21 results on '"Błaszczyk U"'
Search Results
2. Determination of heterocyclic aromatic amines (HAs) content in samples of household-prepared meat dishes
- Author
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Warzecha, L., Janoszka, B., Błaszczyk, U., Stróżyk, M., Bodzek, D., and Dobosz, C.
- Published
- 2004
- Full Text
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3. Clean-up procedures for the analysis of heterocyclic aromatic amines (aminoazaarenes) from heat-treated meat samples
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Janoszka, B, Błaszczyk, U, Warzecha, L, Strózyk, M, Damasiewicz-Bodzek, A, and Bodzek, D
- Published
- 2001
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4. Killer Yeasts And Their Application
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Błaszczyk Urszula
- Subjects
antagonism ,killer yeasts ,killer toxin ,antagonizm ,drożdże killerowe ,toksyna killerowa ,Microbiology ,QR1-502 - Abstract
A killer phenotype, associated with the production and secretion of killer toxins, is widespread among yeasts and in competitive conditions gives an advantage to killer yeast strains in relation to other, sensitive microorganisms colonizing the same ecological niche. Killer toxins are proteins, usually glycoproteins, that are able to kill strains of susceptible yeasts. Each killer toxin has unique properties that vary depending on the strain of yeast that produces it. These differences concern the location of genes that encode toxins, molecular weight, as well as mechanisms of action. Some strains of killer yeast are characterized by a wide range of antagonistic activity, inhibit the development of a number of yeast strains, as well as molds, and have been studied for many years in terms of their biotechnological potential. Killer yeast and its toxins can find potential application in many fields: in the production of food and beverages, especially during wine fermentation and maturation, in biological control of plant pathogens, in yeast biotyping and as new antifungal agents.
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- 2019
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5. Antioxidant enzyme activities in rabbits under oxidative stress induced by high fat diet
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Skowron Marta, Zalejska-Fiolka Jolanta, Błaszczyk Urszula, Chwalińska Ewa, Owczarek Aleksander, and Birkner Ewa
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rabbit ,oil ,antioxidant enzymes ,mda ,oxidative stress ,Veterinary medicine ,SF600-1100 - Abstract
The aim of this study was to investigate whether the type and form of oil (raw/non-oxidised (N) or post-frying/oxidised (O)) consumed in high-fat diets affect the oxidative status of an organism, as observed by malondialdehyde (MDA) concentration as an oxidative factor and antioxidant enzyme activity.
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- 2018
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6. Effect of Wickerhamomyces anomalus and Pichia membranifaciens killer toxins on fermentation and chemical composition of apple wines produced from high-sugar juices
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Błaszczyk, U., Sroka, P., Satora, P., and Robert Dulinski
7. The research and development process of high-performance, spunlace non-woven fabric production at Novita s.A
- Author
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Waldemar Woźniak, Sąsiadek, M., Jankowski, B., Błaszczyk, U., and Stryjski, R.
8. Mixed Cultures of Saccharomyces kudravzevii and S. cerevisiae Modify the Fermentation Process and Improve the Aroma Profile of Semi-Sweet White Wines.
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Błaszczyk U, Satora P, and Noga Ł
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- Saccharomyces cerevisiae, Fermentation, Odorants analysis, Ethanol analysis, Wine analysis, Saccharomyces
- Abstract
The purpose of the study was to evaluate the impact of the Saccharomyces cerevisiae and S. kudriavzevii mixed culture on the fermentation, chemical and aromatic composition of semi-sweet white wines. The variables tested in the experiment were the initial ratio of yeast in mixed cultures and the time of inoculation of the S. kudriavzevii co-culture. The addition of S. kudriavzevii to the inoculum did not significantly change the chemical composition of the wines obtained. No reduction in ethanol yield was found in mixed culture fermented wines; however, in some variants of the experiment, the ethanol content was higher. The mixed cultures of S. cerevisiae and S. kudriavzevii increased the level of volatile compounds in white grape wines. Wines fermented with the co-culture of S. kudriavzevii were characterized by a more diversified ester profile. The mixed cultures of S. cerevisiae and S. kudriavzevii raised the levels of terpenes in white wines. The most promising results were obtained for mixed culture variants, in which S. kudriavzevii was sequentially inoculated on the sixth day of fermentation.
