Glucose-Induced Regulation of NHEs Activity and SGLTs Expression Involves the PKA Signaling Pathway

The effect of glucose on the intracellular pH (pH(i)) recovery rate (dpH(i)/dt) and Na(+)-glucose transporter (SGLT) localization was investigated in HEK-293 cells, a cell line that expresses endogenous NHE1, NHE3, SGLT1, and SGLT2 proteins. The activity of the Na(+)/H(+) exchangers (NHEs) was evaluated by using fluorescence microscopy. The total and membrane protein expression levels were analyzed by immunoblotting. In cells cultivated in 5 mM glucose, the pH(i) recovery rate was 0.169 +/- A 0.020 (n = 6). This value did not change in response to the acute presence of glucose at 2 or 10 mM, but decreased with 25 mM glucose, an effect that was not observed with 25 mM mannitol. Conversely, the chronic effect of high glucose (25 mM) increased the pH(i) recovery rate (similar to 40%, P < 0.05), without changes in the total levels of NHE1, NHE3, or SGLT1 expression, but increasing the total cellular (similar to 50%, P < 0.05) and the plasma membrane (similar to 100%, P < 0.01) content of SGLT2. Treatment with H-89 (10(-6) M) prevented the stimulatory effect of chronic glucose treatment on the pH(i) recovery rate and SGLT2 expression in the plasma membrane. Our results indicate that the effect of chronic treatment with a high glucose concentration is associated with increased NHEs activity and plasma membrane expression of SGLT2 in a protein kinase A-dependent way. The present results reveal mechanisms of glucotoxicity and may contribute to understanding the diabetes-induced damage of this renal epithelial cell.

The sleep-wake cycle in adult rats following pilocarpine-induced temporal lobe epilepsy

The relationship between sleep and epilepsy is both complex and clinically significant. Temporal lobe epilepsy (TLE) influences sleep architecture, while sleep plays an important role in facilitating and/or inhibiting possible epileptic seizures. The pilocarpine experimental model reproduces several features of human temporal lobe epilepsy and is one of the most widely used models in basic research. The aim of the present study was to characterize, behaviorally and electrophysiologically, the phases of sleep-wake cycles (SWC) in male rats with pilocarpine-induced epilepsy. Epileptic rats presented spikes in all phases of the SWC as well as atypical cortical synchronization during attentive wakefulness and paradoxical sleep. The architecture of the sleep-wake phases was altered in epileptic rats, as was the integrity of the SWC. Because our findings reproduce many relevant features observed in patients with epilepsy, this model is suitable to study sleep dysfunction in epilepsy. (C) 2009 Elsevier Inc. All rights reserved.

Effect of short-term creatine supplementation on markers of skeletal muscle damage after strenuous contractile activity

The protective effect of short-term creatine supplementation (CrS) upon markers of strenuous contractile activity-induced damage in human and rat skeletal muscles was investigated. Eight Ironman triathletes were randomized into the placebo (Pl; n = 4) and creatine-supplemented (CrS; n = 4) groups. Five days prior to the Ironman competition, the CrS group received creatine monohydrate (20 g day(-1)) plus maltodextrin (50 g) divided in two equal doses. The Pl group received maltodextrin (50 g day(-1)) only. The effect of CrS (5 g day(-1)/kg body weight for 5 days) was also evaluated in a protocol of strenuous contractile activity induced by electrical stimulation in rats. Blood samples were collected before and 36 and 60 h after the competition and were used to determine plasma activities of creatine kinase (CK), lactate dehydrogenase (LDH), aldolase (ALD), glutamic oxaloacetic acid transaminase (GOT), glutamic pyruvic acid transaminase (GPT), and C-reactive protein (CRP) level. In rats, plasma activities of CK and LDH, muscle vascular permeability (MVP) using Evans blue dye, muscle force and fatigue were evaluated. Activities of CK, ALD, LDH, GOT, GTP, and levels of CRP were increased in the Pl group after the competition as compared to basal values. CrS decreased plasma activities of CK, LDH, and ALD, and prevented the rise of GOT and GPT plasma activities. In rats, CrS delayed the fatigue, preserved the force, and prevented the rise of LDH and CK plasma activities and MVP in the gastrocnemius muscle. CrS presented a protective effect on muscle injury induced by strenuous contractile activities.

