Allopregnanolone Potentiates the Glutamate-Mediated Seizures Induced by 4-Aminopyridine in Rat Hippocampus in vivo

Excitatory and inhibitory neurotransmission in the central nervous system can be modulated by neurosteroids. We previously found that in rat hippocampal slices allopregnanolone (3α-hydroxy-5α-pregnan-20-one), a positive GABA_A receptor modulator, suppresses the epileptic discharges induced by 4-aminopyridine (4-AP), a convulsant K⁺ channel blocker that stimulates glutamate release. Here, we tested the action of allopregnanolone on the epileptogenic and excitotoxic effects of the intrahippocampal administration of 4-AP in vivo. Drugs were perfused by a microdialysis cannula-electrode in the dorsal hippocampus and the EEG was recorded. Extracellular levels of aspartate, glutamate and GABA were analyzed by HPLC in the microdialysis fractions, and 24 h after the experiment the hippocampus was studied histologically. 4-AP induced intense epileptic discharges, increased the extracellular levels of aspartate, glutamate, and GABA by 383, 420, and 245%, respectively, and produced a notable neurodegeneration in CA1 and CA3 areas. Allopregnanolone administration alone did not affect the electrical activity, amino acids levels or cellular morphology, but when co-infused with 4-AP incremented 55–77% the duration of the epileptic discharges, and potentiated 32–49% the release of glutamate in comparison with 4-AP alone. The 4-AP-induced neurodegeneration was not modified by allopregnanolone. The NMDA receptor antagonist MK-801 protected against the epilepsy and neurodegeneration produced by 4-AP, and allopregnanolone did not affect this protection. We conclude that, differently from the observations in vitro, allopregnanolone potentiated the stimulatory effect of 4-AP on glutamate release and that this may explain the potentiation of the epileptogenic effect of 4-AP in vivo.     

XopR, a type III effector secreted by Xanthomonas oryzae pv. oryzae, suppresses microbe-associated molecular pattern-triggered immunity in Arabidopsis thaliana

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. The XopR protein, secreted into plant cells through the type III secretion apparatus, is widely conserved in xanthomonads and is predicted to play important roles in bacterial pathogenicity. Here, we examined the function of XopR by constructing transgenic Arabidopsis thaliana plants expressing it under control of the dexamethasone (DEX)-inducible promoter. In the transgenic plants treated with DEX, slightly delayed growth and variegation on leaves were observed. Induction of four microbe-associated molecular pattern (MAMP)-specific early-defense genes by a nonpathogenic X. campestris pv. campestris hrcC deletion mutant were strongly suppressed in the XopR-expressing plants. XopR expression also reduced the deposition of callose, an immune response induced by flg22. When transiently expressed in Nicotiana benthamiana, a XopR::Citrine fusion gene product localized to the plasma membrane. The deletion of XopR in X. oryzae pv. oryzae resulted in reduced pathogenicity on host rice plants. Collectively, these results suggest that XopR inhibits basal defense responses in plants rapidly after MAMP recognition.     

Resveratrol mobilizes Ca2+ from intracellular stores and induces c-Jun N-terminal kinase activation in tumoral AR42J cells

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a phytoalexin naturally found in grapes and red wine, is a redox-active compound endowed with significant positive activities. In this study, the effects of resveratrol on intracellular free Ca(2+) concentration ([Ca(2+)](c)) and on cell viability in tumoral AR42J pancreatic cells are examined. The results show that resveratrol (100 μM and 1 mM) induced changes in [Ca(2+)](c), that consisted of single or short lasting spikes followed by a slow reduction toward a value close to the resting level. Lower concentrations of resveratrol (1 and 10 μM) did not show detectable effects on [Ca(2+)](c). Depletion of intracellular Ca(2+) stores by stimulation of cells with 1 nM CCK-8, 20 pM CCK-8 or 1 μM thapsigargin, blocked Ca(2+) responses evoked by resveratrol. Conversely, prior stimulation of cells with resveratrol inhibited Ca(2+) mobilization in response to a secondary application of CCK-8 or thapsigargin. In addition, resveratrol inhibited oscillations in [Ca(2+)](c) evoked by a physiological concentration of CCK-8 (20 pM). On the other hand, incubation of cells in the presence of resveratrol induced a reduction of cell viability. Finally, incubation of AR42J cells in the presence of resveratrol led to activation of c-Jun N-terminal kinase (JNK), a mitogen-activated protein kinase responsive to stress stimuli. Activation of JNK was reduced in the absence of extracellular Ca(2+). In summary, the results show that resveratrol releases Ca(2+) from intracellular stores, most probably from the endoplasmic reticulum, and reduces AR42J cells viability. Reorganization of cell's survival/death processes in the presence of resveratrol may involve Ca(2+)-mediated JNK activation.     

