Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Dynamics of activities of matrix metalloproteinases-9 and -2, and the tissue inhibitors of MMPs in fetal fluid compartments during gestation and at parturition in the mare

During late gestation in the mare, rapid fetal growth is accompanied by considerable placental growth and further invasion of the endometrium by microvilli. This growth requires extensive remodeling of the extracellular matrix (ECM). In early pregnancy, we know that matrix metalloproteinase (MMP)-9 and -2 are involved in the endometrial invasion during endometrial cup formation. The present study investigated whether MMPs are found in fetal fluids later in gestation and during parturition, and if there was a difference in their activities between normal and preterm delivery. Amniotic fluids were collected from pony mares during the latter half of gestation, and amniotic and allantoic fluids from pony and thoroughbred mares at foaling. The fluids were analysed for the activity of MMP-9 and -2, and TIMPs using zymography techniques. There was an increase (P = 0.002) in activity of latent MMP-9 when approaching normal foaling, and a decrease (P < 0.001) during foaling. MMP-2 activity did not change through gestation, or during foaling. When comparing samples from pregnancies resulting in preterm deliveries with samples from foaling mares, the activity of MMP-9 was lower (P < 0.001) and MMP-2 activity was higher (P = 0.004) during foaling than preceding preterm delivery. The activity of MMP-9 was lower (P = 0.002) prior to preterm delivery than before delivery of a live foal at term, whereas no difference (P = 0.07) was demonstrated for latent MMP-2 activity when comparing the same groups. The activity of TIMP-2 was higher (P < 0.001) in the pre-parturient period before normal foaling than preceding preterm delivery. These results suggest that MMPs may have a role as markers for high risk pregnancy in the mare.     

Glyphidohaptor safiensis n. sp. (Monogenea: Ancyrocephalidae) from the white-spotted rabbitfish Siganus canaliculatus (Park) (Perciformes: Siganidae) off Oman,with notes on its phylogenetic position within the Ancyrocephalidae Bychowsky & Nagibina, 1968 (sensu lato)

A new ancyrocephalid monogenean is described from the gills of wild White-spottedrabbitfish Siganus canaliculatus (Park) based on morphological and molecular analyses. Glyphidohaptor safiensis n. sp. can be distinguished from other members of the genus by the shape of the accessory piece of the male copulatory organ (MCO). Unlike its congeners, the rod-shaped accessory piece of G. safiensis n. sp. is distally broad and flattened. The MCO of G. safiensis n. sp. is curved, enclosed in a heavy sheath with a terminal flap. Partial large subunit (LSU), partial small subunit (SSU) and the partial SSU, entire internal transcribed spacer region 1 (ITS1) and partial 5.8S rDNA of the new species and two species of Tetrancistrum Goto & Kikuchi, 1917 from the same host and locality were sequenced and subjected to phylogenetic analysis. The LSU rDNA analysis grouped G. safiensis n. sp. with Tetrancistrum sp. from the gills of Siganus fuscescens Houttuyn from Australia, indicating a possible misidentification of the latter. Sequences of the SSU rDNA of the new species were most similar to those for Pseudohaliotrema sphincteroporus Yamaguti, 1953, demonstrating the close relatedness of the genera Pseudohaliotrema Yamaguti, 1953 and Glyphidohaptor Kritsky, Galli & Yang, 2007 within the Ancyrocephalidae. The comparison of the partial SSU (424 bp) and entire ITS1 and partial 5.8S rDNA (246 bp) sequences obtained for G. safiensis n. sp. with the only available sequence of another member of Glyphidohaptor Kritsky, Galli & Yang, 2007, G. pletocirra Paperna, 1972 (HE601931-HE601933) yielded on average 1.08% dissimilarity (a difference of 7 bases), with a p-distance of 0.010 ± 0.004%. This is the first record of a species of Glyphidohaptor from S. canaliculatus and from the Persian Gulf, the Gulf of Oman and the Arabian Sea.

     

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

Background

Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy.

Methods

Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors.

Results

Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors.

Conclusions

In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.     

Indigo: an annotated bibliography

This bibliography lists and comments on publications describing the history, preparation, chemistry and some applications of indigo and related dyes. Note that the commentary by the author is in italics after the bibliographic information.     

F2-Isoprostanes,tocopherols and normal pregnancy

This study investigates oxidative stress and antioxidants in normal human pregnancy and post-partum period. Thirty-seven healthy women with normal pregnancies were included. Both urinary and serum samples were collected throughout the pregnancy and post-partum period. Oxidative stress was estimated by measuring the reliable in vivo marker, namely 8-iso-prostaglandin F_2α (8-iso-PGF_2α, an F₂-isoprostane) and antioxidant status was evaluated by measuring α- and γ-tocopherol in serum samples. Pregnancy was associated with successively increased levels of 8-iso-PGF_2α with advancing gestational age. The median post-partum value corresponded to the values observed in early gestation and a significant decrease was observed from late pregnancy to the post-partum period. Lipid-adjusted α- and γ-tocopherol levels decreased with advancing gestational age. This longitudinal study suggests that mild oxidative stress is involved in normal human pregnancy.     

