Interleukin-4 inhibits caspase-3 by regulating several proteins in the Fas pathway during initial stages of human T helper 2 cell differentiation

Interleukin-4 (IL-4) is the main cytokine that polarizes activated na?ve CD4+ T cells in the T helper 2 (Th2) direction. IL-4 also regulates the subsequent stages of Th2 cell-mediated diseases, such as allergies. We conducted a proteomics study to identify IL-4-induced differences during the initial stages of T helper cell differentiation. Primary CD4+ T lymphocytes were isolated from human cord blood, activated through CD3 and CD28, and cultured in the presence or absence of IL-4. Soluble proteins were separated by two-dimensional electrophoresis and visualized by staining with autoradiography, which indicated that at least 20 proteins might be regulated by IL-4. From this minimum of 20 stained proteins, altogether 35 proteins were identified using tandem mass spectrometry. Interestingly the fragmented form of GDP dissociation inhibitor expressed in lymphocytes/Rho GDP dissociation inhibitor 2 (Ly-GDI), a known target of Caspase-3, was observed to be down-regulated in IL-4-treated cells. It was shown in further studies that IL-4 decreases Caspase-3 activity and cell death in these cells. Neutralizing Fas-Fas ligand interaction led to decreased Caspase-3 activity and lowered Ly-GDI fragmentation. We further characterized the effects of IL-4 on the expression of main regulators in the Fas-mediated pathway. We demonstrated that IL-4 decreases expression of Fas receptor and increases expression of Bid, Bcl-2, and Bcl-xL. Importantly IL-4 significantly up-regulated the short form of c-FLIP, although the levels of c-FLIP long were unaltered after IL-4 induction. Taken together, our results indicate that IL-4 inhibits caspase activity during the initial stages of human Th2 cell differentiation by regulating expression of several key players in the Fas-induced pathway.     

Generalist Life Cycle Aids Persistence of Alexandrium ostenfeldii (Dinophyceae) in Seasonal Coastal Habitats of the Baltic Sea

In seasonal environments, strong gradients of environmental parameters can shape life cycles of phytoplankton. Depending on the rate of environmental fluctuation, specialist or generalist strategies may be favored, potentially affecting life cycle transitions. The present study examined life cycle transitions of the toxin producing Baltic dinoflagellate Alexandrium ostenfeldii and their regulation by environmental factors (temperature and nutrients). This investigation aimed to determine whether genetic recombination of different strains is required for resting cyst formation and whether newly formed cysts are dormant. Field data (temperature and salinity) and sediment surface samples were collected from a site with recurrent blooms and germination and encystment experiments were conducted under controlled laboratory conditions. Results indicate a lack of seasonal germination pattern, set by an endogenous rhythm, as commonly found with other dinoflagellates from the Baltic Sea. Germination of quiescent cysts was triggered by temperatures exceeding 10°C and combined nutrient limitation of nitrogen and phosphorus or a drop in temperature from 16 to 10°C triggered encystment most efficiently. Genetic recombination was not mandatory for the formation of resting cysts, but supported higher numbers of resistant cysts and enhanced germination capacity after a resting period. Findings from this study confirm that A. ostenfeldii follows a generalist germination and cyst formation strategy, driven by strong seasonality, which may support its persistence and possibly expansion in marginal environments in the future, if higher temperatures facilitate a longer growth season.     

Biotin induces tetramerization of a recombinant monomeric avidin. A model for protein-protein interactions

Chicken avidin, a homotetramer that binds four molecules of biotin was converted to a monomeric form by successive mutations of interface residues to alanine. The major contribution to monomer formation was the mutation of two aspartic acid residues, which together account for ten hydrogen bonding interactions at the 1-4 interface. Mutation of these residues, together with the three hydrophobic residues at the 1-3 interface, led to stable monomer formation in the absence of biotin. Upon addition of biotin, the monomeric avidin reassociated to the tetramer, which exhibited properties similar to those of native avidin, with respect to biotin binding, thermostability, and protease resistance. To our knowledge, these unexpected results represent the first example of a small monovalent ligand that induces oligomerization of a monomeric protein. This study may suggest a biological role for low molecular weight ligands in inducing oligomerization and in maintaining the stability of multimeric protein assemblies.     

