Research Journal of Biotechnology

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Research Journal of Biotechnology

In silico docking analysis of pyrrolysyl tRNA synthetase polyspecificity towards the genetically encoded Phenylalanine analogs

Nadarajan Saravanan Prabhu and Yun Hyungdon

Genetic incorporation of unnatural amino acids such as analogs of phenylalanine, histidine, tryptophan and 22nd proteinogeneic amino acid pyrrolysine is accomplished by pyrrolysyl tRNA synthetases engineered through rational engineering and directed evolutionary approaches. It is demonstrated that the E. coli phenylalanine tRNA synthetase shares active similarity with pyrrolysyl tRNA synthetases of Methanosarcina sp. and further engineering has been done to incorporate Phenylalanine analogs. Until now around 50 new engineered variants were reported for the genetic incorporation of more than 100 unnatural amino acids. In order to incorporate specific unnatural amino acid, unique engineered pyrrolysyl tRNA synthetase is required. This is the major limitation of this study which can be overcome by using polyspecific tRNA synthetase. In this study, we carried out high throughput in silico analysis to predict the polyspecificity of 10 different tRNA synthetases that encode 45 different phenylalanine analogs. Among the 10 tRNA synthetases, 3 different tRNA synthetases show polyspecificity that can encode 45 different phenylalanine analogs.

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Acute and Subacute Toxicity of Ethyl Acetate Fraction of Cochliobolus spicifer (Nelson) isolated from Phoenix dactylifera (Linnaeus) on Balb/c Mice

Abutaha Nael, AL-Zharani Mohammed, AL Mekhlafi Fahd A., AL-Tamimi Jameel, Mashaly Ashraf M.A. and Wadaan Muhammad A.

The aim of this study was to assess the toxicological effects of the ethyl acetate (ETAC) fraction of Cochliobolus spicifer (Nelson) isolated from Phoenix Dactylifera (Linnaeus) using Balb/c mice as an animal model. An acute toxicity study of the C. spicifer EtoAC fraction was carried out in male Balb/c mice using a range of doses of the extract (1,000, 2,000 and 4,000 mg/kg body weight). These doses were injected intraperitoneally and the mice were observed for mortality for 24 hrs after exposure. A sub-acute toxicity investigation was performed using a dose of 125 mg/kg body weight. These doses were injected intraperitoneally at 24 hour intervals for 14 days. Pathological alterations in the kidneys and liver were inspected histologically. In the acute toxicity study, the Balb/c mice did not show mortality within 24 hrs of exposure. In the subacute toxicity study, blood sugar levels were significantly reduced (p < 0.05) at a dose of 125 mg/kg body weight. The microscopic investigation of the kidney architecture of control and treated mice showed a normal cellular appearance. The histopathological study of the liver revealed hepatocytes, infiltration with massive inflammatory cells, dilation in the central vein and cytoplasmic vacuolization. The C. spicifer fraction isolated from P. dactylifera was well tolerated when intraperitonally injected at a highest dose of 4000 mg/kg body weight but toxic when injected for 14 days at a dose of 125 mg/kg.

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Morphological, Pathological and Molecular Diversity of Colletotrichum Capsici inciting Fruit Rot in Chilli (Capsicum Annuum L.)

Srideepthi R., Lakshmisahitya U., Peddakasim D., Suneetha P. and Krishna M.S.R.

