1.  Journal of  Cell and Tissue Research 15(3): 5133- 5140  (2015) 

Hydrophobic loading and releasing of milk protein carrier systems for tissue culture based screenings 

 Kimpel, F. and Schmitt, J.J. 

Department of Food Technology, Fulda University of Applied Sciences, Leipziger Str. 123, 36037, Fulda, Germany.  

Abstract: Hydrophobic test substances are one of the major problems in cell culture based screenings for new functional molecules. As these substances are not soluble in water based buffers or tissue culture media, they are not able to reach the surface of the testing cells. Therefore, hydrophobic test substances need to be associated to carrier systems which are dispersible in water. The substances need to reach the tissue surfaces together with these carrier systems to attach to the lipophilic surface parts of the cell membranes. Encapsulation of the hydrophobic molecules in lipid structures like emulsions or liposomes cannot be applied, because those structures entrap the molecules and hardly present them to the cell surface membranes. This work introduces a solution to this problem by associating a hydrophobic test molecule to hydrophobic but water dispersible milk protein carrier systems. Those systems were nano structures of casein (micelles), isolated acid casein, sodium caseinate, β-lactoglobulin rich whey protein isolate and isolated α-lactalbumin. It was shown that acid casein and sodium caseinate can carry high amounts of the used hydrophobic model substance (β-carotene) but do not entirely release it. Whey protein isolate carries 5 times less molecules but releases them completely in lipophilic environments. Nano structures of casein diminish the carrying capacity of casein. 

Key words: Milk proteins, β-Carotene, Nano structures.

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2.  Journal of  Cell and Tissue Research 15(3): 5141- 5150  (2015) 

MX2 gene expression and prediction of  B-cell  epitopes  of  its protein to detect early pregnancy in buffalo 

Buragohain, L.,  Nanda, T., Kumar, R., Gupta, S.S. and Balhara, A.K. 

Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar 125 004, Haryana.  

Abstract: The current research was conducted on the expression of Myxovirus resistance 2 (MX2) gene in the peripheral blood of buffalo heifers. The present study reports changes in the expression of Interferon- tau (IFN- τ)  stimulated MX2 gene in blood leukocyte on day17 in pregnant (n=10) and non-pregnant (n=6) buffalo heifers. The synthesized cDNA from extracted RNA of blood cells were used for partial amplification (739bp) of MX2 transcript by PCR with gene specific primers. The PCR products were subjected to agarose gel electrophoresis which showed a marked up-regulation of MX2 gene on day 17 of pregnant group and negligible expression in non-pregnant groups. Further with bioinformatics tools, structural, molecular and biological functional studies were done, followed by prediction of  most antigenic B-cell linear epitopes of Mx2 protein. Chemical  synthesis of these epitopes would help to develop an antibody against it. So, MX2 could  be a  potent pregnancy  specific  marker  which  could  be  used  to  diagnose early pregnancy in buffalo before beginning of next estrous cycle. 

 3. Journal of  Cell and Tissue Research 15(3): 5151- 5157  (2015) 

Cadmium induced ultrastructural changes in the ciliate, Stylonychia mytilus (Ciliophora, hypotrichida)  

 Makhija, S., Gupta, R., Toteja, R., Abraham, J.S.  and Sripoorna, S. 

Ciliate Biology Laboratory, Acharya Narendra Dev College, University of Delhi, Kalkaji, New Delhi 110019. 

Abstract: Transmission electron microscopy (TEM) revealed several ultrastructural changes induced by exposure to cadmium in ciliated protozoan, Stylonychia mytilus. The main cytoplasmic alterations in the post treated cells were:-condensation of the ground cytoplasm, mitochondrial disorganization, cytoplasmic vacuolization, accumulation of electron dense bodies and autophagosome formation. At the nuclear level, there was condensation of macronuclear chromatin and the nucleolar organization underwent major alteration. In the micronuclei, there was convolution of micronuclear surface and retraction of chromatin from the nuclear envelope. These modifications were compared with the changes induced by cisplatin, heat shock treatment, ageing and starvation. 

 4. Journal of  Cell and Tissue Research 15(3): 5159- 5167 (2015) 

Neurocytotoxicity consequences of heat and mild  cold stress in human neural precursor cells and its lineages

 Vishwakarma, S.K.,  Bardia, A., Rahamathulla, S.,  Raju,  N.,  Fathima,  N., Tiwari, S.K., Paspala, S.A.B.,  Sandhya, A.,  Vishnupriya, S. and  Khan, A.A.

 Central Laboratory for Stem Cell Research & Translational Medicine, CLRD, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad 500058.

Abstract: Temperature mediated stress associated with stroke is a life threatening illnesses which results in severe central nervous system (CNS) dysfunction. A complex cascade of cellular and molecular mechanisms happens due to trigger of thermal stress in the body. The present study was undertaken to illustrate how apparently subtle differences in the cellular responses occurs due to heat and mild cold stress on proliferating homogenous population of human NPCs and its lineages. Proliferating CD133+ve enriched human NPCs were exposed to mild cold shock at 33°C and heat shock at 42°C  for 3 days. Neurospheres derived from control cultures at day 14 were dissociated and cultured in neuronal differentiation medium under heat and mild-cold stress for 3 days to assess the changes in molecular and neuronal cell phenotype. Both the stress conditions resulted in increased apoptosis during long-term in culture of developing neurospheres. Reduced neuronal differentiation was observed with aberrent neuronal cell phenotype during both types of stress. The G1 phase of cell cycle was found most severely affected during mild cold stress (33°C) for 14 days of exposure. However, heat stress at 42°C resulted in induced cell cycle activity. Cells under heat and mild cold stress resulted in reduced induction of 72kDa Hsp-70 mRNA transcript in differentiated cells as compared to undifferentiated cells. The present study provided evidence of cellular and molecular alterations generated by heat and mild cold stress in developing neurospheres and lineage differentiation of human NPCs during in vitro culture. 

