Signaling events in the hypoxic induction of alcohol dehydrogenase gene in Arabidopsis

Expression of the alcohol dehydrogenase gene (ADH) of Arabidopsis is induced during hypoxia. Because many plants increase their ethylene production in response to hypoxic stress, we examined in this report whether ethylene is involved in the hypoxic induction of ADH in Arabidopsis. We found that the hypoxic induction of ADH can be partially inhibited by aminooxy acetic acid, an inhibitor of ethylene biosynthesis. This partial inhibition can be reversed by the addition of 1-aminocyclopropane-1-carboxylic acid, a direct precursor of ethylene. In addition, the hypoxic induction of the ADH gene is also reduced in etr1-1 and ein2-1, two ethylene insensitive mutants in ethylene-signaling pathways, whereas the addition of exogenous ethylene or an increase in cellular ethylene alone does not induce ADH under normoxic conditions. Kinetic analyses of ADH mRNA accumulation indicated that an ethylene signal is required for the induction of ADH during later stages of hypoxia. Therefore, we conclude that ethylene is needed, but not sufficient for, the induction of ADH in Arabidopsis during hypoxia.     

Hypoxia stimulates p16 expression and association with cdk4

Exposure of CV-1P cells to hypoxic conditions causes cell proliferation inhibition concomitant with the accumulation of pRb in the hypophosphorylated, growth suppressive form. This is in part due to inhibition of pRb-directed cdk4 and cdk2 activity. In this study we attempted to elucidate the mechanism by which cdk4 is inactivated under hypoxic conditions. After 18 h of hypoxia, CV-1P cells are inhibited from progressing from G(1) phase into the S phase of the cell cycle. This occurs in conjunction with dephosphorylation of serine-795, which is a putative substrate of cdk4. The amounts of cdk4, cdk6, and the D type cyclins are not affected by 18 h of hypoxia. The levels of cdki p16, p18, p19, and p57 under aerobic or hypoxic conditions were analyzed and although the levels of most cdki are unaffected by hypoxic conditions, the level of p16 increases significantly by 18 h of hypoxia. The mechanism by which cdk4 activity is inhibited under hypoxic conditions may be mediated through p16 association with cdk4. Immunoprecipitation analysis shows that p16 binds to cdk4 under hypoxic conditions but does not in cells maintained under aerobic conditions. Thus p16 may be involved in hypoxia-induced growth inhibition.     

Salicylic acid induces apoptosis in colon carcinoma cells grown in-vitro: influence of oxygen and salicylic acid concentration

In solid tumors the hypoxic environment can promote tumor progression and resistance to therapy. Recently, acetylsalicylic acid a major component of analgesic drugs and its metabolite salicylic acid (SA) have been shown to reduce the risk of colon cancer, but the mechanisms of action remain still unclear. Here we elucidate the effects of physiologically relevant concentrations of SA on colon carcinoma cells (CaCo-2) grown under normoxic and hypoxic conditions. Western blotting, caspase-3/7 apoptosis assays, MTS cell-proliferation assays, LDH cytotoxicity assays and hydrogen peroxide measurements were performed to investigate the effects of 1 and 10μM SA on CaCo-2 cells grown under normoxic conditions and cells exposed to hypoxia. Under normoxic conditions, SA did not influence cell proliferation or LDH release of CaCo-2 cells. However, caspase-3/7 activity was significantly increased. Under hypoxia, cell proliferation was reduced and LDH release and caspase-3/7 activities were increased. None of these parameters was altered by the addition of SA under hypoxic conditions. Hypoxia increased hydrogen peroxide concentrations 300-fold and SA significantly augmented the release of hydrogen peroxide under normoxic, but not under hypoxic conditions. Phosphorylation of the pro-survival kinases akt and erk1/2 was not changed by SA under hypoxic conditions, whereas under normoxia SA reduced phosphorylation of erk1/2 after 2 hours. We conclude that in colon carcinoma cells effects of SA on apoptosis and cellular signaling are dependent on the availability of oxygen.     

Protective effect of malonic acid in hypoxic hypoxia]

Malonic acid injection causes an increase in the survival of rats with acute hypoxic hypoxia. Endogenic malonic acid is supposed to be of great importance in stimulating tissue resistance to hypoxia.     

