Search results for: enzyme inhibitors

Authors: Sakina Fatima, Joseph-Patrick W. E. Clarke, Patricia A. Thibault, Subha Kalyaanamoorthy, Michael Levin, Aravindhan Ganesan

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Heteronuclear ribonucleoprotein (hnRNPA1 or A1) is associated with the pathology of different diseases, including neurological disorders and cancers. In particular, the aggregation and dysfunction of A1 have been identified as a critical driver for neurodegeneration (NDG) in Multiple Sclerosis (MS). Structurally, A1 includes a low-complexity domain (LCD) and two RNA-recognition motifs (RRMs), and their interdomain coordination may play a crucial role in A1 aggregation. Previous studies propose that RNA-inhibitors or nucleoside analogs that bind to RRMs can potentially prevent A1 self-association. Therefore, molecular-level understanding of the structures, dynamics, and nucleotide interactions with A1 RRMs can be useful for developing therapeutics for NDG in MS. In this work, a combination of computational modelling and biochemical experiments were employed to analyze a set of RNA-A1 RRM complexes. Initially, the atomistic models of RNA-RRM complexes were constructed by modifying known crystal structures (e.g., PDBs: 4YOE and 5MPG), and through molecular docking calculations. The complexes were optimized using molecular dynamics simulations (200-400 ns), and their binding free energies were computed. The binding affinities of the selected complexes were validated using a thermal shift assay. Further, the most important molecular interactions that contributed to the overall stability of the RNA-A1 RRM complexes were deduced. The results highlight that adenine and guanine are the most suitable nucleotides for high-affinity binding with A1. These insights will be useful in the rational design of nucleotide-analogs for targeting A1 RRMs.

Keywords: hnRNPA1, molecular docking, molecular dynamics, RNA-binding proteins

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Authors: M. A. Siti Faridah, K. Muhammad, H. M. Ghazali, Y. A. Yusof

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This study was conducted to investigate the effects of three variables namely types of cell wall degrading enzymes (Viscozyme L, Pectinex Ultra SP-L, Rapidase PAC, Rohament CL and Rohapect PTE) at varying concentrations (0.25-3% v/w) and times (30 min-24 h) on the zinc (Zn-) amaranth purees. Liquefaction treatment of the Zn-amaranth purees with Viscozyme (1% v/w at pH 5 and 45ºC for 3 h) was found to be the best procedure, which produced Zn-amaranth puree with low viscosity (8.60 mPas). Zn-amaranth purees were also found to have the highest metallo-chlorophyll derivative contents (0.16 mg/g), free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) values (12.49 mM (TE)/g fresh weight) and ferric reducing antioxidant power (FRAP) values (4.57 mM (TE)/g fresh weight) within 3 h of liquefaction. Other physicochemical properties of the enzyme-liquefied Zn-amaranth purees indicated that lightness (L*) (12.54), greenness a*/b* (-0.30), reducing sugar (103.88 mg/mL) and soluble dietary fibre (5.94%) of the purees were higher compared to that of nonenzyme-liquefied amaranth purees.

Keywords: amaranth, antioxidant, chlorophyll derivative, enzymatic liquefaction

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Authors: Hamide Amani, Afshin FarahBakhsh, Iman Farahbakhsh

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In this paper, an amprometric biosensor based on surface modified polypyrrole (PPy) has been developed for the quantitative estimation of urea in aqueous solutions. The incorporation of urease (Urs) into a bipolymeric substrate consisting of PPy was performed by entrapment to the polymeric matrix, PPy acts as amperometric transducer in these biosensors. To increase the membrane conductivity, multi-walled carbon nanotubes (MWCNT) were added to the PPy solution. The entrapped MWCNT in PPy film and the bipolymer layers were prepared for construction of Pt/PPy/MWCNT/Urs. Two different configurations of working electrodes were evaluated to investigate the potential use of the modified membranes in biosensors. The evaluation of two different configurations of working electrodes suggested that the second configuration, which was composed of an electrode-mediator-(pyrrole and multi-walled carbon nanotube) structure and enzyme, is the best candidate for biosensor applications.

Keywords: urea biosensor, polypyrrole, multi-walled carbon nanotube, urease

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Authors: Margarita Ivanova, Julia Dao, Andrew Friedman, Neil Kasaci, Rekha Gopal, Ozlem Goker-Alpan

