This document reports on an in silico analysis of chloroperoxidase and lignin peroxidase proteins from the pathogenic fungus Macrophomina phaseolina. The authors analyzed the physicochemical properties, secondary structures, 3D structures, motifs, and active sites of 7 chloroperoxidase and 3 lignin peroxidase proteins from M. phaseolina. They identified conserved regions and residues in motifs that could be targets for site-directed mutagenesis to disrupt the lignin degradation activity of these proteins and thereby protect economically important plant species.
Abstract— MicroRNAs (miRNAs) function on post-transcriptional gene silencing and regulate the gene expression by degrading the transcripts of their targets, leading to down-regulation of the target genes. Plant miRNAs have been reported to play important roles in developmental control, hormone secretion, cell proliferation, and response to environmental stresses. In this review, we have reviewed miRNA expression and its potential role in regulating cell differentiation in Arabidopsis and summarized the miRNAs regulated cell differentiation during root, shoot, leave, and embryo development. We have further described practical application of expression of miRNAs in plant molecular breeding.
Flow Cytometric Analysis for Ploidy and DNA Content of Banana Variants Induce...paperpublications3
Abstract: Nuclear DNA content of mutated banana plants was determined by using flow cytometric techniques. It is a powerful tool for large scale screening of ploidy levels. Nuclei were isolated from young leaves from (banana mutants & Glycine plants) supplemented with Propidium- iodide (PI) and RNAse. "Glycine max" used as internal reference standard for identifying the nuclear DNA content by FCM. For ploidy estimation DAPI was used. The results showed differences in DNA content between variants indicating the effect of gamma-irradiation on the genotype of these plants. Variants of short plant stature or stunted growth showed great differences in DNA content compared to control (non-irradiated). The phenotypic variations observed at high doses were likely due to changes in the DNA sequences at the chromosomal level. Nuclear DNA contents decreased with an increase of gamma-dose from 20 Gy to 60 Gy. However, there were no significant differences between DNA content at 20 Gy and 30 Gy and also between 40 Gy and 60 Gy, while they were differed significantly from the control. The results showed no significant differences in ploidy level between all samples used (3n); while all selected mutants (variants) showed differences in DNA content.
Molecular characterization of rice (Oryza sativa L.) genotypes using target r...Innspub Net
In the present investigation, based on the seven rice putative candidate iron transporter genes, novel TRAP markers were developed. These markers were successfully employed in the molecular diversity study among 30 rice genotypes representing improved rice cultivars and land races with varied grain iron content (7.38 - 30.58 ppm). Totally, thirty TRAP primer combinations were screened, which generated 703 bands out of which 654 were polymorphic (93%) with an average of 21.8 bands per primer combination. The average polymorphic information content (PIC) values ranged from 0.09 (Osysl4b+ME05) to 0.25 (Osnramp5c+ME05, Osnramp1b+ME02 and Osysl4a +ME02). Gene diversity (H ˆ
) ranged from 0.10 (Osysl4b+ME05) to 0.31 (Osnramp1b + ME02 and Osysl4a +ME02). The Jaccard dissimilarity ranged from 0.15 to 0.52, explaining 37% of genetic variation (Table 4). Grouping of genotypes based on UPGMA and principal coordinate analysis (PCoA) were found comparable and grouping of genotypes into a different cluster was found mainly on the basis of pedigree relationships. TRAP markers revealed well resolved relationships among rice genotypes. The information generated from this study will helps to select parental combinations for breeding high iron content
rice varieties.
Analysis of Seed Proteins in Groundnut Cultivars (Arachis hypogaea L.)IJERA Editor
The seed protein contents and protein banding pattern were studied in commonly cultivated groundnut cultivars.
The groundnut cultivars such as ICGV00351, TMV-7, CO-4,CO-6 and TG-374 were used for quantitative and
qualitative analysis of seed proteins. The protein contents varied among the different varieties of groundnut. The
maximum protein content was observed in CO-6 followed by CO-4, TMV-7, ICGV00351 and TG-374. There
was a slight differences in protein content among the different cultivars. All the five cultivars of groundnut were
subjected to SDS-PAGE analysis. The results revealed that the variation in total number of bands and MW-Rf
values. The maximum number of MW-Rf value was noticed in TG-374 and ICGV00351, and the minimum
MW-Rf value was 11 recorded in CO-6 and TMV-7.
Abstract— MicroRNAs (miRNAs) function on post-transcriptional gene silencing and regulate the gene expression by degrading the transcripts of their targets, leading to down-regulation of the target genes. Plant miRNAs have been reported to play important roles in developmental control, hormone secretion, cell proliferation, and response to environmental stresses. In this review, we have reviewed miRNA expression and its potential role in regulating cell differentiation in Arabidopsis and summarized the miRNAs regulated cell differentiation during root, shoot, leave, and embryo development. We have further described practical application of expression of miRNAs in plant molecular breeding.
Flow Cytometric Analysis for Ploidy and DNA Content of Banana Variants Induce...paperpublications3
Abstract: Nuclear DNA content of mutated banana plants was determined by using flow cytometric techniques. It is a powerful tool for large scale screening of ploidy levels. Nuclei were isolated from young leaves from (banana mutants & Glycine plants) supplemented with Propidium- iodide (PI) and RNAse. "Glycine max" used as internal reference standard for identifying the nuclear DNA content by FCM. For ploidy estimation DAPI was used. The results showed differences in DNA content between variants indicating the effect of gamma-irradiation on the genotype of these plants. Variants of short plant stature or stunted growth showed great differences in DNA content compared to control (non-irradiated). The phenotypic variations observed at high doses were likely due to changes in the DNA sequences at the chromosomal level. Nuclear DNA contents decreased with an increase of gamma-dose from 20 Gy to 60 Gy. However, there were no significant differences between DNA content at 20 Gy and 30 Gy and also between 40 Gy and 60 Gy, while they were differed significantly from the control. The results showed no significant differences in ploidy level between all samples used (3n); while all selected mutants (variants) showed differences in DNA content.
