Optimizing action of synthetic peptide Selank on active avoidance conditioning test in rats

The action of a synthetic peptide Selank on learning and memory in active avoidance conditioning test was studied in rats with initially low learning ability and normal animals. The peptide was administered repeatedly 15 min prior the training session (4 days). The effect of Selank (300 micrograms/kg) was compared to that of Pyracetam (400 mg/kg). Selank was found to significantly improve learning in rats with low level ability even after a single administration on the first day of training session. The effect progressively increased with repeated Selank treatment: the total numbers of reactions and correct reactions increased, and the number of errors decreased (p < 0.05). In normal rats, the effect was maximal on the third day of treatment and training, i.e., after the completed initial consolidation. Some distinguishing features were revealed in the dynamics of activatory effects of Selank and Pyracetam. These data together with the evidence for ansiolytic effect of Selank show that this drug is promising for optimization of mnestic functions under conditions of high emotional stress.     

Hypoxia inactivates inducible nitric oxide synthase in mouse macrophages by disrupting its interaction with alpha-actinin 4

Nitric oxide, produced in macrophages by the high output isoform inducible NO synthase (iNOS), is associated with cytotoxic effects and modulation of Th1 inflammatory/immune responses. Ischemia and reperfusion lead to generation of high NO levels that contribute to irreversible tissue damage. Ischemia and reperfusion, as well as their in vitro simulation by hypoxia and reoxygenation, induce the expression of iNOS in macrophages. However, the molecular regulation of iNOS expression and activity in hypoxia and reoxygenation has hardly been studied. We show in this study that IFN-gamma induced iNOS protein expression (by 50-fold from control, p < 0.01) and nitrite accumulation (71.6 +/- 14 micro M, p < 0.01 relative to control), and that hypoxia inhibited NO production (7.6 +/- 1.7 micro M, p < 0.01) without altering iNOS protein expression. Only prolonged reoxygenation restored NO production, thus ruling out the possibility that lack of oxygen, as a substrate, was the cause of hypoxia-induced iNOS inactivation. Hypoxia did not change the ratio between iNOS monomers and dimers, which are essential for iNOS activity, but the dimers were unable to produce NO, despite the exogenous addition of all cofactors and oxygen. Using immunoprecipitation, mass spectroscopy, and confocal microscopy, we demonstrated in normoxia, but not in hypoxia, an interaction between iNOS and alpha-actinin 4, an adapter protein that anchors enzymes to the actin cytoskeleton. Furthermore, hypoxia caused displacement of iNOS from the submembranal zones. We suggest that the intracellular localization and interactions of iNOS with the cytoskeleton are crucial for its activity, and that hypoxia inactivates iNOS by disrupting these interactions.     

Semax and Selank Inhibit the Enkephalin-Degrading Enzymes of Human Serum

Dose-dependent effect of synthetic heptapeptides Semax (Met-Glu-His-Phe-Pro-Gly-Pro) and Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) on the enkephalin-degrading enzymes of human serum was demonstrated. The inhibitory effects of Semax (IC₅₀10 M) and Selank (IC₅₀20 M) are more pronounced than that of puromycin (IC₅₀10 mM), bacitracin, and some other inhibitors of peptidases. Beside the heptapeptides, their pentapeptide fragments also possessed an inhibitory effect; tri-, tetra- and hexapeptide fragments did not display such an effect. As the above enzymes take part in degradation of not only enkephalins but also other regulatory peptides, it can be assumed that one of the mechanisms of biological activity of Semax and Selank is related to this inhibitory activity of theirs.     

