Effect of Hyperoxia on Serine Phosphorylation of Apoptotic Proteins in Mitochondrial Membranes of the Cerebral Cortex of Newborn Piglets

Previous studies have shown that hyperoxia results in cerebral cortical neuronal apoptosis. Studies have also shown that phosphorylation of anti-apoptotic proteins Bcl-2 and Bcl-xl results in loss of their anti-apoptotic potential leading to alteration in mitochondrial membrane permeability and the release of apoptogenic proteins in the neuronal cell of the newborn piglets. The present study tests the hypothesis that cerebral hyperoxia will result in increased serine phosphorylation of apoptotic proteins Bcl-2, Bcl-xl, Bax, and Bad in the mitochondrial membranes of the cerebral cortex of newborn piglets. Twelve newborn piglets were divided into normoxic (Nx, n = 6) exposed to an FiO₂ of 0.21 for 1 h and hyperoxic (Hyx, n = 6) exposed to FiO₂ of 1.0 for 1 h. In the Hyx group, PaO₂ was maintained above 400 mmHg while the Nx group was kept at 80–100 mmHg. Cerebral cortical tissue was harvested and mitochondrial fractions were isolated. Mitochondrial membrane proteins were separated using 12% SDS-PAGE, and probed with anti-serine phosphorylated Bcl-2, Bcl-xl, Bax, and Bad antibodies. Protein bands were detected, analyzed by imaging densitometry and density expressed as absorbance (OD × mm²). Phosphorylated Bcl-2 (p-Bcl-2) protein density (OD × mm²) was 81.81 ± 9.24 in Nx and 158.34 ± 10.66 in Hyx (P < 0.05). Phosphorylated Bcl-xl (p-Bcl-xl) protein density was 52.98 ± 3.59 in Nx and 99.62 ± 18.22 in Hyx (P < 0.05). Phosphorylated Bax (p-Bax) protein was 161.13 ± 6.27 in Nx and 174.21 ± 15.95 in Hyx (P = NS). Phosphorylated Bad (p-Bad) protein was 166.24 ± 9.47 in Nx 155.38 ± 12.32 in Hyx (P = NS). The data show that there is a significant increase in serine phosphorylation of Bcl-2 and Bcl-xl proteins while phosphorylation of Bad and Bax proteins were not altered during hyperoxia in the mitochondrial fraction of the cerebral cortex of newborn piglets. We conclude that hyperoxia results in differential post-translational modification of anti-apoptotic proteins Bcl-2 and Bcl-xl as compared to pro-apoptotic proteins Bax and Bad in mitochondria. We speculate that phosphorylation of Bcl-2 and Bcl-xl will result in loss of their anti-apoptotic potential by preventing their dimerization with Bax leading to activation of the caspase cascade of neuronal death.     

Differential Expression of Apoptotic Proteins Following Hypoxia-induced CREB Phosphorylation in the Cerebral Cortex of Newborn Piglets

The present study investigates the correlation between the hypoxia-induced phosphorylation of cyclic AMP response element binding protein and the expression of apoptotic proteins (proapoptotic proteins Bax and Bad and antiapoptotic proteins Bcl-2 and Bcl-xl) during hypoxia in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx) and hypoxic (Hx, FiO₂ = 0.06 for 1 h) groups. Cerebral tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Ser¹³³ phosphorylation of cyclic AMP response element binding (CREB) protein was determined by Western blot analysis using a specific anti-phosphorylated Ser¹³³-CREB protein antibody. The expression of apoptotic proteins was determined by using specific anti-Bax, anti-Bad, anti-Bcl-2 and anti-Bcl-xl antibodies. ATP and PCr values (μmoles/g brain) in Hx were significantly different from Nx (ATP: 4.40 ± 0.39 in Nx vs. 1.19 ± 0.44 in Hx, P < 0.05 vs. Nx; PCr: 3.60 ± 0.40 in Nx vs. 0.70 ± 0.31 in Hx, P < 0.05 vs. Nx). Ser¹³³ phosphorylated CREB protein (OD × mm²) was 74.55 ± 4.75 in Nx and 127.13 ± 19.36 in Hx (P < 0.05 vs. Nx). The expression of proapoptotic proteins Bax and Bad increased and strongly correlated with the increase in CREB protein phosphorylation (correlation coefficient r = 0.82 and r = 0.85, respectively). The expression of antiapoptotic proteins Bcl-2 and Bcl-xl did not show correlation with CREB protein phosphorylation. We conclude that cerebral hypoxia results in differential regulation of CREB protein-mediated expression of proapoptotic and antiapoptotic proteins in the cerebral cortex of newborn piglets. We propose that the increased expression of proapoptotic vs antiapoptotic genes will lead to an increased potential for apoptotic programmed cell death in the Hx newborn brain.     