Anthracnose is a well-known disease caused by Colletotrichum capsici/truncatum resulting in both qualitative and quantitative yield loss of a commercial vegetable crop chilli. Variability among the C. truncatum species with respect to geographical distribution remained as a major constraint to chilli production. Hence present study was aimed at identifying the genetic variability existing among the species within Andhra Pradesh region. Eight different isolates were characterized and confirmed as C. capsici with species specific marker. Variations among the isolates exist in their cultural and morphological features including aerial fluffy cotton to submerged felty type colonies at a growth rate of 3.8-9.8 mm per day while conidial size varied from 18.1-22.36 μm in length to 2.8-4.0 μm in width. Mode of virulence and pathogenic ability of eight isolates were clustered into high (C.c1, C.c4 and C.c7), moderate (C.c2, C.c5 and C.c6) and least (C.c3 and C.c8) based on their disease score. Genetic relationship was analyzed using 40 randomly amplified polymorphic DNA (RAPD) primers. Based on similarity coefficient by un-weighted pair group method with arithmetic average (UPGMA), isolates were distributed into two clusters (I and II) confirming their genetic diversity. No correlation among pathological and RAPD grouping of isolates was observed. Results showed that RAPD-PCR is a valuable technique for evaluating inherited variation within C. capsici species.

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Screening, Biochemical and Molecular identification of novel Streptomyces fumigatiscleroticus VIT-SP4 derived cocktail pectinase from border industrial pectin enriched places of Andhra Pradesh and Tamil Nadu

Praveen Kumar G. and Suneetha V.

The present study is concerned about the isolation of novel cocktail pectinolytic actinomycete from different pectin-rich fruit industrial residue dump sites in the border places Gudiapala, Naraharipet of Andhra Pradesh and Kangeyanellur, Mettukulam of Tamil Nadu States of India. Hundred soil samples were screened by primary and selective enrichment procedures. Isolates were analyzed for pectin lyase (PL), polygalacturonase (PG) and pectin esterase (PE) activity. The strain (designated as VIT-SP4) was able to produce maximum pectinase of solid state fermentation (pectin substrate orange peel was degraded within 72 h) and was taken for further study. The potential strain was identified by morphological, biochemical and molecular characterization. The results revealed that the potential strain was Streptomyces fumigatiscleroticus VIT-SP4 based on sequencing of nucleotide sequence of 16S rRNA gene. The sequence was successfully submitted to Nucleotide sequence database (GenBank).The novel natural-dye assisted calcium alginate immobilization technique has been investigated for crude pectinase for long term usage. The study revealed the better stability and reusability of immobilized pectinase in comparison with free pectinase. Streptomyces fumigatiscleroticus VIT-SP4 strain could be potential strain for cocktail pectinase production commercially in industrial field in mere future.

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Evaluation of Morphological Changes in Histopathological Images of Ovarian and Breast Cancer Tissues and its Correlation with Biochemical Parameters

Anuranjeeta, Sharma Shiru, Shukla K.K. and Anshu Aseem

Ovarian and breast cancers have become the foremost cause of fatality and are frequently diagnosed among women. Several factors have been found to be responsible for uncontrolled proliferation of epithelial cells of ovary and breast. Lipid peroxidation (LPO) is the result of increased oxidative stress in the cells that may be involved in many pathological deregulations including cancer, cell injury and aging. Various biomolecules like lipid, proteins etc. are considered to be damaged due to over production of reactive oxygen species (ROS) as a consequence of LPO. Malondialdehyde (MDA) is an important indicator of the lipid damage which is a by-product of LPO. The aim of the work is to do comparative evaluation of morphological features of dysplastic change of cells such as enlarge size, irregular shape, hyperchromatic nuclei etc. and its correlation with biochemical parameters. For the determination of MDA level in serum of ovarian and breast cancer patients, LPO assay was performed. The study comprised of 55 controls, 75 ovarian and 75 breast cancer patients. The results reveal that MDA levels, CA-125, WBC, are increase, while RBC, Hb and platelets count decreased in cancer patients as compared to normal. For comparing morphological changes in cells, 5 normal and 5 cancerous tissues have been evaluated for both types of cancers. It has been found that cancerous cells have increased value of count of cells, total area, average size, area fraction, perimeter, major axis length, minor axis length, however circularity value is decrease as compared to normal cells. These results suggest that the high MDA levels in serum are the consequence of increased oxidative stress which may cause DNA damage. Further, high oxidative stress in cancer patients may be correlated with the morphological changes in cancer cells.

