Expression of PI3-K,PKB and GSK-3β in the skeletal muscle tissue of gestational diabetes mellitus

Objective:To analyze the expression of phosphatidylinositol 3 kinase(PI3-K),protein kinase B(PKB)and glycogen synthase kinase 3 beta(GSK-3β)in skeletal muscle tissue of gestational diabetes mellitus(GDM).Methods:A total of 90 cases of pregnant women were divided into observation group and control group according to the occurrence of GDM with 45 cases in either,and the expression of PI3-K,PKB,GSK-3βmRNA expression in skeletal muscle tissue was compared between two groups.Results:The total PI3-K p85 protein was significantly higher in the observation group compared with the control group,the activity of PI3-K was lower than that of the latter;The total PKB,GSK-3βprotein in skeletal tissue had no significant difference between two groups,while the serine phosphorylation levels of PKB and GSK-3βwere significantly lower in observation group compared with the control group.Conclusions:The downregulation of PI3-K,PKB and GSK-3βin skeletal tissue of GDM caused by phosphorylation dysfunction of signaling molecules is the reason for insulin resistance and transporter function decline which lead to GDM.     

Two-state dynamics of the SH3–SH2 tandem of Abl kinase and the allosteric role of the N-cap

The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3–SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3–SH2 connector, which involve a phosphorylation site. We also show that the SH3–SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3–SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization.Tyrosine kinases are involved in a wide variety of key signaling processes and are therefore tightly regulated by the cell. Indeed, numerous pathologies, ranging from cancer to neurodegeneration, result from or are associated with deficiencies in kinase regulation. Consequently, these enzymes are a prominent target for novel pharmacological strategies against human disease.This study focuses on Abelson murine-leukemia viral-oncogene homolog-1 (Abl), which is one of the most ubiquitously conserved tyrosine kinases. Abl is present in all metazoa, where it plays an essential role in processes as diverse as cytoskeleton reorganization, DNA repair, and regulation of apoptosis (1, 2). Accordingly, when constitutively active forms of Abl are present in normal cells, these are transformed into cancer cells (3). For example, in human white blood cells, a chromosomal abnormality leads to the fusion of the bcr and abl genes, which together encode for a cytoplasm-targeted deregulated form of Abl; Bcr-Abl interferes with the cell cycle, resulting in uncontrolled cell proliferation, and is the principal cause of chronic myeloid leukemia (CML) (4).The architecture of Abl kinases resembles that of other tyrosine kinase families such as Src and Tec (5). It consists of a Src-homology-3 (SH3) module, which is an interaction domain specialized for recognition of xPxxP sequence motifs; a Src-homology-2 (SH2) module, which recognizes phosphorylated tyrosines; and the catalytic domain, which binds and cleaves ATP, and mediates tyrosine phosphorylation in protein targets. Additional elements in Abl are not conserved in Src or Tec and vary among isoforms. The Abl-1b splice variant, which is our focus, features a long, seemingly unstructured N-terminal extension preceding the SH3 domain, known as the N-cap, which becomes myristoylated posttranslationally. Following the catalytic domain is another long stretch, containing various sequence motifs for DNA or cytoskeleton recognition, among others.The SH3 and SH2 domains not only function as interaction modules, but also as allosteric inhibitors of the catalytic domain. In autoinhibited Abl, the three domains are assembled into a compact arrangement, in which the SH3–SH2 tandem appears to mechanically clamp the N- and C-lobes of the catalytic domain in an inactive configuration (Fig. 1A) (6, 7), without directly occluding the active site. The domain-domain linkers appear to be key elements in this assembly. The SH2 domain docks directly onto the C-lobe of the catalytic domain, whereas its linker to the N-lobe engages the SH3 domain. Simultaneously, the portion of N-cap most proximal to the SH3 domain binds to the short SH3–SH2 connector, while the myristoyl group, at the very N terminus, inserts itself into a hydrophobic cavity within the C-lobe of the catalytic domain. Interestingly, Src-family kinases adopt the same compact domain organization in their down-regulated form (8), although their equivalent to N-cap serves as a membrane anchor and is not involved in autoinhibition. Instead, a C-terminal extension from the catalytic domain containing a phosphorylated tyrosine associates with the SH2 module and seemingly locks the complex in the autoinhibited state. Reversible phosphorylation of this C-terminal tail is a major regulatory mechanism of Src family kinases (9, 10).Open in a separate windowFig. 1.(A) Crystal structure of Abl in the autoinhibited state (PDB ID code 2FO0). The SH2 domain docks onto the C-lobe of the catalytic domain, whereas the SH3 domain engages the SH2-kinase linker. This inhibitory configuration of the SH3–SH2 tandem is referred to as the on conformer throughout the text. (B) Pseudodihedral angles used to describe the relative orientation of the regulatory domains during the simulations, encompassing residues in the C-terminal β-strand of the SH3 domain, the SH3–SH2 connector, and the N-terminal β-strand of the SH2 domain (Methods).Activation of both Src and Abl is enabled by the disengagement or reconfiguration of the intramolecular interactions just described (7, 11, 12). Regulation can therefore be thought as a reversible equilibrium whereby the SH3, SH2, and catalytic domains are either dissociated or self-assembled in one or more configurations. External factors, such as competing interactions involving one or both regulatory domains, will bias this equilibrium in one or the other direction (13). For Abl in particular, the significance of this mechanism is underscored by the fact that mutations that impair the correct assembly of the autoinhibited complex, either in the SH3–SH2 tandem or in the domain-domain linkers, confer CML cells with resistance against inhibitory drugs designed to target the catalytic domain of the Bcr-Abl oncogene (14). This outcome has prompted considerable interest in the mechanisms of allosteric regulation of Abl and other tyrosine kinases and in the development of compounds designed to interfere with these mechanisms (15–19).In this paper, we use molecular simulations, free energy calculations, and differential scanning calorimetry (DSC) to study the determining factors of a necessary step in the assembly of the autoinhibited form of Abl, namely the organization of the SH3 and SH2 domains into a conformation conducive to its association with the SH2–kinase linker (KL). Our results show that the conformational dynamics of the Abl SH3–SH2 tandem are clearly distinct from those of Src and related kinases. This finding enables us to reconcile seemingly contradictory structural and biophysical data on the mechanism of Abl regulation. Our analysis also enables us to formulate a mechanistic hypothesis for the role of the N-cap. Finally, we also examine the impact of activating mutations within the SH3–SH2 unit, particularly in the short connector between the domains.     

