Cu-Zn superoxide dismutase inhibits lactate dehydrogenase release and protects against cell death in murine fibroblasts pretreated with ultraviolet radiation

The effects of adding Cu-Zn superoxide dismutase (Cu-Zn SOD) to culture medium of the murine fibroblast cell line, L-929, pretreated with UV-B (312 nm, 480 mJ/cm(2)) have been investigated. Cell injury was monitored by the release of lactate dehydrogenase (LDH) into the medium, and cell death by the trypan blue exclusion test. UV-B radiation induced cell death by apoptosis, as demonstrated by DNA fragmentation. Over the range 0.1-0.3 microm Cu-Zn SOD, a significant dose-dependent protection against cell death was obtained of the UV-B exposed cells. Cell death correlated with the amount of LDH released into the medium, and Cu-Zn SOD treatment inhibited this. Heat-denatured Cu-Zn SOD did not affect either cell viability or the release of LDH from the cells. Endogenous Cu-Zn SOD activity, monitored by chemiluminescence, decreased by 20% in UV-B-irradiated cells; the addition of 0.3 microm exogenous Cu-Zn SOD to the medium did not affect intracellular Cu-Zn SOD activity. These results establish that Cu-Zn SOD added to extracellular medium can protect cells against injury caused by UV-B exposure.     

Contributory role of VEGF overexpression in endothelin-1-induced cardiomyocyte hypertrophy

Although endothelin-1 (ET-1) stimulates vascular endothelial growth factor (VEGF) expression in a variety of cells, including endothelial cells and vascular smooth muscle cells, the effects of ET-1 on expression of VEGF and its receptors in cardiomyocytes are unknown. In the present study, we found that treatment of neonatal rat cardiomyocytes with ET-1 for 24 h resulted in upregulation of VEGF and its two principal receptors, fetal liver kinase 1 and fms-like tyrosine kinase 1, in a concentration-dependent manner (10(-12) to 10(-6) M). ET-1 treatment also caused significant cardiomyocyte hypertrophy, as indicated by increases in cell surface area and [(14)C]leucine uptake by cardiomyocytes. Treatment with TA-0201 (10(-6) M), an ET(A)-selective blocker, eliminated ET-1-induced overexpression of VEGF and its receptors as well as cardiomyocyte hypertrophy. Treatment with VEGF neutralizing peptides (5-10 mug/ml) partially but significantly inhibited ET-1-induced cardiomyocyte hypertrophy. These results suggest that ET-1 treatment of cardiomyocytes promotes overexpression of VEGF and its receptors via activation of ET(A) receptors, and consequently the upregulated VEGF signaling system appears to contribute, at least in part, to ET-1-induced cardiomyocyte hypertrophy.     

Systemic alpha-adrenergic and nitric oxide inhibition on basal limb blood flow: effects of endurance training in middle-aged and older adults

Endurance training improves endothelium-dependent vasodilation, yet it does not increase basal blood flow in the legs. We determined the effects of a 3-mo aerobic exercise intervention on basal leg blood flow and alpha-adrenergic vasoconstriction and nitric oxide (NO) release in seven apparently healthy middle-aged and older adults (60 +/- 3 yr). Basal femoral artery blood flow (via Doppler ultrasound) (pretraining: 354 +/- 29; posttraining: 335 +/- 34 ml/min) and vascular conductance did not change significantly with the exercise training. Before the exercise intervention, femoral artery blood flow increased 32 +/- 16% with systemic alpha-adrenergic blockade (with phentolamine) (P < 0.05), and the addition of nitric oxide synthase (NOS) inhibition using N(G)-monomethyl-L-arginine (L-NMMA) did not affect femoral artery blood flow. After training was completed, femoral artery blood flow increased 47 +/- 7% with alpha-adrenergic blockade (P < 0.01) and then decreased 18 +/- 7% with the subsequent administration of L-NMMA (P < 0.05). Leg vascular conductance showed a greater alpha-adrenergic blockade-induced vasodilation (+1.7 +/- 0.5 to +3.0 +/- 0.5 units, P < 0.05) as well as NOS inhibition-induced vasoconstriction (-0.8 +/- 0.4 to -2.7 +/- 0.7 units, P < 0.05) after the exercise intervention. Resting plasma norepinephrine concentration significantly increased after the training. These results suggest that regular aerobic exercise training enhances NO bioavailability in middle-aged and older adults and that basal limb blood flow does not change with exercise training because of the contrasting influences of sympathetic nervous system activity and endothelium-derived vasodilation on the vasculature.     