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- 2022
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9. Relationship between Postural Stability, Lead Content, and Selected Parameters of Oxidative Stress.
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Wąsik M, Miśkiewicz-Orczyk K, Słota M, Lisowska G, Kasperczyk A, Bellanti F, Dobrakowski M, Błaszczyk U, Bułdak RJ, and Kasperczyk S
- Subjects
- Humans, Male, Proprioception, Oxidative Stress, Erythrocytes, Lead, Posture
- Abstract
This study attempts to determine whether the increased blood lead concentration affects the posturographic test and to determine the relationship between the parameters of posture stability and selected parameters of oxidative stress. The study population consisted of 268 male employees and was divided into two equal subgroups, depending on the lead content in the blood. A posturographic examination was performed. Concentrations of lead, cadmium, zinc protoporphyrin, selected essential elements, and selected markers of oxidative stress in the blood were tested. Higher blood lead concentrations positively affected the values of the sway results: the field and the mean velocity of the center of the feet pressure in posturography. The absolute value of the proprioception ratio was similar in both subgroups. The content of malondialdehyde shows a statistically significantly higher value in a subgroup with high blood lead concentration and exhibits significant correlations only with some of the posturography parameters. The lipofuscin content in erythrocytes correlates with the results of the posturography test. Zinc protoporphyrin, total oxidant status, total antioxidant capacity, selected minerals, and metals did not correlate with the results of the posturography test. In conclusion, posturographic results correlate only with selected markers of oxidative stress, so it can be assumed that the effect on the body balance is only partial.
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- 2022
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10. Successful correction of hyperglycemia is critical for weight loss and a decrease in cardiovascular risk in obese patients.
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Zalejska-Fiolka J, Birková A, Hubková B, Wielkoszyński T, Čižmárová B, Szlachta B, Fiolka R, Błaszczyk U, Wylęgała A, Kasperczyk S, Grzanka A, Mareková M, and Toborek M
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- Body Mass Index, Glucose, Heart Disease Risk Factors, Humans, Lipids, Obesity metabolism, Risk Factors, Weight Loss, Atherosclerosis, Cardiovascular Diseases etiology, Cardiovascular Diseases prevention & control, Hyperglycemia complications, Insulin Resistance physiology
- Abstract
Weight loss is recommended for obese patients with cardiovascular risk; however, it remains questionable how hyperglycemia affects this process. To address this problem, we aimed to determine the association between weight loss, lipid profile, and body mass parameters in obese normoglycemic and hyperglycemic patients. Obese (body mass index30 kg/m
2 ) normoglycemic and hyperglycemic volunteers were placed on a weight reduction program that included a balanced, low-calorie diet and moderate exercise for 6 months. Participants were assessed for serum glucose, β-cell functions, insulin resistance, lipid metabolism, lipoprotein profile, and body mass parameters. This weight reduction program fully normalized serum glucose levels only in a subpopulation of patients. These individuals also exhibited a significant reduction in body weight, and significant improvement in serum lipid profile and insulin resistance. In contrast, the patients that remained hyperglycemic were characterized by persistent insulin resistance, increased levels of atherogenic fractions of LDL and HDL lipoproteins, and elevated values of a modified Atherogenic Index of Plasma. Correlation analysis indicated a strong positive association between the modified Atherogenic Index of Plasma with atherogenic lipid profile, insulin resistance, and body mass parameters, indicating its usefulness in clinical studies in obese patients. Overall, our data indicate that successful treatment of hyperglycemia facilitates weight loss and improves the composition of blood lipids, while persisting hyperglycemia negatively affects the weight loss process and maintains an atherogenic lipid profile. Because hyperglycemia predisposes to cardiovascular disorders, its correction should be the primary goal during weight reduction therapy., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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11. Application of Bioactive Coatings with Killer Yeasts to Control Post-Harvest Apple Decay Caused by Botrytis cinerea and Penicillium italicum .