Louis Pasteur,the Father of Bacteriology

In 1885 in a Paris hospital one mighthave seen an old man keeping anxious watchover the life of a peasant lad.Eagerly heobserved every sympton.For two weeks he hadscarcely eaten or slept.Life was preciousto him,and this was to be his crowning ach-ievement in snatching men from death.Wouldthe crucial test of this new treatment fail?Or would the dread malady of rabies comeunder the control of preventive medicine,even as anthrax and other contagious diseases     

Politics,culture, and the legitimacy of disease: the case of Buerger’s disease

Thromboangiitis obliterans (TAO) or Buerger’s disease is a rare form of vasculitis with distinctive clinical and pathological features that carries significant morbidity, often leading to amputation, and is strongly associated with tobacco smoking. Despite its distinctive clinicopathological characteristics, the existence of TAO as an entity sui generis was challenged for many years as it languished in relative obscurity. Then, as societal attitudes towards smoking changed, TAO not only became accepted as a disease entity, it quite literally became a poster child to illustrate the ills of smoking. Herein, we examine the history of TAO to illustrate the power of societal attitudes and politics in shaping medicine.     

A Prospective,Randomized, Multicenter,Controlled Study of the Safety of Seprafilm® Adhesion Barrier in Abdominopelvic Surgery of the Intestine

