Molecular characterization and identification of markers for toxic and non-toxic varieties of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. using RAPD,AFLP and SSR markers

Jatropha curcas L., a multipurpose shrub has acquired significant economic importance for its seed oil which can be converted to biodiesel, is emerging as an alternative to petro-diesel. The deoiled seed cake remains after oil extraction is toxic and cannot be used as a feed despite having best nutritional contents. No quantitative and qualitative differences were observed between toxic and non-toxic varieties of J. curcas except for phorbol esters content. Development of molecular marker will enable to differentiate non-toxic from toxic variety in a mixed population and also help in improvement of the species through marker assisted breeding programs. The present investigation was undertaken to characterize the toxic and non-toxic varieties at molecular level and to develop PCR based molecular markers for distinguishing non-toxic from toxic or vice versa. The polymorphic markers were successfully identified specific to non-toxic and toxic variety using RAPD and AFLP techniques. Totally 371 RAPD, 1,442 AFLP markers were analyzed and 56 (15.09%) RAPD, 238 (16.49%) AFLP markers were found specific to either of the varieties. Genetic similarity between non-toxic and toxic verity was found to be 0.92 by RAPD and 0.90 by AFLP fingerprinting. In the present study out of 12 microsatellite markers analyzed, seven markers were found polymorphic. Among these seven, jcms21 showed homozygous allele in the toxic variety. The study demonstrated that both RAPD and AFLP techniques were equally competitive in identifying polymorphic markers and differentiating both the varieties of J. curcas. Polymorphism of SSR markers prevailed between the varieties of J. curcas. These RAPD and AFLP identified markers will help in selective cultivation of specific variety and along with SSRs these markers can be exploited for further improvement of the species through breeding and Marker Assisted Selection (MAS).     

Effects of benzoylphenylurea on chitin synthesis and orientation in the cuticle of the Drosophila larva

Chitin is an essential constituent of the insect exoskeleton, the cuticle, which is an extracellular matrix (ECM) covering the animal. It is produced by the glycosyltransferase chitin synthase at the apical plasma membrane of epidermal and tracheal cells. To fulfil its role in cuticle elasticity and stiffness it associates with proteins, thereby adopting a stereotypic arrangement of helicoidally stacked sheets, which run parallel to the surface of the animal. One approach to understand the mechanisms of chitin synthesis and organisation is to dissect these processes genetically. However, since only a few genes coding for factors involved in chitin synthesis and organisation have been identified to date using the model arthropod Drosophila melanogaster insight arising from mutant analysis is rather limited. To collect new data on the role of chitin during insect cuticle differentiation, we have analysed the effects of chitin synthesis inhibitors on Drosophila embryogenesis. For this purpose, we have chosen the benzoylphenylurea diflubenzuron and lufenuron that are widely used as insect growth regulators. Our data allow mainly two important conclusions. First, correct organisation of chitin seems to directly depend on the amount of chitin synthesised. Second, chitin synthesis and organisation are cell-autonomous processes as insecticide-treated larvae display a mosaic of cuticle defects. As benzoylphenylurea are used not only as insecticides but also as anti-diabetic drugs, the study of their impact on Drosophila cuticle differentiation may be fruitful for understanding their mode of action on a cellular pathway that is seemingly conserved between vertebrates and invertebrates.     

Structural variability in manganese(II) complexes of N,N′-bis(2-pyridinylmethylene) ethane (and propane) diamine ligands