- Author
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Błaszczyk U, Wyrzykowska S, and Gąstoł M
- Abstract
A new method was proposed to produce alginate bio-films containing Pichia membranifaciens and Wickerhamomyces anomalus killer yeast to control the post-harvest fungal decay in organic apples caused by Botrytis cinerea and Penicillium italicum . Coatings with W. anomalus killer yeast effectively controlled the growth of P. italicum during storage at 22 °C. W. anomalus killer yeast incorporated in alginate reduced the P. italicum incidence from 90% (control) to 35% after 14 days of storage at 22 °C. Alginate biofilms with W. anomalus or P. membranifaciens also limited the incidence of the fungal decay of apples inoculated with B. cinerea compared with the control fruits, although the antagonistic capability against B. cinerea was lower than against P. italicum . The survival of W. anomalus cells in alginate coating was higher than P. membranifaciens . The incorporation of killer yeasts into alginate had no significant effect on the mechanical properties (tensile strength, percent elongation at break) of alginate coating, however, they increased the thickness of the biofilm. The bioactive coating reduced the fruit weight loss and had no significant effects on the fruit firmness during storage at 2 °C. As organic apples, produced without any synthetic fungicides, are especially prone to fungal decay during storage, the proposed alginate biofilms containing killer yeast seem to be a very promising solution by offering non-chemical, biological control of post-harvest pathogens.
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- 2022
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12. Loss of Skeletal Muscle Mass and Intracellular Water as Undesired Outcomes of Weight Reduction in Obese Hyperglycemic Women: A Short-Term Longitudinal Study.
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Zalejska-Fiolka J, Birková A, Wielkoszyński T, Hubková B, Szlachta B, Fiolka R, Błaszczyk U, Kuzan A, Gamian A, Mareková M, and Toborek M
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- Adipose Tissue metabolism, Female, Humans, Longitudinal Studies, Muscle, Skeletal metabolism, Obesity metabolism, Weight Loss physiology, Body Composition physiology, Water metabolism
- Abstract
The current study is focused on the influence of hyperglycemia on weight loss in obese premenopausal women. Specifically, the study evaluated the impact of a six-month individualized low-calorie diet combined with moderate exercise on weight reduction and glucose metabolism in obese women with normoglycemia compared to obese women with moderate hyperglycemia. The results indicated that patients with normoglycemia achieved a successful weight loss, which was connected to a decrease in adipose tissue and reflected by diminished content of visceral fat area (VFA) and percent body fat. In contrast, weight reduction in patients with hyperglycemia was connected not only to the loss of VFA but also to undesired decrease in skeletal muscle mass as well as intracellular and total body water. These unfavorable outcomes were observed despite normalization of glucose metabolism reflected by statistically significant lowering glucose, fructosamine, advanced glycation end-products, and HOMA-IR levels. Overall, the obtained results indicate the importance of the measurement of the carbohydrate profile in obese women and the need for an early introduction of weight reduction strategies before the development of hyperglycemia.
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- 2022
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13. Prognostic Value of the Modified Atherogenic Index of Plasma during Body Mass Reduction in Polish Obese/Overweight People.
