Sustained delivery of exogenous melatonin influences biomarkers of oxidative stress and total antioxidant capacity in summer-stressed anestrous water buffalo (Bubalus bubalis)

High ambient temperature during summer in tropical and subtropical countries predisposes water buffaloes (Bubalus bubalis) to develop oxidative stress having antigonadotropic and antisteroidogenic actions. Melatonin is a regulator of seasonal reproduction in photoperiodic species and highly effective antioxidant and free radical scavenger. Therefore, a study was designed to evaluate the effect of sustained-release melatonin on biomarkers of oxidative stress i.e., the serum malondialdehyde (MDA) and nitric oxide (NO), and the total antioxidant capacity (TAC). For the study, postpartum buffaloes diagnosed as summer anestrus (absence of overt signs of estrus, concurrent rectal examination, and RIA for serum progesterone) were grouped as treated (single subcutaneous injection of melatonin at 18 mg/50 kg body weight dissolved in sterilized corn oil as vehicle, n = 20) and untreated (subcutaneous sterilized corn oil, n = 8). Blood sampling for estimation of serum TAC and MDA (mmol/L) and NO (μmol/L) was carried out at 4 days of interval from 8 days before treatment till 28 days after treatment or for the ensuing entire cycle length. Results showed serum TAC concentration was higher in the treatment group with a significant (P < 0.05) increasing trend, whereas MDA and NO revealed a significant (P < 0.05) decline. Serum MDA and NO were higher in control compared with those of treatment group. Moreover, buffaloes in the treatment group showed 90% estrus induction with 18.06 ± 1.57 days mean interval from treatment to the onset of estrus. These results report that melatonin has a protective effect by elevating antioxidant status and reducing oxidative stress resulting in the induction of cyclicity in summer-stressed anestrous buffaloes.     

Development of PEGylated serum albumin with multiple reduced thiols as a long-circulating scavenger of reactive oxygen species for the treatment of fulminant hepatic failure in mice

Reactive oxygen species (ROS) are involved in the pathophysiology of fulminant hepatic failure. Therefore, we developed polyethylene glycol-conjugated bovine serum albumin with multiple reduced thiols (PEG-BSA-SH) for the treatment of fulminant hepatic failure. As a long-circulating ROS scavenger, PEG-BSA-SH effectively scavenged highly reactive oxygen species and hydrogen peroxide in buffer solution. PEG-BSA-SH showed a long circulation time in the plasma after intravenous injection into mice. Fulminant hepatic failure was induced by intraperitoneal injection of lipopolysaccharide and d-galactosamine (LPS/d-GalN) into mice. The LPS/d-GalN-induced elevation of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels was significantly inhibited by a bolus intravenous injection of PEG-BSA-SH. Furthermore, the changes in hepatic lipid peroxide and hepatic blood flow were effectively suppressed by PEG-BSA-SH. In contrast, l-cysteine, glutathione, and dithiothreitol, three traditional reduced thiols, had no statistically significant effects on the serum levels of ALT or AST. These findings indicate that PEG-BSA-SH is a promising ROS scavenger and useful in the treatment of fulminant hepatic failure.     

Quantitative and qualitative assessment on the suitability of seed oil from water plant (Trichilia emetica) for soap making

Despite widespread and its local available as a naturalized hedge and shade plant, the potential of Trichilia emetica was not utilized in soap making by the majority of local community in various parts of Dodoma, Tanzania. This study aimed to assess the quantity (yields) and quality (Acid Values (AVs), %Free Fatty Acids (%FFAs) and Saponification Values (SVs) of seed oil from water plant (T. emetica), suitable for soap making application. Solvent extraction method was used during oil extraction, where by 50gm of preheated and powdered seed materials were immersed in 250 ml of n-hexane in 1:5 (w/v) to dissolve the oil contained in the seed cake. The oil was collected by vaporizing solvent out through Rotary evaporation at 60 °C. Also standard titration methods were used to obtain SVs, AVs and %FFAs of the extracted oil. Results showed that T. emetica seeds contained higher quantity of oil (48.4%−50.2%) than many reported commercial plant seed oils. Also, the study found higher AV (7.4 mgKOH/g−7.8 mgKOH/g), %FFA (3.7% to 3.9%) and SVs (189.5 mgKOH/g − 191.4 mgKOH/g) than the maximum acceptable limits of 0.50 mg KOH/g, 0.020% and 175 mgKOH/g − 187 mgKOH/g prescribed by ASTM standards (2002). The obtained results showed that, T. emetica seeds yielded high oil quantity with low qualities due to higher levels of acidity. But high SVs guarantees the possibility of using T. emetica seed oil in soap making. However, the oil requires purification in order to bring levels of acidity to acceptable standards and guarantee its normal use in soap making.     