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Semi-solid state fermentation: A promising method for production and optimization of tannase from Bacillus gottheilii M2S2

Subbalaxmi Selvaraj and Vytla Ramachandra Murty

Tannase is an important enzyme which finds commercial applications in food industry to reduce the level of tannins in fruit juices, preparation of instantaneous tea and production of gallic acid. Various low cost tannin rich residues such as coffee husk, tamarind seed powder, tea leaves and Triphala powder were studied in semi-solid state fermentation process. Triphala was found to be a prominent substrate which has exhibited maximum tannase activity of 29 ± 0.35 U/L. Thereafter, sequential statistical approach was used to optimize tannase production with Triphala in shake flask. The classical one-variable-at-a-time approach determined moistening media, tannic acid and inoculum volume which significantly influenced the tannase production. A central composite design showed that the optimal values of these factors were 6.2 mL, 1% (w/v) and 6.4 mL respectively. Subsequently, a 7-fold increase in corresponding tannase yield (106 ± 0.61 U/L) was obtained, compared with that produced in the submerged fermentation. The crude tannase showed optimum activity at 40°C and pH 4.0. Vmax and Km values were 1.404 µmol/ml.min and 1.24 mM respectively.

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Studies on the mitochondrial genomics in Salmo trutta caspius population in three Rivers of South Caspian Sea

Rezaei Abolhasan, Akhshabi Sheyda and Jamalzadeh Hamid Reza

Mitochondrial DNA is suitable for phylogenetic studies. Hence in this study, mitochondrial DNA genes in Salmo trutta caspius were sequenced and deposited in Genbank. Six sequences of S.t. caspius mitochondrial ATPase 6 gene with accession nos. LC011387.1, LC154842- LC154847 and the full length of the mitochondrial genome in S.t. caspius (accession no. LC011387.1) were deposited in Genbank. Sequences were aligned between S.t. caspius and Salmo trutta by BLAST program. Although results showed a high homology between both sequences, but single nucleotide variations between ND genes in S.t. caspius and Salmo trutta were observed. The Salmo mitochondrial genome contains one non-coding control region (D-Loop) and two genes for ribosomal RNA, 22 genes encoding tRNAs and thirteen protein-coding genes. S.t. caspius, S. trutta and Salmo salar sequences were converted into a circular map in an online system named Organellar Genome DRAW ( Maps created by OGDRAW were different for S.t. caspius, S. trutta and S. salar. Moreover, Maximum Parsimony method was conducted for evolutionary analysis using MEGA 6.0 program tree between S.t. caspius and other salmonids. Results showed that S.t. caspius and others are covered in a group. Results of evolutionary analysis using Tajima's test were conducted in MEGA 6.0 program for three sequencing including S.t. caspius, S.t. specimen voucher NO. 00 and S.t. fario used as an outgroup for equality of evolutionary rate. Comparison of partial mitochondrial sequence in Iranian S.t. caspius population was performed between three regions [Tonekabon (Cheshmekileh Roud), Ramsar (Safa Roud) and Talesh (Nav roud)]. The results showed that except for one single nucleotide mutation in the partial sequence of mitochondria of S.t. caspius [Ramsar (Safa Roud)], there has been 100% homology between them. In conclusion, the mitochondrial genome homology of S.t. caspius and other salmonids was high, though some SNPs were observed between S.t. caspius and other salmonid species.

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Molecular cloning, sequence characterization of a novel pepper gene MDH and its effect on cytoplasmic male sterility