INTRODUCTION: Seprafilm® adhesion barrier (Seprafilm®) has been proven to prevent adhesion formation after abdominal and pelvic surgery. This article reports safety results, including the postoperative incidence of abdominal and pelvic abscess and pulmonary embolism, from a large, multicenter trial designed to evaluate the safety and effectiveness of Seprafilm® for reduction of adhesion-related postoperative bowel obstruction after abdominopelvic surgery. METHODS: A total of 1,791 patients participated in this prospective, randomized, multicenter, multinational, single-blind, controlled study in patients undergoing abdominopelvic surgery, the majority of whom had inflammatory bowel disease. Just before closure of the abdomen, patients were randomized to a Seprafilm® or no-treatment control group. Patients received an average of 4.4 and as many as 10 Seprafilm® adhesion barriers applied to organs and tissue surfaces that sustained direct surgical trauma and to suspected adhesiogenic surfaces. Complications that occurred within the first month after surgery were evaluated. RESULTS: During the safety evaluation period, the difference between the Seprafilm® and control groups for the incidence of abscess (4 vs. 3 percent, respectively) or pulmonary embolism (<1 percent in both groups) was not statistically significant (P > 0.05). Foreign body reaction was not reported in either group. Fistula (2 vs. <1 percent) and peritonitis (2 vs. <1 percent) occurred more frequently (P 0.05) in the Seprafilm® group. In a subpopulation of patients in whom Seprafilm® was wrapped around a fresh bowel anastomosis, leak-related events, which included anastomotic leak, fistula, peritonitis, abscess, and sepsis, occurred more frequently (P 0.05). There were no other differences in the incidence, severity, or causative relationship of complications between study groups. CONCLUSIONS: This study confirmed the safety of Seprafilm® adhesion barrier with respect to abdominal abscess, pelvic abscess, and pulmonary embolism when administered to patients undergoing abdominopelvic surgery. Foreign body reaction was not reported for any patient. However, wrapping the suture or staple line of a fresh bowel anastomosis with Seprafilm® should be avoided, because the data suggest that this practice may increase the risk of sequelae associated with anastomotic leak.     

Revealing the role of phospholipase Cβ3 in the regulation of VEGF-induced vascular permeability

VEGF induces vascular permeability (VP) in ischemic diseases and cancer, leading to many pathophysiological consequences. The molecular mechanisms by which VEGF acts to induce hyperpermeability are poorly understood and in vivo models that easily facilitate real-time, genetic studies of VP do not exist. In the present study, we report a heat-inducible VEGF transgenic zebrafish (Danio rerio) model through which VP can be monitored in real time. Using this approach with morpholino-mediated gene knock-down and knockout mice, we describe a novel role of phospholipase Cβ3 as a negative regulator of VEGF-mediated VP by regulating intracellular Ca(2+) release. Our results suggest an important effect of PLCβ3 on VP and provide a new model with which to identify genetic regulators of VP crucial to several disease processes.     

Abstracts from the Proceedings of the Xth Scientific Meeting of the Argentine Society of Arterial Hypertension May 8–11, 2003 La Plata,Buenos Aires,Argentina

Administration of angiotensin II (Ang II) into the cerebral ventricles (icv) of rats elicits vasopressin release and an increase in blood pressure. The effect of atrial natriuretic factor (ANF) on these actions of ANG II was studied in conscious spontaneously hypertensive rats. The magnitude and time course of the blood pressure increase following ANG II (50 and 100 ng) were not altered by ANF, icv. However, vasopressin levels which were stimulated from 10.8 ± 1.5 to 62.1 ± 6.4 pq/ml by ANG II (100 ng) were significantly suppressed by combined administration of ANG II (100 ng) and ANF (3 ug/kg) (33.0 ± 4.3 pg/ml). The injection of ANF alone into the cerebral ventricles had no effect on resting blood pressure or vasopressin levels. Peripheral administration of ANF was unable to attenuate the ANG II-induced vasopressin release. These data suggest that there exists a central interaction of ANF and ANG II within the brain which cannot be mimicked by peripheral administration of ANF.     

Compromising the phosphodependent regulation of the GABAAR β3 subunit reproduces the core phenotypes of autism spectrum disorders

