O2 Deprivation Research Articles

Axotomy along with Hypoxia Enhances the Neuronal NADPH-d/NOS Expression in Lower Brain Stem Motor Neurons of Adult Rats

This study was aimed to determine whether axotomy coupled with hypoxia would exert a more profound effect on injury-induced neuronal nitric oxide synthase (NOS) expression. In this connection, the vagus and the hypoglossal nerves of adult rats were transected unilaterally in the same animal, and half of the operated animals were subjected to hypoxia treatment. Both the neuronal NOS immunohistochemistry and the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry were used to assess the neuronal NOS expression. The present results have shown that the number of NADPH-d/NOS-positive [NADPH-d/NOS(1)] neurons in the hypoglossal nucleus (HN) peaked at 14 days after axotomy, while that in dorsal motor nucleus of vagus (DMN) and nucleus ambiguus (NA) was progressively increased up to 60 days. The up-regulation of NADPH-d/NOS in HN and DMN was more pronounced in hypoxic than in normoxic animals, a feature that was not evident in the NA. Quantitative analysis showed that the number of surviving motoneurons in normoxic animals was significantly higher than those subjected to hypoxia at 14 days postaxotomy in HN and at all postaxotomy time points in DMN. The difference may be attributed to their different functional components. Since O2 deprivation leads to poor cellular function, the stronger expression of NADPH-d/NOS and the more drastic neuronal loss following nerve transection in the hypoxic animals compared with the controls suggest that hypoxia plays an important role in peripheral neuropathies in which NO is implicated.

Major difference in the expression of δ‐ and μ‐opioid receptors between turtle and rat brain

The reptilian turtle brain has a remarkably higher endurance for anoxia than mammalian brains. Since the response to O(2) deprivation is dependent in a major way on the expression and regulation of membrane proteins, differences in such proteins may play a role in the species-related differences in hypoxic responses. Because opioid system is involved in the regulation of hypoxic responses, we asked whether there are differences between rat and turtle brains in terms of opioid receptor expression. In this work, we compared the expression and distribution of delta-and mu-opioid receptors in the turtle and rat brains. Our results show that (1) the dissociation constant (K(d)) for delta-receptor binding was approximately four times lower and B(max) was more than double in the turtle brain homogenates than in rat ones; (2) the delta-receptor binding density was heterogeneously distributed in the turtle brain, with a higher density in the rostral regions than in the brainstem and spinal cord, and was generally much higher than in rat brains from the cortex to spinal cord; (3) the delta-opioid receptors in the rat brains were mostly located in the cortex, caudate putamen, and amygdala with an extremely low density in most subcortical (e.g., hippocampus and thalamus) and almost all brainstem regions; and (4) in sharp contrast to delta-opioid receptors, mu-opioid receptor density was much lower in all turtle brain regions compared with the rat ones. Our results demonstrate that the turtle brain is actually an organ of delta-opioid receptors, whereas the rat brain has predominantly mu-opioid receptors. Because we have recently found that delta-opioid receptors protect neurons against glutamate and hypoxic stress, we speculate that the unique pattern of delta-receptor receptor expression and distribution plays a critical role in the tolerance of turtle brain to stressful situations characterized by glutamate excitotoxicity.

Invited review: Adaptive responses of skeletal muscle to intermittent hypoxia: the known and the unknown.

Intermittent hypoxia (IH) describes conditions of repeated, transient reductions in O2 that may trigger unique adaptations. Rest periods during IH may avoid potentially detrimental effects of long-term O2 deprivation. For skeletal muscle, IH can occur in conditions of obstructive sleep apnea, transient altitude exposures (with or without exercise), intermittent claudication, cardiopulmonary resuscitation, neonatal blood flow obstruction, and diving responses of marine animals. Although it is likely that adaptations in these conditions vary, some patterns emerge. Low levels of hypoxia shift metabolic enzyme activity toward greater aerobic poise; extreme hypoxia shifts metabolism toward greater anaerobic potential. Some conditions of IH may also inhibit lactate release during exercise. Many related cellular phenomena could be involved in the response, including activation of specific O2 sensors, reactive oxygen and nitrogen species, preconditioning, hypoxia-induced transcription factors, regulation of ion channels, and influences of paracrine/hormonal stimuli. The net effect of a variety of adaptive programs to IH may be to preserve contractile function and cell integrity in hypoxia or anoxia, a response that does not always translate into improvements in exercise performance.