Molecular characterization of rice (Oryza sativa L.) genotypes using target r...Innspub Net
In the present investigation, based on the seven rice putative candidate iron transporter genes, novel TRAP markers were developed. These markers were successfully employed in the molecular diversity study among 30 rice genotypes representing improved rice cultivars and land races with varied grain iron content (7.38 - 30.58 ppm). Totally, thirty TRAP primer combinations were screened, which generated 703 bands out of which 654 were polymorphic (93%) with an average of 21.8 bands per primer combination. The average polymorphic information content (PIC) values ranged from 0.09 (Osysl4b+ME05) to 0.25 (Osnramp5c+ME05, Osnramp1b+ME02 and Osysl4a +ME02). Gene diversity (H ˆ
) ranged from 0.10 (Osysl4b+ME05) to 0.31 (Osnramp1b + ME02 and Osysl4a +ME02). The Jaccard dissimilarity ranged from 0.15 to 0.52, explaining 37% of genetic variation (Table 4). Grouping of genotypes based on UPGMA and principal coordinate analysis (PCoA) were found comparable and grouping of genotypes into a different cluster was found mainly on the basis of pedigree relationships. TRAP markers revealed well resolved relationships among rice genotypes. The information generated from this study will helps to select parental combinations for breeding high iron content
rice varieties.
Analysis of Seed Proteins in Groundnut Cultivars (Arachis hypogaea L.)IJERA Editor
The seed protein contents and protein banding pattern were studied in commonly cultivated groundnut cultivars.
The groundnut cultivars such as ICGV00351, TMV-7, CO-4,CO-6 and TG-374 were used for quantitative and
qualitative analysis of seed proteins. The protein contents varied among the different varieties of groundnut. The
maximum protein content was observed in CO-6 followed by CO-4, TMV-7, ICGV00351 and TG-374. There
was a slight differences in protein content among the different cultivars. All the five cultivars of groundnut were
subjected to SDS-PAGE analysis. The results revealed that the variation in total number of bands and MW-Rf
values. The maximum number of MW-Rf value was noticed in TG-374 and ICGV00351, and the minimum
MW-Rf value was 11 recorded in CO-6 and TMV-7.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Dr. Ravi S Pandey-Resume for Assistant Professor/ Research Scientist in Bioch...Dr. Swami Gyan Prakash
Dear Member of Recruiter Committee,
I am writing this letter for a position of Assistant Professor/ Research Scientist in Biochemistry, Clinical-Biochemistry, Biotechnology and Molecular Biology. In brief, I am PhD in Medicinal Biochemistry and completed several successful projects as a Postdoctoral Scientist in different discipline of Biochemistry like Molecular Biology of Aging, Alternative splicing in human diseases, Epigenetic regulation in liver and breast cancer from India and USA. I came back to India in April 2010 and worked as a CSIR-Pool Scientist in the area of chromatin remodeling in breast cancer. My tenure has been completed recently. Thus, I am looking for a faculty position of Assistant Professor/ Research Scientist in Biochemistry, Clinical-Biochemistry, Biotechnology and Molecular Biology.
I am highly interested to trend the graduate students in Biochemistry, Biotechnology and Molecular Biology. Besides, I am very much motivated to lead projects in the area of Cancer Biology. Thus, I respectfully submit this letter of application, for I believe my experiences and commitment for teaching and research make me well qualified to meet the needs of Assistant Professor/ Research Scientist in Biochemistry, Clinical-Biochemistry, Biotechnology and Molecular Biology.
I am well acquainted with the molecular techniques associated with DNA, RNA and proteins research. Besides, I am expertise in planning and execution of experiments, mentoring PhD students, interpreting data, as well as writing and data preparation for manuscript publication. During my doctorate and post doctorate time, I taught graduate and postgraduate students on behalf of my mentors. I am well organized, goal oriented; self motivated research scientist and committed to train the student in area of Clinical Biochemistry and Molecular Biology along with lead research work in the area of Cancer Biology. I would appreciate for an interview opportunity to discuss about my background, qualification and expertise that may fit for the position.
Effect of Clariodeoglomusclariodeorum on morphology and abundant of carrot ro...AI Publications
The roothairs are important components of the root for absorbing nutrients for plants and also secreting the plant-produced secretes. Morphology and their number are influenced by various environmental and internal factors and are regulated by them. Mycorrhizal fungi are established through the root and their presence in the root can affect root’s physical and chemical properties.The aim of this study was to evaluate the effect of the presence of the fungus in the roots on the characteristics of the capillary roots.In order to remove the effect of other microorganisms on the results of the experiment, this experiment was performed in vitro on the roots of the carrot secondary phloem tissue culture. In this experiment, the fungus could affect the characteristics of the root hairs: their number (22% decrease in the root hairs number in the root hair area of the root) and their length (A decrease of 21.3% in the length of capillary roots in mycorrhizal plants). These changes in the characteristics of capillary roots were also caused by the presence of fungal structures in the roots as well as by the decrease in the production of strigolactones. In this study, changes in the production of strigolactones calculated by using their effect on seed germination of Phelipancheaegyptiaca.
Slide show..prof.s.p.singh.invited talk. udapur international conference.02 j...Saurashtra University
In Invited Talk by Prof. Satya P. Singh on:
Attributes of Plant Growth Promoting Rhizobacteria in biocontrol and combating salinity stress
An International Conference on Recent Trends in Plant Sciences held at M L Sukhadia University, Udaipur, Rajasthan, India
Anticancer drug discovery using multicellular tumor spheroid modelsHasnat Tariq
Cancer, drug discovery, tumor spheroids, organoids, 3D tumor spheroids, 3D scaffold-based models, Scaffold-free models, 3D Scaffolds, Hanging drop, Low adhesion microplate, Magnetic levitation and bio printing, bioprinting, anticancer,, tumor models, Drug screening assays, flow cytometry, expansion microscopy.