Alterations in the efficacy of neuronal inhibition mediated by GABA_A receptors (GABA_ARs) containing β3 subunits are continually implicated in autism spectrum disorders (ASDs). In vitro, the plasma membrane stability of GABA_ARs is potentiated via phosphorylation of serine residues 408 and 409 (S408/9) in the β3 subunit, an effect that is mimicked by their mutation to alanines. To assess if modifications in β3 subunit expression contribute to ASDs, we have created a mouse in which S408/9 have been mutated to alanines (S408/9A). S408/9A homozygotes exhibited increased phasic, but decreased tonic, inhibition, events that correlated with alterations in the membrane stability and synaptic accumulation of the receptor subtypes that mediate these distinct forms of inhibition. S408/9A mice exhibited alterations in dendritic spine structure, increased repetitive behavior, and decreased social interaction, hallmarks of ASDs. ASDs are frequently comorbid with epilepsy, and consistent with this comorbidity, S408/9A mice exhibited a marked increase in sensitivity to seizures induced by the convulsant kainic acid. To assess the relevance of our studies using S408/9A mice for the pathophysiology of ASDs, we measured S408/9 phosphorylation in Fmr1 KO mice, a model of fragile X syndrome, the most common monogenetic cause of ASDs. Phosphorylation of S408/9 was selectively and significantly enhanced in Fmr1 KO mice. Collectively, our results suggest that alterations in phosphorylation and/or activity of β3-containing GABA_ARs may directly contribute to the pathophysiology of ASDs.GABA_A receptors (GABA_ARs) are Cl⁻ selective ligand-gated ion channels that mediate phasic and tonic inhibition in the adult brain. Consistent with their roles in limiting neuronal excitability, benzodiazepines, barbiturates, general anesthetics, and neurosteroids exert their anxiolytic, anticonvulsant, hypnotic, and sedative effects via potentiating GABA_AR activity (1). GABA_ARs are heteropentamers constructed from α1–6, β1–3, γ1–3, δ, ε, θ, and π subunits. Phasic inhibition is principally mediated by receptors assembled from α1–3, β1–3, and γ2 subunits, whereas those receptors that mediate tonic inhibition contain α4–6, β1–3, and δ subunits (2). Studies using KO mice have shown that the β3 subunit is an essential component of receptor subtypes that mediate phasic and tonic inhibition (3). Together with the Fmr1 gene (Fragile X mental retardation), mutations to the 15q11–13 locus, where the GABA_AR β3 gene resides, are the leading monogenetic causes of autism spectrum disorders (ASDs) (4). Moreover, β3 subunit mutations have been described in seizure disorders, and alterations in subunit expression levels have also been reported in ASDs (3, 5).In vitro studies have revealed that the β3 subunit plays a critical role in regulating the plasma membrane accumulation and synaptic targeting of GABA_ARs via phosphorylation of the intracellular serine residues 408 and 409 (S408/9) (6, 7). S408/9 are substrates of cAMP-dependent PKA, PKC, Ca²⁺-calmodulin type 2-dependent protein kinases (Cam KIIs), and cGMP-dependent protein kinase, and they are principally dephosphorylated by protein phosphatase 2A (8). S408/9 are the principal mediators of high-affinity binding to the clathrin adaptor molecule AP2 within the β3 subunit, and thereby facilitate GABA_AR endocytosis (9). Phosphorylation of S408/9 reduces the affinity of the β3 subunit for AP2 by 100-fold, and mutation of S408/9 to alanine residues (S408/9A) has been shown to mimic the effects of phosphorylation on AP2 binding to the β3 subunit (9, 10). Accordingly, overexpression of the mutant β3 S408/9A subunit in cultured hippocampal neurons leads to an increase in the number and size of inhibitory synapses (7).Studies in animal models of ASDs have reported modifications in the expression levels of some GABA_AR mRNAs and proteins (11, 12). However, the mechanisms underlying these alterations in subunit expression and if they contribute to ASDs remain to be addressed. Therefore, in this study, we have analyzed the role that modified β3 subunit phosphorylation may play in the pathophysiology of ASDs. To test this role, we created a mouse in which the principal sites of phosphodependent regulation within the receptor β3 subunit, S408/9, have been mutated to S408/9A, a mutation that mimics the effects of their phosphorylation. S408/9A mice exhibited increased phasic but decreased tonic inhibition events, which correlated with alterations in the membrane stability of the receptor subtypes that mediate these distinct forms of inhibition. S408/9A mice exhibited alterations in dendritic spine structure, increased repetitive-like behavior, and decreased social interaction, which are hallmarks of ASDs. Therefore, our results provide evidence that alterations in the activity of GABA_ARs containing β3 subunits directly contribute to ASDs.     

