Hydration and packing are crucial to amyloidogenesis as revealed by pressure studies on transthyretin variants that either protect or worsen amyloid disease

The formation of amyloid aggregates is the hallmark of the amyloidogenic diseases. Transthyretin (TTR) is involved in senile systemic amyloidosis (wild-type protein) and familial amyloidotic polyneuropathy (point mutants). Through the use of high hydrostatic pressure (HHP), we compare the stability among wild-type (wt) TTR, two disease-associated mutations (V30M and L55P) and a trans-suppressor mutation (T119M). Our data show that the amyloidogenic conformation, easily populated in the disease-associated mutant L55P, can be induced by a cycle of compression-decompression with the wt protein rendering the latter highly amyloidogenic. After decompression, the recovered wt structure has weaker subunit interactions (loosened tetramer, T(4)(*)) and presents a stability similar to L55P, suggesting that HHP induces a defective fold in the wt protein, converting it to an altered conformation already present in the aggressive mutant, L55P. On the other hand, glucose, a chemical chaperone, can mimic the trans-suppression mutation by stabilizing the native state and by decreasing the amyloidogenic potential of the wt TTR at pH 5.0. The sequence of pressure stability observed was: L55P相似文献   

Composite surface for blocking bacterial adsorption on protein biochips

The design and fabrication of protein biochips requires characterization of blocking agents that minimize nonspecific binding of proteins or organisms. Nonspecific adsorption of Escherichia coli, Listeria innocua, and Listeria monocytogenes is prevented by bovine serum albumin (BSA) or biotinylated BSA adsorbed on SiO(2) surfaces of a biochip that had been modified with a C(18) coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Because streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins, including antibodies. Hence, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfacing protein, avidin or streptavidin, which in turns anchors biotinylated antibody. In our case, the antibody is C11E9, an IgG-type antibody that binds Listeria spp. Nonspecific adsorption of another bacterium, Escherichia coli, is also minimized due to the blocking action of the BSA. The blocking characteristics of BSA adsorbed on C(18)-derivatized SiO(2) surfaces for construction of a protein biochip for electronic detection of pathogenic organisms is investigated.     

Effects of NF-kappa B inhibition on mesenteric ischemia-reperfusion injury

Mesenteric ischemia-reperfusion injury is a serious complication of shock. Because activation of nuclear factor-kappaB (NF-kappaB) has been implicated in this process, we treated rats with vehicle or the IkappaB-alpha inhibitor BAY 11-7085 (25 mg/kg ip) 1 h before mesenteric ischemia-reperfusion (45 min of ischemia followed by reperfusion at 30 min or 6 h) and examined the ileal injury response. Vehicle-treated rats subjected to ischemia-reperfusion exhibited severe mucosal injury, increased myeloperoxidase (MPO) activity, increased expression of interleukin-6 and intercellular adhesion molecule 1 protein, and a biphasic peak of NF-kappaB DNA-binding activity during the 30-min and 6-h reperfusion courses. In contrast, BAY 11-7085-pretreated rats subjected to ischemia-reperfusion exhibited less histological injury and less interleukin-6 and intercellular adhesion molecule 1 protein expression at 30 min of reperfusion but more histological injury at 6 h of reperfusion than vehicle-treated rats subjected to ischemia-reperfusion. Studies with phosphorylation site-specific antibodies demonstrated that IkappaB-alpha phosphorylation at Ser(32),Ser(36) was induced at 30 min of reperfusion, whereas tyrosine phosphorylation of IkappaB-alpha was induced at 6 h of reperfusion. BAY 11-7085 inhibited the former, but not the latter, phosphorylation pathway, whereas alpha-melanocyte-stimulating hormone, which is effective in limiting late ischemia-reperfusion injury to the intestine, inhibited tyrosine phosphorylation of IkappaB-alpha. Thus NF-kappaB appears to play an important role in the generation and resolution of intestinal ischemia-reperfusion injury through different activation pathways.     

