Mammalian brain plasma membranes : Isolation, enzymatic, and chemical characterization

Two variants of a simplified procedure for the isolation of plasma membrane fractions from monkey and rat brains, are described. The preparations show marked enrichments in the marker enzymes, (Na⁺-K⁺) adenosine triphosphatase, acetylcholinesterase, 5′-nucleotidase and adenylate cyclase. Lipid analysis and a protein electrophoretic pattern are presented. An enzymatic check has been made to assess for contamination by other cellular organelles. The amino acid composition of brain membrane proteins show a resemblance to the reported composition of erythrocyte ghost proteins but differ from myelin proteins.     

Plant lectin can target receptors containing sialic Acid, exemplified by podoplanin, to inhibit transformed cell growth and migration

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.     

Design,parallel synthesis,and crystal structures of biphenyl antithrombotics as selective inhibitors of tissue factor FVIIa complex. Part 1: Exploration of S2 pocket pharmacophores

Factor VIIa (FVIIa), a serine protease enzyme, coupled with tissue factor (TF) plays an important role in a number of thrombosis-related disorders. Inhibition of TF·FVIIa occurs early in the coagulation cascade and might provide some safety advantages over other related enzymes. We report here a novel series of substituted biphenyl derivatives that are highly potent and selective TF·FVIIa inhibitors. Parallel synthesis coupled with structure-based drug design allowed us to explore the S2 pocket of the enzyme active site. A number of compounds with IC₅₀ value of <10 nM were synthesized. The X-ray crystal structures of some of these compounds complexed with TF·FVIIa were determined and results were applied to design the next round of inhibitors. All the potent inhibitors were tested for inhibition against a panel of related enzymes and selectivity of 17,600 over thrombin, 450 over trypsin, 685 over FXa, and 76 over plasmin was achieved. Two groups, vinyl 36b and 2-furan 36ab, were identified as the optimum binding substituents on the phenyl ring in the S2 pocket. Compounds with these two substituents are the most potent compounds in this series with good selectivity over related serine proteases. These compounds will be further explored for structure–activity relationship.     

A Computational,Tissue-Realistic Model of Pressure Ulcer Formation in Individuals with Spinal Cord Injury

People with spinal cord injury (SCI) are predisposed to pressure ulcers (PU). PU remain a significant burden in cost of care and quality of life despite improved mechanistic understanding and advanced interventions. An agent-based model (ABM) of ischemia/reperfusion-induced inflammation and PU (the PUABM) was created, calibrated to serial images of post-SCI PU, and used to investigate potential treatments in silico. Tissue-level features of the PUABM recapitulated visual patterns of ulcer formation in individuals with SCI. These morphological features, along with simulated cell counts and mediator concentrations, suggested that the influence of inflammatory dynamics caused simulations to be committed to “better” vs. “worse” outcomes by 4 days of simulated time and prior to ulcer formation. Sensitivity analysis of model parameters suggested that increasing oxygen availability would reduce PU incidence. Using the PUABM, in silico trials of anti-inflammatory treatments such as corticosteroids and a neutralizing antibody targeted at Damage-Associated Molecular Pattern molecules (DAMPs) suggested that, at best, early application at a sufficiently high dose could attenuate local inflammation and reduce pressure-associated tissue damage, but could not reduce PU incidence. The PUABM thus shows promise as an adjunct for mechanistic understanding, diagnosis, and design of therapies in the setting of PU.     

Carbon demand,utilization, and metabolic diversity of bacterioplankton in the frontal regimes of the Indian sector of the Southern Ocean

Bacterial production, respiration and metabolic diversity were measured up to 120 m depth in the Sub-Antarctic Front (SAF) and Polar Fronts I and II (PFI and PFII) of the Indian Ocean sector of the Southern Ocean during 2010 Austral Summer. Prokaryotic cell count was maximum at PFI and PFII (~109 cells L−1) and minimum at SAF (~107 cells L−1). Furthermore, integrated bacterial production was higher at PFI (1.07 mg C m−2 h−1) and PFII (0.72 mg C m−2 h−1) compared to SAF (0.61 mg C m−2 h−1). At PFII, integrated bacterial growth efficiency was higher (8.96) compared to PFI (7.42) and SAF (7.17), signifying that the net contribution of PFII to the microbial loop could be relatively pronounced. Enhanced cell numbers and production at polar fronts indicate that the dissolved organic matter could be converted to secondary biomass through the microbial loop. However, integrated bacterial respiration rate at PFII (0.83 mg C m−2 h−1) was lower than that at PFI (1.84 mg C m−2 h−1) resulting in higher growth efficiency at PFII. Metabolic flexibility at SAF was clearly brought about by utilization of carboxylic acids like D-malic acid and itaconic acid, and carbohydrates like N-acetyl D-glucosamine, D-cellobiose and D-lactose. Utilization of amino acids like glycyl L-glutamic acid and L-threonine, and an amine, phenylethylamine, was critical in determining the metabolic variability at PFI. PFII hosted microbes that utilized phenolic compounds (2-hydroxy benzoic acid and 4-hydroxy benzoic acid) and polymers (like Tween 80). Utilization of polyols over carbohydrates in polar waters indicates a niche with lesser influence of the Antarctic melt waters on the bacterioplankton metabolism.     

