Sugar hydrolases of the infant rat intestine and their arrangement of the brush border membrane.

Lactase and maltase, the predominant sugar hydrolases associated with the intestinal brush bordermembrane of the suckling rat, were purified essentially free of the other to near homogeneity (lactase at specific activity 23, maltase at specific activity 58), and their specific physiocochemical properties determined. Antisera prepared to each showed by immunodiffusion a single common precipitin line with pure enzyme and solubilized proteins of the brush border membrane. Brush border membranes were purified 26--35-fold from infant rat intestine. Membranes prepared from 10-day-old rats contained 32% protein, 43% lipid and 25% carbohydrate with lactase and maltase estimated to comprise in excess of 10% and 2%, respectively, of the membrane protein. Immunotitration curves of lactase and maltase showed equivalent antibody binding by the membrane-bound and free enzyme forms. Furthermore, antibody binding to one enzyme did not affect the immunotitration curve or the extractability (by papain or Triton X-100) of the other membrane-bound enzyme. It was concluded that the lactase and maltase molecules are attached singly on the external membrane surface in a spatially independent manner with their antigenic sites as freely available to antibody binding as exhibited by their papain-solubilized counterparts.     

Neurospora endoexonuclease and its inactive (precursor?) form.

Two nuclease activities which were shown previously to copurify from extracts of log-phase Neurospora mycleia, a single-strand specific endonuclease activity (with DNA and RNA), and a strand nonspecific exonuclease activity (with DNA only) have been found to be associated with a single polypeptide. The enzyme has therefore been classified as an endoexonuclease. In logphase extracts, about 75% of this enzyme was found to exist in an inactive form which was activated in vitro either by endogenous phenylmethylsulfonyl fluoride sensitive proteinase(s) or by exogenous trypsin. The inactive form of endoexonuclease has been purified 45-fold in 15% yield free of the active enzyme. On electrophoresis in 6 M urea--polyacrylamide gels, it migrated at a much slower rate than the active enzyme, indicating that it is a less acidic and(or) larger protein than the active nuclease. The strong adsorption of this inactive enzyme on octyl-Sepharose suggests that the protein may have a relatively large hydrophobic domain. The protein may be a precursor of the active enzyme (a pronuclease) or a strong complex of enzyme with a proteinaceous inhibitor that is not dissociated in 6 M urea or during a variety of chromatographic procedures.     

Apoptosis Repressor with a CARD Domain (ARC) Restrains Bax-Mediated Pathogenesis in Dystrophic Skeletal Muscle

Myofiber wasting in muscular dystrophy has largely been ascribed to necrotic cell death, despite reports identifying apoptotic markers in dystrophic muscle. Here we set out to identify the contribution of canonical apoptotic pathways to skeletal muscle degeneration in muscular dystrophy by genetically deleting a known inhibitor of apoptosis, apoptosis repressor with a card domain (Arc), in dystrophic mouse models. Nol3 (Arc protein) genetic deletion in the dystrophic Sgcd or Lama2 null backgrounds showed exacerbated skeletal muscle pathology with decreased muscle performance compared with single null dystrophic littermate controls. The enhanced severity of the dystrophic phenotype associated with Nol3 deletion was caspase independent but dependent on the mitochondria permeability transition pore (MPTP), as the inhibitor Debio-025 partially rescued skeletal muscle pathology in Nol3 ^-/- Sgcd ^-/- double targeted mice. Mechanistically, Nol3 ^-/- Sgcd ^-/- mice showed elevated total and mitochondrial Bax protein levels, as well as greater mitochondrial swelling, suggesting that Arc normally restrains the cell death effects of Bax in skeletal muscle. Indeed, knockdown of Arc in mouse embryonic fibroblasts caused an increased sensitivity to cell death that was fully blocked in Bax Bak1 (genes encoding Bax and Bak) double null fibroblasts. Thus Arc deficiency in dystrophic muscle exacerbates disease pathogenesis due to a Bax-mediated sensitization of mitochondria-dependent death mechanisms.     