Cell cycle progression and cell division are sensitive to hypoxia in Drosophila melanogaster embryos.

We and others recently demonstrated that Drosophila melanogaster embryos arrest development and embryonic cells cease dividing when they are deprived of O2. To further characterize the behavior of these embryos in response to O2 deprivation and to define the O2-sensitive checkpoints in the cell cycle, embryos undergoing nuclear cycles 3-13 were subjected to O2 deprivation and examined by confocal microscopy under control, hypoxic, and reoxygenation conditions. In vivo, real-time analysis of embryos carrying green fluorescent protein-kinesin demonstrated that cells arrest at two major points of the cell cycle, either at the interphase (before DNA duplication) or at metaphase, depending on the cell cycle phase at which O2 deprivation was induced. Immunoblot analysis of embryos whose cell divisions are synchronized by inducible String (cdc25 homolog) demonstrated that cyclin B was degraded during low O2 conditions in interphase-arrested embryos but not in those arrested in metaphase. Embryos resumed cell cycle activity within ~20 min of reoxygenation, with very little apparent change in cell cycle kinetics. We conclude that there are specific points during the embryonic cell cycle that are sensitive to the O2 level in D. melanogaster. Given the fact that O2 deprivation also influences the growth and development of other species, we suggest that similar hypoxia-sensitive cell cycle checkpoints may also exist in mammalian cells.

Mutation in pre-mRNA adenosine deaminase markedly attenuates neuronal tolerance to O2 deprivation in Drosophila melanogaster.

O2 deprivation can produce many devastating clinical conditions such as myocardial infarct and stroke. The molecular mechanisms underlying the inherent tissue susceptibility or tolerance to O2 lack are, however, not well defined. Since the fruit fly, Drosophila melanogaster, is extraordinarily tolerant to O2 deprivation, we have performed a genetic screen in the Drosophila to search for loss-of-function mutants that are sensitive to low O2. Here we report on the genetic and molecular characterization of one of the genes identified from this screen, named hypnos-2. This gene encodes a Drosophila pre-mRNA adenosine deaminase (dADAR) and is expressed almost exclusively in the adult central nervous system. Disruption of the dADAR gene results in totally unedited sodium (Para), calcium (Dmca1A), and chloride (DrosGluCl-alpha) channels, a very prolonged recovery from anoxic stupor, a vulnerability to heat shock and increased O2 demands, and neuronal degeneration in aged flies. These data clearly demonstrate that, through the editing of ion channels as targets, dADAR, for which there are mammalian homologues, is essential for adaptation to altered environmental stresses such as O2 deprivation and for the prevention of premature neuronal degeneration.

Differential vulnerability of cortical and cerebellar neurons in primary culture to oxygen glucose deprivation followed by reoxygenation.