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology
Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium l...Premier Publishers
The auxins liability often limits expected effects on in vitro culture of plant tissues. In this regard, β-cyclodextrine was used as protective substance to auxins in order to improve rooting and in vitro bulbing of lily (Lilium longiflorum L.). This oligosaccharide was added at a concentration of 10, 20 and 30 mg/l to the rooting medium containing mineral and vitamin additives of Murashige and Skoog (MS), 1.5 mg/l of indole-3-butyric acid (IBA), 30 g/l of sucrose and 6 g/l of agar. After 30 days of culture, in order to enhance in vitro bulbe formation, a liquid medium supplemented with 6-benzylaminopuine (BAP) was added to form a double phase (solid / liquid) media. The results obtained showed that β-cyclodextrin improves the rooting of lily in vitro shoots. Indeed, the average number of roots increases from 5.64 to 7.20 roots per vitro-plant in the medium supplemented with β-cyclodextrin and root length increased from 1.72 to 2.2 cm compared to control.
Cancer is the leading cause of death in worldwide whose treatments are costly and has different physical and emotional side effects. Parsporal inclusion proteins from B. thuringiensis having specific cytotoxic activity against specific cancer cells can be used for cancer treatment which might have relatively lower side effects and might be cost effective. We have reported in silico analysis that is physico-chemical characteristics, secondary structure, 3-D structure, motifs and phylogenetic relationship among these 19 parasporin proteins to characterize them and predict their cancer cell killing mechanism.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Dr. Ravi S Pandey-Resume for Assistant Professor/ Research Scientist in Bioch...Dr. Swami Gyan Prakash
Dear Member of Recruiter Committee,
I am writing this letter for a position of Assistant Professor/ Research Scientist in Biochemistry, Clinical-Biochemistry, Biotechnology and Molecular Biology. In brief, I am PhD in Medicinal Biochemistry and completed several successful projects as a Postdoctoral Scientist in different discipline of Biochemistry like Molecular Biology of Aging, Alternative splicing in human diseases, Epigenetic regulation in liver and breast cancer from India and USA. I came back to India in April 2010 and worked as a CSIR-Pool Scientist in the area of chromatin remodeling in breast cancer. My tenure has been completed recently. Thus, I am looking for a faculty position of Assistant Professor/ Research Scientist in Biochemistry, Clinical-Biochemistry, Biotechnology and Molecular Biology.
I am highly interested to trend the graduate students in Biochemistry, Biotechnology and Molecular Biology. Besides, I am very much motivated to lead projects in the area of Cancer Biology. Thus, I respectfully submit this letter of application, for I believe my experiences and commitment for teaching and research make me well qualified to meet the needs of Assistant Professor/ Research Scientist in Biochemistry, Clinical-Biochemistry, Biotechnology and Molecular Biology.
I am well acquainted with the molecular techniques associated with DNA, RNA and proteins research. Besides, I am expertise in planning and execution of experiments, mentoring PhD students, interpreting data, as well as writing and data preparation for manuscript publication. During my doctorate and post doctorate time, I taught graduate and postgraduate students on behalf of my mentors. I am well organized, goal oriented; self motivated research scientist and committed to train the student in area of Clinical Biochemistry and Molecular Biology along with lead research work in the area of Cancer Biology. I would appreciate for an interview opportunity to discuss about my background, qualification and expertise that may fit for the position.
Effect of Clariodeoglomusclariodeorum on morphology and abundant of carrot ro...AI Publications
The roothairs are important components of the root for absorbing nutrients for plants and also secreting the plant-produced secretes. Morphology and their number are influenced by various environmental and internal factors and are regulated by them. Mycorrhizal fungi are established through the root and their presence in the root can affect root’s physical and chemical properties.The aim of this study was to evaluate the effect of the presence of the fungus in the roots on the characteristics of the capillary roots.In order to remove the effect of other microorganisms on the results of the experiment, this experiment was performed in vitro on the roots of the carrot secondary phloem tissue culture. In this experiment, the fungus could affect the characteristics of the root hairs: their number (22% decrease in the root hairs number in the root hair area of the root) and their length (A decrease of 21.3% in the length of capillary roots in mycorrhizal plants). These changes in the characteristics of capillary roots were also caused by the presence of fungal structures in the roots as well as by the decrease in the production of strigolactones. In this study, changes in the production of strigolactones calculated by using their effect on seed germination of Phelipancheaegyptiaca.
Slide show..prof.s.p.singh.invited talk. udapur international conference.02 j...Saurashtra University
In Invited Talk by Prof. Satya P. Singh on:
Attributes of Plant Growth Promoting Rhizobacteria in biocontrol and combating salinity stress
An International Conference on Recent Trends in Plant Sciences held at M L Sukhadia University, Udaipur, Rajasthan, India
Anticancer drug discovery using multicellular tumor spheroid modelsHasnat Tariq
Cancer, drug discovery, tumor spheroids, organoids, 3D tumor spheroids, 3D scaffold-based models, Scaffold-free models, 3D Scaffolds, Hanging drop, Low adhesion microplate, Magnetic levitation and bio printing, bioprinting, anticancer,, tumor models, Drug screening assays, flow cytometry, expansion microscopy.
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology
Effects of β-cyclodextrin on in vitro rooting and bulbing of lilium (Lilium l...Premier Publishers
The auxins liability often limits expected effects on in vitro culture of plant tissues. In this regard, β-cyclodextrine was used as protective substance to auxins in order to improve rooting and in vitro bulbing of lily (Lilium longiflorum L.). This oligosaccharide was added at a concentration of 10, 20 and 30 mg/l to the rooting medium containing mineral and vitamin additives of Murashige and Skoog (MS), 1.5 mg/l of indole-3-butyric acid (IBA), 30 g/l of sucrose and 6 g/l of agar. After 30 days of culture, in order to enhance in vitro bulbe formation, a liquid medium supplemented with 6-benzylaminopuine (BAP) was added to form a double phase (solid / liquid) media. The results obtained showed that β-cyclodextrin improves the rooting of lily in vitro shoots. Indeed, the average number of roots increases from 5.64 to 7.20 roots per vitro-plant in the medium supplemented with β-cyclodextrin and root length increased from 1.72 to 2.2 cm compared to control.
