Genetic and biochemical analysis of the twin-arginine translocation pathway in halophilic archaea

The twin-arginine translocation (Tat) pathway is present in a wide variety of prokaryotes and is capable of exporting partially or fully folded proteins from the cytoplasm. Although diverse classes of proteins are transported via the Tat pathway, in most organisms it facilitates the secretion of a relatively small number of substrates compared to the Sec pathway. However, computational evidence suggests that haloarchaea route nearly all secreted proteins to the Tat pathway. We have expanded previous computational analyses of the haloarchaeal Tat pathway and initiated in vivo characterization of the Tat machinery in a model haloarchaeon, Haloferax volcanii. Consistent with the predicted usage of the this pathway in the haloarchaea, we determined that three of the four identified tat genes in Haloferax volcanii are essential for viability when grown aerobically in complex medium. This represents the first report of an organism that requires the Tat pathway for viability when grown under such conditions. Deletion of the nonessential gene had no effect on the secretion of a verified substrate of the Tat pathway. The two TatA paralogs TatAo and TatAt were detected in both the membrane and cytoplasm and could be copurified from the latter fraction. Using size exclusion chromatography to further characterize cytoplasmic and membrane TatA proteins, we find these proteins present in high-molecular-weight complexes in both cellular fractions.     

Got Milk? Breastfeeding and Milk Analysis of a Mother on Chronic Hemodialysis

Purpose

Women on dialysis rarely become pregnant. However, the overall rate of successful pregnancies is increasing in this patient population and breastfeeding becomes an option for mothers on dialysis. In this study we performed a systematic breast milk composition analysis of a mother on chronic hemodialysis (HD).

Methods

Specimens of breast milk and blood were collected in regular intervals before and after HD from a 39-year old woman starting on day 10 postpartum. Samples were analyzed for electrolytes, retention solutes, nutrients and other laboratory measurements. Breast milk samples from low-risk mothers matched for postpartum age were used as controls.

Results

Significantly higher levels of creatinine and urea were found in pre-HD breast milk when compared to post-HD. A similar post-dialytic decrease was only found for uric acid but not for any other investigated parameter. Conversely, sodium and chloride were significantly increased in post-HD samples. Compared to controls creatinine and urea were significantly higher in pre-HD samples while the difference remained only significant for post-HD creatinine. Phosphate was significantly lower in pre- and post-HD breast milk when compared to controls, whereas calcium showed no significant differences. In terms of nutrient components glucose levels showed a strong trend for a decrease, whereas protein, triglycerides and cholesterol did not differ. Similarly, no significant differences were found in iron, potassium and magnesium content.

Conclusion

To the best of our knowledge this is the first report on a breastfeeding mother on chronic dialysis. Although we found differences in creatinine, urea, sodium, chloride and phosphate, our general analysis showed high similarity of our patient’s breast milk to samples from low-risk control mothers. Significant variations in breast milk composition between pre- and post-HD samples suggest that breastfeeding might be preferably performed after dialysis treatment. In summary, our findings indicate that breastfeeding can be considered a viable option for newborns of mothers on dialysis.     

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.     

Conservation of amino acid transporters in fungi, plants and animals

When comparing the transporters of three completely sequenced eukaryotic genomes--Saccharomyces cerevisiae, Arabidopsis thaliana and Homo sapiens--transporter types can be distinguished according to phylogeny, substrate spectrum, transport mechanism and cell specificity. The known amino acid transporters belong to five different superfamilies. Two preferentially Na(+)-coupled transporter superfamilies are not represented in the yeast and Arabidopsis genomes, whereas the other three groups, which often function as H(+)-coupled systems, have members in all investigated genomes. Additional superfamilies exist for organellar transport, including mitochondrial and plastidic carriers. When used in combination with phylogenetic analyses, functional comparison might aid our prediction of physiological functions for related but uncharacterized open reading frames.     

