Rab3 Proteins Involved in Vesicle Biogenesis and Priming in Embryonic Mouse Chromaffin Cells

The four Rab3 paralogs A–D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) (rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD^?/?) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD^?/? animals large dense‐core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD^?/? cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short‐term (4–6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state.     

Seasonal population dynamics and trophic role of planktonic nanoflagellates in coastal surface waters of the Southern Baltic Sea

We investigated the temporal dynamics and trophic role of different nanoflagellates in surface waters of the Gulf of Gdańsk (Baltic Sea) between April and October 2007. Two 18S rRNA gene clone libraries were constructed from samples collected in spring and summer, and weekly changes in the abundances of five phylogenetic groups were studied by fluorescence in situ hybridization with newly designed probes. Stramenopiles affiliated with MAST‐6 and Pedinellales were most numerous in spring but rare in summer. Both groups formed short‐lived blooms during a sudden drop of salinity due to riverine influx (from 7.1 to 6.2 practical salinity units). The analysis of food vacuole content suggested that MAST‐6 nanoflagellates were herbivorous, whereas bacterivory was found both in plastidic and aplastidic pedinellid populations. Members of an uncultured lineage of aplastidic, bacterivorous cercozoans distantly related to Ebria tripartita were more abundant in summer when water temperatures exceeded 17°C. Multicellular trophonts and/or free‐living single cell stages of two lineages of Group 1 parasitic Syndiniales (alveolates) were present in spring and early summer. One of these alveolate populations repeatedly peaked before and after the freshwater influx, but was conspicuously absent throughout the period of decreased salinity. Our results indicate that nanoflagellate populations in coastal surface waters may form short‐lived blooms that can only be detected by high‐frequency sampling, and that may be related both to seasonal development and to sporadic (e.g. mixing) events. In view of their trophic diversity we moreover suggest that nanoflagellates in eutrophic coastal waters should not be regarded as a single functional unit.     

Leucine‐to‐carbon empirical conversion factor experiments: does bacterial community structure have an influence?

The suitability of applying empirical conversion factors (eCFs) to determine bacterial biomass production remains unclear because seawater cultures are usually overtaken by phylotypes that are not abundant in situ. While eCFs vary across environments, it has not been tested whether differences in eCFs are driven by changes in bacterial community composition or by in situ environmental conditions. We carried out seawater cultures throughout a year to analyse the correlation between eCFs and bacterial community structure, analysed by catalysed reporter deposition fluorescence in situ hybridization. Gammaproteobacteria usually dominated seawater cultures, but their abundance exhibited a wide range (25–73% of cell counts) and significantly increased with inorganic nutrient enrichment. Flavobacteria were less abundant but increased up to 40% of cells counts in winter seawater cultures, when in situ chlorophyll a was high. The correlations between eCFs and the abundance of the main broad phylogenetic groups (Gamma‐, Alphaproteobacteria and Flavobacteria) were significant, albeit weak, while more specific groups (Alteromonadaceae and Rhodobacteraceae) were not significantly correlated. Our results show that the frequent development of the fast‐growing group Alteromonadaceae in seawater cultures does not strongly drive the observed variations in eCFs. Rather, the results imply that environmental conditions and the growth of specific phylotypes interact to determine eCFs.     

Exopolysaccharide Synthesized by Lactobacillus reuteri Decreases the Ability of Enterotoxigenic Escherichia coli To Bind to Porcine Erythrocytes

