Role of partial protein unfolding in alcohol‐induced protein aggregation

Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease‐related as well as nondisease‐related proteins. Here we probed the biophysical mechanisms underlying alcohol‐induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site‐specific optical probes, two‐dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins. Proteins 2010. © Wiley‐Liss, Inc.     

Mountain biodiversity conservation and management: a paradigm shift in policies and practices in the Hindu Kush-Himalayas

Mountains occupy 24% of the global land surface area and are home to 12% of the world’s population. They have ecological, aesthetic, and socioeconomic significance, not only for people living in mountain areas, but for those living beyond. Mountains need specific attention for their contribution to global goods and services, especially by developing and implementing mountain specific policies. Conservation policies have evolved from the protection of charismatic species, to habitat and ecosystem/landscape conservation, and, finally, to people-oriented conservation approaches. This paper, with particular reference to paradigm shifts in the Hindu Kush-Himalayan (HKH) region, discusses the evolution of conservation policies, developments in conservation practices, the status of protected area management, wetland conservation initiatives and the landscape approach, community-based conservation initiatives, and the convergence of policies and practices. In the HKH region, conservation efforts now adopt participatory approaches, implement policies of decentralised governance for biodiversity management, and empower local communities in biodiversity management. The paradigm shift in the policies and practices related to conservation has been gradual and has included the acceptance of communities as an integral part of national level conservation initiatives, together with the integration of many global conventions. There are many successful pilots in the HKH region that deserve upscaling by the countries from the region. Realising the importance of mountains as hotspots of biodiversity, and due to their role as providers of global goods and services, the Convention on Biological Diversity adopted the Programme of Work on Mountain Biodiversity. Such a decision specific to mountains provides enormous opportunities for both conservation and development. Recent challenges posed by climate change need to be integrated into overall biodiversity conservation and management agendas, especially in mountain areas. The HKH region has been identified as a blank spot for data by the Inter-Governmental Panel on Climate Change, indicating the need to develop regional database and sharing mechanisms. This is a tall task, but one that holds enormous opportunity for the HKH countries and institutions with regional mandates to address the emerging challenges of climate change on biodiversity conservation by reducing scientific uncertainty.     

15NH/D-SOLEXSY experiment for accurate measurement of amide solvent exchange rates: application to denatured drkN SH3

Amide solvent exchange rates are regarded as a valuable source of information on structure/dynamics of unfolded (disordered) proteins. Proton-based saturation transfer experiments, normally used to measure solvent exchange, are known to meet some serious difficulties. The problems mainly arise from the need to (1) manipulate water magnetization and (2) discriminate between multiple magnetization transfer pathways that occur within the proton pool. Some of these issues are specific to unfolded proteins. For example, the compensation scheme used to cancel the Overhauser effect in the popular CLEANEX experiment is not designed for use with unfolded proteins. In this report we describe an alternative experimental strategy, where amide ¹⁵N is used as a probe of solvent exchange. The experiment is performed in 50% H₂O–50% D₂O solvent and is based on the (HACACO)NH pulse sequence. The resulting spectral map is fully equivalent to the conventional HSQC. To fulfill its purpose, the experiment monitors the conversion of deuterated species, ¹⁵N^D, into protonated species, ¹⁵N^H, as effected by the solvent exchange. Conceptually, this experiment is similar to EXSY which prompted the name of ¹⁵N^H/D-SOLEXSY (SOLvent EXchange SpectroscopY). Of note, our experimental scheme, which relies on nitrogen rather than proton to monitor solvent exchange, is free of the complications described above. The developed pulse sequence was used to measure solvent exchange rates in the chemically denatured state of the drkN SH3 domain. The results were found to correlate well with the CLEANEX-PM data, r = 0.97, thus providing a measure of validation for both techniques. When the experimentally measured exchange rates are converted into protection factors, most of the values fall in the range 0.5–2, consistent with random-coil behavior. However, elevated values, ca. 5, are obtained for residues R38 and A39, as well as the side-chain indole of W36. This is surprising, given that high protection factors imply hydrogen bonding or hydrophobic burial not expected to occur in a chemically denatured state of a protein. We, therefore, hypothesized that elevated protection factors are an artefact arising from the calculation of the reference (random-coil) exchange rates. To confirm this hypothesis, we prepared samples of several short peptides derived from the sequence of the drkN SH3 domain; these samples were used to directly measure the reference exchange rates. The revised protection factors obtained in this manner proved to be close to 1.0. These results also have implications for the more compact unfolded state of drkN SH3, which appears to be fully permeable to water as well, with no manifestations of hydrophobic burial.     

