Research Journal of Biotechnology

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Research Journal of Biotechnology

High frequency in vitro multiplication from cotyledonary node explants of an endangered medicinal plant Desmodium gangeticum L. (DC)

An efficient, rapid and reproducible plant regeneration protocol was developed for Desmodium gangeticum (L.) DC an endangered medicinal plant. Cotyledonary node explants from 10 day-old aseptic seedlings were cultured on full and half-strength Murashige and Skoog (MS) and Gamborg’s (B5) media containing the cytokinins 6- benzyl aminopurine (BA), kinetin (KIN) and thiadiazuran (TDZ). BA was found to be the most effective for shoot proliferation. The highest response (98% of explants) and the maximum number of shoots (12.4 per explant) were induced on MS medium supplemented with 4.44 μM BA. Repeated subcultures of cotyledonary nodes for three months on the above medium enabled mass multiplication of shoots without any evidence of decline. The highest rooting (98%) and maximum number of roots (8) per shoot were obtained when shoots were dipped in indol-3-butyric acid (IBA) solution (492.12 μM) for 30 minutes and then cultured on half-strength MS medium. Plantlets were successfully acclimatized in soilrite with survival rate of 96%. Regenerated plants grew normally in the field without showing any morphological variation.

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Enhancing the milk production and immunity in Holstein Friesian crossbred cow by supplementing novel nano zinc oxide

Rajendran D., Kumar G., Ramakrishnan S. and Thomas K. Shibi

An experiment was conducted to improve the milk production and immunity by supplementing nano zinc oxide for a period of 75 days in lactating crossbred Holstein Friesian cow affected with subclinical mastitis. Total of 48 lactating (2-4th lactation) crossbred Holstein Friesian cow were divided into four groups (T1 to T4) having 12 replicates. The experimental groups are as follows: T1 is healthy unaffected; all other groups (T2 to T4) were affected by subclinical mastitis. T2 was supplemented with inorganic zinc (macro sized particle). Cows in T3 and T4 groups were supplemented with zinc methionine and nano zinc oxide @ 60 ppm of Zn respectively. The results revealed that fortnightly average milk production before and after first to fifth fortnight of the trial period was significantly (P<0.01) higher in healthy (T1) cows. The significant (P<0.01) improvement was noticed in T3 and T4 from 3rd and 2nd fortnight, respectively. Zinc oxide supplemented group (T2) did not improve its milk production throughout the trial period. The somatic cell count (SCC) was significantly (P<0.01) low in healthy animals (below 0.2 million cells per ml) than subclinical mastitis cow before start of experiment. The T2 (Zinc oxide supplemented group) showed only after fifth fortnight below 0.2 million cells per ml. However, T3 and T4 reached 0.2 million cells per ml as early as third and second fortnight respectively. Average serum zinc (μmol/l) content of healthy and subclinical mastitis cross bred Holstein Friesian cows before start of experiment was significantly (P<0.01) higher in healthy animals and after start of experiment it was significantly higher (P<0.01) in nano ZnO supplemented (T4) group. Zinc oxide supplemented group (T2) improved its level after third fortnight but it remained significantly lower than that of nano ZnO supplemented (T4) group. As a result, increase in milk production, immunity and suppression of subclinical mastitis (reduction in SCC values) by nano zinc oxide supplementation to dairy animal could be useful feeding strategies.

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Comparative study on 11β – hydroxysteroid dehydrogenase 1

