Enzyme Protein Content Research Articles

English

In screening for marine bacterial polysaccharide lytic enzymes, the most potent isolate was identified as Bacillus cereus NRC-20. This strain showed high alginase, dextrinase, pectinase and carboxymethyle-cellulase productivity when production medium was adjusted at pH 6.0 and contains (g/l): 3.0 sodium alginate, 3.0 malt extract, 30.0 jatropha cake and inoculated with 8% (v/v) of a 24 h old cell suspension and incubated at 30°C for 48 h on a rotary shaker at 200 rpm. These enzymes mixture were precipitated by 60% acetone and purified by using sephadex G-100, whereas SDS-PAGE suggested a molecular weight of approximately 17, 20, 89 and 279 KDa, respectively. The optimum partially purified enzymes activity when enzyme protein concentration was 27.4 mg/ml and substrate concentration 5.0 g/l shown after 90 min of incubation at 30°C and pH 5 and stable at 60°C for 60 min. The enzymes activities were enhanced in the presence of 0.02 M Na+ and K+ ions and completely inactivated by Mg+2, Cu+2, Co+2 and Pb+2 The enzymes mixture affected the matrix integrity of different microbial biofilms artificially grown on stainless steel sheets of Bacillus subtilis NRRL B-4219, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27953 and Escherichia coli ATCC 25922. Key words: Bacillus cereus, polysaccharide lytic enzymes production, fermentation parameters, enzyme characterization, biofilm removal.

Exogenous nitric oxide improves chilling tolerance of Chinese cabbage seedlings by affecting antioxidant enzymes in leaves

The effects of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, on growth of Chinese cabbage seedlings and antioxidant enzyme activity in leaves were studied under chilling stress. The results showed that the growth of Chinese cabbage seedlings was strongly inhibited when plants were exposed to chilling, whereas spraying SNP in shoots significantly alleviated the inhibition of growth from chilling stress. The plant height, root length, fresh and dry weight of chilling plants all increased. Chilling caused increases of membrane permeability and antioxidant enzymes activity with the exception of catalase after 8 days of treatment. Meanwhile, chilling also resulted in the decrease of chlorophyll content, accumulation of malondialdehyde (MDA) and protein in leaves. Interestingly, application SNP further enhanced the antioxidant enzyme activities, the chlorophyll and protein content, on the contrary, reduced the member permeability and MDA content. Therefore, it was concluded that NO protected plants from oxidative damage and promoted the growth by enhancing activity of antioxidant enzymes in leaves sufficiently to lower membrane injury. However, exogenous NO had no significant effects on seedling in normal conditions.

Effect of delayed processing on nutrient composition, ph and organoleptic quality of pond raised tilapia (Oreochromis shiranus) stored at ambient temperature

Fish are a nutrient rich food but highly perishable due to its high water activity , protein content, neutral pH and presence of autolytic enzymes . This explains why fresh fish quality deteriorates rapidly if not properly stored after catch such as use of low temperature . The implication is that delayed processing of fresh fish negatively influences consumer taste of fish . Further, fresh fish that are not processed on time lose nutrients due to spoilage. In Malawi, most fresh fish are sold in the open due to lack of storage facilities such as refrigerators – a condition that accelerates spoilage of fish . This study was carried out to assess the effect of delayed processing on nutrient composition, pH level and organoleptic quality of pond raised tilapia - Oreochromis shiranus which is the commonest grown tilapia in Malawi. Thirty freshly pond harvested O. shiranus fish (average weight 100g ) were cleaned then deep fried in edible cooking vegetable oil at time intervals of 0, 6 and 12 hours from catch and stored at ambient temperature. A pre -trained sensory evaluation panel consisting six members described changes in the sensory quality of the fresh and fried fish at every sampling time interval. Crude protein, crude fat, ash, moisture content and pH were also determined at each sampling time. Determined c rude protein and crude fat decreased while ash content increased with storage time though these were not significantly different from base line (P>0.05). Moisture content decreased from 0 (fresh) to 6 hours then increased again up to 12 hours of storage. Ash and moisture content correlated positively (0.93 and 0.79% respectively) , whilst crude protein, crude fat and pH were negatively correlated ( -0.91, -0.05 and -0.97, respectively) to sensory quality demerit scores. pH decreased from 5.94 at 0 hours to 5.92 after 6 hours then increased sharply to 5.98 after 12 hours of ambient fish storage. Consumers started to significantly (P<0.05) dislike the fish after 7 hours and eventually rejected the samples after 12 hours of ambient storage time. The study recommends that for optimum consumer satisfaction and nutritional benefit, fresh O. shiranus should be processed at most 7 hours after harvesting .

