The mechanism of antioxidant activity of IRFI005 as a synthetic hydrophilic analogue of vitamin E

Developing a rational strategy to control intracellular reactive oxygen species (ROS) requires understanding the mechanism of antioxidant activity. In this investigation the properties of a novel synthetic analog of vitamin E (IRFI005) with potent antioxidant activity are described. A mechanism is proposed for its efficient radical-scavenging effects. Cellular antioxidant and antitoxicity assays showed IRFI005 to freely permeate across cellular membranes, enabling it to be an effective suppressor of intracellular ROS and to protect cells against toxicity induced by free radical generating compounds. The free radical-scavenging activity of IRFI005 examined by UV–Vis and electron spin resonance (ESR) techniques clearly confirmed a “two electrons and/or H-atom” donation mechanism for each molecule of IRFI005. Reducing power assay as well as semi-empirical calculations revealed that under physiological conditions (pH∼7) almost all IRFI005 molecules are in the anionic state (IRFI005⁻). Data indicated that the electron donating ability of IRFI005⁻ was dominant at physiological pH because of higher stability of quinine-IRFI005⁻ and less barrier energy of IRFI005⁻ than neutral IRFI005. Consequently, the efficient cellular protection of IRFI005 against toxic free radicals can be explained by a two electron-transfer process, because of reduced inter-frontier molecular orbital energy gap barrier at physiological pH. Our findings suggest that hydrophilic vitamin E-like antioxidants are good candidates in designing novel therapeutic strategies for inhibition of oxidative stress associated with different human diseases.     

Response Surface Methodology for Optimizing the Bovine Serum Albumin Fibrillation

Amyloid fibrils are considered as nanostructures that could be formed by ordered self-assembly of the partially-folded states of many different peptides or proteins. In this study, bovine serum albumin was used as a model protein whose ordered aggregation (fibrillation) was optimized. Response surface methodology (RSM) was used in a design that contained a total of 30 experimental trials. The first 24 were organized in a factorial design and from 25 to 30 involved the replications of the central points. Data obtained from RSM were subjected to the analysis of variance (ANOVA) and analyzed using a second order polynomial equation. Subsequent testing of the suggested experimental parameters was done in vitro with Congo red spectrophotometric assay. Protein concentration, pH, temperature and time of incubation were the variables used in this study. Responses were assessed by measuring absorbance in 540?nm (characteristic of amyloid formation) and maximal wavelength. Concomitant effects of variables were assessed in surface plots that each considered two of the variables. Interestingly, the pattern obtained by monitoring absorbance at 540?nm and absorbance in maximal wavelength were identical in most cases. We are reporting the optimum concentration of protein, pH, temperature and time at 5?mg?ml^?1, 3.02 and 72?°C and 48?h, respectively. Our findings suggest that use of Congo red spectrophotometric test, as a simple and affordable assay could be suggested as a first test for assessing fibril formation of proteins. Absorbance in maximal wavelength is recommended as a significant indication of fibril formation.     

The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C3H7OCS2Na (I), C4H9OCS2Na (II) and C5H11OCS2Na (III), were synthesized, by the reaction between CS2 with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K(i) values of 9.8, 7.2 and 6.1 microM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S-) of the anion xanthate, which leads to similar values of K(i) for all three xanthates. Different K(i) values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

A procedure for the purification of beta-lactoglobulin from bovine milk using gel filtration chromatography at low pH

A simple and rapid procedure for the purification of beta-lactoglobulin (β-LG) from bovine milk is described. The procedure exploits the major difference in molecular mass of β-LG and other whey components and the existence of the former in monomeric form at acidic pH. Gel filtration of whey was carried out using a Bio-Gel P10 column at pH 3.0. Residual caseins and other milk proteins were excluded from the gel and β-LG and alpha-lactalbumin (α-LA) emerged as two fully resolved peaks. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested that β-LG was purified to apparent homogeneity, while absorption, fluorescence, and circular dichroism spectroscopy indicated the native-like conformation of the protein. Western blot analysis revealed that the antibodies raised against the purified β-LG in rabbits also readily react with the commercial bovine protein. This procedure requires only 4-5 hr for the purification of about 10 mg of β-LG from a single run while using a small column (2.3 cm x 83 cm) of Bio-Gel P10 and has the potential for scaling up.     

Influence of different types of effectors on the kinetic parameters of suicide inactivation of catalase by hydrogen peroxide

The effects of cyanide and azide ions (class A), sodium-n-dodecyl sulphate (SDS) and 2-mercaptoethanol (class B), 3-aminotriazole (class C) and NADPH (class D) on the initial activity (ai), inactivation rate constant (ki) and the partition ratio (r) of bovine liver catalase reaction with its suicide substrate, hydrogen peroxide, were studied in 50 mM sodium phosphate buffer, pH 7.0 at 27 degrees C. The above kinetic parameters were determined by processing the progress curve data. In class A, which contains fast and reversible inhibitors of catalase, a proportional decrease in ai and ki was observed by inhibitors, so that the r remained constant. In class B, which contains slow and irreversible inactivators, a decrease in ai and constancy of ki and r were observed when catalase was incubated in the presence of such inactivators for a determined time. In class C, containing effector which can combine with intermediate compound I, ai was relatively unchanged but an increase in ki and a decrease in r were observed. In class D, containing effector which reduces compound I to ferricatalase, ai was not affected significantly but some decrease in ki was detected which was linked with an increase in r. These results demonstrate that different classes of effectors affect the determined kinetic parameters of catalase in various ways. Thus, determination of such parameters by simple kinetic experiments can be carried out for classification of the agents which have an effect on the kinetics of catalase.     

