Subtype selective NMDA receptor antagonists: evaluation of some novel alkynyl analogues

A benzylpiperidine analogue with an acetylenic linker, 5-(3-[4-(4-fluorobenzyl)-piperidin-1-yl]-prop-1-ynyl)-1,3-dihydrobenzimidazol-2-one (3), was identified as a chemical lead with excellent activity at the NR1A/2B receptor (IC50=3 nM). Efforts to optimize this activity led to focused modifications around the structural motif of 3. The synthesis and SAR studies are discussed.     

Capsicum annuum proteinase inhibitor ingestion negatively impacts the growth of sorghum pest Chilo partellus and promotes differential protease expression

BackgroundChilo partellus is an important insect pest infesting sorghum and maize. The larvae internalize in the stem, rendering difficulties in pest management. We investigated the effects of Capsicum annuum proteinase inhibitors (CanPIs) on C. partellus larvae by in-vitro and in-vivo experiments.MethodsRecombinant CanPI-7 (with four-Inhibitory Repeat Domains, IRDs), -22 (two-IRDs) and insect proteinase activities were estimated by proteinase assays, dot blot assays and in gel activity assays. Feeding bioassays of lab reared C. partellus with CanPI-7 and -22 were performed. C. partellus proteinase gene expression was done by RT-PCR. In-silico structure prediction of proteinases and CanPI IRDs was carried out, their validation and molecular docking was done for estimating the interaction strength.ResultsLarval proteinases of C. partellus showed higher activity at alkaline pH and expressed few proteinase isoforms. Both CanPIs showed strong inhibition of C. partellus larval proteinases. Feeding bioassays of C. partellus with CanPIs revealed a dose dependent retardation of larval growth, reduction of pupal mass and fecundity, while larval and pupal periods increased significantly. Ingestion of CanPIs resulted in differential up-regulation of C. partellus proteinase isoforms, which were sensitive to CanPI-7 but were insensitive to CanPI-22. In-silico interaction studies indicated the strong interaction of IRD-9 (of CanPI-22) with Chilo proteinases tested.ConclusionsOf the two PIs tested, CanPI-7 prevents induction of inhibitor insensitive proteinases in C. partellus so it can be explored for developing C. partellus tolerance in sorghum.General significanceIngestion of CanPIs, effectively retards C. partellus growth; while differentially regulating the proteinases.     

Effect of potassium chloride and calcium chloride induced stress on in vitro cultures of Bacopa monnieri (L.) Pennell and accumulation of medicinally important bacoside A

Growth, osmotic adjustment, antioxidant enzyme defense and principle medicinal component bacoside A was studied in in vitro raised shoots of Bacopa monnieri under different concentrations of KCl and CaCl₂ (0, 50, 100, 150 or 200 mM). Significant reduction was observed in shoot number per culture; shoot length, fresh weight, dry weight and tissue water content (TWC) when shoots were exposed to increasing KCl and CaCl₂ concentrations (50–200 mM) as compared to control. Minimum damage to the membrane as assessed by malondialdehyde (MDA) content was noticed in control in contrast to sharp increase in KCl and CaCl₂ stressed shoots. Higher amounts of free proline, glycine betaine and total soluble sugars (TSS) accumulated in KCl and CaCl₂ exposed shoots compared to the controls. Among different concentrations of KCl and CaCl₂, increasing concentration of CaCl₂ showed more increase in osmolyte accumulation. Na⁺ content decreased with increasing concentrations of KCl and CaCl₂. Accumulation of K⁺ increased significantly in KCl (50–100 mM) stressed shoots as compared to control, while it decreased in CaCl₂ treated shoots indicating that it prevents the uptake of K⁺ ions. Ca²⁺ accumulation significantly increased with increasing concentrations of CaCl₂ up to 150 mM but decreased at higher concentrations. Shoots treated with KCl and CaCl₂ (0–100 mM) showed higher antioxidant enzyme (SOD, CAT, APX and GPX) activities but KCl suppressed the activities at higher concentrations. Accumulation of bacoside A was enhanced with an increase in KCl and CaCl₂ concentration up to 100 mM. It appears from the data that accumulation of osmolytes, and elevated activities of antioxidant enzymes play an important role in osmotic adjustment in shoot cultures of Bacopa and the two salts tested have a positive effect on bacoside accumulation.     

Evaluation of Genotype MTBDRsl Assay to Detect Drug Resistance Associated with Fluoroquinolones,Aminoglycosides and Ethambutol on Clinical Sediments

Background

The emergence of resistant tuberculosis (TB) is a major setback to the global control of the disease as the treatment of such resistance is complex and expensive. Use of direct detection of mutations by molecular methods could facilitate rapid diagnosis of resistance to offset diagnostic delays. We evaluated the performance of the Genotype MTBDRsl (Hain Life Sciences) for the detection of second line resistant TB directly from stored smear positive sputum sediments.

Methodology/Principal Findings

The assay showed a diverse range of sensitivity and specificity, 91.26% [95% CI, 84–96] and 95.5% [95% CI, 87–99] for FQ (PPV ∼97% & NPV ∼ 87.67%), 56.19% [95%CI, 46–66] and 81% [95%CI, 66–91] for EMB (PPV ∼ 88.06% & NPV ∼ 43.21%) and 100% for SLD. Diagnostic accuracy for FQ, SLD and EMB was 94%, 100% and 63.51%, respectively. 1.17% (2/170) were heteroresistance strains, where the heteroresistance was linked to rrs gene. A varying rate of validity was observed 100% (170/170) for FQ, 94.11% (160/170) for EMB, 88.23% (150/170) for SLD.