Manganese(II) complexes, Mn₂L¹₃(ClO₄)₄, MnL¹(H₂O)₂(ClO₄)₂, MnL²(H₂O)₂(ClO₄)₂, and {(μ-Cl)MnL²(PF₆)}₂ based on N,N′-bis(2-pyridinylmethylene) ethanediamine (L¹) and N,N′-bis(2-pyridinylmethylene) propanediamine (L²) ligands have been prepared and characterized. The single crystal X-ray diffraction analysis of Mn₂L²₃(ClO₄)₄ shows that each of the two Mn(II) ion centers with a Mn-Mn distance of 7.15 Å are coordinated by one ligand while a common third ligand bridges the metal centers. Solid-state magnetic susceptibility measurements as well as DFT calculations confirm that each of the manganese centers is high-spin S = 5/2. The electronic structure obtained shows no orbital overlap between the Mn(II) centers indicating that the observed weak antiferromagentism is a result of through space interactions between the two Mn(II) centers. Under different reaction conditions, L¹ and Mn(II) yielded a one-dimensional polymer, MnL¹(H₂O)₂(ClO₄)₂. Ligand L² when reacted with manganese(II) perchlorate gives contrarily to L¹ mononuclear MnL²(H₂O)₂(ClO₄)₂ complex. The analysis of the structural properties of the MnL²(H₂O)₂(ClO₄)₂ lead to the design of dinuclear complex {(μ-Cl)MnL²(PF₆)} where two chlorine atoms were utilized as bridging moieties. This complex has a rhomboidal Mn₂Cl₂ core with a Mn-Mn distance of 3.726 Å. At room temperature {(μ-Cl)MnL²(PF₆)} is ferromagnetic with observed μ_eff = 4.04 μ_B per Mn(II) ion. With cooling, μ_eff grows reaching 4.81 μ_B per Mn(II) ion at 8 K, and then undergoes ferromagnetic-to-antiferromagnetic phase transition.     

Biosorption of Cd(II) and Pb(II) onto brown seaweed, Lobophora variegata (Lamouroux): kinetic and equilibrium studies

The present work deals with the biosorption performance of raw and chemically modified biomass of the brown seaweed Lobophora variegata for removal of Cd(II) and Pb(II) from aqueous solution. The biosorption capacity was significantly altered by pH of the solution delineating that the higher the pH, the higher the Cd(II) and Pb(II) removal. Kinetic and isotherm experiments were carried out at the optimal pH 5.0. The metal removal rates were conspicuously rapid wherein 90% of the total sorption occurred within 90 min. Biomass treated with CaCl₂ demonstrated the highest potential for the sorption of the metal ions with the maximum uptake capacities i.e. 1.71 and 1.79 mmol g⁻¹ for Cd(II) and Pb(II), respectively. Kinetic data were satisfactorily manifested by a pseudo-second order chemical sorption process. The process mechanism consisting of both surface adsorption and pore diffusion was found to be complex. The sorption data have been analyzed and fitted to sorption isotherm of the Freundlich, Langmuir, and Redlich–Peterson models. The regression coefficient for both Langmuir and Redlich–Peterson isotherms were higher than those secured for Freundlich isotherm implying that the biosorption system is possibly monolayer coverage of the L. variegata surface by the cadmium and lead ions. FT-IR studies revealed that Cd(II) and Pb(II) binding to L. variegata occurred primarily through biomass carboxyl groups accompanied by momentous interactions of the biomass amino and amide groups. In this study, we have observed that L. variegata had maximum biosorption capacity for Cd(II) and Pb(II) reported so far for any marine algae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Total synthesis of (±)-elegansidiol, (±)-farnesiferol B,and (±)-farnesiferol D

The racemic total synthesis of elegansidiol, farnesiferol B, and farnesiferol D has been obtained following a Diels–Alder approach. Gillman addition, cross metathesis reaction are the other key steps involved in the target synthesis.     

A database for inborn errors of metabolism in the Indian state of Andhra Pradesh

A Database for Inborn Errors of Metabolism (IEM) in the Indian State of Andhra Pradesh (DIEMISAP), is a continuously updated literature depository containing the extensive information on the regional prevalence and heterogeneity of Inborn Errors of metabolism. We report the construction of a database, a flat file and secondary data resource developed using Microsoft Front Page as the core engine. The database contains 18 summaries regarding 43 IEM disorders reported in the AP population, both in general and region wise, with references and links to the IEM disorder databases available on web and institutes involved in IEM research, in India. Summaries can be accessed from the reference given against an alphabetically arranged list of IEM disorders. The DIEMISAP is a useful user friendly and extendable online resource for information on prevalence of IEM in AP. AVAILABILITY: http://biochem.uohyd.ernet.in.     