Accuracy of cytologic interpretation of pancreatic neoplasms by fine needle aspiration and pancreatic duct brushings

OBJECTIVE: To investigate the accuracy of fine needle aspiration (FNA) specimens and pancreatic duct brushings in the detection of pancreatic lesions and to compare the results with follow-up biopsy and/or surgical interpretation. STUDY DESIGN: We reviewed a total of 57 specimens (37/20), 37 FNA specimens and 20 pancreatic duct brushings, from 45 patients treated at Froedtert Memorial Lutheran Hospital, affiliated with the Medical College of Wisconsin, Milwaukee, over a 4-year period. The FNA and brushing samples were categorized as follows: positive for malignancy (21/3 = 24), suspicious for malignancy (8/7 = 15) and atypical (8/10 = 18). The results were then correlated with the tissue diagnosis. RESULTS: The 24 cytologic samples positive for malignancy included 23 (20/3) pancreatic ductal carcinoma (CA) and 1 (1/0) neuroendocrine CA; in the suspicious category, 11 (6/5) were pancreatic ductal CA; 2 (0/2) mucinous neoplasms and (2/0) neuroendocrine neoplasms; in the atypical category; 2 (2/0) suggestive of mucinous neoplasia, 1 (1/0) suggestive of serous neoplasia and 9 (2/7) favor reactive; and 6 (3/3) without further categorization. Tissue diagnoses were available in 26 cases: 12 (10/2) cases positive for malignancy, 8 (5/3) suspicious for malignancy and 6 (5/1) atypical. The 12 cytologically positive cases confirmed by histology showed 10 ductal CA, 1 neuroendocrine CA and 1 negative. All 8 cases (100%) suspicious for malignancy revealed positive results, including 5 ductal CA, 1 neuroendocrine neoplasm, 1 mucinous cystic neoplasm and 1 lymphoma. Of the 6 atypical lesions, 1 showed ductal CA, 2 mucinous cystic neoplasm and 3 chronic pancreatitis. CONCLUSION: Pancreatic FNA and duct brushings [table: see text] are accurate methods in identifying pancreatic lesions, particularly ductal CA. Accuracy can be improved in the case of mucinous and other lesions with adequate cellularity of the smear and recognizing the limitations of brush samples in the case of mucinous cystic lesions. False negative results may occur in cases of poor representation of malignant cells or poor sampling.     

Kinetics of denaturation of human and chicken hemoglobins in the presence of co-solvents

The stability of four hemoglobins (Hb) in dimer forms (low concentration) were investigated by the kinetics of denaturation. The rate constants of denaturation were obtained by variation of 280 nm absorption versus time in 10 mM Tris-HCl, 10 mM EDTA, pH 8.0 at 45 degrees C in the absence and presence of 0.5 M ethanol, dimethyl sulfoxide (DMSO), formamide, and glycerol. The results show the trend of rate constants in different co-solvents in the following order: chicken hemolysate < human hemolysate and chicken Hb D < chicken Hb A. The buried surface area was calculated for Hb samples in the absence of co-solvents. Accordingly, the trend points out that: chicken Hb D > chicken Hb A > human Hb A. These results suggest that both chicken hemolysate and chicken Hb D are relatively more stable than human and chicken Hb A, respectively. However, the denaturation rate constants of Hb in different co-solvents have designated the following order: ethanol > DMSO > formamide > glycerol. As a matter of fact, this phenomenon is an indication of an increase in the denaturation capacity (DC) and hydrophobicity, and a decrease in the surface tension of the solution in the preceding co-solvents.     

A lethal phenotype associated with tissue plasminogen deficiency in humans

The role of plasminogen in preventing thrombosis requires activation by tissue plasminogen activator (t-PA) encoded by PLAT. While case–control associations have been pursued for common variants in PLAT, no disease-causing mutations have been reported. We describe a consanguineous family with two children who died shortly after birth due to complications related to severe hydranencephaly and diaphragmatic hernia. A combined exome/autozygome analysis was carried out with informed consent. We identified a homozygous null mutation in PLAT that abrogated t-PA level in patient cells. This is the first reported human knockout mutation of PLAT. The apparent association with hydranencephaly, diaphragmatic hernia and postnatal lethality requires further validation.     

Inhibition of beta-amylase activity by calcium, magnesium and zinc ions determined by spectrophotometry and isothermal titration calorimetry

The inhibition effect of metal ions on beta amylase activity was studied. The inhibitor-binding constant (Ki) was determined by spectrophotometric and isothermal titration calorimetric (ITC) methods. The binding of calcium, magnesium and zinc ion as inhibitors at the active site of barley beta amylase was studied at pH = 4.8 (sodium acetate 16 mM) and T = 300K. The Ki and enthalpy of binding for calcium (13.4, 13.1 mM and -14.3 kJ/mol), magnesium (18.6, 17.8mM and -17.7 kJ/mol) and zinc (17.5, 17.7 mM and -20.0 kJ/mol) were found by spectrophotometric and ITC methods respectively.