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In Fabry disease (FD), α-galactosidase A (α-Gal A) deficiency leads to the accumulation of globotriaosylceramide (Lyso-Gb3 and Gb3), triggering a pathologic cascade that causes the severity of organs damage. The heart is one of the several organs with high sensitivity to the α-Gal A deficiency. A subgroup of patients with significant residual of α-Gal A activity with primary cardiac involvement is occasionally referred to as “cardiac variant.” The cardiovascular complications are most frequently encountered, contributing substantially to morbidity, and are the leading cause of premature death in male and female patients with FD. The deposition of Lyso-Gb-3 and Gb-3 within the myocardium affects cardiac function with resultant progressive cardiovascular pathology. Gb-3 and Lyso-Gb-3 accumulation at the cellular level trigger a cascade of events leading to end-stage fibrosis. In the cardiac tissue, Lyso-Gb-3 deposition is associated with the increased release of inflammatory factors and transforming growth factors. Infiltration of lymphocytes and macrophages into endomyocardial tissue indicates that inflammation plays a significant role in cardiac damage. Moreover, accumulated data suggest that chronic inflammation leads to multisystemic FD pathology even under enzyme replacement therapy (ERT). NF-κB activation plays a subsequent role in the inflammatory response to cardiac dysfunction and advanced heart failure in the general population. TNFalpha/NF-κB signaling protects the myocardial evoking by ischemic preconditioning; however, this protective effect depends on the concentration of TNF-α. Thus, we hypothesize that TNF-α is a critical factor in determining the grade of cardio-pathology. Cardiac hypertrophy corresponds to the expansion of the coronary vasculature to maintain a sufficient supply of nutrients and oxygen. Coronary activation of angiogenesis and fibrosis plays a vital role in cardiac vascularization, hypertrophy, and tissue remodeling. We suggest that the interaction between the inflammatory pathways and cardiac vascularization is a bi-directional process controlled by secreted cytokines and growth factors. The co-coordination of these two processes has never been explored in FD. In a cohort of 40 patients with FD, biomarkers associated with inflammation and cardio hypertrophic remodeling were studied. FD patients were categorized into three groups based on LVmass/DSA, LVEF, and ECG abnormalities: FD with no cardio complication, FD with moderate cardio complication, and severe cardio complication. Serum levels of NF-kB, TNFalpha, Il-6, Il-2, MCP1, ING-gamma, VEGF, IGF-1, TGFβ, and FGF2 were quantified by enzyme-linked immunosorbent assays (ELISA). Among the biomarkers, MCP-1, INF-gamma, VEGF, TNF-alpha, and TGF-beta were elevated in FD patients. Some of these biomarkers also have the potential to correlate with cardio pathology in FD. Conclusion: The study provides information about the role of inflammatory pathways and biomarkers of cardio hypertrophic remodeling in FD patients. This study will also reveal the mechanisms that link intracellular accumulation of Lyso-GB-3 and Gb3 to the development of cardiomyopathy with myocardial thickening and resultant fibrosis.

Keywords: biomarkers, Fabry disease, inflammation, growth factors

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Authors: Manju Verma, Parag A. Deshpande

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Electronic structure calculations of hydrogen terminated metal-porphyrin graphene were carried out to explore the catalytic activity for CO2 hydration reaction. A ruthenium atom was substituted in place of carbon atom of graphene and ruthenium chelated carbon atoms were replaced by four nitrogen atoms in metal-porphyrin graphene system. Ruthenium atom created the active site for CO2 hydration reaction. Ruthenium-porphyrin graphene followed the mechanism of carbonic anhydrase enzyme for CO2 conversion to HCO3- ion. CO2 hydration reaction over ruthenium-porphyrin graphene proceeded via the elementary steps: OH- formation from H2O dissociation, CO2 bending in presence of nucleophilic attack of OH- ion, HCO3- ion formation from proton migration, HCO3- ion desorption by H2O addition. Proton transfer to yield HCO3- ion was observed as a rate limiting step from free energy landscape.

Keywords: ruthenium-porphyrin graphene, CO2 hydration, carbonic anhydrase, heterogeneous catalyst, density functional theory

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Authors: Ben Amar Cheba, Taha Ibrahim Zaghloul, Mohamad Hisham El-Massry, Ahmad Rafik El-Mahdy

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Seventy two bacterial strains with ability to degrade chitin were isolated during a screening program. One of the most potent isolates (strain R2) was identified as Bacillus sp. using conventional methods as well as 16S rRNA technique and submitted in the Gen Bank sequence database as Bacillus sp. R2 with a given accession number DQ 923161. This strain was able to produce high levels of extracellular chitinase. The chitinase of Bacillus sp. R2 hydrolyzed several chitinous substrates preferentially and showed a maximum activity toward the β chitin such as Calmar pen and squid bone chitins with the folds 1.47 and 1.23 respectively. The enzyme also exhibited a substrate binding capacity of more than 70% for squid chitin, shrimp shell colloidal chitin, chitosan and prawn shell chitin. The chitinase showed a moderate antifungal activity against many phytopathogenic fungi such as Aspergillus niger, A. flavus, Penicillium degitatum and Fusarium calmorum.This strain could be a suitable candidate for chitinase production on an industrial scale for using as promising antifungal biopestecide.

Keywords: antifungal activity, Bacillus sp. R2, chitinase, substrate specificity

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Authors: Abbassi Daloii Asieh, Yazdani Hoda

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Background and Objective: evidence supports systemic inflammation in obesity and insulin resistance. Apelin that is secreted by adipose tissue plays an important role in the inflammation process and appear act as an anti-inflammatory cytokines. The aim of this study was the effect of eight weeks aerobic training and nitro-L-arginine-methyl ester (L-NAME) on plasma apelin in male’s rats. Materials and Methods: For this purpose, 24 male Wistar rats aged 20 months were randomly assigned into four groups: Control, training, training and L-NAME and L-NAME. Training intervention was eight weeks aerobic exercise (5 time/weekly) at 75-80 (%) of maximal oxygen consumption. All rats were killed 72 hours after lasted exercise session; blood samples collected and plasma were stored. Data was analyzed by one way ANOVA and Tucky Test. A p value less than 0.05 was considered statistically signigcant. Results: The results showed that after eight weeks of endurance training exercise Apelin plasma compared to the control group did not change significantly. Also, the results showed that there was significant difference in plasma Apelin between groups(P > 0/05). Also, the results showed no significant difference between the insulin levels and glucose of four groups (P > 0/05). Conclusion: It seems that aerobic exercise plasma Apelin levels in male rats is not affected. On the other hand, nitric oxide inhibitors can reduce levels of plasma Apelin.