This study investigated the therapeutic potential of bacterial polysaccharides by employing a model system based on enteroxigenic Escherichia coli (ETEC)-induced hemagglutination of erythrocytes. Exopolysaccharides produced by strains of Lactobacillus reuteri inhibited ETEC-induced hemagglutination of porcine erythrocytes. No effect was observed for dextran produced from Weissella cibaria and commercially available oligo- and polysaccharides.Lactic acid bacteria (LAB) are important in food production due to their positive contribution to flavor and preservation of the final product. Some of these food-grade microorganisms are also valuable for their ability to synthesize exopolysaccharides (EPSs). EPSs are high-molecular-weight sugar polymers, which remain attached to the microorganism as capsular EPS or become excreted into the environment in the form of slime or ropy EPS (26, 30). LAB utilize two distinct biosynthetic pathways to produce EPSs. Heteropolysaccharides (HePSs) comprised of two to eight repeating units of monosaccharides are assembled by cell wall-bound glycosyltransferases in low quantities from intracellular sugar nucleotide precursors (4). Extracellular glycansucrases (glucan- or fructansucrases) synthesize homopolysaccharides (HoPSs) consisting of either glucose or fructose from sucrose, and their yield can be as high as 40 g liter⁻¹ (13, 19). EPS formation by glycansucrases has been reported for lactobacilli of the species Lactobacillus reuteri, L. pontis, L. panis, L. acidophilus, and L. frumenti (28, 29). HoPS synthesis and the corresponding genes have been especially well characterized in L. reuteri (27). All EPSs used in this study were HoPSs formed in the presence of sucrose. In addition to HoPS synthesis, glycansucrases are capable of producing oligosaccharides (OSs). Several OSs are found to have prebiotic properties as they can benefit host health by acting as nondigestible food ingredients and by enhancing the growth of desired microbial members of the gastrointestinal microbiota (7). Emerging research efforts have investigated potential applications of OSs as antiadhesive agents in preventing pathogen colonization. Shoaf et al. (24) suggested that commercial oligo- and polysaccharides reduce the adherence of enteropathogenic Escherichia coli (EPEC) to cell lines. Martín-Sosa et al. (16) reported antiadhesive properties of human milk oligosaccharides against human strains of enterotoxigenic Escherichia coli (ETEC) and uropathogenic E. coli. Similarly, Martín et al. (15) confirmed the binding of milk oligosaccharides to ETEC isolated from calves. In contrast, adhesion studies with EPS are limited.The inhibition of ETEC is of special interest to the swine industry because ETEC is the primary cause for diarrhea in neo- and postnatal piglets and results in substantial economic losses. We therefore aimed to test for antiadhesive properties of EPSs synthesized by LAB and commercially available prebiotics against porcine ETEC strains. Hemagglutination assays were used; these assays are generally accepted as an effective model system for testing ETEC adherence as hemagglutination resembles the attachment of K88-positive bacteria to the gut wall (10).     

The association between leukocytes and sperm quality is concentration dependent

Background  

To evaluate the association between leukocytes (polymorphonuclear granulocytes -PMNL) and semen parameters at different leukocyte concentrations.     

Crystal structures of DPP-IV (CD26) from rat kidney exhibit flexible accommodation of peptidase-selective inhibitors

Dipeptidyl peptidase IV (DPP-IV) belongs to a family of serine peptidases, and due to its indirect regulatory role in plasma glucose modulation, DPP-IV has become an attractive pharmaceutical target for diabetes therapy. DPP-IV inactivates the glucagon-like peptide (GLP-1) and several other naturally produced bioactive peptides that contain preferentially a proline or alanine residue in the second amino acid sequence position by cleaving the N-terminal dipeptide. To elucidate the details of the active site for structure-based drug design, we crystallized a natural source preparation of DPP-IV isolated from rat kidney and determined its three-dimensional structure using X-ray diffraction techniques. With a high degree of similarity to structures of human DPP-IV, the active site architecture provides important details for the design of inhibitory compounds, and structures of inhibitor-protein complexes offer detailed insight into three-dimensional structure-activity relationships that include a conformational change of Tyr548. Such accommodation is exemplified by the response to chemical substitution on 2-cyanopyrrolidine inhibitors at the 5 position, which conveys inhibitory selectivity for DPP-IV over closely related homologues. A similar conformational change is also observed in the complex with an unrelated synthetic inhibitor containing a xanthine core that is also selective for DPP-IV. These results suggest the conformational flexibility of Tyr548 is unique among protein family members and may be utilized in drug design to achieve peptidase selectivity.     

