Presence Of Reductase Research Articles

Milk Quality Parameters of Raw Milk in Ecuador between 2010 and 2020: A Systematic Literature Review and Meta-Analysis.

With the objective of evaluating the quality parameters of raw milk in Ecuador between 2010 and 2020, a systematic review and meta-analysis of 73 studies on raw milk produced in different regions of Ecuador was performed. Under the random effects model, effect size and heterogeneity were determined vs. climatic region both among analyses and studies, with Cochran’s Q, I2 and Tau (π) statistics. For all the variables, it was observed that there was great heterogeneity (I2 > 90%) among the studies; additionally, it was found that climatic region had an influence only among the variables arsenic, mercury, pH and total solids, and it was greater in the coastal region than the Inter-Andean region. The mean values of the physicochemical characteristics of the milk (titratable acidity, ash, cryoscopy, fat, lactose, pH, protein, non-fat solids and total solids) in the great majority of these studies were within the range allowed by Ecuadorian regulations. As for the hygienic quality of raw milk (total bacterial count, somatic cell count and presence of reductase), although the mean values were within those determined by local legislation, it should be noted that the range established by Ecuadorian regulations is relatively much higher compared to other regulations, which possibly means that there is a high presence of bacteria and somatic cells in raw milk. Finally, the presence of several adulterants (added water) and contaminants (AFM1, antibiotics and heavy metals) was confirmed in the milk, in addition to other substances such as eprinomectin, zearalenone and ptaquilosides, whose presence can be very dangerous, because they can be hepatotoxic, immunotoxic and even carcinogenic. In conclusion, there is great variability among the studies reviewed, with the physicochemical characteristics being the most compliant with Ecuadorian legislation; the hygienic characteristics, adulterants and contaminants of raw milk require greater attention by producers and local authorities, so that they do not harm the health of consumers and the profitability of producers in Ecuador.

Textile Industry Wastewaters From Jetpur, Gujarat, India, Are Dominated by Shewanellaceae, Bacteroidaceae, and Pseudomonadaceae Harboring Genes Encoding Catalytic Enzymes for Textile Dye Degradation

Textile industries play an important role in uplifting the national economies worldwide. Nevertheless, they generate a huge amount of intensive colored effluent, which is a serious threat to the environment. The microbial communities present in these highly polluted environmental sites help in remediating pollutants naturally. However, little is known about their genes and enzymes in the textile wastewater systems. In this study, we explored the microbial community structure and their functional capability in three different wastewater systems, i.e., industry sites, effluent treatment plant (ETP), and common effluent treatment plant (CETP). Our findings based on shotgun metagenomics highlight the varied bacterial diversity at the three industry sites. Overall, the major dominant phyla in the industry site and CETP samples were Proteobacteria and Bacteroidetes, while in the ETP site, Firmicutes, Cyanobacteria, and Proteobacteria were predominant. The final discharge sample site was having a higher proportion of the Proteobacteria and Bacteroidetes. Aeromonas caviae, Desulfovibrio desulfuricans, Klebsiella pneumoniae, Pseudomonas stutzeri, Shewanella decolorationis, Shewanella oneidensis, Shewanella putrefaciens, and Vibrio cholera were the abundant species across the three sites. Furthermore, this research study identified the key microbial genes encoding enzymes having a known role in textile dye and aromatic compound degradation. Functional annotation of the shotgun metagenome samples indicates the presence of reductase, azoreductase, nitrate/nitrite reductase, and oxidoreductase enzyme encoding genes. Our findings provide the shotgun metagenomics-based approach for mining the textile dye degrading genes and genomic insights into the bioremediation of textile industrial effluent.