Deletion of betaine-homocysteine S-methyltransferase in mice perturbs choline and 1-carbon metabolism, resulting in fatty liver and hepatocellular carcinomas

Betaine-homocysteine S-methyltransferase (BHMT) uses betaine to catalyze the conversion of homocysteine (Hcy) to methionine. There are common genetic polymorphisms in the BHMT gene in humans that can alter its enzymatic activity. We generated the first Bhmt(-/-) mouse to model the functional effects of mutations that result in reduced BHMT activity. Deletion of Bhmt resulted in a 6-fold increase (p < 0.01) in hepatic and an 8-fold increase (p < 0.01) in plasma total Hcy concentrations. Deletion of Bhmt resulted in a 43% reduction in hepatic S-adenosylmethionine (AdoMet) (p < 0.01) and a 3-fold increase in hepatic S-adenosylhomocysteine (AdoHcy) (p < 0.01) concentrations, resulting in a 75% reduction in methylation potential (AdoMet:AdoHcy) (p < 0.01). Bhmt(-/-) mice accumulated betaine in most tissues, including a 21-fold increase in the liver concentration compared with wild type (WT) (p < 0.01). These mice had lower concentrations of choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, and sphingomyelin in several tissues. At 5 weeks of age, Bhmt(-/-) mice had 36% lower total hepatic phospholipid concentrations and a 6-fold increase in hepatic triacyglycerol concentrations compared with WT (p < 0.01), which was due to a decrease in the secretion of very low density lipoproteins. At 1 year of age, 64% of Bhmt(-/-) mice had visible hepatic tumors. Histopathological analysis revealed that Bhmt(-/-) mice developed hepatocellular carcinoma or carcinoma precursors. These results indicate that BHMT has an important role in Hcy, choline, and one-carbon homeostasis. A lack of Bhmt also affects susceptibility to fatty liver and hepatocellular carcinoma. We suggest that functional polymorphisms in BHMT that significantly reduce activity may have similar effects in humans.     

Compensatory and antiamnestic effects of heptapeptide Selank in monkeys

The work presents results of study of role of heptapeptide Selank—an anxiolytic from group of biologically active peptides—in compensation of disturbed psychic and homeostatic functions in monkeys. New data have been obtained which indicate that an intranasal administration of Selank produces long-term changes of the monkey behavior disturbed during neurosis: elimination of fear and aggression and an increase of explorative activity as well as facilitation of handling reactions and communicational relations. It has been established that on the background of the Selank there occurs a long compensation of disturbed psychic functions (processes of memory) and of homeostatic parameters. It has been shown that unlike the earlier studied neurohormones (thyroliberin and ACTH_4?10), the antistress Selank effects do not depend on the type of neurotic disturbances and have long-term compensatory character. Comparison of the data obtained on monkeys with results of similar studies on the more low-organized mammals (rodents) allow suggesting that the new peptide preparation Selank is a promising agent for correction of various psychoemotional disturbances (alarm-and depression-like disorders).     

Peptides semax and selank affect the behavior of rats with 6-OHDA induced PD-like parkinsonism

Parkinson’s disease (PD) is the second most common severe neurodegenerative disorder that is characterized by progressive degeneration of dopaminergic neurons (DA neurons) in the substantia nigra pars compacta (SNpc) region of the brain. In the present study, we investigated the effects of the synthetic regulatory peptides Semax (analog of an ACTH 4-10 fragment (ACTH4-10)) and Selank (analog of immunomodulatory taftsin) on behavior of rats with 6-hydroxidopamine (6-OHDA) induced PD-like parkinsonism. It was showed that both peptides did not affect motor activity of rats in elevated cross shaped maze and passive defensive behavior of the animals. At the same time, Selank decreased level of anxiety of rats with toxic damage of DA neurons in elevated cross shaped maze. Previously such effects of Selank were revealed in healthy rodents (rats and mice) with different models of psycho-emotional stress. Therefore, toxic damage of substantia nigra does not affect the response of the rat organism on this peptide.     