Keywords: aerobic training, L-NAME, plasma Apelin, male’s rats

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Authors: Swarnali Chakma, Akihiko Hokugo

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Research on three case studies reviewed here explains many aspects and complications of evacuation behavior during an emergency period. The scenario and phenomenon of the disaster were different, but the similarities are that after receiving the warning peoples does not take it seriously. Many individuals evacuated after taking some kind of action, for example; return to home, searching for family members, prepared valuable things etc. Based on a review of the literature, the data identified a number of factors that help explain evacuation behavior during the disaster. In the case of Japan, cultural inhibitors impact people’s behavior; for example, following the traffic rules, some people lost their time to skip because of the slow-moving car makes overcrowded traffic and some of them were washed away by the tsunami. In terms of Bangladeshi culture, women did not want to evacuate without men because staying men and women who do not know each other under the same roof together is not regular practice or comfortable. From these three case studies, it is observed that early warning plays an important role in cyclones, typhoons and earthquakes. A high level of trust from residents in the warning system is important to real evacuation. It is necessary to raise awareness of disaster and provide information on the vulnerability to cyclones, typhoons and earthquakes hazards at community levels. The local level may help decision makers and other stakeholders to make a better decision regarding an effective disaster management.

Keywords: disaster management, emergency period, evacuation, shelter, typhoon

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Authors: Yazun Jarrar

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Smoking has effect on platelet aggregation and the activity of anti-platelet drugs. The chemical 20-hydroxyeicosatetraenoic acid (20-HETE) is a cardiotoxic arachidonic acid metabolite which increases platelet aggregation. In this study, we investigated the influence of cigarette smoking on 20-HETE levels and protein expression of 20-HETE producing enzyme CYP4A11 in isolated platelets from smoker and non-smoker volunteers. The protein expression and 20-HETE levels were analyzed using immunoblot and High-Performance Liquid Chromatography with Mass Spectrometry (HPL-MS) assays. The results showed that 20-HETE level was higher significantly among smokers than non-smokers (t-test, p-value<0.05). The protein expression of CYP4A11 was significantly higher (t-test, p-value<0.05) among the platelets of smokers. We concluded that cigarette smoking increased the level of platelet activator 20-HETE through increasing the protein expression of CYP4A11. These findings may increase the understanding of smoking-drug interaction during antiplatelets therapy.

Keywords: smoking, 20-HETE, CYP4A11, platelet

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Authors: Husham Bayazed

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A growing number of studies indicate that atherosclerotic coronary artery disease (CAD) has a complex pathogenesis that extends beyond cholesterol intimal infiltration. The atherosclerosis process may involve an immune micro-environmental condition driven by local activation of the adaptive and innate immunity arrays, resulting in the formation of atherosclerotic plaques. Therefore, despite the wide usage of lipid-lowering agents, these devastating coronary diseases are not averted either at primary or secondary prevention levels. Many trials have recently shown an interest in the immune targeting of the inflammatory process of atherosclerotic plaques, with the promised improvement in atherosclerotic cardiovascular disease outcomes. This recently includes the immune-modulatory drug “Canakinumab” as an anti-interleukin-1 beta monoclonal antibody in addition to "Colchicine,” which's established as a broad-effect drug in the management of other inflammatory conditions. Recent trials and studies highlight the importance of inflammation and immune reactions in the pathogenesis of atherosclerosis and plaque formation. This provides an insight to discuss and extend the therapies from old lipid-lowering drugs (statins) to anti-inflammatory drugs (colchicine) and new targeted immune-modulatory therapies like inhibitors of IL-1 beta (canakinumab) currently under investigation.

Keywords: atherosclerotic plagues, immune microenvironment, lipid-lowering agents, and immunomodulatory drugs

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Authors: Tesfaye Alamirew, Delele Worku, Solomon W. Fanta, Nigus Gabbiye

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Recently graphen based nanocomposites are become an emerging research areas for fabrication of enzymatic biosensors due to their property of large surface area, conductivity and biocompatibility. This review summarizes recent research reports of graphen based nanocomposites for the fabrication of glucose and ethanol enzymatic biosensors. The newly fabricated enzyme free microwave treated nitrogen doped graphen (MN-d-GR) had provided highest sensitivity towards glucose and GCE/rGO/AuNPs/ADH composite had provided far highest sensitivity towards ethanol compared to other reported graphen based nanocomposites. The MWCNT/GO/GOx and GCE/ErGO/PTH/ADH nanocomposites had also enhanced wide linear range for glucose and ethanol detection respectively. Generally, graphen based nanocomposite enzymatic biosensors had fast direct electron transfer rate, highest sensitivity and wide linear detection ranges during glucose and ethanol sensing.

Keywords: glucose, ethanol, enzymatic biosensor, graphen, nanocomposite

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Authors: Aleksandra Twarda, Dobrosława Krzemień, Grzegorz Dubin, Tad A. Holak

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Stabilin-2 belongs to the group of scavenger receptors and plays a crucial role in clearance of more than 10 ligands from the bloodstream, including hyaluronic acid, products of degradation of extracellular matrix and metabolic products. The Link domain, a defining feature of stabilin-2, has a sequence similar to Link domains in other hyaluronic acid receptors, such as CD44 or TSG-6, and is responsible for most of ligands binding. Present knowledge of signal transduction by stabilin-2, as well as ligands’ recognition and binding mechanism, is limited. Until now, no experimental structures have been solved for any segments of stabilin-2. It has recently been demonstrated that the stabilin-2 knock-out or blocking of the receptor by an antibody effectively opposes cancer metastasis by elevating the level of circulating hyaluronic acid. Moreover, loss of expression of stabilin-2 in a peri-tumourous liver correlates with increased survival. Solving of the crystal structure of stabilin-2 and elucidation of the binding mechanism of hyaluronic acid could enable the precise characterization of the interactions in the binding site. These results may allow for designing specific small-molecule inhibitors of stabilin-2 that could be used in cancer therapy. To carry out screening for crystallization of stabilin-2, we cloned constructs of the Link domain of various lengths with or without surrounding domains. The folding properties of the constructs were checked by nuclear magnetic resonance (NMR). It is planned to show the binding of hyaluronic acid to the Link domain using several biochemical methods, i.a. NMR, isothermal titration calorimetry and fluorescence polarization assay.