Effect of commercially available green and black tea beverages on drug-metabolizing enzymes and oxidative stress in Wistar rats

The effect of commercially available green tea (GT) and black tea (BT) drinks on drugmetabolizing enzymes (DME) and oxidative stress in rats was investigated. Male Wistar rats were fed a laboratory chow diet and GT or BT drink for 5weeks. Control rats received de-ionized water instead of the tea drinks. Rats received the GT and BT drinks treatment for 5weeks showed a significant increase in hepatic microsomal cytochrome P450 (CYP) 1A1 and CYP1A2, and a significant decrease in CYP2C, CYP2E1 and CYP3A enzyme activities. Results of immunoblot analyses of enzyme protein contents showed the same trend with enzyme activity. Significant increase in UDP-glucuronosyltransferase activity and reduced glutathione content in liver and lungs were observed in rats treated with both tea drinks. A lower lipid peroxide level in lungs was observed in rats treated with GT drink. Electrophoretic mobility shift assay revealed that both tea drinks decreased pregnane X receptor binding to DNA and increased nuclear factor-erythroid 2 p45-related factor 2 binding to DNA. These results suggest that feeding of both tea drinks to rats modulated DME activities and reduced oxidative stress in liver and lungs. GT drink is more effective on reducing oxidative stress than BT drink.

Study of enzyme activities and protein content of beluga (Huso huso) semen before and after cryopreservation.

Knowledge gained regarding the biochemical processes that occur during sperm collection, processing and freezing-thawing might improve current sperm cryopreservation techniques. In our present study, we determined the effect of cryopreservation on the total protein concentration (TP) and the activities of certain enzymes in semen samples from the beluga (Huso huso). The TP content of the seminal plasma of fresh semen was 0.47 ± 0.026 g/l, and the TP after cryopreservation was 1.86 ± 0.6 g/l. The activities of acid phosphatase (0.82 ± 0.042 U/l), lactate dehydrogenase (234.4 ± 19.4 U/l), arylsulfatase (143.1 ± 32.5 U/l) and β-N-acetylglucosaminidase (58.39 ± 4.14 U/l) in the seminal plasma of fresh semen were significantly lower than those in the supernatant of frozen-thawed semen samples (7.43 ± 0.64, 3224.6 ± 167.2, 422.6 ± 21.3 and 90.2 ± 5.37 U/l respectively). These parameters may be useful as biomarkers for estimating damage to the cell membrane of spermatozoa caused by freezing-thawing.

Evaluation of ∆2-pioglitazone, an analogue of pioglitazone, on colon cancer cell survival: Evidence of drug treatment association with autophagy and activation of the Nrf2/Keap1 pathway.

Thiazolidinediones have been shown to exhibit anti-proliferative effects against cancer cells derived from diverse tissue origins both in vivo and in vitro. We studied the anti-proliferative impact of 5-{4-(2-(5-ethyl-pyridin-2-yl)-ethoxy)-benzylidene}-thiazolidine-2,4-dione (∆2-pioglitazone), an analogue of pioglitazone, which binds to the nuclear peroxisome proliferator activated receptor-γ without activating it, on human adenocarcinoma-derived HT29 and HCT116 cells. In HTC116 cells, exposure to ∆2-pioglitazone reduced cell growth, but HT29 cells reached the plateau phase of growth after three days. ∆2-pioglitazone treatment did not trigger cells to enter apoptosis but enhanced the autophagy process. The effect of ∆2-pioglitazone treatment was related to the increase of oxygen and nitric oxide-derived species production and decreased glutathione content. Moreover, pre-treatment with an antioxidant before addition of ∆2-pioglitazone limited cell growth inhibition, reduced the production of reactive species and attenuated autophagy within the cells. The impact of the drug was associated with activation of the Nrf2/Keap1 pathway as demonstrated by the increased protein content of several antioxidant enzymes, notably heme-oxygenase-1.

