Research Journal of Biotechnology

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Research Journal of Biotechnology





Effects of natamycin on the elimination of fungal contamination in rice and Arabidopsis thaliana tissue cultures for Agrobacterium-mediated transformation

Liao Pengfei 1, 2, Zhao Junxing2 and Li Shaobo2*

Fungal contamination is an intractable problem in scientific plant tissue culture laboratory. To explore whether natamycin can effectively and safely eliminate the fungal contamination in plant tissue cultures for genetic transformation, we tested the antifungal activities of natamycin against five common filamentous fungi and six fungi derived from tissue culture contaminants and its phytotoxic effects on the tissue cultures of rice and Arabidopsis thaliana. Our results show that natamycin exhibited high and broad-spectrum antifungal activity and that 10 mg L-1 natamycin inhibited the growth of all the tested fungi without adversely affecting seed germination, seedling root growth, callus induction and proliferation. Moreover, the addition of natamycin in co-cultivation medium did not negatively influence the growth of Agrobacterium tumefaciens and genetic transformation efficiency. In conclusion, our results manifest that natamycin can optimally control the fungal contamination in plant genetic transformation mediated by Agrobacterium tumefaciens at the concentration of 10 mg L-1.

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Immunological Analysis of Glycoprotein C of Herpes Virus of Turkey’s expressed in Kluyveromyces lactis

Sudhakar Smitha * and Nair A. J.

At the beginning of the 20th century intensification of the poultry economy gave birth to a new pathogen, a highly evolving Herpes virus that caused Marek’s disease. Vaccination with purified Herpes virus of Turkey’s (HVT) inactivated viral preparations. A membrane fraction isolated from virus-infected chicken embryo fibroblasts can confer immunity against Marek’s disease. HVT-induced membrane antigens could be used as immunogens against Marek’s Disease Virus Type1. In this study glycoprotein C of HVT vaccine strain FC126 was amplified, cloned and expressed in pKLAC vector. The expressed glycoprotein was purified and was injected to field chickens.The serum from chicken was used for immunodiffusion analysis against MDV1 sample. A well developed precipitin line gave clear cut evidence that the in vitro expressed glycoprotein C was capable of eliciting an immune response in chickens against MDV1.This principle can be effectively used for production of recombinant vaccines against MDV1 based on HVT envelope glycoproteins.

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Immunomodulatory properties of Cissampelos leaf extract in normal and fructose-induced hyperglycaemic rats

Basumata Caroline

The primary target of most of the immunomodulatory compounds is believed to be the macrophages which play a key role in the generation of an immune response. Activated macrophages produce not only effector molecules like free radicals and nitric oxide, but also produce cytokines like TNF- , IL1, IL6, IL12, etc. These cytokines mediate the effect or response of other immune cell population. The present study revealed that percent phagocytosis of peritoneal macrophages was significantly enhanced (p<0.001) in normal and hyperglycaemic Cissampelos pareira Linn. (Menispermaceae) leaf extract treated rats. The ranges were 50-52 in normal and 48-52 in hyperglycaemic 100 mg extract treated groups and they were 60-64, 45-60 respectively in the 300mg extract treated rats. Likewise, the haemagglutination antibody titre was also significantly enhanced in the treated animals (64-256 in 300mg groups). Since macrophages have major role in modulating immune response, the results of the present study indicate possible role of Cissampelos pareira Linn. (Menispermaceae) leaf extract in stimulation of immune response. It may be useful in preventing oxidative damage of organs in diabetes.

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An in silico based understanding of drug resistance and residual deletion in tubulin protein from sequential and structural perspective

SelvaaKumar Chellasamy1* and Sudheer M. M. Mohammed2

Tubulin being the fundamental unit of microtubule is actively involved in cell division, transportation and chromosomal segregation. Through this study we tried to investigate the significance of eight residual insert (TVVPGGDL) in S–loop region of α-tubulin which is absent in the S-loop of β-tubulin. This study reports hydrogen bonding interactions between the S-loop and the M-loop in α-tubulin whereas salt bridge is observed between S-loop and the H1-S2 loop of β-tubulin. These residual interactions clearly explain the flexibility/rigidness of the H1-S2 and M loops in α and β subunits which are actively involved in lateral interactions. An increase in bulky residues in the H1-S2 loop along with their interactions with S-loop was found to be responsible for restricted mobility of the H1-S2 loop in β-subunits. This constraint of the H1-S2 loop in association with S-loop brings in weak lateral interaction between β-β subunits in comparison with α-α subunit interaction. Next, the available drug resistant mutant from human β–tubulin protein was investigated using Amino Acid Substitution tools (AAS) like PANTHER, I-Mutant2.0 and Polyphen2. Generated report confirms only the residual mutants at the functionally significant positions like H1-S2 loop, helix H5, T6 loop, B7, B7-H9 loop, H9-B8, H10-B9 and helix H12 to be deleterious. Thus our finding explains the significance of eight residual insert in α-subunit which assist in better lateral interaction through their residual contact with M-loop and the role of residual substitution in drug resistance in β–tubulin. Here we hypothesize that the flexibility of the H1-S2 loop in association with the S-loop determines the strength of lateral interactions. Thus the better understanding of the flexibility of the tubulin protein and residual interactions could assist in better drug designing against cancer.