Keywords: stabilin-2, Link domain, X-ray crystallography, NMR, hyaluronic acid, cancer

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Authors: Jagdish S. Patel, Piyush Chudasama

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Context: Over-expression of P-glycoprotein (P-gp) plays critical role in absorption of many drug candidates which results into lower bioavailability of the drug. P-glycoprotein also over expresses in many pathological conditions like diabetes, affecting the drug therapy. Modulation of P-gp expression using inhibitors can help in designing novel formulation enhancing the bioavailability of the drug in question. Objectives: The main focus of the study was to develop advanced glycation end products (AGEs) induced P-gp over expression in Caco-2 cells. Curcumin, piperine and epigallocatechin gallate were used to evaluate their P-gp inhibitory action using combinatorial approach. Materials and methods: Methylglyoxal (MG) induced P-gp over expression was checked in Caco-2 cells using real time PCR. P-gp inhibitory effects of the phytochemicals were measured after induction with MG alone and in combination of any two compounds. Cytotoxicity of each of the phytochemical was evaluated using MTT assay. Results: Induction with MG (100mM) significantly induced the over expression of P-glycoprotein in Caco-2 cells after 24 hr. Curcumin, piperine and epigallocatechin gallate alone significantly reduced the level of P-gp within 6 hr of treatment period monitored by real time PCR. The combination of any two phytochemical also down regulated the expression of P-gp in cells. Combinations of Curcumin and epigallocatechin gallate have shown significant down regulation when compared with other two combinations. Conclusions: Combinatorial approach for down regulating the expression of P-gp, in pathological conditions like diabetes, has demonstrated promising approach for therapeutic purpose.

Keywords: p-glycoprotein, curcumin, piperine, epigallocatechin gallate, p-gp inhibition

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Authors: Ayodeji O. Falade, Leonard V. Mabinya, Uchechukwu U. Nwodo, Anthony I. Okoh

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Given the high utility of peroxidase in several industrial processes, the search for novel microorganisms with enhanced peroxidase production capacity is of keen interest. This study investigated the process conditions for optimum peroxidase production by Ensifer sp, new ligninolytic proteobacteria with peroxidase production potential. Also, some agricultural residues were valorized for peroxidase production under solid state fermentation. Peroxidase production was optimum at an initial medium pH 7, incubation temperature of 30 °C and agitation speed of 100 rpm using alkali lignin fermentation medium supplemented with guaiacol as the most effective inducer and ammonium sulphate as the best inorganic nitrogen. Optimum peroxidase production by Ensifer sp. was attained at 48 h with specific productivity of 12.76 ± 1.09 U mg⁻¹. Interestingly, probable laccase production was observed with optimum specific productivity of 12.76 ± 0.45 U mg⁻¹ at 72 h. The highest peroxidase yield was observed with sawdust as solid substrate under solid state fermentation. In conclusion, Ensifer sp. possesses the capacity for enhanced peroxidase production that can be exploited for various biotechnological applications.

Keywords: catalase-peroxidase, enzyme production, peroxidase, polymerase chain reaction, proteobacteria

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Authors: Osama Sassi, Manal Eltorki, Iftikhar Ahmad

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Poor integrity is one of the major causes of leaks and accidents in gas transmission pipelines. To ensure safe operation, it is must to have efficient and effective pipeline integrity management (PIM) system. The corrosion management is one of the important aspects of successful pipeline integrity management program together design, material selection, operations, risk evaluation and communication aspects to maintain pipelines in a fit-for-service condition. The objective of a corrosion management plan is to design corrosion mitigation, monitoring, and inspection strategy, and for maintenance in a timely manner. This paper presents the experience of corrosion management of a gas transmission pipeline from Wafa field to Mellitah gas plant in Libya. The pipeline is 525.5 km long and having 32 inches diameter. It is a buried pipeline. External corrosion on pipeline is controlled with a combination of coatings and cathodic protection while internal corrosion is controlled with a combination of chemical inhibitors, periodic cleaning and process control. The monitoring and inspection techniques provide a way to measure the effectiveness of corrosion control systems and provide an early warning when changing conditions may be causing a corrosion problem. This paper describes corrosion management system used in Mellitah Oil & Gas BV for its gas transmission pipeline based on standard practices of corrosion mitigation and inspection.

Keywords: corrosion mitigation on gas transmission pipelines, pipeline integrity management, corrosion management of gas pipelines, prevention and inspection of corrosion

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Authors: Debora B. Moretti, Wiolene M. Nordi, Thaline Maira P. Cruz, José Eurico P. Cyrino, Raul Machado-Neto

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Enzyme activity was evaluated in the intestine of juvenile dourado (Salminus brasiliensis) fed with diets containing 0, 10 or 20% of lyophilized bovine colostrum (LBC) inclusion for either 30 or 60 days. The intestinal enzymes acid and alkaline phosphatase (ACP and ALP, respectively), non-specific esterase (NSE), lipase (LIP), dipeptidyl aminopeptidase IV (DAP IV) and leucine aminopeptidase (LAP) were studied using histochemistry in four intestinal segments (S1, S2, S3 and posterior intestine). Weak proteolitic activity was observed in all intestinal segments for DAP IV and LAP. The activity of NSE and LIP was also weak in all intestines, except for the moderate activity of NSE in the S2 of 20% LBC group after 30 days and in the S1 of 0% LBC group after 60 days. The ACP was detected only in the S2 and S3 of the 10% LBC group after 30 days. Moderate and strong staining was observed in the first three intestinal segments for ALP and weak activity in the posterior intestine. The activity of DAP IV, LAP and ALP were also present in the cytoplasm of the enterocytes. In the present results, bovine colostrum feeding did not cause alterations in activity of intestinal enzymes.

Keywords: carnivorous fish, enterocyte, intestinal epithelium, teleost

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Authors: Rashad Al-Hindi

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The study aimed at investigating and screening of some contaminants in some honey-based products. Sixty-nine 'honey paste' samples marketed in Jeddah, Saudi Arabia, were subjected to physicochemical studies and screening of aflatoxins and pesticide residues. The physicochemical parameters studied were mainly: moisture content, total sugars, total ash, total nitrogen, fibres, total acidity as citric acid and pH. These parameters were investigated using standard methods of analysis. Mycotoxins (aflatoxins) and pesticide residues were by an enzyme-linked immunosorbent assay (ELISA) according to official methods. Results revealed that mean values of the examined criteria were: 15.44±0.36%; 74±4.30%; 0.40±0.062%; 0.22±0.05%; 6.93±1.30%; 2.53±0.161 mmol/kg; 4.10±0.158, respectively. Overall results proved that all tested honey pastes samples were free from mycotoxins (aflatoxins) and pesticide residues. Therefore, we conclude that 'honey pastes' marketed in Jeddah city, Saudi Arabia were safe for human consumption.

Keywords: aflatoxins, honey mixtures, pesticide residues, physicochemical

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Authors: Biswabandhu Bankura, Srikanta Guria, Madhusudan Das

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Purpose: Thyroid peroxidase (TPO) is the key enzyme in the biosynthesis of thyroid hormones. We aimed to identify the spectrum of mutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of the disease. Methods: 200 hypothyroid patients (case) and their corresponding sex and age matched 200 normal individuals (control) were screened depending on their clinical manifestations. Genomic DNA was isolated from peripheral blood samples and TPO gene (Exon 7 to Exon 14) was amplified by PCR. The PCR products were subjected to sequencing to identify mutations. Results: Single nucleotide changes such as Glu 641 Lys, Asp 668 Asn, Thr 725 Pro, Asp 620 Asn, Ser 398 Thr, and Ala 373 Ser were found. Changes in the TPO were assayed in vitro to compare mutant and wild-type activities. Five mutants were enzymatically inactive in the guaiacol and iodide assays. This is a strong indication that the mutations are present at crucial positions of the TPO gene, resulting in inactivated TPO. Key Findings: The results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population of West Bengal.

Keywords: thyroid peroxidase, hypothyroidism, mutation, in vitro assay, transfection

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Authors: Biswabandhu Bankura, Srikanta Guria, Madhusudan Das

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Purpose: Thyroid peroxidase (TPO) is the key enzyme in the biosynthesis of thyroid hormones. We aimed to identify the spectrum of mutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of the disease. Methods: 200 hypothyroid patients (case) and their corresponding sex and age matched 200 normal individuals (control) were screened depending on their clinical manifestations. Genomic DNA was isolated from peripheral blood samples and TPO gene (Exon 7 to Exon 14) was amplified by PCR. The PCR products were subjected to sequencing to identify mutations. Results: Single nucleotide changes such as Glu 641 Lys, Asp 668 Asn, Thr 725 Pro, Asp 620 Asn, Ser 398 Thr, and Ala 373 Ser were found. Changes in the TPO were assayed in vitro to compare mutant and wild-type activities. Five mutants were enzymatically inactive in the guaiacol and iodide assays. This is a strong indication that the mutations are present at crucial positions of the TPO gene, resulting in inactivated TPO. Key Findings: The results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population of West Bengal.

Keywords: thyroid peroxidase, hypothyroidism, mutation, in vitro assay, transfection

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Authors: Sevil Arabaci Tamer, Gonul Gurol, Ibrahim Tekeoglu, Halil Harman, Ihsan Hakki Ciftci

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Neuroserpin (NSP) is a serine protease inhibitor and member of the serpin family. It is expressed in developing and adult nervous systems, and acts as an inhibitor of protease tissue plasminogen activator (tPA) and a regulator of neuronal growth and plasticity. Also NSP displays anti-inflammatory activity. But, its role in rheumatoid arthritis had never been studied before. So, the aim of the present study was to investigate the effect of neuroserpin in patients with RA. A total of 50 frozen (-20 ºC) serum samples 40 of them belonged to patients with RA, and 10 sample belonged to healthy subjects, were enrolled prospectively. We used DAS-28 to evaluate disease activity. The following clinical data gathered from the original patients' charts. Serum neuroserpin levels were measured by enzyme-linked immunosorbent assay. Our preliminary study results demonstrate, for the first time, that NSP levels are significantly different in RA patients relative to healthy subjects (P = 0.014). So, NSP contribute to pathological condition of RA. Thus, we believe that serum NSP levels can be as a marker in patients with RA. However other inflammatory diseases should be further investigated.

Keywords: neuroserpin, rheumatoid arthritis, tPA, tPA inhibitor

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Authors: Yeng Min Yi, Rosli Md Illias, Salehhuddin Hamdan

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Industrial biocatalysis is mainly based on the use of cell free or intracellular enzyme systems. However, the expensive cost and relatively lower operational stability of free enzymes limit practical use in industries. Cell surface display system can be used as a cost-efficient alternative to overcome the laborious purification and substrate transport limitation. In this research, TibA autotransporter from E. coli was used to display Aspergillus fumigatus xylanase (xyn). The amplified xyn was fused in between N-terminal signal peptide and C-terminal β-barrel of TibA. The cloned was transformed and expressed in E. coli BL21 (DE3). Outer membrane localization of TibA-xyn fusion protein was confirmed by SDS PAGE and western blot with expected size of 62.5 kDa. Functional display of xyn was examined by activity assay. Cell surface displayed xyn exhibited the highest activity at 37 °c, 0.3 mM IPTG. As a summary, TibA displaying system has the potential for further industrial applications. Moreover, this is the first report of the display of xylanase using TibA on the surface of E. coli.

Keywords: biocatalysis, cell surface display, Escherichia coli, TibA autotransporter

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Authors: H. Noureddine, P. Rao, R. Guru, A. Chandratreya

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The nice guidance for elective total knee replacements states that patients should be given mechanical thrombo-prophylaxis, and if no contraindications chemical thromboprophylaxis in the form of Dabigatran etexilate, Rivaroxiban, UFH, LMWH, or Fondaparinux sodium (CG92, 1.5.14, January 2010). In Practice administering oral agents has been the dominant practice as it reduces the nursing needs, and shortens hospital stay and is generally received better by patients. However, there are well documented associated bleeding risks, and their effects are difficult to reverse in case of major bleeding. Our experience with oral factor 10 inhibitors used for thromboprophylaxis was marked with several patients developing complications necessitating return to the theatre for wound washouts. This has led us to try a different protocol for thromboprophylaxis that we applied on our patients undergoing total and unicondylar knee replacements. We applied mechanical thromboprophylaxis in the form of intermittent pneumatic pressure devices, and chemical thromboprophylaxis in the form of a dose of prophylactic LMWH pre-op, then 150 mg of Aspirin to start 24 hours after the surgery and to continue for 6 weeks, alongside GI cover with PPIs or antihistamines. We also administered local anaesthetics intra-operatively in line with the ERAS protocol thus encouraging early mobilization. We have identified a cohort of 133 patients who underwent one of the aforementioned procedures in the same trust, and by the same surgeon, where this protocol was applied and examined their medical notes retrospectively with a mean follow-up period of 14 months, to identify the rate and percentage of patients who had thrombo-embolic events in the post-operative period.

Keywords: aspirin, heparin, knee arthroplasty, thromboprophylaxis

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Authors: Hosein Maghsoudi

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Rheumatois arthritis (RA) is progressive inflammatory autoimmune diseases that primarily affect the joints, characterized by synovial hyperplasia and inflammatory cell infiltration, deformed and painful joints, which can lead tissue destruction, functional disability systemic complications, and early dead and socioeconomic costs. The cause of rheumatoid arthritis is unknown, but genetic and environmental factors are contributory and the prognosis is guarded. However, advances in understanding the pathogenesis of the disease have fostered the development of new therapeutics, with improved outcomes. The current treatment strategy, which reflects this progress, is to initiate aggressive therapy soon after diagnosis and to escalate the therapy, guided by an assessment of disease activity, in pursuit of clinical remission. The pathobiology of RA is multifaceted and involves T cells, B cells, fibroblast-like synoviocyte (FLSc) and the complex interaction of many pro-inflammatory cytokine. Novel biologic agents that target tumor necrosis or interlukin (IL)-1 and Il-6, in addition T- and B-cells inhibitors, have resulted in favorable clinical outcomes in patients with RA. Despite this, at least 30% of RA patients are résistance to available therapies, suggesting novel mediators should be identified that can target other disease-specific pathway or cell lineage. Among the inflammatory cell population that might participated in RA pathogenesis, FLSc are crucial in initiaing and driving RA in concert of cartilage and bone by secreting metalloproteinase (MMPs) into the synovial fluid and by direct invasion into extracellular matrix (ECM), further exacerbating joint damage. Invasion of fibroblast-like synoviocytes (FLSc) is critical in the pathogenesis of rheumatoid-arthritis. The metalloproteinase (MMPs) and activator of Toll-like receptor 4 (TLR4)/nuclear factor- κB pthway play a critical role in RA-FLS invasion induced by lipopolysaccharide (LPS). The present study aimed to explore the anti-invasion activity of Glycyrrhizic Acid as a pharmacologically safe phytochemical agent with potent anti-inflammatory properties on IL-1beta and TNF-alpha signalling pathways in Bovine fibroblast-like synoviocyte ex- vitro, on LPS-stimulated bovine FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Results showed that Glycyrrhizic Acid suppressed LPS-stimulated bovine FLS migration and invasion by inhibition MMP-9 expression and activity. In addition our results revealed that Glycyrrhizic Acid inhibited the transcriptional activity of MMP-9 by suppression the nbinding activity of NF- κB in the MMP-9 promoter pathway. The extract of licorice (Glycyrrhiza glabra L.) has been widely used for many centuries in the traditional Chinese medicine as native anti-allergic agent. Glycyrrhizin (GL), a triterpenoidsaponin, extracted from the roots of licorice is the most effective compound for inflammation and allergic diseases in human body. The biological and pharmacological studies revealed that GL possesses many pharmacological effects, such as anti-inflammatory, anti-viral and liver protective effects, and the biological effects, such as induction of cytokines (interferon-γ and IL-12), chemokines as well as extrathymic T and anti-type 2 T cells. GL is known in the traditional Chinese medicine for its anti-inflammatory effect, which is originally described by Finney in 1959. The mechanism of the GL-induced anti-inflammatory effect is based on different pathways of the GL-induced selective inhibition of the prostaglandin E2 production, the CK-II- mediated activation of both GL-binding lipoxygenas (gbLOX; 17) and PLA2, an anti-thrombin action of GL and production of the reactive oxygen species (ROS; GL exerts liver protection properties by inhibiting PLA2 or by the hydroxyl radical trapping action, leading to the lowering of serum alanine and aspartate transaminase levels. The present study was undertaken to examine the possible mechanism of anti-inflammatory properties GL on IL-1beta and TNF-alpha signalling pathways in bovine fibroblast-like synoviocyte ex-vivo, on LPS-stimulated bovine FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Our results clearly showed that treatment of bovine fibroblast-like synoviocyte with GL suppressed LPS-induced cell migration and invasion. Furthermore, it revealed that GL inhibited the transcription activity of MMP-9 by suppressing the binding activity of NF-κB in the MM-9 promoter. MMP-9 is an important ECM-degrading enzyme and overexpression of MMPs in important of RA-FLSs. LPS can stimulate bovine FLS to secret MMPs, and this induction is regulated at the transcription and translational levels. In this study, LPS treatment of bovine FLS caused an increase in MMP-2 and MMP-9 levels. The increase in MMP-9 expression and secretion was inhibited by ex- vitro. Furthermore, these effects were mimicked by MMP-9 siRNA. These result therefore indicate the the inhibition of LPS-induced bovine FLS invasion by GL occurs primarily by inhibiting MMP-9 expression and activity. Next we analyzed the functional significance of NF-κB transcription of MMP-9 activation in Bovine FLSs. Results from EMSA showed that GL suppressed LPS-induced NF-κB binding to the MMP-9 promotor, as NF-κB regulates transcriptional activation of multiple inflammatory cytokines, we predicted that GL might target NF-κB to suppress MMP-9 transcription by LPS. Myeloid differentiation-factor 88 (MyD88) and TIR-domain containing adaptor protein (TIRAP) are critical proteins in the LPS-induced NF-κB and apoptotic signaling pathways, GL inhibited the expression of TLR4 and MYD88. These results demonstrated that GL suppress LPS-induced MMP-9 expression through the inhibition of the induced TLR4/NFκB signaling pathway. Taken together, our results provide evidence that GL exerts anti-inflammatory effects by inhibition LPS-induced bovine FLSs migration and invasion, and the mechanisms may involve the suppression of TLR4/NFκB –mediated MMP-9 expression. Although further work is needed to clarify the complicated mechanism of GL-induced anti-invasion of bovine FLSs, GL might be used as a further anti-invasion drug with therapeutic efficacy in the treatment of immune-mediated inflammatory disease such as RA.