Mechanism of Post-Translational Modification by Tyrosine Phosphorylation of Apoptotic Proteins During Hypoxia in the Cerebral Cortex of Newborn Piglets

The present study aims to investigate the mechanism of phosphorylation of apoptotic proteins and tests the hypothesis that the hypoxia-induced increased tyrosine phosphorylation of apoptotic proteins Bcl-2 and Bcl-xl is Ca²⁺-influx-dependent. Piglets were divided in normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-pretreated with clonidine (Clo + Hx, n = 4) groups. Hypoxic animals were exposed to an FiO₂ of 0.06 for 1 h. Clonidine (12.5 μg/kg, IV) was administered to piglets 30 min prior to hypoxia. Hypoxia was confirmed by ATP and phosphocreatinine (PCr) levels. Cytosol was isolated and separated by 12% SDS–PAGE and probed with tyrosine phosphorylated (p) -Bax, Bad, Bcl-2 and Bcl-xl antibodies and bands were detected. The ATP levels (μmol/g brain) in the Nx, Hx, Clo + Hx were 4.3 ± 1.0 (P < 0.05 vs. Hx, Clo-Hx), 0.9 ± 0.8 and 1.5 ± 0.3, respectively. The PCr levels in the Nx, Hx, Clo + Hx were 2.7 ± 0.7 (P < 0.05 vs. Hx, Clo-Hx), 0.9 ± 0.2 and 0.9 ± 0.9, respectively. Ca²⁺-influx (pmoles/mg protein) was 4.96 ± 0.94 in Nx, 11.11 ± 2.38 in Hx, and 6.23 ± 2.07 in Clo + Hx (P < 0.05 Nx vs. Hx and Hx vs. Clo + Hx). p-Bcl-2 density was 21.1 ± 1.1 Nx, 58.9 ± 9.6 Hx and 29.5 ± 6.4 Clo + Hx (P < 0.05 vs. Hx). p-Bcl-xl density was 29.6 ± 1.5 Nx, 50.6 ± 7.4 Hx and 32.1 ± 0.1 Clo + Hx (P < 0.05 vs. Hx). p-Bax density was 38.6 ± 16.2 Nx, 46.1 ± 5.5 Hx and 41.6 ± 1.9 Clo + Hx groups (P = NS). p-Bad was 66.7 ± 12.8 Nx, 71.2 ± 6.8 Hx and 78.7 ± 22.5 Clo + Hx groups (P = NS). Results showed that clonidine administration prior to hypoxia prevents the hypoxia-induced increased nuclear Ca²⁺-influx and increased phosphorylation of Bcl-2 and Bcl-xl while phosphorylation of Bad and Bax was not altered. We conclude that post-translational modification of anti-apoptotic proteins Bcl-2 and Bcl-xl during hypoxia is nuclear Ca²⁺-influx-dependent. We propose that blockade of nuclear Ca²⁺-influx that prevents phosphorylation of antiapoptotic proteins may become a neuroprotective strategy.     