 5. Journal of  Cell and Tissue Research 15(3): 5169- 5173 (2015) 

Effect of extender and extension  rate on acrosomal integrity of  malabari buck spermatozoa 

 Lekshmi Bhai, K.,  Metilda, J., Behera, S., Harshan, H. M., Aravinda Ghosh, K.N. and Raghavan, K.C. 

Department of Animal Reproduction, Gynaecology and Obstetrics, College of  Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur 680651, Kerala.

Abstract: The objective of the study was to find out the effect of extension rate on the acrosomal integrity of Malabari buck spermatozoa in two different extenders. 48 ejaculates collected from two Malabari bucks were pooled and divided into four groups. Semen in group I and II were extended with egg yolk based extender and Andromed respectively at the rate of 400 million/ml. Extension rate of group III and IV was 800 million/ml with egg yolk based extender and Andromed, respectively. Intactness of acrosome, assessed prior to freezing and 24 hours after freezing showed better results at the extension rate of 400 million/ml with both the extenders. Andromed showed better acrosomal integrity at both the extension rates. 

 6.  Journal of  Cell and Tissue Research 15(3): 5175- 5180 (2015) 

Estimation of C-reactive protein and total serum immunoglobulin concentrations  after uterine immunomodulation in repeat breeding cows 

 Sahoo, S. and  Mohanty, D.N. 

Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry, OUAT, Bhubaneswar, 751003, Orissa.  

Abstract: Twenty one repeat breeding cows were screened for subclinical endometritis and they were assigned equally to one control group and two treatmentgroups. Cows in the control group were infused with 50 ml of normal saline intra-uterine. Out of the two treatment groups, cows in group I were treated with 20 ml of colostrum as intra uterine medication. Cows in treatment group II were infused with 10 ml of non pathogenic E.coli (pure attenuated culture obtained from Himedia Product code – ATCC 25922). C-reactive protein concentrations and total immunoglobulin concentrations were calculated on day 0, day 7th, day 14th and day 21st of estrus cycle from the blood sample. The estimation of  C-reactive protein in all the experimental groups revealed a range between1.2 – 4.8 mg/dl with the negative values obtained for 21 day of estimation indicating improvement of uterine health. Comparison of immunoglobulin values for different days in all the treatment protocol revealed a significant (p<0.05) difference within various days of sampling. 

 7.  Journal of  Cell and Tissue Research 15(3): 5181- 5186 (2015) 

Cloning  and expression of plant codon optimized CRY1AC gene in Saccharomyces cerevisiae 

 Mohan, T.C., Kumara Swamy, G.K., Krishnaraj, P.U., Misra, H.S. and  Kuruvinashetti, M.S. 

Department of Biotechnology, University of Agricultural Sciences, Dharwad 580 005 (Karnataka)

Abstract: A cry1Ac gene was cloned from a native isolate of B. thuringiensis D1. The complete nucleotide sequence of the gene was deduced and the native gene was modified for its optimal expression in plants. The modified gene, cry1Acm, having optimal codon composition for effective expression in plant was cloned into the yeast expression vector pYES2/CT, and its expression was confirmed by qualitative ELISA. Bioassays using total proteins from transgenic yeast, and the intensity of immunodetected protein band indicated higher levels of Cry1Acm expression than the native Cry1Ac protein in Saccharomyces cerevisiae.  

8.  Journal of  Cell and Tissue Research 15(3): 5187- 5192 (2015) 

OAS1 gene as potential biomarker to detect early pregnancy in buffalo 

 Gupta, S.S., Nanda, T., Buragohain, L.,  Kumar, R. and Balhara, A.K. 

Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar 125 004, Haryana.   

Abstract: The present study hypothesized a marked up-regulation of OAS1 (2,5Oligoadenylate Synthetase 1)  gene in early days of pregnancy in buffalo. The experiment was designed with Buffalo heifers (n=13) assigned as pregnant (PH, n=7) or non-pregnant cyclic (NPH, n=6)  groups and buffalo cows assigned as pregnant (PC, n=7) or non-pregnant cyclic (NPC,  n=9)  groups with a control group of Buffalo bulls (n=6). Blood was collected from these animals on day 0, 7, 14, 17, 21, 28 and 35 by accepting the artificial insemination (AI) day as day 0. The total RNA was extracted and converted to corresponding cDNA. Gene specific primers were used for partial amplification (413 bp) of OAS1 transcript by PCR. The PCR products were subjected to agarose  gel electrophoresis which showed a clear cut up-regulation of OAS1 gene from day 7 to 17 and down-regulation from day 17 to 28, having peak expression on day 14, 17 and 21. In brief, OAS1 gene expression can be used to diagnose early pregnancy after 14 day of conception and, hence, designing tool for early pregnancy diagnosis in buffalo. 

 9.  Journal of  Cell and Tissue Research 15(3): 5193-5199 (2015) 

Haemolymph  esterase and peroxidase spectra  in  bivoltine silkworm (Bombyx mori l.) genotypes and their utility  

 Maqbool, A., Dar, H.U., Ahmad, M., Zaffar, G., Malik, G.N. and Mir, S.A. 

Division of Sericulture Mirgund,  Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir Shalimar campus,  Srinagar 191 121.  

