Aspartate modulates the ethanol-induced oxidative stress and glutathione utilizing enzymes in rat testes

In order to develop a preventive strategy against ethanol-induced oxidative damages on various tissues and organs, we have examined the protective effect of aspartate on the pathogenesis of testes in the ethanol treated animals. Adult male Sprague-Dawley rats were given ethanol in an amount of 36% of total calories via Lieber-DeCarli liquid diet for 6 weeks without or with aspartate (2 mM in the diet). The control group was pair-fed the diet containing isocaloric dextrin-maltose instead of ethanol. The pathogenesis of testes at post- 6 weeks of experiments were carried out by histochemistry and biochemical parameters for oxidative stress such as the level of thiobarbituric acid reactive substances (TBARS) and the activities of glutathione utilizing enzymes were also examined. Chronic ethanol administration resulted in the increased amount of thiobarbituric acid reactive substances (TBARS) in the testes, which was significantly lessened by concurrent aspartate treatment (p < 0.05). In addition to this, liver function test indicated by alkaline phosphatase activity in serum showed that the ethanol-induced hepatotoxicity was significantly ameliorated by aspartate administration. And the activities of glucose-6-phosphate dehydrogenase and glutathione transferase in testis cytosol were decreased in the ethanol treated rats (p < 0.01 and < 0.005, respectively). These data suggest that aspartate may attenuate the ethanol-induced oxidative tissue damage in rat testes possibly through a redox-related protective effect on peroxidation.     

Amelioration of oxidative stress,inflammation and tumor promotion by Tin oxide-Sodium alginate-Polyethylene glycol-Allyl isothiocyanate nanocomposites on the 1,2-Dimethylhydrazine induced colon carcinogenesis in rats

BackgroundColorectal cancer (CRC) is a frequently diagnosed cancer that primarily affects the digestive system and an imperative cause of mortalities worldwide.ObjectiveIn this work, we formulated the Tin oxide-Sodium alginate-Polyethylene glycol-Allyl isothiocyanate nanocomposites (SAP-Ally-NCs) and investigated its anticancer role against the DMH-provoked CRC in rats.MethodologyThe formulated SAP-Ally-NCs were characterized different techniques. The CRC was provoked to the rats via injecting 20 mg/kg of DMH and then administered with the formulated SAP-Ally-NCs for 16 weeks. The bodyweight changes and the polyp’s incidences were detected and tabulated. The status of lipid peroxidation and antioxidant enzymes were studied by standard techniques. The inflammatory markers and xenobiotic enzymes level was scrutinized using respective kits. The mRNA expressions of various signaling molecules were examined by RT-PCR. The liver and colon tissues were examined microscopically to detect the histological changes.ResultsThe formulated SAP-Ally-NCs treatment appreciably improved the body weight gain and suppressed the polyp’s incidences in the DMH-challenged animals. SAP-Ally-NCs treated animals were demonstrated the notable reduction in the lipid peroxidation and inflammatory cytokines and elevated the antioxidant enzymes i.e. CAT and SOD activity. SAP-Ally-NCs administered animals exhibited the noticeable reduction in the expression of PCNA, cyclin-D1, iNOS, and COX-2 in the colon tissues. The histological findings also unveiled the therapeutic role of SAP-Ally-NCs.ConclusionIn conclusion, the SAP-Ally-NCs demonstrated the potent anticancer action against the DMH-provoked CRC in rats. In future, it could be a potent chemotherapeutic agent to the CRC.     

Centrifugal precipitation chromatography,a powerful technique for the isolation of active enzymes from tea leaves (Camellia sinensis)

Centrifugal precipitation chromatography was developed approximately 10 years ago. In contrast to other counter-current chromatographic techniques, the centrifugal precipitation chromatography system is operated with two mutually miscible solutions separated by a cut-off membrane. Centrifugal precipitation chromatography was firstly introduced for the separation of proteins using an ammonium sulfate gradient. In this study we describe a novel approach using solvent-based protein precipitation for the isolation of active plant enzymes from tea leaves (Camellia sinensis) by centrifugal precipitation chromatography. We developed a gradient based on acetone and Tris-buffer, because the biological activity of carotenases in tea leaves cannot be preserved in the presence of ammonium sulfate. Parameters such as the critical solvent concentration, flow rate, buffer concentration, and sample load were determined and/or optimized. Subsequently, the newly developed separation protocol was successfully used for the isolation of active carotenoid cleavage enzymes from tea leaves. The isolated enzymes showed high enzymatic activities and purities and could be directly used for enzymatic assays and structure elucidation.     

