EUS-guided gastroenterostomy using a novel through-the-scope exchangeable dual-balloon enteroclysis catheter: a potentially secure and scalable approach.

Video 1Clinical case of dual-balloon through-the-scope exchangeable enteroclysis catheter-assisted EUS-guided gastroenterostomy.

Glutaredoxin attenuates glutathione levels via deglutathionylation of Otub1 and subsequent destabilization of system xC.

Glutathione (GSH) is a critical component of the cellular redox system that combats oxidative stress. The glutamate-cystine antiporter, system xC-, is a key player in GSH synthesis that allows for the uptake of cystine, the rate-limiting building block of GSH. It is unclear whether GSH or GSH-dependent protein oxidation [protein S-glutathionylation (PSSG)] regulates the activity of system xC-. We demonstrate that an environment of enhanced PSSG promotes GSH increases via a system xC--dependent mechanism. Absence of the deglutathionylase, glutaredoxin (GLRX), augmented SLC7A11 protein and led to significant increases of GSH content. S-glutathionylation of C23 or C204 of the deubiquitinase OTUB1 promoted interaction with the E2-conjugating enzyme UBCH5A, leading to diminished ubiquitination and proteasomal degradation of SLC7A11 and augmentation of GSH, effects that were reversed by GLRX. These findings demonstrate an intricate link between GLRX and GSH via S-glutathionylation of OTUB1 and system xC- and illuminate a previously unknown feed-forward regulatory mechanism whereby enhanced GSH protein oxidation augments cellular GSH.

Structural and functional fine mapping of cysteines in mammalian glutaredoxin reveal their differential oxidation susceptibility.

Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity. To date, GLRX cysteines that are oxidatively modified and their relative susceptibilities remain unknown. We utilized molecular modeling approaches, activity assays using recombinant GLRX, coupled with site-directed mutagenesis of each cysteine both individually and in combination to address the oxidizibility of GLRX cysteines. These approaches reveal that C8 and C83 are targets for S-glutathionylation and oxidation by hydrogen peroxide in vitro. In silico modeling and experimental validation confirm a prominent role of C8 for dimer formation and aggregation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased activity of GLRX and resistance to oxidative inactivation and aggregation. Results from these integrated computational and experimental studies provide insights into the relative oxidizability of GLRX's cysteines and have implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.

Oxidation of peroxiredoxin-4 induces oligomerization and promotes interaction with proteins governing protein folding and endoplasmic reticulum stress.

Peroxiredoxins (PRDXs) catalyze the reduction of hydrogen peroxide (H2O2). PRDX4 is the only peroxiredoxin located within the endoplasmic reticulum (ER) and is the most highly expressed H2O2 scavenger in the ER. PRDX4 has emerged as an important player in numerous diseases, such as fibrosis and metabolic syndromes, and its overoxidation is a potential indicator of ER redox stress. It is unclear how overoxidation of PRDX4 governs its oligomerization state and interacting partners. Herein, we addressed these questions via nonreducing Western blots, mass spectrometry, and site-directed mutagenesis. We report that the oxidation of PRDX4 in lung epithelial cells treated with tertbutyl hydroperoxide caused a shift of PRDX4 from monomer/dimer to high molecular weight (HMW) species, which contain PRDX4 modified with sulfonic acid residues (PRDX4-SO3), as well as of a complement of ER-associated proteins, including protein disulfide isomerases important in protein folding, thioredoxin domain-containing protein 5, and heat shock protein A5, a key regulator of the ER stress response. Mutation of any of the four cysteines in PRDX4 altered the HMW species in response to tertbutyl hydroperoxide as well as the secretion of PRDX4. We also demonstrate that the expression of ER oxidoreductase 1 alpha, which generates H2O2 in the ER, increased PRDX4 HMW formation and secretion. These results suggest a link between SO3 modification in the formation of HMW PRDX4 complexes in cells, whereas the association of key regulators of ER homeostasis with HMW oxidized PRDX4 point to a putative role of PRDX4 in regulating ER stress responses.

Optogenetic "low-theta" pacing of the septohippocampal circuit is sufficient for spatial goal finding and is influenced by behavioral state and cognitive demand.