Genome-Wide Analysis of DNA Methylation Dynamics during Early Human Development

DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5–10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.     

Evaluation of Neuronal Apoptosis Precursors in an Experimental Model of Acute Normovolemic Hemodilution

Background

The effects of acute anemia on neuronal cells and the safe limits of hematocrit are not well established. The objective of this study was to evaluate neuronal pro- and anti-apoptotic Bax and Bcl-x proteins, caspase-3 and -9 activity, and DNA fragmentation after acute normovolemic hemodilution (ANH).

Methods

Twenty-four pigs were anesthetized and randomized into 4 groups: Sham, ANH to 15% hematocrit (ANH15%), ANH to 10% hematocrit (ANH10%) and hypoxia (Hx). ANH was achieved by simultaneous blood withdrawal and hydroxyethyl starch infusion. Hx consisted of ventilation with a 6% inspired oxygen fraction for 60 minutes. Bax and Bcl-x proteins as well as DNA fragmentation were evaluated in cortical nuclear and mitochondrial fractions. Caspase-3 and -9 activity was evaluated in the cortical mitochondrial and hippocampal cytosolic fractions. The data were compared using analysis of variance followed by Tukey’s test (P<0.05).

Results

No changes were observed in Bax protein expression after hemodilution in the ANH15% and ANH10% groups compared to the Sham group. Bax expression in the Hx group was increased in the nuclear and mitochondrial fractions compared to all other groups. No significant difference was observed in Bcl-x expression. Caspase-3 and -9 activity in the cytosolic and mitochondrial fractions was different in the Hx group compared to all other groups. No statistical significance in DNA fragmentation was found among the Sham, ANH15% or ANH10% groups.

Conclusion

ANH to 10 and 15% hematocrit did not induce alterations in apoptosis precursors, suggesting that cerebral oxygenation was preserved during these anemic states.     

Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice

Aims

Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is highly expressed in the liver and is involved in both the detoxification of carbonyl compounds and ascorbic acid biosynthesis. By comparison with wild-type mice, Akr1a-knockout (Akr1a^−/−) mice and human Akrla-transgenic (Akr1a^tg/+) mice experience different anesthetic actions from pentobarbital—prolonged in Akr1a-knockout (Akr1a^−/−) mice and shortened in human Akrla-transgenic (Akr1a^tg/+) mice.

Main methods

We investigated this alteration in the anesthetic efficacy of pentobarbital in Akr1a genetically modified mice.

Key findings

Neither the cytosolic protein of wild-type mouse liver nor purified rat AKR1A directly reduced pentobarbital. Ascorbic acid administration neutralized the prolonged duration of the loss of the righting reflex (LORR) in Akr1a^−/− mice, but preincubation of pentobarbital with ascorbic acid prior to administration did not change the anesthetic effect. Those results indicated that ascorbic acid does not directly reduce pentobarbital. Enzymatic activities and levels of the proteins of some cytochrome P450s that make up a potent detoxification system for pentobarbital showed no changes in the genetically modified mice examined. Thus, ascorbic acid also had no effect on the detoxification system in the liver. The prolonged duration of LORR in the Akr1a^−/− mice caused by pentobarbital and the neutralization of the anesthetic effect by ascorbic acid together with other results imply that ascorbic acid alters the responses of the neuronal system to anesthetics.

Significance

Pentobarbital action is increased under conditions of ascorbic acid deficiency, and this may have to be taken into account when anesthetizing malnourished patients.