Bilayer orientation of membrane-bound NH2 groups across microsomal vesicles and their role in the function of gastric K+-stimulated adenosine triphosphatase

The transversal distribution of the free NH₂ groups associated with phosphatidyl ethanolamine and the intrinsic membrane proteins of the purified pig gastric microsomes was quantitated and their relations to the function of the gastric K⁺-stimulated ATPase was investigated. Three different chemical probes such as 2,4,6-trinitrobenzene sulfonic acid (TNBS), 1-fluoro-2,4-dinitrobenzene (FDNB), and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) were used for the study. The structure-function relationship of the membrane NH₂ groups was studied after modification with the probes under various conditions and relating the inhibition of the K⁺-stimulated ATPase to the ATPase-dependent H⁺ accumulation by the gastric microsomal vesicles. TNBS (2 mm) inhibits nearly completely the K⁺-stimulated ATPase and the vesicular dye accumulation, both in presence and absence of valinomycin plus K⁺. Both the K⁺-ATPase and dye uptake were largely (about 50%) protected against TNBS inhibition if the treatment with TNBS was carried out in presence of 2 mm ATP. TNBS and FDNB labeled 70% of the total microsomal PE; the intra- and extravesicular orientation being 48 and 22%, respectively. The presence or absence of ATP did not have any effect on the TNBS labeling of microsomal PE. ATP, however, significantly (P < 0.05) reduced the labeling of protein-bound NH₂ groups of gastric microsomes by TNBS. The intra- and extravesicular orientation of the protein NH₂ groups were 60 and 40%, respectively. Eighteen percent of the total protein-NH₂ appeared to be associated with the K⁺-stimulated ATPase; the rest being associated with non-ATPase proteins of the microsomes. About half (50%) of the total free NH₂ groups of the K⁺-stimulated ATPase were exposed to the vesicle exterior and were found to play critical roles in gastric ATPase function. The generation of florescence after MDPF conjugation of gastric microsomes was largely (50%) inhibited by ATP. ATP also protected completely the MDPF inhibition of gastric K⁺-stimulated ATPase and dye uptake.     

Endogenous protease mediated manifestation of a Ca2+-stimulated ATPase in purified dog gastric microsomes

An endogenous soluble protease has been demonstrated to unmask a Ca²⁺-stimulated ATPase activity in purified dog gastric microsomes. The presence of ATP during protease treatment appears essential for the manifestation of the gastric Ca²⁺-stimulated ATPase activity. The endogenous protease appears to have trypsin-like activity, since soybean trypsin inhibitor completely blocks the protease effect. Manifestation of the Ca²⁺-stimulated ATPase occurs without affecting the microsomal (H⁺ +K⁺)-ATPase activity and associated H⁺ uptake ability. The unmasked Ca²⁺-stimulated ATPase appears insensitive to calmodulin. Possible roles of the enzyme in the regulation of gastric H⁺ transport have been discussed.     

Biotic elicitors enhance diosgenin production in Helicteres isora L. suspension cultures via up-regulation of CAS and HMGR genes

In an attempt to find an alternative and potent source of diosgenin, a steroidal saponin in great demand for its pharmaceutical importance, Helicteres isora suspension cultures were explored for diosgenin extraction. The effect of biotic elicitors on the biosynthesis of diosgenin, in suspension cultures of H. isora was studied. Bacterial as well as fungal elicitors such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus niger were applied at varying concentrations to investigate their effects on diosgenin content. The HPLC based quantification of the treated samples proved that amongst the biotic elicitors, E. coli (1.5%) proved best with a 9.1-fold increase in diosgenin content over respective control cultures. Further, the scaling-up of the suspension culture to shake-flask and ultimately to bioreactor level were carried out for production of diosgenin. During all the scaling-up stages, diosgenin yield obtained was in the range between 7.91 and 8.64 mg l−1, where diosgenin content was increased with volume of the medium. The quantitative real-time PCR (qRT-PCR) analysis showed biotic elicitors induced the expression levels of regulatory genes in diosgenin biosynthetic pathway, the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and cycloartenol synthase (CAS), which can be positively correlated with elicited diosgenin contents in those cultures. The study holds significance as H. isora represents a cleaner and easy source of diosgenin where unlike other traditional sources, it is not admixed with other steroidal saponins, and the scaled-up levels of diosgenin achieved herein have the potential to be explored commercially.     