PAR(2) and Temporomandibular Joint Inflammation in the Rat

The proteinase-activated receptor 2 (PAR(2)) is a putative therapeutic target for arthritis. We hypothesized that the early pro-inflammatory effects secondary to its activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR(2) in retrogradely labeled trigeminal ganglion neurons. Furthermore, PAR(2) immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance-P-containing peripheral sensory nerve fibers. The intra-articular injection of PAR(2) agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx, and induction of mechanical allodynia. The pharmacological blockade of natural killer 1 (NK(1)) receptors abolished PAR(2)-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR(2) activation is proinflammatory in the TMJ, through a neurogenic mechanism involving NK(1) receptors. This suggests that PAR(2) is an important component of innate neuro-immune response in the rat TMJ.

TUMOR NECROSIS FACTOR IS NOT ASSOCIATED WITH INTESTINAL ISCHEMIA/REPERFUSION-INDUCED LUNG INFLAMMATION

Intestinal ischemia-reperfusion (I/R) injury may cause acute systemic and lung inflammation. Here, we revisited the role of TNF-alpha in an intestinal I/R model in mice, showing that this cytokine is not required for the local and remote inflammatory response upon intestinal I/R injury using neutralizing TNF-alpha antibodies and TNF ligand-deficient mice. We demonstrate increased neutrophil recruitment in the lung as assessed by myeloperoxidase activity and augmented IL-6, granulocyte colony-stimulating factor, and KC levels, whereas TNF-alpha levels in serum were not increased and only minimally elevated in intestine and lung upon intestinal I/R injury. Importantly, TNF-alpha antibody neutralization neither diminished neutrophil recruitment nor any of the cytokines and chemokines evaluated. In addition, the inflammatory response was not abrogated in TNF and TNF receptors 1 and 2-deficient mice. However, in view of the damage on the intestinal barrier upon intestinal I/R with systemic bacterial translocation, we asked whether Toll-like receptor (TLR) activation is driving the inflammatory response. In fact, the inflammatory lung response is dramatically reduced in TLR2/4-deficient mice, confirming an important role of TLR receptor signaling causing the inflammatory lung response. In conclusion, endogenous TNF-alpha is not or minimally elevated and plays no role as a mediator for the inflammatory response upon ischemic tissue injury. By contrast, TLR2/4 signaling induces an orchestrated cytokine/chemokine response leading to local and remote pulmonary inflammation, and therefore disruption of TLR signaling may represent an alternative therapeutic target.

Effects of Low-Level Laser Therapy (LLLT) in the Development of Exercise-Induced Skeletal Muscle Fatigue and Changes in Biochemical Markers Related to Postexercise Recovery

STUDY DESIGN: Randomized crossover double-blinded placebo-controlled trial. OBJECTIVE: To investigate if low-level laser therapy (LLLT) can affect biceps muscle performance, fatigue development, and biochemical markers of postexercise recovery. BACKGROUND: Cell and animal studies have suggested that LLLT can reduce oxidative stress and inflammatory responses in muscle tissue. But it remains uncertain whether these findings can translate into humans in sport and exercise situations. METHODS: Nine healthy male volleyball players participated in the study. They received either active LLLT (cluster probe with 5 laser diodes; A = 810 nm; 200 mW power output; 30 seconds of irradiation, applied in 2 locations over the biceps of the nondominant arm; 60 J of total energy) or placebo LLLT using an identical cluster probe. The intervention or placebo were applied 3 minutes before the performance of exercise. All subjects performed voluntary elbow flexion repetitions with a workload of 75% of their maximal voluntary contraction force until exhaustion. RESULTS: Active LLLT increased the number of repetitions by 14.5% (mean +/- SD, 39.6 +/- 4.3 versus 34.6 +/- 5.6; P = .037) and the elapsed time before exhaustion by 8.0% (P = .034), when compared to the placebo treatment. The biochemical markers also indicated that recovery may be positively affected by LLLT, as indicated by postexercise blood lactate levels (P<.01), creatine kinase activity (P = .017), and C-reactive protein levels (P = .047), showing a faster recovery with LLLT application prior to the exercise. CONCLUSION: We conclude that pre-exercise irradiation of the biceps with an LLLT dose of 6 J per application location, applied in 2 locations, increased endurance for repeated elbow flexion against resistance and decreased postexercise levels of blood lactate, creatine kinase, and C-reactive protein. LEVEL OF EVIDENCE: Performance enhancement, level 1b. J Orthop Sports Phys Ther 2010;40(8):524-532. doi:10.2519/jospt.2010.3294

Effect of Cluster Multi-Diode Light Emitting Diode Therapy (LEDT) on Exercise-Induced Skeletal Muscle Fatigue and Skeletal Muscle Recovery in Humans