Hippocampal theta oscillations show prominent changes in frequency and amplitude depending on behavioral state or cognitive demands. How these dynamic changes in theta oscillations contribute to the spatial and temporal organization of hippocampal cells, and ultimately behavior, remain unclear. We used low-theta frequency optogenetic stimulation to pace coordination of cellular and network activity between the medial septum (MS) and hippocampus during baseline and MS stimulation while rats were at rest or performing a spatial accuracy task with a visible or hidden goal zone. Hippocampal receptivity to pan-neuronal septal stimulation at low-theta frequency was primarily determined by speed and secondarily by task demands. Competition between artificial and endogenous field potentials at theta frequency attenuated hippocampal phase preference relative to local theta, but the spike-timing activity of hippocampal pyramidal cells was effectively driven by artificial septal output, particularly during the hidden goal task. Notwithstanding temporal reorganization by artificial theta stimulation, place field properties were unchanged and alterations to spatial behavior were limited to goal zone approximation. Our results indicate that even a low-theta frequency timing signal in the septohippocampal circuit is sufficient for spatial goal finding behavior. The results also advance a mechanistic understanding of how endogenous or artificial somatodendritic timing signals relate to displacement computations during navigation and spatial memory.

Direct cysteine sulfenylation drives activation of the Src kinase.

The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Recent studies indicate that Src is additionally regulated by redox-dependent mechanisms, involving oxidative modification(s) of cysteines within the Src protein, although the nature and molecular-level impact of Src cysteine oxidation are unknown. Using a combination of biochemical and cell-based studies, we establish the critical importance of two Src cysteine residues, Cys-185 and Cys-277, as targets for H2O2-mediated sulfenylation (Cys-SOH) in redox-dependent kinase activation in response to NADPH oxidase-dependent signaling. Molecular dynamics and metadynamics simulations reveal the structural impact of sulfenylation of these cysteines, indicating that Cys-277-SOH enables solvent exposure of Tyr-416 to promote its (auto)phosphorylation, and that Cys-185-SOH destabilizes pTyr-527 binding to the SH2 domain. These redox-dependent Src activation mechanisms offer opportunities for development of Src-selective inhibitors in treatment of diseases where Src is aberrantly activated.

Mechanisms of enhanced basal tone of brain parenchymal arterioles during early postischemic reperfusion: role of ET-1-induced peroxynitrite generation.

The contributions of vasoconstrictors (endothelin-1 (ET-1), peroxynitrite) and endothelium-dependent vasodilatory mechanisms to basal tone were investigated in parenchymal arterioles (PAs) after early postischemic reperfusion. Transient middle cerebral artery occlusion (tMCAO) was induced for 2 hours with 30 minutes reperfusion in male Wistar rats and compared with ischemia alone (permanent MCAO (pMCAO); 2.5 hours) or sham controls. Changes in lumen diameter of isolated and pressurized PAs were compared. Quantitative PCR was used to measure endothelin type B (ETB) receptors. Constriction to intravascular pressure ('basal tone') was not affected by tMCAO or pMCAO. However, constriction to inhibitors of endothelial cell, small- (SK) and intermediate- (IK) conductance, Ca(2+)-sensitive K(+) channels (apamin and TRAM-34, respectively) were significantly enhanced in PAs from tMCAO compared with pMCAO or sham. Addition of the ETB agonist sarafotoxin caused constriction in PAs from tMCAO but not from sham animals (21 ± 4% versus 3 ± 3% at 1 nmol/L; P<0.01) that was inhibited by the peroxynitrite scavenger FeTMPyP (5,10,15,20-tetrakis (N-methyl-4'-pyridyl) porphinato iron (III) chloride) (100 µmol/L). Expression of ETB receptors was not found on PA smooth muscle, suggesting that constriction to sarafotoxin after tMCAO was due to peroxynitrite and not ETB receptor expression. The maintenance of basal tone in PAs after tMCAO may restrict flow to the ischemic region and contribute to infarct expansion.

Downregulation of GluA2 AMPA receptor subunits reduces the dendritic arborization of developing spinal motoneurons.