Phylogeographical Structure in Mitochondrial DNA of Legume Pod Borer (Maruca vitrata) Population in Tropical Asia and Sub-Saharan Africa

This study was undertaken to assess the genetic diversity and host plant races of M. vitrata population in South and Southeast Asia and sub-Saharan Africa. The cytochrome c oxidase subunit 1 (cox1) gene was used to understand the phylogenetic relationship of geographically different M. vitrata population, but previous studies did not include population from Southeast Asia, the probable center of origin for Maruca, and from east Africa. Extensive sampling was done from different host plant species in target countries. Reference populations from Oceania and Latin America were used. An amplicon of 658 bp was produced by polymerase chain reaction, and 64 haplotypes were identified in 686 M. vitrata individuals. Phylogenetic analysis showed no difference among the M. vitrata population from different host plants. However, the results suggested that M. vitrata has formed two putative subspecies (which cannot be differentiated based on morphological characters) in Asia and sub-Saharan Africa, as indicated by the high pairwise F_ST values (0.44–0.85). The extremely high F_ST values (≥0.93) of Maruca population in Latin America and Oceania compared to Asian and African population seem to indicate a different species. On the continental or larger geographical region basis, the genetic differentiation is significantly correlated with the geographical distance. In addition, two putative species of Maruca, including M. vitrata occur in Australia, Indonesia and Papua New Guinea. The negative Tajima’s D and Fu’s F_S values showed the recent demographic expansion of Maruca population. The haplotype network and Automatic Barcode Gap Discovery analyses confirmed the results of phylogenetic analysis. Thus, this study confirmed the presence of three putative Maruca species, including one in Latin America, one in Oceania (including Indonesia) and M. vitrata in Asia, Africa and Oceania. Hence, the genetic differences in Maruca population should be carefully considered while designing the pest management strategies in different regions.     

Biotemplates in the green synthesis of silver nanoparticles

This article recapitulates the scientific advancement towards the greener synthesis of silver nanoparticles. Applications of noble metals have increased throughout human civilization, and the uses for nano-sized particles are even more remarkable. “Green” nanoparticle synthesis has been achieved using environmentally acceptable solvent systems and eco-friendly reducing and capping agents. Numerous microorganisms and plant extracts have been applied to synthesize inorganic nanostructures either intracellularly or extracellularly. The use of nanoparticles derived from noble metals has spread to many areas including jewelery, medical fields, electronics, water treatment and sport utilities, thus improving the longevity and comfort in human life. The application of nanoparticles as delivery vehicles for bactericidal agents represents a new paradigm in the design of antibacterial therapeutics. Orientation, size and physical properties of nanoparticles influences the performance and reproducibility of a potential device, thus making the synthesis and assembly of shape- and size-controlled nanocrystals an essential component for any practical application. This need has motivated researchers to explore different synthesis protocols.     

Nutritional and hormonal effects on storage proteins in the silkworm,Bombyx mori L.

Starvation of 48 h old fifth instar larvae depressed storage protein titres initially for 48 h but retained the levels comparable to control thereafter, possibly due to nutrients obtained during the 48 h feeding after fourth ecdysis. After an initial decline ligated larvae accumulated maximum storage proteins in haemolymph. This is because of inhibitory juvenile hormone titre at the basal level besides the appropriate release of 20-hydroxyecdysone from the ectopic source(s). Injection of methoprene (10 Μg/larva) repressed accumulation of storage proteins while 20-hydroxyecdysone (10 Μg/larva) increased the same. P-soyatose injection to starved and ligated larvae accelerated storage protein accumulation in haemolymph, signalling nutrient indispensability for initiation of storage protein synthesis at the appropriate time of last instar development inBombyx mori.     

Oxidation of methionine residues in recombinant human interleukin-1 receptor antagonist: implications of conformational stability on protein oxidation kinetics

Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.     

Self-assembly of a peptide sequence,EKKE, composed of exclusively charged amino acids: Role of charge in morphology and lead binding

The self-assembly of peptides is influenced by their amino acid sequence and other factors including pH, charge, temperature, and solvent. Herein, we explore whether a four-residue sequence, EKKE, consisting of exclusively charged amino acids shows the propensity to form self-assembled ordered nanostructures and whether the overall charge plays any role in morphological and functional properties. From a combination of experimental data provided by Thioflavin T fluorescence, Congo red absorbance, circular dichroism spectroscopy, dynamic light scattering, field emission-scanning electron microscopy, atomic force microscopy, and confocal microscopy, it is clear that the all-polar peptide and charged EKKE sequence shows a pH-dependent tendency to form amyloid-like structures, and the self-assembled entities under acidic, basic and neutral conditions exhibit morphological variation. Additionally, the ability of the self-assembled amyloid nanostructures to bind to the toxic metal, lead (Pb²⁺), was demonstrated from the analysis of the ultraviolet absorbance and X-ray photoelectron spectroscopy data. The modulation at the sequence level for the amyloid-forming EKKE scaffold can further extend its potential role not only in the remediation of other toxic metals but also towards biomedical applications.