Abstract: The electrophoretic spectra of haemolymph esterase and peroxidase isoenzyme systems were studied in twelve bivoltine silkworm (Bombyx mori L.) genotypes viz. NB4D2, SH6, Meigitsu, CSR4, Sheiki II, Pampore-5, J122, J2M, 14M, JBEL, NJ3 and B38. The biochemical analysis was done using native-polyacrylamide gel electrophoresis. The banding pattern of esterase revealed moderate polymorphism among the genotypes. A total of six bands and six isozymic forms were observed. The relative mobility values ranged from 0.68 to 0.94 and the intensity scores ranged from 8 in B38 to 13 in NB4D2. However,  a great deal of polymorphism was observed  in the banding pattern of peroxidase. In all, nine bands and eleven isozymic forms were realized in this isoenzyme system. The relative mobility ranged from 0.39 to 0.94 and the intensity scores showed a range of 8 in NJ3 and B38 to 13 in NB4D2. Out of fifteen bands observed, only three were monomorphic while twelve were polymorphic. Moreover, the enzyme systems exhibited an appreciable range of relative mobility and activity suggesting that these enzyme systems could be used as markers for genotype identification. Further, significant and positive correlation was found between the isozymes and some yield parameters. Thus, these isozyme systems could be used in Marker Assisted Selection programmes for the improvement of silkworm breeds. 

 10.    Journal of  Cell and Tissue Research 15(3): 5201-5207 (2015) 

Electrophoretic analysis of alkaline phosphatase and amylase in some bivoltine silkworm (Bombyx  mori l.) breeds and their role in genotypic characterization 

 Maqbool, A., Dar, H.U.,  Ahmad, M.,   Malik, G.N.,  Zaffar, G., Mir, S.A. And Mir, M.A. 

Division of Sericulture Mirgund, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar campus, Srinagar 191121 (J & K).    

Abstract: The electrophoretic profiles of alkaline phosphatase and amylase were realized using native-polyacrylamide gel electrophoresis in twelve bivoltine silkworm (Bombyx mori L.) genotypes viz. NB4D2, SH6, Meigitsu, CSR4, Sheiki II, Pampore-5, J122, J2M, 14M, JBEL, NJ3 and B38. The banding pattern of alkaline phosphatase showed a moderate degree of polymorphism. In all four bands and four isozymic forms were observed for the enzyme. The relative mobility ranged from 0.39-0.70 and the intensity score ranged from 3 in B38 to 8 in NB4D2. The profile of amylase revealed a total of four bands with good amount of qualitative polymorphism as seven isozymic forms were observed. The relative mobility ranged from 0.43-0.72 and the intensity score ranged from 1 in NB4D2 and SH6 to 3 in 14M, JBEL, NJ3 and B38.  Out of eight bands observed for these two isozymic systems, six were polymorphic. Moreover, the enzymes showed an appreciable range of relative mobility and activity, suggesting their usefulness as genetic markers for genotype identification. Further, the isozymes were also found associated with yield parameters viz. single cocoon weight, single shell weight, cocoon yield/10,000 larvae by number and cocoon yield/10,000 larvae by weight. Hence, these enzymes can be utilized in Marker Assisted Selection programmes for the improvement of silkworms.  

 11.    Journal of  Cell and Tissue Research 15(3): 5209-5213 (2015) 

Salmonellosis - a potential threat to poultry: A mini review 

 Dalai, N., Shekhar, S., Padhy,  A., Praveen, P.K. and Sahu,  A.R. 

Department of Veterinary Physiology and Biochemistry, Public Health and Epidemiology, Arawali Veterinary College, Sikar 332 001,  Rajasthan. E. mail: niru.vets@gmail.com     

Abstract: Salmonella is the chief causative agent to cause mortality and reduced production in poultry industry. Fowl typhoid and pullorum disease are the two main bacterial diseases which is caused by Salmonella gallinarum and Salmonella pullorum respectively and having zoonotic importance. Enteritis, bacteremia, reduced egg production, diarrhea, inappetance are the main signs and symptoms caused by them. Diagnosis is possible through identification and isolation of the organism through culture and some serological tests such as ELISA, PCR etc. Preventive measures, Vaccination and proper antibiotic treatment can reduce the mortality and chance of occurrence of the disease. 

 12.    Journal of  Cell and Tissue Research 15(3): 5215-5220 (2015) 

Effect of vitamin e on the quality of frozen buck  semen  

 Hazarika, S.B., Bhuyan, D., Deka, B.C., Sinha, S., Biswas, R.K.,  Dutta, D.J., Das, A., Borah, P.  and Dewry, R.K.  

Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary Science, Assam Agriculture University, Ghy 22 . 

Abstract: A total of 30 ejaculates from five adult Beetal bucks were collected to study the effect of different concentrations of vitamin E on quality of frozen buck semen. In the experiment each of 30 ejaculate was split into four equal parts and the first three parts were extended separately with Tris extender containing different concentrations of Vitamin E (2 mM, 6 mM and 8 mM) and in After removal of seminal plasma, the semen was primarily extended with Tris extender (1:5) considering the volume of semen prior to removal of seminal plasma and then split into four parts and finally extended with equal volume of Tris extender. The fourth part of the ejaculates was kept as control (0 mM). The semen characteristics viz. sperm motility, live sperm, live intact acrosome, HOST reacted sperm were recorded after equilibration and after freezing. Extracellular release of ALT and AST was recorded only after freezing. The mean sperm motility, live sperm, live intact acrosome, HOST-reacted sperm and extracellular release of ALT and AST was significantly higher (P<0.05) in Tris extender containing 2 mM concentration of Vitamin E than that containing 6 mM , 8 mM and control (0 mM). The percentages of sperm motility, live sperm, live intact acrosome, HOST-reacted sperm were 84.83 ± 0.17, 88.80 ± 0.35, 82.85 ± 0.21 and 72.53 ± 0.21, and 65.17 ± 0.30, 70.15 ± 0.35, 48.73 ± 0.36 and 55.47 ± 0.32 per cent after equilibration and after freezing with 2 mM concentration of Vitamin E. Extracellular release of ALT and AST was significantly (P<0.05) lower after freezing with 2 mM concentration of Vitamin E than others. It was concluded that supplementation of Tris extender with 2 mM Vitamin E resulted in significant improvement in quality of frozen Beetal buck semen as compared to other concentrations of vitamin E in the extender. 