Molecular profiling of antimicrobial resistance and integron association of multidrug-resistant clinical isolates of Shigella species from Faisalabad, Pakistan

Bacillary dysentery, common in developing countries, is usually caused by Shigella species. A major problem in shigellosis is the rapid emergence of multidrug-resistant strains. This is the first detailed molecular study on drug resistance of Shigella isolates from the Faisalabad region of Pakistan. Ninety-five Shigella isolates obtained after screening of 2500 stool samples were evaluated for in vitro resistance to commonly used antimicrobial agents; the presence or absence of 20 of the most relevant drug resistance genes; and the prevalence of integrons 1, 2, and 3. Shigella flexneri was found to be the most prevalent and most resistant species. Collectively, high resistance was found towards ampicillin (96.84%), tetracycline (93.68%), streptomycin (77.89%), and chloramphenicol (72.63%). Significant emerging resistance was detected towards the modern frontline drugs ciprofloxacin (12.63%), cefradine (17.89%), ceftriaxone (20.00%), cefoperazone (22.10%), and cefixime (28.42%). Prevalence rates for bla(TEM), bla(CTX-M), gyrA, gyrB, qnrS, aadA1, strAB, tetA, tetB, catA, and catP were 78.94%, 12.63%, 20.00%, 21.05%, 21.05%, 67.36%, 42.10%, 12.63%, 53.68%, 33.68%, and 25.26%, respectively. Class 2 integrons (42.10%) were more common in the local isolates. Simultaneous detection of class 1 and 2 integrons in some isolates and a rapidly emerging resistance to modern frontline drugs are the major findings of this study.     

OsNRAMP5, a major player for constitutive iron and manganese uptake in rice

Manganese (Mn) and iron (Fe) are essential mineral micronutrients for plants and their deficiency and or toxicity represents a serious agricultural problem. In rice the information about genes involved in Mn uptake from soil is scarce. Recently, we showed that OsNRAMP5 is a plasma membrane protein involved in Mn and Fe transport. The concentration of Mn in roots, shoots and xylem sap of OsNRAMP5 RNAi (OsNRAMP5i) plants was significantly reduced compared with WT plants. The expression of OsNRAMP5 is not controlled by Fe deficiency in root and was also observed in pistil, ovary, lemma and palea. These data show that rice would utilize OsNRAMP5 for constitutive Fe and Mn uptake, while OsNRAMP5 would also play a role in Fe and Mn transport during flowering and seed development.     

Using gene expression to predict differences in the secretome of human omental vs. subcutaneous adipose tissue

The objective of this study was to characterize differences in the secretome of human omental compared with subcutaneous adipose tissue using global gene expression profiling. Gene expression was measured using Affymetrix microarrays (Affymetrix, Santa Clara, CA) in subcutaneous and omental adipose tissue in two independent experiments (n = 5 and n = 3 independent subjects; n = 16 arrays in total, 2 for each subject). Predictive bioinformatic algorithms were employed to identify secreted proteins. Microarray analysis identified 22 gene probe sets whose expression was significantly different with a fold change (FC) greater than 5 in expression in both experiments between omental and subcutaneous adipose tissue. Using bioinformatic predictive programs 11 of these 22 probe sets potentially coded for secreted proteins. Pathway network analysis of the secreted proteins showed that three of the proteins are part of a common pathway network. These proteins gremlin 1 (GREM1), pleiotrophin (PTN), and secretory leukocyte peptidase inhibitor (SLPI) are expressed respectively 43×, 23×, and 5× in omental adipose tissue relative to subcutaneous adipose tissue as determined by real-time PCR. The presence of GREM1, PTN, and SLPI protein in human adipose tissue was confirmed by western blotting. All three proteins are expressed in the human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell line. The expression of GREM1, PTN, and SLPI changed with the differentiation of the preadipocytes into mature adipocytes. Gene expression coupled with predictive bioinformatic algorithms have identified several genes coding for secreted proteins which are expressed differently in omental adipose tissue compared to subcutaneous adipose tissue proving a valid alternative approach to help further define the adipocyte secretome.     

Lack of correlation between outcomes of membrane repair assay and correction of dystrophic changes in experimental therapeutic strategy in dysferlinopathy

Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fulfilling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.     

Conditional Astroglial Rictor Overexpression Induces Malignant Glioma in Mice

Background

Hyperactivation of the mTORC2 signaling pathway has been shown to contribute to the oncogenic properties of gliomas. Moreover, overexpression of the mTORC2 regulatory subunit Rictor has been associated with increased proliferation and invasive character of these tumor cells.