Tang N. N.1 and Le L.1,2*

11β – Hydroxysteroid dehydrogenase 1 (11βHSD1) belongs to the human short-chain dehydrogenase /reductase super-family which catalyzes the conversion of cortisone to cortisol in humans. High 11βHSD1 activity is known to closely link to certain metabolic syndromes, including insulin resistance. 11βHSD1 inhibitors act as competitors of cortisol and prevent the binding of the substrate to 11βHSD1, suppressing 11βHSD1 activity and increasing hepatic insulin sensitivity. 11βHSD1 inhibition is an important therapeutic target of type 2 diabetes drug design. In this study, a comparative analysis of human 11βHSD1 homologous sequences was intensively analyzed using bioinformatics tools. The different level of similarities among these homologous sequences to human 11βHSD1 was considered as a critical standard to suggest potential animals for clinical testing. The result of the phylogenetic tree shows that the human 11βHSD1 sub-family evolved into two distinct pathways with two groups of homologous proteins. Based on group one, 22 selected proteins were further analyzed on the level of sequence similarity. As a consequence, there were 8 potential models for animal testing (Bos taurus; Ovis aries; Oryctolagus cuniculus; Mesocricetus auratus; Sus scrofa; Heterocephalus glaber, Rattus norvegicus; Mus musculus; and Myotis lucifugus) that displayed a high level of sequence similarity to human 11βHSD1. Key residues that vary from humans to each selected species will also be discussed in detail.

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Immobilization of pectinase on co-precipitated magnetic nanoparticles for enhanced stability and activity

Muthulingam Seenuvasan1, Carlin Geor Malar1, Sridhar Preethi1, Nagarajan Balaji1, Jayaraj Iyyappan1, Madhava Anil Kumar1 and Kannaiyan Sathish Kumar2*

The covalent binding of the pectinase enzyme onto magnetic nanoparticles (MNPs) via glutaraldehyde activation was investigated. The size, structure and morphology of the nanoparticles were characterized using transmission electron microscopy (TEM) and X-ray diffraction (XRD) analysis. The average particle size for both naked and pectinase bound MNPs from the XRD pattern using Debye-Scherrer relation reports that binding process did not cause any significant change in size of MNPs. The pectinase binding onto the nanoparticles were confirmed by Fourier transform infrared (FT-IR) spectroscopy. The maximum activity of immobilized pectinase was obtained at its weight ratio of about 16.2 x10-3 mg bound pectinase/mg MNPs. The characteristics performance of the immobilized MNPs was compared with the naked MNPs by pH, temperature, reusability, storage ability and kinetic studies.

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TDZ enhances multiple shoot production from nodal explants of Tinospora cordifolia-a commercially important medicinal plant species of NE India

Choudhury Shally Sultana1* and Handique P. J. 2

Tinospora cordifolia is an important and commercially exploited medicinal plant species found in natural habitats of NE India. In an experiment of in vitro propagation of this species, the formation of multiple shoots in high frequency was achieved in nodal explants culture. MS basal medium was used throughout the experiments with different combinations and concentrations of BAP, Kinetin and Thidiazuron(TDZ).Combination of BAP(2mg/l), Kinetin(4mg/l) and TDZ(0.20mg/l) in MS medium gave a maximum average of 10.29 nos. of shoots per explants within 30 days of inoculation. Individual shoots from about 45 days old multiple shoot cultures were separated and implanted in half strength MS medium supplemented with IBA (2mg/l) for rooting. Root initiation was noticed after 5-6 days and it became profuse after 5 weeks. The rooted plantlets weretransplanted ex-vitro and raised in pots under green house conditions for one month followed by their field transfer. The survival percentage was found to be 80%. By the protocol described here one can get at least 10-12 rooted plants from one explant in 90 days. The rapidity (i.e. within 90 days ) of multiplication of true-to type plants (i.e. within 90 days) and efficient transplantation of T.cordifolia can be useful in conservation and propagation of elite plants for commercial exploitation.