Mechanism of Increased Tyrosine (Tyr<Superscript>99</Superscript>) Phosphorylation of Calmodulin During Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of nNOS-Derived Nitric Oxide

The present study aims to investigate the mechanism of calmodulin modification during hypoxia and tests the hypothesis that hypoxia-induced increase in Tyr⁹⁹ phosphorylation of calmodulin in the cerebral cortex of newborn piglets is mediated by NO derived from nNOS. Fifteen piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, F_iO₂ of 0.07 for 1 h, n = 5) and hypoxic-pretreated with nNOSi (Hx-nNOSi, n = 5) groups. nNOS inhibitor I (selectivity >2,500 vs. eNOS and >500 vs. iNOS) was administered (0.4 mg/kg, I.V.) 30 min prior to hypoxia. Cortical membranes were isolated and tyrosine phosphorylation (Tyr⁹⁹ and total) of calmodulin determined by Western blot using anti-phospho-(pTyr⁹⁹)-calmodulin and anti-pTyr antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by densitometry and expressed as absorbance. The pTyr⁹⁹ calmodulin (ODxmm²) was 78.55 ± 10.76 in Nx, 165.05 ± 12.26 in Hx (P < 0.05 vs. Nx) and 96.97 ± 13.18 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Expression of total tyrosine phosphorylated calmodulin was 69.24 ± 13.69 in Nx, 156.17 ± 16.34 in Hx (P < 0.05 vs. Nx) and 74.18 ± 3.9 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). The data show that administration of nNOS inhibitor prevented the hypoxia-induced increased Tyr⁹⁹ phosphorylation of calmodulin. Total tyrosine phosphorylation of calmodulin was similar to Tyr⁹⁹ phosphorylation. We conclude that the mechanism of hypoxia-induced modification (Tyr⁹⁹ phosphorylation) of calmodulin is mediated by NO derived from nNOS. We speculate that Tyr⁹⁹ phosphorylated calmodulin, as compared to non-phosphorylated, binds with a higher affinity at the calmodulin binding site of nNOS leading to increased activation of nNOS and increased generation of NO.     

Mechanism of Activation of Caspase-9 and Caspase-3 during Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of Nuclear Ca<Superscript>2+</Superscript>-Influx

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, the executioner of programmed cell death. We have also shown that cerebral hypoxia results in high affinity Ca²⁺–ATPase-dependent increase in nuclear Ca²⁺-influx in the cerebral cortex of newborn piglets. The present study tests the hypothesis that inhibiting nuclear Ca²⁺-influx by pretreatment with clonidine, an inhibitor of high affinity Ca²⁺–ATPase, will prevent the hypoxia-induced increase in caspase-9 and caspase-3 activity in the cerebral cortex of newborn piglets. Thirteen newborn piglets were divided into three groups, normoxic (Nx, n = 4), hypoxic (Hx, n = 4), and hypoxic treated with clonidine (100 mg/kg) (Hx–Cl, n = 5). Anesthetized, ventilated animals were exposed to an FiO₂ of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Caspase-9 and -3 activity were determined spectrofluoro-metrically using specific fluorogenic synthetic substrates. ATP (μmoles/g brain) was 4.6 ± 0.3 in Nx, 1.7±0.4 in Hx (P < 0.05 vs. Nx), and 1.5 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). PCr (μmoles/g brain) was 3.6 ± 0.4 in Nx, 1.1 ± 0.3 in Hx (P < 0.05 vs. Nx), and 1.0 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). Caspase-9 activity (nmoles/mg protein/h) was 0.548 ± 0.0642 in Nx and increased to 0.808 ± 0.080 (P < 0.05 vs. Nx and Hx–Cl) in the Hx and 0.562 ± 0.050 in the Hx–Cl group (p = NS vs. Nx). Caspase-3 activity (nmoles/mg protein/h) was 22.0 ± 1.3 in Nx and 32 ± 6.3 in Hx (P < 0.05 vs. Nx) and 18.8 ± 3.2 in the Hx–Cl group (P < 0.05 vs. Hx). The data demonstrate that clonidine administration prior to hypoxia prevents the hypoxia-induced increase in the activity of caspase-9 and caspase-3. We conclude that the high afinity Ca²⁺–ATPase-dependent increased nuclear Ca²⁺ during hypoxia results in increased caspase-9 and caspase-3 activity.     

