Deep insemination with sex-sorted Cashmere goat sperm processed in the presence of antioxidants.

Flow cytometrically sex-sorted sperm have been widely used for improving reproductive management in the dairy industry. However, the industrial application of this technology in other domestic species is largely limited by the lower fertility after insemination. The aim of this study was to investigate effects of antioxidant supplementation during the sex-sorting and freezing process on the quality and functions of sorted sperm from Liaoning Cashmere goats. We tested the effects of antioxidant supplementation during sex-sorting and freezing process, including ascorbic acid-2-glucoside AA-2G, glutathione, melatonin and vitamin C (VC), on the quality and functions of sex-sorted fresh and frozen-thawed sperm. Based on these experiments, we performed deep insemination with sex-sorted sperm using our improved strategy, in comparison to unsorted sperm. In Experiment 1, compared with control group and other antioxidants, AA-2G supplementation significantly alleviated the degradation of motility and viability of fresh sperm after sorting and showed the highest percentage of sperm with normal morphology. In addition, AA-2G supplementation showed an evident protection against the sorting process-induced membrane and acrosome damage. In Experiment 2, AA-2G supplementation was most effective in protecting motility, while melatonin supplementation appears to facilitate the degradation of quality of frozen-thawed sex-sorted sperm. In Experiment 3, we performed deep insemination with sperm that were sorted and frozen in the presence of AA-2G and obtained a satisfying pregnancy rate comparable to that from unsorted sperm. The results showed that AA-2G supplementation efficiently protects quality and function of both fresh and frozen-thawed sex-sorted sperm of Cashmere goats, thus obtaining a satisfying pregnancy outcome.

Folic acid reduces doxorubicin-induced cardiomyopathy by modulating endothelial nitric oxide synthase.

The use of doxorubicin (DOXO) as a chemotherapeutic drug has been hampered by cardiotoxicity leading to cardiomyopathy and heart failure. Folic acid (FA) is a modulator of endothelial nitric oxide (NO) synthase (eNOS), which in turn is an important player in diseases associated with NO insufficiency or NOS dysregulation, such as pressure overload and myocardial infarction. However, the role of FA in DOXO-induced cardiomyopathy is poorly understood. The aim of this study was to test the hypothesis that FA prevents DOXO-induced cardiomyopathy by modulating eNOS and mitochondrial structure and function. Male C57BL/6 mice were randomized to a single dose of DOXO (20 mg/kg intraperitoneal) or sham. FA supplementation (10 mg/day per oral) was started 7 days before DOXO injection and continued thereafter. DOXO resulted in 70% mortality after 10 days, with the surviving mice demonstrating a 30% reduction in stroke volume compared with sham groups. Pre-treatment with FA reduced mortality to 45% and improved stroke volume (both P < 0.05 versus DOXO). These effects of FA were underlain by blunting of DOXO-induced cardiomyocyte atrophy, apoptosis, interstitial fibrosis and impairment of mitochondrial function. Mechanistically, pre-treatment with FA prevented DOXO-induced increases in superoxide anion production by reducing the eNOS monomer:dimer ratio and eNOS S-glutathionylation, and attenuated DOXO-induced decreases in superoxide dismutase, eNOS phosphorylation and NO production. Enhancing eNOS function by restoring its coupling and subsequently reducing oxidative stress with FA may be a novel therapeutic approach to attenuate DOXO-induced cardiomyopathy.

Fyn arrests swainsonine-induced apoptosis in 293T cells via Akt and its phosphorylation.

Swainsonine (SW), an extract from Astragalus membranaceus, represents a new class of compounds that inhibit growth and induce apoptosis in a cancer model. In this study, we demonstrated the effect of Fyn on SW-induced apoptosis in 293T cells. Western blotting was used to measure the expression of the apoptosis-related factors caspase-3, Bcl-2, Bax, and the key factor Akt (also known as protein kinase B). Apoptosis increased dramatically after treatment with SW. Unlike the control group, after transfection with Fyn, the expression of Bcl-2, in contrast to Bax, was markedly upregulated. The results also showed that the protein expression levels of Akt and phosphorylated Akt were markedly increased. Our results establish that Fyn can arrest SW-induced apoptosis via the activity of Akt and its effective phosphorylation in 293T cells.