Linking Phospholipase Mobility to Activity by Single‐Molecule Wide‐Field Microscopy

Many of the biological processes taking place in cells are mediated by enzymatic reactions occurring in the cell membrane. Understanding interfacial enzymatic catalysis is therefore crucial to the understanding of cellular function. Unfortunately, a full picture of the overall mechanism of interfacial enzymatic catalysis, and particularly the important diffusion processes therein, remains unresolved. Herein we demonstrate that single‐molecule wide‐field fluorescence microscopy can yield important new information on these processes. We image phospholipase enzymes acting upon bilayers of their natural phospholipid substrate, tracking the diffusion of thousands of individual enzymes while simultaneously visualising local structural changes to the substrate layer. We study several enzyme types with different affinities and catalytic activities towards the substrate. Analysis of the trajectories of each enzyme type allows us successfully to correlate the mobility of phospholipase with its catalytic activity at the substrate. The methods introduced herein represent a promising new approach to the study of interfacial/heterogeneous catalysis systems.     

A New Microplate Screening Method for the Simultaneous Activity Quantification of Feruloyl Esterases, Tannases, and Chlorogenate Esterases

Feruloyl, chlorogenate esterases, and tannases are enzymes useful in phenolic modifications of pharmaceutical relevance as protectors against several degenerative human diseases. Therefore, there is a growing interest in discovering new sources of these enzymes. However, traditional methods for their activity measurements are time-consuming and poorly adapted for high-throughput screening. In this study, a successful new microplate high-throughput screening method for the simultaneous quantification of all mentioned activities is demonstrated. This method allows the detection of activities as low as 1.7 mU ml(-1). Furthermore, reaction rates increased proportionally with the amount of enzyme added, and no interferences with the other commercial hydrolases tested were found. The utility of the method was demonstrated after simultaneously screening feruloyl, chlorogenate esterase, and tannase activities in solid state fermentation extracts obtained during the kinetics of production of 20 fungal strains. Among these, seven strains were positive for at least one of the esterase activities tested. This result shows the potential for the rapid routine screening assays for multiple samples of moderate low to high enzymatic levels.     

Increased gamma-aminobutyrate aminotransferase activity in brain of patients with Alzheimer's disease

In order to search for more proximal factors in the pathogenesis of Alzheimer's disease, we studied the activities of various enzyme in the brains of patients, as well as control cases, by postmortem autopsy. In addition to the findings already known, such as the increase in prolyl endopeptidase (post-proline cleaving enzyme, PPCE) activity and the decrease in kallikrein activity, we found, anew, an increase in aminobutyrate aminotransferase (GABA-T) activity in the Alzheimer brain. This may be an important impetus for the reduction of gamma-aminobutyric acid (GABA) in the brain, one of the neurotransmitters. It has to be determined whether the former two abnormalities offer a background for such an abnormality of the neurotransmitter.     

LC/ESI/TOF-MS Characterization,Anxiolytic and Antidepressant-like Effects of Mitragyna speciosa Korth Extract in Diabetic Rats

In this study, the attenuative effects of the hydro-alcoholic extract from Mitragyna speciosa (MSE) against diabetes-induced anxiety and depression-like behaviors were examined. In addition, UPLC/ESI/TOF-MS analysis was performed to identify the phytochemical nature of MSE. DM was induced using a combination of high fructose/streptozotocin, and the diabetic rats were treated with MSE (50 and 200 mg/kg) for 5 weeks. After treatment, the animals were subjected to a forced swim test, open field test and elevated plus-maze tests. Additionally, proinflammatory cytokines and oxidative stress parameters were evaluated in the brain tissues of the rats. UPLC/ESI/TOF-MS analysis revealed that MSE is abundantly rich in polyphenolic constituents, notably flavonoid and phenolic glycosides. Behavioral tests and biochemical analyses indicated that diabetic rats showed significantly increased anxiety and depressive-like behavioral deficits, brain oxidative stress and pro-inflammatory cytokines levels (IL-1β, IL-6 and TNF-α). Treatment with MSE (50 and 200 mg/kg) significantly attenuated increased blood glucose level, depressive and anxiety-like behaviors in diabetic rats. Additionally, the antioxidant enzymes activities were markedly increased in MSE-treated animals, while TNF-α, IL-1β and IL-6 cytokines were notably suppressed. Taken together, these results suggested that MSE has potentials as antidepressant and anxiolytic-like effects and improves the brain oxido-inflammatory status in diabetic rats.     