Deng Ming-hua, Lv Jun-heng, Zhao Kai and Huo Jin-long

Malate dehydrogenase (MDH) is a key enzyme in the glycometabolism, it catalyzes the conversion of oxaloacetate and malate utilizing the NAD/NADH coenzyme system and also prevails in the energy metabolism reaction. The complete coding sequence (CDS) of the MDH gene was amplified using a reverse transcriptase PCR based on the conserved sequence information of the tomato and other Solanaceae plants and known highly homologous pepper ESTs. 332 amino acids were encoded by the 999-long cDNA of the pepper MDH gene. Nucleotide sequence analysis showed that the encoded amino acids were highly homologous with the seven species: Solanum tuberosum (98%), Solanum lycopersicum (98%), Nicotiana tabacun (96%), Solanum chilense (96%), Morus notabilis (96%), Phaseolus vulgaris (94%) and Theobroma cacao (95%). The result of the tissues expression showed that the pepper MDH gene is over expressed in placenta, moderately in pericarp and seed, weakly in leaf and hardly expressed in stem and flower. During the abortion stages, expression levels of MDH in anthers of a sterile line were lower than that in the maintainer and the MDH gene expression level of transcripts in the maintainer line is much more stable than that in the sterile line. The lower expression levels of MDH and the instability expression trend may disturb the balanced of energy metabolism in the sterile line which indicated that stable transcripts of MDH are necessary to maintain energy metabolism at a normal level. The expression levels of MDH were regulated by the restorer gene and became stable. The restorer gene likely plays an important role in keeping the balance of the energy metabolism within normal levels during microspore development.

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The change of nucleic acid content in the melon regeneration process

Huijun Zhang and Jinfeng Chen

Melon inbred line M16 cotyledon node is used as explants in this study and is cultured on a MS medium supplemented with 1.0mg/L 6-BA and 0.6mg/L IAA to induce adventitious buds. The result shows that in the process of melon adventitious buds produce, the content of DNA and nucleic acid first rises slowly and then reduces, rises again during the period of cotyledon embryo; RNA first rises and then reduces. The ratio of DNA/RNA rises slowly after the 7th day. It is clear that nucleic acid content is closely related with melon’s regeneration.

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Development of High Yielding Quality Protein Maize (QPM) Hybrids and their Molecular Detection

Lenka Devraj and Tripathy Swapan K.

A set of 12x12 diallel crosses (without reciprocals) along with six QPM and six Non-QPM parents and a standard check (Vivek QPM 9) were tested for morpho-economic traits including seed yield and both quantitative and qualitative assay of seed protein. Seed protein in QPM inbreds was qualitatively rich in tryptophan and lysine content (0.81% and 3.29%) and it was nearly double as compared to Non-QPM parental inbreds (0.41% and 1.68%). BQPM 7-4 had shown tremendously high tryptophan and lysine content (0.96% and 3.82%). BQPM 7-4/BM 3-10 yielded highest (97.62q/ha), but considering seed yield and protein quality, BQPM 7-4 x BQPM 3-7, BQPM 3-7 x BQPM 9-2 and BQPM 3-7 x BM 3-10 may be selected as promising single cross QPM hybrids as these qualify the minimum standard for QPM with nearly 30% higher seed yield potential than Vivek QPM 9. These hybrids can be of immense value for commercial cultivation in the farmer’s field.

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Cloning expression of autophagy-related proteins of rainbow trout (Oncorhynchus mykiss) and production of antisera for immunological assays

Huang Tianqing and Han Ying

Autophagy is an important characteristic of biological phenomenon existing extensively in cell cycles and its mechanism had been well studied in mammalian and plant cells but researches about lower vertebrate fishes were scare. Rainbow trout (Oncorhynchus mykiss) is the appropriate model for biochemical and biophysical research of salmon fishes. In order to explore autophagy mechanism of rainbow trout deeply, rainbow trout LC3B and ATG12 genes had been cloned and the 3D structures of RBT-LC3B and RBT-ATG12 proteins were predicted. The CDS regions of these two genes were amplified and inserted into pET-32a vector. The recombinant proteins were expressed in Escherichia coli Rosetta strain using IPTG and auto-induction procedures. The expressed proteins were identified at a molecular weight of 30kDa and 28kDa respectively. The specific proteins were purified by Ni-NTA column. Antisera of RBT autophagy proteins were got after immune rabbits. The specificity of antisera was verified by detecting autophagy-related proteins expressions in rainbow trout gonads and the activity was verified by ELISA. The antisera were specific and efficient and can be used for immunological research of rainbow trout.