Hypoxia-induced Activation of Epidermal Growth Factor Receptor (EGFR) Kinase in the Cerebral Cortex of Newborn Piglets: The Role of Nitric Oxide

The present study aims to investigate the mechanism of EGFR kinase activation during hypoxia and tests the hypothesis that hypoxia-induced increased activation of EGFR kinase in the cerebral cortical membrane fraction of newborn piglets is mediated by nitric oxide (NO) derived from neuronal nitric oxide synthase (nNOS). Fifteen newborn piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-treated with nNOS inhibitor (Hx-nNOSi, n = 5). Hypoxia was induced by an FiO2 of 0.07 for 60 min. nNOS inhibitor I (selectivity >2,500 vs. endothelial NOS, eNOS, and >500 vs. inducible NOS, iNOS) was administered (0.4 mg/kg, i. v.) 30 min prior to hypoxia. EGFR kinase tyrosine phosphorylation at Tyr1173, an index of activation of EGFR kinase, was determined by Western blot analysis using an anti-phospho (pTyr¹¹⁷³)-EGFR kinase antibody. Protein bands were analyzed by imaging densitometry and expressed as absorbance (OD × mm²). EGFR kinase activity was determined radiochemically using immunopurified enzyme. EGFR kinase activity was expressed as pmols/mg protein/hr. Density of phosphor (pTyr¹¹⁷³)-EGFR kinase (OD × mm²) was 60.2 ± 9.8 in Nx, 177.0 ± 26.9 in Hx (P < 0.05 vs. Nx) and 79.9 ± 15.7 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Activity of EGFR kinase (pmoles/mg protein/hr) was 4,603 ± 155 in Nx, 8,493 ± 427 in Hx (P < 0.05 vs. Nx) and 4,516 ± 104 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Pretreatment with nNOS inhibitor prevented the hypoxia-induced increased phosphorylation and increased activity of EGFR kinase. We conclude that the mechanism of hypoxia-induced increased activation of EGFR kinase is mediated by nNOS-derived NO.     

Effect of Hypoxia on Expression of Apoptotic Proteins in Nuclear,Mitochondrial and Cytosolic Fractions of the Cerebral Cortex of Newborn Piglets: The Role of Nuclear Ca<Superscript>++</Superscript>-influx

We have shown that hypoxia results in increased influx of nuclear Ca++ and increased expression of nuclear apoptotic proteins. The present study tests the hypothesis that hypoxia alters the distribution of pro-apoptotic proteins Bad and Bax, and the anti-apoptotic proteins Bcl-xl, and Bcl-2 in the nuclear, mitochondrial and cytosolic compartments of the cerebral cortex of newborn piglets and the administration of Clonidine, an inhibitor of high affinity nuclear Ca++ -ATPase, will prevent the hypoxia-induced increase in apoptotic proteins' expression. Studies were conducted in 19 newborn piglets, 6 normoxic (Nx), 7 hypoxic and 6 Clonidine-treated hypoxic (Hx-Clo). Tissue hypoxia was documented biochemically by measuring cerebral tissue ATP and phosphocreatine (PCr) levels. Bax and Bad protein expression increased in all the three compartments during hypoxia, while there was no significant change in the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. In Clonidine pretreated hypoxic group, the hypoxia-induced increased expression of pro-apoptotic proteins Bad and Bax was prevented in all the three fractions. We conclude that hypoxia results in increased expression of pro-apoptotic proteins in nuclear, mitochondrial and cytosolic compartments and that the increased expression of pro-apoptotic proteins during hypoxia is nuclear Ca++ -influx-dependent. We propose that during hypoxia the increased ratio of (pro-apoptotic Bad and Bax/anti-apoptotic Bcl-xl and Bcl-2) in all the three compartments, will lead to altered mitochondrial and nuclear membrane permeability as well as caspase-9 activation in the cytosolic compartment.     

