Optimization,purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2°C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn²⁺ and strongly inhibited by Ba²⁺. All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.     

Marker-assisted improvement of bacterial blight resistance in parental lines of Pusa RH10, a superfine grain aromatic rice hybrid

Pusa RH10, the widely cultivated superfine grain aromatic rice hybrid, and its parental lines Pusa6B and PRR78 are susceptible to bacterial blight (BB) disease caused by Xanthomonas oryzae pv. oryzae. Pusa1460, a Basmati rice variety, was utilized as the donor for introgressing BB resistance genes xa13 and Xa21 into Pusa6B and PRR78 using a marker-assisted backcross breeding program. The markers RG136 and pTA248 linked to BB resistance genes xa13 and Xa21, respectively, were used for foreground selection. Seventy-four STMS markers polymorphic between Pusa6B and Pusa1460, and 54 STMS markers polymorphic between PRR78 and Pusa1460, were utilized for background selection to recover the recurrent parent genome ranging from 85.14 to 97.30% and 87.04 to 92.81% in the 10 best BC₂F₅ families of Pusa6B and PRR78, respectively. RM6100, an STMS marker linked to fertility restorer gene (Rf), was used for marker-assisted selection of Rf gene in an improved version of PRR78. The extent of donor segments in the improved version of Pusa6B was estimated to be <0.97 and <2.15 Mb in the genomic regions flanking xa13 and Xa21, respectively, whereas in improved PRR78, it was estimated to be <2.07 and <3.45 Mb in the corresponding genomic regions. Improved lines of Pusa6B and PRR78 showed yield advantages of up to 8.24 and 5.23%, respectively. The performance of the BB-resistant version of Pusa RH10 produced by intercrossing the improved parental lines was on a par with or superior to the original Pusa RH10.     

Characterization of a glycoprotein isolated from a continuous tumor-cell line of human lung carcinoma

A glycoprotein with a molecular weight of 62 000 has been isolated from a tumor-cell line, A549, and purified to homogeneity by gel chromatography. The glycoprotein contained sialic acid, galactose, mannose, N-acetylglucosamine and a relatively high amount of glutamic acid and proline. The data indicated that the overall composition of this glycoprotein was different from that of the glycoprotein of Mr 62 000 isolated from lung lavage of patients with alveolar proteinosis. The glycoprotein did not react with the antiserum raised against glycoprotein of Mr 62 000 isolated from lung lavage of patients with alveolar proteinosis.     

Phytoremediation of 137Cs: factors and consequences in the environment

Radionuclide contamination is a concerning threat due to unexpected nuclear disasters and authorized discharge of radioactive elements, both in the past and in present times. Use of atomic power for energy generation is associated with unresolved issues concerning storage of residues and contaminants. For example, the nuclear accidents in Chernobyl 1986 and Fukushima 2011 resulted in considerable deposition of cesium (Cs) in soil, along with other radionuclides. Among Cs radioactive variants, the anthropogenic radioisotope ¹³⁷Cs (t_½?=?30.16 years) is of serious environmental concern, owing to its rapid incorporation into biological systems and emission of β and γ radiation during the decaying process. To remediate contaminated areas, mostly conventional techniques are applied that are not eco-friendly. Hence, an alternative green technology, i.e., phytoremediation, should in future be considered and implemented. This sustainable technology generates limited secondary waste and its objectives are to utilize hyper-accumulating plants to extract, stabilize, degrade, and filter the radionuclides. The review highlights plant mechanisms for up-taking radionuclides and influences of different environmental factors involved in the process, while considering its long-term effects.

