Ameliorating reactive oxygen species-induced <Emphasis Type="Italic">in vitro</Emphasis> lipid peroxidation in brain,liver, mitochondria and DNA damage by <Emphasis Type="Italic">Zingiber officinale</Emphasis> Roscoe

Iron is an essential nutrient for a number of cellular activities. However, excess cellular iron can be toxic by producing reactive oxygen species (ROS) such as superoxide anion (O₂⁻) and hydroxyl radical (HO^·) that damage proteins, lipids and DNA. Mutagenic and genotoxic end products of lipid peroxidation can induce the decline of mitochondrial respiration and are associated with various human ailments including aging, neurodegenerative disorders, cancer etc. Zingiber officinale Roscoe (ginger) is a widely used spice around the world. The protective effect of aqueous ethanol extract of Z. officinale against ROS-induced in vitro lipid peroxidation and DNA damage was evaluated in this study. The lipid peroxidation was induced by hydroxyl radical generated from Fenton’s reaction in rat liver and brain homogenates and mitochondrial fraction (isolated from rat liver). The DNA protection was evaluated using H₂O₂-induced changes in pBR-322 plasmid and Fenton reaction-induced DNA fragmentation in rat liver. The results indicated that Z. officinale significantly (P<0.001) protected the lipid peroxidation in all the tissue homogenate/mitochondria. The extract at 2 and 0.5 mg/ml could protect 92 % of the lipid peroxidation in brain homogenate and liver mitochondria respectively. The percent inhibition of lipid peroxidation at 1mg/ml of Z. officinale in the liver homogenate was 94 %. However, the extract could partially alleviate the DNA damage. The protective mechanism can be correlated to the radical scavenging property of Z. officinale. The results of the study suggest the possible nutraceutical role of Z. officinale against the oxidative stress induced human ailments.     

Kinetics of Inhibition of Monoamine Oxidase Using <Emphasis Type="Italic">Cymbopogon martinii</Emphasis> (Roxb.) Wats.: A Potential Antidepressant Herbal Ingredient with Antioxidant Activity

The study was designed to evaluate the antioxidant activity and effect of Cymbopogon martinii (Roxb.) Wats. (Poaceae) leaves on the activity of monoamine oxidase and kinetics of enzyme inhibition. Ethanol extract of C. martinii and rat brain mitochondrial monoamine oxidase preparation ware used to study the kinetics of enzyme inhibition using double reciprocal Lineweaver–Burk plot. The DPPH was used as a source of free radical to evaluate antioxidant potential. It is observed that, the ethanolic extract of C. martinii inhibits the monoamine oxidase activity with competitive mode of inhibition. The V _max (0.01 mM/min) remained constant while, K _m varied from 21.00 ± 1.1, 43.33 ± 1.5 and 83.33 ± 1.4 mM for 100–500 μg/ml concentration of C. martinii. The K _i values were calculated to be 90.00 ± 0.87, 75.00 ± 0.69, 68.18 ± 0.68 μg for 100–500 μg/ml concentration of C. martini. It also shows a significant DPPH (1,1-diphenyl-2-picryl hydrazine) radical scavenging (IC₅₀ = 0.34 ± 0.05 mg/ml) and reducing activity (IC₅₀ = 0.70 ± 0.22 mg/ml). The C. martini can be considered as a possible source of MAO inhibitor used in the treatment of depression and other neurological disorders.     

Evaluation of antioxidant profile and activity of amalaki (Emblica officinalis), spirulina and wheat grass

Aqueous and alcoholic extracts of amalki (Emblica officinalis), spirulina and wheatgrass were prepared and analyzed for antioxidant vitamin content (vitamin C and E), total phenolic compounds. Antioxidant status, reducing power and effect on glutathione S-transferase (GST) activity were evaluated in vitro. Vitamin C content of crude amalaki powder was found to be 5.38 mg/g, while very less amount 0.22 mg/g was detected in wheat grass. Amalki was rich in vitamin E like activity, total phenolic content, reducing power and antioxidant activity. Total antioxidant activity of aqueous extract of amalki, spirulina and wheat grass at 1mg/ml concentration were 7.78, 1.33 and 0.278 mmol/l respectively. At similar concentrations the total antioxidant activity of alcoholic extract of amalaki, spirulina and wheat grass was 6.67, 1.73 and 0.380 mmol/l respectively. Amalki was also found to be rich source of phenolic compounds (241mg/g gallic acid equivalent). Alcoholic extract of wheat grass showed 50 % inhibition in FeCl₂- ascorbic acid induced lipid peroxidation of rat liver homogenates in vitro. Both aqueous and alcoholic extracts of amalaki inhibited activity of rat liver glutathione S-transferase (GST) in vitro in dose dependant manner. Since GST acts as powerful drug metabolizing enzyme its inhibition by amalaki offers possibility of its use for lowering therapeutic dose of herbal preparations. The aqueous extracts of both amalki and spirulina also showed protection against t-BOOH induced cytotoxicity and production of ROS in cultured C₆ glial cells.     

Thyroidal regulation of substrate kinetics properties of cytochrome oxidase in rat liver mitochondria

Effects of thyroidectomy (T_x) and subsequent treatment with 3,5,3’-triiodothyronine (T₃), and combined treatment (T_R) with T₃ + thyroxine (T₄) on substrate kinetics properties of cytochrome oxidase of rat liver mitochondria were examined. T_x resulted in lowering of cytochromes content with decrease in the enzyme activity, and K_m and V_max. T₃ and T_R regimens restored the cytochromes contents and the V_max values to normal. In control, T₃ and T_R groups the enzyme activity resolved in two kinetic components; in T_x group three kinetic components were evident. The K_m values for all components decreased significantly in the experimental groups with concomitant increase in catalytic efficiency, K_cat/K_m. Significant alterations in the contents of total phospholipid and of cholesterol were noted while the changes in the phospholipids composition were only of restricted nature. Regression analysis revealed that total phospholipid, cholesterol and phosphatidylcholine, phosphatidylethanolamine play significant role in fine tuning the enzyme activity.     

Differential pH sensitivity of tissue superoxide dismutases

Superoxide dismutase (SOD) activities in the human and rat RBCs and rat liver, kidney, brain and heart mitochondria as well as cytosolic fractions were determined by the pyrogallol assay procedure with slight modifications. Measurements were carried out in 0.1 M potassium phosphate buffer pH 8.0 and 9.2 to assess the pH stability of the SODs from various systems. Under these conditions the SODs from different systems including RBCs exhibited differential pH stability i.e. they displayed differential susceptibility at pH 9.2. Even in a given tissue, the mitochondrial and cytosolic SODs contents show a tissue-specific pattern. Our results also suggest that measurements carried out at pH 8.0 may give more realistic estimates of SOD activities.     

Effect of streptozotocin-induced diabetes on oxidative energy metabolism in rat liver mitochondria—A comparative study of early and late effects

The reports in the literature on effects of diabetes on mitochondrial energy-linked functions are conflicting. Hence we carried out systematic studies to evaluate the effects at the early and the late stages of the disease using STZ-diabetic rat as a model. At the end of one week, after induction of diabetes, respiration rates with glutamate and succinate as the substrates increased; respiration rates with other substrates e.g. β-hydroxybutyrate, pyruvate + malate and ascorbate + TMPD were not affected despite substantial decrease in the β-hydroxybutyrate dehydrogenase activity and cytochrome b and c+c₁ contents. Insulin treatment brought about increase in the cytochrome contents beyond control values. The ATPase activity was generally low in the diabetic animals and was not restored by insulin treatment. At the end of one month, the respiratory activities with all the substrates were generally low. Insulin treatment either restored or stimulated the respiration rates beyond control values. The content of cytochromes was differentially affected in the diabetic animals, but insulin treatment caused significant increase beyond control levels. The pattern for ATPase activity was similar to the early effects. At both the stages i.e. early and late stages of diabetes the mitochondria were tightly coupled. The ADP/O ratios were in normal expected ranges and the respiratory control ratios were comparable with the control groups. Insulin treatment resulted in apparent restoration of respiratory activity. However, the effects on the cytochromes and dehydrogenases activities were differential. Taken together the two observations would suggest that the mitochondria were not re-instated to normality despite apparent restoration of respiratory function.     

Method evaluation study of a new generation of vitamin D assays

Introduction

Recently several diagnostic manufacturers have launched new 25-hydroxy-vitamin D (25[OH]D) assays, which are aligned to the National Institute of Standards and Technology (NIST) Standard Reference Materials (SRM) (NIST, Gaithersburg, Maryland). The aim of this study was to compare the performance of one liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, one enzyme linked immunosorbent assay (ELISA), and one recalibrated and previous version of a chemiluminescence immunoassay (CLIA).

Material and methods

Serum-aliquots of 198 patient samples from routine 25(OH)D analysis were measured by the ClinMass® LC-MS/MS Complete Kit (RECIPE Chemicals + Instruments GmbH, Munich, Germany), the ORGENTEC 25(OH)D₃/D₂ ELISA (ORGENTEC Diagnostika GmbH, Mainz, Germany), the recalibrated Immunodiagnostic Systems (IDS)-iSYS 25(OH)D^S and the previous used IDS-iSYS 25(OH)D CLIA (Immunodiagnostic Systems Ltd, Boldon, United Kingdom). Bland-Altman and Deming regression analyses were calculated for methods comparison of all tested 25(OH)D assays. The LC-MS/MS method was defined as the reference method. Within-run and between-run precision measurements were performed for all methods with three different concentration levels.

Results

Compared to the LC-MS/MS method, the new IDS-iSYS 25(OH)D^S and ORGENTEC 25(OH)D₃/D₂ assay demonstrated mean relative biases of 16.3% and 17.8%. The IDS-iSYS 25(OH)D assay showed the lowest mean bias of 1.5%. Deming regression analyses of the recalibrated IDS-iSYS 25(OH)D^S and the ORGENTEC 25(OH)D₃/D₂ assay showed proportional differences, when compared to the reference method. All assays showed a within-run and between-run imprecision of ≤ 20% at each of the evaluated concentration levels.

Conclusions

The evaluated standardized immunoassays and LC-MS/MS are useful methods for measuring 25(OH)D serum-levels in clinical laboratories.Key words: vitamin D, immunoassays, liquid chromatography-tandem mass spectrometry, reference standards, quality improvement     

A Simple Economical Method for Assay of Atherogenic Small Dense Low-Density Lipoprotein-Cholesterol (sdLDL-C)

In the present study, we report a simple and economical precipitation method for the quantitative determination of small, dense LDL-cholesterol (sdLDL-C) in serum that is considered to be an emerging risk factor for cardiovascular disease. This method consisted of precipitation of lipoproteins of density <1.044 g/ml using heparin-MnCl₂ and quantification of cholesterol existed in the supernatant using reagents for routine cholesterol assay instead of the costly direct low density lipoprotein-cholesterol assay kit. The supernatant contained sdLDL and high-density lipoprotein (HDL) that was confirmed by polyacrylamide gel electrophoresis. sdLDL-C concentration can be calculated by subtracting the HDL-C value from the total cholesterol concentration of the supernatant. sdLDL-C values obtained by this modified method were similar to those obtained by direct assay of sdLDL-C and there was significant correlation between the two methods. In conclusion, this method is highly economical, do not require special equipments and is useful to evaluate atherogenic risk.     

Comparative evaluation of ultramicro—and macro-chemo enzyme based assays of glucose, cholesterol and triglycerides

A comparison of the absorbance, enzyme/substrate concentration, reaction efficiency and sensitivity has been made for enzyme-based clinical chemistry assays, using a conventional colorimeter versus a strip-microwell reader, in order to establish the value of ultra-microchemical procedure, with reaction volume 87 μl (light path length=0.25 cm). By utilizing commercial kits available for the quantitation of serum glucose, cholesterol and triglycerides, it has been established that the micro method is highly cost effective (9–30 fold), reproducible and sensitive. Comparison of blood drawn by a finger prick (capillary) and venipuncture for normal and pathological specimens show reproducibility between different laboratory technologists and in reference with the values reported by an accredited reference laboratory. Since the micro method uses very little serum, it is most suitable for analyses of small smaples, from large population-based field trials. However, the assay range has to be titrated for each commercial kit to establish the enzyme/substrate equivalence.     

An oxalate oxidase from grain sorghum leaf: Use in determination of urinary oxalate

An Oxalate oxidase (Oxalate: O₂ oxidoreductase, EC 1,2,3,4) has been purified to apparent homogeneity from leaves of 10-day old seedling plants of grain sorghum hybrid CSH-5. The enzyme exhibited maximum activity at pH 5.0 and 40°C. The rate of H₂O₂ formation was linear upto 2 min. The enzyme was strongly stimulated by Cu⁺⁺. The enzyme has greater resistance towards various cations and anions found in urine, compared to moss, barley, banana peel and bleet stem oxalate oxidases. This improved characteristic of the enzyme make it better suited for its use in the determination of urinary oxalate. A simple method of measuring oxalate in urine using this enzyme preparation is described.     

Immunomodulation of ovalbumin-specific IgG and other classes of antibody response by honey in mice

It was reported earlier that intraperitoneal administration of honey had immunosuppressive activity on elicitation of allergen-specific murine antibody response as evaluated by passive cutaneous anaphylaxis and double immunodiffusion methods. In this study, the immunomudulatory effect of honey is evaluated by enzyme linked immunosorbent assay (ELISA) using ovalbumin as model allergen. It was found that ovalbumin (OVA)-specific IgG antibody responses elicited with various doses of OVA were significantly suppressed by rock bee honey (p<0.01). Honey was also found to have inhibited the production of OVA-specific IgM, IgA, IgG₁, and IgG_2b whereas that of IgG_2a and IgG₃ were not affected. Furthermore, honey also suppressed the OVA-specific total IgG antibody response in various inbred mice with different genetic background. In addition, the suppressive activity of honey was examined in different groups of mice by injecting honey at different time intervals, before and after immunization with OVA. The anti-OVA IgG antibody response was suppressed significantly when honey was injected 12 hours prior/latter to OVA injection. These results confirm the suppressive activity of honey on antibody response and suggest possible clinical application.     

Purification and characterization of chymotrypsin-like enzyme from rat plasma

A chymotrypsin-like enzyme was purified from rat plasma, involving ammonium sulfate fractionation and chromatographgy on CM-sephadex and red sepharose. The purified enzyme effectively hydrolysed the ester substrates for chymotrypsin (N-acetyl L-tyrosine ethyl ester and N-acetyl L-tryptophan ethyl ester). The Km values for the two substrates were 2.2×10^?3M and 9.0×10^?3M respectively. The hydrolytic activity of the enzyme was inhibited by phenylmethyl sulfonyl fluoride and tosylphenylalanine chloromethylketone, suggesting the presence of serine and histidine at the active centre. The enzyme exhibited anionic nature and possessed a high molecular weight (MW 71,000) as observed by gel exclusion chromatography on Sephadex G-200. The enzyme was stable upon exposure to pH 7.0–9.0, but was inactivated upon heat treatment at 60°C for 5 min.     

Effect of buspirone: An anxiolytic drug on blood glucose in humans

The present study investigated the effect of an antianxiety drug, buspirone on blood glucose and plasma insulin level concerning the role of 5-HT_1A receptors in blood glucose regulation in healthy humans. Twelve healthy male volunteers were administered single oral doses of buspirone (10 mg) or placebo, in a randomized, crossover way, followed by oral glucose load (75 gm in 200 ml) at reported T_max i.e. the time of peak plasma concentration of the respective administered drug. The blood samples were collected as predose, postdose and post oral glucose load at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hr to investigate the effect of buspirone or placebo at basal blood glucose and plasma insulin level and after oral glucose load induced (postprandial) blood glucose and plasma insulin level. Blood glucose and plasma insulin concentrations were estimated by glucose hexokinase method and enzyme linked immunosorbent assay (ELISA) method respectively. The concentration of blood glucose was significantly (p<0.05) decreased after oral glucose load following administration of buspirone in comparison with placebo however no significant change was observed in the fasting blood glucose and plasma insulin (fasting and oral glucose load induced) level. In conclusions, the present study findings show that buspirone produced a significant alteration in blood glucose level in healthy humans. In addition, study results also indicate that the involvement of serotonergic (5-HT, receptors) mechanism of blood glucose regulation in humans is different from animals.     

In vivo assay to identify bacteria with β-glucosidase activity

Backgroundβ-Glucosidase assay is performed with purified or semipurified enzymes extracted from cell lysis. However, in screening studies, to find bacteria with β-glucosidase activity among many tested bacteria, a fast method without cell lysis is desirable. In that objective, we report an in vivo β-glucosidase assay as a fast method to find a β-glucosidase producer strain.ResultsThe method consists in growing the strains for testing in a medium supplemented with the artificial substrate p-nitrophenyl-β-glucopyranoside (pNPG). The presence of β-glucosidases converts the substrate to p-nitrophenol (pNP), a molecule that can be easily measured in the supernatant spectrophotometrically at 405 nm. The assay was evaluated using two Bifidobacterium strains: Bifidobacterium longum B7254 strain that lacks β-glucosidase activity and Bifidobacterium pseudocatenulatum B7003 strain that shows β-glucosidase activity. The addition of sodium carbonate during pNP measurement increases the sensitivity of pNP detection and avoids the masking of absorbance by the culture medium. Furthermore, we show that pNP is a stable enzymatic product, not metabolized by bacteria, but with an inhibitory effect on cell growth. The β-glucosidase activity was measured as units of enzyme per gram per minute per dry cell weight. This method also allowed the identification of Lactobacillus strains with higher β-glucosidase activity among several lactobacillus species.ConclusionThis in vivo β-glucosidase assay can be used as an enzymatic test on living cells without cell disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria not only with β-glucosidase activity but also with high β-glucosidase activity.     

MEASUREMENT OF SOD ACTIVITY BY ~（19）F NUCLEAR MAGNETIC RESONANCE（NMR）RELAXATION RATE

ＭＥＡＳＵＲＥＭＥＮＴＯＦＳＯＤＡＣＴＩＶＩＴＹＢＹ~（１９）ＦＮＵＣＬＥＡＲＭＡＧＮＥＴＩＣＲＥＳＯＮＡＮＣＥ（ＮＭＲ）ＲＥＬＡＸＡＴＩＯＮＲＡＴＥＺｈａｏＢａｏ－Ｌｕ；ＣｈｅｎＹｕｎ－ＺｕｎａｎｄＸｉｎＷｅｎ－Ｊｕａｎ（ＩｎｓｔｉｔｕｔｅｏｆＢｉ?..     

The use of lysosomal enzymuria in the early detection and monitoring of the progression of diabetic nephropathy

Recent acquisitions on the early detection and monitoring of the progression of diabetic complications (nephropathy) using the techniques of enzymology (lysosomal enzymes) are reviewed. it appears that the kidney is the principal source of urinary lysosomal enzymes. Urinary samples for lysosomal enzyme determination can be either 24-hour or spot-collection. The use of synthetic substrates (4-methylumbelliferyl substrates) provides an easy, inexpensive, sensitive and highly reproducible method of lysosomal enzyme assay. It is recommended that more than one enzyme be assayed in the process. The use of fractional enzyme excretion (FEE) ratios is further recommended. The urinary lysosomal glycosidases investigated and found to be of particular diagnostic value in the early detection of diabetic nephropathy include N-acetyl-β-D-glucosaminidase (β-hexosaminidase, NAG), β-glucuronidase and β-galactosidase, with NAG being the most useful indicator. Urinary NAG can be used in monitoring the progression of diabetic nephropathy. The fluorimetric assay of lysosomal glycosidases is particuarly recommended in developing countries since it is simple, sensitive and inexpensive.     

Redistribution of glucose-6-phosphate dehydrogenase in response to cerebral ischemia in rat brain

Glucose-6-phosphate dehydrogenase (G6PD), a cytoplasmic enzyme, plays a protective role during oxidative stress in eucaryotic cells, since they provide coenzymes and substrates to the primary antioxidant enzymes. The redistribution of G6PD in the hippocampus was studied post-ischemia (PI). There was a characteristic localisation of G6PD in pyramidal cell layers of the rat hippocampus. In hippocampus CA1 cells were stained weakly whereas CA3 cells showed strong histochemical staining. Ischemia induced up-regulation of G6PD in the hippocampus was in a specific manner. First, the activity increased in the whole hippocampus (at 4 hours PI) which persisted 6 hrs PI in CA1 area. However G6PD activity decreased in the CA3 area & dentate gyrus. At 10 & 24 hrs PI, activity decreased in CA1 area but normalised in CA3 area & dentate gyrus compared to controls. This suggests that the sensitive CA1 neurons are transiently capable of generating an anti-oxidative arsenal to cope with the oxidative stress in the first few hours PI. We can conclude that the brain contains inducible endogenous mechanisms that are capable of enhancing the ability of neurons to withstand lethal ischemic challenge. (Presently working in Vardhman Mahavir Medical College & Safdarjung Hospital, New Delhi)     

Induction of Multiple Sclerosis and Response to Tyrosine Kinase Inhibitors

The goal of this work is to determine the role of the autoimmune cells in multiple sclerosis (MS) induction and the immunomodulatory mechanism of therapy with tyrosine kinase inhibitors (TKIs) in MS attenuation. Samples (5 × 10⁵ cells per well) of C6 and primary rat astrocytes were stimulated with 10 ng/mL of platelet-derived growth factor (PDGFbb) as a positive control forming a mouse model of MS. PDGFbb was added to the astrocytes in the absence or presence of 0.1 and 1 μM of imatinib. Proliferation of C6 and primary rat astrocytes samples were assessed for samples staging by the addition of 1 μCi of ³H-thymidine per well. Samples of RAW 264.7 cells were stimulated for 48 h with 10 ng/mL of PDGFbb in the absence or presence of 0.1 and 1 μM of sorafenib. Tumour necrotic factor (TNF) levels in culture supernatants from RAW 264.7 cells were measured by ELISA. The histologic grade (H_G) and the level of TNF of the mouse model of MS was 1/5 and 5 times respectively of those in the control one to clarify that MS induction is due to a major decrease in H_G inversely proportional to the accompanied increase in TNF level perpetuating local inflammation and demyelination in MS lesion. The addition of 0.1 and 1 μM doses of imatinib increased H_G of the mouse model of MS by 6 and 11 times respectively while 0.1 and 1 μM doses of sorafenib decreased TNF level to be 1/2 and 1/5 of that in the mouse model of MS respectively restoring normal rate of TNF level of normal lesion to show that H_Gand TNF level would be strongly inversely correlated (r = −0.99) in attenuating MS effectively by TKIs therapy but not in an inverse proportion as in MS induction.     

Enhanced production of glutaminase-free l-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines

BackgroundThe increasing rate of breast cancer globally requires extraordinary efforts to discover new effective sources of chemotherapy with fewer side effects. Glutaminase-free l-asparaginase is a vital chemotherapeutic agent for various tumor malignancies. Microorganisms from extreme sources, such as marine bacteria, might have high l-asparaginase productivity and efficiency with exceptional antitumor action toward breast cancer cell lines.Resultsl-Asparaginase-producing bacteria, Bacillus velezensis isolated from marine sediments, were identified by 16S rRNA sequencing. l-Asparaginase production by immobilized cells was 61.04% higher than that by free cells fermentation. The significant productivity of enzyme occurred at 72 h, pH 6.5, 37°C, 100 rpm. Optimum carbon and nitrogen sources for enzyme production were glucose and NH₄Cl, respectively. l-Asparaginase was free from glutaminase activity, which was crucial medically in terms of their severe side effects. The molecular weight of the purified enzyme is 39.7 KDa by SDS-PAGE analysis and was ideally active at pH 7.5 and 37°C. Notwithstanding, the highest stability of the enzyme was found at pH 8.5 and 70°C for 1 h. The enzyme kinetic parameters displayed V_max at 41.49 μmol/mL/min and a K_m of 3.6 × 10⁻⁵ M, which serve as a proof of the affinity to its substrate. The anticancer activity of the enzyme against breast adenocarcinoma cell lines demonstrated significant activity toward MDA-MB-231 cells when compared with MCF-7 cells with IC₅₀ values of 12.6 ± 1.2 μg/mL and 17.3 ± 2.8 μg/mL, respectively.ConclusionThis study provides the first potential of glutaminase-free l-asparaginase production from the marine bacterium Bacillus velezensis as a prospect anticancer pharmaceutical agent for two different breast cancer cell lines.How to cite: Mostafa Y, Alrumman S, Alamri S, et al. Enhanced production of glutaminase-free L-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines. Electron J Biotechnol 2019;42. https://doi.org/10.1016/j.ejbt.2019.10.001.     

Detection of enzymes dehydrogenases and proteases inBrugia malayi filarial parasites

Lymphatic filariasis caused mainly by infection fromW. bancrofti andB. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis ofB. malayi mf, infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key enzymes of carbohydrate metabolism viz., Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose-6-phosphate dehydrogenase (G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high in mf lysate compared to their activity in lysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel to their activity inlysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel electrophoresis (PAGE) using 7.5% non-gradient gel showed the presence of two isoforms of each of the three enzymes (MDH, ME & G6PDH) in mf lysate, while only one form of each enzyme was present in L₃ larval and adult worm lysates. Further proteolytic enzyme activity was demonstrated both in microfilarial and infective larval lysates ofB. malayi. While both mf and L₃ larval lysates showed optimal protease activity at alkaline pH of 9.0, the mf lysate showed increased activity also at pH 3.0. The infective larval lysate was markedly inhibited by Tosylamide-L-Phenylalanine chloromethyl ketone (TPCK), a thiol protease inhibitor, while the protease activity in mf lysate was significantly inhibited by both TPCK and a serine protease inhibitor Phenyl Methyl Sulphonyl Flouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and the L₃ larval lysate had 6 protease molecules of 18, 25, 37, 49, 70 and 200 kDa size.