Surfacing activity and food utilization in the air-breathing fish polyacanthus cupanus exposed to constant Po2

The influence of feeding on the surfacing frequency of the air-breathing fishPolyacanthus cupanus was investigated. Feeding increased the surfacing and swimming activities. Food conversion, maintenance requirements and energy spent in swimming and surfacing were measured under different feeding regimes.     

RAPD based genetic stability analysis among micropropagated,synthetic seed derived and hardened plants of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.): a threatened Indian medicinal herb

Bacopa monnieri (L.), a highly endangered miracle medicinal herb with global interest, is one of the popular ancient Indian ayurvedic plants. With ever increasing demand for Bacopa based formulations in pharmaceutical industries, there is a need to preserve the stocks of the plant through biotechnological approaches. Randomly amplified polymorphic DNA (RAPD) fingerprinting approach was applied to analyze the genetic stability of 19 different B.monnieri plants randomly selected after micropropagation, regrowth from alginate encapsulated uninodal cuttings (before and after storage at 4°C) and hardening with the mother plant (wild type). 16 arbitrary decamer primers amplified a total of 334 reproducible distinct DNA fragments ranging from 180 to 1,500 bp, of which 262 (78.4%) were monomorphic and the rest (21.5%) were polymorphic with an average of 20.8 bands per primer. The extent of polymorphism was low to moderate. Primers OPAK 14, OPM 15 and OPD 13 generated 69, 46 and 42% polymorphic patterns. Primers OPA 04, OPU 13 and OPD 08 generated 100% monomorphic pattern. Similarity matrix based on Jaccard’s coefficient revealed that pair wise values between the wild type and its analyzed plants ranged from 0.00 to 0.92 and among the micropropagated, synthetic seed derived and hardened plants, the range of genetic distance is from 0.67 to 0.92. Unweighted pair group method with arithmetic averages cluster analysis resulted in one loose group of the wild type with three subgroups. The present study paves the way for the identification and maintenance of genetically uniform B. monnieri plants micropropagated in the lab, plants regrown from synthetic seeds and hardened in the field.     

Targeting and sensing cancer cells with ZnO nanoprobes in vitro

A new class of zinc oxide quantum dots (ZnO QDs) was investigated as nanoprobes for targeting cancer cells in vitro. ZnO nanoparticles were synthesized using conventional sol–gel method and encapsulated using trimethoxy aminopropyl silane. Transferrin, the ligand targeting the cancer cells, was conjugated to the ZnO QDs. In vitro imaging studies using MDA-MB-231 showed the biocompatible ZnO nanoprobe selectively binding to the cell surface receptor and internalizing through receptor-mediated endocytosis. Time-lapsed photobleaching studies indicate the ZnO QDs to be resistant to photobleaching, making them suitable for long term imaging purpose. Investigation of the ZnO nanoprobe as a platform for sensitive bioassays indicates that it can be used as an alternative fluoroprobe for cancer cell targeting and sensing applications.     

Transgenic indica rice cv. ADT 43 expressing a Δ<Superscript>1</Superscript>-pyrroline-5-carboxylate synthetase (<Emphasis Type="Italic">P5CS</Emphasis>) gene from <Emphasis Type="Italic">Vigna aconitifolia</Emphasis> demonstrates salt tolerance

To develop salt tolerant rice, the P5CS gene of Vigna aconitifolia, encoding for proline synthesis, was introduced into the popular indica rice cultivar ADT 43. Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1301/P5CS, carrying the proline synthesis encoding gene P5CS, was co-cultivated with embryogenic callus of rice. Adding 100 μM acetosyringone to the Linsmaier and Skoog (LS) liquid and solid co-culture medium, along with 30 mg/l hygromycin and 250 mg/l timentin, contributed to significantly higher efficiency of transformation. Southern blot analysis of T₁ independent transformants revealed that the copy number of transgene varied between one and three. When transgenic plants were subjected to salt stress, these plants grew well in the presence of up to 200 mM NaCl, while control plants died within 10 days under these treatment conditions. These transgenic plants grew under salt stress for a period of 4 weeks, and were capable of flowering and set seed. T₁ plants segregated into 3:1 ratio suggesting Mendelian segregation pattern of inheritance of the P5CS transgene.     