AMPA receptors lacking the GluA2 subunit allow a significant influx of Ca(2+) ions. Although Ca(2+)-permeable AMPA receptors are a familiar feature at early stages of development, the functional significance of these receptors during the maturation of the nervous system remains to be established. Chicken lumbar motoneurons express Ca(2+)-permeable AMPA receptors at E6 but the Ca(2+) permeability of AMPA receptors decreases ∼3-fold by E11. Considering that activity-dependent changes in intracellular Ca(2+) regulates dendritic outgrowth, in this study we investigated whether downregulation of GluA2 expression during a critical period of development alters the dendritic arborization of spinal motoneurons in ovo. We use an avian replication-competent retroviral vector RCASBP (B) carrying the marker red fluorescent protein (RFP) and a GluA2 RNAi construct to downregulate GluA2 expression. Chicken embryos were infected at E2 with one of the following constructs: RCASBP(B)-RFP, RCASBP(B)-RFP-scrambled RNAi, or RCASBP(B)-RFP-GluA2 RNAi. Infection of chicken embryos at E2 resulted in widespread expression of RFP throughout the spinal cord with ≥60% of Islet1/2-positive motoneurons infected, resulting in a significant reduction in GluA2 protein expression. Downregulation of GluA2 expression had no effect on the dendritic arborization of E6 motoneurons. However, downregulation of GluA2 expression caused a significant reduction in the dendritic arborization of E11 motoneurons. Neither motoneuron survival nor maturation of network activity was affected by changes in GluA2 expression. These findings demonstrate that increased GluA2 expression and changes in the Ca(2+) permeability of AMPA receptors regulate the dendritic arborization of spinal cord motoneurons during a critical period of development.

COL1A1 oligodeoxynucleotides decoy: biochemical and morphologic effects in an acute wound repair model.

Type I collagen is an integral component of granulation tissue and scar, that is highly dependent on TGFß1, a member of a pro-fibrotic family of cytokines, for its promotion and deposition. Blocking COL1A1 gene transcription obstructs type I collagen synthesis, hindering the progress of granulation tissue deposition and fibrosis. Local injections of a double stranded oligodeoxynucleotide (dsODN) decoy, containing the TGFß1 regulatory element that is located in the distal promoter of the COL1A1 gene, were investigated in a rat polyvinyl alcohol (PVA) sponge granulation tissue implant model. The effects on the granulation tissue deposition by dsODN decoy therapy were evaluated by the synthesis of types I and III collagens as well as ED-A (cellular) fibronectin. Fluorescently labeled dsODN was used to identify the distribution of the decoy molecules in the sponge implant relative to the observed histological effects. Morphological alterations in cells and changes in the organization of connective tissue were documented and evaluated. Collagen levels were reduced by half in implants treated with 10 nM dsODN decoy compared to scrambled dsODN-treated implants. Histologically, dsODN decoy treated implants had an increased cellular density without a corresponding increase in deposited connective tissue. Polarized light birefringence pattern of Sirius red-stained sections showed less collagen fibers accumulating between fibroblasts. The highest concentration of fluorescently labeled dsODN was identified within the interior margin of sponge implants, correlating to increased cellular density and an altered birefringence patterns. In conclusion, 10 nM dsODN decoy therapy reduced collagen deposition and altered the organization of granulation tissue, supporting its potential as a localized anti-fibrotic therapy for limiting fibrotic conditions.

GnRH stimulates expression of PACAP in the pituitary gonadotropes via both the PKA and PKC signaling systems.

Recent studies have demonstrated a clear role for pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of gonadotropin biosynthesis and secretion, both alone and in conjunction with GnRH. First defined as a hypothalamic releasing factor, PACAP subsequently has been identified in the gonadotrope subpopulation of the anterior pituitary gland, suggesting that PACAP may act as an autocrine-paracrine factor in this tissue. In initial studies, we determined that GnRH markedly stimulated endogenous PACAP mRNA levels and promoter-reporter activity in the mature gonadotrope cell line, LbetaT2. GnRH-stimulated rat PACAP promoter activity was blunted with deletion from position -915 to -402 and eliminated with further truncation to position -77 relative to the transcriptional start site. Site-directed mutagenesis demonstrated a functional requirement for a cAMP response element (CRE)-like site at position -205 and an activating protein-1 (AP-1)-like site at position -275, both of which bound CRE binding protein and AP-1 family members on EMSA. Treatment with pharmacological activators or inhibitors of second messenger signaling pathways implicated the protein kinase A, protein kinase C, and MAPK pathways in the GnRH response. In support of these in vitro data, we demonstrate that JunB binds to the rat PACAP gene promoter by chromatin immunoprecipitation assay and that small interfering RNA knockdown of JunB, cFos, and CRE binding protein factors blunts PACAP expression. In summary, these results further elucidate the complex functional interactions between PACAP and GnRH in the anterior pituitary. Specifically, these studies demonstrate that GnRH-stimulated PACAP gene expression is mediated via multiple signaling pathways acting on CRE/AP-1 sites in the proximal gene promoter. Because both PACAP and GnRH regulate gonadotropin biosynthesis and secretion, these results provide important insight into the critical fine tuning of gonadotrope function and, thereby, the maintenance of normal reproductive function.