Enzyme‐Responsive Silica Mesoporous Supports Capped with Azopyridinium Salts for Controlled Delivery Applications

The preparation of a new capped silica mesoporous material, Rh-Azo-S, for on-command delivery applications in the presence of target enzymes is described. The material consists of nanometric mesoporous MCM-41-like supports loaded with Rhodamine B and capped with an azopyridine derivative. The material was designed to show "zero delivery" and to display a cargo release in the presence of reductases and esterases, which are usually present in the colon, mainly due to intestinal microflora. The opening and cargo release of Rh-Azo-S in vitro studies were assessed and seen to occur in the presence of these enzymes, whereas no delivery was noted in the presence of pepsine. Moreover, Rh-Azo-S nanoparticles were used to study controlled Rhodamine B dye delivery in intracellular media. HeLa cells were employed for testing the "non"-toxicity of nanoparticles. Moreover, delivery of the dye in these cells, through internalization and enzyme-mediated gate opening, was confirmed by confocal microscopy. Furthermore, the nanoparticles capped with the Azo group and loaded with a cytotoxic camptothecin (CPT) were also prepared (solid CPT-Azo-S) and used as delivery nanodevices in HeLa cells. When this solid was employed, the cell viability decreased significantly due to internalization of the nanoparticles and delivery of the cytotoxic agent.

Fractionation of Liver-Microsomes with Polyethylene-Glycol and Purification of NADH-Cytochrome b 5 Oxidoreductase and Cytochrome b 5

A simplified, rapid procedure for the purification of NADH-cytochrome b 5 oxidoreductase and cytochrome b 5 from either rat or rabbit liver is described. Microsomes were prepared by fractionation with polyethylene glycol and solubilized with Triton X-100. Cytochrome b 5 was purified by a two-column procedure, anion exchange chromatography using DEAE-cellulose, and hydrophobic chromatography on phenyl-Sepharose. The final preparation of cytochrome b 5 was purified more than a 120-fold from rat or rabbit liver microsomes, with specific content of about 50 nmol per mg protein, and overall yield of 22 to 32%. Only a single band with mol wt of 18,600 was found on sodium dodecyl sulfate (SDS)-gels or on Western blots using a polyclonal antibody raised against the purified b 5. NADH-cytochrome b 5 oxidoreductase was purified by a three-column procedure, DEAE-cellulose, hydroxylapatite, and ADP-agarose. The final product was purified more than 400-fold from rat or rabbit liver microsomes with a yield of about 25% and find specific activity of about 1600 μmol ferricyanide reduced per minute per milligram of protein. A single band with mol wt of 33,100 was found on SDS-gels. The reductase catalyzed reduction of ferricyanide, dichlorophenol-indophenol, and cytochrome b 5. Cytochrome c was reduced in the presence of reductase plus cytochrome b 5, and this was inhibited by the anti-b 5 IgG. The reductase catalyzed a rapid rate of reduction of ferric-ATP, which was slightly elevated by cytochrome b 5. Ferric-histidine and ferric-ammonium sulfate were slowly reduced by reductase; addition of cytochrome b 5 markedly stimulated reduction of these ferric complexes but inhibited reduction of ferric-EDTA. The procedures described result in highly purified preparations of reductase and cytochrome b 5 with relatively good yield and require fewer chromatographic steps than previously reported.

Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.

Mechanism(s) for the metabolism of mitoxantrone: Electron spin resonance and electrochemical studies

Mitoxantrone has been reported to lack certain properties that characterize quinone containing antitumor agents that undergo enzymatic reduction. These properties are the stimulation of NADPH oxidation, the stimulation of oxygen consumption by microsomes and reductases and, the absence of oxygen free radicals during these reactions. Having these properties implies the presence of a futile redox cycle that requires the generation and the oxidation of a semiquinone free radical. It would follow that if mitoxantrone does not redox cycle in the presence of reductases, then the semiquinone free radical is not produced or, if it is formed, it reacts quickly to form diamagnetic products. However, using liver microsomes, there are reports of the formation of the mitoxantrone free radial anion. In this paper we investigated the mitoxantrone free radical anion generated electrochemically and found that in the presence of oxygen it behaved like other semiquinones. That is, it is oxidized to the parent compound (presumably generating oxygen free radicals), indicating the ability to redox cycle. The reduction potential to generate such free radical in aqueous medium is very high (−0.79 V) when compared to diaziquone (−0.36 V) and Adriamycin (−0.6 V). This suggests that mitoxantrone may not be a substrate for reductases. Under reductive conditions with purified NADPH cytochrome P-450 reductase which very easily reduces diaziquone and Adriamycin, mitoxantrone was not reduced. However, under the same conditions, mitoxantrone was oxidized by the prototype oxidase horseradish peroxidase with the production of a mitoxantrone free radical. This oxidation was accompanied by a drastic change in color and the formation of a dark precipitate. Because microsomes contain a variety of enzymes, we suggest that the previously observed free radical in microsomes is probably due to the oxidation of mitoxantrone. In this theory, this product is probably a polymer which would not require oxygen to be formed. Thus, under oxidative conditions, the mitoxantrone free radical cation will also display impaired redox activity.