ATG12对缺氧缺血性脑病小鼠神经细胞凋亡和自噬的影响

目的探讨自噬相关蛋白12 （ATG12）对缺氧缺血性脑病（HIE）小鼠细胞凋亡和自噬的影响及分子机制。 方法通过尾静脉注射腺相关病毒构建ATG12低表达小鼠模型,将40只小鼠分为假手术组、HIE模型组、对照病毒模型（NC-HIE）组和ATG低表达病毒模型（ATG12 shRNA-HIE）组,HIE模型组小鼠左侧颈动脉结扎后低氧（8﹪氧气+92﹪氮气）处理2.5?h,假手术组不予结扎和低氧处理。缺氧处理后,荧光定量PCR检测脑组织ATG12 mRNA表达水平。比色法检测各组小鼠大脑神经细胞SOD和MDA水平;通过Tunel法检测各组小鼠大脑神经细胞凋亡水平;通过Western Blot检测各组小鼠大脑神经细胞LC3A/B、ATG12和SQSTM1/?p62蛋白表达水平。采用t检验和单因素方差分析对实验数据进行统计分析。 结果与假手术组小鼠脑组织ATG12 mRNA水平（1.00±0.14）相比,HIE模型组小鼠脑组织ATG12 mRNA水平（5.23±0.37）显著升高（t?= 33.60,P?< 0.01）;与假手术组小鼠脑组织超氧化物歧化酶（SOD）活性[（103.60±4.84）?U/?mgprot]和丙二醛（MDA）含量[（42.40±3.17）?μmol/?mgprot]比较,HIE模型组小鼠脑组织SOD活性[（62.60±3.44）?U/?mgprot]显著降低,MDA含量[（83.80±4.39）?μmol/?mgprot]显著升高,与NC-HIE组小鼠脑组织SOD活性[（61.20±4.39）?U/mgprot]和MDA含量[（85.20± 2.70）?μmol/?mgprot]比较,ATG12 shRNA-?HIE组小鼠脑组织SOD活性[（93.80± 5.43）?U/?mgprot]显著升高,MDA含量[（49.20±3.49）?μmol/mgprot]显著降低,差异具有统计学意义（F?= 222.7,P?< 0.01;F?=?415.8,P?相似文献   

Iron homeostasis is maintained in the brain, but not the liver, following mild hypoxia

Alterations in iron metabolism or oxidative damage in response to hypoxic incidents have been examined following re-oxygenation of the hypoxic tissue. To understand the consequences of decreased tissue oxygen on iron load, metal-catalyzed redox activity and oxidative modifications in isolation from re-oxygenation, the present study exposed mice to either normoxia, or mild hypoxia (380 Torr; approximately 10% normobaric oxygen) where the tissue was not allowed to re-oxygenate prior to examination. Brain, liver and skeletal muscle were examined for Fe3+ load, metal-catalyzed redox activity and oxidative modifications to proteins (N(epsilon)-(carboxymethyl)lysine), lipids (4-hydroxynonenal pyrrole) and nucleic acids (8-hydroxyguanosine). Hypoxia induced a 43% increase in the iron content of the liver (P < 0.001) as determined by ICP-MS and a 3.8-fold increase in Fe3+ load (P < 0.001) as determined by Perl's stain. There was a corresponding 2-fold increase in metal-catalyzed redox activity (P < 0.01) in the liver, but no change in the expression of oxidative markers. In contrast, non-significant increases in Fe3+ and metal-catalyzed redox activity were observed in the cerebral cortex, and molecular and granular layers of the hippocampus and cerebellum. Interestingly, hypoxia significantly decreased oxidative modifications to proteins and lipids, but not nucleic acids in most brain regions examined. In addition, hypoxia did not alter the Fe content of skeletal muscle, or the contents of Zn, Cu, Ni or Mn in liver, skeletal muscle, cerebral cortex or hippocampus. Together, these results indicate that there is a tighter regulation of iron metabolism in the brain than the liver, which limits the redistribution of Fe3+ following hypoxia.     

Effects of Hypoxia on the Activity of the Dopaminergic Neuron System in the Rat Striatum as Studied by In Vivo Brain Microdialysis

The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4-dihy-droxyphenylethylamine (dopamine) were examined in the rat Striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage-dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O₂ in N₂) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 mUM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism.     