Effect of Hyperoxia on Cortical Neuronal Nuclear Function and Programmed Cell Death Mechanisms

There is growing concern over detrimental neurologic effects to human newborns caused by increased inspired oxygen concentrations. We hypothesize that hyperoxia (FiO₂ > 0.95) results in increased high-affinity Ca²⁺-ATPase activity, Ca²⁺-influx, and proapoptotic protein expression in cortical neuronal nuclei of newborn piglets. Neuronal cerebral energy metabolism was documented by determining ATP and phosphocreatine levels. Neuronal nuclear conjugated dienes and fluorescent compounds were measured as indices of lipid peroxidation. High-affinity Ca²⁺-ATPase activity and ATP-dependent Ca²⁺-influx were determined to document neuronal nuclear membrane function. Hyperoxia resulted in increases in lipid peroxidation, high-affinity Ca²⁺-ATPase activity, ATP-dependent Ca²⁺-influx, and Bax/Bcl-2 ratio in the cortical neuronal nuclei of newborn piglets. We conclude that hyperoxia results in modification of neuronal nuclear membrane function leading to increased nuclear Ca²⁺-influx, and propose that hyperoxia-induced increases in intranuclear Ca²⁺ activates the Ca²⁺/calmodulin-dependent protein kinase pathway, triggering increased CREB protein-mediated apoptotic protein expression in hyperoxic neurons.     

Effects of Magnesium Sulfate Administration During Hypoxia on CaM Kinase IV and Protein Tyrosine Kinase Activities in the Cerebral Cortex of Newborn Piglets

The present study tested the hypothesis that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in Ca²⁺/Calmodulin-dependent-kinase (CaM Kinase) IV and Protein Tyrosine Kinase (PTK ) activities. Animals were randomly divided into normoxic (Nx), hypoxic (Hx) and magnesium-pretreated hypoxic (Mg²⁺-Hx) groups. Cerebral hypoxia was confirmed biochemically by measuring ATP and phosphocreatine (PCr) levels. CaM Kinase IV and PTK activities were determined in Nx, Hx and Mg²⁺-Hx newborn piglets. There was a significant difference between CaM kinase IV activity (pmoles/mg protein/min) in Nx (270 ± 49), Mg²⁺-Hx (317 ± 82) and Hx (574 ± 41, P < 0.05 vs. Nx and Mg²⁺-Hx) groups. Similarly, there was a significant difference between Protein Tyrosine Kinase activity (pmoles/mg protein/h) in normoxic (378 ± 68), Mg²⁺-Hx (455 ± 67) and Hx (922 ± 66, P < 0.05 vs. Nx and Mg²⁺-Hx ) groups. We conclude that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in CaM Kinase IV and Protein Tyrosine Kinase activities. We propose that by blocking the NMDA receptor ion-channel mediated Ca²⁺-flux, magnesium sulfate administration inhibits the Ca²⁺/calmodulin-dependent activation of CaMKIV and prevents the generation of nitric oxide free radicals and the subsequent increase in PTK activity. As a result, phosphorylation of CREB and Bcl-2 family of proteins is prevented leading to prevention of programmed cell death.     

Early Regional Response of Apoptotic Activity in Newborn Piglet Brain Following Hypoxia and Ischemia

Responses of selected neuroregulatory proteins that promote (Caspase 3 and Bax) or inhibit (Bcl-2, high Bcl-2/Bax ratio) apoptotic cell death were measured in the brain of piglets subjected to precisely controlled hypoxic and ischemic insults: 1 h hypoxia (decreasing FiO₂ from 21 to 6%) or ischemia (ligation of carotid arteries and hemorrhage), followed by 0, 2 and 4 h recovery with 21% FiO₂. Protein expression was measured in cortex, hippocampus and striatum by Western blot. There were no significant differences in expression of Caspase-3 between sham operated, hypoxic and ischemic groups. There were significant regional differences in expression of Bcl-2 and Bax in response to hypoxia and ischemia. The changes in Bcl-2/Bax ratio were similar for hypoxia and ischemia except for striatum at zero time recovery, with ischemia giving lower ratios than hypoxia. The Bcl-2/Bax ratio was also lower for the striatum than for the other regions of the brain, suggesting this region is the more susceptible to apoptotic injury.     