Normal protein content but abnormally inhibited enzyme activity in muscle carnitine palmitoyltransferase II deficiency

The biochemical consequences of the disease causing mutations of muscle carnitine palmitoyltransferase II (CPT II) deficiency are still enigmatic. Therefore, CPT II was characterized in muscle biopsies of nine patients with genetically proven muscle CPT II deficiency. Total CPT activity (CPT I+CPT II) of patients was not significantly different from that of controls. Remaining activities upon inhibition by malonyl-CoA and Triton X-100 were significantly reduced in patients. Immunohistochemically CPT II protein was predominantly expressed in type-I-fibers with the same intensity in patients as in controls. Western blot showed the same CPT II staining intensity ratio in patients and controls. CPT I and CPT II protein concentrations estimated by ELISA were not significantly different in patients and in controls. Citrate synthase activity in patients was significantly increased. Total CPT activity significantly correlated with both CPT I and CPT II protein concentrations in patients and controls. This implies (i) that normal total CPT activity in patients with muscle CPT II deficiency is not due to compensatory increase of CPT I activity and that (ii) the mutant CPT II is enzymatically active. The data further support the notion that in muscle CPT II deficiency enzyme activity and protein content are not reduced, but rather abnormally inhibited when fatty acid metabolism is stressed.

Novel effects of rosiglitazone on SMAD2 and SMAD3 signaling in white adipose tissue of diabetic rats

The effects of the proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone (ROSI) on the transforming growth factor (TGF)-β/SMAD signaling pathway in white adipose tissue (WAT) of diabetic rats were assessed. Six-week-old, male ZDF rats were fed a chow diet with (ZDF ROSI) or without (ZDF chow) ROSI (diet, 100 mg/kg) for 6 weeks. Subcutaneous (scWAT) and retroperitoneal (rpWAT) adipose tissues were excised to quantify the protein content/phosphorylation. ZDF ROSI animals showed enhanced glucose tolerance and mitochondrial protein content in both depots. The protein content of enzymes involved in fatty acid handling was increased in scWAT of ZDF ROSI animals. ZDF ROSI exhibited decreased phosphorylation of SMAD2 and SMAD3 exclusively in scWAT, along with increases in inhibitory SMAD7 and the E3 ubiquitin ligase SMURF2. In contrast, ROSI increased the protein content of SMAD4, TGF-β receptor I and II, and SMAD Anchor for Receptor Activation in scWAT. For the first time, the fact that ROSI inhibits SMAD2 and SMAD3 signaling in a depot-specific manner in diabetic rats was demonstrated. In scWAT, ROSI reduced SMAD2 and SMAD3 phosphorylation, likely through the inhibitory actions of SMAD7 and SMURF2. Induction of proximal components of the SMAD pathway may constitute a feedback mechanism to counteract ROSI-induced lipid synthesis in scWAT.

Thermo-Protective Role of 28-Homobrassinolide in <i>Brassica juncea</i> Plants

The present work was undertaken to study the effects of 28-homobrassinolide on growth, stress markers, antioxidative enzyme [superoxide dismutase (EC 1.15.1.1), guaiacol peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6) ] activities and protein content in 10 days old seedlings of Brassica juncea L. treated with different degrees (4°C, 44°C) of temperature. 28-homobrassinolide at 10-9 M concentration lowered temperature stress. Different degrees of temperature treatment alone decreased the enzyme activities and protein concentration of seedlings. However, pre-sowing treatments of 28-homobrassinolide lowered the temperature stress and enhanced the contents of MDA and proline, activities of enzymes and protein concentration of seedlings.