Effectiveness of Inactivated Influenza Vaccines in Preventing Influenza-Associated Deaths and Hospitalizations among Ontario Residents Aged ≥65 Years: Estimates with Generalized Linear Models Accounting for Healthy Vaccinee Effects

Background

Estimates of the effectiveness of influenza vaccines in older adults may be biased because of difficulties identifying and adjusting for confounders of the vaccine-outcome association. We estimated vaccine effectiveness for prevention of serious influenza complications among older persons by using methods to account for underlying differences in risk for these complications.

Methods

We conducted a retrospective cohort study among Ontario residents aged ≥65 years from September 1993 through September 2008. We linked weekly vaccination, hospitalization, and death records for 1.4 million community-dwelling persons aged ≥65 years. Vaccine effectiveness was estimated by comparing ratios of outcome rates during weeks of high versus low influenza activity (defined by viral surveillance data) among vaccinated and unvaccinated subjects by using log-linear regression models that accounted for temperature and time trends with natural spline functions. Effectiveness was estimated for three influenza-associated outcomes: all-cause deaths, deaths occurring within 30 days of pneumonia/influenza hospitalizations, and pneumonia/influenza hospitalizations.

Results

During weeks when 5% of respiratory specimens tested positive for influenza A, vaccine effectiveness among persons aged ≥65 years was 22% (95% confidence interval [CI], −6%–42%) for all influenza-associated deaths, 25% (95% CI, 13%–37%) for deaths occurring within 30 days after an influenza-associated pneumonia/influenza hospitalization, and 19% (95% CI, 4%–31%) for influenza-associated pneumonia/influenza hospitalizations. Because small proportions of deaths, deaths after pneumonia/influenza hospitalizations, and pneumonia/influenza hospitalizations were associated with influenza virus circulation, we estimated that vaccination prevented 1.6%, 4.8%, and 4.1% of these outcomes, respectively.

Conclusions

By using confounding-reducing techniques with 15 years of provincial-level data including vaccination and health outcomes, we estimated that influenza vaccination prevented ∼4% of influenza-associated hospitalizations and deaths occurring after hospitalizations among older adults in Ontario.     

Structural Basis for Highly Effective HIV-1 Neutralization by CD4-Mimetic Miniproteins Revealed by 1.5 Å Cocrystal Structure of gp120 and M48U1

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Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

The Role of TLR2, TLR4 and CD14 Genetic Polymorphisms in Gastric Carcinogenesis: A Case-Control Study and Meta-Analysis

Background

In addition to Helicobacter pylori infection, host genetic factors contribute to gastric cancer (GC). Recognition of H. pylori is known to involve Toll-like receptors (TLR), which subsequently leads to activation of NF-κB. Thus, the overall aim of this study was to estimate for the first time the pooled effect size of polymorphisms in TLR2, TLR4 and CD14 on GC development through a meta-analysis.

Methods

A case-control study comprising 284 ethnic Chinese individuals (70 non-cardia GC cases and 214 functional dyspepsia controls) was conducted for the genotyping of TLR2 -196 to -174del, CD14 -260 C/T and TLR4 rs11536889 using PCR, RT-PCR and mass spectrometry. Case-control studies of TLR2, TLR4 and CD14 polymorphisms and GC were searched up to June 2012. Pooled odds ratios and 95% confidence intervals were obtained by means of the random effects model.

Results

In our ethnic Chinese case-control study, the TLR4 rs11536889 C allele increased the risk of GC (OR: 1.89, 95%CI: 1.23–2.92) while the CD14 -260 T allele was protective (OR: 0.62, 95%CI: 0.42–0.91). TLR2 -196 to -174 increased the risk of GC only in H. pylori-infected individuals (OR: 3.10, 95%CI: 1.27–7.60). In the meta-analysis, TLR4 Asp299Gly showed borderline results in the general analysis (pooled OR: 1.58, 95%CI: 0.98–2.60), nevertheless, stratified analysis by ethnicity showed that the mutant allele was a definitive risk factor for GC in Western populations (pooled OR: 1.87, 95%CI: 1.31–2.65). There was a potential association between the TLR2 -196 to -174 deletion allele and GC in Japanese (pooled OR: 1.18, 95%CI: 0.96–1.45). TLR4 Thr399Ile did not provide significant results.