Blooms of single bacterial species in a coastal lagoon of the southwestern Atlantic Ocean

We investigated seasonal differences in community structure and activity (leucine incorporation) of the planktonic bacterial assemblage in the freshwater and brackish-water zones of a shallow coastal lagoon of the southwestern Atlantic Ocean. Alphaproteobacteria formed the dominant microbial group in both zones throughout the sampling period. After an intrusion of marine water, members of the SAR11 lineage became abundant in the brackish-water zone. These bacteria were apparently distributed over the lagoon during the following months until they constituted almost 30% of all prokaryotic cells at both sampling sites. At the first sampling date (March 2003) a single alphaproteobacterial species unrelated to SAR11, Sphingomonas echinoides, dominated the microbial assemblages in both zones of the lagoon concomitantly with a bloom of filamentous cyanobacteria. Pronounced maxima of leucine incorporation were observed once in each zone of the lagoon. In the freshwater zone, this highly active microbial assemblage was a mix of the typical bacteria lineages expected in aquatic systems. By contrast, a single bacterial genotype with >99% similarity to the facultative pathogen gammaproteobacterial species Stenotrophomonas maltophilia formed >90% of the bacterial assemblage (>10(7) cell ml(-1)) in the brackish-water zone at the time point of highest bacterial leucine incorporation. Moreover, these bacteria were equally dominant, albeit less active, in the freshwater zone. Thus, the pelagic zone of the studied lagoon harbored repeated short-term blooms of single bacterial species. This finding may have consequences for environmental protection.     

The potential of a jumping spider, Phidippus clarus, as a biocontrol agent

Spiders, particularly assemblages of species, have been shown to be effective in reducing pest insects and crop damage in field crops and orchards. We investigated the potential for a single jumping spider species to reduce pests in a greenhouse setting. We placed three treatments in large enclosures: 1) control treatment of only sweet basil, Ocimum basilicum L.; 2) sweet basil and a phytophagous pest, fourlined plant bug, Poecilocapsus lineatus (F.) (Heteroptera: Miridae); and 3) sweet basil, fourlined plant bug, and jumping spider Phidippus clarus (Keyserling 1884). After 1 wk, jumping spiders reduced the number of plant bugs. Plants exposed to plant bugs alone were significantly shorter than either control plants or plants exposed to plant bugs and spiders. Chlorophyll concentration did not significantly differ across treatments. We discuss the feasibility of using P. clarus and similar salticids in biocontrol.     

Multimodal functional cardiac MRI in creatine kinase-deficient mice reveals subtle abnormalities in myocardial perfusion and mechanics

A decrease in the supply of ATP from the creatine kinase (CK) system is thought to contribute to the evolution of heart failure. However, previous studies on mice with a combined knockout of the mitochondrial and cytosolic CK (CK(-/-)) have not revealed overt left ventricular dysfunction. The aim of this study was to employ novel MRI techniques to measure maximal myocardial velocity (V(max)) and myocardial perfusion and thus determine whether abnormalities in the myocardial phenotype existed in CK(-/-) mice, both at baseline and 4 wk after myocardial infarction (MI). As a result, myocardial hypertrophy was seen in all CK(-/-) mice, but ejection fraction (EF) remained normal. V(max), however, was significantly reduced in the CK(-/-) mice [wild-type, 2.32 +/- 0.09 vs. CK(-/-), 1.43 +/- 0.16 cm/s, P < 0.05; and wild-type MI, 1.53 +/- 0.11 vs. CK(-/-) MI, 1.26 +/- 0.11 cm/s, P = not significant (NS), P < 0.05 vs. baseline]. Myocardial perfusion was also lower in the CK(-/-) mice (wild-type, 6.68 +/- 0.27 vs. CK(-/-), 4.12 +/- 0.63 ml/g.min, P < 0.05; and wild-type MI, 3.97 +/- 0.65 vs. CK(-/-) MI, 3.71 +/- 0.57 ml/g.min, P = NS, P < 0.05 vs. baseline), paralleled by a significantly reduced capillary density (histology). In conclusion, myocardial function in transgenic mice may appear normal when only gross indexes of performance such as EF are assessed. However, the use of a combination of novel MRI techniques to measure myocardial perfusion and mechanics allowed the abnormalities in the CK(-/-) phenotype to be detected. The myocardium in CK-deficient mice is characterized by reduced perfusion and reduced maximal contraction velocity, suggesting that the myocardial hypertrophy seen in these mice cannot fully compensate for the absence of the CK system.