Improved mannanase production from Penicillium humicola and application for hydrolysis property

Objectives The aim of this research is to produce β-mannanases from a new microbial source using wastes in their nutrition medium. The enzyme produced can be used for the production of galactomanno-oligosaccharides, which are very useful in the health and medical fields. Materials and methods Seven fungal strains and five bacterial strains were tested for the production of β-mannanases. Enzyme activity, protein content, and biomass production were determined in all the cultures produced using standard methods. Optimization studies to maximize enzyme production from the most potent microorganisms, including culture conditions and medium compositions, were also carried out. Preliminary studies for the production of galactomanno-oligosaccharides from locust bean gum using partially purified enzymes were also carried out and followed by thin-layer chromatography techniques and Somogyi methods. Results and conclusion The highest mannanase activities were produced by Penicillium humicola (8.8 U/ml) and Penicillium spp. v (7.75 U/ml) in shaking cultures after 10 days using gum locust bean as a carbon source. Among 13 carbon sources examined, coffee residue and ceratonia seeds were the best carbon sources (10.3 and 8.9 U/ml, respectively) for P. humicola, whereas the best nitrogen source was a mixture of peptone, urea, and ammonium sulfate for the same microorganism. The optimum temperature and pH for enzyme reaction was 55 and 5.5°C, respectively. The enzyme was thermostable and retained 80% of its activity after 1 h at 50°C. The highest reducing sugar of 8900 μg/ml was obtained from locust bean gum hydrolytes after 28 h.

Study of Biosynthesis & Characterization of Microbial α-Amylase by Using Banana Peel Waste

Amylase is very essential for the conversion of starches into oligosaccharides. Alpha- amylase (1, 4-alpha-glucan-glucanohydrolases) are extracellular enzymes which hydrolyze 1, 4-Glycosidic bonds and also are endoenzymes split the substrate in the interior of the molecule. Bacillus subtilis gave maximum production of alpha amylase as compared with the other strain. The study showed that the protein content and enzyme activity are high in different samples. Soil sample was collected from sugarcane waste dumping site, Muradnagar, Ghaziabad, India. Sample were inoculated in two different media using banana peel waste as an alternative source of carbon, which is an economic and profitable due to the inherent nature of the banana waste itself, for α-amylase production by Bacillus subtilis as standard strain and by isolated culture from soil sample. The highest α-amylase enzyme production and protein content were detected in Media - II. We report that α-amylase activity was detected in the crude sample of Media - II at pH 7 and temperature 35◦C using maltose substrate. The isolated culture from soil sample shows high activity of α-amylase enzyme as compared to Bacillus subtilis standard strain. The α-amylase enzyme was used in the starch processing food, detergent, textile, paper, clinical and analytical industry. © 2015 iGlobal Research and Publishing Foundation. All rights reserved.

Lipoic acid prevents fructose-induced changes in liver carbohydrate metabolism: Role of oxidative stress

BackgroundFructose administration rapidly induces oxidative stress that triggers compensatory hepatic metabolic changes. We evaluated the effect of an antioxidant, R/S-α-lipoic acid on fructose-induced oxidative stress and carbohydrate metabolism changes. MethodsWistar rats were fed a standard commercial diet, the same diet plus 10% fructose in drinking water, or injected with R/S-α-lipoic acid (35mg/kg, i.p.) (control+L and fructose+L). Three weeks thereafter, blood samples were drawn to measure glucose, triglycerides, insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) and Matsuda indices. In the liver, we measured gene expression, protein content and activity of several enzymes, and metabolite concentration. ResultsComparable body weight changes and calorie intake were recorded in all groups after the treatments. Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR and lower Matsuda indices compared to control animals. Fructose fed rats showed increased fructokinase gene expression, protein content and activity, glucokinase and glucose-6-phosphatase gene expression and activity, glycogen storage, glucose-6-phosphate dehydrogenase mRNA and enzyme activity, NAD(P)H oxidase subunits (gp91phox and p22phox) gene expression and protein concentration and phosphofructokinase-2 protein content than control rats. All these changes were prevented by R/S-α-lipoic acid co-administration. ConclusionsFructose induces hepatic metabolic changes that presumably begin with increased fructose phosphorylation by fructokinase, followed by adaptive changes that attempt to switch the substrate flow from mitochondrial metabolism to energy storage. These changes can be effectively prevented by R/S-α-lipoic acid co-administration. General significanceControl of oxidative stress could be a useful strategy to prevent the transition from impaired glucose tolerance to type 2 diabetes.

