Membrane integration of poliovirus 2B viroporin

Virus infections can result in a variety of cellular injuries, and these often involve the permeabilization of host membranes by viral proteins of the viroporin family. Prototypical viroporin 2B is responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. 2B protein can be localized at the endoplasmic reticulum (ER) and the Golgi complex, inducing membrane remodeling and the blockade of glycoprotein trafficking. These findings suggest that 2B has the potential to integrate into the ER membrane, but specific information regarding its biogenesis and mechanism of membrane insertion is lacking. Here, we report experimental results of in vitro translation-glycosylation compatible with the translocon-mediated insertion of the 2B product into the ER membrane as a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. A similar topology was found when 2B was synthesized in cultured cells. In addition, the in vitro translation of several truncated versions of the 2B protein suggests that the two hydrophobic regions cooperate to insert into the ER-derived microsomal membranes.     

Comparison and evaluation of the Binax EIA and Biotest EIA urinary antigen kits for detection of Legionella pneumophila antigen in urine samples

The Binax and the Biotest urinary antigen kits for detection of L. pneumophila antigen were compared by testing of selected 67 urine samples obtained from EWGLI as reference samples in External Quality Assessment Scheme. Thirty nine were positive with the Binax kit (100% of sensitivity), and 33 were positive with the Biotest (84.6% of sensitivity). The test specificities were 100% for the both kits. It was concluded that the Binax kit was more suitable for the routine diagnosis of Legionella infections than the Biotest kit.     

Identification of novel Ssl0352 protein (NdhS), essential for efficient operation of cyclic electron transport around photosystem I, in NADPH:plastoquinone oxidoreductase (NDH-1) complexes of Synechocystis sp. PCC 6803

Cyanobacterial NADPH:plastoquinone oxidoreductase, or type I NAD(P)H dehydrogenase, or the NDH-1 complex is involved in plastoquinone reduction and cyclic electron transfer (CET) around photosystem I. CET, in turn, produces extra ATP for cell metabolism particularly under stressful conditions. Despite significant achievements in the study of cyanobacterial NDH-1 complexes during the past few years, the entire subunit composition still remains elusive. To identify missing subunits, we screened a transposon-tagged library of Synechocystis 6803 cells grown under high light. Two NDH-1-mediated CET (NDH-CET)-defective mutants were tagged in the same ssl0352 gene encoding a short unknown protein. To clarify the function of Ssl0352, the ssl0352 deletion mutant and another mutant with Ssl0352 fused to yellow fluorescent protein (YFP) and the His(6) tag were constructed. Immunoblotting, mass spectrometry, and confocal microscopy analyses revealed that the Ssl0352 protein resides in the thylakoid membrane and associates with the NDH-1L and NDH-1M complexes. We conclude that Ssl0352 is a novel subunit of cyanobacterial NDH-1 complexes and designate it NdhS. Deletion of the ssl0352 gene considerably impaired the NDH-CET activity and also retarded cell growth under high light conditions, indicating that NdhS is essential for efficient operation of NDH-CET. However, the assembly of the NDH-1L and NDH-1M complexes and their content in the cells were not affected in the mutant. NdhS contains a Src homology 3-like domain and might be involved in interaction of the NDH-1 complex with an electron donor.     

Derivatives of rhodamine 19 as mild mitochondria-targeted cationic uncouplers

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.     

Metabolomic linkage reveals functional interaction between glucose-dependent insulinotropic polypeptide and ghrelin in humans

The gastric peptide ghrelin promotes energy storage, appetite, and food intake. Nutrient intake strongly suppresses circulating ghrelin via molecular mechanisms possibly involving insulin and gastrointestinal hormones. On the basis of the growing evidence that glucose-dependent insulinotropic polypeptide (GIP) is involved in the control of fuel metabolism, we hypothesized that GIP and/or insulin, directly or via changes in plasma metabolites, might affect circulating ghrelin. Fourteen obese subjects were infused with GIP (2.0 pmol·kg(-1)·min(-1)) or placebo in the fasting state during either euglycemic hyperinsulinemic (EC) or hyperglycemic hyperinsulinemic clamps (HC). Apart from analysis of plasma ghrelin and insulin levels, GC-TOF/MS analysis was applied to create a hormone-metabolite network for each experiment. The GIP and insulin effects on circulating ghrelin were analyzed within the framework of those networks. In the HC, ghrelin levels decreased in the absence (19.2% vs. baseline, P = 0.028) as well as in the presence of GIP (33.8%, P = 0.018). Ghrelin levels were significantly lower during HC with GIP than with placebo, despite insulin levels not differing significantly. In the GIP network combining data on GIP-infusion, EC+GIP and HC+GIP experiments, ghrelin was integrated into hormone-metabolite networks through a connection to a group of long-chain fatty acids. In contrast, ghrelin was excluded from the network of experiments without GIP. GIP decreased circulating ghrelin and might have affected the ghrelin system via modification of long-chain fatty acid pools. These observations were independent of insulin and offer potential mechanistic underpinnings for the involvement of GIP in systemic control of energy metabolism.     

Seasonal abundance and feeding patterns of copepods Temora longicornis, Centropages hamatus and Acartia spp. in the White Sea (66��N)

We have studied the seasonal dynamics of abundance and feeding characteristics of three species of calanoid copepods (Acartia spp., Centropages hamatus and Temora longicornis) in the White Sea from the surface water layer (0–10 m), in order to assess their role in the pelagic food web and to determine the major factors governing their population dynamics during the productive season. These species dominated in the upper water layer (0–10 m) from June through September, producing up to 3 generations per year. Data on the food spectra revealed all species to be omnivorous; but some inter- and intraspecific differences were observed. Generally, copepods consumed diatoms, dinoflagellates and microzooplankton. The omnivory index ‘UC’ (i.e., fatty acid unsaturation coefficient) varied from 0.2 to 0.6, which implied ingestion of phytoplankton. The different degree of selectivity on the same food items by the studied species was observed, and therefore, successful surviving strategy with minimal overlapping could be assumed. In total, the populations of the three studied copepod species grazed up to 2.15 g C m⁻² day⁻¹ and released up to 0.68 g C m⁻² day⁻¹ in faecal pellets. They consumed up to 50% of particulate organic carbon, or up to 85% of phytoplankton standing stock (in terms of Chl. a), and thus played a significant role in the transformation of particulate organic matter. Seasonal changes in abundance of the studied species depended mostly on water temperature in the early summer, but were also affected by food availability (Chl. a concentration) during the productive season.     

Lipopolysaccharide needs soluble CD14 to interact with TLR4 in human monocytes depleted of membrane CD14

Toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. TLR4 recognizes lipopolysaccharide (LPS) in monocytes/macrophages with the help of other molecules like CD14 and MD-2, which indicates that the functional LPS receptor forms a large complex. The functional relationship between the components has been the subject of debate, as have the modifications induced by the ligand in the expression of some of these components. Moreover, as for other members of this family of receptors, the possible direct interaction of receptors and their ligands is a matter of discussion. In this paper we address the question of whether the expression of some of the components influences the expression of the rest. Human monocytes in which CD14 has been downregulated through interference in the turnover of the molecule at the Golgi level, show normal membrane TLR4 expression, when compared with control cells. On the other hand, LPS alters membrane TLR4 expression by monocytes devoid of membrane CD14 only in the presence of human serum. The effect of serum is blocked by anti-CD14 monoclonal antibodies, which strongly suggests a functional role for soluble CD14/LPS complexes in the interaction with TLR4. Our data add information on the relationship between the components of the LPS receptor and the characteristics of the interaction of LPS and TLR4 in cells devoid of membrane CD14.