The effects of glucose and O2 deprivation (OGD) on the survival of cortical and cerebellar neurons were examined to characterize the biochemical mechanisms involved in OGD and OGD followed by reoxygenation. To this aim, neurons were kept for different time periods in a hypoxic chamber with a controlled atmosphere of 95% N(2) and 5% CO2 in a glucose-free medium. After OGD, reoxygenation was achieved by exposing the cells to normal O2 and glucose levels. Neither MTT, an index of mitochondrial oxidative phosphorylation, nor malondialdehyde (MDA) production, a parameter measuring lipid peroxidation, were affected by 1 hr of OGD in cortical neurons. When OGD was followed by 24 hr of reoxygenation, MTT levels were reduced by 40% and MDA was significantly increased, whereas cellular ATP content did not change. Cerebellar granule cells, on the other hand, did not show any reduction of mitochondrial activity after exposure to 1 hr OGD or to 1 hr OGD plus 24 hr of reoxygenation. When OGD was prolonged for 2 hr, a significant reduction of the mitochondrial activity and of cellular ATP content occurred, coupled to a significant MDA increase in cerebellar granule cells, whereas in cortical neurons a reduction of MTT levels after 2 hr OGD was not accompanied by a decrease of cellular ATP content nor by an increase of MDA production. Moreover, 24 hr of reoxygenation further reinforced lipid peroxidation, LDH release, propidium iodide positive neurons and the reduction of ATP content in cerebellar granule cells. The results of the present study collectively show that cortical and cerebellar neurons display different levels of vulnerability to reoxygenation followed by OGD. Furthermore, the impairment of mitochondrial activity and the consequent overproduction of free radicals in neurons were observed for the first time occurring not only during the reoxygenation phase, but already beginning during the OGD phase.

A Ca2+‐dependent cysteine protease is associated with anoxia‐induced root tip death in maize

Imposition of anoxia on maize (Zea mays cv. B73) seedlings for 48 h or longer led to the death of the root tip. The necrosis extended into the root axis during postanoxic treatment, leading to the mortality of 30-50% of the seedlings. Using zymography, protease profiles in the root tissues of anoxic seedlings were studied. O2 deprivation for 24 h or longer repressed pre-existing protease activities and induced a novel soluble enzyme in the roots. The anoxia-induced protease (AIP) activity was predominant in the root apex at 24 h of anoxia and, subsequently, became the most abundant soluble activity in the root axis as well. The induction of AIP and its in vitro renaturation were Ca(2+)-dependent. Inhibitor sensitivity studies indicated that AIP is a cysteine protease. In SDS-acrylamide gels, the enzyme activity migrated as a 23.5 kDa polypeptide. The anoxic induction of the activity was repressed by cycloheximide treatment, suggesting that new protein synthesis was required for the AIP appearance. Excision of the root tip (de-tipping) before anoxia led to a superior recovery of seedlings from stress injury. De-tipped seedlings showed lesser root damage and an increased production of lateral roots compared to intact seedlings. Furthermore, the superior anoxia tolerance of de-tipped seedlings was associated with a decreased AIP activity. Thus, the appearance of AIP activity in the root tip at 24 h of anoxia was spatially and temporally associated with the root tissue death. These studies further indicate that the root tip elimination early during anoxia may provide an adaptive advantage.

Nitrogenase activity in Alnus incana root nodules. Responses to O(2) and short-term N(2) deprivation.

O(2) and host-microsymbiont interactions are key factors affecting the physiology of N(2)-fixing symbioses. To determine the relationship among nitrogenase activity of Frankia-Alnus incana root nodules, O(2) concentration, and short-term N(2) deprivation, intact nodulated roots were exposed to various O(2) pressures (pO(2)) and Ar:O(2) in a continuous flow-through system. Nitrogenase activity (H(2) production) occurred at a maximal rate at 20% O(2). Exposure to short-term N(2) deprivation in Ar:O(2) carried out at either 17%, 21%, or 25% O(2) caused a decline in the nitrogenase activity at 21% and 25% O(2) by 12% and 25%, respectively. At 21% O(2), nitrogenase activity recovered to initial activity within 60 min. The decline rate was correlated with the degree of inhibition of N(2) fixation. Respiration (net CO(2) evolution) decreased in response to the N(2) deprivation at all pO(2) values and did not recover during the time in Ar:O(2). Increasing the pO(2) from 21% to 25% and decreasing the pO(2) from 21% to 17% during the decline further decreased rather than stimulated nitrogenase activity, showing that the decline was not due to O(2) limitation. The decline was possibly due to a temporary disturbance in the supply of reductant to nitrogenase with a partial O(2) inhibition of nitrogenase at 25% O(2). These results are consistent with a fixed O(2) diffusion barrier in A. incana root nodules, and show that A. incana nodules differ from legume nodules in the response of the nitrogenase activity to O(2) and N(2) deprivation.