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Zalejska-Fiolka J, Hubková B, Birková A, Veliká B, Puchalska B, Kasperczyk S, Błaszczyk U, Fiolka R, Bożek A, Maksym B, Mareková M, and Birkner E
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- Adult, Atherosclerosis blood, Atherosclerosis epidemiology, Biomarkers, Body Composition, Cholesterol, HDL blood, Cholesterol, LDL blood, Diet, Exercise, Female, Humans, Male, Middle Aged, Obesity epidemiology, Obesity therapy, Poland epidemiology, Prognosis, Risk Factors, Weight Reduction Programs methods, Cardiovascular Diseases epidemiology, Lipids blood, Overweight epidemiology, Overweight therapy, Weight Loss physiology
- Abstract
Although weight loss is recommended for obese patients, it remains questionable how much weight loss is optimal. A novel index that accurately determines the risk of cardiovascular diseases (CVDs) in terms of weight loss is needed. The modified Atherogenic Index of Plasma (AIP), presented here is unique in the literature. It is calculated based on data for anti-atherogenic, high-density lipoprotein cholesterol (HDL-C) fractions, instead of the total HDL-C. This study investigates whether weight loss correlates with CVD risk, and whether the modified AIP allows more accurate diagnostics in obese/overweight people. According to the increase or decrease of AIP during weight loss, 52 Polish patients were subdivided into two groups: group I (increased AIP; n = 16) and group II (decreased AIP; n = 36). The patients' body mass composition and fasting serum lipid parameters (total cholesterol, triglycerides, HDL-C, and LDL-C (low-density lipoprotein cholesterol)), and cholesterol in 21 lipoprotein sub-fractions were determined. Over six months, all patients reduced their body mass by about 10%. There were no significant differences in anthropometric measures between groups. Increases in large and intermediate HDL-C fractions 1 to 6 and decreases in smaller fractions 7 to 10 were observed in group II. In group I, HDL-C fractions 1 and 10 decreased, while cholesterol in other fractions increased. Increases were observed in the antiatherogenic HDL-C of 52% of group II and 4% of group I. As for atherogenic HDL-C, a decrease of 24% was observed in group II and an increase of 9% in group I. In group I, increases of very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and large LDL fractions were noticed, and the reverse in group II. The results show that the modified AIP is a more accurate indicator of CVD risk than existing indices, and that uncontrolled weight reduction does not necessarily have a beneficial influence, and may adversely affect the cardiovascular system., Competing Interests: The authors declare no conflict of interest.
- Published
- 2018
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14. [The influence of methionine and vitamin E on oxidative stress in rats’ liver exposed to sodium fluoride]
- Author
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Stolecka D, Błaszczyk U, Zalejska-Fiolka J, Romuk E, Owczarek A, and Birkner E
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- Animals, Antioxidants pharmacology, Catalase, Glutathione Peroxidase, Glutathione Reductase, Glutathione Transferase, Liver enzymology, Liver metabolism, Male, Rats, Rats, Wistar, Sodium Fluoride toxicity, Superoxide Dismutase, Superoxide Dismutase-1, Liver drug effects, Methionine pharmacology, Oxidative Stress drug effects, Vitamin E pharmacology
- Abstract
Background: Fluorine influences many processes occurring in the organism. Controversies over the evaluation of the biological effects of this substance are due to a small difference between tolerable and toxic fluorine doses. One of the main mechanisms of the fluorine toxic action is its ability to induce oxidative stress via reactive oxygen species generation and antioxidant defense system impairment. It is important to evaluate possible interactions between fluorine and other substances that may increase or decrease its toxicity., Material and Methods: The study lasted for 35 days. Twenty-four rats were divided into 4 groups: the control, with sodium fluoride (NaF) in the diet, with sodium fluoride, methionine and vitamin E (NaF+M+E) in the diet, with sodium fluoride and vitamin E (NaF+E) in the diet. The biochemical analysis conducted in animal liver homogenates included determination of activities of: total superoxide dismutase (t-SOD), superoxide dismutase with copper and zinc (CuZnSOD), superoxide dismutase with manganese (MnSOD), glutathione peroxidase (GPX), catalase (CAT), glutathione reductase (GR), glutathione S-transferase (GST) and the malondialdehyde (MDA) concentration., Results: The activities of CuZn- SOD, GPX, CAT and MDA concentration were changed significantly. There were no differences in the activities of t-SOD, MnSOD, GR and GST among the experiment., Conclusions: In the conducted experiment, the run-out of enzymatic protection of liver by decreasing of the activities of antioxidant enzymes (CAT and GPX) and increasing the MDA concentration in NaF group was observed. The addition of vitamin E and methionine does not significantly stimulate the enzymatic antioxidant system, however, it causes of MDA concentration decreases. Med Pr 2018;69(4):403–412, (This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.)
- Published
- 2018
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15. [Effect of α-lipoic acid on free radical processes in serum of rats on high fat diet].