Calcium carbonate does not affect nilotinib pharmacokinetics in healthy volunteers.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Gastric upset is a common side effect of nilotinib therapy, and calcium carbonate is frequently used concomitantly, either as antacid or as calcium supplementation. With the increasing number of oral agents in cancer therapy, oral drug-drug interactions are becoming more relevant. Nilotinib has already been shown to be absorbed to a much lesser extent when co-administered with proton pump inhibitors. Because exposure to sub-therapeutic concentrations of anticancer drugs such as nilotinib may result in selection of resistant clones and ultimately relapse, we studied the effect of a calcium carbonate supplement (Tums Ultra 1000®) on nilotinib pharmacokinetics. WHAT THIS STUDY ADDS: Calcium carbonate may be co-administered with nilotinib without significantly affecting the pharmacokinetics of nilotinib and potentially impacting efficacy. PURPOSE: Nilotinib is a second-generation oral tyrosine kinase inhibitor with superior efficacy compared with imatinib mesylate in the treatment for chronic phase chronic myelogenous leukemia. Calcium carbonate is commonly used as a source of calcium supplementation or as antacid to ameliorate the gastrointestinal side effects associated with nilotinib, which could have unknown effects on nilotinib absorption. The purpose of this study was to provide information on the effect of calcium carbonate on the PK of nilotinib in healthy volunteers. METHODS: Healthy subjects were enrolled in a two-period, open-label, single-institution, randomized, cross-over, fixed-schedule study. In one period, each subject received 400 mg of nilotinib p.o. In the other period, 4,000 mg of calcium carbonate (4 X Tums Ultra 1000®) was administered p.o. 15 min prior to the nilotinib dose. Plasma samples were collected at specified timepoints, concentrations of nilotinib were quantitated by LC-MS, and data were analyzed non-compartmentally. RESULTS: Eleven subjects were evaluable. Calcium supplementation did not significantly affect nilotinib pharmacokinetic parameters including area under the plasma concentration versus time curve (18.4 µg/mL h alone vs. 16.9 µg/mL h with calcium carbonate, p = 0.83; 80 % power); maximum plasma concentration (C(max)) (0.670 µg/mL alone vs. 6.18 µg/mL with calcium carbonate, p = 0.97); or half-life (18.9 h alone vs. 17.2 h with calcium carbonate, p = 0.18). CONCLUSIONS: Our results indicate that the use of calcium carbonate does not significantly affect nilotinib pharmacokinetics.

Pathogenetic role of eNOS uncoupling in cardiopulmonary disorders.

The homodimeric flavohemeprotein endothelial nitric oxide synthase (eNOS) oxidizes l-arginine to l-citrulline and nitric oxide (NO), which acutely vasodilates blood vessels and inhibits platelet aggregation. Chronically, eNOS has a major role in the regulation of blood pressure and prevention of atherosclerosis by decreasing leukocyte adhesion and smooth muscle proliferation. However, a disturbed vascular redox balance results in eNOS damage and uncoupling of oxygen activation from l-arginine conversion. Uncoupled eNOS monomerizes and generates reactive oxygen species (ROS) rather than NO. Indeed, eNOS uncoupling has been suggested as one of the main pathomechanisms in a broad range of cardiovascular and pulmonary disorders such as atherosclerosis, ventricular remodeling, and pulmonary hypertension. Therefore, modulating uncoupled eNOS, in particular eNOS-dependent ROS generation, is an attractive therapeutic approach to preventing and/or treating cardiopulmonary disorders, including protective effects during cardiothoracic surgery. This review provides a comprehensive overview of the pathogenetic role of uncoupled eNOS in both cardiovascular and pulmonary disorders. In addition, the related therapeutic possibilities such as supplementation with the eNOS substrate l-arginine, volatile NO, and direct NO donors as well as eNOS modulators such as the eNOS cofactor tetrahydrobiopterin and folic acid are discussed in detail.

