Mechanosensitive membrane channels in action

The tension-driven gating process of MscL from Mycobacterium tuberculosis, Tb-MscL, has been addressed at near-atomic detail using coarse-grained molecular dynamics simulations. To perform the simulations, a novel coarse-grained peptide model based on a thermodynamic parameterization of the amino-acid side chains has been applied. Both the wild-type Tb-MscL and its gain-of-function mutant V21D embedded in a solvated lipid bilayer have been studied. To mimic hypoosmotic shock conditions, simulations were performed at increasing levels of membrane tension approaching the rupture threshold of the lipid bilayer. Both the wild-type and the mutant channel are found to undergo significant conformational changes in accordance with an irislike expansion mechanism, reaching a conducting state on a microsecond timescale. The most pronounced expansion of the pore has been observed for the V21D mutant, which is consistent with the experimentally shown gain-of-function phenotype of the V21D mutant.     

Influence of Humic Substances on Bacterial and Viral Dynamics in Freshwaters

Bacterial and viral abundances were measured in 24 lakes with dissolved organic carbon (DOC) concentrations ranging from 3 to 19 mg of C liter⁻¹. In addition, a laboratory experiment was performed to test the effects of different sources of carbon (i.e., glucose and fulvic acids) and nutrients on the dynamics of viruses and bacteria. In the lake survey, no correlation was found between virus abundance and DOC concentration, yet there was a significant positive correlation between bacterial abundance and DOC concentration. A negative correlation was found between the virus-to-bacteria ratio and DOC level. These results are in agreement with our findings in the laboratory, where virus counts were significantly lower in treatments with fulvic acid additions than in a control (mean, 67.4% ± 6.5% of the control). Virus counts did not differ significantly among the control and treatments with glucose, indicating that it was the type of organic carbon and not quantity which had an impact on viruses. Results from this study suggest that the way viruses control bacterial assemblages in humic lakes is different from the mechanism in clear water systems.     

SH2-Bbeta is a Rac-binding protein that regulates cell motility.

The Src homology 2 (SH2) domain-containing protein SH2-Bbeta binds to and is a substrate of the growth hormone (GH) and cytokine receptor-associated tyrosine kinase JAK2. SH2-Bbeta also binds, via its SH2 domain, to multiple activated growth factor receptor tyrosine kinases. We have previously implicated SH2-Bbeta in GH and platelet-derived growth factor regulation of the actin cytoskeleton. We extend these findings by establishing a potentiating effect of SH2-Bbeta on GH-dependent cell motility and defining regions of SH2-Bbeta required for this potentiation. Time-lapse video microscopy, phagokinetic, and/or wounding assays demonstrate reduced movement of cells overexpressing SH2-Bbeta lacking an intact SH2 domain because of a point mutation or a C-terminal truncation. An N-terminal proline-rich domain (amino acids 85-106) of SH2-Bbeta is required for inhibition of cellular motility by SH2 domain-deficient mutants. Co-immunoprecipitation experiments indicate that Rac binds to this domain. GH is shown to activate endogenous Rac, and dominant negative mutants of SH2-Bbeta are shown to inhibit membrane ruffling induced by constitutively active Rac. These findings suggest that SH2-Bbeta is an adapter protein that facilitates actin rearrangement and cellular motility by recruiting Rac and potentially Rac-regulating, Rac effector, or other actin-regulating proteins to activated cytokine (e.g. GH) and growth factor receptors.     

SH2-B family members differentially regulate JAK family tyrosine kinases.

Activation of JAK tyrosine kinases is an essential step in cell signaling by multiple hormones, cytokines, and growth factors, including growth hormone (GH) and interferon-gamma. Previously, we identified SH2-B beta as a potent activator of JAK2 (Rui, L., and Carter-Su, C. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 7172-7177). Here, we investigated whether the activation of JAK2 by SH2-B beta is specific to JAK2 and SH2-B beta or extends to other JAKs or other members of the SH2-B beta family. When SH2-B beta was overexpressed with JAK1 or JAK3, SH2-B beta failed to increase their activity. However, SH2-B beta bound to both and was tyrosyl-phosphorylated by JAK1. In contrast to SH2-B beta, APS decreased tyrosyl phosphorylation of GH-stimulated JAK2 as well as Stat5B, a substrate of JAK2. APS also decreased tyrosyl phosphorylation of JAK1, but did not affect the activity or tyrosyl phosphorylation of JAK3. Overexpressed APS bound to and was tyrosyl-phosphorylated by all three JAKs. Consistent with these data, in 3T3-F442A adipocytes, endogenous APS was tyrosyl-phosphorylated in response to GH and interferon-gamma. These results suggest that 1) SH2-B beta specifically activates JAK2, 2) APS negatively regulates both JAK2 and JAK1, and 3) both SH2-B beta and APS may serve as adapter proteins for all three JAKs independent of any role they have in JAK activity.     