Decomposition of woody debris in Mediterranean ecosystems: the role of wood chemical and anatomical traits

Plant and Soil - Data about woody debris (WD) decomposition are very scarce for the Mediterranean basin. The specific aim of this work is to explore the relationships between WD traits with the...     

Microbial Dynamics and Diversity of Bacillus thuringiensis in Textile Effluent Polluted and Non-polluted Rice Field Soils of Orissa, India

Microbial diversity was assessed in the soils of non-polluted rice fields of Central Rice Research Institute and Choudwar, and textile effluent contaminated (about 30 years) rice fields of Choudwar about 4 years after cessation of pollution. The soils contained 0.62–1.01 % organic C and 0.07–0.12 % total N, and measured 6.18–8.24 pH and 0.6–2.68 mS/cm Eh which were more in the polluted Choudwar soil. The microbial populations (×10⁶ cfu/g soil) in the soils were: heterotrophs 1.21–10.9, spore formers 0.9–2.43, Gram (−)ve bacteria 4.11–8.0, nitrifiers 0.72–1.5, denitrifiers 0.72–2.43, phosphate solubilizers 0.14–0.9, asymbiotic nitrogen fixers 0.34–0.59, actinomycetes 0.07–0.11, fungi 0–0.5 and Bacillus thuringiensis (Bt) 0.4–0.61 which predominated in the polluted soil of Choudwar. The fungi were scarce in the polluted rice fields. The Bt isolates belonged to three motile and one non-motile group. Two motile Bt isolates were phenotyped as Bt subsp. sotto and israelensis, whereas, the non-motile isolate was Bt subsp. wahuensis. All Bt isolates produced extracellular protease, lipase and amylase enzymes. The microbial guilds had positive correlation among themselves, as well as, with soil physico-chemical characters but the fungi had negative relation and the nitrogen fixers were unrelated with the biotic and abiotic components.     

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. ¹³C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.     

Host-induced silencing of Mi-msp-1 confers resistance to root-knot nematode Meloidogyne incognita in eggplant

RNA interference (RNAi)-based host-induced gene silencing (HIGS) is emerging as a novel, efficient and target-specific tool to combat phytonematode infection in crop plants. Mi-msp-1, an effector gene expressed in the subventral pharyngeal gland cells of Meloidogyne incognita plays an important role in the parasitic process. Mi-msp-1 effector is conserved in few of the species of root-knot nematodes (RKNs) and does not share considerable homology with the other phytonematodes, thereby making it a suitable target for HIGS with minimal off-target effects. Six putative eggplant transformants harbouring a single copy RNAi transgene of Mi-msp-1 was generated. Stable expression of the transgene was detected in T₁, T₂ and T₃ transgenic lines for which a detrimental effect on RKN penetration, development and reproduction was documented upon challenge infection with nematode juveniles. The post-parasitic nematode stages extracted from the transgenic plants showed long-term RNAi effect in terms of targeted downregulation of Mi-msp-1. These findings suggest that HIGS of Mi-msp-1 enhances nematode resistance in eggplant and protect the plant against RKN parasitism at very early stage.

     

Protein kinase C-dependent phosphorylation and mitochondrial translocation of aldose reductase

Although aldose reductase (AR) is a critical participant in osmoregulation, and the metabolism of glucose and aldehydes derived from lipid peroxidation, post-translational mechanisms regulating its activity have not been identified. In this paper, we report that stimulation of protein kinase C (PKC) in several cell types induces phosphorylation of AR and translocation of the phosphorylated protein to the mitochondria. In vitro, recombinant AR was directly phosphorylated by activated PKC, suggesting that AR may be an in vivo PKC substrate. Together, these observations reveal a novel link between PKC activation and the regulation of glucose and aldehyde metabolism.     

Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.