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eISSN: 1682-2978
pISSN: 1682-296x

Editor-in-Chief:  Akhtar Jamal Khan
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Research Article
RuBisCO of Microalgae as Potential Targets for Nutraceutical Peptides: A Computational Study
Gurudeeban Selvaraj, Satyavani Kaliamurthi, Zeynep Elibol Cakmak and Turgay Cakmak
Background and Objective: Microalgae are autotrophic organisms abundantly occur in the aquatic environment. Microalgal biomass is used as an economical precursor for biodiesel feedstock and therapeutic bioactive constituents. The RuBisCO is a souvenir in photosynthetic organisms. However, the information about the biomedical application of RuBisCO and its peptides is insufficient. Therefore, this study aimed to evaluate the therapeutic potential of RuBisCO and its peptides through the computational tools. Materials and Methods: Fourteen RuBisCO large subunit sequences of algae were retrieved from UniProt and assessed in the BIOPEP server. The biological activity, catalyst action and calculation of bioactive peptides tools were accustomed to verify the frequency of incidence of fragments, proteolytic cleavage and the incidence of elite enzymes with the specified action. The physiochemical parameters of the chosen sequences were performed with Protpram tool. Results: The outcome demonstrated Chaetoceros calcitrans exhibits the most effective prospect as a supply of DPP-IV inhibiting peptides, Chlorella pyrenoidosa for antihypertensive and Aphanizomenon flos-aquae for antioxidative, activating ubiquitin and anti-amnestic peptides. High range of bioactive peptides with biological activity in the RuBisCO sequences of Aphanizomenon flos-aquae, Dunaliella salina, Chlorella pyrenoidosa and Chlorella vulgaris related to a high content of glycine and proline. Papain and proteinase K, a catalyst with wide specificity release significant active fragments than bromelain and chymotrypsin. The RuBisCO in selected microalgae showed potential for bioactive peptides linked with an elevated level of glycine and proline that are most rich in biologically active peptides. Conclusion: Further, experimental studies support the utilization of microalgae RuBisCO as a conventional economical source of bioactive peptides for a human.
Research Article
Genome-wide Identification and Analysis of Human and Avian 5'-AMP-Activated Protein Kinase Gamma Subunit Genes
Wuyi Liu
Background and Objective: The regulatory gamma subunits of AMP-activated protein kinase (AMPK) are encoded by the 5'-AMP-activated protein kinase gamma subunit (PRKAG) genes. These genes are dominantly expressed in skeletal muscle and many reports of the animal counterparts suggest that these subunit genes play key roles in the regulation of energy metabolism in the skeletal muscle tissues. This study was designed to identify and analyze human and avian PRKAG genes in a genome scale. Materials and Methods: In the study, all the putative PRKAG gene sequences were used to construct ML (maximum likelihood) phylogenetic trees with package MEGA version 6.06 under JTT+I+G model. After phylogenetic analyses, the putative PRKAG gene were further subjected to the enrichment analyses of gene ontology (GO) and pathway annotations. In this study, there were totally 58 putative PRKAG genes and 31 unique PRKAG genes identified from the human and avian genomes. Results: Phylogenetic analyses indicated that among all the three sub-families of PRKAG genes (i.e. PRKAG1, PRKAG2 and PRKAG3), those protein sequences of PRKAG genes from four avian genomes formed monotonous phylogenetic clusters, whereas all the human protein sequences of PRKAG genes formed sole phylogenetic clusters. Furthermore, functional enrichment analyses of GO and pathway annotations revealed that PRKAG genes were functionally enriched in energy metabolism related signaling pathways and biological processes with significant p-values observed. Conclusion: The dataset and results of this study will facilitate further study on PRKAG genes in domestic animals.