Cancer is the leading cause of death in worldwide whose treatments are costly and has different physical and emotional side effects. Parsporal inclusion proteins from B. thuringiensis having specific cytotoxic activity against specific cancer cells can be used for cancer treatment which might have relatively lower side effects and might be cost effective. We have reported in silico analysis that is physico-chemical characteristics, secondary structure, 3-D structure, motifs and phylogenetic relationship among these 19 parasporin proteins to characterize them and predict their cancer cell killing mechanism.
S. pyogenes, its virulence, antibiotic, phytochemicalsUniversité Laval
Streptococcus pyogenes (group A β-hemolytic Streptococcus (GABHS)) is the common cause of acute bacterial pharyngitis also known as strep throat or sore throat. Recorded cases of GAS bacterial pharyngitis are 15-36% in children and 5-15% in adults. School aged children in seasons of spring and winter harbor the bacterium up to 20% whereas this rate is much lower in adults. On a global scale, over 616 million new cases of GAS pharyngitis occur every year. Not only is this bacterium responsible for hundred millions of pharyngitis cases but also approximately 10,000 to 15,000 cases of invasive GAS cases are reported annually in the United States, accounting for 10% to 13% mortality rate (http://www.cdc.gov/ncidod/dbmd/abcs/forinvasiveandnoninvasivediseaseincidence). Acute infections can lead to rheumatic fever and post-streptococcal glomerulonephritis (kidney inflammation), which distress children worldwide with disability and death, if antibiotic treatment fails or if the disease is left unattended. Rheumatic fever and rheumatic heart disease are known to be the leading causes of cardiovascular death during the first five decades of life in underdeveloped countries mainly concerning children. Tissues contributing to the GAS nosocomial infections are upper respiratory tract, skin, vaginal and anal area, although latter cases are rare.
GAS has several surface proteins and produces numerous extracellular products that facilitate permeation and successive evasion of the host’s immune system. Streptococcal pharyngitis results from the proliferation of GAS in the pharynx. Virulence associated factors enable S. pyogenes to attach to host tissues, elude the immune response, and spread by penetrating the host tissue layers followed by colonization.
Abstract: Here the review converses the "molecular cross-talk" of biofuel production mechanisms for Staphylococcus aureus. Staphylococcus aureus is a leading cause of bacterial infections globally in both healthcare and community settings. The succes of this bacterium is the of an expansive repertoire of virulence factors in combination with acquired antibiotic resistance and propensity for biofilm formation. S. aureus leverages these factors to adapt to and subvert the host immune response. With the burgeoning fiels of immunometabolism, is has become clear that the metabolic program of leukocytes dictates their inflammatory status and overall effectiveness is clearing an infection. The treatment of S. aureus infections become complicated due to the capacity of S. aureus “multidrug-resistant” occurs because of biofilm formationon the surfaces depending on biotic and abiotic factors, genetic factors, and numerous environmental, which vary from species to species. A broad range of molecular phenomenon contributes a high range of recalcitrance that is insisting on the biofilm formation. The previous published literature illustrated that all strains of Staphylococcal sp. contain the “ica locus” and several can form biofilms in vitro condition. Absences of “ica locus” results diminish of capability to produce biofuels, along with "PIA gene", or mediate "N-acetyl glucosaminyl transferase activity” in vitro condition.
Cloning and cDNA Synthesis of PE & PPE Gene of Mycobacterium Strain H37rv5AI Publications
Pathogenic, slow-growing Mycobacterium tuberculosis and other Mycobacterium tuberculosis complex (MTBC) species include the pe/ppe genes. In the pathogen-host connection, these genes are crucial. Despite the fact that the activities of most PE/PPE family proteins are unknown, mounting evidence shows they play a role in M. tuberculosis infection. The role of PE/PPE proteins, which are thought to be involved in the ESX system's action, is investigated. During complicated host-pathogen processes, we also discuss how PE/ PPE proteins are involved in host-pathogen interactions, immune response regulation, and cell fate determination. Finally, we discuss future PE/PPE protein research priorities as well as how present information might be used to develop more accurate diagnostics and vaccines for worldwide TB control. The pe and ppe genes, which are thought to be critical in TB pathogenesis, are only found in mycobacteria. However, nothing is known about how these genes' expression is regulated. Understanding the regulatory control of genes present only in mycobacteria, such as the pe and ppe gene families, might help researchers figure out why the illness is so effective. A transposon mutagenesis method was employed to better understand pe and ppe control. Rv1403c, a previously unknown transcriptional regulator, was discovered as a consequence of this study.
Inhibition of Aldose Activity by Essential Phytochemicals of Cymbopogon citra...CSCJournals
The ambiguity of whether aldose reductase, an enzyme of polyol pathway, is linked to diabetes and its complication has been receded based on the recent studies made on the inhibition of its (Aldose reductase) activity. In our current study, we have used an in silico approach (molecular docking) to analyze the effect of essential phytochemicals obtained from Cymbopogon citratus on the aldose reductase activity. C.citratus is grown extensively in tropical countries including India for perfumery and pharmaceuticals. The essential phytochemicals of C.citratus like Myrcene, Citral, and Geraniol have been used as ligand for the molecular docking analysis with Aldose reductase as receptor. The docking analysis showed Myrcene, with binding energy of -8.76 Kcal/mol is best amongst Citral and Geraniol which are having binding energies of -7.24 Kcal/mol and -7.93 Kcal/mol respectively for inhibiting the activity of Aldose reductase.
