Development of EST-SSR markers for genetic diversity analysis in coconut (Cocos nucifera L.)

Molecular Biology Reports - Genetic improvement in coconut relies on exploiting the vast existing diversity among coconut accessions. Robust molecular markers are a pre-requisite for efficient...     

A prawn transglutaminase: Molecular characterization and biochemical properties

In this study, we report the bioinformatics characterization, gene expression, transglutaminase activity and coagulation assays of transglutaminase (TGase) of freshwater prawn Macrobrachium rosenbergii identified from the constructed cDNA library by GS FLX™ technology. Even though, TGase have sequence similarity, they differ extensively in their substrate specificity and are thought to play an important in variety of functions such as development, tissue differentiation and immune responses etc. Gene expression studies show that MrTGase is widely distributed in the tissues such as heart, muscle, intestine, brain, etc., but higher amounts are found in hemocyte. Results of TGase mRNA relative expression in hemocyte, before and after infected with white spot syndrome baculovirus (WSBV) and Vibrio harveyi show that the gene expression initially increases up to 24 h and then it falls down. Coagulation assay results showed that the endogenous TGase is involved in the rapid assembly of a specific, plasma clotting protein. Structural studies show that MrTGase contains a typical TGc domain between 323 and 424, and two putative integrin-binding motifs at Arg¹⁸⁰–Gly¹⁸¹–Asp¹⁸² and Arg²⁶⁹–Gly²⁷⁰–Asp²⁷¹. The predicted 3D model of MrTGase contains 47.04% coils (366 amino acid residues), 26.74% extended strand (208 residues), 21.72% α-helix (169 residues) and 4.5% beta turns (35 residues). BLASTp analysis of MrTGase exhibited high sequence similarities with other crustacean TGase, with the highest observed in white shrimp (77.1%). Moreover, the phylogenetic analysis also showed that MrTGase clustered with the other members of crustacean TGase. Overall, these results suggested that MrTGase is a major and functional TGase of M. rosenbergii for haemolymph coagulation and also in spread of infection.     

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l^?1 citric acid, 100 µM acetosyringone, and 100 mg l^?1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l^?1 hygromycin and the embryos were germinated in basal medium containing 20 mg l^?1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.     

Lysine residues of IGF-I are substrates for transglutaminases and modulate downstream IGF-I signalling

Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these K→R IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these K→R IGF-I analogues on IGF-I mediated actions. IGF-I analogues with K→R substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.     

Benzyloxybenzylammonium chlorides: Simple amine salts that display anticonvulsant activity

Several antiepileptic drugs exert their activities by inhibiting Na⁺ currents. Recent studies demonstrated that compounds containing a biaryl-linked motif (Ar-X-Ar′) modulate Na⁺ currents. We, and others, have reported that compounds with an embedded benzyloxyphenyl unit (ArOCH₂Ar′, OCH₂ = X) exhibit potent anticonvulsant activities. Here, we show that benzyloxybenzylammonium chlorides (⁺H₃NCH₂C₆H₄OCH₂Ar′ Cl^?) displayed notable activities in animal seizure models. Electrophysiological studies of 4-(2′-trifluoromethoxybenzyloxy)benzylammonium chloride (9) using embryonic cortical neurons demonstrated that 9 promoted both fast and slow inactivation of Na⁺ channels. These findings suggest that the potent anticonvulsant activities of the earlier compounds were due, in part, to the benzyloxyphenyl motif and provide support for the use of the biaryl-linked pharmacophore in future drug design efforts.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Comparative Evaluation of Bivalent Malaria Rapid Diagnostic Tests versus Traditional Methods in Field with Special Reference to Heat Stability Testing in Central India

Background

Malaria presents a diagnostic challenge in areas where both Plasmodium falciparum and P.vivax are co-endemic. Bivalent Rapid Diagnostic tests (RDTs) showed promise as diagnostic tools for P.falciparum and P.vivax. To assist national malaria control programme in the selection of RDTs, commercially available seven malaria RDTs were evaluated in terms of their performance with special reference to heat stability.

Methodology/Principal Findings

This study was undertaken in four forested districts of central India (July, 2011– March, 2012). All RDTs were tested simultaneously in field along with microscopy as gold standard. These RDTs were stored in their original packing at 25°C before transport to the field or they were stored at 35°C and 45°C upto 100 days for testing the performance of RDTs at high temperature. In all 2841 patients with fever were screened for malaria of which 26% were positive for P.falciparum, and 17% for P.vivax. The highest sensitivity of any RDT for P.falciparum was 98% (95% CI; 95.9–98.8) and lowest sensitivity was 76% (95% CI; 71.7–79.6). For P.vivax highest and lowest sensitivity for any RDT was 80% (95% CI; 94.9 - 83.9) and 20% (95% CI; 15.6–24.5) respectively. Heat stability experiments showed that most RDTs for P.falciparum showed high sensitivity at 45°C upto 90 days. While for P.vivax only two RDTs maintained good sensitivity upto day 90 when compared with RDTs kept at room temperature. Agreement between observers was excellent for positive and negative readings for both P.falciparum and P.vivax (Kappa >0.6–0.9).

Conclusion

This is first field evaluation of RDTs regarding their temperature stability. Although RDTs are useful as diagnostic tool for P.falciparum and P.vivax even at high temperature, the quality of RDTs should be regulated and monitored more closely.