Search PubMed⌕ Search

Biomedical subjects

Gabriel G Haddad

Publications and source records attributed to Gabriel G Haddad.

29 records · Page 2Linked to original sources
PubMed2003

Expression of Drosophila trehalose-phosphate synthase in HEK-293 cells increases hypoxia tolerance.

Increasing hypoxia tolerance in mammalian cells is potentially of major importance, but it has not been feasible thus far. The disaccharide trehalose, which accumulates dramatically during heat shock, enhances thermotolerance and reduces aggregation of denatured proteins. Previous studies from our laboratory showed that over-expression of Drosophila trehalose-phosphate synthase (dtps1) increases the trehalose level and anoxia tolerance in flies. To determine whether trehalose can protect against anoxic injury in mammalian cells, we transfected the dtps1 gene into human HEK-293 cells using the recombinant plasmid pcDNA3.1(-)-dtps1 and obtained more than 20 stable cell strains. Glucose starvation in culture showed that HEK-293 cells transfected with pcDNA3.1(-)-dtps1 (HEK-dtps1) do not metabolize intracellular trehalose, and, interestingly, these cells accumulated intracellular trehalose during hypoxic exposure. In contrast to HEK-293 cells transfected with pcDNA3.1(-) (HEK-v), cells with trehalose were more resistant to low oxygen stress (1% O2). To elucidate how trehalose protects cells from anoxic injury, we assayed protein solubility and the amount of ubiquitinated proteins. There was three times more insoluble protein in HEK-v than in HEK-dtps1 after 3 days of exposure to low O2. The amount of Na+-K+ ATPase present in the insoluble proteins dramatically increased in HEK-v cells after 2 and 3 days of exposure, whereas there was no significant change in HEK-dtps1 cells. Ubiquitinated proteins increased dramatically in HEK-v cells after 2 and 3 days of exposure but not in HEK-dtps1 cells over the same period. Our results indicate that increased trehalose in mammalian cells following transfection by the Drosophila tps1 gene protects cells from hypoxic injury. The mechanism of this protection is likely related to a decrease in protein denaturation, through protein-trehalose interactions, resulting in enhanced cellular recovery from hypoxic stress.

Adaptation, Physiological↗
PubMed2003

Understanding the molecular responses to hypoxia using Drosophila as a genetic model.

We have previously discovered that Drosophila melanogaster could recover from extended periods of anoxia (0% oxygen) with no apparent consequential injuries. We have since employed forward and reverse genetic approaches to decipher the molecular basis for anoxia tolerance. In so doing, we have identified several independent mutant lines that demonstrated increased sensitivity to anoxia. Characterization of one of these mutants resulted in the identification of a dADAR gene that plays a role in the sensitivity to low levels of oxygen. We have also used microarrays to study all known Drosophila genes, the expression of which may be altered by hyoxia. Microarrays experiments have generated a large body of information that is being currently analyzed. We believe that these undertakings will provide insight into the genetic mechanisms of hypoxia tolerance and ischemic injuries.

Adenosine Deaminase↗
PubMed2003

Calcium influx via L- and N-type calcium channels activates a transient large-conductance Ca2+-activated K+ current in mouse neocortical pyramidal neurons.

Ca2+-activated K+ currents and their Ca2+ sources through high-threshold voltage-activated Ca2+ channels were studied using whole-cell patch-clamp recordings from freshly dissociated mouse neocortical pyramidal neurons. In the presence of 4-aminopyridine, depolarizing pulses evoked transient outward currents and several components of sustained currents in a subgroup of cells. The fast transient current and a component of the sustained currents were Ca2+ dependent and sensitive to charybdotoxin and iberiotoxin but not to apamin, suggesting that they were mediated by large-conductance Ca2+-activated K+ (BK) channels. Thus, mouse neocortical neurons contain both inactivating and noninactivating populations of BK channels. Blockade of either L-type Ca2+ channels by nifedipine or N-type Ca2+ channels by omega-conotoxin GVIA reduced the fast transient BK current. These data suggest that the transient BK current is activated by Ca2+ entry through both N- and L-type Ca2+ channels. The physiological role of the fast transient BK current was also examined using current-clamp techniques. Iberiotoxin broadened action potentials (APs), indicating a role of BK current in AP repolarization. Similarly, both the extracellular Ca2+ channel blocker Cd2+ and the intracellular Ca2+ chelator BAPTA blocked the transient component of the outward current and broadened APs in a subgroup of cells. Our results indicate that the outward current in pyramidal mouse neurons is composed of multiple components. A fast transient BK current is activated by Ca2+ entry through high-threshold voltage-activated Ca2+ channels (L- and N-type), and together with other voltage-gated K+ currents, this transient BK current plays a role in AP repolarization.

Animals↗
PubMed2003

Chronic hypoxia in development selectively alters the activities of key enzymes of glucose oxidative metabolism in brain regions.