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Cichoń M, Błaszczyk U, and Zalejska-Fiolka J
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- Animals, Creatinine blood, Free Radicals blood, Lipid Peroxides blood, Male, Malondialdehyde blood, Rats, Rats, Wistar, Antioxidants pharmacology, Diet, High-Fat adverse effects, Lipid Peroxidation drug effects, Thioctic Acid pharmacology
- Abstract
Background: Oils are often fried which reduces their beneficial biological and nutritional properties, contributing to disturbances in homeostasis. Some antioxidant substances can improve stability of oils. The aim of the study was to examine the effect of α-lipoic acid (ALA) on the concentration of sulfhydryl groups, lipid peroxides, malondialdehyde, creatinine and urea in serum of rats fed high fat diet for 3 months., Material and Methods: Thirty six Wistar rats were equally divided into 6 groups: the control group on standard breeding diet (SB), oxidized oil (OU) group on SB with 10% oxidized oil, ALA10 group on SB with ALA 10 mg/kg of body weight (b.w.), OU+ALA10 group on SB with oxidized oil and ALA (10 mg/kg b.w.), ALA50 group on SB with ALA in a dose of 50 mg/kg b.w., OU+ALA50 group on SB with oxidized oil and ALA (50 mg/kg b.w.). Oil was oxidized in 180°C for 6 h., Results: We observed decrease in concentration of protein sulfhydryl (PSH) groups in all study groups except for ALA10 vs. control group (C) and increase in OU+ALA10 and OU+ALA50 vs. OU; increase in the lipid hydroperoxide (LHP) concentration in OU, OU+ALA10 and OU+ALA50 vs. C and decrease in all study groups vs. OU; increase of malondialdehyde (MDA) in OU vs. all other groups. And also increase in creatinine and urea concentration in OU group., Conclusions: High fat diet rich in oxidized oil intensifies the lipid peroxidation process and oxidation of sulfhydryl groups. It can also impair kidney function. Administration of lipoic acid in a dose of 10 mg/kg b.w. inhibits the lipid peroxidation and protects sulfhydryl groups. Med Pr 2017;68(3):391-399., (This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.)
- Published
- 2017
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16. The Influence of α-Lipoic Acid and Garlic Administration on Biomarkers of Oxidative Stress and Inflammation in Rabbits Exposed to Oxidized Nutrition Oils.
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Zalejska-Fiolka J, Wielkoszyński T, Rokicki W Jr, Dąbrowska N, Strzelczyk JK, Kasperczyk A, Owczarek A, Błaszczyk U, Kasperczyk S, Stawiarska-Pięta B, Birkner E, and Gamian A
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- Administration, Oral, Animals, Antioxidants administration & dosage, Biomarkers blood, Cytokines immunology, Dietary Supplements, Inflammation chemically induced, Inflammation prevention & control, Male, Oxidation-Reduction, Plant Oils, Rabbits, Garlic chemistry, Inflammation immunology, Inflammation Mediators immunology, Oxidative Stress immunology, Plant Extracts administration & dosage, Thioctic Acid administration & dosage
- Abstract
We hypothesized that addition of substances with antioxidant activity could decrease the concentrations of biomarkers of oxidative stress and inflammatory process, thus inhibiting nonalcoholic steatohepatitis development. We investigated the influence of α-lipoic acid (ALA) and garlic administration on the development of adverse changes in rabbit liver and serum under oxidative stress conditions induced with HFD from oxidized oils. We determined 8-hydroxy-2'-deoxyguanosine (8 OHdG) and malondialdehyde (MDA) in liver homogenates, total oxidant status (TOS), lipid peroxides (LOO) and tumor necrosis factor alpha (TNFα) in blood serum, and TNFα and IL-1α genes expression in liver. The results indicate that the intake of dietary ALA and garlic was significantly associated with decreases of 8 OHdG and MDA levels in rabbits' liver tissue as well as TOS and LOO levels in rabbits' serum. Similarly, TNFα and IL-1α gene expressions were suppressed due to ALA and garlic supplementation. The histopathological analysis confirmed that HFD results in liver disorder leading to steatosis. This adverse effect of HFD was ameliorated by the supplementation of ALA and garlic. The obtained results indicate a beneficial effect of ALA and garlic administration by reducing the oxidative stress intensity and the levels of some proinflammatory cytokines in rabbits fed HFD.