Mechanisms regulating adrenomedullin gene expression in the left ventricle: role of mechanical load

Xenin is a 25 amino acid peptide produced by specific endocrine cells of the duodenal mucosa. Xenin has multiple biological actions in the gastrointestinal tract. It modulates intestinal motility, affects exocrine pancreatic secretion, and gastric secretion of acid. In the present investigation, we studied plasma concentration of xenin in volunteers after modified sham feeding and after meals of different composition. Plasma xenin concentrations were determined by radioimmunoassay in unextracted plasmas and after acidic extraction using C-18 Sep-Pak chromatography and after neutral extraction using affinity filtration. Both extraction methods were followed by C 18 r.p. HPLC chromatography. Xenin plasma concentrations in unextracted and in extracted plasma rose significantly after modified sham feeding when the food was brought to the volunteers from another room immediately before sham feeding started. When the volunteers had the opportunity to observe the preparation of the meal, xenin plasma concentrations during fasting were high and no further rise was observed after sham feeding. Isocaloric feeding resulted in elevated xenin concentrations in unextracted plasma and after high-pressure liquid chromatography. The methods of extraction, acidic or neutral, did not affect the results. CONCLUSION: Cephalic factors, investigated by modified sham feeding, stimulate release of xenin into the circulation. Xenin may participate in the central nervous regulation of gastrointestinal function.     

Increased Visceral Adipose Tissue as a Potential Risk Factor in Patients with Embolic Stroke of Undetermined Source (ESUS)

Purpose

The etiology of an ischemic stroke remains undetermined in 20–35% of cases and many patients do not have any of the conventional risk factors. Increased visceral adipose tissue (VAT) is a suggested new risk factor for both carotid artery atherosclerosis (CAA) and atrial fibrillation (AF), but its role in the remaining stroke population is unknown. We assessed the amount of VAT in patients with embolic stroke of undetermined source (ESUS) after excluding major-risk cardioembolic sources, occlusive atherosclerosis, and lacunar stroke.

Methods

Altogether 58 patients (mean age 57.7±10.2 years, 44 men) with ischemic stroke of unknown etiology but without CAA, known AF or small vessel disease underwent computed tomography angiography and assessment of VAT. For comparison VAT values from three different reference populations were used. Conventional risk factors (smoking, hypertension, diabetes, increased total and LDL-cholesterol, decreased HDL-cholesterol) were also registered.

Results

Mean VAT area was significantly higher in stroke patients (205±103 cm² for men and 168±99 cm² for women) compared to all reference populations (P<0.01). 50% of male and 57% of female patients had an increased VAT area. In male patients, VAT was significantly higher despite similar body mass index (BMI). Increased VAT was more common than any of the conventional risk factors.

Conclusion

Increased VAT was found in over half of our patients with ESUS suggesting it may have a role in the pathogenesis of thromboembolism in this selected group of patients.     

Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP–CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein–protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP–CaM interaction, including a 3D model of the complex between full-length proteins.     

Characterization of oil and starch accumulation in tubers of Cyperus esculentus var. sativus (Cyperaceae): A novel model system to study oil reserves in nonseed tissues

? Premise of the study: Storage oil (triacylglycerol) accumulates in tissues such as the embryo and endosperm of seeds and the fruit mesocarp, but seldom in underground organs. As a rare exception, cultivated variants of yellow nutsedge (Cyperus esculentus) contain high amounts of both oil and starch in the mature tubers. ? Methods: Biochemical analyses and light and electron microscopy were used to study the accumulation patterns of storage nutrients in developing nutsedge tubers. ? Key results: During the initial phase of tuber development, the conducting rhizome tissue is transformed into a storage compartment, then massive storage reserves accumulate in the tuber. At the beginning of tuber development, a large sugar load coincided with the onset of starch accumulation. Oil accumulation started later, concomitant with a substantial drop in the sugar content. Initially, oil accumulated at a lower rate compared to starch, but the rate later increased; after 6 wk, oil made up 24% of tuber dry mass, while starch made up 32%. Protein concentration changed only a small amount throughout this development. Oil and starch accumulated in the same cells throughout the tubers in a sequential fashion during tuber development. ? Conclusions: The developmental pattern in the build up of storage nutrients in the tubers highlights nutsedge as a novel model plant, having potential to significantly widen our understanding on how synthesis of storage reserves, and in particular oils, is regulated and directed in nonseed tissues such as tubers and roots.     

Adenoviruses use lactoferrin as a bridge for CAR-independent binding to and infection of epithelial cells

Most adenoviruses bind to the coxsackie- and adenovirus receptor (CAR). Surprisingly, CAR is not expressed apically on polarized cells and is thus not easily available to viruses. Consequently, alternative mechanisms for entry of coxsackievirus and adenovirus into cells have been suggested. We have found that tear fluid promotes adenovirus infection, and we have identified human lactoferrin (HLf) as the tear fluid component responsible for this effect. HLf alone was found to promote binding of adenovirus to epithelial cells in a dose-dependent manner and also infection of epithelial cells by adenovirus. HLf was also found to promote gene delivery from an adenovirus-based vector. The mechanism takes place at the binding stage and functions independently of CAR. Thus, we have identified a novel binding mechanism whereby adenovirus hijacks HLf, a component of the innate immune system, and uses it as a bridge for attachment to host cells.