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Optimization and Cultural Characterization of Bacillus RV.B2.90 producing Alkalophilic Thermophilic Protease

Vijayalakshmi S., Venkat Kumar S. and Thankamani V.*

A newly isolated alkalophilic, thermophilic Bacillus RV.B2.90 was characterized and tested for the production of extracellular protease in 50 – 300 ml shake flask experiments. The strain was identified as Bacillus cereus by biochemical and 16S rRNA sequencing. Maximum amount of enzyme produced in basal medium at exponential phase was 1284 U/ml. The optimized medium could increase enzyme production by 30% to 4189 U/ml. Growth and enzyme production occurred in media at all initial pH ranging from 7.0 – 12.0, the maximum enzyme production occurred in the medium with initial pH 8.0. The pH increased with growth. Depending on the starting pH 7.0 – 12.0 the duration of incubation required for change of pH to 9.0-10.0 varied markedly 40 – 12 hrs. This phase coincided with maximal protease production at each pH. Production of enzyme increased significantly with temperature of incubation from 37-70˚C, with 1284 U/ml at 37˚C and 1984 U/ml at 70˚C. At 90˚C the growth and enzyme production showed a steep decline to 400 U/ml. With respect to other growth parameters, the optimum agitation speed was 50 rpm, the best carbon sources were xylose and arabinose that enhanced enzyme production to 4189 U/ml and 4016 U/ml, the best nitrogen sources were casein and malt extract showing 3946 U/ml and 3182 U/ml. Inorganic nitrogen sources did not have significant effect on enzyme production. Sodium chloride at 4% (w/v) was found to be optimum for enzyme production. Among the 5 amino acids added to the medium, histidine increased enzyme production to 1853 U/ml (about 30%). Among the salts added to the medium, K+, Mg2+ and Ca2+ were found to increase the enzyme production. The isolate was found to be highly alkalophilic, thermophilic, thermo stable and moderately halophilic protease producing organism.

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Standardization of Genomic DNA Isolation and Molecular Identification of Rhizophora Sp. Using Non-Coding trn L Gene

Gurudeeban S., Ramanathan T.* and Satyavani K.

DNA extraction is difficult in mangrove plants because of phenolic compounds and secondary metabolites that interfere with DNA isolation procedures and subsequent applications such as DNA restriction, amplification and cloning. We developed a simple, rapid and efficient method for isolating genomic DNA from leaves of Rhizophora mucronata and Rhizophora apiculata that is free from polysaccharides and polyphenols. This newly developed protocol includes the use of 2 M NaCl, 0.1 g of polyvinylpyrrolidone (PVP), 3% mercaptoethanol, 1/10 volume of sodium acetate and 70% ethanol in the extraction as well as reducing the centrifugation times during the separation and precipitation of the DNA. Isolated genomic DNA showed high purity and high quantity. The purity of isolated genomic DNA was confirmed by agarose gel electrophoresis. The use of tRNA Leu (trn L) genes also as RFLP markers to precise the genetic diversity and results reveal the degree of polymorphism and to detect similarities among genotypes. The successful double stranded amplification and partial sequences were obtained for the study plants, the size of the trn L (UAA) intron ranging from 587bp for R.mucronata (GenBank Acc.No.: JF318960) and 538bp for R. apiculata (GenBank Acc.No.: JF318959).

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Buccal micronucleus cytome (BMCyt) assay and chromosomal aberration analysis of tannery workers exposed to Chromium III (Cr III) in Vellore population

Sundaramoorthy Rajiv, Joshua Stalin, Nishu Sekar, Elizabeth Diana Anthony, Reshma Susan Reji and Valsala Gopalakrishnan Abilash*

The aim of the study was to investigate the adverse effects of occupational exposure to trivalent chromium (Cr) in tannery workers in Vellore population. Trivalent chromium used in tanning industry is an environmental contaminant that affects towards the humans, plants and animals. The carcinogenic potential of metals is a major issue in defining human health risk from exposure. In this study 20 tannery workers and equally age and sex matched 10 controls with smoking prevalences and alcohol consumption were enrolled for DNA damage analysis in buccal cells by Micronucleus assay (MN) and Chromosome Aberration (CA) analysis used lymphocyte culture. Workers showed a significant increase in MN cells and CA when compared to controls with respect to their smoking habits, alcohol consumption, age and years of exposure. The current study suggested that chronic occupational exposure to Cr could lead to increased levels of DNA damage.

