Maternal and paternal hybrid triploids of tetras

Hybrid maternal triploids were generated by crossing grey-body and red or yellow-tailed Buenos Aires tetra Hemigrammus caudovittatus (BT) males with albino or black Gymnocorymbus ternetzi [widow tetra (WT)] females and retaining the second polar body by heat shock (HS) or cold shock (CS). Paternal triploids were also generated using 2·5% polyethylene glycol (PEG) incubated BT semen to facilitate the entry of two sperm into an egg of WT. Optimum temperature for CS was identified as 6° C and the optimum age for thermal shocking the zygote as 3 min after fertilization. At hatching, survival was 25, 17 and 5% for the HS, CS and paternal triploids, respectively. At maturity, it was further reduced to >2% and the maturity inordinately delayed. With the presence of undifferentiated 'steriles' and almost total absence of females, the expected sex ratio was distorted. Triploid males produced spermatocytes, spermatids but not spermatozoa. Triploidy was confirmed by phenotypic markers, karyotyping, erythrocyte measurement and molecular markers. The fusiform body shape of WT was a dominant phenotypic trait over the typical piscine slender body shape of BT. Polymerase chain reaction products of the genomic DNA of the triploids amplified by OPF6 primer were 300, 450 and 1000 bp length, characteristic of BT, and 500 and 800 bp, typical of WT and thereby confirmed the biparental genomic contribution to the triploids. Analyses of genomic DNA of selected progenies using DMRT-1 marker showed that (1) like the diploid BT males, the hybrid triploid males were also true genetic males, and amplified 237 and 300 bp products but (2) the triploid steriles amplified all the three products of 100, 237 and 300 bp indicating that they were mosaics but the diploid steriles failed to amplify.     

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.     

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.     

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.     

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.     

Isolation and sequence analysis of a small cryptic plasmid pRK10 from a corrosion inhibitor degrading strain Serratia marcescens ACE2

The nucleotide sequence of a smallest cryptic plasmid pRK10 of Serratia marcescens ACE2 was determined. When compared to the all other plasmids reported so far from S. marcescens in sizes of over 70 kb, pRK10 is only 4241 bp long with 53% G + C content and has five coding sequences representing a coding percentage of 65.41. This small plasmid consists of one Tdh gene, four mobilization genes, mobCABD, and an origin of replication homologous to those of ColE1-type plasmids. Analysis of the five open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization containing sequences homologous to the bom region and mobCABD genes of ColE1 and Tdh from Acinetobacter baumannii str. AYE. Results also indicate that pRK10 does not encode any gene for antibiotic/heavy metal resistance. Copy number and incompatibility of the plasmid with plasmids of ColE1 origin of replication was determined and it is quite stable in its natural host as well as in Escherichia coli DH5α. This relatively small plasmid will be useful for construction of shuttle vectors to facilitate the genetic analysis.     

Evaluation of cetyltrimethylammonium bromide as a potential short-term preservative agent for stripped goat skin

The aim of the present study was to evaluate cetyltrimethylammonium bromide (CTAB) as a short-term preservative agent for stripped goat skin, as an alternative to common salt (sodium chloride), the presently used preservative in the leather industry. Bacteria were isolated from the stripped goat skin at various time intervals and 16S rDNA sequencing results showed the presence of both Gram positive and Gram negative bacteria in stripped goat skin. All the bacterial isolates were inhibited by CTAB with MICs of 0.5–24.5 mg l⁻¹. Application of CTAB against microbial consortium on stripped goat skin showed that skin sample kept in 5% CTAB solution for 10 min did not contain any bacterial growth even after 12 days storage at room temperature. Scanning electron microscopy analysis and physical testing of skin preserved using CTAB did not reveal any bacterial attack on fiber structure of skin. This study shows that CTAB can be applied as a viable alternative to NaCl which generates a huge amount of pollution there by reducing pollution from leather processing.     

Biological synthesis of gold nanocubes from Bacillus licheniformis

Microorganisms play an important role in the eco-friendly synthesis of metal nanoparticles. This study illustrates the synthesis of gold nanocubes using the bacterium Bacillus licheniformis after 48 h of incubation at room temperature. The morphology of the samples was analyzed using scanning electron microscopy (SEM) and the particles formed were characterized to be nanocubes. The size of gold nanocubes in aqueous solution has been calculated using UV–Vis spectroscopy, XRD and SEM measurements. The nanoparticles are found to be polydisperse nanocubes in the size range 10–100 nm.     

Gold nano particle decorated graphene core first generation PAMAM dendrimer for label free electrochemical DNA hybridization sensing

A novel first generation (G1) poly(amidoamine) dendrimer (PAMAM) with graphene core (GG1PAMAM) was synthesized for the first time. Single layer of GG1PAMAM was immobilized covalently on mercaptopropionic acid (MPA) monolayer on Au transducer. This allows cost effective and easy deposition of single layer graphene on the Au transducer surface than the advanced vacuum techniques used in the literature. Au nano particles (17.5 nm) then decorated the GG1PAMAM and used for electrochemical DNA hybridization sensing. The sensor discriminates selectively and sensitively the complementary double stranded DNA (dsDNA, hybridized), non-complementary DNA (ssDNA, un-hybridized) and single nucleotide polymorphism (SNP) surfaces. Interactions of the MPA, GG1PAMAM and the Au nano particles were characterized by Ultra Violet (UV), Fourier Transform Infrared (FTIR), Raman spectroscopy (RS), Thermo gravimetric analysis (TGA), Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Cyclic Voltmetric (CV), Impedance spectroscopy (IS) and Differntial Pulse Voltammetry (DPV) techniques. The sensor showed linear range 1×10(-6) to 1×10(-12) M with lowest detection limit 1 pM which is 1000 times lower than G1PAMAM without graphene core.     

Arginine and Lysine interactions with �� residues in metalloproteins

Metalloproteins have many different functions in cells such as enzymes; signal transduction, transport and storage proteins. About one third of all proteins require metals to carry out their functions. In the present study we have analyzed the roles played by Arg and Lys (cationic side chains) interactions with π (Phe, Tyr or Trp) residues and their role in the structural stability of metalloproteins. These interactions might play an important role in the global conformational stability in metalloproteins. In spite of its lower natural occurrence (1.76%) the number of Trp residues involved in energetically significant interactions is higher in metalloproteins.