Effects of Type II diabetes on capillary hemodynamics in skeletal muscle

Microcirculatory red blood cell (RBC) hemodynamics are impaired within skeletal muscle of Type I diabetic rats (Kindig CA, Sexton WL, Fedde MR, and Poole DC. Respir Physiol 111: 163-175, 1998). Whether muscle microcirculatory dysfunction occurs in Type II diabetes, the more prevalent form of the disease, is unknown. We hypothesized that Type II diabetes would reduce the proportion of capillaries supporting continuous RBC flow and RBC hemodynamics within the spinotrapezius muscle of the Goto-Kakizaki Type II diabetic rat (GK). With the use of intravital microscopy, muscle capillary diameter (d(c)), capillary lineal density, capillary tube hematocrit (Hct(cap)), RBC flux (F(RBC)), and velocity (V(RBC)) were measured in healthy male Wistar (control: n = 5, blood glucose, 105 +/- 5 mg/dl) and male GK (n = 7, blood glucose, 263 +/- 34 mg/dl) rats under resting conditions. Mean arterial pressure did not differ between groups (P > 0.05). Sarcomere length was set to a physiological length ( approximately 2.7 mum) to ensure that muscle stretching did not alter capillary hemodynamics; d(c) was not different between control and GK rats (P > 0.05), but the percentage of RBC-perfused capillaries (control: 93 +/- 3; GK: 66 +/- 5 %), Hct(cap), V(RBC), F(RBC), and O(2) delivery per unit of muscle were all decreased in GK rats (P < 0.05). This study indicates that Type II diabetes reduces both convective O(2) delivery and diffusive O(2) transport properties within muscle microcirculation. If these microcirculatory deficits are present during exercise, it may provide a basis for the reduced O(2) exchange characteristic of Type II diabetic patients.     

Inflammation drives dysbiosis and bacterial invasion in murine models of ileal Crohn's disease

Background and Aims

Understanding the interplay between genetic susceptibility, the microbiome, the environment and the immune system in Crohn’s Disease (CD) is essential for developing optimal therapeutic strategies. We sought to examine the dynamics of the relationship between inflammation, the ileal microbiome, and host genetics in murine models of ileitis.

Methods

We induced ileal inflammation of graded severity in C57BL6 mice by gavage with Toxoplasma gondii, Giardia muris, low dose indomethacin (LDI;0.1 mg/mouse), or high dose indomethacin (HDI;1 mg/mouse). The composition and spatial distribution of the mucosal microbiome was evaluated by 16S rDNA pyrosequencing and fluorescence in situ hybridization. Mucosal E. coli were enumerated by quantitative PCR, and characterized by phylogroup, genotype and pathotype.

Results

Moderate to severe ileitis induced by T. gondii (day 8) and HDI caused a consistent shift from >95% Gram + Firmicutes to >95% Gram - Proteobacteria. This was accompanied by reduced microbial diversity and mucosal invasion by adherent and invasive E. coli, mirroring the dysbiosis of ileal CD. In contrast, dysbiosis and bacterial invasion did not develop in mice with mild ileitis induced by Giardia muris. Superimposition of genetic susceptibility and T. Gondii infection revealed greatest dysbiosis and bacterial invasion in the CD-susceptible genotype, NOD2^−/−, and reduced dysbiosis in ileitis-resistant CCR2^−/− mice. Abrogating inflammation with the CD therapeutic anti-TNF-α-mAb tempered dysbiosis and bacterial invasion.

Conclusions

Acute ileitis induces dysbiosis and proliferation of mucosally invasive E. coli, irrespective of trigger and genotype. The identification of CCR2 as a target for therapeutic intervention, and discovery that host genotype and therapeutic blockade of inflammation impact the threshold and extent of ileal dysbiosis are of high relevance to developing effective therapies for CD.     

Trichothecenes and zearalenone production by fusarium species isolated from Argentinean black beans

Isolates of Fusarium species obtained from freshly harvested bean grains for human consumption collected from different Argentinean regions, were investigated for their ability to biosynthesise trichothecenes and zearalenone either on rice grains or beans. Low incidence of toxigenic fungi was observed. These mycotoxigenic species produced several toxins when grown on rice but none or little amount when cultured on beans. The results of this report suggest that contamination of Argentinean beans with Fusarium mycotoxins will not be common and therefore people would be at low mycotoxicosis risk through consumption of beans.     