Identification of genes encoding atypical odorant-binding proteins in Aedes albopictus (Diptera: Culicidae).

Insect odorant-binding proteins (OBPs) are a diverse gene family that encode proteins thought to function as molecular chaperones by binding semiochemicals and transporting them through the aqueous lymph of insect sensilla. Between 66 and 68 genes have been classified as OBPs in both Anopheles gambiae (Giles) and Aedes aegypti L. based on bioninformatics criteria. We have cloned and sequenced from a subtracted cDNA library three OBPs in Aedes albopcitus (Skuse). BLASTP and phylogenetic analysis of deduced amino acid sequences identified a unique putative ortholog in Ae. aegypti for each Ae. albopictus OBP. Comparison of these putative Ae. aegypti orthologs with the results of previous bioinformatics analyses of OBP genes in Ae. aegypti highlight the potential variability of bioinformatics analyses and suggest that the OBP gene family of Culicids is even more diverse than previously described. Alignment of deduced amino acid sequences and phylogenetic analysis identified the N-terminal region of Culicid OBPs that is associated with aedine-specific diversification. Analysis of tissue-specific expression indicates that two of the Ae. albopictus OBPs are expressed both in preadult stages and in the hemolymph of adults, suggesting that the proteins encoded by these genes may be involved in the transport of hydrophobic ligands in the hemolymph. The other Ae. albopictus OBP is expressed exclusively in antennae and leg, suggesting a chemosensory function. These results are discussed within the context of the evolution and functional diversification of OBPs in mosquitoes.

Functional effects of the hypertrophic cardiomyopathy R403Q mutation are different in an alpha- or beta-myosin heavy chain backbone.

The R403Q mutation in the beta-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly alpha-MHC as opposed to the beta-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q alpha-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous alpha-MHC was largely replaced with transgenically encoded beta-MHC. A His(6) tag was cloned at the N terminus of the alpha-and beta-MHC to facilitate protein isolation by Ni(2+)-chelating chromatography. Characterization of the R403Q alpha-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a beta-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q alpha-S1 is approximately 30% higher than for wild type, whereas the enzymatic activity for R403Q beta-S1 is reduced by approximately 10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.

Tissue stretch decreases soluble TGF-beta1 and type-1 procollagen in mouse subcutaneous connective tissue: evidence from ex vivo and in vivo models.

Transforming growth factor beta 1 (TGF-beta1) plays a key role in connective tissue remodeling, scarring, and fibrosis. The effects of mechanical forces on TGF-beta1 and collagen deposition are not well understood. We tested the hypothesis that brief (10 min) static tissue stretch attenuates TGF-beta1-mediated new collagen deposition in response to injury. We used two different models: (1) an ex vivo model in which excised mouse subcutaneous tissue (N = 44 animals) was kept in organ culture for 4 days and either stretched (20% strain for 10 min 1 day after excision) or not stretched; culture media was assayed by ELISA for TGF-beta1; (2) an in vivo model in which mice (N = 22 animals) underwent unilateral subcutaneous microsurgical injury on the back, then were randomized to stretch (20-30% strain for 10 min twice a day for 7 days) or no stretch; subcutaneous tissues of the back were immunohistochemically stained for Type-1 procollagen. In the ex vivo model, TGF-beta1 protein was lower in stretched versus non-stretched tissue (repeated measures ANOVA, P < 0.01). In the in vivo model, microinjury resulted in a significant increase in Type-1 procollagen in the absence of stretch (P < 0.001), but not in the presence of stretch (P = 0.21). Thus, brief tissue stretch attenuated the increase in both soluble TGF-beta1 (ex vivo) and Type-1 procollagen (in vivo) following tissue injury. These results have potential relevance to the mechanisms of treatments applying brief mechanical stretch to tissues (e.g., physical therapy, respiratory therapy, mechanical ventilation, massage, yoga, acupuncture).