Arachidonate release and c-fos expression in various models of hypoxia and hypoxia–hypoglycemia in retinoic acid differentiated neuroblastoma cells

Hypoxia-hypoglycemia has played an important role in inducing both phospholipase A2 activation and the expression of the early gene c-fos, in the neuroblastoma cell line SK-N-BE, after it has been differentiated by retinoic acid. Under hypoxic-hypoglycemic conditions, arachidonic acid release has found to be significant after 30 min, whereas c-fos expression has required at least 4 h. This model has been obtained by adding glycolytic inhibitor 2-deoxyglucose to the culture and by placing cells in an atmosphere containing 100% N2 for different time periods. This condition has been compared with two different models: NaCN and nitrogen have been used as hypoxic stimuli, without inhibiting the glycolytic pathway, but the same cell cultures have been used. Cell viability and the fall of cellular ATP levels have been evaluated in all the models, in order to monitor and compare the hypoxic cellular damage. Phospholipase A2 activation has been found to be significant in all conditions, even if to a different extent; but only hypoxia combined with the inhibition of the glycolytic pathway, has induced a significant expression of c-fos. It is very difficult to study hypoxic stimuli in 'in vitro' systems. Our study has compared three different models and the one combining gaseous hypoxia and hypoglycemic conditions seems to be very effective in stimulating early events involved in hypoxic phenomena such as phospholipase activation and the expression of the early gene c-fos.     

Protection from hypoxic injury in cultured hepatocytes by glycine,alanine, and serine

Summary Isolated hepatocytes from rat liver in primary culture rapidly lost viability under hypoxic conditions. In the presence of glycine, L-alanine or L-serine loss of viability under hypoxic conditions was greatly retarded. Glycine and L-serine already showed significant protection from hypoxic injury at a concentration of 0.1 mM; at 10 mM, all three amino acids offered almost complete protection. Beside these standard amino acids, 1-aminocyclopropane-1-carboxylic acid (ACPC) and sarcosine significantly decreased hypoxic injury of the hepatocytes, although to a lesser extent. Other amino acids tested provided only slight protection or had no effect on hypoxic injury of the hepatocytes. In the presence of the protective amino acids neither the ATP content nor the lactate production of the hypoxic hepatocytes were significantly affected. The addition of glycine, L-alanine and L-serine led to marked membrane alterations (blebs). These alterations, however, occurred without loss of viability and were reversible upon reoxygenation after up to 4 h of hypoxia.Abbreviations LDH lactate dehydrogenase - ACPC 1-amino-cyclopropane-1-carboxylic acid - HEPES 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid     

Inhibition of synthesis of nucleic acids, proteins and respiration in isolated thymocytes by anthracyclines

The effect of anthracycline antibiotics such as carminomycin, daunomycin (rubomycin) and adriamycin on respiration and synthesis of nucleic acids and protein was studied comparatively. The anthracyclines inhibited the processes. By their efficacy in that respect they could be arranged in the following order: carminomycin greater than rubomycin greater than adriamycin. Thus, 50 per cent inhibition of nucleic acid synthesis in the thymocytes required 0.027, 0.044 and 0,173 mM of carminomycin, rubomycin and adriamycin respectively. Protein synthesis and respiration in the thymocytes were less sensitive to the effect of the anthracyclines than synthesis of nucleic acids. The study results were compared with the literature data on the effect of the compounds on respiration and synthesis of nucleic acids and protein in tumour and bacterial cells.     