Semax and selank inhibit the enkephalin-degrading enzymes from human serum]]

A dose-dependent effect of synthetic heptapeptides Semax (Met-Glu-His-Phe-Pro-Gly-Pro) and Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) on the enkephalin-degrading enzymes of human serum was demonstrated. The inhibitory effects of Semax (IC50 10 microM) and Selank (IC50 20 microM) are more pronounced than those of puromycin (IC50 10 mM), bacitracin, and some other inhibitors of peptidases. Beside the heptapeptides, their pentapeptide fragments also possessed an inhibitory effect; tri-, tetra-, and hexapeptide fragments did not display such an effect. As the above enzymes take part in degradation of not only enkephalins but also other regulatory peptides, it can be assumed that one of the mechanisms of biological activity of Semax and Selank is related to this inhibitory activity of theirs.     

A microarray analysis of the hypoxia-induced modulation of gene expression in human adipocytes

The effect of hypoxia on global gene expression in human adipocytes has been examined using DNA microarrays. Adipocytes (Zen-Bio, day 12 post-differentiation) were exposed to hypoxia (1% O(2)) or 'normoxia' (21% O(2)) for 24 h and extracted RNA probed with Agilent arrays containing 41,152 probes. A total of 1346 probes were differentially expressed (>2.0-fold change, P < 0.01) in response to hypoxia; 650 genes were up-regulated (including LEP, IL6, VEGF, ANGPTL4) and 650 down-regulated (including ADIPOQ, UCP2). Major genes not previously identified as hypoxia-sensitive in adipocytes include AQP3, FABP3, FABP5 and PPARGC1A. Ingenuity analysis indicated that several pathways and functions were modulated by hypoxia, including glucose utilization, lipid oxidation and cell death. Network analysis indicated a down-regulation of p38/MAPK and PGC-1α signalling in the adipocytes. It is concluded that hypoxia has extensive effects on human adipocyte gene expression, consistent with low O(2) tension underlying adipose tissue dysfunction in obesity.     

The role of Bax/Bcl-2 and pro-caspase peptides in hypoxia/reperfusion-dependent regulation of MAPK(ERK): discordant proteomic effect of MAPK(p38)

BACKGROUND: The molecular regulation of MAPKs and apoptosis was investigated in a model of hypoxia-tolerance. Survival of neurons in Chrysemys picta bellii, an anoxia-tolerant turtle, involves a reduction in energy metabolism. The biochemical/physiological mechanisms of anoxia tolerance have been examined at the level of ion transport and ATP turnover. However, changes in the phosphorylation state of key enzymes and kinases, mainly, MAPKs, may occur during anoxia, thereby reversible protein phosphorylation could be a critical factor and major mechanism of metabolic reorganization for enduring anaerobiosis. METHODS: If a turtle were to undergo hypoxia akin to that experienced in its native habitat, it was placed in a glass aquarium filled with water to within a half inch of the top. After the turtle was anesthetized, through extended hypoxia or anesthesia, the animal was sacrificed by decapitation. The brain was then excised and placed in anoxic artificial cerebrospinal fluid. Total protein extraction was performed by homogenizing brain in a buffer, followed by threonine and tyrosine phosphorylation determination of MAPKs, and caspase activity. RESULTS: MAPK(p38) was decreased after reoxygenation following 1 day and 1 week hypoxia. The effect of hypoxia on the phosphorylation of MAPK(ERK) was biphasic: Enhancement at 5h and inhibition at 6 weeks. Pro-caspases 8/9 were unchanged by hypoxia until increasing at 6 weeks. Both pro-caspases were upregulated by reoxygenation at 1 day or 6 weeks hypoxia. Neither hypoxia nor reoxygenation induced the cleavage of pro-caspases 8/9 into p20 and p10, respectively. Furthermore, hypoxia induced Bax at 3 days and 1 week, and reoxygenation increased Bax #8776; 4-fold at 1 day. Although the expression of Bcl-2 was slightly increased by hypoxia, [Bcl-2] was 3-4-fold smaller in comparison with Bax. CONCLUSION: These results indicate that hypoxia up-regulates MAPK(ERK) but not MAPK(p38;) hypoxia/reperfusion increases the expression of caspases and pro-apoptotic cofactors. The patterns of MAPK regulation suggest the significance of these kinases in cellular adaptation to oxygen deprivation with biomedical correlations, and thereby identify novel natural responsive signaling cofactors in Chrysemys picta bellii with potential pharmacologic and clinical applications.     