Luminescent Strategies for Label‐Free G‐Quadruplex‐Based Enzyme Activity Sensing

By catalyzing highly specific and tightly controlled chemical reactions, enzymes are essential to maintaining normal cellular physiology. However, aberrant enzymatic activity can be linked to the pathogenesis of various diseases. Therefore, the unusual activity of particular enzymes can represent testable biomarkers for the diagnosis or screening of certain diseases. In recent years, G‐quadruplex‐based platforms have attracted wide attention for the monitoring of enzymatic activities. In this Personal Account, we discuss our group's works on the development of G‐quadruplex‐based sensing system for enzyme activities by using mainly iridium(III) complexes as luminescent label‐free probes. These studies showcase the versatility of the G‐quadruplex for developing assays for a variety of different enzymes.     

Untersuchungen über Enzymaktivitäten der Leberzelle bei Vitamin-E-Mangel

The enzymatic elongation of acyl-CoA esters by malonyl-CoA, respectively de novo synthesis of fatty acids from malonyl-CoA by liver microsomes and non particulate fraction of α-tocopherol deficient rats is diminished versus controls. However, liver microsomes of vitamin E deficient rats synthesize more eicosatetraenic acid from γ-linolenic acid and more γ-linolenic acid from linolic acid than do those of tocopherol supplemented animals. It has often been shown that the liver phosphatides of tocopherol deficient rats contain more arachidonic acid than those of controls, a fact which can be explained now by increased activity of the enzymes involved in the biosynthesis of arachidonic acid. Certain polyenic fatty acids are more rapidly synthesized in the absence of naturally occuring antioxidants. Some enzymes of the respiratory chain have also been examined. No vitamin E deficiency effect has been found on enzymes such as gluconate-6-P-dehydrogenase, isocitrate-dehydrogenase, malate-dehydrogenase and lactate dehydrogenase contained in the non particulate fraction. Sonicated mitochondria of tocopherol deficient rats show a greater activity towards cytochrome, oxydase and β-hydroxy-acetyl-CoA-dehydrogenase than controls, possibly due to ultrastractural alteration of this particle.     

Selecting Better Biocatalysts by Complementing Recoded Bacteria**

In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded organisms addicted to non-canonical amino acids (ncAAs) to evolve biocatalysts that can provide these building blocks from synthetic precursors. We exemplify our platform by engineering carbamoylases that display catalytic efficiencies more than five orders of magnitude higher than those observed for the wild-type enzyme for ncAA-precursors. As growth rates of bacteria under selective conditions correlate with enzymatic activities, we were able to elicit improved variants from populations by performing serial passaging. By requiring minimal human intervention and no specialized equipment, we surmise that our strategy will become a versatile tool for the in vivo directed evolution of diverse biocatalysts.     

Beneficial Effects of Laurel (Laurus nobilis L.) and Myrtle (Myrtus communis L.) Extract on Rat Health

Polyphenols of Laurel and Myrtle exhibit structural diversity, which affects bioavailability, metabolism, and bioactivity. The gut microbiota plays a key role in modulating the production, bioavailability and, thus the biological activities of phenolic metabolites, particularly after the intake of food containing high-molecular-weight polyphenols. The aim of this study was to investigate whether the polyphenolic components of Laurel and Myrtle aqueous extract have beneficial effects on rat health. The growth of lactic acid bacteria (LAB), β-glucuronidase, β-glucosidase, β-galactosidase activity, pH value, body weight change and food efficacy ratio after intragastric treatment of rats with Laurel and Myrtle extract at doses of 50 and 100 mg/kg for two weeks were investigated. The endogenous populations of colonic probiotic bacteria (Lactobacilli and Bifidobacteria) were counted on selective media. According to the obtained data, Laurel extract in the applied dose of 50 and 100 and Myrtle extract (100 mg/kg) positively affects the rats health by increasing the number of colonies of Lactobacilli and Bifidobacteria compared to the control group, causes changes in glycolytic enzymatic activity and minor change in antioxidative tissue activity. In addition, high doses of Laurel increase food efficiency ratio, while Myrtle has the same effect at a lower dose.     

