Helicobacter pylori specific immune response induced by conservative flagellin linear B-cell epitope

AIM:To testify the immunogenicity of a conservative B-cell linear epitope of Helicobacter pylori (H pylori) flagellin A. METHODS: Different programs were used to analyze the secondary structure, molecular hydropathy, and surface accessibility of H pylori flagellin A. Linear B-cell epitopes were estimated based on the structural and physiochemical information. Analysis of residue divergence was proposed to screen a conservative linear epitope. The 29-peptide (Pep29mer) synthesized by chemical method, including the predicted conservative B-cell epitope and a known K2d compatible T-cell epitope, was used to immunize mice, and then H pylori-specific antibodies were detected by ELISA. RESULTS: Based on the analyses of divergent amino acid residues, structural and physiochemical characteristics, it was strongly suggested that the short fragment NDSDGR was the core of a conservative linear epitope in flagellin A. Animals immunized by Pep29mer acquired efficient immune response. In detail, serum H pylori-specific IgA and IgGl increased significantly in immunized group, while IgG2a only had an insignificant change. H pylori-specific IgA in gastrointestinal flushing fluid also increased significantly. CONCLUSION: The conservative short fragment NDSDGR is the core of a linear B-cell epitope of flagellin A.     

Rosiglitazone improves insulin sensitivity and glucose tolerance in subjects with impaired glucose tolerance

OBJECTIVE: This study was designed to evaluate the effects of rosiglitazone (ROS) on insulin sensitivity, beta-cell function, and glycaemic response to glucose challenge and meal in subjects with impaired glucose tolerance (IGT). METHODS: Thirty patients with IGT (ages between 30 and 75 years and BMI (body mass index) < or = 27 kg/m2) were randomly assigned to receive either placebo (n = 15) or ROS (4 mg/day) (n = 15). All participants underwent a 75-g oral glucose tolerance test (OGTT), meal test, and frequently sampled intravenous glucose tolerance test (FSIGT) before and after the 12-week treatment. RESULTS: After 12 weeks of ROS treatment, there were significant increases in total cholesterol (TC) (4.25 +/- 0.22 vs 4.80 +/- 0.17 mmol/l, P < 0.001), high-density lipoprotein cholesterol (HDL-C) (1.25 +/- 0.07 vs 1.43 +/- 0.06 mmol/l, P < 0.05), and low-density lipoprotein cholesterol (LDL-C) (2.70 +/- 0.15 vs 3.37 +/- 0.17 mmol/l, P < 0.05) without changes in triglyceride concentration, TC/HDL-C and LDL-C/HDL-C ratio. Although the acute insulin response (AIR) to intravenous glucose and disposition index (measured as the ability of pancreatic beta-cell compensation in the presence of insulin resistance) remained unchanged, the insulin sensitivity (SI) and glucose effectiveness (SG) were remarkably elevated (0.38 +/- 0.06 vs 0.54 +/- 0.09 x 10(-5) min(-1)/pmol, P < 0.05; 0.017 +/- 0.002 vs 0.021 +/- 0.001 min(-1), P < 0.05, respectively) in the ROS group. The glucose, insulin, and c-peptide areas under curve (AUC) in response to OGTT and the glucose and insulin AUC during meal were significantly ameliorated in the ROS group. Five out of 15 (33%) and two out of 15 (13%) subjects treated with ROS and placebo, respectively, reversed to normal response during OGTT (P < 0.05). CONCLUSION: Rosiglitazone treatment significantly improved insulin resistance and reduced postchallenge glucose and insulin concentrations in patients with impaired glucose tolerance without remarkable effects on beta-cell secretory function.     

RIM genes differentially contribute to organizing presynaptic release sites

Tight coupling of Ca(2+) channels to the presynaptic active zone is critical for fast synchronous neurotransmitter release. RIMs are multidomain proteins that tether Ca(2+) channels to active zones, dock and prime synaptic vesicles for release, and mediate presynaptic plasticity. Here, we use conditional knockout mice targeting all RIM isoforms expressed by the Rims1 and Rims2 genes to examine the contributions and mechanism of action of different RIMs in neurotransmitter release. We show that acute single deletions of each Rims gene decreased release and impaired vesicle priming but did not alter the extracellular Ca(2+)-responsiveness of release (which for Rims gene mutants is a measure of presynaptic Ca(2+) influx). Moreover, single deletions did not affect the synchronization of release (which depends on the close proximity of Ca(2+) channels to release sites). In contrast, deletion of both Rims genes severely impaired the Ca(2+) responsiveness and synchronization of release. RIM proteins may act on Ca(2+) channels in two modes: They tether Ca(2+) channels to active zones, and they directly modulate Ca(2+)-channel inactivation. The first mechanism is essential for localizing presynaptic Ca(2+) influx to nerve terminals, but the role of the second mechanism remains unknown. Strikingly, we find that although the RIM2 C(2)B domain by itself significantly decreased Ca(2+)-channel inactivation in transfected HEK293 cells, it did not rescue any aspect of the RIM knockout phenotype in cultured neurons. Thus, RIMs primarily act in release as physical Ca(2+)-channel tethers and not as Ca(2+)-channel modulators. Different RIM proteins compensate for each other in recruiting Ca(2+) channels to active zones, but contribute independently and incrementally to vesicle priming.     