- Published
- 2015
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17. Analysis of azaarenes in pan fried meat and its gravy by liquid chromatography with fluorescence detection.
- Author
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Błaszczyk U and Janoszka B
- Abstract
A method for analysis of six azaarenes (benzo[h]quinoline, benzo[a]acridine, benzo[c]acridine, dibenzo[a,c]acridine, dibenzo[a,j]acridine and dibenzo[a,h]acridine) in thermally treated high-protein food has been described. The clean-up procedure used based on alkaline hydrolysis, tandem solid phase extraction on columns filled with Extrelut - diatomaceous earth and cation exchanger (propyl sulfonic acid), enabled a selective isolation of carcinogenic compounds belonging to benzoacridines and dibenzoacridines from samples of cooked meat and its gravy. The isolated fractions of aza-PAHs were analysed by high-performance liquid chromatography with fluorescence detection. The detection limits for the azaarenes were between 0.0001ng and 0.005ng loaded on column. The recoveries for the four-ring and five-ring azaarenes were from 55% to 67%. Two types of dishes prepared from pork by pan-frying were investigated. Total contents of the benzoacridines and dibenzoacridines determined in cooked meat were 1.57 and 2.50ng/g in collar and chop samples, respectively; their gravies contained 0.34 and 0.59ng of these azaarenes per g of cooked meat., (Copyright © 2007 Elsevier Ltd. All rights reserved.)
- Published
- 2008
- Full Text
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18. Effect of garlic supplementation on erythrocytes antioxidant parameters, lipid peroxidation, and atherosclerotic plaque formation process in oxidized oil-fed rabbits.
- Author
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Zalejska-Fiolka J, Kasperczyk A, Kasperczyk S, Błaszczyk U, and Birkner E
- Subjects
- Animals, Brassica rapa, Fatty Acids, Monounsaturated, Glutathione Peroxidase blood, Lipid Peroxidation, Male, Malondialdehyde blood, Oxidation-Reduction, Plant Oils, Rabbits, Rapeseed Oil, Superoxide Dismutase blood, Antioxidants analysis, Coronary Artery Disease prevention & control, Erythrocytes metabolism, Garlic
- Abstract
Effect of garlic supplementation on blood antioxidant status, lipid peroxidation, and coronary plaque formation process was investigated in oxidized oil-fed rabbits. Eighteen adult male mixed European rabbits were given a balanced diet (21 g% protein, 34 g% fat, 45 g% carbohydrate), which contained isocaloristic addition of nonoxidized or oxidized rapeseed oil in the presence and absence of garlic. The experiment lasted 24 weeks. At the beginning and every 6 weeks, rabbits were weighed, and blood was taken. To evaluate the antioxidant status of the rabbits, erythrocytes malondialdehyde (MDA) concentration, total superoxide dismutase (t-SOD), and glutathione peroxidase (GPX) activations were determined. After the experiment was completed, aortas were dissected for histological examinations. Changes in the contents of the above parameters and histological examinations showed that oxidized rapeseed, oil administered to rabbits, caused the development of atherosclerotic changes and disturbed antioxidant status. The addition of garlic in such diets inhibited atherosclerotic changes in the aorta wall, and it is related to the homeostatic activity of antioxidative enzymes and lipid peroxidation.
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- 2007
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19. Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by differential scanning calorimetry.
- Author
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Błaszczyk U and Wasylewski Z
- Subjects
- Calorimetry, Differential Scanning, Circular Dichroism, DNA chemistry, DNA genetics, Nucleic Acid Denaturation, Peptide Fragments chemistry, Peptide Fragments metabolism, Protein Denaturation, Protein Folding, Thermodynamics, Transition Temperature, Cyclic AMP metabolism, Cyclic AMP Receptor Protein chemistry, Cyclic AMP Receptor Protein metabolism, DNA metabolism, DNA-Binding Proteins chemistry, DNA-Binding Proteins metabolism, Escherichia coli chemistry
- Abstract
The cyclic AMP receptor protein (CRP) regulates the expression of many genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. In this study, we have used differential scanning calorimetry (DSC) and circular dichroism (CD) to measure the enthalpy change and melting temperature of the apo-CRP and CRP complexes with cAMP or DNA sequences lac, gal, and palindromic ICAP. DSC and CD measurements showed irreversible thermal denaturation process of CRP. Enthalpy of dissociation of the protein-DNA complex, as measured by DSC, depends on the DNA sequence. The thermal transition of the protein in CRP-DNA complexes, measured by CD, indicates that the protein stability in the complex is also DNA sequence-dependent.