Enhanced expression ofpgdSgene for high production of poly-γ-glutamic aicd with lower molecular weight inBacillus licheniformisWX-02

BACKGROUND The poly-γ-glutamic acid (γ-PGA) hydrolase of PgdS affects the molecular weight and yield of γ-PGA in Bacillus subtilis, while the effects of PgdS on the γ-PGA synthesis in Bacillus licheniformis have not been investigated. RESULTS By heterologous expression in Escherichia coli, the PgdS was characterized. The purified PgdS had an optimal γ-PGA degradation activity from pH 5.5 to 6.5, with an appropriate reaction temperature from 37 °C to 45 °C. The enzyme activity could be enhanced by Zn2+, Mn2+, Ca2+ and SDS, while inhibited by Hg2+ or Cu2+. By enhanced expression of pgdS gene in vivo, the enzyme activity, protein concentration and gene transcriptional level of PgdS were improved, and γ-PGA molecular weight decreased from 1000–1200 kDa to 600–800 kDa. Moreover, the γ-PGA yield reached 20.16 g L-1, increased by 54% than the native strain (13.11 g L−1). Gene transcript levels of glutamate transporters and poly-γ-glutamate synthetase were confirmed to be improved, which might be the reason for the increased γ-PGA production. CONCLUSION Enhanced expression of pgdS gene in B. licheniformis WX-02 helped to reduce the molecular weight and improve the yield of γ-PGA. This study provided a novel strategy to regulate the molecular weight and yield of γ-PGA. © 2013 Society of Chemical Industry

Photosynthesis, ion accumulation, antioxidants activities and yield responses of different cotton genotypes to mixed salt stress

This study analyzes the effects of soil salinity on photosynthetic character, osmoregulation, content of pigment, K+/Na+ ratio, lipoxygenase and antioxidants activities in functional leaves during the flowering and boll-forming stages of two cotton cultivars, namely, CCRI-44 (salt-tolerant) and Sumian12 (salt-sensitive), grown under different soil salinity conditions. In the control plants, non-significant differences were found in gas exchange, saturation irradiance (SI) and carotenoid (Car) content between the two cultivars. However, it showed higher K+/Na+ ratio, antioxidant enzyme activities, soluble sugar and protein contents, and lower chlorophyll (Chl) content and yield in CCRI-44. Salinity stresses remarkably increased soluble sugar and protein contents, lipoxygenase and the antioxidant activities, but decreased K+/Na+ ratio, Chl and Car contents, SI, photosynthetic capacities and yield, the extent being considerably larger in Sumian12 than CCRI-44. Although the soluble sugar, protein contents and the antioxidant activities of Sumian12 elevated more evidently under salt stresses, those variables never reached the levels of CCRI-44. Thus, CCRI-44 could maintain higher seed cotton yield than Sumian12 by sustaining higher osmoregulation and antioxidative abilities, which led to higher photosynthetic capacity. Hence, the salt-tolerant cotton cultivars could harmonize the relationship between CO2 assimilation (source) and the seed cotton yield (sink) under the experimental conditions. Key words: Cotton (Gossypium hirsutum L.), mixed salt stress, salt tolerance, photosynthesis, ion accumulation, antioxidant enzyme activity, seed cotton yield.