Carboxyl terminus of Hsp70‐interacting protein (CHIP) is required to modulate cardiac hypertrophy and attenuate autophagy during exercise

The carboxyl terminus of Hsp70‐interacting protein (CHIP) is a ubiquitin ligase/cochaperone critical for the maintenance of cardiac function. Mice lacking CHIP (CHIP?/?) suffer decreased survival, enhanced myocardial injury and increased arrhythmias compared with wild‐type controls following challenge with cardiac ischaemia reperfusion injury. Recent evidence implicates a role for CHIP in chaperone‐assisted selective autophagy, a process that is associated with exercise‐induced cardioprotection. To determine whether CHIP is involved in cardiac autophagy, we challenged CHIP?/? mice with voluntary exercise. CHIP?/? mice respond to exercise with an enhanced autophagic response that is associated with an exaggerated cardiac hypertrophy phenotype. No impairment of function was identified in the CHIP?/? mice by serial echocardiography over the 5 weeks of running, indicating that the cardiac hypertrophy was physiologic not pathologic in nature. It was further determined that CHIP plays a role in inhibiting Akt signalling and autophagy determined by autophagic flux in cardiomyocytes and in the intact heart. Taken together, cardiac CHIP appears to play a role in regulating autophagy during the development of cardiac hypertrophy, possibly by its role in supporting Akt signalling, induced by voluntary running in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

The relation between cartilage biomarkers (C2C,C1,2C,CS846, and CPII) and the long-term outcome of rheumatoid arthritis patients within the CAMERA trial

Introduction  

The aim of this study was to investigate whether serum biomarker levels of C2C, C1,2C, CS846, and CPII can predict the long-term course of disease activity and radiographic progression early in the disease course of rheumatoid arthritis (RA).     

Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig) proteins

Background

Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²⁺. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²⁺ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold.

Principal Findings

We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9^th (Lig A9) and 10^th repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²⁺ with dissociation constants of 2–4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold.

Conclusions

We demonstrate that the Lig are Ca²⁺-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²⁺. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²⁺-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²⁺ binding.     

Proteomic analysis of human biopsy samples by single two-dimensional electrophoresis: Coomassie,silver, mass spectrometry,and Western blotting

Proteomic analysis of myocardial tissue from patient populations is critical to our understanding of cardiac disease, but has been limited until now by the small size of biopsies (approximately 20-50 microg), and complicated by the difference in relative abundance of contractile proteins over other cellular components. Here we describe an approach to analysis of myocardial biopsies from patients undergoing coronary artery bypass surgery. First, individual biopsies are selectively extracted, producing subfractions that correspond to the contractile proteins and the cytosolic proteins. Two-dimensional electrophoresis separated proteins are detected by first staining with Coomassie blue then silver, to permit a wider range of accurate quantification. Western blotting of two-dimensional separated samples, to validate peptide mass fingerprinting data, previously required additional gel separations for transfer since staining protocols are not compatible with transfer to membranes or immunoblotting. An existing silver destaining protocol was adapted to allow removal of silver from a whole gel, followed by transfer and Western blotting. An existing Coomassie blue removal protocol was also adapted to permit Western blotting of gels stained with Coomassie blue and silver. Together, these techniques permit peptide mass fingerprinting concurrent with Western blotting of a single protein spot from a single biopsy, eliminating the need for repeated gel separations, and improving spot alignment between immunoblots and stained gels. In the end, this approach may allow a more complete characterization of protein changes in small human biopsies, and also reduce the number of repeated gel separations necessary for a standard proteomic analysis.     

Connexin 30.3 is expressed in the kidney but not regulated by dietary salt or high blood pressure

Several isoforms of connexin (Cx) proteins have been identified in a variety of tissues where they play a role in intercellular communication, either as the components of gap junctions or as large, nonselective pores known as hemichannels. This investigation seeks to identify the localization and regulation of Cx30.3 in mouse, rat, and rabbit kidney using a Cx30.3(+/lacZ) transgenic approach and immunofluorescence. Cx30.3 was detected in all three species and predominantly in the renal medulla. Both the nuclear lacZ staining indicative of Cx30.3 expression and indirect immunohistochemistry provided the same results. Cx30.3 immunolabeling was mainly punctate in the mouse, typical for gap junctions. In contrast, it showed continuous apical plasma membrane localization in certain tubule segments in the rat and rabbit kidney, suggesting that it may also function as hemichannels. In the cortex, Cx30.3 was localized in the intercalated cells of the cortical collecting duct, because the immunoreactive cells did not label for AQP2, a marker for principal cells. In the medulla, dense Cx30.3 staining was confined to the ascending thin limbs of the loop of Henle, because the immunoreactive cells did not label for AQP1, a marker of the descending thin limbs. Immunoblotting studies indicated that Cx30.3 expression was unchanged in response to either high or low salt intake or in spontaneously hypertensive rats. Cx30.3 appears to be constitutively expressed in certain renal tubular segments and cells and its role in overall kidney function remains to be investigated.