 13.    Journal of  Cell and Tissue Research 15(3): 5221-5225 (2015) 

Comparative evaluation of different therapeutic regimes in primary  ketotic buffaloes 

 Kumar, A., Kumar, T., Kumar, P., Sindhu, N., Charaya, G., Goel, P.,  Sridhar, and  Surbhi 

Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana - 125 004. 

Abstract: The investigation was conducted to evaluate the therapeutic efficacy of different drugs in twenty four (n=24) buffaloes suffering from primary ketosis (screened out of 145 buffaloes). Primary ketotic animals were diagnosed on the basis of clinical signs (selective anorexia, drastic reduction in milk yield and absence of any other concurrent diseases) and two positive urine tests (Rothera’s test and Keto-Diastix strip test). These twenty four animals were divided into three groups. Group A buffaloes (n = 8) were treated with propylene glycol 220 g b.i.d. orally on 1st day followed by 100g once a day along with dexamethasone (7 mg/100 kg BW I/M.). Group B (n = 8) were treated with dextrose 20 percent (1 litre) intravenously along with triamcinolone acetonide @ 0.1 mg/kg BW I/M once daily. Group C (n = 8) were treated with dextrose 20 percent (1 litre) intravenously along with isoflupredone acetate @ 1.0 mg/30 kg BW I/M once daily. Hemato-biochemical study revealed significant alterations in the values of various parameters before and after the treatment.  The recovery time in group A animals was 4.88 ± 0.29 days, in group B animals was 4.12 ± 0.22 days and in group C animals was 3.00 ± 0.26 days. So comparative evaluation of three drugs combinations revealed better results for the therapeutic regimen dextrose (20 percent) along with isofluperadone acetate then combination of dextrose 20 percent along with triamcinolone acetonide and treatment of propylene glycol with dexamethasone.  

 14.    Journal of  Cell and Tissue Research 15(3): 5227-5232(2015) 

Isolation and identification of photorhabdus luminescens an insect pathogenic bacteria associated with  entomopathogenic nematode  

Chowdhury, R., Meena, C.M.,  Kumar, S. and Suresh. C.K. 

Department of Plant Biotechnology, University of Agricultural Sciences, GKVK, Bengaluru 560065, (Karnataka).  

Abstract: Photorhabdus luminescens is a gram negative symbiotic bacterium belonging to the family Enterobacteriaceae, and is a lethal pathogen of insects. It lives in the gut of an entomopathogenic nematode of the family Heterorhabditidae. When the nematode infects an insect, P. luminescensis released into the blood stream and rapidly kills the insect host by producing toxins. So the present study was carried out to isolate and characterize the bacterium Photorhabdus luminescens from entomopathogenic nematodes Heterorhabditis indica (Karnataka strain) collected from soils of V C farm Mandya (Karnataka agroclimatic Zone-6), Karnataka, India. A total of twenty nematodes were isolated from 30 soil samples by the use of 300 modified Galleria traps. The bacterium was isolated by streaking the heamolymph of the dead larva of Galleria on NBTA plates. The identification and characterization of the isolates was done by microscopic observation, biochemical and physiological characters like Gelatin liquefaction test, Catalase test, Lactose fermentation test, Urease test and Colony morphology studies on Nutrient agar, NBTA and Mac Conkey agar media. A total of eleven strains were characterized as P. luminescens and their molecular diversity analysis was done using Random Amplified Polymorphic DNA (RAPD) markers.

 15.    Journal of  Cell and Tissue Research 15(3): 5233-5239 (2015)

Molecular characterization of chloropyrifos degrading bacteria and gene 

Paramar, K., Tomar, R.S., Parakhia, M.V.,  Malviya. B.J., Rathod, V. M., Bhatt, A.J., Bhalara, R.B. and Golakiya, B.A. 

Department of Biotechnology, Junagadh Agricultural University, Junagadh  362 001, Gujarat. 

Abstract: Pesticide encompasses a variety of different types of chemicals including herbicides, insecticides, fungicides, and rodenticides. In which organo phosphate pesticides are widely used now a days. Microbial degradation become a suitable option because they use chlorpyrifos as a carbon source and degrade it. The experimental material was comprised of 45 chlorpyrifos degrading bacteria which were reduced down to 19 microbes by screening at 500ppm. These 19 chlorpyrifos degrading isolates were further characterized by molecular and their ability to degrade chlorpyrifos.  All isolates were characterized by RAPD and based on the RAPD result all 19 bacterial isolates were grouped in to 2 main clusters with 55% similarity. Characterization of chloropyrifos degrading gene was carried out by using specific primer. Out of 19 isolates, 7 cntained mpd gene and 10 isolates were containing opd gene while remaining 02 isolates which did not show amplification with opd/ mpd primer so it may contain other unreported gene responsible for chloropyrifos degradation. Isolates were identified based on 16s rRNA sequence and one highly efficient bacterium selected for genome characterization.  

 16.    Journal of  Cell and Tissue Research 15(3): 5241-5246 (2015) 

Plant growth promoting traits and  in vitro antagonism of drought tolerant endophytic bacteria  isolated from grasses of kutch 

 Akbari Disheeta. L., Akbari, L.F., Bhadania Roshani, A.,  Parkhiya, M.V. And Golakiya, B.A. 

Department of Biotechnology, College of Agriculture, Junagadh Agriculture University, Junagadh 362001.  

Abstract: A total forty two unique bacterial endophytic strains were isolated from Eleusineindica, Sporobolusfertilis and Cenchrusciliaris. The eight isolates were screened as a drought tolerant endophytic bacteria. The two isolates were positive for phosphate solubilisation, all the endophytic isolates were positive to siderophore and six isolates were positive to ACC deaminase activity. All isolates were able to produce IAA and EPS. The SM 19 isolate produced 36.70 µgml-1 IAA at 30% PEG 6000 concentration. While The EI 49 isolate produced 124.21 µgml-1 of exopolysaccharide at 30% PEG6000. These bacteria seem to have developed mechanisms to cope with drought stress. The EI 41 and EI 47 isolates could successfully inhibited growth of Helminthosporium sativum. 