Research Article
Differential In vitro Direct Regeneration of Tomato Genotypes on Various Combinations of Growth Regulators
Nadia M El-Shafey, Nada Hassan, Salah El-Din A Khodary and Abdelfattah Badr
Background and Objective: Tomato (Lycopersicon esculentum L.) is one of the most extensively consumed crops. Optimizing the conditions of its in vitro regeneration besides extending the technology to a wider range of cultivars will enhance transformation results. The current study aimed at evaluating the effect of genotype and growth regulators in both of pre-culture and regeneration media on direct shoot regeneration from cotyledonary explants. Attention is particularly given to study of the interaction between the genotype, growth regulators and media type. Materials and Methods: Cotyledonary leaf explants from five tomato genotypes (National Gene Bank’s accession numbers, E1: 13139, E2: 13163, E3: 13145, F4: 12676 and E5: 12702) were cultured on two different pre-culture media (PCM) for 2 days and then sub-cultured on three different shoot induction media (SIM) consisting of Murashig and Skoog (MS) media supplemented with various combinations of growth regulators. Results: SIM3 (MS medium supplemented with 1.0 mg L–1 6-benzylaminopurine and 1.0 mg L–1 zeatin) was the most effective in scoring high regeneration frequency and shoot number as well as the highest shoot length. Although PCM1 (0.5 mg L–1 indol-3-acetic acid and 0.5 mg L–1 kinetin) was less effective in direct regeneration from most of the investigated tomato genotypes, it worked effectively with E3 by exhibiting the highest shoot number (13±0.08) directly regenerated/explant, high shoot length (0.87±0.23 cm) and high regeneration frequency (86%) after transferring to SIM3. The genotype F4 came after E3 by exhibiting high shoot number and length as well as the highest regeneration frequency, in addition to scoring neither callus nor adventitious roots, while E1, E2 and E5 were superior in callus induction and adventitious roots. Conclusion: The differential response of the investigated genotypes to the types and combinations of growth regulators depends on the endogenous levels of hormones in the tested tomato genotypes.
Research Article
Characterization of a Naphthalene Catabolic Gene Cluster and Heterologous Expression of Naphthalene Dioxygenase Genes from Rhodococcus ruber OA1
Ying Sun, Zhenglong Wang, Shuyao Zhang, Xiaodan Li, Huaxin Gao and Chunyang Zhang
Background and Objective: Naphthalene is a Polycyclic Aromatic Hydrocarbon (PAH) pollutant which is toxic and widespread in the environment. Rhodococcus ruber OA1 is a gram-positive bacterium which can utilize naphthalene as the sole carbon and energy source for growth, but no molecular biological research has been conducted on its naphthalene catabolic gene cluster. The objective of this study was to characterize the naphthalene catabolic gene cluster and naphthalene dioxygenase (NDO) from R. ruber OA1. Methodology: The gene cluster for naphthalene degradation from R. ruber OA1 was identified by in situ hybridization and sequence analysis. Then the genes narAa and narAb encoding the large and small subunit of naphthalene dioxygenase, as part of the cluster were sub-cloned and heterologously expressed to confirm their functions. Results: In situ hybridization and sequence analysis revealed a 43,754 bp fragment in the cloned plasmid, including the gene cluster narAaAbBC encoding the proteins responsible for converting naphthalene to salicylaldehyde. After the narAa and narAb genes were sub-cloned and expressed, the enzymatic activity was assayed, which revealed that narAa and narAb were functional in Escherichia coli BL21 (DE3). Sub-cloning and heterologous expression of the rub1 gene revealed that the rub1 gene product was not indispensable for naphthalene degradation. Conclusion: A gene cluster for naphthalene biodegradation was identified in R. ruber OA1 for the first time. The narAa and narAb genes were sub-cloned and heterologously expressed to confirm their functions.
Research Article
Expression of Mouse Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) in Pichia pastoris
Nasrin Madadi, Faezeh Ghasemi, Mohammad Soukhtanloo, Majid Mojarad, Farnaz Zahedi Avval and Baratali Mashkani
Background and Objectives: Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factors involved in proliferation and differentiation of bone marrow precursor cells into granulocytes, monocyte that used in treatment of patient with cancer, therefore, it was decided to express murine GM-CSF as a recombinant protein in Pichia pastoris (P. pastoris). Materials and Methods: The protein sequence of mouse GM-CSF was obtained from Uniprot database and ordered for synthesis after back translation and optimization of coding sequence. It was subsequently cloned into pPICZαA expression vector expressed in Pichia pastoris. Expression and activity of GM-CSF was confirmed by SDS-PAGE, dot blot, Western blotting in the culture supernatant. Biological activity of the product was confirmed by promoting FDC-P1 cell growth. Non-linear regression analysis was used for fitting a curve onto the cell proliferation data plotted against the growth factor dilutions and calculation of EC50 value. Results: Sequence optimization improved CAI from 0.66 in the native to 0.86 in the optimized GM-CSF sequence through replacing 24% of the nucleotides. In dot blot analysis, GM-CSF expression reached its peak on 5 days and started to decline after 7 days. The GM-CSF protein was appeared as bands with apparent molecular mass nearly 16-17 kDa. It was estimated that 18.5 μg mL–1 of active murine GM-CSF was secreted into the culture supernatant. Conclusion: The recombinant GM-CSF protein was expressed in Pichia pastoris yeast and was biologically active in the FDC-P1 cell line proliferation.

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