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An efficient protocol for genomic DNA isolation from field grown mature leaves of Penniseteum gluacum[(L) R. Br.]

Padaria Chatrath J.,* Choudhary M. and Tarafdar A.

Pearl millet, Penniseteum gluacum [(L) R. Br.] is a rich source of novel genes for tolerance to different abiotic stresses as salinity, drought and high temperature. To carry out work on genomics of P. gluacum for isolating abiotic stress responsive novel genes, high quantity of DNA in purified form is required. Field grown leaf samples of P. gluacum are rich in secondary metabolites and polysaccharides which greatly interfere during DNA isolation and purification process leading to poor quality and quantity of isolated DNA. Such DNA preparations hinder the downstream molecular biology studies as restriction, PCR amplification and cloning. Here we report an improvement in quality and yield of the extracted DNA from leaves of field grown pearl millet plants through the modification in the cetyl trimethylammonium bromide (CTAB) DNA extraction buffer. This modified buffer included 2% CTAB, 2% polyvinylpyrrolidone and 0.5% β-mercaptoethanol with rest of the constituents as same as CTAB method described by Doyle and Doyle5. The isolated DNA proved amenable to downstream molecular studies like PCR amplification and restriction digestion.

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In Vitro Clonal Propagation of Vernonia anthelmintica (L.) Willd.- An Oil-Yielding Herb

Kure S. R., Mulani R. M. and Surwase B. S.*

A method for in vitro regeneration of Vernonia anthelmintica (L.) Willd. of Asteraceae from shoot tip explants derived from mature plants is outlined. The explants when cultured on Murashige and Skoog (MS) medium supplemented with 1, 2, 3, 4 and 5 mgl-1 levels of cytokinins viz. N6-benzyladenine (BA) and Kinetin (Kn) either alone or in combination with auxins i.e. α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) or silver nitrate (AgNO3) or copper sulphate ( CuSO4) induced multiple shoots. However, the best condition for shoot growth was with 2 mgl-1 BA and 0.5 mgl-1 copper sulphate in MS medium. An average of 9.9 shoots was obtained per explant after 30 days of culture. The rooting of in vitro developed shoots (5~6 cm length ) was best in ½ MS medium alone containing 3% sucrose with 80% of response and micropropagated plants were successfully acclim¬atized (70%) within 6 weeks after rooting. The standardized protocol reported in this study may help in conservation of this plant species which is currently exploited from the nature.

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Optimization of bacteriocin-like substance production by Bacillus cereus XH25 using response surface methodology

Zhong X.T.,1 Zhang Q.,1, 2 Lin K.,1, 2 Yuan C.H.,1 Li K.,1 Jia B.H.,1, 2 Che Z.M.1 and Xiang W.L.*1, 2

Response surface methodology was employed to optimize medium composition of bacteriocin-like substance production by Bacillus cereus XH25. The initial pH of medium, soluble starch and (NH4)2SO4 were found to influence significantly on the bacteriocin-like substance production. The steepest ascent method was undertaken to access the optimal region of the above three significant factors and then their concentrations were optimized by the central composite design and the response surface method. The results indicated that the quadratic term of the three factors and the interactions between soluble starch and initial pH value, (NH4)2SO4 and initial pH value had a significant effect on the production of bacteriocin-like substance. The optimized medium composition was soluble starch 15.32 g/L, (NH4)2SO4 6.02 g/L and initial pH value 6.15 which tamped a 21.69% increase of antibacterial activity against Staphylococcus aureus ATCC 9144 when compared with the original medium. The predicted value 12.76 mm was verified subsequently by validation experiments value 12.90 mm. The excellent correlation between predicted and observed values further confirmed the adequacy and accuracy of the model obtained. The results obtained in current work provided a basis for large scale fermentation of bacteriocin-like substance by B. cereus XH25.

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The effect of lignin and Sapindus saponin on enzymatic hydrolysis of cellobiose

Bu Lingxi, Zhao Danqing and Jiang Jianxin*

This study examines the role of lignin from furfural residues and natural saponin from Sapindus mokorossi on the enzymatic hydrolysis of cellobiose. Lignin and Sapindus saponin play an active role in enzyme hydrolysis of the cellobiose substrate. Addition of Sapindus saponin to the hydrolysis reaction increases the glucose yield. In the absence of saponin, the glucose yield is the highest with the addition of 10% lignin. The adsorption rate of the enzyme on lignin is rapid in the sample with Sapindus saponin. Lignin content shows to have no apparent effect on cellobiose hydrolysis with low enzyme loading. After hydrolysis for 4 h, the enzyme activity did not significantly improve when the lignin content was increased from 5% to 20%. Addition of Sapindus saponin and 20% lignin resulted in the highest β-glucosidase activity among the samples studied. In the cellobiose hydrolysis system, lignin, which was only solid, has the potential to promote the enzymatic reaction of cellobiose hydrolysis.