 17.    Journal of  Cell and Tissue Research 15(3): 5247-5254 (2015) 

Separation and identification of phytochemicals from Lilium polyphyllum d. don (Kshirkakoli), an ingredient  of ashtavarga 

Raval, S.S., Mandavia, M. K., Mahatma, M.K. and Golakiya, B.A. 

Department of Biochemistry, Junagadh Agricultural University, Junagadh 362 001, Gujarat. 

Abstract: The primary goal of present study was qualitative analysis and mass detection of Lilium polyphyllum D.Don (Kshirkakoli) with antiaging ethno-botanical records in northern India. The dried root samples were extracted with 9:1 methanol/water (MeOH/H2O) solution. Sepbox 2D-2000 a multicolumn 2D-HPLC was used to separate the root extract with C 18 analytical column and mobile phase consisting acetonitrile (ACN)/MeOH/H2O for each gradient resulted in 103 fraction. The method provides rapid and reproducible results. Screening was carried out by UV detector data. Among them 3 fractions were selected for gas chromatography/mass spectrometry (GC/MS). Total 26 compounds with different similarity index (SI) were identified through GC/MS analysis. Highest similarity index (89%) of R-(-)-Cyclohexylethylamine and lowest similarity index (71%) of Macdougallin was observed. 

 18.    Journal of  Cell and Tissue Research 15(3): 5255-5266 (2015) 

Protein profile and isoenzymes in response to water stress in pearl millet 

 Anatala, T.J., Mandavia, M.K., Dave, R.A., Kothari, V.V., Gajera, H.P., Ramani, H.R., Patel, S.V. and Golakiya, B.A.. 

Department of Biotechnology, Junagadh Agricultural University, Junagadh 362 001 

Abstract: Single pearl millet J-2454 genotype was tolerant to drought. The water stress was imposed on 23 days seedling up to six days. After 29 days, the physiological parameters viz. seed weight, seed germination percentage, soil moisture content, relative water content (RWC) and shoot length (cm) were studied. Also biochemical parameters like total protein, proline, glycine betaine and different isoenzymes viz. peroxidase, polyphenol oxidase (PPO), superoxide dismutase (SOD) were considered. Leaf  protein  profile  of  control  and  water  stressed  plants  have  been  compared by 2D gel electrophoresis. The results showed that physiological and biochemical parameters  did not show  any significant change. Different level of bands was detected in all three different isoenzymes. All bands had monomorphic banding pattern. Coomassie staining of the gels allowed visualization of around 1271 well resolved spots within the 4-7 pH and 10–110 kDa ranges. 

 19.    Journal of  Cell and Tissue Research 15(3): 5267-5273 (2015) 

Comparative  in vitro study of thrombolytic effect of Punica granatum and Azadirachta  indica 

Jena, D.,  Behera, P.C. and Bisoi, P.C. 

Department of Biochemistry, College of Veterinary Sciences and Animal Husbandry, Orissa University of Agriculture and Technology, Bhubaneswar 751003, Odisha.   

Abstract: The aim of the  study was to investigate the thrombolytic activity of Punica granatum and Azadirachta indica over canine, bovine, caprine and avian blood clot using streptokinase and water as positive and negative control respectively. Using an in vitro thrombolytic model, the clot lysis activity of A. indica was observed as 48.06±0.59%, 50.01±1%, 47.6±0.69% and 28.03±0.35% and of P. granatum as 44.73±0.65%, 48.53±0.56%, 43.83±0.6% and 47.17±0.32% in cattle, dog, goat, poultry respectively. The percentage (%) clot lysis was statistically significant (p<0.05) when compared with vehicle control whereas streptokinase showed clot lysis 43.13±0.9%, 58.4±0.72%, 55.07±0.58% and 44.67±0.65%  respectively. We observed that these herbal extracts possess thrombolytic properties that could lyse blood clots in vitro. However, in vivo clot dissolving properties and active component(s) of these extracts for clot lysis are observed to be protein in nature as the protein extract of Azadirachta indica exhibits different protein bands between 16.6 to 90.4 kda where as that of Punica granatum exhibits only 2 bands of 10.9 and 17.3 kDa MW. So the individual components from these bands can be used for further research.

 20.    Journal of  Cell and Tissue Research 15(3): 5275-5281 (2015)

Separation of phytochemicals from peucedanum nagpurense by using sepbox and gcms 

Dobaria Jalpa, D.,  Patel, S.V., Raval,  S.S., Mandavia, M.K.  and Golakiya, B.A. 

 Department of Biotechnology and Biochemistry, Junagadh Agricultural University, Junagadh-362 002, Gujarat. 

Abstract: The aim of present study was to investigate the phytochemical present in the root of Peucedanum nagpurense (Tejraj), the endangered herb with antiaging ethno-botanical records in northern India. Dry root samples extracted with 90% methanol, separated with Sepbox 2D-2000-a multicolumn HPLC for quantification using C 18 analytical column with mobile phase consisting solvent system of ACN/MeOH/H2O for each gradient run. The method proved to be reproducible and rapid, produces 292 fractions, high qualitative results. Multimode reader with photodiode detector provides wavelength based results of all fractions. Two fractions were selected for GC-MS in which 2-Methoxy-4-vinylphenol was found common in both fraction and total of 42 compounds with similarity index were identified through GC-MS analysis.  

 21.    Journal of  Cell and Tissue Research 15(3): 5283-5288 (2015) 

New thermostable lipase by Paenibacillus barengoltzii MS11 from hot water spring of Himachal Pradesh, India 

 Verma, A., Sharma, A. and  Shirkot, P. 