Keywords: glycyrrhizic acid, bovine fibroblast-like synoviocyte, tlr4/nf-κb, metalloproteinase-9

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Authors: Hatem Shreha

Abstract:

Aromatase inhibitors administered before sexual differentiation of the gonads can induce sex reversal in female chickens (phenotypic male). To analyze the process of sex reversal, we have followed for several months the changes induced by Fadrozole, a nonsteroidal aromatase inhibitor on the morphology of female sex-reversed and female sex-reversed supplemented with L-tyrosine which was previously shown to stimulate the release of Gn Rh. Fadrozole (1mg/egg) was injected into eggs on day four of incubation, phenotypic males and phenotypic males treated with L-tyrosine and males hatched from eggs injected Fadrozole were sacrificed by slaughtering at 16 weeks old and the remaining chicks were sacrificed at 28 weeks old. Both sexes from control chickens were sacrificed at the same age (16 &28 weeks). Hatchability, behavior, body weight, shank length, comb weight, testes weight, blood cells count and wattle weight of sex reversal were tested at 16 and 28 weeks. The results showed that body weight, comb weight, wattles weight and shank length of sex-reversed females were significantly different from control female. Behavior of phenotypic males and phenotypic males fed on L- tyrosine showed aggressive sexual behavior like that of control males and absence of laying behavior. In conclusion our results confirm that Fedrazole injection in eggs before sex differentiation produce a male behavior and morphological index of male in female chicken.

Keywords: sex reversal, fadrozole, phenotypic male, L- tyrosine

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Authors: A. A. Al-Saleh, E. A. Ismail, A. A. Metwalli

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Effects of camel and cow casein hydrolysates by trypsin enzyme on rheological and sensory properties and growth of starter culture of the yoghurts made from cow milk have been investigated. The hydrolysates strongly decreased the fermentation and coagulation time of the yoghurts. The rate of pH decrease was higher with camel casein hydrolysate in comparison with cow casein hydrolysate at all concentrations used (0.5; 1.0 and 1.5%). Viscosities of the yoghurt made with hydrolysates significantly (p<0.05) decreased compared to control samples. The addition of the hydrolysates significantly (p <0.05) increased the hardness and adhesiveness of the yoghurts. No significant differences in water holding capacity of control and treated samples were obsereved at 0.5 and 1.0% casein hydrolysate addition. However, increasing casein hydrolysate addition to 1.5% decreased water holding capacity of yoghurt samples. The sensory evaluation scores of the yoghurts were significantly (p<0.05) improved with the addition of casein hydrolysates.

Keywords: yoghurt, camel casein hydrolysates, cow casein hydrolysate, sensory evaluation

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Authors: Nermin Isik, Ozlem Derinbay Ekici, Oguzhan Avci

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The aim of this study was to determination of role infection in neonatal calves in Central Anatolian, Turkey. A total 300 fecal samples were collected from diarrheic neonatal calves, aged between 0–90 days from Konya, Karaman, and Aksaray from January to April 2014. Fecal specimens from calves with clinically diarrheic symptoms were examined for the presence of Bovine Coronavirus, Bovine Rotavirus, Cryptosporidium sp., and E. coli by commercially available capture direct enzyme linked immunosorbent assay (ELISA) kit and Modified Ziehl Neelsen method (MZN). Calves were grouped according to their age as follows: 1-14, 15-29, and 30-90 days. Cryptosporidium sp. infection was detected in 52.8%, 58.8%, and 39.2% by ELISA and 33.9%, 47%, 26.7% by MZN in the respective age groups. The seroprevalance of Rotavirus (12.5 %, 40 %, 12.5 %), Coronavirus (2.5%, 0%, 3.5%) and E. coli (5%, 4.7%, 8.9%) infections were determined according to the age groups respectively. Cryptosporidium sp. was the most detected enteropathogen (52 %) of calves and coronavirus was the least detected (2 %). The detection rate of the mixed enfection was 12.3%. In conclusion, it must be evaluated by mix infections in calves with diarrhea. These results will provide an important contribution against the factors that cause diarrhea

Keywords: cryptosporidium sp., bovine coronavirus, bovine rotavirus, E.coli, calve, ELISA

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Authors: A. M. Gadanya, B. A. Ahmad, U. Maigatari

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Effect of oral administration of Gadagi tea on superoxide dismutase activity was assessed on twenty (20) male subjects (aged 21-40years). Ten (10) male non Gadagi tea consumers (aged 20-26 years), were used as control. Blood samples were collected from the subjects and analysed for serum superoxide dismutase activity using R&D Enzyme Linked Immunosorbent Assay method (ELISA). The subjects were grouped into four based on age i.e group I (21-25 years), group II (26-30 years), and also based on duration of the tea consumption, i.e group A (5-9 years) , group B (10-14 years), group C (15-19 years) and group D (20-24 years). The subjects in group I (0.12 U mg-l +0.05), group II (0.11 U mg-l +0.01), group III (0.14 U mg-l +0.08) and group IV (0.17 U mg-l +0.11) showed increased activity of serum superoxide dismutase when compared with the control subjects (0.88 U mg-l +0.02) (P<0.05). There was no statistical significant difference in superoxide dismutase activity within the case groups (P<0.05), based on age and duration of consumption of the tea. Thus, Gadagi tea consumption could increase serum superoxide dismutase activity in humans.

Keywords: “Gadagi” tea, Serum, Superoxide dismutase, Humans.

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Authors: Ali Bayram, Burak Uz, Remzi Yiğiter

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The impairment of lipid metabolism in the central nervous system has been suggested as a critical factor of Alzheimer’s disease (AD) pathogenesis. Homo sapiens acyl-coenyme A synthetase short-chain family member 2 (ACSS2) gene encodes the enzyme acetyl-Coenzyme A synthetase (AMP forming; AceCS) providing acetyl-coenzyme A (Ac-CoA) for various physiological processes, such as cholesterol and fatty acid synthesis, as well as the citric acid cycle. We investigated ACSS2, transcript variant 1 (ACSS2*1), mRNA levels in the peripheral blood mononuclear cells (PBMC) of patients with AD and compared them with the controls. The study group comprised 50 patients with the diagnosis of AD who have applied to Gaziantep University Faculty of Medicine, and Department of Neurology. 49 healthy individuals without any neurodegenerative disease are included as controls. ACSS2 mRNA expression in PBMC of AD/control patients was 0.495 (95% confidence interval: 0.410-0.598), p= .000000001902). Further studies are needed to better clarify this association.

Keywords: Alzheimer’s disease, ACSS2 Genes, mRNA expression, RT-PCR

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Authors: Nada Alqadri, Omaima Nasir

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Natural compounds are an important source of potential inhibitors; they have a lot of pharma potential with less adverse effects. The effective antiviral activities of natural products have been proved in different studies. The outbreak of COVID-19 in Wuhan, Hubei, in December 2019, coronavirus has had a significant impact on people's health and lives. Based on previous studies, natural products can be introduced as preventive and therapeutic agents in the fight against COVID-19; considering that no food or supplement has been authorized to prevent COVID-19, individuals continue to search for and consume specific herbs, foods, and commercial supplements for this purpose. This study will be aimed to estimate the uses of herbal and natural products during the COVID-19 infection to determine their usage reasons and evaluate their potential side effects. An online cross-sectional survey of different participants will be conducted and will be a focus on respondents’ chronic disease histories, socio-dmographic characteristics, and frequency and trends of using these products. Descriptive and univariate analyses will be performed to determine prevalence and associations between various products used and respondents’ socio-demographic data. Relationships will be tested using Pearson’s chi-square test or an exact probability test. Our main findings will give evidence of beneficial uses of natural products and herbal medicine as prophylactic and will be a vigorous approach to stop or at least slow down COVID-19 infection and transmission. This will be of great interest of public health, and the results of our study will lend health officials better control on the current pandemic.

Keywords: COVID-19, herbs, natural products, saudi arabia

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Authors: Kasim Sakran Abass

Abstract:

This main aim of this study was to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There were significantly higher esterases activities in dilution 1:10 in all blood samples from quail, duck, and chick compared to other dilutions (1:5, 1:15, 1:20, and 1:25). Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration–inhibition curves were determined for the inhibitor in the presence of dilutions 1:5, 1:10 plus 1:15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Among the thiol esters (dilution 1:5) was observed to have the highest specificity constant (kcat/Km), and the Km and kcat values were 176 μM and 16,765 s−1, respectively for S-phenyl thioacetate ester, while detected in (dilution 1:15) the lowest specificity constant (kcat/Km), and the Km and kcat values were 943 μM and 1154 s−1, respectively for acetylthiocholine iodide ester.

Keywords: esterase, animal, dilution, pesticides

Procedia PDF Downloads 519