Partition separation and characterization of the polyhydroxyalkanoates synthase produced from recombinant Escherichia coli using an aqueous two-phase system

Polyhydroxyalkanoates (PHAs) are renewable and biodegradable polyesters which can be synthesized either by numerous of microorganisms in vivo or synthase in vitro. The synthesis of PHAs in vitro requires an efficient separation for high yield of purified enzyme. The recombinant Escherichia coli harboring phaC gene derived from Ralstonia eutropha H16 was cultivated in the chemically defined medium for overexpression of synthase in the present work. The purification and characteristics of PHA synthase from clarified feedstock by using aqueous two-phase systems (ATPS) was investigated. The optimized concentration of ATPS for partitioning PHA synthase contained polyethylene glycol 6000 (30%, w/w) and potassium phosphate (8%, w/w) with 3.25 volume ratio in the absence of NaCl at pH 8.7 and 4°C. The results showed that the partition coefficient of enzyme activity and protein content are 6.07 and 0.22, respectively. The specific activity, selectivity, purification fold and recovery of phaC(Re) achieved 1.76 U mg⁻¹, 29.05, 16.23 and 95.32%, respectively. Several metal ions demonstrated a significant effect on activity of purified enzyme. The purified enzyme displayed maximum relative activity as operating condition at pH value of 7.5 and 37°C. As compared to conventional purification processes, ATPS can be a promising technique applied for rapid recovery of PHA synthase and preparation of large quantity of PHA synthase on synthesis of P(3HB) in vitro.

Changes in Antioxidative Enzyme Activity, Glycogen, Lipid, Protein, and Malondialdehyde Content in Cadmium-Treated Galleria mellonella Larvae

Abstract In this study, we analyzed the effects of cadmium (1.25, 2.5, 5, 10, 20, and 40 mg per 100 g of nutrient) on total protein, lipid, and glycogen levels, lipid peroxidation (MDA), superoxide dismutase (SOD) and catalase (CAT) activities, and metal accumulation in Galleria mellonella L. High concentrations of Cd (20 and 40 mg) significantly lowered total protein, lipid, and glycogen levels. MDA level significantly increased when the nutrient contained 5 mg and more of Cd, whereas SOD activity significantly increased at Cd concentrations starting from 1.25 mg. CAT activity significantly decreased at all concentrations. Metal accumulation also significantly increased in a dose-dependent manner. These changes observed on activity of antioxidant enzymes, energy reserves, MDA content, and accumulation levels of Cd can be used as a reliable biomarker of environmental heavy metal pollution in model insect G. mellonella.

Stress modulation response of 24-epibrassinolide against imidacloprid in an elite indica rice variety Pusa Basmati-1

In this study, the response of exogenous application of BRs under the stress induced by neonicotinoid insecticide, imidacloprid (IMI) was evaluated in rice. It was demonstrated that treatment of rice seeds with various concentrations of 24-epibrassinolide (EBL) (10−11, 10−9 and 10−7M), an active form of BRs enhanced the tolerance of seedlings to imidacloprid IMI stress (0.01%, 0.015% and 0.02% IMI). Various morphological, biochemical and physiological parameters such as growth responses, protein, proline and malondialdehyde (MDA) content and activities of antioxidant enzymes were analyzed in seedlings treated with or without EBL and IMI or with their binary combinations. IMI treatment resulted in an adverse effect on growth and protein content of seedlings whereas an enhancement in the level of MDA and proline content was evinced. The activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and monodehydroascorbatereductase (MDHAR) increased after treatment with IMI. Application of different concentrations of EBL along with IMI resulted in an overall improvement in the growth, levels of protein and proline content. EBL induced stress attenuation was also supported by enhancement in the activity of various antioxidant enzymes and reduction in MDA content. Further, the expression of some key antioxidant genes (Cu/Zn-SOD, Fe-SOD, Mn-SOD, APX, CAT and GR) were analyzed under the various treatments of IMI and EBL. EBL treatment resulted in an up-regulation in the expression of most of the genes. However, transcriptional changes for both EBL and IMI treated seedlings indicate their interactions and this could have a possible link in offering EBL induced IMI stress tolerance in rice.