The viable but non-culturable state of wine micro-organisms during storage.

Colony counting and DEFT did not give the same results when wine micro-organisms were enumerated. Both methods were used to monitor the population of acetic acid bacteria (AAB) and lactic acid bacteria (LAB) during wine storage. Results suggest that part of the populations had reached a viable but non-culturable (VBNC) state. These cells were unable to produce colonies but could hydrolyse fluorescent esters and could be counted by DEFT. For AAB, O2 deprivation quickly induced this state. Recovery from this state was very rapid as soon as O2 was available. The response was not so clear for LAB during wine storage. However, a similar state was induced by sulfiting. Moreover, filtration of wine stored in barrels and contaminated by Brettanomyces, AAB and LAB demonstrated that cell size was not homogeneous. Cells which remained in wine after several weeks could pass through a 0.45-microm membrane. However, when they re-entered a growing phase, they were again retained by membrane filtration. During and after the decline phase, wine micro-organisms might survive as smaller cells in a VBNC state.

O(2) deprivation inhibits Ca(2+)-activated K(+) channels via cytosolic factors in mice neocortical neurons.

O(2) deprivation induces membrane depolarization in mammalian central neurons. It is possible that this anoxia-induced depolarization is partly mediated by an inhibition of K(+) channels. We therefore performed experiments using patch-clamp techniques and dissociated neurons from mice neocortex. Three types of K(+) channels were observed in both cell-attached and inside-out configurations, but only one of them was sensitive to lack of O(2). This O(2)-sensitive K(+) channel was identified as a large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), as it exhibited a large conductance of 210 pS under symmetrical K(+) (140 mM) conditions, a strong voltage-dependence of activation, and a marked sensitivity to Ca(2+). A low-O(2) medium (PO(2) = 10-20 mmHg) markedly inhibited this BK(Ca) channel open probability in a voltage-dependent manner in cell-attached patches, but not in inside-out patches, indicating that the effect of O(2) deprivation on BK(Ca) channels of mice neocortical neurons was mediated via cytosol-dependent processes. Lowering intracellular pH (pH(i)), or cytosolic addition of the catalytic subunit of a cAMP-dependent protein kinase A in the presence of Mg-ATP, caused a decrease in BK(Ca) channel activity by reducing the sensitivity of this channel to Ca(2+). In contrast, the reducing agents glutathione and DTT increased single BK(Ca) channel open probability without affecting unitary conductance. We suggest that in neocortical neurons, (a) BK(Ca) is modulated by O(2) deprivation via cytosolic factors and cytosol-dependent processes, and (b) the reduction in channel activity during hypoxia is likely due to reduced Ca(2+) sensitivity resulting from cytosolic alternations such as in pH(i) and phosphorylation. Because of their large conductance and prevalence in the neocortex, BK(Ca) channels may be considered as a target for pharmacological intervention in conditions of acute anoxia or ischemia.

A Drosophila CDK5α-like Molecule and Its Possible Role in Response to O2 Deprivation

Cyclin-dependent kinase 5 activator (Cdk5α) is an activator of Cdk5 kinase activity and its expression is restricted to neurons. The complex of Ckd5/Cdk5α is essential for neurite outgrowth during neuronal differentiation and possibly also for neuronal degeneration. Here we report the isolation and characterization of a Drosophila Cdk5α-like molecule (dCdk5α). The gene encoding this molecule is localized in the Drosophila chromosome region of 31D1-31D2. The expression of this gene is differentially regulated with a very low level at earlier developmental stages and reaches the highest level in the adult. The C-terminal of this molecule shares high homology with the mammalian Cdk5α molecule. Constitutive over-expression of dCdk5α in transgenic flies significantly prolongs their recovery time from 5 min to O2 deprivation or anoxia in older flies (15 days) but not in young ones (4 days). In addition, anoxia up-regulated the expression of this gene. Taken together, the results in this report and others provide a framework for genetically dissecting the functions of Cdk5α/Cdk5 complex in the CNS.

