Tissue Standards Using Stain Free Gels as a Superior Approach for Aging Research
A good loading control is critical for accurate comparison of tissue protein levels in aged and young tissue. After reviewing the literature we discovered that the housekeeping proteins commonly used in Western analysis had significant drawbacks. The purpose of this study was to evaluate the linearity and reproducibility of common housekeeping proteins tubulin, actin and GAPDH compared to measures of total protein staining using Direct Blue and Stain Free gels. We found that measurements of total protein staining were superior to housekeeping proteins both in linearity and reproducibility in young and old mouse, liver, heart, and brain. Among the total protein staining methods, the Stain Free method was superior to Direct Blue, in terms of lower variability and higher accuracy in all tissues, at both ages.
Aims: The presented research paper is aimed to prevalence of apoptosis for the first time by using in silico molecular docking and simulation using caspase-3 zymogen and zinc-coordinated compounds. Antioxidant activities of the coordinated complexes were compared to the positive controls (BHT and ascorbic acid).
Study design: Molecular docking analysis was performed by using Autodock 4.2, setting at 150x140x110 Gridbox, center at -57.616, 31.092, 88.666 with 0.375 Å spacing. Their antioxidant activity was tested by FRAP and DPPH assay.
Place and Duration of Study: Department of Chemistry and department of molecular medicine, Faculty of Medicine, University of Malaya laboratories between January 2014 – July 2014
Methodology: Compounds derived from zinc(II) ion give rise to coordination complexes exhibited CHN, NMR (1H & 13C) and FT-IR spectra consistent with the proposed structures. Based on the x-ray crystal structure, one of the derivatives is a mononuclear square-pyramidal metal complex, with τ value of 0.35. The molecular docking simulation formed between compounds and caspase 3 showed the ligands bind to the active-site gorge well positioned in the active-site gorge.
Results: The residues that have been involved in this protein-ligand interaction docked well by using Autodock 4.2, setting at 150x140x110 Gridbox, center at -57.616, 31.092, 88.666 with 0.375 Å spacing in the hydrophobic pocket. Their antioxidant activity tested revealed their FRAP assay values of 531.11±0.021 and 1886.11±0.008 higher than value of 187.3±2.6 shown by BHT used as standard. The compounds showed IC50 values 21.50±0.009 and 14.80±0.002 lower than ascorbic acid with an IC50 value of 2.26±0.001 μg/mL.
Conclusion: Synthesized zinc(II) complexes have been confirmed to inhibit the activity of caspase 3 both in silico and in vitro and were tested for antioxidant activity by both FRAP and DPPH methods.
Aims: The objective of the present study was to evaluate the anti-obesity effects of unripe Rubus coreanus Miquel (uRC) in 3T3-L1 adipocytes and body weight, epididymal fat and perirenal fat weight, and lipid profiles in diet-induced obese (DIO) C57BL/6 mice.
Methodology: The lipid accumulation in 3T3-L1 adipocytes was carried out Oil Red O staining. And uRC (50 and 100 mg/kg/day) were orally administered for 90 days from the day of feeding with high fat diet (HFD). The serum total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL)-cholesterol and low density lipoprotein(LDL)-cholesterol and glucose levels were measured using Alere cholesterol LDXⓇ system. And the serum glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen(BUN) and creatinine levels were measured using the respective kits.
Results: Our results indicated that treatment with uRC dose-dependently inhibited lipid accumulation in 3T3-L1 adipocytes. Moreover, after oral administration for 12 weeks, uRC (50 and 100mg/kg/day) extract produced a significant decrease in the serum total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, glucose and glutamic-oxaloacetic transaminase (GOT) levels of HFD-induced obese mice. Similarly, uRC extract elevated serum high density lipoprotein (HDL) cholesterol. These results suggest that uRC extract may be a useful resource for the management of obesity.
Conclusion: These results suggest that uRC extract may be a useful resource for the management of obesity.
Radioiodination and Biological Evaluation of Epinephrine as a Possible Adrenergic Receptors Imaging Agent
An adopted method for the preparation of high radiochemical purity [125I] iodoepinephrine was developed in order to characterize the binding properties of adrenergic receptors. Direct radioiodination of epinephrine was carried out using chloramine-T as oxidizing agent. The reaction proceeds well within 15 min at ambient room temperature up to 25+1ºC and afforded a radiochemical yield up to 94%. Different chromatographic techniques (electrophoresis and paper chromatography) were used to evaluate the radiochemical yield and purity of the labeled product. Biodistribution studies were carried out in normal Albino Swiss mice and the results showed rapid and high cardiac uptake of 125I-epinephrine. The result indicates the possibility of using radioiodinated epinephrine as myocardial imaging agent.
Aims: This study was carried out to assess the antimicrobial effect of the volatile oil of Thaumatococcus danielli (Benn.) Benth. leaves against selected enteric pathogens and its possible inhibition against a partially purified and characterized extracellular protease of Shigella dysenteriae.
