Conservation strategies for threatened medicinal plant – Barleria prionitis L. – using in vitro and ex vitro propagation techniques

Over-utilisation and continuous depletion of medicinal plants have affected their supply and loss of genetic diversity. Hence the current study is based on conservation strategies for threatened medicinal plants with special reference to Barleria prionitis L. using in vitro and ex vitro propagation techniques. We have developed here a protocol for plant regeneration of Barleria prionitis L. We have also developed an efficient system of vegetative propagation of Barleria prionitis L. through stem cuttings using revive rooting hormones. These studies can be useful for conservation strategies of this important medicinal plant.     

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.     

Ecdysteroid Stimulated Protein Synthesis in the Male Accessory Reproductive Glands of Spodoptera litura

Summary

20-hydroxyecdysone stimulates protein synthesis in the male accessory reproductive glands of pharate adults of Spodoptera litura both in vivo and in vitro. Juvenile hormone-I has no synergistic effect when given in conjunction with ecdysone in vivo but it inhibits ecdysone-stimulated protein synthesis in vitro.     

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.     

The Chemical Nature of the Polar Functional Group of Oxidized Acyl Chain Uniquely Modifies the Physicochemical Properties of Oxidized Phospholipid-Containing Lipid Particles

Oxidative modification of phospholipids generates a variety of oxidized phospholipid (Ox-PL) species which differ considerably in their chemical compositions and molecular structures. Recent results suggest that even closely related Ox-PL species can have considerably different biological effects. However, the molecular mechanism for this is not yet clear. In truncated Ox-PLs (tOx-PLs) the fatty acyl chain is shorter in length than the parent nonoxidized phospholipid molecules and contains a polar functional group(s). In a previous study we showed that two closely related tOx-PL species having a similar polar functional group and differing only in the length of the oxidized fatty acyl chain exerts significantly different effects on the physicochemical properties of the nonoxidized phospholipid particles containing these lipids (Kar et al., Chem Phys Lipids 164:54–61, 2011). In this study we have characterized the effect of polar functional groups of oxidized fatty acyl chain on the physicochemical properties of the nonoxidized phospholipid particles containing these lipids. Our results show that Ox-PL species differing only in the chemical nature of polar functional groups in their oxidized fatty acyl chain modify the properties of nonoxidized phospholipid particles containing them in a distinctive way. These results indicate that different species of Ox-PLs induce unique changes in the physicochemical properties of lipid particles/membranes containing them and that this may lead to their different biological effects.     

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.     

Deciphering the role of charge,hydration, and hydrophobicity for cytotoxic activities and membrane interactions of bile acid based facial amphiphiles

We synthesized four cationic bile acid based facial amphiphiles featuring trimethyl ammonium head groups. We evaluated the role of these amphiphiles for cytotoxic activities against colon cancer cells and their membrane interactions by varying charge, hydration and hydrophobicity. The singly charged cationic Lithocholic acid based amphiphile (LCA-TMA₁) is most cytotoxic, whereas the triply charged cationic Cholic acid based amphiphile (CA-TMA₃) is least cytotoxic. Light microscopy and Annexin-FITC assay revealed that these facial amphiphiles caused late apoptosis. In addition, we studied the interactions of these amphiphiles with model membrane systems by Prodan-based hydration, DPH-based anisotropy, and differential scanning calorimetry. LCA-TMA₁ is most hydrophobic with a hard charge causing efficient dehydration and maximum perturbations of membranes thereby facilitating translocation and high cytotoxicity against colon cancer cells. In contrast, the highly hydrated and multiple charged CA-TMA₃ caused least membrane perturbations leading to low translocation and less cytotoxicity. As expected, Chenodeoxycholic acid and Deoxycholic acid based amphiphiles (CDCA-TMA₂, DCA-TMA₂) featuring two charged head groups showed intermediate behavior. Thus, we deciphered that charge, hydration, and hydrophobicity of these amphiphiles govern membrane interactions, translocation, and resulting cytoxicity against colon cancer cells.     

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.