Maintenance of 32P uptake and transport in Pinus serotina seedlings under hypoxic growth conditions

In the present study, we examined the effects of long- and short-term hypoxia on net uptake and transport of phosphorus to shoots of pond pine (Pinus serotina Michx.), a moderately flood-tolerant southern pine, and the influence aerenchyma formation might have in maintenance of P uptake and transport. Seedlings were grown under aerobic (250 μM O₂) or hypoxic (≤50 μM O₂) solution conditions for 5.3 weeks in continuously flowing solution culture containing 100 μM P. Intact seedlings were then labeled with ³²P for up to 24 h to determine how short- and long-term hypoxic solution conditions affected rates of unidirectional influx and the accumulation of ³²P in roots and shoots. Seedlings in the long-term hypoxic treatment were grown for 5.3 weeks in hypoxic solution and also labeled in hypoxic uptake solution. The short-term hypoxic treatments included a 24-h hypoxic pretreatment followed by time in labeled hypoxic uptake solution for seedlings grown under aerobic or hypoxic conditions; in the latter case, diffusion of atmospheric O₂ entry into stem and root collar lenticels was blocked, thus removing any influence that aerenchyma formation might have had on enhancing O₂ concentrations of root tissue. Although unidirectional influx rates of ³²P in roots of seedlings grown under long-term hypoxic conditions were 1.4 times those of aerobically grown seedlings, accumulation of ³²P in roots was similar after 24 h in labeled uptake solution. These results suggest that ³²P efflux was also higher under hypoxic conditions. Higher shoot/root fresh weight ratios and lower shoot P concentrations in seedlings grown under hypoxic solution conditions suggest that the “shoot P demand” per unit root should be high. Yet accumulation of ³²P in shoots was reduced by 50% after 24 h in hypoxic uptake solution. Both short-term hypoxic treatments decreased accumulation of ³²P in roots by more than 50%. Short-term hypoxia decreased shoot accumulation in seedlings grown under aerobic and hypoxic conditions by 84 and 50%. respectively. Short- and long-term hypoxic conditions increased the percentage of root ³²P in the nucleic acid and chelated-P pools, resulting in a significantly smaller percentage of ³²P in the soluble inorganic phosphate (p_i) pool, the pool available for transport to the shoot. However, a reduction in pool size or in labeling of the pool available for transport cannot fully account for the large reduction in accumulation of ³²P in shoots, particularly in the short-term hypoxic treatment of aerobically grown seedlings. Our results suggest that both influx and transport of ³²P to shoots of pond pine seedlings are O₂-dependent processes, and that the transport of ³²P to shoots may be more sensitive to hypoxic solution conditions than influx at the cortical and epidermal plasmalemma, with aerenchyma formation supporting a substantial amount of both ³²P uptake and transport.     

Functional morphology of thymocytes during physiological fluctuations in hormone balance

Some metabolic parameters of thymocytes of noninbred female rats were evaluated by quantitative histochemistry during the estrous cycle. There were cyclic variations in the activity of NAD . H and NADP H-diaphorases as well as in the content of DNA and RNA in thymocytes of all parts of the thymus. The greatest range of variations in the content of nucleic acids was recorded in thymocytes of medulla. The activity of acid phosphatase in thymocytes remained unchanged over the period of the estrous cycle.     

Post-irradiation hypoxic incubation of X-irradiated MOLT-4 cells reduces apoptotic cell death by changing the intracellular redox state and modulating SAPK/JNK pathways

To elucidate radiobiological effects of hypoxia on X-ray-induced apoptosis, MOLT-4 cells were treated under four set of conditions: (1) both X irradiation and incubation under normoxia, (2) X irradiation under hypoxia and subsequent incubation under normoxia, (3) X irradiation under normoxia and subsequent incubation under hypoxia, and (4) both X irradiation and incubation under hypoxia, and the induction of apoptosis was examined by fluorescence microscopy. About 28–33% apoptosis was observed in cells treated under conditions 1 and 2, but this value was significantly reduced to around 18–20% in cells treated under conditions 3 and 4, suggesting that post-irradiation hypoxic incubation rather than hypoxic irradiation mainly caused the reduction of apoptosis. The activation and expression of apoptosis signal-related molecules SAPK/JNK, Fas and caspase-3 were also suppressed by hypoxic incubation. Effects of hypoxic incubation were canceled when cells were treated under conditions 3 and 4 with an oxygen-mimicking hypoxic cell radiosensitizer, whereas the addition of N-acetyl-L-cysteine again reduced the induction of apoptosis. From these results it was concluded that hypoxia reduced the induction of apoptosis by changing the intracellular redox state, followed by the regulation of apoptotic signals in X-irradiated MOLT-4 cells.     

