New biochemical insights into the dynamics of water molecules at the GMP or IMP binding site of human GMPR enzyme: A molecular dynamics study

Human GMP reductase (hGMPR) enzyme is involved in a cellular metabolic pathway, converting GMP into IMP, and also it is an important target for anti-leukemic agents. Present computational investigations explain dynamical behavior of water molecules during the conformational transition process from GMP to IMP using molecular dynamics simulations. Residues at substrate-binding site of cancerous protein (PDB Id. 2C6Q) are mostly more dynamic in nature than the normal protein (PDB Id. 2BLE). Nineteen conserved water molecules are identified at the GMP/IMP binding site and are classified as (i) conserved stable dynamic and (ii) infrequent dynamic. Water molecules W11, W14, and W16 are classified as conserved stable dynamic due to their immobile character, whereas remaining water molecules (W1, W2, W3, W4, W5, W7, W8, W9, W10, W12, W13, W15, W17, W18, and W19) are infrequent with dynamic nature. Entrance or displacement of these infrequent water molecules at GMP/IMP sites may occur due to forward and backward movement of reference residues involving ligands. Four water molecules of hGMPR-I and nine water molecules of hGMPR-II are observed in repetitive transitions from GMP to IMP pathway, which indicates discrimination between two isoforms of hGMPRs. Water molecules in cancerous protein are more dynamic and unstable compared to normal protein. These water molecules execute rare dynamical events at GMP binding site and could assist in detailed understanding of conformational transitions that influence the hGMPR's biological functionality. The present study should be of interest to the experimental community engaged in leukemia research and drug discovery for CML cancer.     

Myeloperoxidase-dependent Lipid Peroxidation Promotes the Oxidative Modification of Cytosolic Proteins in Phagocytic Neutrophils

Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.     

Biotechnological interventions in sea buckthorn (Hippophae L.): current status and future prospects

Sea buckthorn (Hippophae L., Elaeagnaceae) is an economically and ecologically important medicinal plant comprising of species which are winter hardy, dioecious, wind-pollinated multipurpose shrubs bearing yellow or orange berries with nitrogen-fixing ability. It grows widely in cold regions of Indian Himalayas, China, Russia, Europe and many other countries. It is commonly known as ‘cold desert gold’ due to its high potential as a bio-resource for land reclamation, reducing soil erosion and its multifarious uses. The wild populations are being used for harvesting economic benefits with negligible plantation efforts. Although this plant has many excellent traits, it is still in an early phase of domestication. This woody plant is prone to many pests and diseases which destroy the plants and halt its commercial production. Limited progress has been made for improvement of sea buckthorn through breeding programs due to long juvenile period and lack of QTL linkage map, which makes screening of mapping populations a time-consuming and labor-intensive task. Conventional propagation methods, i.e. seeds, softwood and hardwood cuttings, and suckers are in place but are cumbersome and season dependent. Therefore, application of modern tools of biotechnology needs to be standardized for harnessing maximum benefits from this nutraceutical plant. Improvement of this genus through genetic transformation requires an efficient regeneration system, which is yet to be standardized. Taxonomic status of the genus is controversial and requires more inputs. Taxonomic delineation of species and subspecies and also the breeding programs can be more robustly addressed using molecular markers. This review summarizes the progress made and suggests some future directions of research for this important fruit species.     

Indicator microbes correlate with pathogenic bacteria, yeasts and helminthes in sand at a subtropical recreational beach site

Aims: Research into the relationship between pathogens, faecal indicator microbes and environmental factors in beach sand has been limited, yet vital to the understanding of the microbial relationship between sand and the water column and to the improvement of criteria for better human health protection at beaches. The objectives of this study were to evaluate the presence and distribution of pathogens in various zones of beach sand (subtidal, intertidal and supratidal) and to assess their relationship with environmental parameters and indicator microbes at a non‐point source subtropical marine beach. Methods and Results: In this exploratory study in subtropical Miami (Florida, USA), beach sand samples were collected and analysed over the course of 6 days for several pathogens, microbial source tracking markers and indicator microbes. An inverse correlation between moisture content and most indicator microbes was found. Significant associations were identified between some indicator microbes and pathogens (such as nematode larvae and yeasts in the genus Candida), which are from classes of microbes that are rarely evaluated in the context of recreational beach use. Conclusions: Results indicate that indicator microbes may predict the presence of some of the pathogens, in particular helminthes, yeasts and the bacterial pathogen Staphylococcus aureus including methicillin‐resistant forms. Indicator microbes may thus be useful for monitoring beach sand and water quality at non‐point source beaches. Significance and Impact of the Study: The presence of both indicator microbes and pathogens in beach sand provides one possible explanation for human health effects reported at non‐point sources beaches.     

