Anti-platelet activity of a three-finger toxin (3FTx) from Indian monocled cobra (Naja kaouthia) venom

A low molecular weight anti-platelet peptide (6.9 kDa) has been purified from Naja kaouthia venom and was named KT-6.9. MALDI-TOF/TOF mass spectrometry analysis revealed the homology of KT-6.9 peptide sequence with many three finger toxin family members. KT-6.9 inhibited human platelet aggregation process in a dose dependent manner. It has inhibited ADP, thrombin and arachidonic acid induced platelet aggregation process in dose dependent manner, but did not inhibit collagen and ristocetin induced platelet aggregation. Strong inhibition (70%) of the ADP induced platelet aggregation by KT-6.9 suggests competition with ADP for its receptors on platelet surface. Anti-platelet activity of KT-6.9 was found to be 25 times stronger than that of anti-platelet drug clopidogrel. Binding of KT-6.9 to platelet surface was confirmed by surface plasma resonance analysis using BIAcore X100. Binding was also observed by a modified sandwich ELISA method using anti-KT-6.9 antibodies. KT-6.9 is probably the first 3FTx from Indian monocled cobra venom reported as a platelet aggregation inhibitor.     

Effects of Lipids on the Rheological Properties of Aqueous Soybean Protein Dispersions

The flow properties of soybean protein–lipid–water suspension systems and coagulated gels were related to a protein–lipid interaction. For powdered soybean lecithin-added soybean protein suspension systems and their heat-induced gels, the yield stress (σ_y) and the consistency index (K) increased with increasing amounts of added lipid, but the flow behavior index (n) and the thixotropy index (TI) decreased. On the other hand, there were only small changes in the magnitudes of the thixotropic parameters and the viscometric parameters (σ_y, K and n) after adding soybean oil at various concentrations to soybean protein dispersions. These facts suggested that the formation of a protein–phospholipid complex increased the effective particle size, and that the intermolecular entanglements and linkages among the protein molecules or among the protein-phospholipid complexes were weakened by the addition of polar lipids. The thixotropy index defined in this study is available for characterizing the stress decay that occurs within soybean protein dispersions and heat-induced gels as they are sheared.     

DOPA Production with Enterobacter cloacae NB 320 by Transamination Reaction

Taxonomical investigation was performed on the bacterium, strain NB 320 isolated from soil, and it was identified as Enterobacter cloacae. This bacterium produced the enzyme which catalyzed the transamination reaction between 3,4-dihydroxyphenyl pyruvate and an amino acid to form l-Dopa.

The optimum culture conditions for the enzyme production were studied along with the characteristics of the enzyme. The enzyme of the strain was different in some properties from that of Alcaligenes faecalis IAM 1015 which had been already studied. The former utilized glutamate as an amino donor best among the amino acids tested for transamination and was induced by the addition of glutamine and asparagine. Intact cells of the strain did not catalyze the reaction unless they were treated with sonication or with a detergent.     

The Action of Peptidases from Aspergillus sojae on Soybean Proteins

To elucidate the mechanism of light-activation of pyruvate P_L dikinase in maize leaf, the inactive form was purified to homogeneity from dark-treated leaves using an activation system to locate it. The purification procedure included ammonium sulfate-fractionation followed by conventional chromatography.

The homogeneous enzyme after maximal activation had a specific activity comparable to that of the active enzyme obtained from non-dark-treated plants. The enzyme was indistinguishable from the active one in its molecular size and charge and in the amino acid composition of its acid-hydrolysate.     