Effects of fatty acid provision during severe hypoxia on routine and maximal performance of the in situ tilapia heart

The ability to maintain stable cardiac function during environmental hypoxia exposure is crucial for hypoxia tolerance in animals and depends upon the maintenance of cardiac energy balance as well as the state of the heart’s extracellular environment (e.g., availability of metabolic fuels). Hypoxic depression of plasma [non-esterified fatty acids] (NEFA), an important cardiac aerobic fuel, is a common response in many species of hypoxia-tolerant fishes, including tilapia. We tested the hypothesis that decreased plasma [NEFA] is important for maintaining stable cardiac function during and following hypoxia exposure, based on the premise that continued reliance upon cardiac fatty acid metabolism under such conditions could impair cardiac function. We examined the effect of severe hypoxia exposure (PO₂ < 0.2 kPa) on routine and maximum performance of the in situ perfused tilapia heart under conditions of routine (400 μmol L^?1) and low (75 μmol L^?1) [palmitate], which mimicked the in vivo levels of plasma [NEFA] found in normoxic and hypoxic tilapia, respectively. Under both concentrations of palmitate, the in situ tilapia heart showed exceptional hypoxic performance as a result of a high maximum glycolytic potential, confirming our previous results using a perfusate without fatty acids. We additionally provide evidence suggesting that non-contractile ATP demand is depressed in tilapia heart during hypoxia exposure. Cardiac performance during and following severe hypoxia exposure was unaffected by the level of palmitate. Thus, we conclude that hypoxic depression of plasma [NEFA] in fishes does not play a role in cardiac hypoxia tolerance.     

Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival

Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.     

Effect of hypokinesia on nucleic acid and protein metabolism in the lymphoid organs of rats

Metabolism of nucleic acids and protein by lymphoid cells of the rat spleen and thymus was studied under conditions of 22-day hypokinesia. It was shown that in the course of hypokinesia the loss of cellular mass by the spleen and thymus was associated with varied biochemical changes in the remaining lymphoid cells. The thymocytes showed a significant activation of nucleic acid and protein biosynthesis. Meanwhile in spleen lymphocytes, DNA and RNA metabolism was inhibited with no appreciable changes in protein metabolism. Potential mechanisms of changes in metabolism of thymus and spleen lymphocytes under long-term hypokinesia are discussed.     

The effect of graded hypoxia on the embryonic chick heart

Embryonic ventricular function in the chick was measured in response to graded levels of hypoxia. Myocardial contractility, as measured by cinephotoanalysis and expressed as shortening fraction, was significantly depressed after 1 hour of moderate hypoxia (6% O2) and after 5 hours of milder (16% O2 and 11% O2) levels of hypoxia (P less than .05). Microscopy confirmed associated myocyte damage with cell death noted after 5 hours of moderate hypoxic stress. Heart rate change was not related to the severity of hypoxia. The greatest level of tachycardia was noted with conditions of mildest hypoxia (16% O2). The data confirm that cardiac contractility, as measured by shortening fraction, is depressed on exposure to hypoxia, with impairment of function related to the severity of the hypoxic conditions.     

Characteristics of the human EEG during cognitive-mnemenic activity under hypoxic conditions

The amplitude-frequency and spatiotemporal characteristics of the EEGs of subjects performing various cognitive-mnemenic activities under the conditions of graduated hypoxia were studied. The quickness and correctness of test performance were significantly decreased beginning from the sixth minute of hypoxia as compared to normoxic conditions. The amplitude and mean period of the dominant EEG activity in this functional state were higher than in the same tests performed under normoxic conditions and lower than in the case of hypoxia not accompanied by the performance of tests. The spatiotemporal characteristics of the EEG under hypoxic conditions displayed both the characteristics typical of hypoxia (a decrease in EEG cross-correlation within anterior cortical regions) and those typical of cognitive-mnemenic activity (an increase in the correlation between the EEGs of distant zones of anterior and posterior cortical regions). It is assumed that the “intermediate” EEG pattern observed in subjects performing cognitive-mnemenic tests under hypoxic conditions reflects opposite effects of hypoxia and intellectual effort on the functional activity of brain neurons.