Methodology/Principal Findings

To determine whether Rictor overexpression was sufficient to induce glioma formation in mice, we inserted a Cre-lox-regulated human Rictor transgene into the murine ROSA26 locus. This floxed Rictor strain was crossed with mice expressing the Cre recombinase driven from the glial fibrillary acidic protein (GFAP) promoter whose expression is limited to the glial cell compartment. Double transgenic GFAP-Cre/Rictor^loxP/loxP mice developed multifocal infiltrating glioma containing elevated mTORC2 activity and typically involved the subventricular zone (SVZ) and lateral ventricle. Analysis of Rictor-dependent signaling in these tumors demonstrated that in addition to elevated mTORC2 activity, an mTORC2-independent marker of cortical actin network function, was also elevated. Upon histological examination of the neoplasms, many displayed oligodendroglioma-like phenotypes and expressed markers associated with oligodendroglial lineage tumors. To determine whether upstream oncogenic EGFRvIII signaling would alter tumor phenotypes observed in the GFAP-Cre/Rictor^loxP/loxP mice, transgenic GFAP-EGFRvIII; GFAP-Cre/Rictor^loxP/loxP mice were generated. These mice developed mixed astrocytic-oligodendroglial tumors, however glioma formation was accelerated and correlated with increased mTORC2 activity. Additionally, the subventricular zone within the GFAP-Cre/Rictor^loxP/loxP mouse brain was markedly expanded, and a further proliferation within this compartment of the brain was observed in transgenic GFAP-EGFRvIII; GFAP-Cre/Rictor^loxP/loxP mice.

Conclusion/Significance

These data collectively establish Rictor as a novel oncoprotein and support the role of dysregulated Rictor expression in gliomagenesis via mTOR-dependent and mTOR-independent mechanisms. Furthermore, oncogenic EGFRvIII signaling appears to potentiate the in vivo proliferative capacity of GFAP-Cre/Rictor^loxP/loxP gliomas.     

Significantly Reducing Post-Tonsillectomy Haemorrhage Requiring Surgery by Suturing the Faucial Pillars: A Retrospective Analysis

Background

The tonsillectomy is one of the most frequently performed surgical procedures. Given the comparatively frequent postsurgical bleeding associated with this procedure, particular attention has been paid to reduction of the postoperative bleeding rate. In 2006, we introduced routine suturing of the faucial pillars at our clinic to reduce postoperative haemorrhage.

Methods

Two groups from the years 2003–2005 (n = 1000) and 2007–2009 (n = 1000) have been compared. We included all patients who had an elective tonsillectomy due to a benign, non-acute inflammatory tonsil illness. In the years 2007–2009, we additionally sutured the faucial pillars after completing haemostasis. For primary haemostasis we used suture ligation and bipolar diathermy.

Results

The rate of bleeding requiring second surgery for haemostasis was 3.6% in 2003–2005 but only 2.0% in 2007–2009 (absolute risk reduction 1.6% (95% CI 0.22%–2.45%, p = 0.04)). The median surgery time—including adenoidectomy and paracentesis surgery—increased from 25 to 31 minutes (p<0.01).

Conclusions

We have been able to substantiate that suturing of the faucial pillars nearly halves the rate of postoperative haemorrhage. Surgery takes 8 minutes longer on average. Bleeding occurs later, mostly after 24 h. The limitations of this study relate to its retrospective character and all the potential biases related to observational studies.     

Comparative GC–FID and GC–MS analysis of the mono and sesquiterpene secondary metabolites produced by the field grown and micropropagated plants of <Emphasis Type="Italic">Artemisia amygdalina</Emphasis> Decne

The essential oil obtained by hydrodistillation from the leaves of micropropagated plants of Artemisia amygdalina was analyzed by capillary GC–FID and GC–MS and compared with that obtained from the leaves of field growing parent plants. The oil yield from the micropropagated plants was lower (0.05% v/w) than the oil yield obtained from field-grown plants (0.2% v/w). The major constituents of the field-grown plants were p-cymene (21.0%), 1,8-cineole (24.9%), α-terpineol (5.9%), β-caryophyllene (4.7%), germacrene D (4.0%), while as the major constituents from the micropropagated plants were p-cymene (11.3%),1,8-cineole (10.2%), borneol (7.9%), α-longipinene (5.5%), α-copaene (5.5%) and β-caryophyllene (17%). The essential oil from field-grown plant was dominated by the presence of oxygenated monoterpenes (41.5%), monoterpene hydrocarbons (35.9%) and sesquiterpene hydrocarbons (16.3%) while as the essential oil of micropropagated plants was characterized by sesquiterpene hydrocarbons (40.0%), oxygenated monoterpenes (25.2%) and monoterpene hydrocarbons (21.6%).