Hydrogen peroxide signaling through tumor necrosis factor receptor 1 leads to selective activation of c-Jun N-terminal kinase

Binding of tumor necrosis factor-alpha (TNFalpha) to its receptor, TNF-R1, results in the activation of inhibitor of kappaB kinase (IKK) and c-Jun N-terminal kinase (JNK) pathways that are coordinately regulated and important in survival and death. We demonstrated previously that in response to hydrogen peroxide (H2O2), the ability of TNFalpha to activate IKK in mouse lung epithelial cells (C10) was inhibited and that H2O2 alone was sufficient to activate JNK and induce cell death. In the current study, we investigated the involvement of TNF-R1 in H2O2-induced JNK activation. In lung fibroblasts from TNF-R1-deficient mice the ability of H2O2 to activate JNK was inhibited compared with fibroblasts from control mice. Additionally, in C10 cells expressing a mutant form of TNF-R1, H2O2-induced JNK activation was also inhibited. Immunoprecipitation of TNF-R1 revealed that in response to H2O2, the adapter proteins, TRADD and TRAF2, and JNK were recruited to the receptor. However, expression of the adaptor protein RIP, which is essential for IKK activation by TNFalpha, was decreased in cells exposed to H2O2, and its chaperone Hsp90 was cleaved. Furthermore, data demonstrating that expression of TRAF2 was not affected by H2O2 and that overexpression of TRAF2 was sufficient to activate JNK provide an explanation for the inability of H2O2 to activate IKK and for the selective activation of JNK by H2O2. Our data demonstrate that oxidative stress interferes with IKK activation while promoting JNK signaling, creating a signaling imbalance that may favor apoptosis.     

Study on quantitative structure-toxicity relationships of benzene derivatives acting by narcosis

Hydrophobicity (logP) as well as quantiative structure-toxicity relationships (QSTRs) of some benzene derivatives acting by narcosis have been established based on narcotic mechanisms of action and toxicity data to the fathead minnow, Daphnia magna and Vibrio fischeri using information-theoretic topological index (Id). Excellent results are obtained in multiparametric regression upon introduction of dummy parameters (indicator variables). Consistent increase in R(2)(A) values indicated that inspite of collinarity between Id and one of the indicator variables (I(3-6)) the proposed models are statistically significant.     

K(+) versus Na(+) ions in a K channel selectivity filter: a simulation study

Molecular dynamics simulations of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal significant differences in interactions of the selectivity filter with K(+) compared with Na(+) ions. K(+) ions and water molecules within the filter undergo concerted single-file motion in which they translocate between adjacent sites within the filter on a nanosecond timescale. In contrast, Na(+) ions remain bound to sites within the filter and do not exhibit translocation on a nanosecond timescale. Furthermore, entry of a K(+) ion into the filter from the extracellular mouth is observed, whereas this does not occur for a Na(+) ion. Whereas K(+) ions prefer to sit within a cage of eight oxygen atoms of the filter, Na(+) ions prefer to interact with a ring of four oxygen atoms plus two water molecules. These differences in interactions in the selectivity filter may contribute to the selectivity of KcsA for K(+) ions (in addition to the differences in dehydration energy between K(+) and Na(+)) and the block of KcsA by internal Na(+) ions. In our simulations the selectivity filter exhibits significant flexibility in response to changes in ion/protein interactions, with a somewhat greater distortion induced by Na(+) than by K(+) ions.     

Some aspects of chemical ionization mass spectroscopy using ammonia as reagent gas: A valuable technique for biomedical and natural products studies

The use of ammonia as reagent gas increases considerably the utility of chemical ionization mass spectroscopic (ci-ms) analysis: compounds of biological interest, such as steroid hormones, bile acids, prostaglandins, phospholipids, alkaloids, antibiotics, etc., display strong pseudomolecular ions (mostly M⁺ + 18). The need for derivatization and/or chromatographic purification of many types of compounds is sharply reduced. Ammonium carbonate or ¹⁵NH₄Cl can be introduced into the direct probe for obtaining satisfactory ci-ms(NH₃) spectra. Bile salts and some bile acid conjugates can be studied without derivatization. Potassium penicillanate gives a strong peak corresponding to the free acid + NH₄⁺. Deproteinized blood samples provide a detailed picture of individual components, such as triglycerides, lysolecithins, cholesterol esters, etc. Frag-mentation patterns for structural information can be generated by adding argon to ammonia. One shortcoming of the ci-ms(NH₃) method is the progressive replacement of halogen with hydrogen.     