Hypoxia increases calcium flux through cortical neuron glutamate receptors via protein kinase C

The effects of 30 s to 10 min hypoxia (PO2-10 mmHg) on glutamate receptor activity were studied in murine cortical neurons. Receptor activity was assessed as a rise in intracellular calcium concentration ([Ca2+]i) following a 10 s application of 1 mm glutamate or 100 micro mN-methy-d-aspartate (NMDA) in the presence of 0.1 mm Mg2+ and 10 micro m glycine. Change in [Ca2+]i elicited by glutamate increased 26% (n = 192, p < 0.001) and that to NMDA by 74% (n = 9, p < 0.01) during a 100-s period of hypoxia. After 10 min hypoxia, responses to glutamate were 62% smaller than those in normoxia, with increased basal intracellular [Ca2+]i predicting reduced receptor activity. When neurons were exposed to NMDA after 10 min of hypoxia, [Ca2+]i increases were 12% smaller than after 100 s hypoxia, but still 53% larger than in oxygenated neurons (n = 9, p = 0.01). Neurons expressed relatively similar amounts of NR2A, -B, -C, and -D subunits. The phosphorylation of NMDA NR1 subunits increased during hypoxia. Pre-treatment of neurons with a protein kinase C (PKC) inhibitor (chelerythrine, 10 micro m) prevented increases in N-methy-d-aspartate receptor (NMDAR) activity during hypoxia and reduced the phosphorylation of NR1 subunits. These results suggest that enhancement of glutamate receptor activity during the first minutes of hypoxia is mediated by phosphorylation of NMDARs by PKC and that other mechanisms, possibly involving intracellular calcium, limit glutamate receptor-mediated calcium influx during longer periods of hypoxia.     

Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.     

Iron homeostasis is maintained in the brain,but not the liver,following mild hypoxia

Abstract

Alterations in iron metabolism or oxidative damage in response to hypoxic incidents have been examined following re-oxygenation of the hypoxic tissue. To understand the consequences of decreased tissue oxygen on iron load, metal-catalyzed redox activity and oxidative modifications in isolation from re-oxygenation, the present study exposed mice to either normoxia, or mild hypoxia (380 Torr; ～10% normobaric oxygen) where the tissue was not allowed to re-oxygenate prior to examination. Brain, liver and skeletal muscle were examined for Fe³⁺ load, metal-catalyzed redox activity and oxidative modifications to proteins (N^?-(carboxymethyl)lysine), lipids (4-hydroxynonenal pyrrole) and nucleic acids (8-hydroxyguanosine). Hypoxia induced a 43% increase in the iron content of the liver (P < 0.001) as determined by ICP-MS and a 3.8-fold increase in Fe³⁺ load (P < 0.001) as determined by Perl's stain. There was a corresponding 2-fold increase in metal-catalyzed redox activity (P < 0.01) in the liver, but no change in the expression of oxidative markers. In contrast, non-significant increases in Fe³⁺ and metal-catalyzed redox activity were observed in the cerebral cortex, and molecular and granular layers of the hippocampus and cerebellum. Interestingly, hypoxia significantly decreased oxidative modifications to proteins and lipids, but not nucleic acids in most brain regions examined. In addition, hypoxia did not alter the Fe content of skeletal muscle, or the contents of Zn, Cu, Ni or Mn in liver, skeletal muscle, cerebral cortex or hippocampus. Together, these results indicate that there is a tighter regulation of iron metabolism in the brain than the liver, which limits the redistribution of Fe³⁺ following hypoxia.     