Oligodeoxynucleotide decoy therapy blocks type 1 procollagen transcription and the prolyl hydroxylase beta subunit translation.

Persistent transforming growth factor-beta1 (TGF-beta1) exposure to lungs increases type 1 collagen synthesis and deposition resulting in excess fibrosis which leads to morbidity and possibly death. We now report using human embryonic lung fibroblasts in the presence of TGF-beta1, a novel double-stranded (ds) DNA decoy with phosphorothioate (PT) linkages, containing the TGF-beta cis-element found in the distal promoter region of the COL1A1 gene which silences COL1A1 gene expression. In a cell-free protein translation system, we have previously reported that collagen synthesis was inhibited by disulfide isomerase, the prolyl-4-hydroxylase (P-4-H) beta subunit. By comparative proteomics dsdecoy therapy increased the levels of disulfide isomerase, the P-4-H beta subunit. These findings taken together support the notion that the dsdecoy inhibits type 1 collagen synthesis at both the transcriptional and translational levels.

Therapies for bleomycin induced lung fibrosis through regulation of TGF-beta1 induced collagen gene expression.

This review describes normal and abnormal wound healing, the latter characterized by excessive fibrosis and scarring, which for lung can result in morbidity and sometimes mortality. The cells, the extracellular matrix (ECM) proteins, and the growth factors regulating the synthesis, degradation, and deposition of the ECM proteins will be discussed. Therapeutics with particular emphasis given to gene therapies and their effects on specific signaling pathways are described. Bleomycin (BM), a potent antineoplastic antibiotic increases TGF-beta1 transcription, TGF-beta1 gene expression, and TGF-beta protein. Like TGF-beta1, BM acts through the same distal promoter cis-element of the COL1A1 gene causing increased COL1 synthesis and lung fibrosis. Lung fibroblasts exist as subpopulations with one subset predominantly responding to fibrogenic stimuli which could be a specific cell therapeutic target for the onset and development of pulmonary fibrosis.

Phenotypic expression of human hepatoma cells in culture.

Hepatomas thrive in a hypoxic environment resulting in the induction of a cluster of hypoxia related genes. The protein phenotypic expression include hypoxia inducible factor-alpha, prolyl-4-hydroxylase, vascular endothelear growth factor and erythropoietin. The present study was undertaken to determine if human hepatoma cells when cultured for 72 h in the presence of serum under normoxia would maintain their cancerous phenotypic expression of certain hypoxia inducible genes. Our positive results affords an in vitro model system to test hypoxia inhibitors on the expression and the intracellular compartmentalization or the secretion of these hypoxia-inducible proteins.

Patterns of variation among distinct alleles of the Flag silk gene from Nephila clavipes.

Spider silk proteins and their genes are very attractive to researchers in a wide range of disciplines because they permit linking many levels of organization. However, hypotheses of silk gene evolution have been built primarily upon single sequences of each gene each species, and little is known about allelic variation within a species. Silk genes are known for their repeat structure with high levels of homogenization of nucleotide and amino acid sequence among repeated units. One common explanation for this homogeneity is gene convergence. To test this model, we sequenced multiple alleles of one intron-exon segment from the Flag gene from four populations of the spider Nephila clavipes and compared the new sequences to a published sequence. Our analysis revealed very high levels of heterozygosity in this gene, with no pattern of population differentiation. There was no evidence of gene convergence within any of these alleles, with high levels of nucleotide and amino acid substitution among the repeating motifs. Our data suggest that minimally, there is relaxed selection on mutations in this gene and that there may actually be positive selection for heterozygosity.

Modulation of pituitary adenylate cyclase-activating polypeptide (PACAP) expression in explant-cultured guinea pig cardiac neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) expression was quantified in explant-cultured guinea pig cardiac ganglia neurons. In explant culture, both the percentage of PACAP-immunoreactive neurons and pro-PACAP transcript levels increased significantly. Treatment with neurturin or glial-derived neurotrophic factor significantly suppressed the percentage of PACAP-IR neurons, but not pro-PACAP transcript levels.