Conclusions

TLR4 rs11536889 and CD14 -260 C/T are associated with non-cardia GC in Chinese. Based on our meta-analysis, the TLR signalling pathway is involved in gastric carcinogenesis, TLR4 Asp299Gly and TLR2 -196 to -174del showing associations with GC in an ethnic-specific manner.     

Left Atrial Structure and Function in Heart Failure with Preserved Ejection Fraction: A RELAX Substudy

Given the emerging recognition of left atrial structure and function as an important marker of disease in heart failure with preserved ejection fraction (HF-pEF), we investigated the association between left atrial volume and function with markers of disease severity and cardiac structure in HF-pEF. We studied 100 patients enrolled in the PhosphdiesteRasE-5 Inhibition to Improve CLinical Status and EXercise Capacity in Diastolic Heart Failure (RELAX) trial who underwent cardiac magnetic resonance (CMR), cardiopulmonary exercise testing, and blood collection before randomization. Maximal left atrial volume index (LAVi; N = 100), left atrial emptying fraction (LAEF; N = 99; including passive and active components (LAEF_P, LAEF_A; N = 80, 79, respectively) were quantified by CMR. After adjustment for multiple testing, maximal LAVi was only associated with age (ρ = 0.39), transmitral filling patterns (medial E/e’ ρ = 0.43), and N-terminal pro-BNP (NT-proBNP; ρ = 0.65; all p<0.05). Lower LAEF was associated with older age, higher transmitral E/A ratio and higher NT-proBNP. Peak VO₂ and V_E/VCO₂ slope were not associated with left atrial structure or function. After adjustment for age, sex, transmitral E/A ratio, CMR LV mass, LV ejection fraction, and creatinine clearance, NT-proBNP remained associated with maximal LAVi (β = 0.028, p = 0.0007) and total LAEF (β = -0.033, p = 0.001). Passive and active LAEF were most strongly associated with age and NT-proBNP, but not gas exchange or other markers of ventricular structure or filling properties. Left atrial volume and emptying function are associated most strongly with NT-proBNP and diastolic filling properties, but not significantly with gas exchange, in HFpEF. Further research to explore the relevance of left atrial structure and function in HF-pEF is warranted.     

Toward a Low‐Cost Artificial Leaf: Driving Carbon‐Based and Bifunctional Catalyst Electrodes with Solution‐Processed Perovskite Photovoltaics

Molecular hydrogen can be generated renewably by water splitting with an “artificial‐leaf device”, which essentially comprises two electrocatalyst electrodes immersed in water and powered by photovoltaics. Ideally, this device should operate efficiently and be fabricated with cost‐efficient means using earth‐abundant materials. Here, a lightweight electrocatalyst electrode, comprising large surface‐area NiCo₂O₄ nanorods that are firmly anchored onto a carbon–paper current collector via a dense network of nitrogen‐doped carbon nanotubes is presented. This electrocatalyst electrode is bifunctional in that it can efficiently operate as both anode and cathode in the same alkaline solution, as quantified by a delivered current density of 10 mA cm^?2 at an overpotential of 400 mV for each of the oxygen and hydrogen evolution reactions. By driving two such identical electrodes with a solution‐processed thin‐film perovskite photovoltaic assembly, a wired artificial‐leaf device is obtained that features a Faradaic H₂ evolution efficiency of 100%, and a solar‐to‐hydrogen conversion efficiency of 6.2%. A detailed cost analysis is presented, which implies that the material‐payback time of this device is of the order of 100 days.