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Pretreatment with Pterocarpus marasupium extracts protecting mice against 6 Gray electron beam radiation

Sujata B.,1 Vidya S.M.,1* Ganesh S.2 and Rao C. V.1

Radiation can induce the acute and chronic radiation sickness via physical, chemical and biological mechanisms. In this study, we investigated the radioprotective effects of Pterocarpus marsupium ethanolic (PM) on electron beam radiated mice. Mice were given Pterocarpus marsupium at three different concentrations (100, 200 and 400 mg/kg B.W) orally one daily for 15 consecutive days before exposure to 6 Gy electron beam radiation. Pterocarpus treated mice after irradiated by 6Gy significantly increased the spleen and thymus index compared to 6 Gy irradiation+DDW mice. Besides, the Pterocarpus treatment facilitated the recovery of white blood cells (WBC). Moreover, the irradiated mice showed a depletion in superoxide dismutase (SOD), catalase, glutathione-S-transferase (GST), glutathione, activities and total anti-oxidative capacity, while elevation in the malondialdehyde (MDA) contents. PM treated mice after irradiation increased activities of SOD and catalase and decreased the MDA levels in liver. In addition, the reduced numbers of micronuclei (MN) of polychromatic erythrocytes (PCEs) in the bone marrow after mice were irradiated by 6 Gy of electron beam radiation. Findings indicated that Pterocarpus marsupium had the therapeutic effects on mice against hematopoietic suppression and anti-oxidative damage caused by irradiation.

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Selective Isolation and Molecular Identification of l-glutaminase producing Bacteria from Marine Sediments

Kiruthika J.1* and Saraswathy N.2

L-Glutaminase (L-glutamine amidohydrolase E. C. 3.5.1.2) is a therapeutic enzyme which catalyses the conversion of L-glutamine to L-glutamic acid and ammonia. It has gained importance due to its potential application as anti-cancer and flavor enhancing agent. With an aim to isolate a potential L-glutaminase producing strain, soil samples were collected from different river banks of Kerala and Tamil Nadu, India. Among the several strains, based on the enzyme activity, one of the potent isolate was selected and identified. The morphological and biochemical characterization revealed that the potent isolate belongs to the genus Vibrio. Further, 16s rRNA gene (1.4 Kbp) of the potent isolate was amplified and analyzed which showed that it had about 99% similarity with Vibrio sp. IMSF-06 (NCBI accession no. GQ907023) and the next homologue was Vibrio azureus; HNS029 (NCBI accession no. JN128263). Thus, the strain was ultimately identified as Vibrio azureus strain JK 79 (GenBank Acession Number JQ820323).

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An overview on Sickle Cell Disease Profile

Kaur M.,1* Dangi C.B.S.1 and Singh M.2

Sickle cell disease (SCD) is a very devastating condition caused by an autosomal recessive inherited haemoglobinopathy. This disease affects millions of peoples globally which results in serious complications due to vaso-occlusive phenomenon and haemolysis. This genetic abnormality is due to substitution of amino acid valine for the glutamic acid at the sixth position of beta chain of haemoglobin. This disease was described about one hundred year ago. The haemoglobin S (hbS) produced as result of this defect is poorly soluble and polymerized when deoxygenated. Symptoms of sickle cell disease are chronic anaemia, pain full crises, acute chest syndrome, stroke and susceptibility to bacterial infection. In recent years measures like prenatal screening, better medical care, parent education, immunization and penicillin prophylaxis have successfully reduced morbidity and mortality and have increased tremendously life expectancy of affected individuals. Three principal current therapeutics modalities available for childhood SCD are blood transfusion, hydroxy urea and bone marrow transplantation. Genetic counselling, continued medical education for health professionals about sickle cell disease, its complications and management is necessary. World health organization has actively promoted several national screening programs with dual goals of informing reproductive choice and thereby reducing the number of severely affected children.

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Association of Chronic Hepatitis C with Dermatologic Manifestations

Al Akeel Raid

HCV infection (HCV) results in various clinical manifestations, leading to dermatological and mucocutaneous manifestations. Common among these are dermatological conditions and systemic disorders affecting the skin. HCV is the cause of or is associated with various dermatologic disorders. In patients with such disorders, HCV infection must be sought routinely because antiviral therapy may be beneficial in some of them. Treatment of dermatological manifestations of HCV through eradication of the virus is therefore important in effective patient management, although this alone is not a major justification for HCV treatment.

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