Glycine in the conserved motif III modulates the thermostability and oxidative stress resistance of peptide deformylase in Mycobacterium tuberculosis

Peptide deformylase (PDF) catalyses the removal of the N-formyl group from the nascent polypeptide during protein maturation. The PDF of Mycobacterium tuberculosis H37Rv (MtbPDF), overexpressed and purified from Escherichia coli, was characterized as an iron-containing enzyme with stability towards H(2) O(2) and moderate thermostability. Substitution of two conserved residues (G49 and L107) from MtbPDF with the corresponding residues found in human PDF affected its deformylase activity. Among characterized PDFs, glycine (G151) in motif III instead of conserved aspartate is characteristic of M. tuberculosis. Although the G151D mutation in MtbPDF increased its deformylase activity and thermostability, it also affected enzyme stability towards H(2) O(2) . Molecular dynamics and docking results confirmed improved substrate binding and catalysis for the G151D mutant and the study provides another possible molecular basis for the stability of MtbPDF against oxidizing agents.     

Multiple templates-based homology modeling enhances structure quality of AT1 receptor: validation by molecular dynamics and antagonist docking

We present a comparative account on 3D-structures of human type-1 receptor (AT1) for angiotensin II (AngII), modeled using three different methodologies. AngII activates a wide spectrum of signaling responses via the AT1 receptor that mediates physiological control of blood pressure and diverse pathological actions in cardiovascular, renal, and other cell types. Availability of 3D-model of AT1 receptor would significantly enhance the development of new drugs for cardiovascular diseases. However, templates of AT1 receptor with low sequence similarity increase the complexity in straightforward homology modeling, and hence there is a need to evaluate different modeling methodologies in order to use the models for sensitive applications such as rational drug design. Three models were generated for AT1 receptor by, (1) homology modeling with bovine rhodopsin as template, (2) homology modeling with multiple templates and (3) threading using I-TASSER web server. Molecular dynamics (MD) simulation (15 ns) of models in explicit membrane-water system, Ramachandran plot analysis and molecular docking with antagonists led to the conclusion that multiple template-based homology modeling outweighs other methodologies for AT1 modeling.     

Purification and characterization of manganese-dependent alkaline serine protease from Bacillus pumilus TMS55

The purification and characterization of a Mn2+-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be 60 degreesC. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. Mn2+ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants (H2O2, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.     

Role of aromatic stack pairing at the catalytic site of gelonin protein

Aromatic–aromatic interactions play an important role in the enzyme–substrate recognition mechanism and in stabilization of proteins. Gelonin – a ribosome inactivating protein (RIP) from the plant Gelonium multiflorum – belongs to type-I RIPs and shows N-glycosylation activity which has been used as a model to explain the role of aromatic–aromatic stack pairing in RIPs. RIPs have a different substrate binding site and catalytic site. Role of tyrosine residues at the binding site has already been known but the role of tyrosine residues at catalytic site is still unclear. In this study, the role of tyrosine–adenine–tyrosine aromatic stack pairing at the catalytic site was studied by in silico mutation studies using molecular dynamic simulations. Through this study we report that, despite the fact that aromatic stack pairing aids in recognition of adenine at binding site, both the tyrosine residues of stack pairing play a crucial role in the stabilization of adenine at catalytic site. In the absence of both the tyrosine residues, adenine was unstable at catalytic site that results in the inhibition of N-glycosylation activity of gelonin protein. Hence, this study highlights the importance of π–π stack pairing in the N-glycosidic activity of gelonin by determining its role in stabilizing adenine at catalytic site.