Development and validation of a Sensitive bioanalytical method for the quantitative estimation of Pantoprazole in human plasma samples by LC–MS/MS: Application to bioequivalence study

The present study aims at developing a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of pantoprazole sodium (PS) in human plasma using pantoprazole D3 (PSD3) as internal standard (IS). Chromatographic separation was performed on Zorbax SB-C18, 4.6 mm × 75 mm, 3.5 μm, 80 Å column with an isocratic mobile phase composed of 10 mM ammonium acetate (pH 7.10): acetonitrile (30:70, v/v), pumped at 0.6 mL/min. PS and PSD3 were detected with proton adducts at m/z 384.2 → 200.1 and 387.1 → 203.1 in multiple reaction monitoring (MRM) positive mode, respectively. Precipitation method was employed in the extraction of PS and PSD3 from the biological matrix. This method was validated over a linear concentration range of 10.00–3000.00 ng/mL with correlation coefficient (r) ≥ 0.9997. Intra- and inter-day precision of PS were found to be within the range of 1.13–1.54 and 1.76–2.86, respectively. Both analytes were stable throughout freeze/thaw cycles, bench top and postoperative stability studies. This method was successfully utilized in the analysis of blood samples following oral administration of PS (40 mg) in healthy human volunteers.     

Vacuolin-1-modulated exocytosis and cell resealing in mast cells

The small chemical vacuolin-1 induces rapid formation of large vacuoles in various cell types. In epithelial cells, vacuolin-1 has been shown to inhibit Ca²⁺ ionophore-induced exocytosis depending on experimental conditions used but had no effect on repair of damaged membranes. However, it is not known whether vacuolin-1 could inhibit exocytosis induced by immunoreceptor triggering in professional secretory cells and whether there is any correlation between effect of vacuolin-1 on exocytosis and membrane repair in such cells. Here we show that in rat basophilic leukemia (RBL-2H3) cells activated by the high-affinity IgE receptor (FcεRI) triggering vacuolin-1 enhanced exocytosis. Under identical conditions of activation, vacuolin-1 inhibited exocytosis in mouse bone marrow-derived mast cells (BMMCs). This inhibition was not reflected by decreased phosphorylation of the FcεRI α and β subunits, linker for activation of T cells, non-T cell activation linker, Akt and MAP kinase Erk, and uptake of extracellular Ca²⁺, indicating that early activation events are not affected. In both cell types vacuolin-1 led to formation of numerous vacuoles, a process which was inhibited by bafilomycin A1, an inhibitor of vacuolar H⁺-ATPase. Thapsigargin- or Ca²⁺ ionophore A23187-induced exocytosis also showed different sensitivity to the inhibitory effect of vacuolin-1. Pretreatment of the cells with vacuolin-1 followed by permeabilization with bacterial toxin streptolysin O enhanced Ca²⁺-dependent repair of plasma membrane lesions in RBL-2H3 cells but inhibited it in BMMCs. Our data indicate that lysosomal exocytosis exhibits different sensitivity to vacuolin-1 depending on the cell type analyzed and mode of activation. Furthermore, our results support the concept that lysosomal exocytosis is involved in the repair of injured plasma membranes.     

Remarkable enhancement in the DNA-binding ability of C2-fluoro substituted pyrrolo[2,1-c][1,4]benzodiazepines and their anticancer potential

C2-Fluoro substituted DC-81, and its dimers that comprise of two C2-fluoro substituted DC-81 subunits tethered to their C8-position through simple alkane spacers as well as piperazine moiety side-armed with symmetrical alkyloxy spacers have been designed and synthesized. These fluoro substituted pyrrolo[2,1-c][1,4]benzodiazepines have shown remarkable DNA-binding ability and most of them possess promising anticancer activity, having GI₅₀ values in micromolar to nanomolar concentration range. DNA thermal denaturation studies show that some of these compounds (14a–c and 15) increase the ΔT_m values in the range of 28.9–38 °C, and this is further confirmed by the restriction endonuclease studies. This study illustrates the importance of introducing fluoro substitution at the C2-position apart from the incorporation of a piperazine ring in between the alkyloxy linker for enhancement of the DNA-binding ability in comparison to DSB-120 and SJG-136 (ΔT_m = 10.2 and 25.7 °C). Moreover, the variations in the DNA-binding ability with respect to fluoro substitution in this class of dimers has been investigated by molecular modeling studies. Some representative C2-fluoro substituted dimers (8a and 14a) have also exhibited significant anticancer activity in the 60 cancer cell line assay of the National Cancer Institute (NCI).