Conclusions/Significance

Genotype MTBDRsl is simple, rapid, economical assay that can be used to detect commonly known resistance associated with Fluoroquinolone, second line injectable drugs and ethambutol. The assay detects the targeted resistance in less time as compared to phenotypic DST. But due to low NPV to FQ (88%) and EMB (43.21%), the assay results must be interpreted in coordination with the phenotypic DST.     

Comparison of the levels of the growth factors in umbilical cord serum and human milk and its clinical significance

The process of the growth of the fetus begins in the uterus and gets further accelerated following the birth, especially during initial few months. The role of the growth factors in the physiology of the cellular growth is already well established. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) seem to be imperative for angiogenesis, cell development and proliferation as well as maintenance of the tissues. The levels of these factors in the maternal serum during pregnancy as well as during postpartum period are insignificant. Consequently, we hypothesized that the fetus receives moderate supply of these growth factors from the placenta during its stay in the uterus. This supply gets further augmented during the postpartum period through the different source, i.e. mother's milk. To study this physiological transition of the source of the growth factors from the placenta to the breast milk, the concentrations of VEGF and HGF in the cord serum of full term neonates and that in the breast milk of the corresponding mothers were analyzed during ELISA. The human milk, especially the colostrum revealed significantly higher levels of VEGF and HGF (1541.759 ± 119.349 pg/ml and 7129.249 ± 273.472 pg/ml) than cord serum (16.632 ± 0.773 pg/ml and 2581.6 ± 108.275 pg/ml) respectively. The multifold higher levels of VEGF observed in colostrum probably correlates with its high neonatal requirement for the maturation of the gastrointestinal epithelium following birth. The higher levels of both the growth factors in the breast milk than those observed in the cord serum probably explain their higher needs by the neonates for immunological protection, protein synthesis and neurocognitive development. The observations of the present study strengthen the policy of the colostrum feeding, which is promoted by organizations like World Health Organization (WHO). This study further documents the fact that the commercial milk formulae cannot replace the human milk.     

Differential growth, physiological and biochemical responses of niger (Guizotia abyssinica Cass.) cultivars to water-deficit (drought) stress

The present study demonstrates the effect of polyethylene glycol-8000 (PEG) and percent field capacity (FC%)-induced water-deficit stress on growth, water status, productivity and various biochemical parameters in Guizotia abyssinica Cass. cultivars (IGP 76, GA 10, No. 71 and IGPN 2004) at seedling and maturity stages of the plant. Cultivar GA 10 showed higher, IGP 76 and No. 71 moderate, and IGPN 2004 least reduction in percent seed germination given PEG stress treatments (0, 10 and 20%). A similar pattern was observed for decreased growth and water content of the seedlings and plants of the cultivars exposed to both kinds of water-deficit stresses. The productivity (number of capitula per plant, number of seeds per capitula and 1,000-seed weight) and total chlorophyll content of cultivar IGPN 2004 was significantly higher in comparison to other cultivars given different FC% treatments (100, 80 and 60%). Significantly higher accumulation of proline, glycine betaine and total soluble sugars and lower damage to membrane lipids under increased water-deficit stress (i.e., at 80 and 60% FC) conditions in cultivar IGPN 2004 suggested its more tolerance capacity to water-deficit stress in comparison to other cultivars. Besides, antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase and guaiacol peroxidase) partially demonstrated variations in the tolerance of the cultivars to water-deficit stress. The results suggest that cultivar IGPN 2004 could be considered as more tolerant, and IGP 76 and No. 71 as moderately tolerant, whereas GA 10 was more sensitive to water-deficit stress.     

Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb.

This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of “Soma” drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog’s) medium fortified with either IAA or NAA (0.5–2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.Key words: In vitro propagation, Pyridone alkaloid, Cerpegin, Callus, Ceropegia juncea     

Growth and physiological alterations in Niger cultivars under drought stress

Aim of the present study was to determine differential responses in growth and physiology of tolerant (cv. IGPN 2004) and sensitive (cv. GA 10) cultivars of Niger (Guizotia abyssinica Cass.) using in vitro grown calli under water deficit conditions. The calli were subjected to drought stress using PEG-8000 (–0.16,–0.45,–0.87,–1.42 bar) for 15 d and relative growth rate (RGR), percent tissue water content (% TWC), osmolytes (proline–Pro, glycine betaine—GB, total soluble sugars—TSS) accumulation, malondialehyde (MDA) content as well as antioxidant enzyme activities such as superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) were analysed. Our findings showed that RGR and percent TWC was decreased significantly with the intensity of drought stress in both cultivars, but the RGR reduction was least (five folds) in cv. IGPN 2004 than in cv. GA 10 (6.2 folds). In osmolyte accumulation such as Pro and GB, cv. IGPN 2004 was found superior (5.5 and ten folds higher, respectively) to tolerate drought stress than GA 10; however, no change was observed in TSS accumulation. Further, it was noted that cv. IGPN 2004 caused least oxidative damage to the membranes. It also exhibited better SOD, CAT and APX activities and had higher α-tocopherol content. The least reduction in growth and MDA content and higher osmolytes and antioxidant activities in cv. IGPN 2004 revealed more drought stress tolerance at cellular level. It was suggested that increased drought tolerance of cv. IGPN 2004 was coupled with its better maintenance of RGR, percent TWC, reduced lipid peroxidation, more accumulation of osmolytes and higher antioxidant enzymes.