Characterization of Natural Inhibitors of Trypsin and Chymotrypsin by Electrophoresis in Acrylamide-Agarose Gels
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Polyacrylamide is a polymer of controllable molecular weight formed by the polymerization of acrylamide monomers available in one of three forms: solid (powder or micro beads), aqueous solution, or inverse emulsions (in water droplets coated with surfactant and suspended in mineral oil). Residual acrylamide monomer is likely an impurity in most Polyacrylamide preparations, ranging from <1 ppm to 600 ppm. Higher levels of acrylamide monomers are present in the solid form compared to the other two forms. Polyacrylamide is reportedly used in 110 cosmetic formulations, at concentrations ranging from 0.05% to 2.8%. Residual levels of acrylamide in Poly acrylamide can range from < .01 % to 0.1 %, although representative levels were reported at 0.02% to 0.03%. Because of the large sizes of Polyacrylamide polymers, they do not penetrate the skin. Polyacrylamide itself is not significantly toxic. For example, an acute oral toxicity study of Polyacrylamide in rats reported that a single maximum oral dose of 4.0 g/kg body weight was tolerated. In subchronic oral toxicity studies, rats and dogs treated with Polyacrylamide at doses up to 464 mg/kg body weight showed no signs of toxicity. Several 2-year chronic oral toxicity studies in rats and dogs fed diets containing up to 5% Polyacrylamide had no significant adverse effects. Polyacrylamide was not an ocular irritant in animal tests. No compound-related lesions were noted in a three-generation reproductive study in which rats were fed 500 or 2000 ppm Polyacrylamide in their diet. Polyacrylamide was not carcinogenic in several chronic animal studies. Human cutaneous tolerance tests performed to evaluate the irritation of 5% (w/w) Polyacrylamide indicated that the compound was well tolerated. Acrylamide monomer residues do penetrate the skin. Acrylamide tested in a two-generation reproductive study at concentrations up to 5 mg/kg day x in drinking water, was associated with prenatal lethality at the highest dose, with evidence of parental toxicity. The no adverse effects level was close to the 0.5 mg/kg day x dose. Acrylamide tested in a National Toxicology Program (NTP) reproductive and neurotoxicity study at 3, 10, and 30 ppm produced no developmental or female reproductive toxicity. However, impaired fertility in males was observed, as well as minimal neurotoxic effects. Acrylamide neurotoxicity occurs in both the central and peripheral nervous systems, likely through microtubule disruption, which has been suggested as a possible mechanism for genotoxic effects of acrylamide in mammalian systems. Acrylamide was genotoxic in mammalian in vitro and in vivo assays. Acrylamide was a tumor initiator, but not an initiator/promoter, in two different mouse strains at a total dose of 300 mg/kg (6 doses over 2 weeks) resulting in increased lung adenomas and carcinomas without promotion. Acrylamide was tested in two chronic bioassays using rats. In one study, increased incidence of mammary gland tumors, glial cell tumors, thyroid gland follicular tumors, oral tissue tumors, uterine tumors and clitoral gland tumors were noted in female rats. In male rats, the number of tumors in the central nervous system (CNS), thyroid gland, and scrotum were increased with acrylamide exposure. In the second study, using higher doses and a larger number of female rats, glial cell tumors were not increased, nor was there an increase in mammary gland, oral tissue, clitoral gland, or uterine tumors. Tumors of the scrotum in male rats were confirmed, as were the thyroid gland follicular tumors in males and females. Taken together, there was a dose-dependent, but not statistically significant, increase in the number of astrocytomas. Different human lifetime cancer risk predictions have resulted, varying over three orders of magnitude from 2 × 10 3 to 1.9 × 120 6. In the European Union, acrylamide has been limited to 0.1 ppm for leave-on cosmetic products and 0.5 ppm for other cosmetic products. An Australian risk assessment suggested negligable health risks from acrylamide in cosmetics. The Cosmetic Ingredient Review (CIR) Expert Panel acknowledged that acrylamide is a demonstrated neurotoxin in humans and a carcinogen in animal tests, but that neurotoxic levels could not be attained by use of cosmetics. Although there are mechanisms of action of acrylamide that have been proposed for tumor types seen in rat studies that suggest they may be unique to the rat, the Panel was not convinced that these results could be disregarded as a species-specific finding with no relevance to human health and safety. Based on the genotoxicity and carcinogenicity data, the Panel does not believe that acrylamide is a genotoxic carcinogen in the usual manner and that several of the risk assessment approaches have overestimated the human cancer risk. The Panel did conclude, however, that it was appropriate to limit acrylamide levels to 5 ppm in cosmetic formulations.
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<p>Carbohydrate rich food cooked at high temperature can lead to the formation of acrylamide. The aim of this investigation is to analyze the proteomics of mice liver in response to acrylamide intoxication since the liver is the major site of acrylamide detoxification and metabolism. The liver protein pattern from sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) treated mice with acrylamide in drinking water for 9 weeks did not show variations from the control. However, analysis by 2-D gel of liver proteins from mice treated with 0.5 µg acrylamide/kg for 9 weeks showed variations in protein expression. At least 10 protein spots were significantly affected (T > 2) by the acrylamide treatment. The affected proteins were identified using MALDI TOF/TOF and mass searches through Mascot. These proteins were up-regulated or down-regulated depending on their physiological function. Probably the proteins are involved in detoxification of acrylamide or cell protection. The data from this study shows that proteomics is a valuable tool for assessing acrylamide toxicity in the affected tissues.</p>
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Preparative Polyacrylamide-Agarose Gel Electrophoresis of Proteins and RNAS by the Use of New Device
Abstract Quantitatively reproducible results were obtained by using a new device for preparative gel electrophoresis combined with polyacrylamide-agarose composite gel. When an adequate gel-buffer system was selected according to the procedure described in this paper, proteins and RNA's were well separated and recovered. The new device for preparative gel electrophoresis and the method for preparation of polyacrylamide-agarose composite gel are presented together with the elution profiles of the recovered substances.
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Plasmid-mediated beta-lactamases from strains of Escherichia coli and Pseudomonas aeruginosa were separated by isoelectric focusing on a 0.8-mm thin-layer agarose gel with a pH gradient of 3.5 to 9.5. Their banding patterns and isoelectric points were compared with those obtained with a 2.0-mm polyacrylamide gel as the support medium. The agarose method produced banding patterns and isoelectric points which corresponded to the polyacrylamide gel data for most samples. Differences were observed for HMS-1 and PSE-1 beta-lactamases. The HMS-1 sample produced two highly resolvable enzyme bands in agarose gels rather than the single faint enzyme band observed on polyacrylamide gels. The PSE-1 sample showed an isoelectric point shift of 0.2 pH unit between polyacrylamide and agarose gel (pI 5.7 and 5.5, respectively). The short focusing time, lack of toxic hazard, and ease of formulation make agarose a practical medium for the characterization of beta-lactamases.
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The new chromogenic reagent,dibromo-p-chloro-sulfosulfonazo,was synthesized,which shows a good color reaction with cerium rare earth metal,and higher selectivity.The method can be applied to the photometric analysis of cerium in rare earth ore.
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