Oxygen-dependent reduction of a nitroxide free radical by electron paramagnetic resonance monitoring of circulating rat blood

The effects of fraction of inspired oxygen (FiO₂) on the reduction of a nitroxide free radical were studied by X-band electron paramagnetic resonance (EPR) monitoring of circulating rat blood. The decay half-life of the metabolism/elimination phase increased significantly by 24 ± 8% during hyperoxia and decreased significantly by 16 ± 4% during hypoxia.     

Human amnion epithelial cells modulate hyperoxia-induced neonatal lung injury in mice

Background aimsHuman amnion epithelial cells (hAECs) prevent pulmonary inflammation and injury in fetal sheep exposed to intrauterine lipopolysaccharide. We hypothesized that hAECs would similarly mitigate hyperoxia-induced neonatal lung injury.MethodsNewborn mouse pups were randomized to either normoxia (inspired O₂ content (FiO₂) = 0.21, n = 60) or hyperoxia (FiO₂ = 0.85, n = 57). On postnatal days (PND) 5, 6 and 7, hAECs or sterile saline (control) was administered intraperitoneally. All animals were assessed at PND 14.ResultsHyperoxia was associated with lung inflammation, alveolar simplification and reduced postnatal growth. Administration of hAECs to hyperoxia-exposed mice normalized body weight and significantly attenuated some aspects of hyperoxia-induced lung injury (mean linear intercept and septal crest density) and inflammation (interleukin-1α, interleukin-6, transforming growth factor-β and platelet-derived growth factor-β). However, hAECs did not significantly alter changes to alveolar airspace volume, septal tissue volume, tissue-to-airspace ratio, collagen content or leukocyte infiltration induced by hyperoxia.ConclusionsIntraperitoneal administration of hAECs to neonatal mice partially reduced hyperoxia-induced lung inflammation and structural lung damage. These observations suggest that hAECs may be a potential therapy for neonatal lung disease.     

Air pollution induces enhanced mitochondrial oxidative stress in cystic fibrosis airway epithelium

We studied the effects of airborne particulate matters (PM) on cystic fibrosis (CF) epithelium. We noted that PM enhanced human CF bronchial epithelial apoptosis, activated caspase-9 and PARP-1; and reduced mitochondrial membrane potential. Mitochondrial inhibitors (4,4-diisothiocyanatostilbene-2,2'disulfonic acid, rotenone and thenoyltrifluoroacetone) blocked PM-induced generation of reactive oxygen species and apoptosis. PM upregulated pro-apoptotic Bad, Bax, p53 and p21; and enhanced mitochondrial localization of Bax. The anti-apoptotic Bcl-2, Bcl-xl, Mcl-1 and Xiap remained unchanged; however, overexpression of Bcl-xl blocked PM-induced apoptosis. Accordingly, we provide the evidence that PM enhances oxidative stress and mitochondrial signaling mediated apoptosis via the modulation of Bcl family proteins in CF.     

Effect of 7-Nitroindazole Sodium on the Cellular Distribution of Neuronal Nitric Oxide Synthase in the Cerebral Cortex of Hypoxic Newborn Piglets

Cerebral hypoxia results in generation of nitric oxide (NO) free radicals by Ca⁺⁺-dependent activation of neuronal nitric oxide synthase (nNOS). The present study tests the hypothesis that the hypoxia-induced increased expression of nNOS in cortical neurons is mediated by NO. To test this hypothesis the cellular distribution of nNOS was determined immunohistochemically in the cerebral cortex of hypoxic newborn piglets with and without prior exposure to the selective nNOS inhibitor 7-nitroindazole sodium (7-NINA). Studies were conducted in newborn piglets, divided into normoxic (n = 6), normoxic treated with 7-NINA (n = 6), hypoxic (n = 6) and hypoxic pretreated with 7-NINA (n = 6). Hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 h. Cerebral tissue hypoxia was documented by decrease of ATP and phosphocreatine levels in both the hypoxic and 7-NINA pretreated hypoxic groups (P < 0.01). An increase in the number of nNOS immunoreactive neurons was observed in the frontal and parietal cortex of the hypoxic as compared to the normoxic groups (P < 0.05) which was attenuated by pretreatment with 7-NINA (P < 0.05 versus hypoxic). 7-NINA affected neither the cerebral energy metabolism nor the cellular distribution of nNOS in the cerebral cortex of normoxic animals. We conclude that nNOS expression in cortical neurons of hypoxic newborn piglets is NO-mediated. We speculate that nNOS inhibition by 7-NINA will protect against hypoxia-induced NO-mediated neuronal death.     