Hypoxia induces oxidative stress in tissues of a goby, the rotan Perccottus glenii

The effects of hypoxia (0.4 mg O₂/L) for 2, 6 or 10 h and subsequent normoxic recovery on the levels of lipid peroxides, thiobarbituric acid reactive substances, protein carbonyls (CP), free thiols, and the activities of six antioxidant and associated enzymes were measured in the brain, liver, and skeletal muscle of the rotan Perccottus glenii. Hypoxia increased CP content in the brain (5.0–7.4-fold), liver (2.2–3.3-fold) and muscle (3.2–61-fold) relative to controls and the levels remained elevated during recovery. Lipid peroxide content rose within 2 h of hypoxia in all tissues examined with the most marked increase (8.7-fold) in the liver, but decreased again during longer hypoxic exposure except in the muscle. Levels of low-molecular mass thiols were transiently lowered after 2 h hypoxia in all tissues, but were higher compared with controls after longer hypoxic exposure and recovery. Hypoxia decreased protein thiol content in the liver and muscle that return to control levels during recovery. Experimental conditions affected enzyme activities in a different manner. Superoxide dismutase activity rose two-fold in the liver of hypoxic fish, and a similar tendency was seen in muscle glutathione-S-transferase. Activities of other enzymes were decreased or unchanged during hypoxia and elevated in some cases during normoxic recovery. Taken together, these data show that hypoxia resulted in the development of oxidative stress and a compensatory changes of antioxidant enzymes in the tissues.     

Hypoxia and recovery perturb free radical processes and antioxidant potential in common carp (Cyprinus carpio) tissues

The effects of hypoxia exposure and subsequent normoxic recovery on the levels of lipid peroxides (LOOH), thiobarbituric acid reactive substances (TBARS), carbonylproteins, total glutathione levels, and the activities of six antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of the common carp Cyprinus carpio. Hypoxia exposure (25% of normal oxygen level) for 5h generally decreased the levels of oxidative damage products, but in liver TBARS content were elevated. Hypoxia stimulated increases in the activities of catalase (by 1.7-fold) and glutathione peroxidase (GPx) (by 1.3-fold) in brain supporting the idea that anticipatory preparation takes place in order to deal with the oxidative stress that will occur during reoxygenation. In liver, only GPx activity was reduced under hypoxia and reoxygenation while other enzymes were unaffected. Kidney showed decreased activity of GPx under aerobic recovery but superoxide dismutase (SOD) and catalase responded with sharp increases in activities. Skeletal muscle showed minor changes with a reduction in GPx activity under hypoxia exposure and an increase in SOD activity under recovery. Responses by antioxidant defenses in carp organs appear to include preparatory increases during hypoxia by some antioxidant enzymes in brain but a more direct response to oxidative insult during recovery appears to trigger enzyme responses in kidney and skeletal muscle.     

Mild sustained and intermittent hypoxia induce apoptosis in PC-12 cells via different mechanisms

Episodic hypoxia, a characteristic feature of obstructive sleep apnea, induces cellular changes and apoptosis in brain regions associated with neurocognitive function. To investigate whether mild, intermittent hypoxia would induce more extensive neuronal damage than would a similar degree of sustained hypoxia, rat pheochromocytoma PC-12 neuronal cells were subjected to either sustained (5% O₂) or intermittent (alternating 5% O₂ 35 min, 21% O₂ 25 min) hypoxia for 2 or 4 days. Quantitative assessment of apoptosis showed that while mild sustained hypoxia did not significantly increase cell apoptosis at 2 days (1.31 ± 0.29-fold, n = 8; P = NS), a significant increase in apoptosis occurred after 4 days (2.25 ± 0.4-fold, n = 8; P < 0.002), without increased caspase activation. Furthermore, caspase inhibition with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) did not modify sustained hypoxia-induced apoptosis. In contrast, mild, intermittent hypoxia induced significant increases in apoptosis at 2 days (3.72 ± 1.43-fold, n = 8; P < 0.03) and at 4 days (4.57 ± 0.82-fold, n = 8; P < 0.001) that was associated with enhanced caspase activity and attenuated by Z-VAD-FMK pretreatment. We conclude that intermittent hypoxia induces an earlier and more extensive apoptotic response than sustained hypoxia and that this response is at least partially dependent on caspase-mediated pathways. In contrast, caspases do not seem to play a role in sustained hypoxia-induced apoptosis. These findings suggest that different signaling pathways are involved in sustained and intermittent hypoxia-induced cell injury and may contribute to the understanding of differential brain susceptibility to sustained and intermittent hypoxia. episodic hypoxia; neuronal cell death; caspase; hypoxic adaptation     