Characterization of Proteases Production by Varying Carbon Sources from Bacil...
MMK_PUB1
1. Contents lists available at BioMedSciDirect Publications
Journal homepage: www.biomedscidirect.com
International Journal of Biological & Medical Research
Int J Biol Med Res. 2014; 5(3): 4246-4257
In silico Analysis of Chloroperoxidase and Lignin Peroxidase of Pathogenic Fungus
Macrophomina phaseolina
a b a b
Yeasmeen Ali , Mohd Omar Faruk Sikder , Lolo Wal Marzan , Farjana Sharmenb, Md Mursalin Khan ,
a
Md Amzad Hosain , *
a
Department of Genetic Engineering and Biotechnology, University of Chittagong, Chittagong- 4331.
b
Bioinformatician, Basic and Applied Research on Jute Project, Bangladesh Jute Research Institute, Dhaka- 1207.
A R T I C L E I N F O A B S T R A C T
Keywords:
Motif
Botryosphaeriaceae
Physicochemical property
Active site
Secondary structure.
Original Article
Macrophomina phaseolina is a destructive fungus that affects more than 500 plant species
throughout the world. Lignin degradation of the host plant is crucial step for the pathogenesis
of this fungus. Chloroperoxidase and lignin peroxidase are two important enzymes for lignin
degradation. To develop an effective antagonist against this fungus, understanding these
proteins is necessary. In this study, we have reported physico-chemical characteristics,
phylogenic relationship, secondary structure, 3-D structure, motifs of 7 chloroperoxidase and
3 lignin peroxidase proteins. Moreover, pockets as well as conserved residues in the motif
sequence were identified as the target for site-directed mutagenesis of chloroperoxidase and
lignin peroxidase. This mutagenesis or designing target ligands against these proteins may
stoptheligninolyticactivityofM.phaseolina,whichwouldsaveeconomicallyimportantplants.
BioMedSciDirect
Publications
International Journal of
BIOLOGICAL AND MEDICAL RESEARCH
www.biomedscidirect.comInt J Biol Med Res
1. Introduction
Copyright 2010 BioMedSciDirect Publications IJBMR - All rights reserved.ISSN: 0976:6685.c
Macrophomina phaseolina is a Botryosphaeriaceae fungus that
belongs to phylum ascomycota. It is one of the most devastating soil
and seed borne pathogens infecting more than 500 plant species
[1]. The plant species includes jute [2], cotton [3], maize [4], pulses
[5], sunflower [6] etc. This fungus under favorable environmental
conditions can cause diseases like charcoal rot, stem rot, damping
off, seedling blight, collar rot, and root rot in various important
crops[7].
Disease development is considered to be associated with
several factors such as heat stress, soil water deficit, physiological
stress or coarse soil texture [8]. Due to having sclerotia [thick-
walled resistant hyphal mat] in soil and plant debris, it is difficult to
control M. phaseolina [9]. This pathogen is widely distributed
throughout the world especially in tropical and subtropical
countries [10]. In Bangladesh, due to pathogenicity of this fungus
onlyjutefiberproductionrateisreducedby30%[11].
In 2012, the genome sequence as well as gene prediction of M.
phaseolina was accomplished. The genome size was about 49 Mb
and about 14,249 protein-coding genes were predicted [11]. It
possessesligninolyticactivity,whichistheresultofcombinationof
different enzymatic system such as slaccases, lignin peroxidases,
galactoseoxidases,chloroperoxidases,haloperoxidases,andheme
peroxidases. Lignin peroxidase [Lip] is an extracellur enzyme [12],
which with the help of a cofactor, Veratryl alcohol interacts with
lignin polymer. Likewise, chloroperoxidase [CPO] is also an
extraxellur enzyme that requires heme or vanadium as a cofactor
[13]. This enzyme acts on lignin by oxidization of Cl− to
hypochlorous acid [HOCl] or a similarly reactive chlorine
electrophile[14]. However, the mechanism is not still well
understood.
Our study is to analyze the chloroperoxidase and lignin
peroxidase of M. phaseolina, which will help to understand the
pathogenesis of this devastating fungus. We have analyzed 7
chloroperoxidase and 3 lignin peroxidase enzymes and found
different important investigations such as physic-chemical
characteristics, functional motifs, 3D structure of these proteins.
We also showed the active site pockets, different amino acids that
could be structurally and functionally critical as well as
Copyright 2010 BioMedSciDirect Publications. All rights reserved.c
* Corresponding Author : Yeasmeen Ali
Lecturer, Department of Genetic Engineering and Biotechnology,
University of Chittagong, Chittagong- 4331.
E-mail: hossainamzad1971@yahoo.com
2. 4247
Yeasmeen Ali et.al Int J Biol Med Res. 2014; 5(3): 4246-4257
phylogeneticrelationshipamongtheseperoxidases.Allofthese
findings would decipher an effective way to block the activity of
chloroperoxidase and lignin peroxidase of M. phaseolina to protect
differenteconomicallyimportantcropspecies.
MATERIALSANDMETHODS:
Retrieval of Macrophomina phaseolina chloroperoxidase and
ligninperoxidaseproteinsequences:
Protein sequences of M. phaseolina were retrieved from NCBI
protein database in FASTA format. 7 chloroperoxidase and 3 lignin
peroxidase protein sequences were selected for analysis.
[http://www.ncbi.nlm.nih.gov/].