High-dose folic acid pretreatment blunts cardiac dysfunction during ischemia coupled to maintenance of high-energy phosphates and reduces postreperfusion injury.

BACKGROUND: The B vitamin folic acid (FA) is important to mitochondrial protein and nucleic acid synthesis, is an antioxidant, and enhances nitric oxide synthase activity. Here, we tested whether FA reduces myocardial ischemic dysfunction and postreperfusion injury. METHODS AND RESULTS: Wistar rats were pretreated with either FA (10 mg/d) or placebo for 1 week and then underwent in vivo transient left coronary artery occlusion for 30 minutes with or without 90 minutes of reperfusion (total n=131; subgroups used for various analyses). FA (4.5x10(-6) mol/L i.c.) pretreatment and global ischemia/reperfusion (30 minutes/30 minutes) also were performed in vitro (n=28). After 30 minutes of ischemia, global function declined more in controls than in FA-pretreated rats (Delta dP/dtmax, -878+/-586 versus -1956+/-351 mm Hg/s placebo; P=0.03), and regional thickening was better preserved (37.3+/-5.3% versus 5.1+/-0.6% placebo; P=0.004). Anterior wall perfusion fell similarly (-78.4+/-9.3% versus -71.2+/-13.8% placebo at 30 minutes), yet myocardial high-energy phosphates ATP and ADP reduced by ischemia in controls were better preserved by FA pretreatment (ATP: control, 2740+/-58 nmol/g; ischemia, 947+/-55 nmol/g; ischemia plus FA, 1332+/-101 nmol/g; P=0.02). Basal oxypurines (xanthine, hypoxanthine, and urate) rose with FA pretreatment but increased less during ischemia than in controls. Ischemic superoxide generation declined (3124+/-280 cpm/mg FA versus 5898+/-474 cpm/mg placebo; P=0.001). After reperfusion, FA-treated hearts had smaller infarcts (3.8+/-1.2% versus 60.3+/-4.1% placebo area at risk; P<0.002) and less contraction band necrosis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positivity, superoxide, and nitric oxide synthase uncoupling. Infarct size declined similarly with 1 mg/d FA. CONCLUSIONS: FA pretreatment blunts myocardial dysfunction during ischemia and ameliorates postreperfusion injury. This is coupled to preservation of high-energy phosphates, reducing subsequent reactive oxygen species generation, eNOS-uncoupling, and postreperfusion cell death.

Effect of folic acid on endothelial function following acute myocardial infarction.

The aim of this study was to test the influence of high-dose folic acid (10 mg/d) on endothelial function in patients referred for coronary intervention after an acute myocardial infarction (AMI) and determine its relation to homocysteine levels. Flow-mediated dilation (FMD) of the brachial artery was performed in 40 patients after AMI (16 with normal homocysteine levels and 24 patients with elevated levels [>11 micromol/L]). Subjects were randomized to receive first folic acid (10 mg/day; group A) or placebo (group B) for 6 weeks in a double-blind crossover trial with a 2-week washout. Plasma folate, total homocysteine and its subtypes (oxidized, reduced, and protein-bound), FMD, and nitroglycerin-mediated dilation were assessed at baseline and at 6 and 14 weeks. In group A, folic acid improved FMD from 3.98 +/- 0.35% to 6.44 +/- 0.56% (p <0.001). This effect persisted after the crossover with placebo (5.42 +/- 0.59, p = 0.13). In group B, placebo did not increase FMD (4.01 +/- 0.34% vs 4.46 +/- 0.38, p = 0.38); however, a significant increase was observed in the second active treatment period (6.49 +/- 0.56%, p = 0.005). In both groups, improved FMD neither correlated with basal levels of homocysteine and its subtypes nor with changes induced during the folate treatment. Nitroglycerin-mediated dilation did not change significantly in either group. Folic acid increased FMD in both normo- and hyperhomocysteinanemic groups (p = 0.006 and p <0.001). In conclusion, 6-week treatment with high-dose folic acid improves endothelial function in post-AMI patients, independent from homocysteine status. Folic acid can be recommended to improve postinfarction endothelial dysfunction in patients with normo- and hyperhomocysteinemia.