     

Assessment of genetic diversity in 31 species of mangroves and their associates through RAPD and AFLP markers

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity in 31 species of mangroves and mangrove associates. Four AFLP primer combinations resulted in the amplification of 840 bands with an average of 210 bands per primer combination and 11 RAPD primers yielded 319 bands with an average of 29 bands per primer. The percentage of polymorphism detected was too high indicating the high degree of genetic variability in mangrove taxa both at inter- and intra-generic levels. In the dendrogram, species belonging to a particular family/ genus, taxa inhabiting similar habitats or having similar adaptations tended to be together. There were exceptions too; as many unrelated species of mangroves form clusters. The intrafamilial classification and inter-relationships of genera in the family Rhizophoraceae could be confirmed by molecular analysis. Both the markers RAPD and AFLP were found equally informative and useful for a better understanding of the genetic variability and genome relationships among mangroves and their associated species.     

Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads

In the present study, the optimum conditions for the production of xylanase by immobilized spores of Trichoderma reesei SAF3 in calcium alginate beads were determined. The operational stability of the beads during xylanase production under semi-continuous fermentation was also studied. The influence of alginate concentration (1, 2, 3, and 4%) and initial cell loading (100, 200, 300, 400, and 500 beads per flask) on xylanase production was considered. The production of xylanase was found to increase significantly with increasing concentration of alginate and reached a maximum yield of 3.12 ± 0.18 U ml⁻¹ at 2% (w/v). The immobilized cells produced xylanase consistently up to 10 cycles and reached a maximum level at the forth cycle (3.36 ± 0.2 U ml⁻¹).     

Chitosan IFN-γ-pDNA Nanoparticle (CIN) Therapy for Allergic Asthma

Background  

Allergic subjects produce relatively low amounts of IFN-γ, a pleiotropic Th-1 cytokine that downregulates Th2-associated airway inflammation and hyperresponsiveness (AHR), the hallmarks of allergic asthma. Adenovirus-mediated IFN-γ gene transfer reduces AHR, Th2 cytokine levels and lung inflammation in mice, but its use would be limited by the frequency of gene delivery required; therefore, we tested chitosan/IFN-γ pDNA nanoparticles (CIN) for in situ production of IFN-γ and its in vivo effects.     

Click chemistry based rapid one-pot synthesis and evaluation for protease inhibition of new tetracyclic triazole fused benzodiazepine derivatives

We describe herein a one-pot synthesis of novel tetracyclic scaffolds that incorporate a fusion of a proline, 1,2,3-triazole ring with [1,4]-benzodiazepin-8(4H)-one ring systems following click chemistry. The expected peptide bond formation followed by in situ 1,3-dipolar cycloaddition in absence of any catalyst led to the formation of new triazole fused benzodiazepine derivatives.     

Cross-transferability and Polymorphic Potential of Genomic STMS Markers of Brassica Species

The present study was carried out with the objective of evaluating genomic STMS markers developed earlier in Brassica napus, B. oleracea, B. rapa and B. nigra for their use in Brassica juncea and B. carinata. Ninety-six of the 100 STMS markers used under standardized annealing temperatures and gel concentrations produced clear reproducible amplification pattern. For majority of the markers 60 °C annealing temperature and 3.5% metaphor agarose gel were found suitable. High cross-transferability of STMS markers to related Brassica species including B. carinata (91.6%) and B. juncea (87.5%) suggested the possibility of utilizing these markers for genome analysis in the species where no such markers are available. The ‘B’ genome derived markers showed lower level of transferability to the ‘A’ and ‘C’ genome Brassica species. The potential of STMS markers to detect polymorphism among Brassica species and genera was 98.9%. The level of inter-specific polymorphism was much higher than the intea-specific polymorphism. The markers capable of revealing polymorphism among Brassica species and genera would be useful in Brassica introgression breeding programme. The polymorphic markers were found efficient in establishing the expected evolutionary relationships among the six different Brassica species and two related genera. Low level of intra-specific polymorphism revealed by these markers suggested use of a large set of such markers for various applications in Brassica genetics, genomics and breeding.     

Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.)

Background

Despite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.

Results

Considering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (H _E ) and observed (H _o ) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.

Conclusion

UGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.