The significance of lipid composition for membrane activity: new concepts and ways of assessing function

In the last decade or so, it has been realised that membranes do not just have a lipid-bilayer structure in which proteins are embedded or with which they associate. Structures are dynamic and contain areas of heterogeneity which are vital for their formation. In this review, we discuss some of the ways in which these dynamic and heterogeneous structures have implications during stress and in relation to certain human diseases. A particular stress is that of temperature which may instigate adaptation in poikilotherms or appropriate defensive responses during fever in mammals. Recent data emphasise the role of membranes in sensing temperature changes and in controlling a regulatory loop with chaperone proteins. This loop seems to need the existence of specific membrane microdomains and also includes association of chaperone (heat stress) proteins with the membrane. The role of microdomains is then discussed further in relation to various human pathologies such as cardiovascular disease, cancer and neurodegenerative diseases. The concept of modifying membrane lipids (lipid therapy) as a means for treating such pathologies is then introduced. Examples are given when such methods have been shown to have benefit. In order to study membrane microheterogeneity in detail and to elucidate possible molecular mechanisms that account for alteration in membrane function, new methods are needed. In the second part of the review, we discuss ultra-sensitive and ultra-resolution imaging techniques. These include atomic force microscopy, single particle tracking, single particle tracing and various modern fluorescence methods. Finally, we deal with computing simulation of membrane systems. Such methods include coarse-grain techniques and Monte Carlo which offer further advances into molecular dynamics. As computational methods advance they will have more application by revealing the very subtle interactions that take place between the lipid and protein components of membranes - and which are so essential to their function.     

Effects of hypothermia and re-warming on the inflammatory response in a murine multiple hit model of trauma

INTRODUCTION: Although, hypothermia is a frequent event after trauma, it is unclear whether its beneficial or detrimental effects are more important. This study aims to quantify the effects of hypothermia and re-warming on the inflammatory response after fracture/hemorrhage and subsequent fracture stabilization with resuscitation. MATERIALS AND METHODS: Eighty-one male C57Bl/6 mice (aged 8-10 weeks, weighing 22.0+/-3.0 g) underwent femoral fracture and hemorrhage followed by resuscitation and splint fixation of the fracture. Animals were sacrificed 3h after induction of hemorrhage and fracture. Besides a sham group (n=6), four experimental groups were created: A: normothermia (n=12), B: hypothermia after trauma (n=21), C: re-warming after resuscitation and before stabilization (n=21), and D: hypothermia before trauma (n=21). Groups B-D were further subdivided into three subgroups according to the degree of hypothermia (subgroup 1: 35-33 degrees C, subgroup 2: 32.9-30.0 degrees C, and subgroup 3: 29.9-27.0 degrees C). Plasma cytokine (TNF-alpha, IL-6, and IL-10) and chemokine (MCP-1) concentrations were determined by ELISA, pulmonary permeability changes were quantified, and histological analysis of lung and liver tissues was performed. RESULTS: Normothermia resulted in a significantly increased early mortality rate. A significantly increased pro-inflammatory and decreased anti-inflammatory responses were also observed in normothermia as compared to hypothermia. The extent of these changes was most pronounced in the severe hypothermic group. Re-warming after mild hypothermia resulted in a pro-inflammatory response comparable to normothermia. CONCLUSION: Hypothermia has a beneficial effect on early survival after trauma, which appears to be independent of the level of hypothermia and re-warming. Re-warming, however, enhanced the pro-inflammatory response. Further studies with a longer posttraumatic observation period are required to investigate the long term effects of the hypothermia and re-warming-induced changes on the pro- and anti-inflammatory responses.     

Rapid screening of invertebrate predators for multiple prey DNA targets

DNA-based techniques are providing valuable new approaches to tracking predator-prey interactions. The gut contents of invertebrate predators can be analysed using species-specific primers to amplify prey DNA to confirm trophic links. The problem is that each predator needs to be analysed with primers for the tens of potential prey available at a field site, even though the mean number of species detected in each gut may be as few as one or two. Conducting all these PCRs (polymerase chain reactions) is a lengthy process, and effectively precludes the analysis of the hundreds of predators that might be required for a meaningful ecological study. We report a rapid, more sensitive and practical approach. Multiplex PCRs, incorporating fluorescent markers, were found to be effective at amplifying degraded DNA from predators' guts and could amplify mitochondrial DNA fragments from 10+ species simultaneously without 'drop outs'. The combined PCR products were then separated by size on polyacrylamide gels on an ABI377 sequencer. New primers to detect the remains of aphids, earthworms, weevils and molluscs in the guts of carabid predators were developed and characterized. The multiplex-sequencer approach was then applied to field-caught beetles, some of which contained DNA from as many as four different prey at once. The main prey detected in the beetles proved to be earthworms and molluscs, although aphids and weevils were also consumed. The potential of this system for use in food-web research is discussed.     

The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.     

Detection of scavenged material in the guts of predators using monoclonal antibodies: a significant source of error in measurement of predation?

Molecular detection systems used to analyse the gut contents of invertebrate predators have enhanced our understanding of trophic interactions, but do not distinguish between the methods of consumption. Many predators regularly scavenge, which could have profound implications for quantitative analyses of the dynamics of predation. We report the first quantified assessment of the potential error caused by scavenging in post-mortem measurements of predation in a slug-carabid system. An anti-slug monoclonal antibody was able to detect antigens from decayed slugs after surprisingly long periods, significantly longer on relatively sterile peat than on natural soil. On soil the half-life of antibody-detectable slug proteins was 8.2 days while on peat it was 11.5 days. When slugs that had decayed on soil for 100 h were fed to the carabid predator Pterostichus melanarius, slug proteins could still be identified after 6 h (but not 12 h) digestion. Fresh and decayed slug was eaten in equal quantities by the beetles suggesting no aversion to the latter. The results suggest that significant errors may be caused by scavenging leading to inaccurate interpretation of predation rates in the field.