Department of Biotechnology, Dr. Y. S. Parmar University of  Horticulture and Forestry, Nauni, Solan 173230 (H.P.).  

Abstract: A thermophilic bacterium Paenibacillus barengoltzii MS11 producing extracellular thermostable lipase was isolated from Manikaran hot water spring of Himachal Pradesh (India). Till date there is no report in literature about thermostable lipase produced by Paenibacillus barengoltzii. MS11 strain was studied morphologically and biochemically, followed by sequencing of its 16S rRNA gene. BLASTn search analysis of the sequence showed maximum identity with Paenibacillus barengoltzii (NR 042756.1) and the G+C content was found to be 58.7%. Paenibacillus barengoltzii MS11 bacterial isolate was quantitatively screened for lipase activity and the maximum lipase activity was achieved at 48 hours of incubation period, with the pH 7.5 at 65ºC. The lipase enzyme from Paenibacillus barengoltzii MS11 was purified by using precipitate of saturation (NH4)2SO4, and Sephadex G-100 column, and the molecular mass of the enzyme was determined by SDS-PAGE. The study confirmed that the isolated Paenibacillus barengoltzii MS11 to be a true thermophile and could be a source of thermostable lipase which can be exploited for pharmaceutical and industrial applications. 

 22.    Journal of  Cell and Tissue Research 15(3): 5289-5294 (2015) 

Gamma rays induced mutagenesis for identification of new variants via RAPD markers in chrysanthemum (Chrysanthemum morifolium Ramat.) 

 Telem, R.S., Sadhukhan, R., Mandal, N.,  Sarkar, H.K. and Wani, S.H. 

Farm Science Centre (KVK), Senapati Distt., P.O. Kangpokpi 795129  (Manipur).  

Abstract: Rooted cuttings of four local chrysanthemum cultivars were irradiated with 10, 20 and 30 Gy gamma rays to induce favourable variation. Lower doses of gamma irradiation resulted in homeosis and induced encouraging novelties while the higher doses often induced high degree of abnormalities and consequent mortality. Colour novelties induced by the mutagens were isolated in vitro and purified by RAPD techniques. Out of twenty primers screened, eight were selected on the basis of robustness of amplification, reproducibility, scorability of banding patterns and were employed for differentiating mutants.  The Jaccard Coefficients was used to calculate the genetic similarity. The genetic distance in the distance matrix ranged from 0.42 – 0.67 percent. Our findings suggested that by using RAPD markers the lately developed chrysanthemum cultivars are distinct from their parents and the mutants are reliable. 

23.    Journal of  Cell and Tissue Research 15(3): 5295-5299  (2015) 

Single and interactive toxic potential of roundup® and ammonium nitrate on haemato-biochemical parameters in wistar rats 

Dar, A.M., Sultana, M., Mir, H.A., Bader, A.M., Raina, R. and   Prawez, S. 

Division of Veterinary Pharmacology  and Toxicology, MJF College of Veterinary and Animal Science, Chomu 303702, Jaipur (Rajasthan) 

Abstract: The aim of present study was to unravel the single and interactive toxic potential of Roundup® and ammonium nitrate in rats after their oral administration. The animals were randomly divided into four groups with six rats in each group. Group I were orally administered with water and served as control. Group II animals were orally exposed to Roundup® @ 500mg/Kg/day. Animals in group III were orally treated with ammonium nitrate @ 220mg/Kg/day while as group IV received both Roundup® and ammonium nitrate @ 500mg/Kg/day and 220mg/Kg/day respectively. After 28th day of treatment, blood samples were taken and analysed for various haemato-biochemical parameters. Significant decrease was observed in Hb, TEC and PCV in group II and III as compared to control. As compared to these groups, the changes were more severe in group IV which was receiving both Roundup® and ammonium nitrate together. Contrary to this, a significant increase in ALT, AST, ALP, ACP, LDH, BUN, CR were noticed in all groups as compared to  control animals. The total protein content was also  significantly decreased in group II and IV. 

 24.    Journal of  Cell and Tissue Research 15(3): 5301-5308 (2015)

Optimization and validation of Agrobacterium-mediated genetic transformation for commercial Indian bread wheat (Triticum aestivum l.) cultivars using mature embryo 

Parmar, S.S., Jaiwal, P.K., Agarwal, N. and Kaushik, S.K. 

Seed Technology Division, Indian Grassland and Fodder Research Institute, Jhansi 284003 (UP) 

Abstract: An efficient Agrobacterium infiltration method for mature embryonic axis of hexaploid wheat was developed in order to improve the transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), along with other cultural parameters were optimized in a conventional ‘dipping’style method of Agrobacterium inoculation. It was observed that the transient expression of uidA gene is raised with increase in duration of SAAT and acetosyringone concentration. However, this increase in GUS expression was not significant after 5 minutes of SAAT and 200µM of acetosyringone respectively. Finally, to validate the applicability of the optimized protocol, we transformed the commercial hexaploid wheat cultivar PBW343 with cassette containing hpt as selectable marker and our test gene of interest.  PCR and Southern blot analysis of T0 plants with test (dps) gene and hpt gene confirmed the applicability of the optimized parameters.  

 25.    Journal of  Cell and Tissue Research 15(3): 5309-5312  (2015) 

Histological development of conical and lenticullar papillae in the tongue of goat foetii (Capra hircus) 

Dar, Y. M., Sarma, K., Suri, S.,  and Devi, J. 

