Molecular identification of two strains of third-stage larvae of Contracaecum rudolphii sensu lato (Nematoda: Anisakidae) from fish in Poland

Contracaecum sp. larvae (L3) from fish were identified using nucleotide sequences of the internal transcribed spacers ITS-1 and ITS-2 of the ribosomal DNA. The nematode larvae originated from fish in a freshwater situation (crucian carp Carassius carassius, from Selment Wielki Lake in Mazury, northeastern Poland) and a brackish-water region (Caspian round goby Neogobius melanostomus from the Baltic Sea, Gdafisk Bay at the Polish coast). Two strains (Contracaecum rudolphii A and B) of Contracaecum rudolphii senso lato, a parasite common at the adult stage in fish-eating birds, were identified. In fish from the freshwater site, only the strain temporarily designated C. rudolphii B was identified; in the brackish-water region, both strains were found, suggesting that fish serve as paratenic host for both genotypes. Contracaecum rudolphii sensu lato has been recorded in several species of fish-eating birds in Poland, particularly in the great cormorant, Phalacrocorax carbo, in which the abundance is highest. The results, although based on a restricted number of larvae, suggest that the life cycles of both genotypes can be completed in the Polish region and that at least one of them, C. rudolphii B, can develop both in fresh and brackish water.     

Deletion of the protein kinase A/protein kinase G target SMTNL1 promotes an exercise-adapted phenotype in vascular smooth muscle

In vivo protein kinases A and G (PKA and PKG) coordinately phosphorylate a broad range of substrates to mediate their various physiological effects. The functions of many of these substrates have yet to be defined genetically. Herein we show a role for smoothelin-like protein 1 (SMTNL1), a novel in vivo target of PKG/PKA, in mediating vascular adaptations to exercise. Aortas from smtnl1(-/-) mice exhibited strikingly enhanced vasorelaxation before exercise, similar in extent to that achieved after endurance training of wild-type littermates. Additionally, contractile responses to alpha-adrenergic agonists were greatly attenuated. Immunological studies showed SMTNL1 is expressed in smooth muscle and type 2a striated muscle fibers. Consistent with a role in adaptations to exercise, smtnl1(-/-) mice also exhibited increased type 2a fibers before training and better performance after forced endurance training compared smtnl1(+/+) mice. Furthermore, exercise was found to reduce expression of SMTNL1, particularly in female mice. In both muscle types, SMTNL1 is phosphorylated at Ser-301 in response to adrenergic signals. In vitro SMTNL1 suppresses myosin phosphatase activity through a substrate-directed effect, which is relieved by Ser-301 phosphorylation. Our findings suggest roles for SMTNL1 in cGMP/cAMP-mediated adaptations to exercise through mechanisms involving direct modulation of contractile activity.     

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Sulfonamides with the N-alkyl-N′-dialkylguanidine moiety as 5-HT7 receptor ligands

A series of arylsulfonamides containing guanidine incorporated in the structure of secondary amines (piperidine, piperazine) was synthesized on SynPhase Lanterns and evaluated for 5-HT_1A, 5-HT_2A, and 5-HT₇ receptors. The results demonstrated that N-alkyl-N′-dialkylguanidines displayed good 5-HT₇/5-HT_1A selectivity and may be regarded as promising structural core for development of 5-HT₇ ligands.     

Comparison of genomes of eight species of sections <Emphasis Type="Italic">Linum</Emphasis> and <Emphasis Type="Italic">Adenolinum</Emphasis> from the genus<Emphasis Type="Italic"> Linum</Emphasis> based on chromosome banding,molecular markers and RAPD analysis

Karyotypes of species sects. Linum and Adenolinum have been studied using C/DAPI-banding, Ag-NOR staining, FISH with 5S and 26S rDNA and RAPD analysis. C/DAPI-banding patterns enabled identification of all homologous chromosome pairs in the studied karyotypes. The revealed high similarity between species L. grandiflorum (2n = 16) and L. decumbens by chromosome and molecular markers proved their close genome relationship and identified the chromosome number in L. decumbens as 2n = 16. The similarity found for C/DAPI-banding patterns between species with the same chromosome numbers corresponds with the results obtained by RAPD-analysis, showing clusterization of 16-, 18- and 30-chromosome species into three separate groups. 5S rDNA and 26S rDNA were co-localized in NOR-chromosome 1 in the genomes of all species investigated. In 30-chromosome species, there were three separate 5S rDNA sites in chromosomes 3, 8 and 13. In 16-chromosome species, a separate 5S rDNA site was also located in chromosome 3, whereas in 18-chromosome species it was found in the long arm of NOR-chromosome 1. Thus, the difference in localization of rDNA sites in species with 2n = 16, 2n = 30 and 2n = 18 confirms taxonomists opinion, who attributed these species to different sects. Linum and Adenolinum, respectively. The obtained results suggest that species with 2n = 16, 2n = 18 and 2n = 30 originated from a 16-chromosome ancestor.     