Association between random glucose and all-cause mortality: findings from the mortality follow-up of the German National Health Interview and Examination Survey 1998

Background

Random glucose is widely measured in epidemiological studies and in the clinical setting when standardized fasting protocols and oral glucose tolerance testing or HbA_1c measuring are not feasible. The relationship between random glucose and all-cause mortality has hardly been studied so far and was examined in the present study.

Methods

We ascertained mortality status among 5955 persons aged 18–79?years and free of known diabetes when participating in the German National Health Interview and Examination Survey 1998 (mean observation time 11.7?years, 458 deaths). Cox regression was applied to analyze the association of random serum glucose with all-cause mortality taken potential confounders into account. Relative mortality risks were estimated as hazard ratios (HRs) with 95% confidence intervals (CIs) modeling random glucose as categorical or continuous variable.

Results

Compared to random glucose levels of 4.3 -?<?5.3?mmol/L, HRs (95% CIs) were 1.94 (0.85–4.45) for levels <?4.3?mmol/L and 1.16 (0.89–1.50), 1.20 (0.91–1.58), 1.42 (0.88–2.29), 2.02 (1.26–3.25) and 4.71 (2.20–10.10) for levels 5.3 -?<?5.8, 5.8 -?<?6.8, 6.8 -?<?7.8, 7.8 -?<?11.1 and?≥?11.1?mmol/L, adjusted for age, sex, lifestyle, anthropometry and chronic diseases. An additional adjustment for fasting time or HbA_1c yielded similar estimates. Modeling continuous random glucose by restricted cubic spline functions revealed comparable findings.

Conclusions

In the present epidemiological study drawn from the general population, random glucose showed a significant association with all-cause mortality, independent of main potential confounders. Thus, random glucose measures are highly relevant to health risk assessment among people without known diabetes when fasting glucose or HbA_1c are difficult to obtain.

     

Sulfoximines as potent RORγ inverse agonists

Progress in the identification of suitable RORγ inverse agonists as clinical candidates has been hampered by the high lipophilicity that seems required for high potency on this nuclear receptor. In this context, we decided to focus on the replacement of the hydroxymethyl group found on known modulators to determine if more polarity could be tolerated in this position. SAR of the replacement of this moiety is presented in this article leading to the identification of sulfoximine derivatives as potent modulators with pharmacological activity in the in vivo mouse Imiquimod psoriasis model.     

Evolutionary implications of C3–C4 intermediates in the grass Alloteropsis semialata

C₄ photosynthesis is a complex trait resulting from a series of anatomical and biochemical modifications to the ancestral C₃ pathway. It is thought to evolve in a stepwise manner, creating intermediates with different combinations of C₄‐like components. Determining the adaptive value of these components is key to understanding how C₄ photosynthesis can gradually assemble through natural selection. Here, we decompose the photosynthetic phenotypes of numerous individuals of the grass Alloteropsis semialata, the only species known to include both C₃ and C₄ genotypes. Analyses of δ¹³C, physiology and leaf anatomy demonstrate for the first time the existence of physiological C₃–C₄ intermediate individuals in the species. Based on previous phylogenetic analyses, the C₃–C₄ individuals are not hybrids between the C₃ and C₄ genotypes analysed, but instead belong to a distinct genetic lineage, and might have given rise to C₄ descendants. C₃ A. semialata, present in colder climates, likely represents a reversal from a C₃–C₄ intermediate state, indicating that, unlike C₄ photosynthesis, evolution of the C₃–C₄ phenotype is not irreversible.     

Impact of Plant Species and Site on Rhizosphere-Associated Fungi Antagonistic to Verticillium dahliae Kleb.

Fungi with antagonistic activity toward plant pathogens play an essential role in plant growth and health. To analyze the effects of the plant species and the site on the abundance and composition of fungi with antagonistic activity toward Verticillium dahliae, fungi were isolated from oilseed rape and strawberry rhizosphere and bulk soil from three different locations in Germany over two growing seasons. A total of 4,320 microfungi screened for in vitro antagonism toward Verticillium resulted in 911 active isolates. This high proportion of fungi antagonistic toward the pathogen V. dahliae was found for bulk and rhizosphere soil at all sites. A plant- and site-dependent specificity of the composition of antagonistic morphotypes and their genotypic diversity was found. The strawberry rhizosphere was characterized by preferential occurrence of Penicillium and Paecilomyces isolates and low numbers of morphotypes (n = 31) and species (n = 13), while Monographella isolates were most frequently obtained from the rhizosphere of oilseed rape, for which higher numbers of morphotypes (n = 41) and species (n = 17) were found. Trichoderma strains displayed high diversity in all soils, but a high degree of plant specificity was shown by BOX-PCR fingerprints. The diversity of rhizosphere-associated antagonists was lower than that of antagonists in bulk soil, suggesting that some fungi were specifically enriched in each rhizosphere. A broad spectrum of new Verticillium antagonists was identified, and the implications of the data for biocontrol applications are discussed.     

In vitro budding of intralumenal vesicles into late endosomes is regulated by Alix and Tsg101

Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively.