Biochemical Processing

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Purification and characterization of fibrinolytic enzyme from Kiwifruit

Salem A. Habib, Entsar A. Saad*

Abstract

When a blood vessel is injured, the body forms the blood clot (thrombus). When a thrombus causes more than 90% obstruction this can result in anoxia and infarction. Therefore, searching for effective, inexpensive and safe thrombolytic agents from various natural sources is of great interest. Hence, fibrinolytic enzyme production from kiwifruit for blood clot lysis that can be employed as a good therapeutic agent is of importance. The present investigation deals with purification, characterization and therapeutic application of kiwifruit fibrinase. Fibrinase was purified from concentrated homogenat with specific activity of 160 U mg-1 protein. The optimal pH was 6.5. The enzyme pK1 was 5.2 and pK2 was 7.3. The km value for fibrin was 1.5 g/l. The optimal temperature was 30° C. The activation energy was 10.4 KJ/mol. Fibrinase was inactivated at 60, 70 and 80° C with t1/2 values of 34, 21 and 16 min at 60, 70 and 80° C, respectively. Many compounds and metal ions like iodoacetamide, EDTA and Cu2+ inhibited the fibrinase while others like G-6-P, AMP and Ca2+ showed enzyme activation. Fibrinase displayed great affinity towards human blood clot and seems to be a promising thrombolytic agent for clinical use.

Present study was aimed to study the role of EDTA, an non-antibiotic adjuvant, in relaxase enzyme inhibition which is essential to transfer of resistant gene from one bacteria to another bacteria. We also studied the effect of Elores (ceftriaxone plus sulbactam with EDTA, an non-antibiotic adjuvant) in comparison with other antimicrobial drugs. Escherichia coli K12 MTCC-1302 obtained from Institute of Microbial Technology, Chandigarh, India was used in the study. Minimum inhibitory concentration (MIC) was performed according to CLSI method. DNA relaxases activity was measured in the presence of various concentrations of EDTA and drugs at half of MIC. Results demonstrated that a significant inhibition of relaxases enzyme activity was observed from 4.0 mM (71.7±7.4; p>0.05) and continue up to 10 mM of disodium edtate. Such an inhibition was resulted in because of chelation of Mg2+ required for the activity of relaxases. When various drugs were evaluated for their effect on the relaxases activity, only Elores was found to be able to inhibit relaxases activity. None of the drugs of comparison could inhibit relaxeses significantly in comparison to Elores. From the above results, it can be conclude that EDTA, a non-antibiotic adjuvant at 10 mM and above exhibited a significant inhibition of DNA relaxases enzyme activity. Elores, also inhibited DNA relaxases due to presence of EDTA as a non antibiotic adjuvant. Therefore, the inhibition of relaxases is a potentially a novel approach to combat spreading of antibiotic resistant gene. This strategy will improve our understanding on the ecological abundance as well as spreading of resistant gene from one bacteria to another bacteria during hospital stay.

A correlative study of copper, ceruloplasmin and iron in urine as biomarkers for diabetic nephropathy

Ramlinga Reddy, Paul Simon, Arnadi Ramachandrayya Shivashankara*

Abstract

Nephropathy is one of the earliest complications of diabetes mellitus. Search is on for markers of diabetic nephropathy especially the biomarkers of incipient nephropathy. Present study focused on metals iron and copper which are mediators of oxidative stress, the main culprit of pathogenesis of diabetic complications, and ceruloplasmin the antioxidant. In diagnosed diabetic nephropathic patients, diabetic patients without any complications and normal healthy controls, urinary levels of copper, ceruloplasmin and iron were estimated and correlated with microlabuminuria. In all diabetic patients, urinary levels of copper and iron were higher and ceruloplasmin was lower when compared to controls. The changes in copper, iron and ceruloplasmin were highly pronounced in diabetic patients with nephropathy. The urinary levels of copper, iron and ceruloplasmin showed significant positive correlation with albumin excretion in diabetic nephropathic patients. Changes in Copper, Iron and Ceruloplasmin reflect the oxidative stress and role of metals in diabetic nephropathy. Though microalbuminuria is the gold standard parameter for the diagnosis and management of nephropathy, the early normoalbuminuric stages of diabetes and susceptibility for future complications could be detected by these parameters. The present study had limitations in the form of small sample size and considering only a limited number of biochemical parameters. Future studies with larger sample which correlate the biochemical changes with clinical staging of diabetic nephropathy, and assessing the clinical utility of various biochemical parameters in the management of diabetic complications, are required.

Arsenic induced apoptosis in C57Bl/6 mice

Dalal Basudeb*

Abstract

Inorganic arsenic is potent environmental toxicant to which millions of people are exposed over the world, mainly through contaminated water. Exposure of human populations to arsenic has been associated with a high incidence of detrimental and carcinogenic effects. Exposure dependent apoptotic or necrotic effect of arsenic is not clearly understood. Objective of the present study was to investigate arsenic exposure induced apoptotic effect in cells of mice blood. Sodium Arsenite was incubated to cell pellets obtained after removal of erythrocytes followed by addition of various concentrations of arsenic in vitro. 50% of cells showed apoptosis at the higher (10 μM) concentration whereas control population showed 20% apoptotic effect. Our observation revealed that apoptosis mediated cell death occurred more when cells were exposed for higher dose of arsenic.

Assessment of Smad-1 and Spondin-2 as biomarkers for early detection of Nephropathy in type 2 Diabetic patients

Ibrahim A. Emara*, Mohamed S. Shouman

Abstract

Diabetic nephropathy (DN) is the most commoncause of chronic kidney disease (CKD). The aim ofthe study is to determine both risk factors (smad1 and Spondin-2) as early detection of nephropathy. The study included 4 groups of subjects, Group 1 (control subjects), Group 2 (diabetic patients with normoalbuminuria), Group 3 (diabetic patients with microalbuminuria) and Group 4 (diabetic patients with macroalbuminuria). FBG, HbA1c, Insulin, Creatinine, Urea, Microalbumin, creatinine in urine, lipid profile, Smad-1 and Spondin-2 level were determined and ACR was estimated. There is a significant decrease in the GFR in the diabetic with microalbuminuria and macroalbuminuria when compared to the normoalbuminuria diabetic group and control group. Insulin and HOMA-IR showed significant increase in the diabetic patients group with microalbuminuria and macroalbuminuria compared to diabetic patients with normoalbuminuria and control group. Smad-1 andSpondin-2 levels were significantly increased in type 2 DM with microalbuminuria and macroalbuminuria in compared to type 2 diabeteswith microalbuminuria and control group. In conclusion Smad-1 and Spondin-2 may serve asearly predictors for nephropathy in type 2 DM. So, early diagnosis and good glycemic control areeffective in reducing the diabetic nephropathy.

Injectable progestin contraceptive could increase the risk of insulin resistance syndrome in humans

Onyesom Innocent, Ogbodu, D. Ejiroghene* Osioma, Ejovi Avwioroko, J. Oghenetega

Abstract

This study evaluates the biochemical predictors of insulin resistance syndrome and cardiovascular function among DMPA users. Forty-five consenting women were selected and divided into three groups (n=15 per group) comprising DMPA, Cu-IUD and non-contraceptive users. Blood pressure (BP) and body mass index (BMI) were determined. The blood samples collected were processed for the assay of glucose, insulin and lipids. Results show a significantly increased insulin level (26.31±10.06 μIU/ml) among DMPA users compared with the Cu-IUD (15.98 ± 5.77 mmol/L) and non-contraceptive (9.57±3.04 μIU/ml) groups respectively. A significant decrease in HDL-cholesterol level was observed in the DMPA group (0.62±0.10 mmol/L) as compared with the Cu-IUD (0.69±0.06 mmol/L) and non-contraceptive (0.74±0.17mmol/L) users. In conclusion, DMPA induces hyperinsulinemia associated with increase in insulin resistance index, reduction of HDL-cholesterol and elevation in LDLC/ HDL-C ratio. This trend may be worrisome because these are predictors of insulin resistance syndrome. The implications of these altered predictors need to be documented for proper health care delivery and family planning policy.

Lipid profile and liver marker enzymes in male and female culturedadults Clarias gariepinus

Hammed A.M., Wusu A.D., Amosu A.O.*, Fashina-Bombata H.A., Avoseh O.

Abstract

Twelve (12) cultured adult Clarias gariepinus, six (6) male and six (6) female of average weight 795±179.52g were used to investigate the lipid profile and liver marker enzymes; important and efficient biochemical indices used in determining the general well-being of an organism. Concentrations of cholesterol, triglycerides and phospholipids (lipid profile) as well as the liver marker enzymes, i.e. Alanine Aminotransferase (ALT: EC: 2.6.1.2), Aspartate Aminotransferase (AST: EC: 2.6.1.1) and Alkaline Phosphatase (ALP: EC: 3.1.3.1) were determined using enzymatic procedures while statistical analysis was carried out using Statistical Package for Social Science (SPSS version 20). Plasma cholesterol in female species (81.55±16.25mg/dl) and phospholipids (191.97±53.50 mg/dl) were significant (P<0.05) than in male species (49.77±13.91mg/dl) and (153.09±40.08 mg/dl). There was a significant difference (P<0.05) between the male (99.44±22.90mg/dl) and female species (85.92±15.08) triglycerides concentrations. The male species are generally significantly (P<0.05) higher in High Density Lipoproteins (HDL) than the female. ALT concentration was found to be higher than ALP and AST in both sexes, the overall result was found to be below optimal concentration values thereby making the cultured sampled of adult Clarias gariepinus species safe for human consumption.

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