Experimental hyperprolinemia induces mild oxidative stress, metabolic changes, and tissue adaptation in rat liver

The present study investigated the effects of chronic hyperprolinemia on oxidative and metabolic status in liver and serum of rats. Wistar rats received daily subcutaneous injections of proline from their 6th to 28th day of life. Twelve hours after the last injection the rats were sacrificed and liver and serum were collected. Results showed that hyperprolinemia induced a significant reduction in total antioxidant potential and thiobarbituric acid-reactive substances. The activities of the antioxidant enzymes catalase and superoxide dismutase were significantly increased after chronic proline administration, while glutathione (GSH) peroxidase activity, dichlorofluorescin oxidation, GSH, sulfhydryl, and carbonyl content remained unaltered. Histological analyses of the liver revealed that proline treatment induced changes of the hepatic microarchitecture and increased the number of inflammatory cells and the glycogen content. Biochemical determination also demonstrated an increase in glycogen concentration, as well as a higher synthesis of glycogen in liver of hyperprolinemic rats. Regarding to hepatic metabolism, it was observed an increase on glucose oxidation and a decrease on lipid synthesis from glucose. However, hepatic lipid content and serum glucose levels were not changed. Proline administration did not alter the aminotransferases activities and serum markers of hepatic injury. Our findings suggest that hyperprolinemia alters the liver homeostasis possibly by induction of a mild degree of oxidative stress and metabolic changes. The hepatic alterations caused by proline probably do not implicate in substantial hepatic tissue damage, but rather demonstrate a process of adaptation of this tissue to oxidative stress. However, the biological significance of these findings requires additional investigation. J. Cell. Biochem. 113: 174183, 2012. (C) 2011 Wiley Periodicals, Inc.

The effect of sodium fluoride preservative and storage temperature on the stability of cocaine in horse blood, sheep vitreous and deer muscle

The in vitro stability of cocaine in horse blood, sheep vitreous humour (VH) and homogenised deer muscle is described. The stability of cocaine in horse blood was of interest because many toxicology laboratories utilise horse blood for the preparation of calibration and check standards and the latter are typically stored during routine use. The storage stability of cocaine in human VH and muscle has not been previously reported. In the absence of blank human VH and muscle, cocaine stability under varying conditions was demonstrated in animal tissues. Blood and VH were stored with and without addition of NaF at room temperature (RT), 4 degrees C and -18 degrees C for 84 days. Muscle homogenates were prepared in water, water/2% NaF, and phosphate buffer (pH 6.0)/2% NaF, and stored for 31 days at RT, 4 degrees C and -18 degrees C. Cocaine stability in human muscle obtained from cocaine positive forensic cases was assessed following storage at -18 degrees C for 13 months. Cocaine and benzoylecgonine (BZE) were extracted using SPE and quantified by GC-MS/MS. Cocaine was stable for 7 days in refrigerated (4 degrees C) horse blood fortified with 1 and 2% NaF. In the absence of NaF, cocaine was not detectable by day 7 in blood stored at RT and 4 degrees C and had declined by 81% following storage at -18 degrees C. At 4 degrees C the rate of cocaine degradation in blood preserved with 2% NaF was significantly slower than with 1% NaF. The stability of cocaine in horse blood appeared to be less than that reported for human blood, probably attributable to the presence of carboxylesterase in horse plasma. Cocaine stored in VH at -18 degrees C was essentially stable for the study period whereas at 4 degrees C concentrations decreased by >50% in preserved and unpreserved VH stored for longer than 14 days. Fluoride did not significantly affect cocaine stability in VH. The stability of cocaine in muscle tissue homogenates significantly exceeded that in blood and VH at every temperature. In preserved and unpreserved samples stored at 4 degrees C and below, cocaine loss did not exceed 2%. The increased stability of cocaine in muscle was attributed to the low initial pH of post-mortem muscle. In tissue from one human case stored for 13 months at -18 degrees C the muscle cocaine concentration declined by only 15% (range: 5-22%). These findings promote the use of human muscle as a toxicological specimen in which cocaine may be detected for longer compared with blood or VH. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Peptidomic Analysis of HEK293T Cells: Effect of the Proteasome Inhibitor Epoxomicin on Intracellular Peptides