Division of Veterinary Anatomy, FVSc and AH, Sher-e-Kashmir University of Agricultural Sciences & Technology Kashmir, Jammu 181102 

Abstract: The present study was conducted on the tongue of 18 goat foetii divided into three prenatal age groups to study the sequential events in regard to histological development of  conical and lenticular papillae of the same in goat foetii. The primitive conical papillae were seen over the torus linguae in 62 days old goat foetus (CRL= 10.10 cm), which further differentiated at 80 days of gestation (CRL= 15.30 cm), to become fully developed at 146 days of gestation (CRL= 35.50 cm). The primitive lenticular papillae were seen distributed over the lingual epithelium at torus linguae from 62 days of gestational age (CRL= 10.10 cm). These papillae had a broad shape and lined by a basal layer of cuboidal cells with a superficial layer of 6-7 cells. The height and diameter of these papillae were found to be 41.03 ± 2.33 μ and 43.22 ± 4.12 μ in group I and 78.45± 4.83 μ and 98.67± 5.93 μ in goat foetii of group II, respectively. In group III, the mean values for height and diameter of the conical papillae were recorded to be 211.78 ± 9.73 μ and 298.34 ± 22.13 μ, respectively. The same values in regard to the lenticular papillae were found to be 201.67 μ ± 8.91 and 324.11± 29.73 μ, respectively.     

26.    Journal of  Cell and Tissue Research 15(3): 5313-5315  (2015) 

GFP-tagged endophytic bacterium Enterobacter  cloacae strain dsm colonized the wheat 

Akbari Disheeta, L. 

Department of Biotechnology, College of Agriculture, Junagadh Agriculture University, Junagadh 362001.  

Abstract: The Enterobacter cloacae strain DS Mtaxonomically identified using 16S ribosomal DNA sequence was isolated from roots of Cenchrus biflorus. Its endophytic colonization was investigated microscopically using green fluorescent protein introduced by vector pGLO containing ampicillin resistance gene and arabinose sugar. The strain able to colonize wheat tissues and localized in the roots and stems.  

 27.    Journal of  Cell and Tissue Research 15(3): 5317-5321 (2015) 

Real time expression analysis of rac1 gene in gga-mir-142-3p knockdowned developing chicken embryo 

Raja, P., Manesh Kumar, P., Tembhurne, P.A., Ingle, V.C. and Kalorey, D.R. 

Department of Veterinary Microbiology and Animal Biotechnology, Teaching and Research cell, Nagpur Veterinary College, Nagpur  440 001 Maharashtra. 

Abstract: Development of an organism in different stage is controlled largely by a small non coding RNA called MicroRNA (miRNA). Regulation of gene expression in developmental processes is an important aspect of miRNA function.  The genes are expressed at defined stages of development and determine the body plan pattern of the growing embryo. Real time expression analysis of RAC1genes after Knockdown of the gga-miR-142-3p on the immune and visceral organs of developing chicken embryo opens the new avenue towards understanding function of these gene involved in cell structure, size, shape, maturation and  migration. Bioinformatic analysis of targets prediction reveled that RAC1 gene has the complimentary seed sequence for miR-142-3p at their 3’UTR. In the present study, the knock down of gga-miR-142-3p was carried out at 14 days old embryonated eggs of chicken by intravenous inoculation of LNA modified miRNA inhibitor keeping scramble control group. Expression analysis of targeted genes was carried out by using the SYBR green qPCR technology revealed that RAC1 gene was up-regulated in bursa, thymus, spleen, heart, kidney and was down regulated in lungs as compared to scramble control. The histopathological studies of immune organs reveals loss of thymic follicle in the thymus and loss of lymphocytic population in spleen as compared to the scramble control. The result of the present study suggests that gga-miR-142-3p is required for normal developments of immune organs and other organs. An aberrant expression of RAC1 may leads to disturbance in migration of T and B cells resulting in pathological outcomes in the organs during the embryonic developments. 

 28.    Journal of  Cell and Tissue Research 15(3): 5323-5328 (2015) 

Molecular characterization of toll like receptor 2 gene using PCR—RFLP in jersey crossbred cattle 

Mundhe, U.T., Das, D.N., Saravanan, R., Divya, P. and  Nath, S. 

Animal Genetics Laboratory, Dairy Production Section, Southern Regional Station, Indian Council of Agricultural Research- National Dairy Research Institute, Bengaluru 560065 (Karnataka).

Abstract: Present study was carried out to investigate genetic polymorphism of TLR2 gene of exon 1 and exon 2 and their association with subclinical mastitis in Jersey crossbred cattle by PCR- RFLP. Blood samples were collected from 60 randomly selected, lactating Jersey crossbred cattle in which 30 animals were suffering from either subclinical or clinical mastitis from Nandini Sperm Station farm Hessarghatta, Bengaluru and local farm maintained by farmers in and around Namakkal district of Tamil Nad. DNA was isolated by phenol chloroform isoamyl alcohol method. PCR standardization was carried out for amplification of region of exon1, exon 2.2. exon 2.3, exon 2.4, exon 2.5 and exon 2.6 using published primers [1]. PCR – RFLP was carried out using HaeIII, Mn II, Hinc II, EcoRV, PStI and Bsty I restriction enzymes respectively. Sequence analysis showed 10 single nucleotide polymorphism in the coding region of TLR 2 gene, which includes 5 transitions (G11456A exon 2.3), (C12153T, C 12260T, T12471C, T12501C exon 2.5) and 5 transversions (G11391T, T11424A exon 2.3), (A12225C G12228T C12441G exon 2.5). Though exonic regions of the studied gene showed monomorphic patterns, however sequence analysis showed the role of toll like receptor 2 gene towards subclinical mastitis could not be established. 

29.    Journal of  Cell and Tissue Research 15(3): 5329-5334  (2015) 

Histological studies on prenatal skin of developing gaddi sheep foetus 

Razvi, R., Shukla, P., Rajput, R. and  Pathak, V. 

Division of  Veterinary Anatomy, Himachal Pradesh Krishi Vishvvidlaya, Palampur  176 061, Himachal Pradesh.  