Experimental nonalcoholic fatty liver disease in mice leads to cytochrome p450 2a5 upregulation through nuclear factor erythroid 2-like 2 translocation

Mouse cytochrome P450 2A5 (CYP2A5) is upregulated in various liver diseases and a putative common feature for all of these conditions is altered cellular redox status. Nuclear factor erythroid 2-like 2 (Nrf2) is a transcription factor that is post-translationally regulated by oxidative stress and controls the transcription of protective target genes. In the present study, we have characterized the regulation of CYP2A5 by Nrf2 and evaluated gene expression, protein content and activity of anti-oxidant enzymes in the Nrf2+/+ and Nrf2−/− mice model of non-alcoholic fatty liver (NAFLD). After eight weeks of feeding on a high-fat diet, livers from Nrf2−/− mice showed a substantial increase in macro and microvesicular steatosis and a massive increase in the number of neutrophil polymorphs, compared to livers from wild-type mice treated similarly. Livers of Nrf2−/− mice on the high-fat diet exhibited more oxidative stress than their wild-type counterparts as assessed by a significant depletion of reduced glutathione that was coupled with increases in malondialdehyde. Furthermore, results in Nrf2-deficient mice showed that CYP2A5 expression was significantly attenuated in the absence of Nrf2, as was found with the conventional target genes of Nrf2. The treatment of wild-type mice with high-fat diet leaded to nuclear accumulation of Nrf2, and co-immunoprecipitation experiments showed that Nrf2 was bound to Cyp2a5. These findings suggest that the high-fat diet induced alteration in cellular redox status and induction of CYP2A5 was modulated through the redox-sensitive transcription Nrf2.

Modulation of the Rat Hepatic Cytochrome P4501A Subfamily Using Biotin Supplementation

Studies have found that biotin favors glucose and lipid metabolism, and medications containing biotin have been developed. Despite the use of biotin as a pharmacological agent, few studies have addressed toxicity aspects including the possible interaction with cytochrome P450 enzyme family. This study analyzed the effects of pharmacological doses of biotin on the expression and activity of the cytochrome P4501A subfamily involved in the metabolism of xenobiotics. Wistar rats were treated daily with biotin (2 mg/kg, i.p.), while the control groups were treated with saline. All of the rats were sacrificed by cervical dislocation after 1, 3, 5, or 7 days of treatment. CYP1A1 and CYP1A2 mRNAs were modified by biotin while enzyme activity and protein concentration were not affected. The lack of an effect of biotin on CYP1A activity was confirmed using other experimental strategies, including (i) cotreatment of the animals with biotin and a known CYP1A inducer; (ii) the addition of biotin to the reaction mixtures for the measurement of CYP1A1 and CYP1A2 activities; and (iii) the use of an S9 mixture that was prepared from control and biotin-treated rats to analyze the activation of benzo[a]pyrene (BaP) into mutagenic metabolites using the Ames test. The results suggest that biotin does not influence the CYP1A-mediated metabolism of xenobiotics.

Differences in Expression, Content, and Activity of 11β-HSD1 in Adipose Tissue between Obese Men and Women

Cortisol production in adipose tissue is regulated by 11β-HSD1. Objective. To determine whether there are differences in gene expression, enzyme activity, and protein content of the 11β-HSD1 enzyme in VAT (visceral adipose tissue) and SAT (subcutaneous adipose tissue) from obese compared to nonobese adults. Methods. VAT and SAT samples were obtained from 32 obese subjects (BMI > 30 Kg/m2) who underwent bariatric surgery and 15 samples from controls submitted to elective surgery. Fasting serum glucose, insulin, and lipids were measured. The expression of 11β-HSD1 was determined by RT-PCR, the enzyme activity by thin-layer chromatography, and the protein content by Western blot. Results. Obese patients had higher cholesterol, insulin, and HOMA-IR compared to nonobese. There were no differences in VAT or SAT expression of 11β-HSD1 between obese and nonobese patients. However, we found lower 11β-HSD1 activity and protein content in VAT, in obese women versus nonobese women (P < 0.05). BMI and 11β-HSD1 enzyme activity and protein content in VAT correlated inversely in women. Conclusions. Regulation of 11β-HSD1 activity in VAT from obese subjects appears to be gender specific, suggesting the existence of a possible protective mechanism modulating this enzyme activity leading to a decrease in the production of cortisol in this tissue.