Expression of monolysocardiolipin acyltransferase activity is regulated in concert with the level of cardiolipin and cardiolipin biosynthesis in the mammalian heart

Background  

Monolysocardiolipin acyltransferase (MLCL AT) catalyzes the acylation of monolysocardiolipin to cardiolipin in mammalian tissues. We previously reported that cardiac cardiolipin levels, MLCL AT and cardiolipin synthase activities were all elevated in rats made hyperthyroid by thyroxine treatment. In this study, we examined if cardiac mitochondrial MLCL AT activity was dependent upon the biosynthesis and level of cardiolipin in the heart. Rat heart mitochondrial MLCL AT activity was determined under conditions in which the levels of cardiac cardiolipin and cardiolipin synthase activity were either reduced or unaltered using four different disease models in the rat. In addition, these parameters were examined in a murine model of cardiac cell differentiation.     

Modulating effect of graphine oxide loaded hesperidin nanocomposite on the 1,2-dimethylhydrazine provoked colon carcinogenesis in rats via inhibiting the iNOS and COX-2 pathways

Hesperidin is a flavonoid derived from citrus plant peels. It have convinced biological actions, which includes antioxidant possessions, anti-inflammatory outcome, and thus we investigate that hesperidin will encompass chemopreventive probable next to 1,2-dimethylhydrazine (DMH)-provoked experimental colon carcinogenesis in male Wistar rats. Rats were randomly alienated into six groups. Group I rats were considered as control. Group II rats received only DMH. Groups III&IV animals received 20 mg/kg b.w of DMH subcutaneous one time a week, for initial 4 weeks. In adding, groups III & IV animals given DMH along with hesperidin at the dose of 5&10 mg/kg b.w., correspondingly for about 16 weeks. In present study we optimized hesperidin loaded with graphine oxide as a result achieved and was itemized and illustrated by UV Visible spectroscopy (876.25 nm), X-Ray diffraction, Fourier Transform Infrared Spectroscopy and Dynamic light scattering (45.50 nm). Hesperidin to the DMH induced rats drastically diminished the incidence of polyps as contrast to the DMH alone animals. Additionally in hesperidin management over DMH exposed experimental rats, we observed elevated actions of the oxidation inhibitors and diminished planes of LPO in liver and passage along with improved stage of lipids and antioxidants in colon tissues, which be distorted in the DMH unaided rats. Moreover, we experiential tainted actions of Interleukins, tumor necrosis factor and bioactive enzymes in DMH only rats, which are upturned in hesperidin treatment. All remarks are sustained in our histological conclusion. Ultimately, hesperidin might worned as effectual chemopreventive agent adjacent to DMH tempted colon cancer.     

A comparative investigation into the effect of chronic alcohol feeding on the myocardium of normotensive and hypertensive rats: an electrophoretic and biochemical study

We investigated whether the imposition of chronic alcohol in hypertension leads to greater biochemical and cellular abnormalities of the myocardium than those arising in normotension. Fifteen-week-old spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats were fed ethanol-containing diets for six weeks. Particular attention was focused on the composition of contractile proteins identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fractional rate of protein synthesis, and synthesis rates relative to RNA (RNA activity) or DNA (cellular efficiency). In addition, myocardial enzymes and adenine nucleotides were measured. In both SHR and WKY rats chronic ethanol caused a general decrease in the contents of all nine contractile proteins with myosin heavy chain predominantly affected. Fractional rates of mixed (i.e., total) and myofibrillary proteins remained unaltered in both WKY rats and SHR, as were cellular efficiencies. The RNA activity was significantly reduced in ethanol-treated SHR but not in WKY rats. In ethanol-treated SHR, cardiac creatine kinase (CK) and malate dehydrogenase (MDH) activities were increased, AMP levels were elevated, whilst ATP levels and the energy charge were reduced. In WKY rats, the only significant change related to increased aspartate aminotransferase activities in response to alcohol feeding. Although there were only subtle differences between the response of the normotensive and hypertensive rats due to ethanol dosage, the reduced ATP levels and increased CK and MDH activities in SHR may reflect a greater susceptibility to ischaemic damage. Reduced contractile protein content, particularly myosin heavy chain, may contribute to contractile defects, a common feature of subclinical and clinical alcoholic cardiomyopathy.     