Isolation and characterization of a <Emphasis Type="Italic">Glutamate decarboxylase</Emphasis> (GAD) gene and their differential expression in response to abiotic stresses from <Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

Glutamate decarboxylase (GAD) catalyzes the conversion of l-glutamate to γ-aminobutyric acid (GABA). A full-length cDNA encoding GAD (designated as PgGAD) was isolated and characterized from the root of Panax ginseng C. A. Meyer. The length cDNA of PgGAD was 1881 bp and contained a 1491 bp open reading frame (ORF) encoding a glutamate decarboxylase protein of 496 amino acids, possessing a Ser-X-X-Lys active site, which belongs to the GAD group. The deduced amino acid sequence of the PgGAD was classified in the plant GAD family and has 76–85% high similarity with other plants as like petunia, Arabidopsis, tomato. Secondary structure of PgGAD was predicted by using SOPMA software program. Southern blot analysis of genomic DNA suggests that, there is more than one copy of the PgGAD gene. The organ specific gene expression pattern also studied in P. ginseng seedlings, in which the stem showed elevated expression than root, leaf, bud and rhizomes. Along with this, we also confirmed the gene expression of PgGAD under various abiotic stresses like temperature stress, osmotic stress, anoxia, oxidative stress, and mechanical damage. Temporal analysis of gene expression except exposure of oxidative stress revealed an enhanced expression after each stresses. The enzyme activity of PgGAD was stimulated to 2-fold under cold stress.     

Co-Existence of Multidrug-Resistant and -Susceptible Strains of Pseudomonas aeruginosa from a Single Clinical Isolate

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and difficult to treat due to acquired-resistance to multiple antibiotics. A pair of strains, M38100A and M38100B, previously identified from a single clinical isolate of P. aeruginosa was investigated to understand phenotypic and genotypic characteristics. Results revealed that the pair of strains was very similar for serum susceptibility, growth rate in a complex medium (Luria–Bertani), RAPD-genotype profiles, status of genes encoding type III secretion toxins, and no extra-chromosomal DNA. However, antibiotic susceptibility of the strain M38100B showed resistant to all tested-antibiotics while the strain M38100A showed susceptible to the same tested-antibiotics as similar levels of P. aeruginosa PAO1. The strain M38100B exhibited no growth in a minimal medium as a sole carbon and nitrogen source of glutamate while the strain M38100A grew well in the same minimal medium. These results suggest that multidrug resistance of the strain M38100B may be caused by multiple mutations on its genomic DNA and a precursor stage for a homogeneous multidrug resistant population.     

Atrial local Ca signaling and inositol 1,4,5-trisphosphate receptors

In atrial myocytes lacking t-tubules, action potential triggers junctional Ca²⁺ releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca²⁺ signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in atrial myocytes, and show our new findings on the role of IP₃R subtype in the regulation of spontaneous focal Ca²⁺ releases in the compartmentalized areas of atrial myocytes. The Ca²⁺ sparks, representing focal Ca²⁺ releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca²⁺ sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP₃Rs compared with ventricular cells and possess significant levels of type 1 IP₃R (IP₃R1) and type 2 IP₃R (IP₃R2). Over the last decade the roles of atrial IP₃R on the enhancement of Ca²⁺-induced Ca²⁺ release and arrhythmic Ca²⁺ releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca²⁺ imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP₃R1 in the maintenance of peri-nuclear and non-junctional Ca²⁺ sparks via stimulating a posttranslational organization of RyR clusters.     

6-(4′-Aryloxy-phenyl)vinyl-1,2,4-trioxanes: A new series of orally active peroxides effective against multidrug-resistant Plasmodium yoelii in Swiss mice

A new series of 6-(4′-aryloxy-phenyl)vinyl-1,2,4-trioxanes 10a–d, 11a–d, and 12a–d have been synthesized and evaluated for their antimalarial activity against multidrug-resistant Plasmodium yoelii in Swiss mice by oral route. Trioxanes 10b and 10c, the two most active compounds of the series, provided 100% protection to the infected mice at 48 mg/kg × 4 days. Clinically useful drug β-arteether provided 100% and 20% protection at 48 mg/kg × 4 days and 24 mg/kg × 4 days, respectively, in this model.     

Evaluation of amide replacements in CCR5 antagonists as a means to increase intrinsic permeability. Part 2: SAR optimization and pharmacokinetic profile of a homologous azacyle series

Replacement of a secondary amide with a piperidine or azetidine moiety in a series of CCR5 antagonists led to the discovery of compounds with increased intrinsic permeability. This effort led to the identification of a potent CCR5 antagonist which exhibited an improved in vivo pharmacokinetic profile.     

A conceptual framework to analyse the land-use/land-cover changes and its impact on phytodiversity: a case study of North Andaman Islands,India

Phytodiversity is affected both by natural and anthropogenic factors and in Island ecosystems these impacts can devastate or reduce diversity, if the native vegetation is lost. In addition to rich species richness and diversity, Island systems are the sites of high endemism and any threat to these ecosystems will consequently lead to loss and extinction of species. To understand the dynamics including feedbacks of these changes in phytodiversity of North Andaman Islands, a conceptual framework is proposed which focuses on understanding the land-use and land-cover changes and its impact with phytodiversity. In considering land-use and land-cover changes this work highlights the direct and indirect drivers of changes—socio-economic, biophysical and climatic factors. Migration of population, their socio economic needs and government policies were identified as major driving forces threatening the phytodiversity of these Islands. Apart from human beings, natural disasters like tsunami and introduced herbivorous animals like elephants also contributed to forest destruction in these Islands. The integrated analysis based on such framework will provide insights for holistic resource management including ecological conservation.