Fecundity and MHC affects ejaculation tactics and paternity bias in sand lizards

We demonstrate that extending copulation enhances probability of paternity in sand lizards and that determinants of copulation duration depend on a males' mating order (first or second). First males, with no information on presence of rivals, extend copulation when mating with a more fecund female. Second males, however, adjust copula duration in relation to a first male's relatedness with his female, which there is reason to believe can be deduced from the MHC-related odor of the copulatory plug. Male-female relatedness negatively influences a male's probability of paternity, and when second males are in a favored role (i.e., the first male is the one more closely related to the female), second males transfer larger ejaculates, resulting in higher probability of paternity. This result corroborates predictions from recent theoretical models on sperm expenditure theory incorporating cryptic female choice and sexual conflict. More specifically, the results conform to a "random roles" model, which depicts males as being favored by some females and disfavored by others, but not to a "constant-type" model, in which a male is either favored or disfavored uniformly by all females in a population.     

Mating success and oviposition of a butterfly are not affected by non-lethal tissue sampling

Non-lethal methods of tissue sampling are increasingly used for genetic studies of insect species and the effects of this approach have long been assumed to be minimal. Tissue removal has the potential to influence insect reproductive behaviours such as mate recognition, courtship or oviposition but the effects of non-lethal sampling on reproductive success have not been widely and adequately tested. Here, we test potential effects of both wing-clipping and leg removal on reproductive behaviours of the cabbage white butterfly (Pieris rapae). We conducted a total of 93 male and 59 female mating trials, and found no significant differences in mating success between treated (i.e., tissue removed) and control individuals in either sex. We also monitored the number and location of eggs laid by 58 females. We found no significant differences in egg-laying behaviour among leg removed and control individuals. Power analysis indicated that we had sufficient statistical power to detect moderate effects of treatment on both mating and oviposition. Our study provides the most comprehensive examination to date of the effects of non-lethal sampling on reproductive behaviours in a butterfly/insect species, and supports the contention that tissue sampling is non-detrimental. To fully comprehend the general impacts of tissue sampling on butterfly reproductive behaviour however, additional similar studies need to be conducted on a variety of species with differing mating behaviours. Only through meta-analysis, may it be possible to detect more subtle effects of tissue removal which cannot be revealed within a single study due to sample size limitations.     

Inhibition of GSK3 promotes replication and survival of pancreatic beta cells

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.     

Comparative Genomics of Marine Mussels (Mytilus spp.) Gender Associated mtDNA: Rapidly Evolving atp8

The unusual mode of mitochondrial DNA inheritance, with two separate: maternal (F) and paternal (M) lineages, gives unique opportunities to study the evolution of the mitochondrial genome. This system was first discovered in the marine mussels Mytilus. The three related species: Mytilus edulis, Mytilus galloprovincialis and Mytilus trossulus form a complex in which the divergence of M and F lineages pre-dates the speciation. The complete mitochondrial genomes of both lineages were known for all species except Pacific M. trossulus. Here we report, for the first time, the complete sequences of both mitochondrial genomes of Pacific M. trossulus, filling the gap. While the reported M and F genomes are highly diverged (26%), they have similar organisation. The only difference is the translocation of one tRNA gene into the long, mosaic control region of the F genome. Consistent presence of an ORF which most likely represents the atp8 gene was confirmed in both genomes. The predicted protein has characteristics expected of the functional atp8 even though the M and F versions are markedly different in length. Comparative analysis involving all three species led to the conclusion that the cause of a faster evolution of atp8 and Mytilus mtDNA in general is most likely the Compensation-Draft Feedback process coupled with relatively relaxed selection in the M lineage. Thus, we postulate that the adaptive changes may have played a role in the emergence of highly diverged, barely recognizable atp8 in Mytilus mussels.