Aromatase activity after a short-course of letrozole administration in adult men at sea level and at high altitude (with or without excessive erythrocytosis)

Men living at high altitudes in Peru compared to sea level counterparts have erythrocytosis (hemoglobin 16-21?g/dl) or excessive erythrocytosis (hemoglobin>21?g/dl). High testosterone (T) levels in men at high altitude (HA) were associated with excessive erythrocytosis. High androgen levels could be due to a low aromatase activity or to an elevated rate of conversion from precursors to testosterone. The aim of this study was to evaluate aromatase activity and rate of conversion from precursors to testosterone before and after administration of the aromatase enzyme inhibitor letrozole (5?mg/day) for a 5-day period to men at HA and at sea level (SL). The response to short term aromatase inhibition was assessed in 30 adult men living at sea level, 31 native men at HA with erythrocytosis (Hb 16-21?g/dl), and 35 men at HA with excessive erythrocytosis (Hb>21?g/dl). Serum hormone levels, estradiol/testosterone, testosterone/androstenedione, and testosterone/dehydroepiandrosterone sulfate (DHEAS) ratios were measured. Men with erythrocytosis had lower basal serum T/androstenedione ratios than men with excessive erythrocytosis at HA and men at sea level. Men at HA with excessive erythrocytosis had higher T/DHEAS ratios than men with erythrocytosis and than those at sea level before and after letrozole administration. After letrozole administration, both groups of men at high altitude (with erythrocytosis or with excessive erythrocytosis) showed lower aromatase activities than those at sea level. In conclusion, higher serum testosterone levels in men with excessive erythrocytosis were associated with an increased rate of conversion from DHEAS to testosterone rather than to a lower aromatase activity.     

Nerve growth factor induces the expression of chaperone protein calreticulin in human epithelial ovarian cells

Epithelial ovarian cancer is highly angiogenic and high expression of Nerve Growth Factor (NGF), a proangiogenic protein. Calreticulin is a multifunctional protein with anti-angiogenic properties and its translocation to the tumor cell membrane promotes recognition and engulfment by dendritic cells. The aim of this work was to evaluate calreticulin expression in human normal ovaries, benign and borderline tumors, and epithelial ovarian cancer samples and to evaluate whether NGF regulates calreticulin expression in human ovarian surface epithelium and in epithelial ovarian cancer cell lines. Calreticulin mRNA and protein levels were analyzed using RT-PCR, Western blot and immunohistochemistry in 67 human ovarian samples obtained from our Institution. Calreticulin expression induced by NGF stimulation in cell lines was evaluated using RT-PCR, Western blot and immunocytochemistry. We found a significant increase of calreticulin mRNA levels in epithelial ovarian cancer samples as compared to normal ovaries, benign tumors, and borderline tumors. Calreticulin protein levels, evaluated by Western blot, were also increased in epithelial ovarian cancer with respect to benign and borderline tumors. When HOSE and A2780 cell lines were stimulated with Nerve Growth Factor, we found an increase in calreticulin protein levels compared to controls. This effect was reverted by GW441756, a TRKA specific inhibitor. These results suggest that NGF regulates calreticulin protein levels in epithelial ovarian cells through TRKA receptor activation.     