Discovery of novel 2,4-disubstituted pyrimidines as Aurora kinase inhibitors

In order to explore novel Aurora kinase inhibitors, a series of novel 2,4-disubstituted pyrimidines were designed, synthesized and evaluated their in vitro anti-proliferative activities against a panel of cancerous cell lines (A549, HCT-116 and MCF-7). Among them, compound 12a showed the moderate to high anti-proliferative activities against A549 (IC₅₀ = 12.05 ± 0.45 μM), HCT-116 (IC₅₀ = 1.31 ± 0.41 μM) and MCF-7 (IC₅₀ = 20.53 ± 6.13 μM) cells, as well as the Aurora A and Aurora B inhibitory activities with the IC₅₀ values of 309 nM and 293 nM, respectively. Furthermore, compound 12a induced apoptosis by upregulated the pro-apoptotic proteins Bax and decreased the anti-apoptotic protein Bcl-xl in HCT-116 cells. Moreover, the molecular docking study showed that compound 12a had good binding modes with Aurora A and Aurora B and the bioinformatics prediction discovered that compound 12a exhibited good drug likeness using SwissADME. Taken together, these results indicated that 12a may be a potential anticancer compound that was worthy of further development as Aurora kinase inhibitor.     

A quantitative study of the biotransformation of insulin-enhancing VO2+ compounds

Potentiometric (pH titrations) and spectroscopic (electron paramagnetic resonance) methods have been used to determine the thermodynamic stability constants of the various VO²⁺ complexes formed after the interaction of four insulin-enhancing vanadium compounds, [VO(6-mepic)₂], cis-[VO(pic)₂(H₂O)], [VO(acac)₂], and [VO(dhp)₂], where 6-mepic, pic, acac, and dhp indicate the deprotonated forms of 6-methylpicolinic acid, picolinic acid, acetylacetone, and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone, with high molecular mass [human serum apotransferrin (hTf) and human serum albumin (HSA)] and low molecular mass (lactate) components of blood serum. In particular, log β values for the formation of (VO)hTf (13.0 ± 0.5), (VO)₂hTf (25.5 ± 0.5), (VO)HSA (9.1 ± 1.0), (VO) ₂ ^d HSA (20.9 ± 1.0), cis-VO(dhp)₂(hTf) (25.5 ± 0.6), cis-VO(dhp)₂(HSA) (25.9 ± 0.6), (VO)hTf(lact) (14.5 ± 0.8), (VO)₂hTf(lact)₂ (28.5 ± 0.8), (VO)hTf(pic) (15.6 ± 0.8), and (VO)₂hTf(pic)₂ (30.4 ± 0.8) were determined. The values of the stability constants were used to compare the calculated composition of ternary and quinary systems with that recently proposed by some of us through electron paramagnetic resonance and density functional theory methods (Sanna et al. in Inorg. Chem. 49:174–187, 2010) and to predict the distribution of VO²⁺ ion in blood serum when one of the four insulin-enhancing vanadium compounds studied, [VO(carrier)₂], is administered.     