Physico-chemicalcharacterization:
Different properties including number of amino acids,
molecular weight, theoretical isoelectric point [pI], amino acid
composition [%], number of positively [Arg + Lys] and negatively
charged [Asp + Glu] residues, extinction co-efficient, instability
index, aliphatic index and Grand Average of Hydropathicity
[GRAVY] were calculated using ExPASy's ProtParam tool [15]
[http://expasy.org/tools/protparam.html]. The crystallization
tendency of the proteins was determined by using the CRYSTALP2
W E B S E R V E R [ 1 6 ] [ h t t p : / / b i o m i n e -
ws.ece.ualberta.ca/CRYSTALP2.html] that accepts protein
sequences in the FASTA format. The CRYSTALP2 is a kernel-based
method that uses the composition and collocation of amino acids,
hydrophobicity and isoelectric point of the given sequences to
estimatethecrystallizationpropensityoftheproteins.
Characterizationofsecondarystructure:
Secondary structure prediction was performed by SOPMA [17]
[Self-Optimized Prediction Method with Alignment,
http://npsapbil.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/np
sa_sopma.html] tool The input protein sequences were given in
FASTA format. The number of conformational states was adjusted
to four in order to predict Helix, Sheet, Coil and Turn while other
parameters were set as default. By using this software, Alpha helix
[Hh], 310 helix, [Gg], Pi helix [Ii], Beta bridge [Bb], Extended strand
[Ee], Beta turn [Tt], Bend region [Ss], Random coil [Cc], Ambigous
states,andotherstateswerepredicted.
Protein3Dstructureprediction:
The 3D structures of chloroperoxidases and lignin peroxidases
were predicted using the 3D-JIGSAW [version 3] comparative
m o d e l i n g s e r v e r [ 1 8 ] [ h t t p : / /
bmm.cancerresearchuk.org/~3djigsaw/], which accepts
sequences in FASTA format. This program constructs 3D models for
given protein sequences based on the known homologous proteins
structure. It uses HMM for searching homologous templates in
sequencedatabases[PFAM+PDB+nr]andsplitsthequerysequence
into domains. The good templates with maximum coverage of the
queries are then used for modeling and the generated models are
representedasPDBfileformat.
Detectionofmotif:
The motif scan tool [19, 20] [http://myhits.isb-sib.ch/cgi-
bin/motif_scan] was used to identify the motifs and their locations
in the sequence of Lignin peroxidase and chloroperoxidase. These
protein sequences were given as input data [FASTA format] and
scannedagainst'PROSITEPatterns'.Theselectedmotifswerethen
visualized by using Swiss- Pdb Viewer V4.1.0 [21]. Locations,
nature, match score and match details of the motifs were analyzed.
It was aimed to find out the conserved regions of these motifs. At
this point of view, multiple sequence alignment [MSA] of these
motifs was performed using EBI Clustal Omega [22].
[https://www.ebi.ac.uk/Tools/msa/clustalo/]. As expected, a
number of conserved regions were found and their length,
conservedaminoacidswereanalyzed.
Activesiteprediction:
Active site analysis was performed using Computed Atlas of
Surface Topography of Protein [CASTp]. CASTp for automatically
locating and measuring protein pockets and cavities, is based on
precise computational geometry methods, including alpha shape
and discrete flow theory. CASTp identifies and measures pockets
and pocket mouth openings, as well as cavities. The program
specifies the atoms lining pockets, pocket openings, and buried
cavities; the volume and area of pockets and cavities; and the area
and circumference of mouth openings. Most frequently, the largest
pocket/cavity has been the active site, but there are a number of
instructive exceptions [23]. Ligand volume and binding site
volume are somewhat correlated, but the ligand seldom occupies
the entire site. Auxiliary pockets near the active site have been
suggested as additional binding surface for designed ligands [24].
Analysis of active sites is important for the modeled protein as a
precursor to further work on its docking studies, and to shape the
processofmakingagridbeforedocking.
PredictionofEvolutionaryrelationship:
Evolutionary tree among these 10 lignin degrading protein
sequences were aligned using multiple sequence alignment tool
C l u s t a l o m e g a [ 2 5 ] [ h t t p s : / / w w w. e b i . a c . u k
/Tools/services/web_clustalw2/toolform.ebi] , followed by
construction of evolutionary tree using the protein sequences in
FASTA format as input data The alignment parameters were tuned
finely to find out the best alignment and Neighbor Joining [NJ]
methodwasselectedtoconstructthetree.
RESULTS:
Physico-chemicalcharacterization:
Isoelectric point [pI] is a pH in which net charge of protein is
zero. pI of lignin peroxidase 2 and 3 were observed to lie in the
acidic range, while the rest of the proteins occur in alkaline range.
From the study of instability index, it was found that all
peroxidases are stable [except Chloroperoxidase 7], as instability
index value less than 40 indicates stability of a protein
3. sequences and ranked them according to the scores of
ramachandran plot. For each of the proteins, the top ranked 3D
model was selected from the given models [supplementary file, fig
1]. The 3D models generated by 3D JIGSAW were then used further
forthemotifdetectionandpocketfindings.
MotifIdentification:
Chloroperoxidase and lignin peroxidase are found in M.
phaseolina as well as many other fungi, posing a major role in lignin
degradation [11, 31]. Motifs for chloroperoxidase and lignin
peroxidase were identified using Motif scan tool [Fig 1,
Supplementary file, table 4]. If we look into the conserved regions
[Fig 2], a number of residues are revealed which are already known
to have important role in catalysis. These conserverd regions are
predominated by cysteine, proline, aspartate, asparagines,
histidine,alanineandglycine.
Activesiteanalysis:
The active sites of chloroperoxidase and lignin peroxidase were
predicted [fig 3]. Further, in this study, we have also reported the
best active site area of the experimental enzymes as well as the
number of amino acids involved in it; showed the number of
pockets,withtheirareaandvolume[Supplementaryfile,fig2].
In case of the lignin peroxidase, the volume of the pockets is
quite similar and average is 2157 ų. There are some variations in
the area of pockets; for lignin peroxidase 2, it is 1100.9 Å2
[Supplementary file, table 5]. On the other hand, for lignin
peroxidase 3, it is highest 1857.8 Å2. For lignin peroxidase 1, the
area is between the two others-1542.7 Å2. But from the
visualizationwecanseeasimilarpocketstructures.