Structure and dynamics of K channel pore-lining helices: a comparative simulation study

Isolated pore-lining helices derived from three types of K-channel have been analyzed in terms of their structural and dynamic features in nanosecond molecular dynamics (MD) simulations while spanning a lipid bilayer. The helices were 1) M1 and M2 from the bacterial channel KcsA (Streptomyces lividans), 2) S5 and S6 from the voltage-gated (Kv) channel Shaker (Drosophila melanogaster), and 3) M1 and M2 from the inward rectifier channel Kir6.2 (human). In the case of the Kv and Kir channels, for which x-ray structures are not known, both short and long models of each helix were considered. Each helix was incorporated into a lipid bilayer containing 127 palmitoyloleoylphosphatidylcholine molecules, which was solvated with approximately 4000 water molecules, yielding approximately 20, 000 atoms in each system. Nanosecond MD simulations were used to aid the definition of optimal lengths for the helix models from Kv and Kir. Thus the study corresponds to a total simulation time of 10 ns. The inner pore-lining helices (M2 in KcsA and Kir, S6 in Shaker) appear to be slightly more flexible than the outer pore-lining helices. In particular, the Pro-Val-Pro motif of S6 results in flexibility about a molecular hinge, as was suggested by previous in vacuo simulations (, Biopolymers. 39:503-515). Such flexibility may be related to gating in the corresponding intact channel protein molecules. Analysis of H-bonds revealed interactions with both water and lipid molecules in the water/bilayer interfacial region. Such H-bonding interactions may lock the helices in place in the bilayer during the folding of the channel protein (as is implicit in the two-stage model of membrane protein folding). Aromatic residues at the extremities of the helices underwent complex motions on both short (<10 ps) and long (>100 ps) time scales.     

Proline-induced hinges in transmembrane helices: possible roles in ion channel gating

A number of ion channels contain transmembrane (TM) alpha-helices that contain proline-induced molecular hinges. These TM helices include the channel-forming peptide alamethicin (Alm), the S6 helix from voltage-gated potassium (Kv) channels, and the D5 helix from voltage-gated chloride (CLC) channels. For both Alm and KvS6, experimental data implicate hinge-bending motions of the helix in an aspect of channel gating. We have compared the hinge-bending motions of these TM helices in bilayer-like environments by multi-nanosecond MD simulations in an attempt to describe motions of these helices that may underlie possible modes of channel gating. Alm is an alpha-helical channel-forming peptide, which contains a central kink associated with a Gly-x-x-Pro motif in its sequence. Simulations of Alm in a TM orientation for 10 ns in an octane slab indicate that the Gly-x-x-Pro motif acts as a molecular hinge. The S6 helix from Shaker Kv channels contains a Pro-Val-Pro motif. Modeling studies and recent experimental data suggest that the KvS6 helix may be kinked in the vicinity of this motif. Simulations (10 ns) of an isolated KvS6 helix in an octane slab and in a POPC bilayer reveal hinge-bending motions. A pattern-matching approach was used to search for possible hinge-bending motifs in the TM helices of other ion channel proteins. This uncovered a conserved Gly-x-Pro motif in TM helix D5 of CLC channels. MD simulations of a model of hCLC1-D5 spanning an octane slab suggest that this channel also contains a TM helix that undergoes hinge-bending motion. In conclusion, our simulations suggest a model in which hinge-bending motions of TM helices may play a functional role in the gating mechanisms of several different families of ion channels.     

Targeted Intracellular Delivery of Resveratrol to Glioblastoma Cells Using Apolipoprotein E-Containing Reconstituted HDL as a Nanovehicle

The objective of this study is to transport and deliver resveratrol to intracellular sites using apolipoprotein E3 (apoE3). Reconstituted high-density lipoprotein (rHDL) bearing resveratrol (rHDL/res) was prepared using phospholipids and the low-density lipoprotein receptor (LDLr)-binding domain of apoE3. Biophysical characterization revealed that resveratrol was partitioned into the phospholipid bilayer of discoidal rHDL/res particles (~19 nm diameter). Co-immunoprecipitation studies indicated that the LDLr-binding ability of apoE3 was retained. Cellular uptake of resveratrol to intracellular sites was evaluated in glioblastoma A-172 cells by direct fluorescence using chemically synthesized NBD-labeled resveratrol (res/NBD) embedded in rHDL/res. Competition and inhibition studies indicate that the uptake is by receptor mediated endocytosis via the LDLr, with co-localization of apoE3 and res/NBD in late endosomes/lysosomes. We propose that rHDL provides an ideal hydrophobic milieu to sequester resveratrol and that rHDL containing apoE3 serves as an effective “nanovehicle” to transport and deliver resveratrol to targeted intracellular sites.     