Oxygen deprivation stimulates Ca2+-mediated phosphorylation of mRNA cap-binding protein eIF4E in maize roots.

Flooding of maize seedlings causes O2 deprivation that leads to a global reduction in protein synthesis and selective translation of cytoplasmic mRNAs. Since selective translation in animal cells can involve the cap-binding protein eIF4E, we characterized the distinct mRNA cap-binding proteins eIF4E and eIFiso4E of maize. These proteins have 45% deduced amino acid sequence identity and are highly conserved at residues of eIF4E that function in intermolecular interactions in animals. Maize eIF4E is a phosphoprotein. O2 deprivation resulted in a decrease in the isoelectric point of eIF4E, consistent with additional phosphorylation. Modification of eIF4E was mimicked by treatment with caffeine under aerobic conditions and blocked by treatment with ruthenium red under O2 deprivation, implicating Ca2+ as a second messenger in eIF4E modification. In contrast, no isoelectric variants of eIFiso4E were detected. The possible role of cytosolic Ca2+ and pH in regulation of mRNA cap-binding protein activity under O2 deprivation is discussed.

Neuroprotective effects of riluzole: An electrophysiological and histological analysis in an in vitro model of ischemia

The protective effects of riluzole against the neuronal damage caused by O2 and glucose deprivation (ischemia) was investigated in rat cortical slices by recording electrophysiologically the cortico-cortical field potential and by evaluating histologically the severity of neuronal death. Five minutes of ischemia determined an irreversible depression of the amplitude of the field potential. In addition, this insult caused a clear enhancement of the number of death cells that were specifically colored with trypan blue (a vital colorant which stains altered cells). We found that riluzole, which by itself depressed the synaptic transmission, neuroprotected when perfused 15–20 min before and during ischemia. In fact, due to the treatment with riluzole, the ischemia-induced irreversible depression of the field potential recovered and less cells were stained with trypan blue. These findings demonstrate that riluzole prevents neuronal death in an in vitro model of ischemia and suggest a therapeutic use of this drug in order to reduce the pathophysiological outcomes of stroke. Synapse 32:147–152, 1999. © 1999 Wiley-Liss, Inc.

Neuroprotective effects of riluzole: an electrophysiological and histological analysis in an in vitro model of ischemia.

The protective effects of riluzole against the neuronal damage caused by O2 and glucose deprivation (ischemia) was investigated in rat cortical slices by recording electrophysiologically the cortico-cortical field potential and by evaluating histologically the severity of neuronal death. Five minutes of ischemia determined an irreversible depression of the amplitude of the field potential. In addition, this insult caused a clear enhancement of the number of death cells that were specifically colored with trypan blue (a vital colorant which stains altered cells). We found that riluzole, which by itself depressed the synaptic transmission, neuroprotected when perfused 15-20 min before and during ischemia. In fact, due to the treatment with riluzole, the ischemia-induced irreversible depression of the field potential recovered and less cells were stained with trypan blue. These findings demonstrate that riluzole prevents neuronal death in an in vitro model of ischemia and suggest a therapeutic use of this drug in order to reduce the pathophysiological outcomes of stroke.

An electrophysiological analysis of the protective effects of felbamate, lamotrigine, and lidocaine on the functional recovery from in vitro ischemia in rat neocortical slices.