In the analysis of the chloroperoxidases we find similar pocket
sizes and structures too. For the chloroperoxidase 2,
chloroperoxidase 3, chloroperoxidase 4, chloroperoxidase 5 and
chloroperoxidase 6, the average pocket area is 2301.7 Å2 and
average pocket volume is 3428.6 Å3. Among these, the highest area
showedbychloroperoxidase4thatis3072.7Å2andvolumeis5042
Å3. There are two chloroperoxidases show major variations; those
are chloroperoxidase 1 and chloroperoxidase 7. For
chloroperoxidase1,areais697.6Å2andvolumeis907.5Å3.Incase
of chloroperoxidase 7, we identified paramount variation; it is
totally different from other chloroperoxidases regarding the area
[208.4 Å2], volume [203.8 Å3] and pocket. From the visualization,
we can see a similar pocket structure among the chloroperoxidases
otherthanthechloroperoxidase7.
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[Supplementary file, Table 1]. Additionally, Aliphatic index [AI]
refers to the relative volume of a protein occupied by its aliphatic
side chains. The higher the Aliphatic index of proteins, the more
thermally stable the proteins. Aliphatic index of chloroperoxidase
1 [100.11] and 5 [90.84] classifies them as most thermostable,
closely followed by other chloroperoxidases [chloroperoxidase 3,
4, 6 and lignin peroxidase 9]. Grand average of hydropathicity
index [GRAVY] indicates the interaction of the proteins in water.
Chloroperoxidase 1 [0.099] and lignin peroxidase 1 [0.022] are
hydrophobic [due to positive GRAVY values]. GRAVY values of
otherperoxidaseswereobservedwithinawiderangeof-0.055to-
0.54[hydrophilic].
The amino acid composition of each peroxidase sequence was
calculated by using ExPASY's ProtParam tool [supplementary file,
Table 2]. High percentage of alanine [above 9.9] and serine [above
8.5] was found in all three lignin peroxidases compared to other
amino acid. In all chloroperoxidase [except chloroperoxidase 7],
leucine content was found significant. Again, chloroperoxidase 2, 4
and 5 have a glycine content above 8.9. Moreover, a good
percentage of serine was found in chloroperoxidase 3 [9.6] and 7
[9.2]andalsochloroperoxidase7isprolinerich[10.0%].
There are some properties of the proteins such as isoelectric
point, hydrophobicity, and the frequency of certain collocated di
and tripeptides that are pivotal indicators of crystallization [16].
The CRYSTALP2 accounts all such characteristics of given
sequences for estimating the confidence of crystallization.The
higher the confidence, the more probable that protein is
crystallizableandviceversa.Amongthe7chloroperoxidasesand3
lignin peroxidases, chloroperoxidase 5 showed the highest
confidence of crystalizaiton that was 0.61 which was followed by
chloroperoxidase 2 [0.565] and chloroperoxidase 6 [0.523],
respectively [supplementary file, table 1]. In contrast, lignin
peroxidase 3 represented the least confidence of crystallization
[0.221]. Each of the the remaining chloroperoxidases exhibited
higher confidence of crystallization than that of remaining lignin
peroxidases. From the CRYSTALP2 result, it can be assumed that
chloroperoxidases are more likely to be crystallized than lignin
peroxidases. Among all chloroperoxidase, chloroperoxidase 5
representsmaximumpropensityforcrystallization.
Characterizationofsecondarystructure:
SOPMA analysis of secondary structure of chloroperoxidase
andligninperoxidaseproteinsequencesresultsthepredominance
of random coil which is followed by alpha helix, extended strand,
andbetasheet,respectively[supplementaryfile,table3].Incaseof
Chloroperoxidase 1, Lignin Peroxidase 1, it was found that alpha
helixexceedtherandomcoil.
Protein3Dstructure:
The 3D structure of protein is very crucial for comprehending
the protein functions, their sub-cellular localization as well as
protein-protein interactions. Based on homology modeling, 3D-
JIGSAWresultsshowedfive3Dmodelsforeachofthegiven
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Fig 1: Motifs in Chloroperoxidase and Lignin peroxidase. Insets show most common amino acids in motifs.
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Fig 2: Conserved regions in chloroperoxidase [top] and lignin peroxidases [bottom] motifs using boxshade.
Activesiteanalysis:
The active sites of chloroperoxidase and lignin peroxidase
were predicted [fig 3]. Further, in this study, we have also reported
the best active site area of the experimental enzymes as well as the
number of amino acids involved in it; showed the number of
pockets,withtheirareaandvolume[Supplementaryfile,fig2].
In case of the lignin peroxidase, the volume of the pockets is
quite similar and average is 2157 ų. There are some variations in
the area of pockets; for lignin peroxidase 2, it is 1100.9 Å2
[Supplementary file, table 5]. On the other hand, for lignin
peroxidase 3, it is highest 1857.8 Å2. For lignin peroxidase 1, the
area is between the two others-1542.7 Å2. But from the
visualizationwecanseeasimilarpocketstructures.
In the analysis of the chloroperoxidases we find similar pocket
sizes and structures too. For the chloroperoxidase 2,
chloroperoxidase 3, chloroperoxidase 4, chloroperoxidase 5 and
chloroperoxidase 6, the average pocket area is 2301.7 Å2 and
averagepocketvolumeis3428.6Å3.Amongthese,thehighestarea
showedbychloroperoxidase4thatis3072.7Å2andvolumeis
5042 Å3. There are two chloroperoxidases show major
variations; those are chloroperoxidase 1 and chloroperoxidase 7.
Forchloroperoxidase1,areais697.6Å2andvolumeis907.5Å3.In
caseofchloroperoxidase7,weidentifiedparamountvariation;itis
totally different from other chloroperoxidases regarding the area
[208.4 Å2], volume [203.8 Å3] and pocket. From the visualization,
we can see a similar pocket structure among the
chloroperoxidasesotherthanthechloroperoxidase7.