Study Design: The volatile oil was used as antibacterial agent against eight enteric pathogens and as potential inhibitor to a partially purified extracellular protease of Shigella dysenteriae.
Place and Duration of Study: Department of Biochemistry, Faculty of Science, Lagos State University, Ojo Lagos State, Nigeria; between May – September 2013.
Methodology: The volatile oil was extracted by hydrodistillation. The sensitivity, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the volatile oil against the pathogens were determined by disc-diffusion and micro-dilution techniques. The extracellular protease of Shigella dysenteriae was partially purified by (NH4)2SO4 salting-out followed by gel filtration chromatography. Enzyme assay was carried out with casein (as substrate) and volatile oil (as inhibitor).
Results: The average inhibition zones ranged from 16 – 30 mm. Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecalis remained insensitive to the oil. Shigella dysenteriae and Proteus vulgaris showed highest and lowest sensitivities respectively. Highest purification fold of 2.63 with specific activity of 18.4μmol/min/mg protein were obtained after Sephadex G-75 gel filtration of the enzyme extract. The oil competitively inhibited the partially purified extracellular protease of Shigella dysenteriae with Vmax = 8.33 ∆A/min, Km = 0.85mg/ml (no inhibitor) and apparent Km´ = 1.54mg/ml (inhibitor). Optimal activities of this protease were obtained at pH 7.5 and 30ºC.
Conclusion: Though, it may not be clarified whether the oil interacted with the substrate/enzyme. Nevertheless, there was an inhibition of the extracellular protease of Shigella dysenteriae by the volatile oil of Thaumatococcus danielli (Benn.) Benth. It was therefore envisaged that elaborate work on the inhibition of this type of enzyme in pathogens by essential oil would be a platform of arresting infection caused by bacteria.
Partial-purification and Characterization of Angiotensin Converting Enzyme Inhibitory Proteins from the Leaves and Seeds of Moringa oleifera
Aim: To partially-purify, characterize and investigate the inhibition pattern of proteins from the leaves and seeds of Moringa oleifera.
Methodology: Crude proteins obtained from the leaves and seeds were partially-purified by a two-step method: cold acetone precipitation and sephadex G-75 gel filtration chromatography and Angiotensin Converting Enzyme (ACE) inhibitory activities determined using Cushman and Cheung method with some modifications on the assay conditions. The effects of pH, temperature and some digestive enzymes on the inhibitory proteins were determined. The inhibition pattern was also investigated using Lineweaver-Burk plots.
Results: The ACE inhibitory proteins from the leaves and seeds were purified 8.41 and 12.40 folds with a yield of 38 and 35.18%, respectively on sephadex G-75 column. The optimum pH and temperature of the inhibitory proteins were 8.2 and 37.6°C, for leaves and 7.8 and 39°C, for seeds, respectively. The partially-purified ACE inhibitors exhibited a mixed type of inhibition. The Km of inhibitory proteins obtained from the leaves increased from 3.51mM without the inhibitor to 4.79mM and 5.62mM in the presence of 0.5 and 1mg/ml of the inhibitor, respectively, while Vmax decreased from 0.953µmol/min without inhibitor to 0.784µmol/min and 0.629µmol/min, in the presence of 0.5 and 1mg/ml of the inhibitor, respectively, with a Ki of 3.51mM. For the seeds, the Km increased from 2.89mM (without inhibitor) to 5.747mM (at 1mg/ml inhibitor), while Vmax decreased from 0.910 (without inhibitor) to 0.628µmol/min (at 1mg/ml inhibitor), with a Ki of 1.58mg/ml. The digestive enzymes, pepsin and trypsin significantly (P<0.05) reduced the activity of the partially-purified inhibitors from both leaves and seeds compared to captopril. In conclusion, ACE inhibitory proteins from M. oleifera may be beneficial as nutraceutical or drug for blood pressure regulation in hypertensive patients.
The new acyclic Schiff base [L] ligand: 3,3'-(1E,1'E)-1,1'-(2,2'-azanediylbis (ethane-2,1-diyl)bis(azan-1-yl-1-ylidene))bis(ethan-1-yl-1-ylidene) bis(4-hydroxy-6-methyl-2H-pyran-2-one) derived from condensation of one mole diethylene diamine triamine (dien) with two moles of dehydroacetic acid have behaved tetra dentate dibasic chelating agent with all metal ions under study.
Three bimetallic Cr(III), Co(II) and Fe(III) acyclic polydentate complexes [M2L2Cl2]Cl4 ,M=Cr(III) , Fe(III) and [M2L2Cl2], M=Co(II) have been prepared and fully characterized by UV-Vis., FTIR, micro-elemental analysis , as well as the magnetic moments of solid complexes and the measurements of molar conductance in DMSO solution helped us in investigate the chemical structure of bimetallic models . From the results obtained by different techniques, it was found that the proposed structures of the prepared complexes have octahedral structure.