Protocatechuic acid suppresses MPP+ -induced mitochondrial dysfunction and apoptotic cell death in PC12 cells.

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia (A.) oxyphylla, showed antioxidant neuroprotective effect in our previous study. Here, we investigated the effect of PCA on the MPP(+)-induced mitochondrial dysfunction and apoptotic cell death in PC12 cells. The apoptosis in MPP(+)-induced PC12 cells was associated with loss of mitochondrial membrane potential, the formation of reactive oxygen species (ROS), GSH depletion, activation of caspase-3 and down-regulation of Bcl-2. In contrast, treatment of PC12 cells with PCA significantly prevented the above-mentioned mitochondrial dysfunction. Our data pointed to the potential clinical application/use of PCA to overcome neurodegenerative diseases such as Parkinson's disease.

Protocatechuic acid from Alpinia oxyphylla against MPP+-induced neurotoxicity in PC12 cells.

An ethyl acetate extract of Alpinia oxyphylla was found to possess neuroprotective activity against 1-methyl-4-phenylpyridinium ion (MPP(+)) induced apotosis and oxidative stress in cultured PC12 cells. From the extract, a phenolic compound was isolated through bioassay-guided fractionation and identified as protocatechuic acid (PCA) by IR, MS, and (1)H and (13)C NMR spectroscopy. It was the first time which was isolated from the kernels of A. oxyphylla. Exposure of PC12 cells to 1mM MPP(+) may cause significant viability loss and apoptotic cell death. PCA stimulated PC12 cellular proliferation and markedly attenuated MPP(+)-induced apoptotic cell death in a dose-dependent manner. By observing the nuclear morphological changes and flow cytometric analysis, PCA showed its significant effect on protecting PC12 cells against MPP(+)-induced apoptosis. Meanwhile, PCA enhanced the activities of superoxide dismutase (SOD) and catalase (CAT) in PC12 cells. In addition, PCA also dose-dependently reduced the hydrogen peroxide (H(2)O(2))- or sodium nitroprusside (SNP)-induced cell death in PC12 cells. The results suggest that PCA may be one of the primary active components in the kernels of A. oxyphylla and provide a useful therapeutic strategy for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinson's disease.

Catalpol inhibits apoptosis in hydrogen peroxide-induced PC12 cells by preventing cytochrome c release and inactivating of caspase cascade.

In the present study, using a rat pheochromocytoma (PC12) cell line, the effect of catalpol on H2O2-induced apoptosis was studied. The apoptosis in H2O2-induced PC12 cells was accompanied by down-regulation of Bcl-2, up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol and sequential activation of caspase-1 and caspase-3 then leading to cleavage of poly-ADP-ribose polymerase (PARP). Catalpol not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation, PARP cleavage, and eventually protected against H2O2-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with catalpol can block H2O2-induced apoptosis by the regulation of Bcl-2 family members, as well as suppression of cytochrome c release and caspase cascade activation.

Purification and characterization of ginsenoside-beta-glucosidase from ginseng.

The ginsenoside-beta-glucosidase that hydrolyzes the beta-(1-->2)-glucoside of the ginsenoside Rg3 sugar moiety to ginsenoside Rh2 was isolated from the ginseng root, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 59 kDa. The optimum temperature of the ginsenoside-beta-glucosidase was 60 degrees C, and the optimum pH was 5.0. Ca2+ ion had positive effect on ginsenoside-beta-glucosidase, while Cu2+ had negative effect on it. The ginsenoside-beta-glucosidase may be a special beta-glucosidase that is different from the original exocellulase such as beta-glucosidase (EC 3.2.1.21).

[The design of measure and control system for composite treating instrument of gastrointestinal cancer].

This paper introduced a composite method of treating gastrointestinal tract cancer-thermo-chemotherapy. A complete set of intelligentialized real-time measure and control system was well designed. The experiment result showed that this system has a stable performance and good real-time.