The immature brain is more resistant to hypoxia/ischemia than the mature brain. Although chronic hypoxia can induce adaptive-changes on the developing brain, the mechanisms underlying such adaptive changes are poorly understood. To further elucidate some of the adaptive changes during postnatal hypoxia, we determined the activities of four enzymes of glucose oxidative metabolism in eight brain regions of hypoxic and normoxic rats. Litters of Sprague-Dawley rats were put into the hypoxic chamber (oxygen level maintained at 9.5%) with their dams starting on day 3 postnatal (P3). Age-matched normoxic rats were use as control animals. In P10 hypoxic rats, lactate dehydrogenase (LDH) activity in cerebral cortex, striatum, olfactory bulb, hippocampus, hypothalamus, pons and medulla, and cerebellum was significantly increased (by 100%-370%) compared to those in P10 normoxic rats. In P10 hypoxic rats, hexokinase (HK) activity in hypothalamus, hippocampus, olfactory bulb, midbrain, and cerebral cortex was significantly decreased (by 15%-30%). Neither alpha-ketoglutarate dehydrogenase complex (KGDHC, which is believed to have an important role in the regulation of the tricarboxylic acid [TCA] cycle flux) nor citrate synthase (CS) activity was significantly decreased in the eight regions of P10 hypoxic rats compared to those in P10 normoxic rats. In P30 hypoxic rats, LDH activity was only increased in striatum (by 19%), whereas HK activity was only significantly decreased (by 30%) in this region. However, KGDHC activity was significantly decreased in olfactory bulb, hippocampus, hypothalamus, cerebral cortex, and cerebellum (by 20%-40%) in P30 hypoxic rats compared to those in P30 normoxic rats. Similarly, CS activity was decreased, but only in olfactory bulb, hypothalamus, and midbrain (by 9%-21%) in P30 hypoxic rats. Our results suggest that at least some of the mechanisms underlying the hypoxia-induced changes in activities of glycolytic enzymes implicate the upregulation of HIF-1. Moreover, our observation that chronic postnatal hypoxia induces differential effects on brain glycolytic and TCA cycle enzymes may have pathophysiological implications (e.g., decreased in energy metabolism) in childhood diseases (e.g., sudden infant death syndrome) in which hypoxia plays a role.

Aging↗
PubMed2003

Maturation of neuronal excitability in hippocampal neurons of mice chronically exposed to cyclic hypoxia.

To examine the effects of chronic cyclic hypoxia on neuronal excitability and function in mice, we exposed mice to cyclic hypoxia for 8 h daily (9 cycles/h) for approximately 2 wk (starting at 2-3 days of age) and examined the properties of freshly dissociated hippocampal neurons obtained from slices. Compared with control (Con) hippocampal CA1 neurons, exposed neurons (CYC) had similar resting membrane potentials (V(m)) and action potentials (AP). CYC neurons, however, had a lower rheobase than Con neurons. There was also an upregulation of the Na(+) current density (333 +/- 84 pA/pF, n = 18) in CYC compared with that of Con neurons (193 +/- 20 pA/pF, n = 27, P < 0.03). Na(+) channel characteristics were significantly altered by hypoxia. For example, the steady-state inactivation curve was significantly more positive in CYC than in Con (-60 +/- 6 mV, n = 8, for CYC and -71 +/- 3 mV, n = 14, for Con, P < 0.04). The time constant for deactivation (tau(d)) was much shorter in CYC than in Con (at -100 mV, tau(d)=0.83 +/- 0.23 ms in CYC neurons and 2.29 +/- 0.38 ms in Con neurons, P = 0.004). We conclude that the increased neuronal excitability in mice neurons treated with cyclic hypoxia is due to alterations in Na(+) channel characteristics and/or Na(+) channel expression. We hypothesize from these and previous data from our laboratory (Gu XQ and Haddad GG. J Appl Physiol 91: 1245-1250, 2001) that this increased excitability is a reflection of an enhanced central nervous system maturation when exposed to low O(2) conditions in early postnatal life.

Animals↗
PubMed2003

Na+ channel expression and neuronal function in the Na+/H+ exchanger 1 null mutant mouse.

Mice lacking Na(+)/H(+) exchanger 1 (NHE1) suffer from recurrent seizures and die early postnatally. Although the mechanisms for seizures are not well established, our previous electrophysiological work has shown that neuronal excitability and Na(+) current density are increased in hippocampal CA1 neurons of these mutant mice. However, it is unknown whether this increased density is related to altered expression or functional regulation of Na(+) channels. In this work, we asked three questions: is the increased excitability limited to CA1 neurons, is the increased Na(+) current density related to an increased Na(+) channel expression, and, if so, which Na(+) channel subtype(s) is upregulated? Using neurophysiological, autoradiographic, and immunoblotting techniques, we showed that both CA1 and cortical neurons have an increase in membrane excitability and Na(+) current density; Na(+) channel density is selectively upregulated in the hippocampus and cortex (P < 0.05); and Na(+) channel subtype I is significantly increased in the hippocampus and Na(+) channel subtype II is increased in the cortex. Our results demonstrate that mice lacking NHE1 upregulate their Na(+) channel expression in the hippocampal and cortical regions selectively; this leads to an increase in Na(+) current density and membrane excitability. We speculate that neuronal overexcitability due to Na(+) channel upregulation in the hippocampus and cortex forms the basis of epileptic seizures in NHE1 mutant mice.