The response of macroinvertebrate community taxa and functional groups to pollution along a heavily impacted river in Central Europe (Bílina River,Czech Republic)

Macroinvertebrate communities were investigated along a gradient of heavy industrial and municipal pollution in the highland Bílina River (Czech Republic). Physico-chemical determinants and ions were monitored and community analysis performed focusing on taxonomic composition, ecological functioning (feeder and dweller guilds) and water quality metrics, including saprobity index, BMWP and diversity. Impacted sites differed significantly from reference and from recovered stretches. Chemical data revealed two main pollution factors, (1) a “salinity determinant”, described best by conductivity and SO₄²⁻, and (2) an “organic pollution determinant”, represented best by O₂ concentrations and NO₂⁻, all varying locally and temporally. Some metrics and taxa showed significant correlations to abiotic parameters. Functional communities showed a stronger relationship to the “organic pollution determinant”, suggesting that elevated organic pollution had a dominating influence on functional community metrics; though other variables may also have an influence in this multistress environment. On the other hand, there were indications that the taxonomic community was more influenced by ion concentrations (“salinity determinant”). The gradient from reference sites to polluted sites was weaker in the final sampling campaign. The results presented here can be used as a reference for assessing future changes in environmental impact from pollution, being finer and more detailed than assessment according to the EU’s WFD.     

Dynamics in space use of American mink (Neovison vison) in a fishpond area in Northern Germany

Telemetry of American mink Neovison vison revealed seasonal and sexual variations in home-range size, intersexual- and partially intrasexual range overlaps and spatial dynamic of territorial system. From 2003 to 2006, 14 mink (nine males and five females) were radio-tracked in a fishpond area in northern Germany. We observed comparatively large home ranges. Both sexes reduced their large summer home ranges to smaller ones in the winter period. In both seasons, male mink used significantly larger areas than female mink. During the mating season, males considerably enlarged their home ranges and roamed nearly through the entire study area. These variations and diversity of range size were discussed in connection with thermoregulatory behaviour, prey availability and distribution, population density and stability of the social system. Analysing intraspecific static interactions, we observed high intersexual overlapping rates in both the summer and winter periods. But there were no or rather very low levels of intrasexual overlapping. However, in the summer period, large home ranges of neighbouring males also overlapped to some extent, but temporal avoidance of adjacent territory holders was observed. Consequently, our results reveal intrasexual territoriality, with males having larger territories, overlapping those of females. The shifting of stable temporary home ranges observed within one season or between the same seasons of consecutive years demonstrates the highly dynamic nature of spatial behaviour among American mink. All these recorded characteristic features of spatial behaviour should be considered when planning mink control projects and measures for the management of this invasive carnivore.     

The actin binding protein, fesselin, is a member of the synaptopodin family

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and α-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.     

Annual and seasonal space use of different age classes of female wild boar Sus scrofa L.

In a radiotelemetric study, we analysed space use of 24 female specimens (14 family groups and 14 nonreproductive yearling females) out of 23 wild boar groups for periods between 3 and 39 months. Generally, wild boar used relatively small areas, showed high site fidelity but also a strong individual variation of home ranges, indicating a high flexibility in space use. Although age-specific differences were not statistically significant, female yearlings tended to have larger mean annual home ranges (1,185 ha MCP) than animals ranging in family groups (771 ha). Yearlings also showed a stronger shifting from spring to summer home ranges (2,345 m) and a tendency towards larger home range sizes in summer (791 ha MCP), compared to family groups (shift 1,766 m, MCP 461 ha). Yearlings displayed a dislocation of about 1 km of the annual centre in the first year after dividing from the mother. In contrast, in adults older than 2 years, the dislocation of the annual center was only 240 m.     

In vitro characterization of native mammalian smooth-muscle protein synaptopodin 2

An analysis of the primary structure of the actin-binding protein fesselin revealed it to be the avian homologue of mammalian synaptopodin 2 [Schroeter, Beall, Heid, and Chalovich (2008) Biochem. Biophys. Res. Commun. 371, 582-586]. We isolated two synaptopodin 2 isoforms from rabbit stomach that corresponded to known types of human synaptopodin 2. The purification scheme used was that developed for avian fesselin. These synaptopodin 2 forms shared several key functions with fesselin. Both avian fesselin and mammalian synaptopodin 2 bound to Ca(2+)-calmodulin, alpha-actinin and smooth-muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca(2+)-calmodulin-dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of alpha-actinin, to polymerize actin in a Ca(2+)-calmodulin-dependent manner, or to bind to Ca(2+)-calmodulin or myosin. These properties are consistent with the proposed function of synaptopodin 2 in organizing the cytoskeleton.