Studies on cellular adhesion of Xenopus laevis melanophores: modulation of cell-cell and cell-substratum adhesion in vitro by endogenous Xenopus galactoside-binding lectin

We have investigated cell-cell and cell-substratum adhesion of Xenopus laevis neural crest cells at various stages of melanophore differentiation. Single-cell suspensions were obtained by trypsinization and aggregated in a cell-cell adhesion assay. Unpigmented cells did not adhere while the rate of adhesion of melanophores correlated with the degree of melanization. Melanophore cell-cell adhesion decreased significantly in the presence of beta-galactosidase, which suggests that cell-surface galactose is involved. Beta-galactoside-binding lectin has been isolated and purified from embryos at the stage of neural crest migration. When added to aggregating cells smaller, looser clusters formed compared to controls. When lectin was added to cells in stationary culture to test cell-substratum adhesion, melanophores spread more smoothly and formed more regular spacing patterns. These results suggest that this lectin can modulate receptors used in cell-cell and cell-substratum adhesion of melanophores.     

Development and Validation of Real-Time PCR for Rapid Detection of Mecistocirrus digitatus

Hematophagous activity of Mecistocirrus digitatus, which causes substantial blood and weight loss in large ruminants, is an emerging challenge due to the economic loss it brings to the livestock industry. Infected animals are treated with anthelmintic drugs, based on the identification of helminth species and the severity of infection; however, traditional methods such as microscopic identification and the counting of eggs for diagnosis and determination of level of infection are laborious, cumbersome and unreliable. To facilitate the detection of this parasite, a SYBR green-based real-time PCR was standardized and validated for the detection of M. digitatus infection in cattle and buffaloes. Oligonucleotides were designed to amplify partial Internal Transcribed Spacer (ITS)-1 sequence of M. digitatus. The specificity of the primers was confirmed by non-amplification of DNA extracted from other commonly occurring gastrointestinal nematodes in ruminants. Plasmids were ligated with partial ITS-1 sequence of M. digitatus, serially diluted (hundred fold) and used as standards in the real-time PCR assay. The quantification cycle (Cq) values were plotted against the standard DNA concentration to produce a standard curve. The assay was sensitive enough to detect one plasmid containing the M. digitatus DNA. Clinical application of this assay was validated by testing the DNA extracted from the faeces of naturally infected cattle (n = 40) and buffaloes (n = 25). The results were compared with our standard curve to calculate the quantity of M. digitatus in each faecal sample. The Cq value of the assay depicted a strong linear relationship with faecal DNA content, with a regression coefficient of 0.984 and efficiency of 99%. This assay has noteworthy advantages over the conventional methods of diagnosis because it is more specific, sensitive and reliable.     

Interaction between Colletotrichum falcatum pathotypes and biocontrol agents

Abstract

To select efficient antagonistic strain(s) of biocontrol agents against most of the existing pathotypes of Colletotrichum falcatum, an in vitro interaction study was carried out with 13 pathotypes, 12 isolates of Pseudomonas spp. and 6 isolates of Trichoderma spp. Antagonistic pseudomonad strains exhibited greater variation in their activity depending on the virulence of the pathotype. The lower the pathogen virulence, the higher was the antagonistic activity noticed. In general, sub-tropical pathotypes were suppressed at a comparatively higher level than the tropical pathotypes. Among the four efficient P. fluorescens strains selected based on their inhibitory effect against various pathotypes, ARR1G and VPT4 were effective against tropical pathotypes and FP7 showed moderate effect against all the pathotypes. The strain KKM2 was effective against sub-tropical and weaker tropical pathotypes. Strains of Trichoderma spp. did not show much variation in antagonism, but varied in their mode of action in suppressing the pathogen growth. However, based on higher rate of hyperparasitism, T. harzianum strains T5 and T62 were selected against all the pathotypes.     