Antiphospholipid syndrome, “the best prophet of the future”

The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the occurrence of venous and arterial thromboses, often multiple, and obstetric-related adverse events in the presence of antiphospholipid antibodies (aPL). APS, first described in 1983, as thrombosis, abortion and cerebral disease, is nowadays recognised as a systemic disease with a wide constellation of clinical manifestations related to acute and chronic vascular lesions. The presence of aPL is the serological hallmark of APS representing a heterogeneous population of autoantibodies with many antigenic specificities directed to phospholipid-binding proteins, either alone or in combination with phospholipids. Many assays have been developed for aPL detection. Particularly, anticardiolipin antibodies, anti-β2-glycoprotein I antibodies and lupus anticoagulant are essential tools for APS diagnosis. The cumulative evidence indicates that aPL are pathogenic autoantibodies binding to target cells and promoting thrombosis and pregnancy complications through a wide range of pathological mechanisms not yet fully understood. Finally, the recognition of the important role of aPL to assess the individual risk of thrombosis or pregnancy complications has expanded the concept of aPL, and currently aPL profile is regarded as a major risk factor for clinical thrombotic events.     

Immunolocalization of murine type VI 3β-hydroxysteroid dehydrogenase in the adrenal gland,testis, skin,and placenta

The enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is essential for the biosynthesis of all active steroid hormones, such as those secreted from the adrenal gland, testis, ovary, skin and placenta. The 3β-HSD enzymes exist in multiple isoforms in humans and rodents. To date, six different isoforms have been identified in the mouse, and these isoforms are speculated to play different roles in different tissues. We previously showed that the murine type VI 3β-HSD isoform (Hsd3b6) is expressed specifically in the aldosterone-producing zona glomerulosa cells within the adrenal gland and that its overexpression causes abnormally increased aldosterone synthesis, revealing a crucial (or rate-limiting) role of this enzyme in steroidogenesis. However, potential contributions of this enzyme to the steroid hormone synthesis outside the adrenal glands are poorly understood. This paucity of knowledge is partly because of the lack of isoform-specific antibody that can be used for immunohistochemistry. Here, we report the development and characterization of specific antibody to Hsd3b6 and show the results of immunohistochemistry for the adrenal gland, testis, ovary, skin and placenta. As expected, Hsd3b6 immunoreactivities within the adrenal gland were essentially confined to the zona glomerulosa cells, where aldosterone is produced. By contrast, no immunopositive cells were observed in the zona fasciculata, which is where corticosterone is produced. In the gonads, while the ovaries did not show any detectable immunoreactivity to Hsd3b6, the testes displayed intense immunoreactivities within the interstitial Leydig cells, where testosterone is produced. In the skin, positive immunoreactivities to Hsd3b6 were only seen in the sebaceous glands, suggesting a specific role of this enzyme in sebaceous function. Moreover, in the placenta, Hsd3b6 was specifically found in the giant trophoblast cells surrounding the embryonic cavity, which suggests a role for this enzyme in local progesterone production that is required for proper embryonic implantation and/or maintenance of pregnancy. Taken together, our data revealed that Hsd3b6 is localized in multiple specific tissues and cell types, perhaps thereby involved in biosynthesis of a number of tissue-specific steroid hormones with different physiological roles.     

Sequence Heterogeneity of the ORF3 Gene of Porcine Epidemic Diarrhea Viruses Field Samples in Fujian,China, 2010–2012

Twenty-seven field samples that showed positive in PEDV detection were collected from different farms of Fujian province from 2010 to 2012. Their heterogeneity was investigated by analysis of the ORF3 gene because of its potential function as a representation of virulence. According to the results, six Fujian strains in Group 1 showed a different genotype with unique point mutations, which might be used in differentiation between PEDV groups and brought potential antigenic variation. P55 and five reference strains in Group 2 had a long length deletion, showing another genotype and might be involved in the variation of virulence. Phylogenetic analysis revealed that the collected Fujian strains were very distant from the vaccine development strain CV777, which might be the reason why the vaccine was inefficient to control the disease. The results can help to reconsider the strategy of PEDV vaccine management and prevent outbreaks of PEDV-induced diarrhea more efficiently.