Role of satellite cells versus myofibers in muscle hypertrophy induced by inhibition of the myostatin/activin signaling pathway

Myostatin and activin A are structurally related secreted proteins that act to limit skeletal muscle growth. The cellular targets for myostatin and activin A in muscle and the role of satellite cells in mediating muscle hypertrophy induced by inhibition of this signaling pathway have not been fully elucidated. Here we show that myostatin/activin A inhibition can cause muscle hypertrophy in mice lacking either syndecan4 or Pax7, both of which are important for satellite cell function and development. Moreover, we show that muscle hypertrophy after pharmacological blockade of this pathway occurs without significant satellite cell proliferation and fusion to myofibers and without an increase in the number of myonuclei per myofiber. Finally, we show that genetic ablation of Acvr2b, which encodes a high-affinity receptor for myostatin and activin A specifically in myofibers is sufficient to induce muscle hypertrophy. All of these findings are consistent with satellite cells playing little or no role in myostatin/activin A signaling in vivo and render support that inhibition of this signaling pathway can be an effective therapeutic approach for increasing muscle growth even in disease settings characterized by satellite cell dysfunction.     

Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells

We describe a microchip designed to quantify the levels of a dozen cytoplasmic and membrane proteins from single cells. We use the platform to assess protein–protein interactions associated with the EGF-receptor-mediated PI3K signaling pathway. Single-cell sensitivity is achieved by isolating a defined number of cells (n = 0–5) in 2 nL volume chambers, each of which is patterned with two copies of a miniature antibody array. The cells are lysed on-chip, and the levels of released proteins are assayed using the antibody arrays. We investigate three isogenic cell lines representing the cancer glioblastoma multiforme, at the basal level, under EGF stimulation, and under erlotinib inhibition plus EGF stimulation. The measured protein abundances are consistent with previous work, and single-cell analysis uniquely reveals single-cell heterogeneity, and different types and strengths of protein–protein interactions. This platform helps provide a comprehensive picture of altered signal transduction networks in tumor cells and provides insight into the effect of targeted therapies on protein signaling networks.Although signal transduction inhibitors occasionally offer clinical benefit for cancer patients (1), signal flux emanating from oncogenes is often distributed through multiple pathways (2), potentially underlying the failure of most such inhibitors (3). Measuring signal flux through multiple pathways, in response to signal transduction inhibitors, may help uncover network interactions that contribute to therapeutic resistance and that are not predicted by analyzing pathways in isolation (4). The cellular and molecular complexity of a solid tumor microenvironment (5) suggests the need to study signaling in individual cancer cells.Protein–protein interactions within signaling pathways are often elucidated by assessing the levels of relevant pathway proteins in model and tumor-derived cell lines and with various genetic and molecular perturbations. Such interactions, and the implied signaling networks, may also be elucidated via quantitative measurements of multiple pathway-related proteins within single cells (6). At the single-cell level, inhibitory and activating protein–protein relationships, as well as stochastic (single-cell) fluctuations, are revealed. However, most techniques for profiling signaling pathways (7, 8) require large numbers of cells. Single-cell immunostaining (9) is promising, and some flow cytometry (6) techniques are relevant, as discussed below.We describe quantitative, multiplex assays of intracellular signaling proteins from single cancer cells using a platform called the single-cell barcode chip (SCBC). The SCBC is simple in concept: A single or defined number of cells is isolated within an approximately 2 nL volume microchamber that contains an antibody array (10) for the capture and detection of a panel of proteins. The SCBC design (11) permits lysis of each individual trapped cell.Intracellular staining flow cytometry can assay up to 11 phosphoproteins from single cells (6). Our SCBC can profile a similar size panel, but only for approximately 100 single cells per chip. Each protein is assayed twice, yielding some statistical assessment for each experiment. The SCBC is a relatively simple platform and only requires a few hundred cells per assay.We used the SCBC to study signal transduction in glioblastoma multiforme (GBM), a primary malignant brain tumor (12). GBM has been genetically characterized, yet the nature of signaling pathways downstream of key oncogenic mutations, such as epidermal growth factor receptor activating mutation (EGFRvIII) and phosphatase and tensin homolog (PTEN) tumor suppressor gene loss associated with receptor tyrosine kinase (RTK)/PI3K signaling, are incompletely understood (13–15). Single-cell experiments may also help resolve the characteristic heterogeneity of GBM.We interrogated 11 proteins directly or potentially associated with PI3K signaling (see SI Appendix, Methods I) through three isogenic GBM cell lines: U87 (expressing wild-type p53, mutant PTEN, and low levels of wild-type EGFR, no EGFRvIII) (16, 17), U87 EGFRvIII (U87 cells stably expressing EGFRvIII deletion mutant), and U87 EGFRvIII PTEN (U87 cells coexpressing EGFRvIII and PTEN) (18). Fig. 1 diagrams this biology. Each cell line was investigated under conditions of standard cell culture, in response to EGF stimulation, and after erlotinib treatment followed by EGF stimulation. The proteins assayed represented RTKs and proteins signifying activation of PI3K and MAPK signaling. They were (p- denotes phosphorylation) p-Src, p-mammalian target of rapamycin (p-mTOR), p-p70 ribosomal protein S6 kinase (p-p70S6K), p-glycogen synthase kinase-3 (p-GSK-3α/β), p-p38 mitogen activated protein kinase (p-p38α), p-extracellular regulated kinase (p-ERK), p-c-Jun N-terminal kinase (p-JNK2), p-platelet derived growth factor receptor β (p-PDGFRβ), p-vascular endothelial growth factor receptor 2 (p-VEGFR2), tumor protein 53 (P53), and total EGFR.Open in a separate windowFig. 1.The PI3K pathway activated by EGF-stimulated EGFR or by the constitutively activated EGFRvIII. All proteins in light blue with central yellow background were assayed. Orange background proteins were expressed in the cell lines U87 EGFRvIII or U87 EGFRvIII PTEN. The oval, yellow background components are the investigated molecular perturbations.     