Peptides derived from cytosolic, mitochondrial, and nuclear proteins have been detected in extracts of animal tissues and cell lines. To test whether the proteasome is involved in their formation, HEK293T cells were treated with epoxomicin (0.2 or 2 mu M) for 1 h and quantitative peptidomics analysis was performed. Altogether, 147 unique peptides were identified by mass spectrometry sequence analysis. Epoxomicin treatment decreased the levels of the majority of intracellular peptides, consistent with inhibition of the proteasome beta-2 and beta-5 subunits. Treatment with the higher concentration of epoxomicin elevated the levels of some peptides. Most of the elevated peptides resulted from cleavages at acidic residues, suggesting that epoxomicin increased the processing of proteins through the beta-1 subunit. Interestingly, some of the peptides that were elevated by the epoxomicin treatment had hydrophobic residues in P1 cleavage sites. Taken together, these findings suggest that, while the proteasome is the major source of intracellular peptides, other peptide-generating mechanisms exist. Because intracellular peptides are likely to perform intracellular functions, studies using proteasome inhibitors need to be interpreted with caution, as it is possible that the effects of these inhibitors are due to a change in the peptide levels rather than inhibition of protein degradation.

Adaptations of the aging animal to exercise: role of daily supplementation with melatonin

The pineal gland, through melatonin, seems to be of fundamental importance in determining the metabolic adaptations of adipose and muscle tissues to physical training. Evidence shows that pinealectomized animals fail to develop adaptive metabolic changes in response to aerobic exercise and therefore do not exhibit the same performance as control-trained animals. The known prominent reduction in melatonin synthesis in aging animals led us to investigate the metabolic adaptations to physical training in aged animals with and without daily melatonin replacement. Male Wistar rats were assigned to four groups: sedentary control (SC), trained control (TC), sedentary treated with melatonin (SM), and trained treated with melatonin (TM). Melatonin supplementation lasted 16 wk, and the animals were subjected to exercise during the last 8 wk of the experiment. After euthanasia, samples of liver, muscle, and adipose tissues were collected for analysis. Trained animals treated with melatonin presented better results in the following parameters: glucose tolerance, physical capacity, citrate synthase activity, hepatic and muscular glycogen content, body weight, protein expression of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and protein kinase activated by adenosine monophosphate (AMPK) in the liver, as well as the protein expression of the glucose transporter type 4 (GLUT4) and AMPK in the muscle. In conclusion, these results demonstrate that melatonin supplementation in aging animals is of great importance for the required metabolic adaptations induced by aerobic exercise. Adequate levels of circulating melatonin are, therefore, necessary to improve energetic metabolism efficiency, reducing body weight and increasing insulin sensitivity.

Storage of bovine reproductive tissues and RNA extracts on ice for 24h or repeated freeze–thaw cycles do not affect RNA integrity

The aims of this study were to test (i) the effect of time of tissue and RNA extracts storage on ice and (ii) the effect of repeated freeze–thaw cycles on RNA integrity and gene expression of bovine reproductive tissues. Fragments of endometrium (ENDO), corpus luteum (CL) and ampulla (AMP) were subdivided and incubated for 0, 1, 3, 6, 12 or 24 h on ice. RNA extracts were incubated on ice for 0, 3, 12 or 24 h, or exposed to 1, 2, 4 or 6 freeze–thaw cycles. RNA integrity number (RIN) was estimated. Expression of progesterone receptor (PGR) and cyclophilin genes from RNA extracts stored on ice for 0 or 24 h, and 1 or 6 freeze–thaw cycles was measured by qPCR. Tissue and RNA extract incubation on ice, and repeated freeze–thaw cycles did not affect RIN values of RNA from ENDO, CL or AMP. Storage on ice or exposure to freeze–thaw cycles did not affect Cq values for PGR or cyclophilin genes. In conclusion, neither generalized RNA degradation nor specific RNA degradation was affected by storage of tissue or RNA extracts on ice for up to 24 h, or by up to 6 freeze–thaw cycles of RNA extracts obtained from bovine ENDO, CL and AMP.