Effects of chronic ethanol administration on the activities and relative synthetic rates of myelin and synaptosomal plasma membrane-associated sialidase in the rat brain

In an attempt to understand the possible mechanism of chronic ethanol-induced generation of asialoconjugates in the brain and consequent behavioral abnormalities, we have studied the effects of chronic ethanol feeding to rats on the plasma membrane sialidase status in the various subcellular fractions of the brain. We determined sialidase activity using 3H-monosialoganglioside (3H-GM3), 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4-MU-NeuAC) substrates and Amplex Red (Sialidase) kit. We determined the plasma membrane sialidase protein by Western blot using the anti-plasma membrane sialidase. We also determined its relative synthetic rate (RSR) by the 60 min incorporation of intracranially infused [35S]-methionine (50 microCi/100 g) into immunoprecipitable plasma membrane sialidase. Chronic ethanol administration stimulated the sialidase activity in the total brain homogenate as well as the myelin and synaptosomal membrane fractions, respectively, in all the three experimental models. Chronic ethanol also increased the concentration of the rat brain plasma membrane sialidase protein relative to that of glyceraldehyde-3-phosphate dehydrogenase by 2.4-, 1.62- and 1.51-fold in the total brain homogenate, myelin and synaptosomal membrane fractions, respectively. These increases in plasma membrane sialidase activity and its protein content were due to concomitant increases in their relative synthetic rates by 115% (p < 0.01) and 72% (p < 0.01) in the myelin and synaptosomal membrane fractions, respectively. Thus, our studies clearly show that chronic ethanol induced deglycosylation of brain gangliosides is in part, due to specific up-regulation of plasma membrane sialidase in the myelin and synaptosomal membrane fractions of the brain. This increase in plasma membrane sialidase may be responsible for chronic-ethanol-induced physiological and neurological impairment in the brain, presumably due to deglycosylation of gangliosides that are essential for crucial cellular and metabolic activities.     

Heart HIF-1alpha and MAP kinases during hypoxia: are they associated in vivo?

To study the in vivo dynamics of hypoxia-inducible factor 1alpha (HIF-1alpha), master regulator of O(2)-dependent gene expression, and mitogen-activated protein kinases (MAPKs) in the hypoxic myocardium, Sprague-Dawley rats (n = 4 to 6 per group) were exposed to 1-hr hypoxia (10% O(2)), 23-hr hypoxia, and 23-hr hypoxia, followed by reoxygenation. HIF-1alpha increased 15-fold after 1-hr hypoxia, remained constant for 23 hrs, and returned to baseline on reoxygenation. Extracellular signal-regulated kinases (ERK1/2) were unchanged throughout. Phosphorylated p38 increased 4-fold after 1-hr hypoxia and returned to baseline within 23-hr hypoxia. The activity of stress-activated protein kinases/c-Jun NH(2)-terminal kinases (JNKs), measured as phosphorylated c-Jun, increased 3-fold after 1-hr hypoxia and remained sustained afterward. Furthermore, HIF-1alpha was halved in rats that were administered with the p38 inhibitor SB202190 and made hypoxic for 1 hr. In conclusion, although very sensitive to the reoxygenation, HIF-1alpha is overexpressed in vivo in the hypoxic myocardium, and its acute induction by hypoxia is correlated with that of p38.