Abstract: Histological studies of skin from dorsal regions of neck, thorax and loin of body were carried out on eight healthy Gaddi sheep foeti, which were collected from local slaughter houses of Palampur (H.P). After measurement of crown-rump length (CRL), the age of foeti were determined.  Skin tissue samples were collected, fixed (10% neutral buffered formalin), processed, sectioned and finally stained to study their histological developmental changes. Differentiation of epidermis took place from an ectoderm germinal cell layer and rate of differentiation and growth of epidermis was fast in neck dorsal region than other two regions under study. At CRL 16.4 cm appearance of collagen fibers, sweat glands and elastic fibers was also observed. The sebaceous glands were seen at CRL 19.8 cm. At CRL 24.5 increase of collagen fiber synthesis deep in dermis was seen; this process caused the dermis to contain two layers: the superficial layer (papillary layer) and deep layer (reticular layer). All sub-layers of epidermis were developed along with the cells of hair follicles. The number and growth of sweat glands was higher in thorax dorsal region than other two regions under study. The thickness of epidermis and dermis increased with advancement of gestational age. At CRL 42.4 cm, the structure of the skin was complete. The reaction for bound lipids was intense in epidermis and moderate in dermis with Sudan black B stain. Fine reticular fibers were seen around sweat, sebaceous and hair follicles. Strong PAS positive reaction was seen in outer layer and moderate in the inner layer of hair follicles. With the advancement of gestational age the PAS reaction changed from mild to intense and from mild to moderate in epidermis and dermis respectively. 

 30.    Journal of  Cell and Tissue Research 15(3): 5335-5340 (2015) 

Age associated variations in lymphocytic growth hormone secretion in cattle

Rose, M.K. and Gupta, M. 

Department of Veterinary Physiology and Biochemistry, College of Veterinary Sciences Lal Lajpat Rai University of Veterinary and Animal Sciences, Hisar 125004, Haryana. 

Abstract: The lymphocytic growth hormone (GH) and nitric oxide (NO) levels were studied among three different age groups of cattle viz. cyclic adult cows (3-8 years of age), young female calves (2 weeks old) and growing female calves (6 months old). The effects of leptin, sodium nitroprusside (SNP) and N-nitro-L-arginine methyl ester (L-NAME) on the secretion of lymphocytic GH and NO were also studied. Lymphocytic GH in young calves (90.92±6.21 ng/12 million lymphocytes) and growing calves (86.15±9.94 ng/12 million lymphocytes) were significantly higher than in adult cows (59.42±3.92 ng/12 million lymphocytes) without any significant change in lymphocytic NO secretion with age. Incubation of lymphocytes with SNP could significantly increase lymphocytic GH secretion only in adult cows, while in 6 months old calves it significantly reduced the lymphocytic NO secretion. Thus suggesting that in young calves the secretion of lymphocytic GH is independent of leptin and NO dependent mechanism. 

 31.    Journal of  Cell and Tissue Research 15(3): 5341-5346 (2015) 

Strengthening of protocol for sugarcane (Saccharum officinarum l.) through  meristem culture 

Udhutha, J., Mali, S.C., Sahare H.A.  and Parmar R.D. 

Main Sugarcane Research Station, Navsari Agriculture University, Navsari- 396 450 Gujarat.  

Abstract: A rapid micro propagation and acclimatization response of two different varieties of sugarcane (CoN 07072 and CoN 05071) was obtained in this study. The shoot apical meristem of different sizes was cultured on MS medium supplemented with different concentrations and combinations of BAP and kinetin either alone or in combination with each other alongwith GA3. Best shoot formation response in CoN 07072 was obtained on MS medium containing 2mg/l BAP while in CoN 05071 the combination of 1 mg/l BAP with 1 mg/l Kinetin showed best shoot formation response from apical meristem. Meristem of 3.0 mm size proved to be the best size for micropropagation of sugarcane. Excellent multiplication response of In vitro formed shoots was obtained when the concentration of BAP was decreased to 1.5 mg/l in CoN 7072 and 0.25 mg/l BAP & Kin in CoN 05071 (i.e. 0.25 mg/l BAP + 0.25 mg/l Kinetin). MS medium containing 1.0 mg/l NAA and 2.0 mg/l IBA showed 100% rooting response of In vitro regenerated shoots of both the varieties of sugarcane within eight days of inoculation. Best hardening response was obtained in Sand+ Soil + Pressmud (1:1:1) media. 

32.    Journal of  Cell and Tissue Research 15(3): 5347-5350  (2015) 

Producton of double haploid population in two Indica  rice (Orysa sativa l.)  CROSS SAFRI-17XIR-64 and MTU1010 variety 

Sahu, M., Minj, A., Chopkar, R., Jha, Z. and Verulkar, S.B. 

Department of Plant Molecular Biology and Biotechnology, Indira Gandhi Krishi Vishwavidyala, Raipur, 492012, 

Abstract: Anther culture based double haploid (DH) production is a technology which can significantly reduce the time period required for development of new crop variety.  In the present investigation an attempt was made to develop DH using anther culture in rice (Oryza sativa L.). One cross Safri-17xIR-64 and one variety MTU1010 was subject for the study. Parameter like media composition, hormonal treatment etc was standardized for efficient development of DH. The anther were excised and plated on to N6 (Chu.,1978) media supplemented with 3% maltose 0.8% agar , 2 ml/l  2,4-D and the pH was maintained of 5.8. In cross Safri-17xIR64 the callus induction percent was 0.49% and in variety MTU1010 callus induction percent is 0.40%.The induce callus was transfer in 16 different media (T1 to T16 ) for regeneration. Then green callus was transfer to green plant regeneration media. Treatment no.T15 was found to be best as it has produce 106 number of  green  plant & treatment no.T1 has produced 58 albino plants in Safri-17xIR-64. In MTU1010, T1 has generated 9 green plants and 5 albino plants.