A new ecdysteroid, 2-deoxy-5beta,20-dihydroxyecdysone from the fruits of Diploclisia glaucescens

Chemical investigation of ethyl acetate extract of the fruits of Diploclisia glaucescens of the family Menispermaceae furnished a new ecdysteroid 2-deoxy-5beta,20-dihydroxyecdysone, together with 20-hydroxyecdysone, 3-deoxy-1beta,20-dihydroxyecdysone, 2-deoxy-20-hydroxyecdysone, 24-ethyl-20-hydroxyecdysone (makisterone C). Latter two ecdysteroids are reported first time from the family Menispermaceae.     

Chromatographic separation and sensitive determination of teriflunomide,an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive, selective and high throughput liquid chromatography tandem mass spectrometry (LC–ESI-MS/MS) method has been developed for the determination of teriflunomide, an active metabolite of leflunomide in human plasma. Plasma samples were prepared by liquid–liquid extraction of teriflunomide and valsartan as internal standard (IS) in ethyl acetate from 200 μL human plasma. The chromatographic separation was achieved on an Inertsil ODS-3 C18 (50 mm × 4.6 mm, 3 μm) analytical column using isocratic mobile phase, consisting of 20 mM ammonium acetate–methanol (25:75, v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for teriflunomide (m/z 269.0 → 82.0) and IS (m/z 434.1 → 350.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and negative ion mode. The method was validated over a wide dynamic concentration range of 10.1–4001 ng/mL. Matrix effect was assessed by post-column infusion experiment and the mean process efficiency were 91.7% and 88.2% for teriflunomide and IS respectively. The method was rugged and rapid with a total run time of 2.0 min and is applied to a bioequivalence study of 20 mg leflunomide (test and reference) tablet formulation in 12 healthy Indian male subjects under fasting condition.     

First LC-MS/MS electrospray ionization validated method for the quantification of perindopril and its metabolite perindoprilat in human plasma and its application to bioequivalence study

A high throughput bioanalytical method based on solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS), has been developed for the estimation of perindopril and its metabolite perindoprilat, an angiotensin-converting enzyme inhibitor in human plasma. Ramipril was used as internal standard (IS). The extraction of perindopril, perindoprilat and ramipril from the plasma involved treatment with phosphoric acid followed by solid phase extraction (SPE) using hydrophilic lipophilic balance HLB cartridge. The SPE eluate without drying were analyzed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode to quantify perindopril and perindoprilat in human plasma. The total chromatographic run time was 1.5 min with retention time for perindopril, perindoprilat and ramipril at 0.33, 0.35 and 0.30 min. The developed method was validated in human plasma matrix, with a sensitivity of 0.5 ng/ml (CV, 7.67%) for perindopril and 0.3 ng/ml (CV, 4.94%) for perindoprilat. This method was extensively validated for its accuracy, precision, recovery, stability studies and matrix effect especially because the pattern of elution of all the analytes appears as flow injection elution. Sample preparation by this method yielded extremely clean extracts with very good and consistent mean recoveries; 78.29% for perindopril, 76.32% for perindoprilat and 77.72% for IS. The response of the LC-MS/MS method for perindopril and perindoprilat was linear over the range 0.5-350.0 ng/ml for perindopril and 0.3-40 ng/ml for perindoprilat with correlation coefficient, r>/=0.9998 and 0.9996, respectively. The method was successfully applied for bioequivalence studies in human subjects samples with 4 mg immediate release (IR) formulations.     

Ethylenediamine diacetate (EDDA) mediated synthesis of aurones under ultrasound: Their evaluation as inhibitors of SIRT1

An improved synthesis of functionalized aurones has been accomplished via the reaction of benzofuran-3(2H)-one with a range of benzaldehydes in the presence of a mild base EDDA under ultrasound. A number of aurones were synthesized (within 5-30min) and the molecular structure of a representative compound determined by single crystal X-ray diffraction study confirmed Z-geometry of the C-C double bond present within the molecule. Some of the compounds synthesized have shown SIRT1 inhibiting as well as anti proliferative properties against two cancer cell lines in vitro. Compound 3a [(Z)-2-(5-bromo-2-hydroxybenzylidene) benzofuran-3(2H)-one] was identified as a potent inhibitor of SIRT1 (IC(50)=1μM) which showed a dose dependent increase in the acetylation of p53 resulting in induction of apoptosis.     

Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene

Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread.