Melatonin Inhibits AGS Cell Proliferation by Binding to the ATP Binding Site of CDK2 Under Hyperglycemic Conditions.

Cancer cells utilize glucose as their primary energy source. The aggressive nature of cancer cells is therefore enhanced in hyperglycemic conditions. This study has been adopted to investigate the therapeutic potential of melatonin against such aggressive proliferation of AGS cells-a human gastric cancer cell line, under hyperglycemic conditions. AGS cells were incubated with high glucose-containing media, and the effects of melatonin have been evaluated, therein. Cell proliferation, ROS generation, flow-cytometric analysis for cell cycle and apoptosis, wound healing, immunoblotting, zymography, reverse zymography assays, in-silico analysis, and kinase activity assays were performed to evaluate the effects of melatonin. We observed that melatonin inhibited the hyperglycemia-induced cell proliferation in a dose-dependent manner. It further altered the expression and activity of MMP-9 and TIMP-1. Moreover, melatonin inhibited AGS cell proliferation by arresting AGS cells in the G0/G1 phase after binding in the ATP binding site of CDK-2, thereby inhibiting its kinase activity. In association, a significant decrease in the expression of cyclin D1, cyclin E, CDK-4, and CDK-2 were observed. In conclusion, these findings suggest that melatonin has anti-gastric cancer potential. Melatonin could therefore be included in future drug designs for gastric cancer-hyperglycemia co-morbidity treatment.

Dosimetric Evaluation of the Treatment Plan on Indigenous Heterogeneous Phantoms using Analytical Anisotropic Algorithm and Acuros-XB Algorithm for Different Photon Energies.

Background: Modern radiotherapy techniques are using advanced algorithms; however, phantoms used for quality assurance have homogeneous density; accordingly, the development of heterogeneous phantom mimicking human body sites is imperative to examine variation between planned and delivered doses. Objective: This study aimed to analyze the accuracy of planned dose by different algorithms using indigenously developed heterogeneous thoracic phantom (HT). Material and Methods: In this experimental study, computed tomography (CT) of HT was done, and the density of different parts was measured. The plan was generated on CT images of HCP with 6 and 15 Megavoltage (MV) photon beams using different treatment techniques, including three-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiation therapy (IMRT), and volumetric modulated arc therapy (VMAT). Plans were delivered by the linear accelerator, and the dose was measured using the ion chamber (IC) placed in HT; planned and measured doses were compared. Results: Density patterns for different parts of the fabricated phantom, including rib, spine, scapula, lung, chest wall, and heart were 1.849, 1.976, 1.983, 0.173, 0.855, and 0.833 g/cc, respectively. Variation between planned and IC estimated doses with the tolerance (±5%) for all photon energies using different techniques. Acuros-XB (AXB) showed a slightly higher variation between computed and IC estimated doses using HCP compared to the analytical anisotropic algorithm (AAA). Conclusion: The indigenous heterogeneous phantom can accurately simulate the dosimetric scenario for different algorithms (AXB or AAA) and be also utilized for routine patient-specific QA.

Melatonin rescues swim stress induced gastric ulceration by inhibiting matrix metalloproteinase-3 via down-regulation of inflammatory signaling cascade.

AIM: This study investigated the link between forced swim induced acute gastric ulceration, inflammation and MMP-3 along with the possible mechanism of protective efficacy of melatonin. MAIN METHODS: We distributed Balb/c mice into four different groups. Group 1 and 2 were given PBS gavage. Group 3 and 4 were given melatonin (60 mg/kg b.wt.) and omeprazole (25 mg/kg b.wt.), respectively, an hour prior to forced swim. Ulcer index, tissue histology, immunohistochemistry, protein carbonylation, lipid peroxidation, Myeloperoxidase, Zymography, Western blotting, reactive oxygen species (ROS), mitochondrial dehydrogenase, mitochondrial transmembrane potential and bioinformatical analysis were performed. KEY FINDINGS: Our data revealed that gastric ulceration due to forced swim stress is responsible for overproduction of ROS, which may be a prime reason for mitochondrial dysfunction and induction of apoptosis via activation of Caspase-3. ROS is also responsible for p38 phosphorylation which in turn increases the activity of MMP-3 in ulcerated milieu, along with the oxidation of proteins, peroxidation of lipids and altered expression patterns of heat shock protein (HSP)-70. Melatonin is shown to reduce the inflammatory burden in gastric milieu and offers gastroprotection by binding to the active site of MMP-3; thereby inhibiting its activity, as suggested by in silico studies. Melatonin also inhibits the downregulation of HSP-70 and activates p38 dephosphorylation and thereby, it rescues gastric mucosal cells from stress-induced ulceration. SIGNIFICANCE: Our findings suggest that, melatonin imparts its gastroprotective effect by down-regulating the activation of MAPK-ERK pathway along with binding to the active site of MMP-3.

Zymography and Reverse Zymography for Testing Proteases and Their Inhibitors.

Zymography is a powerful technique for the assay of different hydrolases that act upon any biological macromolecule. In particular, zymography is used to assay the activities of serine proteases, e.g., matrix metalloproteinases (MMPs), and reverse zymography is used for tissue inhibitors of metalloproteinases (TIMPs) in multifarious experimental samples. Zymography is a method of electrophoretic separation of proteases under non-reducing conditions in a polyacrylamide gel containing substrate. The resolved proteins are renatured by exchange of the anionic detergent with a nonionic one, and the gel is incubated in a specific buffer for the specific proteases. After staining the gel by Coomassie blue staining solution, the proteolytic activities are visualized as clear colorless bands against a dark background. In contrast, reverse zymography is a parallel technique to detect protease inhibitors. In addition to substrate gelatin, proteases (i.e., MMPs) are also incorporated in proper ratio into the polyacrylamide gel. After electrophoresis, during the developing step, the MMPs specifically digest the substrate in regions where TIMPs are absent. Thus, inhibitors/TIMP is represented as dark zones of inhibition against a transparent background after staining. In this chapter, common troubleshoots during sample preparation, running zymography, and data interpretation are discussed. Notes are specified to enhance the sensitivity of the methods. In conclusion, zymography could be crucial for enzyme assay at the nanogram level and for the improvement of new investigative techniques for diseases such as endometriosis, rheumatoid arthritis, osteoarthritis, tumor invasion, and inflammation.

Fungal Genomic Resources for Strain Identification and Diversity Analysis of 1900 Fungal Species.

Identification and diversity analysis of fungi is greatly challenging. Though internal transcribed spacer (ITS), region-based DNA fingerprinting works as a "gold standard" for most of the fungal species group, it cannot differentiate between all the groups and cryptic species. Therefore, it is of paramount importance to find an alternative approach for strain differentiation. Availability of whole genome sequence data of nearly 2000 fungal species are a promising solution to such requirement. We present whole genome sequence-based world's largest microsatellite database, FungSatDB having >19M loci obtained from >1900 fungal species/strains using >4000 assemblies across globe. Genotyping efficacy of FungSatDB has been evaluated by both in-silico and in-vitro PCR. By in silico PCR, 66 strains of 8 countries representing four continents were successfully differentiated. Genotyping efficacy was also evaluated by in vitro PCR in four fungal species. This approach overcomes limitation of ITS in species, strain signature, and diversity analysis. It can accelerate fungal genomic research endeavors in agriculture, industrial, and environmental management.

Molecular and Cellular Mechanisms of Electronegative Lipoproteins in Cardiovascular Diseases.

Dysregulation of glucose and lipid metabolism increases plasma levels of lipoproteins and triglycerides, resulting in vascular endothelial damage. Remarkably, the oxidation of lipid and lipoprotein particles generates electronegative lipoproteins that mediate cellular deterioration of atherosclerosis. In this review, we examined the core of atherosclerotic plaque, which is enriched by byproducts of lipid metabolism and lipoproteins, such as oxidized low-density lipoproteins (oxLDL) and electronegative subfraction of LDL (LDL(-)). We also summarized the chemical properties, receptors, and molecular mechanisms of LDL(-). In combination with other well-known markers of inflammation, namely metabolic diseases, we concluded that LDL(-) can be used as a novel prognostic tool for these lipid disorders. In addition, through understanding the underlying pathophysiological molecular routes for endothelial dysfunction and inflammation, we may reassess current therapeutics and might gain a new direction to treat atherosclerotic cardiovascular diseases, mainly targeting LDL(-) clearance.

Identifying the Therapeutic Significance of Mesenchymal Stem Cells.

The pleiotropic behavior of mesenchymal stem cells (MSCs) has gained global attention due to their immense potential for immunosuppression and their therapeutic role in immune disorders. MSCs migrate towards inflamed microenvironments, produce anti-inflammatory cytokines and conceal themselves from the innate immune system. These signatures are the reason for the uprising in the sciences of cellular therapy in the last decades. Irrespective of their therapeutic role in immune disorders, some factors limit beneficial effects such as inconsistency of cell characteristics, erratic protocols, deviating dosages, and diverse transfusion patterns. Conclusive protocols for cell culture, differentiation, expansion, and cryopreservation of MSCs are of the utmost importance for a better understanding of MSCs in therapeutic applications. In this review, we address the immunomodulatory properties and immunosuppressive actions of MSCs. Also, we sum up the results of the enhancement, utilization, and therapeutic responses of MSCs in treating inflammatory diseases, metabolic disorders and diabetes.

Role of Ceramidases in Sphingolipid Metabolism and Human Diseases.

Human pathologies such as Alzheimer's disease, type 2 diabetes-induced insulin resistance, cancer, and cardiovascular diseases have altered lipid homeostasis. Among these imbalanced lipids, the bioactive sphingolipids ceramide and sphingosine-1 phosphate (S1P) are pivotal in the pathophysiology of these diseases. Several enzymes within the sphingolipid pathway contribute to the homeostasis of ceramide and S1P. Ceramidase is key in the degradation of ceramide into sphingosine and free fatty acids. In humans, five different ceramidases are known-acid ceramidase, neutral ceramidase, and alkaline ceramidase 1, 2, and 3-which are encoded by five different genes (ASAH1, ASAH2, ACER1, ACER2, and ACER3, respectively). Notably, the neutral ceramidase N-acylsphingosine amidohydrolase 2 (ASAH2) shows considerable differences between humans and animals in terms of tissue expression levels. Besides, the subcellular localization of ASAH2 remains controversial. In this review, we sum up the results obtained for identifying gene divergence, structure, subcellular localization, and manipulating factors and address the role of ASAH2 along with other ceramidases in human diseases.

Author Correction: Insights into the functionality of endophytic actinobacteria with a focus on their biosynthetic potential and secondary metabolites production.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Determination and production of antimicrobial compounds by Aspergillus clavatonanicus strain MJ31, an endophytic fungus from Mirabilis jalapa L. using UPLC-ESI-MS/MS and TD-GC-MS analysis.

Endophytic fungi associated with medicinal plants are reported as potent producers of diverse classes of secondary metabolites. In the present study, an endophytic fungi, Aspergillus clavatonanicus strain MJ31, exhibiting significant antimicrobial activity was isolated from roots of Mirabilis jalapa L., was identified by sequencing three nuclear genes i.e. internal transcribed spacers ribosomal RNA (ITS rRNA), 28S ribosomal RNA (28S rRNA) and translation elongation factor 1- alpha (EF 1α). Ethyl acetate extract of strain MJ31displayed significant antimicrobial potential against Bacillus subtilis, followed by Micrococccus luteus and Staphylococcus aureus with minimum inhibitory concentrations (MIC) of 0.078, 0.156 and 0.312 mg/ml respectively. In addition, the strain was evaluated for its ability to synthesize bioactive compounds by the amplification of polyketide synthase (PKS) and non ribosomal peptide synthetase (NRPS) genes. Further, seven antibiotics (miconazole, ketoconazole, fluconazole, ampicillin, streptomycin, chloramphenicol, and rifampicin) were detected and quantified using UPLC-ESI-MS/MS. Additionally, thermal desorption-gas chromatography mass spectrometry (TD-GC-MS) analysis of strain MJ31 showed the presence of 28 volatile compounds. This is the first report on A. clavatonanicus as an endophyte obtained from M. jalapa. We conclude that A. clavatonanicus strain MJ31 has prolific antimicrobial potential against both plant and human pathogens and can be exploited for the discovery of new antimicrobial compounds and could be an alternate source for the production of secondary metabolites.

Insights into the functionality of endophytic actinobacteria with a focus on their biosynthetic potential and secondary metabolites production.

Endophytic actinobacteria play an important role in growth promotion and development of host plant by producing enormous quantities of novel bioactive natural products. In the present investigation, 169 endophytic actinobacteria were isolated from endospheric tissues of Rhynchotoechum ellipticum. Based on their antimicrobial potential, 81 strains were identified by 16rRNA gene analysis, which were taxonomically grouped into 15 genera. All identified strains were screened for their plant growth promoting attributes and, for the presence of modular polyketide synthases (PKSI, PKSII and nonribosomal peptide synthetase (NRPS) gene clusters to correlate the biosynthetic genes with their functional properties. Expression studies and antioxidant potential for four representative strains were evaluated using qRT-PCR and DPPH assay respectively. Additionally, six antibiotics (erythromycin, ketoconazole, fluconazole, chloramphenicol, rifampicin and miconazole) and nine phenolic compounds (catechin, kaempferol, chebulagic acid, chlorogenic acid, Asiatic acid, ferulic acid, arjunic acid, gallic acid and boswellic acid) were detected and quantified using UHPLC-QqQLIT-MS/MS. Furthermore, three strains (BPSAC77, 121 and 101) showed the presence of the anticancerous compound paclitaxel which was reported for the first time from endophytic actinobacteria. This study provides a holistic picture, that endophytic actinobacteria are rich bacterial resource for bioactive natural products, which has a great prospective in agriculture and pharmaceutical industries.

Antimicrobial Potential, Identification and Phylogenetic Affiliation of Wild Mushrooms from Two Sub-Tropical Semi-Evergreen Indian Forest Ecosystems.

The diversity of wild mushrooms was investigated from two protected forest areas in India and 231 mushroom specimens were morphologically identified. Among them, 76 isolates were screened for their antimicrobial potential against seven bacterial and fungal pathogens. Out of 76 isolates, 45 isolates which displayed significant antimicrobial activities were identified using ITS rRNA gene amplification and subsequently phylogenetically characterized using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Sequencing of the ITS rRNA region classified the isolates into 16 genera belonging to 11 families. In total, 11 RAPD and 10 ISSR primers were selected to evaluate genetic diversity based on their banding profile produced. In total 337 RAPD and 312 ISSR bands were detected, among which percentage of polymorphism ranges from 34.2% to 78.8% and 38.6% to 92.4% by using RAPD and ISSR primers respectively. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of selected two methods were structured similarly, grouping the 46 isolates into two clusters which clearly showed a significant genetic distance among the different strains of wild mushroom, with an similarity coefficient ranges from 0.58 to 1.00 and 0.59 to 1.00 with RAPD and ISSR analysis respectively. This reporthas highlighted both DTR and MNP forests provide a habitat for diverse macrofungal species, therefore having the potential to be used for the discovery of antimicrobials. The report has also demonstrated that both RAPD and ISSR could efficiently differentiate wild mushrooms and could thus be considered as efficient markers for surveying genetic diversity. Additionally, selected six wild edible mushroom strains (Schizophyllum commune BPSM01, Panusgiganteus BPSM27, Pleurotussp. BPSM34, Lentinussp. BPSM37, Pleurotusdjamor BPSM41 and Lentinula sp. BPSM45) were analysed for their nutritional (proteins, carbohydrates, fat and ash content), antioxidant potential. The present findings also suggested that the wild edible mushroom strains do not have only nutritional values but also can be used as an accessible source of natural antioxidants.

Phytohormone production endowed with antagonistic potential and plant growth promoting abilities of culturable endophytic bacteria isolated from Clerodendrum colebrookianum Walp.

In this study, culturable endophytic bacterial isolates obtained from an ethnomedicinal plant Clerodendrum colebrookianum Walp., were assessed for their diversity, in vitro screening for their plant growth promoting (PGP) activities and to use them as inoculant for in vivo PGP activities with biocontrol potential. Totally, 73 isolates were recovered from different tissues of C. colebrookianum were identified by 16S rRNA gene sequencing and phylogenetically analyzed by using BOX-PCR fingerprinting. Out of 73 isolates, 52 exhibited varying extents of antagonistic potential were selected for screening for various PGP traits. Concerning the PGP activities, the percentage of isolates positive for P-solubilisation, indolic compounds production, siderophore and ammonia production were 84.6, 92.3, 78.8 and 98.0 respectively. All isolates were positive for the production of hydrocyanic acid (HCN) and 86.5%, 84.6% and 90.3% of isolates showed significant cellulase, amylase and protease production respectively. Further, the top 10 bacterial isolates based on a bonitur scale with multiple PGP activities were screened for root surface colonization and biofilm formation ability. Out of selected 10 isolates, 9 showed significant potential for root surface colonization on tomato roots. Isolate BPSAC6 identified as Bacillus sp. was most efficient in biofilm formation as assessed with respect to the intensity of crystal violet, which further showed their potential to withstand various biotic and abiotic stresses. Furthermore, Bacillus sp. strain BPSAC6 showed a significant increase in shoot and root height as well as fresh weight after 45 and 60 d of inoculation with tomato seedlings. Additionally, biosynthetic potential of antagonistic isolate was detection by using PKSI, PKSII and NRPS biosynthetic genes. Two isolates Pseudomonas psychrotolerans and Labrys wisconsinensis were reported first time as an endophyte. At last, first time an endophytic bacterial strain Bacillus sp. BPSAC6 was reported to produce altogether three phytohormones (IAA, Kinetin and 6-Benzyladenine). This study is the first report that bacteria isolated from C. colebrookianum has biocontrol as well as PGP abilities endowed with phytohormones production and can be used for the preparation of bioinoculant for plant growth promotion.

Detection of biosynthetic gene and phytohormone production by endophytic actinobacteria associated with Solanum lycopersicum and their plant-growth-promoting effect.

In the present study, fifteen endophytic actinobacterial isolates recovered from Solanum lycopersicum were studied for their antagonistic potential and plant-growth-promoting (PGP) traits. Among them, eight isolates showed significant antagonistic and PGP traits, identified by amplification of the 16S rRNA gene. Isolate number DBT204, identified as Streptomyces sp., showed multiple PGP traits tested in planta and improved a range of growth parameters in seedlings of chili (Capsicum annuum L.) and tomato (S. lycopersicum L.). Further, genes of indole acetic acid (iaaM) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) were successively amplified from five strains. Six antibiotics (trimethoprim, fluconazole, chloramphenicol, nalidixic acid, rifampicin and streptomycin) and two phytohormones [indole acetic acid (IAA) and kinetin (KI)] were detected and quantified in Streptomyces sp. strain DBT204 using UPLC-ESI-MS/MS. The study indicates the potential of these PGP strains for production of phytohormones and shows the presence of biosynthetic genes responsible for production of secondary metabolites. It is the first report showing production of phytohormones (IAA and KI) by endophytic actinobacteria having PGP and biosynthetic potential. We propose Streptomyces sp. strain DBT204 for inoculums production and development of biofertilizers for enhancing growth of chili and tomato seedlings.

Distribution and antimicrobial potential of endophytic fungi associated with ethnomedicinal plant Melastoma malabathricum L.

Distributions of endophytic fungi associated with ethnomedicinal plant Melastoma malabathricum L. was studied and 91 isolates belonging to 18 genera were recovered. The isolates were distributed to sordariomycetes (62.63%), dothideomycetes (19.78%), eurotiomycetes (7.69%), zygomycetes (4.19%), agaricomycetes (1.09%), and mycelia sterilia (4.39%). Based on colony morphology and examination of spores, the isolates were classified into 18 taxa, of which Colletotrichum, Phomopsis and Phoma were dominant, their relative frequencies were 23.07%, 17.58% and 12.08% respectively. The colonization rate of endophytic fungi was determined and found to be significantly higher in leaf segments (50.76%), followed by root (41.53%) and stem tissues (27.69%). All the isolates were screened for antimicrobial activity and revealed that 26.37% endophytic fungi were active against one or more pathogens. Twenty four isolates showing significant antimicrobial activity were identified by sequencing the ITS1-5.8S-ITS2 region of rRNA gene. Results indicated that endophytic fungi associated with leaf were functionally versatile as they showed antimicrobial activity against most of the tested pathogens. The endophytic fungi Diaporthe phaseolorum var. meridionalis (KF193982) inhibited all the tested bacterial pathogens, whereas, Penicillium chermesinum (KM405640) displayed most significant antifungal activity. This seems to be the first hand report to understand the distribution and antimicrobial ability of endophytic fungi from ethno-medicinal plant M. malabathricum.

In Vitro and In Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants.