High-performance liquid chromatography in enzymatic analysis. Opening lecture

During the previous two decades, high-performance liquid chromatography (HPLC) has proven to be an extremely useful technique with which to study the activity of enzymes and this paper will explore some of these uses. The success of the method can be seen not only from the increase in the number of papers utilizing this technique but also from the insights gained from its use on cellular phenomena. Given this success, it is no wonder that HPLC has become the technique of choice for many biologists seeking a more quantitative understanding of biological processes. Based on past experience, there is every reason to expect that the application of HPLC to the assaying of enzymatic activities will usher in another era of fundamental discoveries in the biological sciences. HPLC is particularly well suited to the assay of one activity in the presence of other activities obviating the need for extensive and tedious purification of biological samples. This advantage makes this technique particularly well suited to those who wish to use enzymes as markers for cellular processes, as indicators of metabolic activity and as evidence of gene function. To date, well over 100 activities have been assayed by this method. The method is particularly suited to problem-solving especially in such cases as when the presence of competing reactions prevents the recovery of the expected reaction products. Of the many applications, examples will be given on the use of HPLC for (1) monitoring the activity of an enzyme in a cell-free system, (2) monitoring the flow of metabolites through a multienzyme system and (3) the detection and study of new enzymatic activities. Some generalizations about the use of HPLC methods for the analysis of enzymatic activities will be presented.     

Water and a protic ionic liquid acted as refolding additives for chemically denatured enzymes

In this communication, we present the ability of water and a protic ionic liquid, triethyl ammonium phosphate (TEAP) to act as refolding additives for the urea-induced chemical denaturated state of the two enzymes, α-chymotrypsin and succinylated Con A. We show that the enzymatic activity is regained and in certain circumstances enhanced.     

Comparative characteristics of proteins of cottonplant lines differing in the strength of the fiber

The enzymes and proteins of the fibers of two lines of cotton plant differing in the strength of the fiber have been investigated. It has been shown that the activities of glucan synthetase and peroxidase rise as the fiber matures, while the activities of β-(1-3)-glucanase and cellulase fall. The specific enzymatic activities of peroxidase and glucan synthetase in the L-175 line, distinguished by a stronger fiber, are higher than for the L-466 line with a weaker fiber. The activity of glucanase changes according to the strength of the fiber. In a study of the protein composition of cotton fibers, polypeptides with molecular masses of 28 and 39 kDa were found among the proteins responsible for the strength of the fiber. A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 162 70 71. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 530–536, July–August, 1998.     

The Hypolipidemic and Antioxidant Activity of Wheat Germ and Wheat Germ Protein in High-Fat Diet-Induced Rats

Background: So far, no articles have discussed the hypolipidemic effect of wheat germ protein in in vivo experiments. Objective: In this study, we investigated the effects of wheat germ protein (WGP, 300 mg/kg/day) and wheat germ (WG, 300 mg/kg/day) on cholesterol metabolism, antioxidant activities, and serum and hepatic lipids in rats fed a high-fat diet through gavage. Methodology: We used 4-week-old male Wistar 20 rats in our animal experiment. Biochemical indicators of fecal, serum and liver were tested by kits or chemical methods. We also conducted the cholesterol micellar solubility experiment in vitro. Results: After 28 days of treatment, our results showed that WGP significantly reduced the serum levels of total cholesterol (p < 0.05) and nonhigh-density lipoprotein cholesterol (p < 0.05), improved the enzymatic activities of cholesterol 7-α hydroxylase (p < 0.01) and low-density lipoprotein receptor (p < 0.01) and increased bile acid excretion in feces (p < 0.05). Conclusion: WG did not significantly increase bile acid excretion in feces or decrease serum levels of total cholesterol. Moreover, WGP and WG both presented significant antioxidant activity in vivo (p < 0.05) and caused a significant reduction in cholesterol micellar solubility in vitro (p < 0.001). Therefore, WGP may effectively prevent hyperlipidemia and its complications as WGP treatment enhanced antioxidant activity, decreased the concentration of serum lipids and improved the activity of enzymes involved in cholesterol metabolism.