A theoretical treatment of the formation of complexes in the gas phase was studied, this was done using the HYPERCHEM-6 program in general. The synthesized compounds were tested for antimicrobial activity by cup plate diffusion method. The results indicate the enhanced activity of metal complexes over the parent ligands.
Aim: The aim of this study was to investigate the effect of chronic exposure to petroleum hydrocarbon pollution (PHC) on some biochemical parameters of the fruit juice of Citrus sinensis.
Place and Duration of study: This study was carried out at Ebocha-Egbema and Uvuru Mbaise in Imo state (Niger Delta Area), Nigeria between October 2008 and October 2011.
Methodology: Acidity (pH), concentrations of ascorbic acid (AA), glutathione (GSH), citric acid, glucose and the activity of lactate dehydrogenase (LDH) in the juice of just-ripe orange fruits (Citrus sinensis) from the two environments were investigated by standard methods. The estimated values were analyzed using student t-test and the results expressed as mean ± standard deviation.
Results: The results obtained revealed that there was no significant (p≥0.05) difference in the mean pH values, ascorbic acid and glucose concentrations of the fruit juice from the two areas studied. Mean concentrations of glutathione and citric acid in the juice from Ebocha (0.44±0.09 and 18.80±1.14mg/l) were significantly (p≤0.05) lower than the values in the juice from Uvuru (0.66±0.10 and 21.43±2.02 mg/l), respectively. The results also showed that the mean activity of lactate dehydrogenase was significantly higher in the juice from Ebocha (7.033+/-1.73 U/l) than in that from Uvuru (5.344±1.74 U/l).
Conclusion: The findings of this study are suggestive of a possible alteration in the metabolic activities of Citrus sinensis trees evident in its fruit juice due to the PHC pollution in Ebocha in the Niger Delta.
Objective: The aim of the present study was to investigate the protective effect of curcumin against aflatoxinB1 (AFB1) induced hepatotoxicity.
Materials and Methods: Twenty-eight healthy adult male Wistar rats were divided into four groups. Rats of the first group received basal diet and served as control. Rats in the second group received curcumin orally (15mg/5ml/kg body weight) whereas, rats in the third groups injected with single intraperitoneal injection of AFB1 (3mg/kg BW). Rats in the fourth group received a combination of second and third groups for five weeks.
Results: Biochemical analysis of serum samples indicated a significant increase in aspartate transaminase (AST) and alanine transaminase (ALT) activities and total cholesterol and creatinine concentrations along with significant decrease in protein content of AFB1 intoxicated rats compared to control group. Oral administration of curcumin along with injected AFB1 restored AST, ALT, total cholesterol, creatinine and total protein near to control values. Biochemical analysis of liver antioxidants revealed a significant (P < 0.05) reduction in catalase (CAT) and superoxide dismutase (SOD) activities and hepatic reduced glutathione (GSH) content in rats injected with AFB1 compared to control. On the contrary, oral administration of curcumin along with injected AFB1 enhanced hepatic CAT and SOD activities and GSH concentration towards the control values, suggesting that curcumin could improve the antioxidant status in AFB1 induced oxidative stress. The Biochemical findings were supported by histopathology of liver tissues which indicated vacuolar degeneration and necrotizing changes in liver of rats intoxicated with AFB1 and significant amelioration of these effects in these rats whenever treated with curcumin.
Conclusion: Conclusively, oral administration of curcumin along with AFB1 caused significant inhibition in AFB1-induced hepatotoxicity in rats by increasing the concentration of GSH and activation of antioxidant enzymes.
Aims: The present study was undertaken to find the levels of aspartate transaminases (AST), creatine kinase (CK-MB) and arginase in serum sample of patients under study and check effectiveness of arginase as a marker in acute coronary syndrome (ACS), and to find the correlation between AST and CK-MB with arginase in unstable angina (UA) and myocardial infarction (MI).
Study Design: Case control study carried out at department of Biochemistry, B. J. Medical College Pune, Maharashtra, India, between Sept 2011-Dec 2013.
Methodology: The study comprised of clinically diagnosed 60 patients: 30 of UA and 30 of MI. Age and sex matched 30 patients were studied as controls. Blood sample were collected from individual under study, serum was separated and used to estimate levels of CK-MB, AST and arginase.
Results: The levels of AST, CK-MB and arginase were estimated and the results were compared with that of control. The levels of arginase were elevated significantly (P<0.001) like AST and CK-MB in patients with MI as compared to UA. In the study there was no correlation observed between CK-MB and arginase in patients of UA or controls. The same was true with the levels of AST and arginase. On the other hand a strong positive correlation was found between CK-MB and AST with arginase in MI patients.
Conclusion: It can be concluded that like AST and CK-MB, arginase also increases with progression of ACS. Thus arginase can be used as sensitive marker to differentiate between UA and MI.