liver, and skeletal muscle and alleviated metabolic dysfunction during a 6-week treatment period, but they did not alter healthy metabolism. Our data indicate that HIF-P4H-1 contributed especially to skeletal muscle and WAT metabolism and that its loss lowered body weight and serum cholesterol levels upon aging. In addition, we found HIF-P4H-3 had effects on the liver and WAT and its loss increased body weight, adiposity, liver weight and triglyceride levels, WAT inflammation, and cholesterol levels and resulted in hyperglycemia and insulin resistance, especially during aging. Finally, we demonstrate HIF-P4H-2 affected all tissues studied; its inhibition lowered body and liver weight and serum cholesterol levels and improved glucose tolerance. We found very few HIF target metabolic mRNAs were regulated by the inhibition of three isoenzymes, thus suggesting a potential for selective therapeutic tractability. Altogether, these data provide specifications for the future development of HIF-P4H inhibitors for the treatment of metabolic diseases.     

Production of pectin methylesterase from Erwinia chrysanthemi B374 in Bacillus subtilis

Summary The gene coding for pectin methylesterase (PME) of Erwinia chrysanthemi B374 (pme) was cloned by a polymerase chain reaction. The pme gene was expressed in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the -amylase gene from B. amyloliquefaciens. The cultivation of B. subtilis cells carrying the cloned pme resulted in efficient secretion of PME into the culture medium based on enzymatic and sodium dodecyl sulphate-polyacrylamide gel electrophoresis characterizations. The NH₂-terminal sequence analysis of the secreted PME revealed two different NH₂-termini. Heterologous processing was probably due to a second putative signal peptidase cleavage site at the joint region between the PME and -amylase signal peptide. Offprint requests to: R. Heikinheimo     

Efficient DNA fingerprinting of Clostridium botulinum types A, B, E, and F by amplified fragment length polymorphism analysis

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.     

The colour of fitness: plumage coloration and lifetime reproductive success in the tawny owl

We studied variation in plumage colour and life history in a population of tawny owls (Strix aluco) in southern Finland, using 26 years of data on individually marked male and female owls. Colour was scored on a semi-continuous scale from pale grey to reddish brown. Colour scoring was repeatable and showed a bimodal distribution (grey and brown morph) in both sexes. During the study period, colour composition was stable in the study population in both sexes. The sexes did not mate assortatively with respect to their colour. Colour was a highly heritable trait and was under selection. Grey-coloured male and female owls had a higher lifetime production of fledglings, and grey-coloured male (but not female) owls produced more recruits during their lifetime than brown individuals. Selection on colour was mediated through viability selection and not through fecundity selection. Our results reveal remarkably strong selection on a genetically determined phenotypic trait.     

Contrasting above‐ and belowground organic matter decomposition and carbon and nitrogen dynamics in response to warming in High Arctic tundra

Tundra regions are projected to warm rapidly during the coming decades. The tundra biome holds the largest terrestrial carbon pool, largely contained in frozen permafrost soils. With warming, these permafrost soils may thaw and become available for microbial decomposition, potentially providing a positive feedback to global warming. Warming may directly stimulate microbial metabolism but may also indirectly stimulate organic matter turnover through increased plant productivity by soil priming from root exudates and accelerated litter turnover rates. Here, we assess the impacts of experimental warming on turnover rates of leaf litter, active layer soil and thawed permafrost sediment in two high‐arctic tundra heath sites in NE‐Greenland, either dominated by evergreen or deciduous shrubs. We incubated shrub leaf litter on the surface of control and warmed plots for 1 and 2 years. Active layer soil was collected from the plots to assess the effects of 8 years of field warming on soil carbon stocks. Finally, we incubated open cores filled with newly thawed permafrost soil for 2 years in the active layer of the same plots. After field incubation, we measured basal respiration rates of recovered thawed permafrost cores in the lab. Warming significantly reduced litter mass loss by 26% after 1 year incubation, but differences in litter mass loss among treatments disappeared after 2 years incubation. Warming also reduced litter nitrogen mineralization and decreased the litter carbon to nitrogen ratio. Active layer soil carbon stocks were reduced 15% by warming, while soil dissolved nitrogen was reduced by half in warmed plots. Warming had a positive legacy effect on carbon turnover rates in thawed permafrost cores, with 10% higher respiration rates measured in cores from warmed plots. These results demonstrate that warming may have contrasting effects on above‐ and belowground tundra carbon turnover, possibly governed by microbial resource availability.     

Phenotypic and Genotypic Analysis of Rats with Cerebellar GABAA Receptors Composed from Mutant and Wild-Type α6 Subunits

Abstract: The alcohol-sensitive (ANT) rat line, developed for high behavioral sensitivity to ethanol, also exhibits enhanced sensitivity to benzodiazepines, such as diazepam. The rat line carries a point mutation in the cerebellum-specific γ-aminobutyric acid type A (GABA_A) receptor subunit α6, making their diazepam-insensitive (DIS) receptors sensitive to diazepam. We now report that phenotypes of individual ANT and alcohol-insensitive rats, classified on diazepam sensitivity of cerebellar [³H]Ro 15-4513 binding, correlated well with homozygous wild-type, homozygous mutant, and heterozygous genotypes, although some heterozygotes were biased toward the parental phenotypes. GABA down-modulated DIS [³H]Ro 15-4513 binding in mutant homozygotes but tended to up-modulate it in heterozygotes and wild-type homozygotes. Slopes for GABA inhibition of cerebellar t-butylbicyclophosphoro[³⁵S]thionate binding were larger in mutant than in wild-type homozygotes, with heterozygotes being intermediate. Diazepam displacement of [³H]Ro 15-4513 binding in heterozygotes revealed three components, with their affinities indistinguishable from those in combined wild-type and mutant homozygotes. This lack of interaction in DIS binding between wild-type and mutant α6 subunits was substantiated by experiments on recombinant receptors. The data suggest that the α6 subunit-containing GABA_A receptors in the heterozygotes are formed from individual mutant and wild-type subunits with their relative expression differing from animal to animal.     

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity

The ceIV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 by and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of Ce1V1 was highly similar to that of CeIV of another E. c. subsp. carotovora strain SCRI193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. Ce1Vl mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.