Fig 3: Pockets [active site] of the enzymes
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Evolutionaryrelationship:
Phylogenetic tree shows evolutionary relationship among all the ten proteins. There are two major clades on the tree. All
chloroperoxidases [except chloroperoxidase 1] reside in a common clade whereas, three lignin peroxidases reside in another tree.
Chloroperoxidase1seemstobetheoutgroupofthetree.
Table 1: Physico-chemical parameters of chloroperoxidase and lignin peroxidase:
Fig 4: Phylogenetic relationship among chloroperoxidases and lignin peroxidases
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Table 2: Composition of amino acid of chloroperoxidase and lignin peroxidase (in %):
Table 3: Secondary structure prediction of chloroeroxidase and lignin peroxidase (in %):
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Table 4: Identification of motifs:
Table 5: Active site Prediction
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10. Fig 2: Pockets (active site) in motif of chloroperoxidase and lognin peroxidase.
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11. DISCUSSION:
Instability index and aliphatic index indicate the relative
thermostability of the proteins, whereas GRAVY values show
hydrophilic nature of most of them. lignin peroxidases have high
percentage of alanine and serine. On the other hand,
chloroperoxidases are predominated by leucine, glycine, proline
and serine. These amino acids have important role in protein
structure. Serine is small in size and hence reasonably common to
occur within the tight turns on the protein surface or within the
interior of a protein. Serine side-chain hydroxyl oxygen can form a
hydrogen bond with the protein backbone [26]. Leucine is a
hydrophobic amino acid as it has a branched hydrocarbon side
chains usually buried in folded proteins. The hydrophobic effect
accounts for stabilization of water-soluble proteins [24]. High
percentage of glycine may be responsible for the stability of triple
helicalstructure,sinceincorporationoflargeaminoacidscancause
steric hindrance [27]. Increased proportion of proline residues is
significant not only to act as structural disruptor of the secondary
structuralelementsbutalsotopointoutwardandstabilizethehelix
[28].
From the CRYSTALP2 result, it is clear that chloroperoxidases
are more likely to be crystallized than lignin peroxidases. Among all
chloroperoxidase, chloroperoxidase 5 represents maximum
propensity for crystallization. The knowledge of crystallization
tendency of protein has a great importance in structural biology
that provides valuable insight into the structure-function
relationship of the proteins [16]. Moreover, crystallographic
knowledge of proteins has immense roles in pharmaceutical,
biotechnological and chemical industries for rational drug
designing, protein engineering and other applications. [29].
Secondarystructureanalysisshowedpredominanceofrandomcoil
over other structures. Large number of random coil gives the
protein more flexibility and self-assembly [30]. Protein 3D models
were created using homology modeling that were validated by
Ramachandranplot.
Motif scan tool was used as motif finder in these proteins.
Conserved regions of these motifs are important zone to mitigate
their activities. It is possible to inactivate these proteins by
designing ligands against these conserved sites in 3D protein
structure. Moreover, any type of site directed mutagenesis can be
performedintheseregionstoinactivatetheseproteins.Todoso,we
have to acquire a deep knowledge on it. cysteine, proline, aspartate,
asparagines, histidine, alanine and glycine are major amino acid
that not only have structural role but also important in catalytic
function. Cysteine is important for disulfide linkage as well as for
metal binding [32]. Another important residue is proline. It is
unable to adopt a normal helical conformation because it
introduces kinks into alpha helices. Aspartate and asparagine are
frequently involved in protein active sites. Because of their negative
charge, they can interact with positively charged non-protein
atoms. Since Aspartate has a shorter side-chain, it is slightly more
rigid within protein structures. Thus, it has a slightly stronger
preferencetobeinvolvedinproteinactivesites[26].Histidineisthe
most common amino acid in protein active sites. They
efficiently bind metal ions, often acting together with cysteines.
Alanineplaysimportantroleinsubstraterecognitionorspecificity,
particularly in interactions with non-reactive atoms such as
carbon.TheuniquenessofGlycine[HasRgroup]alsomeansthatit
can play a distinct functional role, such as using its sidechain-less
backbonetobindtophosphates[26].
Predicted active sites were of similar structure in same protein
family. Volume, area and charge density of active sites were
satisfactory. All of the chloroperoxidase proteins [except
chloroperoxidase 1] share a common ancestral point [marked by
arrow, Fig 4] after being diverged from root of the tree. Fungal
lignin peroxidase 1 and 3 are more closely related than lignin
peroxidase 2. Chloroperoxidase 1 seems to be the outgroup of the
tree. Probably this protein has acquired a lot of change in its
sequenceovertimewithoutlosingitscatalyticactivity.
CONCLUSION:
A number of plants for example crop, fiber or other
commercially valuable species are infected by Macrophomina
phaseolina fungus and causes a great lose throughout the world.
For the pathogenesis of this fungus, different genes are involved.
Among these genes, chloroperoxidase and lignin peroxidase are
two important genes that are required for a crucial step that is
lignin degradation. In present study, we have analyzed physico-
chemical characteristics, secondary and tertiary structure as well
as motifs in 7 chloroperoxidase and 3 lignin peroxidase. During
this analysis, we found amino acids like cysteine, aspertate,
asparagine, proline, histidine and glycine, which play vital role
both structurally and functionally. Moreover, we have searched the
active sites in these 10 proteins and found highest pocket area for
chloroperoxidase 4 and lignin peroxidase 3. Study of evolutionary
relationship reveals that all of the proteins shared a common
ancestor. All of these findings help us to understand the characters
of these proteins. This will facilitate to design a potent target
against chloroperoxidase and lignin peroxidase and protect the
fungalsusceptiblecropspecies.
CONFLICTOFINTERESTS
The authors declare that there is no conflict of interests
regardingthepublicationofthispaper.
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