Methyl-β-cyclodextrin potentiates the BITC-induced anti-cancer effect through modulation of the Akt phosphorylation in human colorectal cancer cells

ABSTRACT

Methyl-β-cyclodextrin (MβCD) is an effective agent for the removal of plasma membrane cholesterol. In this study, we investigated the modulating effects of MβCD on the antiproliferation induced by benzyl isothiocyanate (BITC), an ITC compound mainly derived from papaya seeds. We confirmed that MβCD dose-dependently increased the cholesterol level in the medium, possibly through its removal from the plasma membrane of human colorectal cancer cells. The pretreatment with a non-toxic concentration (2.5 mM) of MβCD significantly enhanced the BITC-induced cytotoxicity and apoptosis induction, which was counteracted by the cholesterol supplementation. Although BITC activated the phosphoinositide 3-kinase (PI3K)/Akt pathway, MβCD dose-dependently inhibited the phosphorylation level of Akt. On the contrary, the treatment of MβCD enhanced the phosphorylation of mitogen activated protein kinases, but did not potentiate their BITC-induced phosphorylation. These results suggested that MβCD might potentiate the BITC-induced anti-cancer by cholesterol depletion and thus inhibition of the PI3K/Akt-dependent survival pathway.

Abbreviations: CDs: cyclodextrins; MβCD: methyl-β-cyclodextrin; ITCs: isothiocyanates; BITC: benzyl isothiocyanate; PI3K: phosphoinositide 3-kinase; PDK1: phosphoinositide-dependent kinase-1; MAPK: mitogen activated protein kinase; ERK1/2: extracellular signal-regulated kinase1/2; JNK: c-Jun N-terminal kinase; PI: propidium iodide; FBS: fatal bovine serum; TLC: thin-layer chromatography; PBS(-): phosphate-buffered saline without calcium and magnesium; MEK: MAPK/ERK kinase; PIP2: phosphatidylinositol-4,5-bisphosphate; PIP3: phosphatidylinositol-3,4,5-trisphosphate     

Characterization of glycosomal RING finger proteins of trypanosomatids

The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins-PEX2, PEX10 and PEX12-comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.     

A field study of nitrogen dynamics and spring barley growth as affected by the quality of incorporated residues from white clover and ryegrass

Biofumigation refers to the suppression of soil-borne pests and pathogens by biocidal compounds released by Brassicaceous green manure and rotation crops when glucosinolates (GSLs) in their tissues are hydrolysed. We investigated the effect of environment and ontogeny on the GSL production, and thus biofumigation potential, of eight entries from five Brassica species. The environments included autumn and spring sown field plots (FA and FS) and potted plants grown under ambient conditions (PAM) or in a temperature controlled glasshouse at 20 °C/12 °C (PTC). GSL concentration was measured in the root and shoot tissue at buds-raised, flowering and maturity. Of particular interest was the suitability of the pot-grown plants for screening large numbers of brassicas for GSL production. The type of GSLs present in the tissues and their relative proportions remained relatively constant across environments and at different growth stages, with the exception of an increase in indolyl GSLs in the FS environment suspected of being induced by insect attack. Total GSL concentration generally declined from buds-raised to flowering in all environments, and was lowest at maturity. The exceptions were B. campestris, which had higher GSL concentration at flowering than at buds-raised, and the PTC environment in which most species also showed an increase at flowering. Despite GSL types and their proportions remaining relatively constant, the total GSL concentration in the root and shoot tissue of all entries varied significantly with environment (3–10-fold) and was generally ranked FS>PAM>FA>PTC. Interactions between species and environments meant that the ranking of the Brassica entries for total shoot and root GSL concentration changed with environment. However within three entries from B. napus, the ranking was consistent across the environments. The added effect of environment on phenological development and biomass production further influenced GSL production (the product of GSL concentration and biomass) on a ground area basis. The results suggest that glasshouse environments can be used to determine the types and proportions of GSLs present, and to rank entries within, but not between species for the total concentration in the tissues. However the influence of the environment on both GSL concentration and biomass production suggests that an accurate estimate of GSL production on a ground area basis to assess biofumigation potential will require measurement in the target environment.     