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Molecular Cloning and Characterization of a Novel Human SCAN Zinc-Finger Protein ZNF447 as a Transcription Activator

Li J., Luo K., Wu J., Yang X. and Yu L.*

Zinc finger proteins play important functions in various cellular processes such as transcription and translation, cell proliferation, differentiation and apoptosis. ZNF447 is identified as a novel zinc finger gene containing a SCAN domain at the N-terminus and two C2H2 (Cys2-His2) zinc finger motifs at the C-terminus. ZNF447 is expressed in several human adult tissues, including heart, brain, kidney, thymus, testis and bone marrow and localized in the nucleus. Reporter gene assays showed that ZNF447 is a transcriptional activator and activated the transcriptional activities of NFκB. In comparison, ZNF447n, another isoform of ZNF447 lacking the zinc fingers, is moderately expressed in all the 18 human adult tissues, localizes in the cytoplasm and plays no functions in transcription regulation. ZNF447 can form a homo-dimer with ZNF447n through SCAN domain and helps ZNF447n to enter the nucleus which may make the transcription regulation more complicated.

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Isolation and characterization of cellulose degrading Brevvibacillus brevis and optimization of its cellulase activity at different physico-chemical parameters

Prasad P.1, Tanuja1 and Bedi S. 2

The goal of this research was to investigate the growth and cellulase enzyme production ability of the isolated bacterial strain St-2, identified as Brevibacillus brevis, at different physico-chemical parameters of pH (3-11), temperature (4-60 0C), salinity (1-10%) and incubation period. The enzyme production was analyzed individually by Dinitrosalicylic acid (DNS) method. It was observed that the isolate showed luxuriant to moderate growth over a wide range of pH (5-11), temperature (4-45 0C), salinity (1-6% NaCl concentration) over 5-6 days of incubation period. The optimal pH was 7, temperature 260C and time duration 6 days for maximum enzyme activity in terms of released reducing sugar (CMCase 6.5 mg/mL and FPase 7.0 mg/mL). The enzyme activity was minimum at higher pH 11 (CMCase 4.6 mg/mL and FPase 5.8 mg/mL) and at 45 0C temperature (CMCase 2.6 mg/mL and FPase 3.2 mg/mL) by the bacterial strain.

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Physiological and biochemical characterization of Xantha mutant of Cucumis melo L.

Shao Qin and Yu Zeyuan*

A novel xantha mutant of Cucumis melo L arose spontaneously and displayed a distinctive phenotype of yellowish leaf across its whole growth period. In order to explain this phenomenon, a simple screening protocol was used to identify the xantha mutant that chlorophyll biosynthesis was hampered at conversion from porphobilinogen (PBG) into uroporphyrinogen (UrogenⅢ). Compared with the normal green plant, chlorophyll content decreased (81.5%), the ratio of chlorophyll a / chlorophyll b increased (52.7%) and carotenoids reduced by 70.7% significantly in the mutant. Net photosynthetic rate and fluorescence kinetic parameters demonstrated that the decrease of chlorophyll content significantly influenced the photosynthetic efficiency of the mutant. Under transmission and specimens for scanning electron microscopy, we observed that mutant leaves were found with underdeveloped chloroplasts, containing fewer palisade tissues and aberrant thylakoids and granum. These results indicated that xantha formation of the mutant might be caused by faulty development of chloroplasts.

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Analysis of Quantum Dots for Nano-Bio applications as the Technological Platform of the Future

Kumar D.N.T.*1,2,3,4 and Wei Qufu1

Semiconductor nanocrystals, also called quantum dots (QDs), are versatile inorganic probes with unique photo-physical properties, such as narrow and size-dependent fluorescence (FL) with broad absorption spectra, strong FL intensity and excellent anti-photo bleaching. QDs have been used in a diverse range of biological applications in different domains such as cell labeling, genomic detection, optical sensors, nanosensors, quantum mechanics based Drug Delivery systems and bio-medical imaging. Nanotechnology has the potential to revolutionize medicine, as well as many other seemingly unrelated subjects such as electronics, textiles and energy production.

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