The microtubule depolymerizing agent naphthazarin induces both apoptosis and autophagy in A549 lung cancer cells

Naphthazarin (DHNQ, 5,8-dihydroxy-l,4-naphthoquinone) is a naturally available 1,4-naphthoquinone derivatives. In this study, we focused on elucidating the cytotoxic mechanism of naphthazarin in A549 non-small cell lung carcinoma cells. Naphthazarin reduced the A549 cell viability considerably with an IC₅₀ of 16.4 ± 1.6 μM. Naphthazarin induced cell death in a dose- and time-dependent manner by activating apoptosis and autophagy pathways. Specifically, we found naphthazarin inhibited the PI3K/Akt cell survival signalling pathway, measured by p53 and caspase-3 activation, and PARP cleavage. It also resulted in an increase in the ratio of Bax/Bcl2 protein levels, indicating activation of the mitochondrial apoptotic pathway. Similarly naphthazarin triggered LC3II expression and induced autophagic flux in A549 cells. We demonstrated further that naphthazarin is a microtubule inhibitor in cell-free system and in A549 cells. Naphthazarin treatment depolymerized interphase microtubules and disorganised spindle microtubules and the majority of cells arrested at the G₂/M transition. Together, these data suggest that naphthazarin, a microtubule depolymerizer which activates dual cell death machineries, could be a potential novel chemotherapeutic agent.     

Gating of the Mitochondrial Permeability Transition Pore by Long Chain Fatty Acyl Analogs in Vivo

The role played by long chain fatty acids (LCFA) in promoting energy expenditure is confounded by their dual function as substrates for oxidation and as putative classic uncouplers of mitochondrial oxidative phosphorylation. LCFA analogs of the MEDICA (MEthyl-substituted DICarboxylic Acids) series are neither esterified into lipids nor β-oxidized and may thus simulate the uncoupling activity of natural LCFA in vivo, independently of their substrate role. Treatment of rats or cell lines with MEDICA analogs results in low conductance gating of the mitochondrial permeability transition pore (PTP), with 10–40% decrease in the inner mitochondrial membrane potential. PTP gating by MEDICA analogs is accounted for by inhibition of Raf1 expression and kinase activity, resulting in suppression of the MAPK/RSK1 and the adenylate cyclase/PKA transduction pathways. Suppression of RSK1 and PKA results in a decrease in phosphorylation of their respective downstream targets, Bad(Ser-112) and Bad(Ser-155). Decrease in Bad(Ser-112, Ser-155) phosphorylation results in increased binding of Bad to mitochondrial Bcl2 with concomitant displacement of Bax, followed by PTP gating induced by free mitochondrial Bax. Low conductance PTP gating by LCFA/MEDICA may account for their thyromimetic calorigenic activity in vivo.     

Expression and regulation of α‐transducin in the pig gastrointestinal tract

Taste signalling molecules are found in the gastrointestinal (GI) tract suggesting that they participate to chemosensing. We tested whether fasting and refeeding affect the expression of the taste signalling molecule, α‐transducin (G_αtran), throughout the pig GI tract and the peptide content of G_αtran cells. The highest density of G_αtran‐immunoreactive (IR) cells was in the pylorus, followed by the cardiac mucosa, duodenum, rectum, descending colon, jejunum, caecum, ascending colon and ileum. Most G_αtran‐IR cells contained chromogranin A. In the stomach, many G_αtran‐IR cells contained ghrelin, whereas in the upper small intestine many were gastrin/cholecystokinin‐IR and a few somatostatin‐IR. G_αtran‐IR and G_αgust‐IR colocalized in some cells. Fasting (24 h) resulted in a significant decrease in G_αtran‐IR cells in the cardiac mucosa (29.3 ± 0.8 versus 64.8 ± 1.3, P < 0.05), pylorus (98.8 ± 1.7 versus 190.8 ± 1.9, P < 0.0 l), caecum (8 ± 0.01 versus 15.5 ± 0.5, P < 0.01), descending colon (17.8 ± 0.3 versus 23 ± 0.6, P < 0.05) and rectum (15.3 ± 0.3 versus 27.5 ± 0.7, P < 0.05). Refeeding restored the control level of G_αtran‐IR cells in the cardiac mucosa. In contrast, in the duodenum and jejunum, G_αtran‐IR cells were significantly reduced after refeeding, whereas G_αtran‐IR cells density in the ileum was not changed by fasting/refeeding. These findings provide further support to the concept that taste receptors contribute to luminal chemosensing in the GI tract and suggest they are involved in modulation of food intake and GI function induced by feeding and fasting.