Synthesis of N-tetra-O-acetyl-β-d-glucopyranosyl-N′-(4′,6′-diarylpyrimidin-2′-yl)thioureas

Some 2-amino-4,6-diarylpyrimidines 2 have been prepared from substituted benzylideneacetophenones and guanidine hydrochloride in the presence of alkali by conventional heating in alcoholic medium and microwave heating in solvent-free conditions. N-(2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl)-N′-(4′,6′-diarylpyrimidin-2′-yl)thioureas 4 have been synthesized by reaction of per-O-acetylated glucopyranosyl isothiocyanate 1 and substituted 2-amino-4,6-diarylpyrimidines 2. Two different methods have been used, namely, refluxing in anhydrous dioxane and solvent-free microwave-assisted coupling. The second procedure afforded higher yields in much shorter reaction times. The compounds 2 and 4 were tested for their antibacterial and antifungal activities in vitro against Staphylococcus epidermidis, Enterobacter aerogenes and Candida albicans by disc diffusion method.     

A tetrameric allyl complex of sodium, and computational modeling of the Na-allyl chemical shift

Transmetallation of Li[A′] (A′ = [1,3-(SiMe₃)₂C₃H₃]⁻) with sodium tert-butoxide produces the corresponding sodium salt, which crystallizes from THF/toluene in the form of a cyclic tetramer, {Na[A′](thf)}₄. The Na atoms are in a square planar arrangement, bridged with π-bound allyl ligands; the Na-C distances range from 2.591(3)-2.896(3) Å, with an average of 2.70 Å. The geometries of several model organosodium complexes containing cyclopentadienyl and allyl ligands were optimized with density functional theory methods. The optimized structures were used with the gauge-including atomic orbital (GIAO) method to calculate their ²³Na NMR magnetic shielding values. Unlike the case with NaCp, the chemical shift of unsubstituted Na(C₃H₅) is very sensitive to the presence of coordinated THF (causing a 20 ppm upfield shift); silyl substitution has an even larger effect (30 ppm upfield shift). The observed ²³Na shift of δ −3.3 ppm for Na[A′] in THF-d₈, however, cannot be reliably distinguished from that calculated for the [Na(thf)₄]⁺ cation alone.     

Biofumigation and Enhanced Biodegradation: Opportunity and Challenge in Soilborne Pest and Disease Management

Management of soilborne pests and diseases in cropping systems is often highly challenging—in implementation of acceptable methodologies and in dealing with secondary problems. The phase-out of methyl bromide brings this into particularly sharp focus. There is a need for diversified options and alternatives to fill different roles across the soilborne pest and disease management spectrum, but flexibility is limited, as practicalities demand that they fit into a prophylactic methodology. It is against such a backdrop that expectations and promotion of alternatives must be set. There is also a need to recognize potentially serious problems that may have been masked under historical management regimes, but for which the nature of the system offers little scope to avoid or manage.

Biofumigation is the beneficial use of Brassica green manures that release isothiocyanates chemically similar to methyl isothiocyanate, the active agent from the synthetic fumigant metam sodium, which is used as a substitute for methyl bromide in some systems. A systematic approach to research into biofumigation, specifically aimed at overcoming a long history of empiricism, has seen significant recent advances in both basic and applied knowledge. A key development has been achievement of maximal biofumigation potential through greatly enhanced release of appropriate isothiocyanates into soil. These advances have led to commercial adoption, demonstrating that biofumigation, when applied to appropriate production systems, can have efficacy and offer cost savings. Crucially, these systematically derived research and development findings and their adoption now provide the impetus for self-sustaining further development and market penetration of the concept. Despite this success, biofumigation is not seen as being sufficiently powerful or practical in implementation to be an alternative to methyl bromide on a broad scale and misdirection in that regard could be counterproductive to more appropriately targeted further development.

Enhanced microbial biodegradation is a cryptic phenomenon that can diminish the efficacy of soil-applied pesticides, including isothiocyanates and most other currently available methyl bromide substitutes. Because methyl bromide is not susceptible, the phenomenon has potentially serious implications in intensive production systems switching from methyl bromide to reliance on other compounds that are. It is an intractable problem once induced. Avoidance of its onset is the only feasible management strategy. This has been aided for some particularly vulnerable environments by recent clarification of key risk factors associated with soil type, soil pH, and calcium content.