We used field potential recording techniques to examine whether felbamate (FBM), lamotrigine (LTG), and lidocaine (LID) protect against the irreversible functional damage induced by transient ischemia. Five minutes of ischemia caused a depression of the field potential in rat cortical slices, which did not recover even after more than 1 h of washout. The N-methyl-D-aspartate (NMDA) antagonist ketamine (50 microM) protected against depression of the field caused by ischemia. On the other hand, the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2.3-dione (CNQX) (10 microM) had protective effects only if co-applied with ketamine. We found that either FBM (30-300 microM), which did not modify the amplitude of the field EPSP, or LTG (10-300 microM), which reversibly depressed the excitatory synaptic transmission, had a marked protective effect when superfused before and during the ischemic insult. After FBM (100 microM) and LTG (100 microM), the field EPSP recovered by 84 +/- 1% and 73 +/- 2.7% of control, respectively. Furthermore, LID (30-300 microM) was less effective than FBM and LTG in inducing a functional recovery from the damage caused by ischemia (58 +/- 1.8%). The rank order of potency, based on the maximal protection caused by the three drugs, was FBM > LTG > LID. Our results suggest that a noticeable neuroprotection can be obtained during glucose and O2 deprivation by preventive therapeutic regimens which use the two recently marketed anticonvulsant drugs, FBM and LTG.

Mitochondrial contribution to the anoxic Ca2+ signal in maize suspension-cultured cells

Anoxia induces a rapid elevation of the cytosolic Ca2+ concentration ([Ca2+]cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca2+ release is important for gene activation and survival in O2-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca2+ to the anoxic [Ca2+]cyt elevation in maize cells. Imaging of intramitochondrial Ca2+ levels showed that a majority of mitochondria released their Ca2+ in response to anoxia and took up Ca2+ upon reoxygenation. We also investigated whether the mitochondrial Ca2+ release contributed to the increase in [Ca2+]cyt under anoxia. Analysis of the spatial association between anoxic [Ca2+]cyt changes and the distribution of mitochondrial and other intracellular Ca2+ stores revealed that the largest [Ca2+]cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca2+]cyt under aerobic conditions but prevented a [Ca2+]cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca2+]cyt and participate in the signaling of O2 deprivation.

Molecular and Biochemical Characterization of Cytosolic Phosphoglucomutase in Maize1

Phosphoglucomutase (PGM) catalyzes the interconversion of glucose (Glc)-1- and Glc-6-phosphate in the synthesis and consumption of sucrose. We isolated two maize (Zea mays L.) cDNAs that encode PGM with 98.5% identity in their deduced amino acid sequence. Southern-blot analysis with genomic DNA from lines with different Pgm1 and Pgm2 genotypes suggested that the cDNAs encode the two known cytosolic PGM isozymes, PGM1 and PGM2. The cytosolic PGMs of maize are distinct from a plastidic PGM of spinach (Spinacia oleracea). The deduced amino acid sequences of the cytosolic PGMs contain the conserved phosphate-transfer catalytic center and the metal-ion-binding site of known prokaryotic and eukaryotic PGMs. PGM mRNA was detectable by RNA-blot analysis in all tissues and organs examined except silk. A reduction in PGM mRNA accumulation was detected in roots deprived of O2 for 24 h, along with reduced synthesis of a PGM identified as a 67-kD phosphoprotein on two-dimensional gels. Therefore, PGM is not one of the so-called "anaerobic polypeptides." Nevertheless, the specific activity of PGM was not significantly affected in roots deprived of O2 for 24 h. We propose that PGM is a stable protein and that existing levels are sufficient to maintain the flux of Glc-1-phosphate into glycolysis under O2 deprivation.