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Comparative diversity analysis in advanced breeding lines of basmati rice (Oryza sativa L.) using agro-morphological and SSR markers

Sharma Richa, Salgotra Romesh Kumar and Bhat Javaid Akhter

The basmati rice is nature’s gift to Indian sub-continent and is an economic asset to the farmers of North-western Himalayas. Basmati rice is known to have narrow genetic base subjected to decreasing yield and quality deterioration. In this regard, 30 advanced breeding lines of basmati in F4 generation developed through the crossing of diverse parents along with three check varieties are comparatively evaluated for diversity analysis using both agro-morphological and SSR markers. Analysis of variance showed that all the characters except grain breadth were significantly different (P<0.01) among the genotypes. The first four principal components (PCs) of the PCA analysis contributed 78.14% of the variability. The dendrogram obtained through both agro-morphological and SSR analysis separated the genotypes into four major clusters (I, II, III and IV) each, with the Bray-Curtis distance and Jaccard’s similarity coefficient ranging from 0.83-1.00 and 0.38-0.92 respectively. Out of 36 SSR markers tested, only 14 primer pairs were found polymorphic and a total of 31 alleles were detected with an average of 2.2 alleles per locus. The Mantel test revealed a significant correlation (r = 0.24, p<0.01) between the agro-morphological and SSR matrices. Both methods result in diverse clustering of genotypes largely based on their pedigree and origin, suggesting considerable diversity among the studied genotypes. Hence, both methods proved effective for the diversity analysis of basmati rice and their combined study provides useful information.

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Molecular cloning, sequence characterization and quantitative analysis of the expression of Dahel caspase-1 in Dastarcus helophoroides

Zhang Zhengqing and Li Menglou

Caspase genes family plays a central and evolutionary role in programmed cell death (PCD) which removes unwanted, damaged and dangerous cells to maintain homeostasis. In this study, caspase-1 gene was first cloned and investigated in D. helophoroides and its molecular information, physicochemical property, phylogenetic relationship, expression patterns and enzyme activities were analyzed. The Dahel caspase-1 with full-length of 1,201 bp contains a 996 bp open reading frame (ORF) that encodes a protein of 332 amino acids. The protein is a hydrophilic protein and it is highly homologous to Tribolium castaneum caspase-1 and Bombyx mori caspase-1. The Dahel caspase-1 protein had no N-terminal signal peptide but it contains a big CASc domain (caspase catalytic domain) which consists of a caspase-p20 domain (including a substrate binding site of His amino acid residues (LSHG) and a pentapeptide active-site motif of Cys amino acid residues (QACQG)) and caspase-p10 domain. Comparison of the Dahel caspase-1 expression in different tissues, stages and age groups showed that the gene expression was extremely high in ovary, pupa stage and 40 months’ age group respectively. Moreover, corresponding with the gene expression level variation, the caspase-1 enzyme activity showed increased trend with age growth after adult eclosion. The research provides molecular biology and expression level foundations for future analyzing of caspase family gene in D. helophoroides and other insects.

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An Overview of Medicinal plants for Potential Cardio-Protective Activity

Harishkumar Rajendran, Subramanyam Deepika and Selvaraj Chinnadurai Immanuel

Cardiovascular diseases are life-threatening leading to death. Throughout the developed countries, the increase in burden of cardiovascular disease has placed this disease as the rapid cause than the other non-communicable diseases. They also became the predominant disease in low and middle-income countries including India. Phytomedicine plays an important role in recent days to solve so many health issues. The treatments of these complications, by the way, of traditional knowledge are collectively called Traditional System of medicine. In India, Ayurveda, Siddha and Unani system of medicines are followed from the ancient days. This review mainly focuses on the potential role of some medicinal plants and their phyto-compounds in protecting the heart and reducing the risk factors of cardiovascular disease.

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