Enhanced chemosensitivity to CPT-11 in colorectal carcinoma xenografts by small hairpin RNA interference targeting PLK1

Commonly used drugs for the treatment of colon{} cancer patients like CPT-11 shows severe side effects or induces resistance in clinical settings. Thus, we analyzed a?combination of PLK1 (polo-like kinase 1)-specific short hair RNA (shRNA), a?potent tool to destroy mitosis in cancer cells, together with CPT-11 to enhance drug sensitivity. Cellular proliferation and apoptosis were determined in SW620 colorectal carcinoma cells. Knockdown of cellular PLK1 led to the decreased mRNA and PLK1 protein in RT-PCR and western blot assay. The viability declined (pnohistochemistry, accompanied with TUNEL assay. As we expect, the combination treatment delayed tumor growth (p of PLK1-specific shRNA interference with low-dose CPT-11 triggered a?antitumor efficacy and represented a?potential strategy to treat colon cancer. Keywords: CPT-11, drug resistance, SN-38, PLK1, RNA interference, colorectal carcinoma.     

Reduced release probability prevents vesicle depletion and transmission failure at dynamin mutant synapses

Endocytic recycling of synaptic vesicles after exocytosis is critical for nervous system function. At synapses of cultured neurons that lack the two "neuronal" dynamins, dynamin 1 and 3, smaller excitatory postsynaptic currents are observed due to an impairment of the fission reaction of endocytosis that results in an accumulation of arrested clathrin-coated pits and a greatly reduced synaptic vesicle number. Surprisingly, despite a smaller readily releasable vesicle pool and fewer docked vesicles, a strong facilitation, which correlated with lower vesicle release probability, was observed upon action potential stimulation at such synapses. Furthermore, although network activity in mutant cultures was lower, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity was unexpectedly increased, consistent with the previous report of an enhanced state of synapsin 1 phosphorylation at CaMKII-dependent sites in such neurons. These changes were partially reversed by overnight silencing of synaptic activity with tetrodotoxin, a treatment that allows progression of arrested endocytic pits to synaptic vesicles. Facilitation was also counteracted by CaMKII inhibition. These findings reveal a mechanism aimed at preventing synaptic transmission failure due to vesicle depletion when recycling vesicle traffic is backed up by a defect in dynamin-dependent endocytosis and provide new insight into the coupling between endocytosis and exocytosis.