Demonstration of cytochrome reductases in rat liver peroxisomes: biochemical and immunochemical analyses.

In this study we utilized the analytical cell fractionation approach in combination with immunoblotting techniques and immunoelectron microscopy to test for the presence of NADPH cytochrome P-450 reductase and NADH cytochrome c (b5) reductase in rat liver peroxisomes. Highly purified peroxisomes from clofibrate-treated rats exhibited both NADPH cytochrome P-450 reductase activity and NADH cytochrome c reductase activity (using cytochrome c as an electron acceptor). These activities were inhibited by the respective reductase antibodies made against the endoplasmic reticulum (ER) enzymes. Immunoblot data in combination with immunoelectron microscopy indicated that the peroxisomal NADPH cytochrome P-450 reductase is localized in the matrix of the organelle and has a subunit of molecular weight similar to that of the ER enzyme, whereas the NADH cytochrome c (b5) reductase is localized in the membranes of the peroxisomes. Again, the subunit molecular weight was similar to that of the ER enzyme. The presence of these reductases in peroxisomes further supports the role of this organelle in bile acid synthesis and cholesterol metabolism.

Catalytic activity of cytochrome P-450 isozyme 3a isolated from liver microsomes of ethanol-treated rabbits. Oxidation of alcohols.

Cytochrome P-450 isozyme 3a, isolated from hepatic microsomes of rabbits treated chronically with ethanol, was found to have a unique substrate specificity when compared with isozymes 2, 3b, 3c, and 4. Form 3a has unusually high activity in the p-hydroxylation of aniline and in the oxidation of alcohols to aldehydes. These properties are reflected in the increased activities of these substrates in microsomes from ethanol-treated rabbits as compared to microsomes from untreated animals or those administered phenobarbital or 5,6-benzoflavone. The ethanol-oxidizing activity of isozyme 3a, which requires the presence of NADPH and NADPH cytochrome P-450 reductase and is stimulated by the presence of phospholipid, was shown not to be due to contaminating catalase or an NAD- or NADP-dependent alcohol dehydrogenase. Isozyme 3a catalyzes the oxidation of methanol, 1-propanol, and 1-butanol as well as ethanol; the relationships between the apparent Km values for these alcohols and their octanol-water partition coefficients is in accord with the known hydrophobic nature of the P-450 binding site. Whereas typical substrates of isozyme 2 are known to be metabolized with the stoichiometry predicted of a monooxygenase reaction, with isozyme 3a the sum of acetaldehyde formed from ethanol and of hydrogen peroxide generated is inadequate to account for the NADPH and oxygen consumed. Free hydroxyl radicals appear to mediate the slow oxidation of ethanol in the presence of the reductase alone but not the faster rate catalyzed by P-450 isozyme 3a. The results obtained, however, do not rule out the involvement of hydroxyl radical equivalent generated and bound at the active site of the cytochrome.

A Reduced Triphosphopyridine Nucleotide-linked Cystine Reductase in the Clothes Moth, Tineola bisselliella (Humm)

IN the course of studies on the digestion of wool by insects we have examined the clothes moth for the presence of reductases of disulphide bonds. Enzyme preparations were made by cold homogenization of whole larvae of the clothes moth followed by centrifugation at 30,000 g for 30 min. and dialysis of the supernatant for 20 hr. against cold 0.05 M tris (hydroxymethyl) aminomethane-hydrochloric acid buffer at pH 7.3. Cystine reductase activity was demonstrated by measuring the decrease in absorption at 340 mµ of reduced triphosphopyridine nucleotide (California Foundation) using anaerobic cuvettes in a Beckman DU spectrophotometer. Also the production of SH-groups was measured by a modified Grunert and Phillips colorimetric nitroprusside method1, and confirmed by titration with phenyl mercuric nitrate.