Prevention of liver ischemia reperfusion injury by a combined thyroid hormone and fish oil protocol

Several preconditioning strategies are used to prevent ischemia–reperfusion (IR) liver injury, a deleterious condition associated with tissue resection, transplantation or trauma. Although thyroid hormone (T₃) administration exerts significant protection against liver IR injury in the rat, its clinical application is controversial due to possible adverse effects. Considering that prevention of liver IR injury has also been achieved by n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation to rats, we studied the effect of n-3 PUFA dietary supplementation plus a lower dose of T₃ against IR injury. Male Sprague-Dawley rats receiving fish oil (300 mg/kg) for 3 days followed by a single intraperitoneal dose of 0.05 mg T₃/kg were subjected to 1 h of ischemia followed by 20 h of reperfusion. Parameters of liver injury (serum transaminases, histology) and oxidative stress (liver contents of GSH and oxidized proteins) were correlated with fatty acid composition, NF-κB activity, and tumor necrosis factor-α (TNF-α) and haptoglobin expression. IR significantly modified liver histology; enhanced serum transaminases, TNF-α response or liver oxidative stress; and decreased liver NF-κB activity and haptoglobin expression. Although IR injury was not prevented by either n-3 PUFA supplementation or T₃ administration, substantial decrease in liver injury and oxidative stress was achieved by the combined protocol, which also led to increased liver n-3 PUFA content and decreased n-6/n-3 PUFA ratios, with recovery of NF-κB activity and TNF-α and haptoglobin expression. Prevention of liver IR injury achieved by a combined protocol of T₃ and n-3 PUFA supplementation may represent a novel noninvasive preconditioning strategy with potential clinical application.     

Shared resistance to aging and ALS in neuromuscular junctions of specific muscles

Normal aging and neurodegenerative diseases both lead to structural and functional alterations in synapses. Comparison of synapses that are generally similar but respond differently to insults could provide the basis for discovering mechanisms that underlie susceptibility or resistance to damage. Here, we analyzed skeletal neuromuscular junctions (NMJs) in 16 mouse muscles to seek such differences. We find that muscles respond in one of three ways to aging. In some, including most limb and trunk muscles, age-related alterations to NMJs are progressive and extensive during the second postnatal year. NMJs in other muscles, such as extraocular muscles, are strikingly resistant to change. A third set of muscles, including several muscles of facial expression and the external anal sphinter, succumb to aging but not until the third postnatal year. We asked whether susceptible and resistant muscles differed in rostrocaudal or proximodistal position, source of innervation, motor unit size, or fiber type composition. Of these factors, muscle innervation by brainstem motor neurons correlated best with resistance to age-related decline. Finally, we compared synaptic alterations in normally aging muscles to those in a mouse model of amyotrophic lateral sclerosis (ALS). Patterns of resistance and susceptibility were strikingly correlated in the two conditions. Moreover, damage to NMJs in aged muscles correlated with altered expression and distribution of CRMP4a and TDP-43, which are both altered in motor neurons affected by ALS. Together, these results reveal novel structural, regional and molecular parallels between aging and ALS.     

Delayed administration of VEGF rescues spinal motor neurons from death with a short effective time frame in excitotoxic experimental models in vivo

VEGF (vascular endothelial growth factor) prevents neuronal death in different models of ALS (amyotrophic lateral sclerosis), but few studies have addressed the efficacy of VEGF to protect motor neurons after the onset of symptoms, a critical point when considering VEGF as a potential therapeutic target for ALS. We studied the capability of VEGF to protect motor neurons after an excitotoxic challenge in two models of spinal neurodegeneration in rats induced by AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) administered either chronically with osmotic minipumps or acutely by microdialysis. VEGF was administered through osmotic minipumps in the chronic model or injected intracerebroventricularly in the acute model, and its effects were assessed by immunohistochemical and histological analyses and motor performance tests. In the chronic model, VEGF stopped the progression of the paralysis and protected motor neurons when administered after AMPA before the onset of the motor symptoms, whereas no protection was observed when administered after the onset. VEGF was also protective in the acute model, but with a short time window, since the protection was effective when administered 1 h but not 2 h after AMPA. Our results indicate that while VEGF has an indubitable neuroprotective effect, its therapeutic potential for halting or delaying the progression of motor neuron loss in ALS would likely have a short effective time frame.