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.

     

The Sphagnum microbiome supports bog ecosystem functioning under extreme conditions

Sphagnum‐dominated bogs represent a unique yet widely distributed type of terrestrial ecosystem and strongly contribute to global biosphere functioning. Sphagnum is colonized by highly diverse microbial communities, but less is known about their function. We identified a high functional diversity within the Sphagnum microbiome applying an Illumina‐based metagenomic approach followed by de novo assembly and MG‐RAST annotation. An interenvironmental comparison revealed that the Sphagnum microbiome harbours specific genetic features that distinguish it significantly from microbiomes of higher plants and peat soils. The differential traits especially support ecosystem functioning by a symbiotic lifestyle under poikilohydric and ombrotrophic conditions. To realise a plasticity–stability balance, we found abundant subsystems responsible to cope with oxidative and drought stresses, to exchange (mobile) genetic elements, and genes that encode for resistance to detrimental environmental factors, repair and self‐controlling mechanisms. Multiple microbe–microbe and plant–microbe interactions were also found to play a crucial role as indicated by diverse genes necessary for biofilm formation, interaction via quorum sensing and nutrient exchange. A high proportion of genes involved in nitrogen cycle and recycling of organic material supported the role of bacteria for nutrient supply. 16S rDNA analysis indicated a higher structural diversity than that which had been previously detected using PCR‐dependent techniques. Altogether, the diverse Sphagnum microbiome has the ability to support the life of the host plant and the entire ecosystem under changing environmental conditions. Beyond this, the moss microbiome presents a promising bio‐resource for environmental biotechnology – with respect to novel enzymes or stress‐protecting bacteria.     

Accumulation of germanium (Ge) in plant tissues of grasses is not solely driven by its incorporation in phytoliths

Ge/Si ratios of plant phytoliths have been widely used to trace biogeochemical cycling of Si. However, until recently, information on how much of the Ge and Si transferred from soil to plants is actually stored in phytoliths was lacking. The aim of the present study is to (i) compare the uptake of Si and Ge in three grass species, (ii) localize Ge and Si stored in above-ground plant parts and (iii) evaluate the amounts of Ge and Si sequestrated in phytoliths and plant tissues. Mays (Zea mays), oat (Avena sativa) and reed canary grass (Phalaris arundinacea) were cultivated in the greenhouse on soil and sand to control element supply. Leaf phytoliths were extracted by dry ashing. Total elemental composition of leaves, phytoliths, stems and roots were measured by ICP-MS. For the localization of phytoliths and the determination of Ge and Si within leaf tissues and phytoliths scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy (EDX) and laser ablation inductively coupled mass spectrometry (LA-ICP-MS) was used. The amounts of Si and Ge taken up by the species corresponded with biomass formation and decreased in the order Z. mays > P. arundinacea, A. sativa. Results from LA-ICP-MS revealed that Si was mostly localized in phytoliths, while Ge was disorderly distributed within the leaf tissue. In fact, from the total amounts of Ge accumulated in leaves only 10% was present in phytoliths highlighting the role of organic matter on biogeochemical cycling of Ge and the necessity for using bulk Ge/Si instead of Ge/Si in phytoliths to trace biogeochemical cycling of Si.

     

Lipid Sorting and Multivesicular Endosome Biogenesis

Intracellular organelles, including endosomes, show differences not only in protein but also in lipid composition. It is becoming clear from the work of many laboratories that the mechanisms necessary to achieve such lipid segregation can operate at very different levels, including the membrane biophysical properties, the interactions with other lipids and proteins, and the turnover rates or distribution of metabolic enzymes. In turn, lipids can directly influence the organelle membrane properties by changing biophysical parameters and by recruiting partner effector proteins involved in protein sorting and membrane dynamics. In this review, we will discuss how lipids are sorted in endosomal membranes and how they impact on endosome functions.It is now well established that membranes along the endocytic and secretory pathway show differences not only in protein but also in lipid composition. For example, lipid gradients exist along the biosynthetic pathway with increasing density of cholesterol and sphingolipids from the endoplasmic reticulum (ER) to the plasma membrane (Maxfield and van Meer 2010). Also, phosphoinositides show distributions restricted to relatively well-characterized membrane territories (Di Paolo and De Camilli 2006). Given the facts that lipids are small and contain little structural information when compared with proteins, that they can diffuse rapidly within membranes, and that membranes are connected by membrane flow during transport, it is not always obvious how different lipids are segregated from each other.In this article, we will evoke different mechanisms that may contribute to the heterogeneous lipid composition of endocytic membranes, including physicochemical properties of the membrane, interactions with other proteins or lipids, and synthesis or degradation. In addition, it has also become apparent that peripheral membrane proteins often interact with membranes via diverse lipid-binding motifs, and thus that lipids directly contribute to the distribution of many peripheral membrane proteins. For example, phosphatidylinositol 3-phosphate (PI(3)P) is detected predominantly on early endosomes, where most characterized PI(3)P-binding proteins encoded by the human genome are found as well (Raiborg et al. 2013). We will also discuss how some lipids may regulate protein sorting and membrane transport within the endosomal system.     

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti

Inward-rectifying K⁺ (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K⁺ currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na⁺. Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl⁺ > K⁺ > Rb⁺ > NH₄⁺) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K⁺.     

Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod (Gadus morhua) macrophages

The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1β and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.     

A genetic screen identifies BRCA2 and PALB2 as key regulators of G2 checkpoint maintenance

To identify key connections between DNA-damage repair and checkpoint pathways, we performed RNA interference screens for regulators of the ionizing radiation-induced G2 checkpoint, and we identified the breast cancer gene BRCA2. The checkpoint was also abrogated following depletion of PALB2, an interaction partner of BRCA2. BRCA2 and PALB2 depletion led to premature checkpoint abrogation and earlier activation of the AURORA A-PLK1 checkpoint-recovery pathway. These results indicate that the breast cancer tumour suppressors and homologous recombination repair proteins BRCA2 and PALB2 are main regulators of G2 checkpoint maintenance following DNA-damage.