Action Potentials↗
PubMed2002

Developmental expression and enzymatic activity of pre-mRNA deaminase in Drosophila melanogaster.

Pre-mRNA editing, conducted by adenosine deaminase acting on RNA (ADAR), plays an important role in many biological/physiological processes. This post-transcriptional event creates protein diversity that stems from a single gene via alteration of either genetic codons or alternative splicing sites. Our data demonstrate that both expression of Drosophila ADAR (dADAR) gene and dADAR editing activity are highly regulated during development. The lack of editing activity during embryonic development and the CNS-limited expression of dADAR in the adult may specify its important role in maintaining neuronal function in the Drosophila CNS.

Adenosine Deaminase↗
PubMed2002

Regulation of TGF-beta ligand and receptor expression in neonatal rat lungs exposed to chronic hypoxia.

Long-term effects of hypoxia are largely due to its modulatory effects on proliferation and differentiation of epithelial and endothelial cells, processes also regulated by the transforming growth factor (TGF)-beta system. We investigated the effects of hypoxia on the TGF-beta system in rat lungs from different developmental stages. Sprague-Dawley rats were exposed to 9.5% oxygen during either the first 2 wk of life or adulthood. Analysis revealed an arrest of alveolarization in hypoxic postnatal day 14 rats. Bioactive TGF-beta levels in bronchoalveolar lavage fluid were increased in these animals, and Western blot analysis revealed upregulation of TGF-beta receptor (TbetaR) I and II. None of these changes was observed in hypoxic adults. Hypoxia did, however, lead to decreased expression of TbetaRIII in both postnatal day 14 and adult rats. Immunohistochemical analysis localized TbetaRI-III predominantly to bronchiolar and alveolar epithelium; these patterns did not change with hypoxia. Thus we observed changes in TGF-beta activity and TbetaR isotype expression in rat lung that parallel the arrest in alveolarization seen with chronic hypoxia in early development. These alterations may partly explain the morphological changes observed in hypoxia.

Animals↗
PubMed2001

Role of trehalose phosphate synthase in anoxia tolerance and development in Drosophila melanogaster.

Recent studies have shown that trehalose plays a protective role in yeast in a variety of stresses, including heat, freezing and thawing, dehydration, hyperosmotic shock, and oxidant injury. Because (a) heat shock and anoxia share mechanisms that allow organisms to survive, (b) Drosophila melanogaster is tolerant to anoxia, and (c) trehalose is present in flies and is metabolically active, we asked whether trehalose can protect against anoxic stress. Here we report on a new role of trehalose in anoxia resistance in Drosophila. We first cloned the gene trehalose-6-phosphate synthase (tps1), which synthesizes trehalose, and examined the effect of tps1 overexpression as well as mutation on the resistance of Drosophila to anoxia. Upon induction of tps1, trehalose increased, and this was associated with increased tolerance to anoxia. Furthermore, in vitro experiments showed that trehalose reduced protein aggregation caused by anoxia. Homozygous tps1 mutant (P-element insertion into the third intron of the gene) leads to lethality at an early larval stage, and excision of the P-element rescues totally the phenotype. We conclude that trehalose contributes to anoxia tolerance in flies; this protection is likely to be due to a reduction of protein aggregation.

Adaptation, Physiological↗
PubMedDate not supplied

Calcium and pH homeostasis in neurons during hypoxia and ischemia.

One of the important events during hypoxia or ischemia in the brain (or other organs for that matter, including the myocardium) is the accumulation of Ca2+ ions intracellularly. Although various studies have shown various sources of and routes for Ca2+ entry and accumulation, it is clear now that it is likely that there is a multitude rather than a single mechanism for this accumulation. In this review, we highlight this Ca2+ accumulation during low O2 states and discuss some of the mechanisms leading to accumulation for two main reasons: (a) an accumulation of Ca2+ in the cytosol has been proven to be deleterious for cell function although this accumulation of Ca2+ and consequences represent only a limited view of events that can lead to cell injury during such stress and (b) developing therapeutic strategies involving the reduction or elimination of this accumulation depends, by and large, on the mechanism of entry. In addition to reviewing some of these Ca2+ events, we will also review the relation between pH (H+) and Ca2+ since these two ions and their regulation are tied to each other in a major way. For example, extracellular acidosis, which can occur during ischemia, has a remarkable effect on the function of some of the Ca2+ entry routes.

Animals↗