Effects of temperature and chronic hypoxia on survivorship of the zebra mussel (Dreissena polymorpha) and Asian clam (Corbicula fluminea)

We examined the effects of four levels of chronic hypoxic stress at three temperatures on the survivorship of Dreissena polymorpha and Corbicula fluminea to assess the efficacy of O2 deprivation as a macrofouling control treatment and examine if critical hypoxia limits support reported distribution patterns. At 25°C, the hypoxia tolerance was examined at Po2 = 7.9, 11.9, 15.9, 23.8, and 31.8 Torr (1 Torr = 133.322 Pa) or 5, 7.5, 10, 15, and 20% of full air O2 saturation (Po2 = 159 Torr). At 15°C, the hypoxia tolerance to 7.9, 11.9, and 15.9 Torr was tested and at 7.9 Torr for 5°C treatments. For both species, Po2 and temperature influenced survivorship dramatically with increasing survivorship at higher Po2 and decreasing temperatures. At 25°C, C. fluminea experienced mortality at 7.9, 11.9, and 15.9 Torr, with LT50 values of 144, 216, and 216 h, respectively, versus 288, 384, and 480 h for the 15°C exposures. Dreissena polymorpha treatments had LT50 values of 120, 216, and 216 h at 25°C for the 7.9-, 11.9-, and 15.9-Torr treatments versus 26% mortality after 600 h and 28% mortality after 720 h at 15°C. The 7.9-Torr treatments at 5°C had LT50 values of 480 h for C. fluminea and 1056 h for D. polymorpha. This study showed that both species displayed broad seasonal variation in hypoxia tolerance and that hypoxia limits may be used to assess infestation risk.

Accumulation of γ-aminobutyric acid in nodulated soybean in response to drought stress.

Nitrogen fixation and nodule permeability to O2 diffusion are decreased by drought stress. Since γ-aminobutyric acid (GABA) synthesis is rapidly stimulated by a variety of stress conditions including hypoxia, it was hypothesized that decreased O2 availability in nodules stimulates glutamate decarboxylase (GAD) activity (EC 4.1.1.15), thereby resulting in GABA accumulation. First, the amino acid composition of xylem sap was determined in plants subjected to soil water deficits. While the xylem sap concentration of several amino acids increased when the plant was subjected to a water deficit, the greatest increase was in GABA. GABA accumulation was examined in response to stress induced by hypoxia or the addition of polyethylene glycol (PEG) to the nutrient solution. The exposure of soybean nodules to hypoxia for 6 h enhanced the GABA concentration by 6-fold, but there was no change in GABA concentration in response to the PEG treatment. No major changes in the in vitro GAD activity were measured in nodule cytosol or bacteroids. The present data do not support the hypothesis that decreased nodule O2 permeability and a resulting O2 deprivation inside nodules may stimulate in vitro GAD activity and thus GABA accumulation. However, the data could indicate a possible effect of hypoxia and drought stress on the in vivo activity of GAD.

RNase Activities Are Reduced Concomitantly with Conservation of Total Cellular RNA and Ribosomes in O2-Deprived Seedling Roots of Maize.

The effect of O2 deprivation on the activities of RNases and levels of total cellular RNA and ribosomes in seedling roots of maize (Zea mays L.) was investigated. Sodium dodecyl sulfate-polyacrylamide gels containing RNA were used to distinguish RNase isoenzymes by apparent molecular mass. Since O2 deprivation causes a decrease in cytosolic pH from approximately pH 7.4 to 6.4 and an elevation in cytosolic Ca2+, RNase levels were examined in the physiological range of cytosolic pH and in the presence of Ca2+, Mg2+, Zn2+, ethylenediaminetetracetate, or ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid. The activity of a number of RNases present in aerobic roots was reduced in response to O2 deprivation. Several RNases with a pH optimum of 6.4 were rapidly down-regulated by O2 deprivation. Spectrophotometric assay of extracts revealed that RNase activity was higher at pH 6.4 than at 7.2, and ethylenediaminetetracetate-insensitive RNase activity decreased in response to O2 deprivation. The decrease in RNase activity was correlated with no loss of total cellular RNA or ribosomes, despite a 4-fold decrease in run-on transcription of rRNA in isolated nuclei. Regulation of RNase activity may facilitate the conservation of nontranslating ribosomes and poorly translated mRNAs during O2 deprivation.