- Published
- 2003
- Full Text
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20. Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli.
- Author
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Polit A, Błaszczyk U, and Wasylewski Z
- Subjects
- Allosteric Regulation, Cyclic AMP metabolism, Cyclic AMP Receptor Protein metabolism, DNA metabolism, Escherichia coli, Escherichia coli Proteins metabolism, Fluorescence Polarization, Fluorescence Resonance Energy Transfer, Macromolecular Substances, Models, Molecular, Naphthalenesulfonates chemistry, Protein Conformation drug effects, Spectrometry, Fluorescence, Cyclic AMP chemistry, Cyclic AMP Receptor Protein chemistry, DNA chemistry, Escherichia coli Proteins chemistry
- Abstract
cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP-(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP-(cAMP)4 complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMP binding in the CRP-(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP-(cAMP)2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 A in apo-CRP to 18.7 A in the CRP-(cAMP)2 complex. The binding of cAMP in the CRP-(cAMP)4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to approximately 20 A. The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein.
- Published
- 2003
- Full Text
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21. Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by dynamic light scattering and time-resolved fluorescence anisotropy methods.
- Author
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Błaszczyk U, Polit A, Guz A, and Wasylewski Z
- Subjects
- Chymotrypsin metabolism, Cyclic AMP Receptor Protein chemistry, DNA-Binding Proteins chemistry, DNA-Binding Proteins metabolism, Escherichia coli genetics, Escherichia coli Proteins chemistry, Fluorescence Polarization, Fluorescent Dyes metabolism, Light, Promoter Regions, Genetic, Scattering, Radiation, Time Factors, Cyclic AMP metabolism, Cyclic AMP Receptor Protein metabolism, DNA, Bacterial metabolism, Escherichia coli metabolism, Escherichia coli Proteins metabolism
- Abstract
Cyclic AMP receptor protein (CRP) regulates the expression of more than 100 genes in Escherichia coli when complexed with cyclic AMP. Dynamic light scattering (DLS) and fluorescence decay anisotropy measurements of CRP were performed in solution, in the absence and presence of cAMP. We have also measured the effect of DNA sequences, including lac and gal promoter sequences, on the shape of CRP-DNA complexes. DLS measurements show that upon cAMP binding at low nucleotide concentration, the Stokes radius decreases from the value of 2.8 nm for apo-CRP to the value of 2.7 nm. At higher cAMP concentration, only a very small further decrease was detected. Fluorescence anisotropy decay measurements, with the use of CRP labeled at Cys-178 with 1,5-I-AENS, indicate that apo-CRP exhibits two rotational correlation times. The longer time, theta1 = 23.3 ns, corresponds to the overall motion of the protein, and the shorter time, theta2 = 1.4 ns, exhibits segmental mobility of the C-terminal domain of CRP. Binding of cAMP into CRP induced substantial increase of theta1 to the value of 30.7 ns, whereas theta2 remained unchanged. The DLS measurements indicate that the binding of CRP into a fragment of DNA possessing a sequence of lac promoter induces a larger increase in the Stokes radius of lac-CRP complex than in case of gal-CRP complex. Similarly, a higher change was detected in rotational correlation time, theta1, in the case of lac-CRP complex than in case of gal-CRP. Because the lac and gal promoters are characteristic for the two different classes of CRP-dependent promoters, one can expect that the observed differences in lac-CRP and gal-CRP complexes are important in activation of transcription in Escherichia coli.
- Published
- 2001
- Full Text
- View/download PDF
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