PCR gut analysis reveals that Tenuiphantes tenuis (Araneae: Linyphiidae) is a potentially significant predator of Argentine stem weevil,Listronotus bonariensis (Coleoptera: Curculionidae), in New Zealand pastures

Polymerase chain reaction (PCR) gut analysis was conducted on specimens of the introduced spider Tenuiphantes tenuis collected from dairy pasture in Canterbury, New Zealand. PCR primers were specifically designed to amplify a fragment of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) from Listronotus bonariensis and revealed that this major pasture pest species is consumed in the field by T. tenuis. The field predation rate of L. bonariensis by T. tenuis was estimated from our PCR results together with published data on the degradation of DNA and the density of T. tenuis in Canterbury pastures. We found that T. tenuis is a potentially significant predator of L. bonariensis in New Zealand pastures.     

Nutrient stocks in managed and natural humid tropical fallows

Managed fallows which recover nutrients more rapidly than natural secondary vegetation may improve the performance of shifting agriculture systems operating under inadequately long fallow cycles. Our objective was to construct nutrient balances for the soil, vegetation, and litter compartments of six planted leguminous fallows and natural secondary vegetation during 53 months. The fallows were planted on a previously cultivated Ultisol (Acrisol) in the Peruvian Amazon and included:Centrosema macrocarpum (Centrosema),Pueraria phaseoloides (Pueraria),Stylosanthes guianensis (Stylosanthes),Desmodium ovalifolium (Desmodium),Cajanus cajan (Cajanus), andInga edulis (Inga). In addition, in the natural fallow treatment secondary vegetation was allowed to establish and grow naturally. Quantities of extractable P, K, Ca, and Mg, total N, and organic C in soil to a 45 cm depth, and macrouttrients in aboveground biomass, roots, and litter were estimated at fallow planting, at 8, 17, and 29 months afterward, and at fallow clearing (53 months). Total N stocks increased by 10% in the Stylosanthes, Desmodium, Pueraria, and Inga treatments, but changed little in the Cajanus, Centrosema and natural fallows. This difference was largely due to greater net increases in both soil and vegetation compartments in the former group of treatments. In the Inga, Desmodium, and natural fallows, total stocks of P and K at 53 months were about 40% to 80% greater and 12% greater, respectively, than initial values, but Ca and Mg stocks were reduced by 25% to 40%. In the other treatments, there was generally little change in P stocks, but large (30% to 60%) reductions in K, Ca, and Mg during the course of the fallow. Although there were net decreases of stocks of P, K, Ca, and Mg in soil in all treatments during the fallow, storage of P and K in vegetation and litter in the Inga, Desmodium, and natural fallows offset losses of these nutrients from soil. These treatments also tended to accumulate more Ca and Mg in biomass and litter than the other treatments. These results suggest that leguminous fallow vegetation that accumulates large amounts of biomass may increase N, P, and K stocks, but that incomplete recuperation of Ca and Mg may limit the sustainability of short-rotation fallow-based systems on acidic, infertile soils. ei]Section editor: G R Stewart     

Concordance of allozyme and microsatellite differentiation in a marine fish, but evidence of selection at a microsatellite locus

Previous studies have reported higher levels of divergence for microsatellites than for allozymes in several species, suggested to reflect stabilizing selection on the allozymes. We compared the differentiation patterns of 11 allozyme and nine microsatellite loci using 679 spawning Atlantic herring (Clupea harengus) collected in the Baltic and North Seas to test for differential natural selection on these markers. Observed distributions of F statistics for the two types of markers are conspicuously dissimilar, but we show that these differences can largely be explained by sampling phenomena caused by different allele frequency distributions and degrees of variability. The results show consistently low levels of differentiation for both marker types, with the exception of one outlier microsatellite locus with a notably high F(ST). The aberrant pattern at this locus is primarily due to two alleles occurring at markedly high frequencies in the Baltic, suggesting selection at this locus, or a closely linked one. When excluding this locus, the two marker types show similar, weak differentiation patterns with F(ST) values between the Baltic and the North Seas of 0.001 and 0.002 for allozymes and microsatellites, respectively. This small heterogeneity, and weak isolation by distance, is easier to distinguish statistically with microsatellites than with allozymes that have fewer alleles and skewed frequency distributions. The allozymes, however, also detect surprisingly low levels of divergence. Our results support suggestions that previously described differences between marker types are primarily caused by a small number of outlier loci.