Rutin decreases lipopolysaccharide-induced acute lung injury via inhibition of oxidative stress and the MAPK–NF-κB pathway

Acute lung injury (ALI) is a serious disease with unacceptably high mortality and morbidity rates. Up to now, no effective therapeutic strategy for ALI has been established. Rutin, quercetin-3-rhamnosyl glucoside, expresses a wide range of biological activities and pharmacological effects, such as anti-inflammatory, antihypertensive, anticarcinogenic, vasoprotective, and cardioprotective activities. Pretreatment with rutin inhibited not only histopathological changes in lung tissues but also infiltration of polymorphonuclear granulocytes into bronchoalveolar lavage fluid in lipopolysaccharide (LPS)-induced ALI. In addition, LPS-induced inflammatory responses, including increased secretion of proinflammatory cytokines and lipid peroxidation, were inhibited by rutin in a concentration-dependent manner. Furthermore, rutin suppressed phosphorylation of NF-κB and MAPK and degradation of IκB, an NF-κB inhibitor. Decreased activities of antioxidative enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and heme oxygenase-1 caused by LPS were reversed by rutin. At the same time, we found that ALI amelioration by chelation of extracellular metal ions with rutin is more efficacious than with deferoxamine. These results indicate that the protective mechanism of rutin is through inhibition of MAPK–NF-κB activation and upregulation of antioxidative enzymes.     

How to deal with oxygen radicals stemming from mitochondrial fatty acid oxidation

Oxygen radical formation in mitochondria is an incompletely understood attribute of eukaryotic cells. Recently, a kinetic model was proposed, in which the ratio between electrons entering the respiratory chain via FADH₂ or NADH determines radical formation. During glucose breakdown, the ratio is low; during fatty acid breakdown, the ratio is high (the ratio increasing—asymptotically—with fatty acid length to 0.5, when compared with 0.2 for glucose). Thus, fatty acid oxidation would generate higher levels of radical formation. As a result, breakdown of fatty acids, performed without generation of extra FADH₂ in mitochondria, could be beneficial for the cell, especially in the case of long and very long chained ones. This possibly has been a major factor in the evolution of peroxisomes. Increased radical formation, as proposed by the model, can also shed light on the lack of neuronal fatty acid oxidation and tells us about hurdles during early eukaryotic evolution. We specifically focus on extending and discussing the model in light of recent publications and findings.     

Endothelin-1 stimulates catalase activity through the PKCδ-mediated phosphorylation of serine 167

Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H₂O₂) levels. These changes correlated with an increase in serine-phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be protein kinase Cδ (PKCδ) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H₂O₂ compared to the wild-type enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist tezosentan. S167 is located on the dimeric interface, suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel filtration to examine the multimeric structure of recombinant wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKCδ was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity.     

Determination of free amino acids in burley tobacco by high performance liquid chromatography

A reversed-phase high performance liquid chromatographic method was developed for determining free amino acids in burley tobacco. The test was done by OPA/3-mercaptopropionic acid as the pre-column derivatizing reagent. Chromatographic column was Elitte C¹⁸ column (4.6 mm × 250 mm i.d., 5 μm). Mobile phase A was 18 mol/l NaAc (pH7.2) including 0.002%(v/v) triethylamine and 0.3%(v/v) furanidine. Mobile phase B was 100 mol/l NaAc (pH7.2)–acetonitrile–methanol (v/v = 1:2:2). The column temperature was 40 °C and the flow rate was 1.0 ml/min. The fluorescence detector was used with 350 nm excitation wave length and 450 nm emission wave length. The average recoveries of the method ranged from 95.3–100.7% with the relative standard deviation of 2.32–9.24%. The method is simple, accurate and has good repeatability. The results of the determination of seventeen kinds of free amino acids in burley leaves were produced by the way of different ratios of cake fertilizer and inorganic fertilizer. The results show that Aspartic acid has the highest content however ratio of cake fertilizer and inorganic fertilizer. The contents of most of the free amino acids are increased and then gradually decreased with the increase in organic manure. The contents of most of the free amino acids are very close at 15:85% ratio and 30:70% ratio of cake fertilizer and inorganic fertilizer. The total amount of free amino acids is the highest at 30:70% ratio of cake fertilizer and inorganic fertilizer. Considering comprehensively, the quality of burley leaves is the best at 30:70% ratio of cake fertilizer and inorganic fertilizer.     

Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase

In evaluating the relative expression of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in vivo in states like Down syndrome in which one dismutase is present at increased levels, we measured activities of both enzymes, in tissues of control and transgenic mice constitutively expressing increased levels of CuZnSOD, during exposure to normal and elevated oxygen tensions. Using SOD gel electrophoresis assay, CuZnSOD and MnSOD activities of brain, lung, heart, kidney, and liver from mice exposed to either normal (21%) or elevated (>99% oxygen, 630 torr) oxygen tensions for 120 h were compared. Whereas CuZnSOD activity was elevated in tissues of transgenic relative to control mice under both normoxic or hyperoxic conditions, MnSOD activities in organs of transgenic mice were remarkably similar to those of controls under both conditions. To confirm the accuracy of this method in quantitating MnSOD relative to CuZnSOD expression, two other methods were utilized. In lung, which is the organ exposed to the highest oxygen tension during ambient hyperoxia, a sensitive, specific ELISA for MnSOD was used. Again, MnSOD protein was not different in transgenic relative to control mice during exposure to air or hyperoxia. In addition, lung MnSOD protein was not changed significantly by exposure to hyperoxia in either group. In kidney, a mitochondrion-rich organ, SOD assay, before and after inactivation of CuZnSOD with diethyldithiocarbamate, was used. MnSOD activity was not different in organs from air-exposed transgenic relative to control mice. The data indicated that expression of MnSOD in vivo was not affected by overexpression of the CuZnSOD and, therefore, the two enzymes are probably regulated independently.