Heart Failure Care in Low- and Middle-Income Countries: A Systematic Review and Meta-Analysis

Background

Heart failure places a significant burden on patients and health systems in high-income countries. However, information about its burden in low- and middle-income countries (LMICs) is scant. We thus set out to review both published and unpublished information on the presentation, causes, management, and outcomes of heart failure in LMICs.

Methods and Findings

Medline, Embase, Global Health Database, and World Health Organization regional databases were searched for studies from LMICs published between 1 January 1995 and 30 March 2014. Additional unpublished data were requested from investigators and international heart failure experts. We identified 42 studies that provided relevant information on acute hospital care (25 LMICs; 232,550 patients) and 11 studies on the management of chronic heart failure in primary care or outpatient settings (14 LMICs; 5,358 patients). The mean age of patients studied ranged from 42 y in Cameroon and Ghana to 75 y in Argentina, and mean age in studies largely correlated with the human development index of the country in which they were conducted (r = 0.71, p<0.001). Overall, ischaemic heart disease was the main reported cause of heart failure in all regions except Africa and the Americas, where hypertension was predominant. Taking both those managed acutely in hospital and those in non-acute outpatient or community settings together, 57% (95% confidence interval [CI]: 49%–64%) of patients were treated with angiotensin-converting enzyme inhibitors, 34% (95% CI: 28%–41%) with beta-blockers, and 32% (95% CI: 25%–39%) with mineralocorticoid receptor antagonists. Mean inpatient stay was 10 d, ranging from 3 d in India to 23 d in China. Acute heart failure accounted for 2.2% (range: 0.3%–7.7%) of total hospital admissions, and mean in-hospital mortality was 8% (95% CI: 6%–10%). There was substantial variation between studies (p<0.001 across all variables), and most data were from urban tertiary referral centres. Only one population-based study assessing incidence and/or prevalence of heart failure was identified.

Conclusions

The presentation, underlying causes, management, and outcomes of heart failure vary substantially across LMICs. On average, the use of evidence-based medications tends to be suboptimal. Better strategies for heart failure surveillance and management in LMICs are needed. Please see later in the article for the Editors'' Summary     

Hairy root cultures: A suitable biological system for studying secondary metabolic pathways in plants

Hairy roots, a plant disease caused by Agrobacterium rhizogenes, show distinctive features such as high growth rate, unlimited branching, and biochemical and genetic stability. Hairy roots resemble normal roots in terms of differentiated morphology and biosynthetic machinery, producing similar secondary metabolites compared to wild‐type roots. As a result, hairy roots have been a topic of intense research for the past three decades, fueling innumerable attempts to develop in vitro hairy root cultures for a large number of plants for the commercial‐scale production of secondary metabolites. The same characteristics have now led to further applications, such as using hairy root cultures as experimental systems for secondary metabolic pathway elucidation studies. Although the trend is relatively new, it has already gained momentum. This review summarizes these developments. The following discussion focuses on the rationale and advantages of using hairy root cultures for secondary metabolic pathway elucidation studies, the methods used, and the results that have been obtained so far.     

Fermentation and downstream process for high yield production of Plasmodium falciparum recombinant HRP II protein and its application in diagnosis

Malaria represents the world’s greatest public health problem in terms of number of people affected, levels of morbidity and mortality in tropical and subtropical countries. Malaria parasites are members of the Apicomplexa, family of Plasmodiidae. Histidine-rich protein-II secreted by Plasmodium falciparum is known to be a compelling marker in malaria diagnosis and follow-up. In our present study, we have optimized the batch fermentation and downstream process for large scale production of recombinant P. falciparum HRP-II 62 kDa protein for diagnostic application. The culture broth was effectively induced with IPTG twice at different time intervals to sustain induction for a long period. Batch fermentation resulted in a wet weight of 61.34 g/L and dry cell biomass 12.81 g/L. With the improved downstream process, purified recombinant protein had a yield of 304.60 mg/L. The authenticity of the purified recombinant protein was confirmed via western blotting using indigenously developed HRP-II specific monoclonal antibodies and known positive human clinical sera samples. Further, the reactivity of recombinant HRP-II protein was validated using commercially available immuno chromatographic strips. Indirect ELISA using recombinant purified protein recognized the P. falciparum specific antibodies in suspected human sera samples. Our results clearly suggest that the recombinant HRP-II protein produced via batch fermentation has immense potential for routine diagnostic application.