Endophytic actinomycetes have shown unique plant growth promoting as well as antagonistic activity against fungal phytopathogens. In the present study forty-two endophytic actinomycetes recovered from medicinal plants were evaluated for their antagonistic potential and plant growth-promoting abilities. Twenty-two isolates which showed the inhibitory activity against at least one pathogen were subsequently tested for their plant-growth promoting activities and were compared genotypically using DNA based fingerprinting, including enterobacterial repetitive intergenic consensus (ERIC) and BOX repetitive elements. Genetic relatedness based on both ERIC and BOX-PCR generates specific patterns corresponding to particular genotypes. Exponentially grown antagonistic isolates were used to evaluate phosphate solubilization, siderophores, HCN, ammonia, chitinase, indole-3-acetic acid production, as well as antifungal activities. Out of 22 isolates, the amount of indole-3-acetic acid (IAA) ranging between 10-32 µg/ml was produced by 20 isolates and all isolates were positive for ammonia production ranging between 5.2 to 54 mg/ml. Among 22 isolates tested, the amount of hydroxamate-type siderophores were produced by 16 isolates ranging between 5.2 to 36.4 µg/ml, while catechols-type siderophores produced by 5 isolates ranging from 3.2 to 5.4 µg/ml. Fourteen isolates showed the solubilisation of inorganic phosphorous ranging from 3.2 to 32.6 mg/100ml. Chitinase and HCN production was shown by 19 and 15 different isolates, respectively. In addition, genes of indole acetic acid (iaaM) and chitinase (chiC) were successively amplified from 20 and 19 isolates respectively. The two potential strains Streptomyces sp. (BPSAC34) and Leifsonia xyli (BPSAC24) were tested in vivo and improved a range of growth parameters in chilli (Capsicum annuum L.) under greenhouse conditions. This study is the first published report that actinomycetes can be isolated as endophytes from within these plants and were shown to have antagonistic and plant growth promoting abilities. These results clearly suggest the possibility of using endophytic actinomycetes as bioinoculant for plant growth promotion, nutrient mobilization or as biocontrol agent against fungal phytopathogens for sustainable agriculture.

In silico analysis of Myoglobin in Channa striata.

Myoglobin is a cytoplasmic hemoprotein, expressed solely in cardiac myocytes and oxidative skeletal muscle fibers, that reversibly binds O2 by its heme residue. Myoglobin is an essential oxygen-storage hemoprotein capable of facilitating oxygen transport and modulating nitric oxide homeostasis within cardiac and skeletal myocytes. Functionally, myoglobin is well accepted as an O2- storage protein in muscle, capable of releasing O2 during periods of hypoxia or anoxia. There is no evidence available regarding active sites, ligand binding sites, antigenic determinants and the ASA value of myoglobin in Channa striata. We further document the predicted active sites in the structural model with solvent exposed ASA residues. During this study, the model was built by CPH program and validated through PROCHECK, Verify 3D, ERRAT and ProSA for reliability. The active sites were predicted in the model with further ASA analysis of active site residues. The discussed information thus provides the predicted active sites, ligand binding sites, antigenic determinants and ASA values of myoglobin model in Channa striata.

Computational Analysis of N-acetyl transferase in Tribolium castaneum.

N-acetyl transferase (NAT) is responsible to catalyze the transfer of acetyl groups to arylamines from acetyl-CoA. Aralkylamine Nacetyl transferase (AANAT), which belongs to GCN5-related N-acetyl transferase member, is a globular 23-kDa cytosolic protein that forms a reversible regulatory complex with 14-3-3 proteins, AANAT regulates the daily cycle of melatonin biosynthesis in mammals, making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. There is no evidence available regarding α and ß subunits, active site and their ASA value in Dopamine N-acetyl transferase. Therefore, we describe the development of Dopamine N-acetyl transferase model in Tribolium castaneum. We further document the predicted active sites in the structural model with solvent exposed ASA residues. During this study, the model was built by CPH program and validated through PROCHECK, Verify 3D, ERRAT and ProSA for reliability. The active sites were predicted in the model with further ASA analysis of active site residues. The discussed information thus provides insight to the predicted active site and ASA values of Dopamine N-acetyl transferase model in Tribolium castaneum.

Characterization of Bacillus thuringiensis Cry1 class proteins in relation to their insecticidal action.

Thirty nine Bt Cry1 subgroup protein sequences were retrieved from NCBI and analyzed for physicochemical properties, active site and relationship in relation to their variations in toxicity. Cry1 proteins were found to be hydrophilic and stable. SOSUI server predicted presence of two transmembrane regions in Ag and a single transmembrane region from Aa to Ae. EMBOSS PepWheel tool analysis of the transmembrane regions showed that there were 23 highly conserved residues towards the N terminal which are hydrophobic and more than half of the residues were neutrally charged. No signal peptide was detected which classifies the Cry1 group proteins as non-secretory proteins. Cry1 proteins have very high composition of neutral amino acids and might transform into negative charge after solubilization in alkaline environment (insect midgut). The negatively charged protein might misfold causing difficultly to digest and thereby be toxic to lepidopteran. Active sites of Cry1 proteins with more than 50% neutral amino acids showed wide insecticidal spectrum and further positive correlation (r = 0.7731) was observed between neutral amino acids and insect species affected (Y = -138.21 + 2.907X). Similarity of sequences was found between Cry1 proteins based on their high or low spectrum of insecticidal activity.