Background and Objectives: There are some indications that low-level laser therapy (LLLT) may delay the development of skeletal muscle fatigue during high-intensity exercise. There have also been claims that LED cluster probes may be effective for this application however there are differences between LED and laser sources like spot size, spectral width, power output, etc. In this study we wanted to test if light emitting diode therapy (LEDT) can alter muscle performance, fatigue development and biochemical markers for skeletal muscle recovery in an experimental model of biceps humeri muscle contractions. Study Design/Materials and Methods: Ten male professional volleyball players (23.6 [SD +/- 5.6] years old) entered a randomized double-blinded placebo-controlled crossover trial. Active cluster LEDT (69 LEDs with wavelengths 660/850 nm, 10/30 mW, 30 seconds total irradiation time, 41.7J of total energy irradiated) or an identical placebo LEDT was delivered under double-blinded conditions to the middle of biceps humeri muscle immediately before exercise. All subjects performed voluntary biceps humeri contractions with a workload of 75% of their maximal voluntary contraction force (MVC) until exhaustion. Results: Active LEDT increased the number of biceps humeri contractions by 12.9% (38.60 [SD +/- 9.03] vs. 34.20 [SD +/- 8.68], P = 0.021) and extended the elapsed time to perform contractions by 11.6% (P = 0.036) versus placebo. In addition, post-exercise levels of biochemical markers decreased significantly with active LEDT: Blood Lactate (P = 0.042), Creatine Kinase (P = 0.035), and C-Reative Protein levels (P = 0.030), when compared to placebo LEDT. Conclusion: We conclude that this particular procedure and dose of LEDT immediately before exhaustive biceps humeri contractions, causes a slight delay in the development of skeletal muscle fatigue, decreases post-exercise blood lactate levels and inhibits the release of Creatine Kinase and C-Reative Protein. Lasers Surg. Med. 41:572-577, 2009. (C) 2009 Wiley-Liss, Inc.

Sustained activation of p53 in confluent nucleotide excision repair-deficient cells resistant to ultraviolet-induced apoptosis

p53 activation is one of the main signals after DNA damage, controlling cell cycle arrest, DNA repair and apoptosis. We have previously shown that confluent nucleotide excision repair (NER)-deficient cells are more resistant to apoptosis induced by ultraviolet irradiation (UV). Here, we further investigated the effect of cell confluence on UV-induced apoptosis in normal and NER-deficient (XP-A and XP-C) cells, as well as the effects of treatments with the ATWATR inhibitor caffeine, and the patterns of p53 activation. Strong p53 activation was observed in either proliferating or confluent cells. Caffeine increased apoptosis levels and inhibited p53 activation in proliferating cells, suggesting a protective role for p53. However, in confluent NER-deficient cells no effect of caffeine was observed. Transcription recovery measurements showed decreased recovery in proliferating XPA-deficient cells, but no recovery was observed in confluent cells. The levels of the cyclin/Cdk inhibitor, p21(Waf1/Cip1), correlated well with p53 activation in proliferating cells. Surprisingly, confluent cells also showed similar activation of p21(Waf1/Cip1). These results indicate that reduced apoptosis in confluent cells is associated with the deficiency in DNA damage removal, since this effect is not clearly observed in NER-proficient cells. Moreover, the strong activation of p53 in confluent cells, which barely respond to apoptosis, suggests that this protein, under these conditions, is not linked to UV-induced cell death signaling. (c) 2008 Elsevier B.V. All rights reserved.

Resistance to ultraviolet-induced apoptosis in DNA repair deficient growth arrested human fibroblasts is not related to recovery from RNA transcription blockage

The impact of ultraviolet (UV-C) photoproducts on apoptosis induction was investigated in growth arrested (confluent) and proliferating human primary fibroblasts. Confluent fibroblasts were more resistant to UV-C-induced apoptosis than proliferating cells, and this was observed for normal human cells and for cells from patients with Cockayne and trichothiodystrophy syndromes, deficient in transcription coupled repair. This resistance was sustained for at least seven days and was not due to DNA repair efficiency, as the removal of CPDs in the genome was similar under both growth conditions. There was no correlation between reduced apoptosis and RNA synthesis recovery. Following UV-C treatment, proliferating and confluent fibroblasts showed a similar level of RNA synthesis inhibition and recovery from transcription blockage. These results support the hypothesis that the decrease of DNA replication, in growth arrested cells, protects cell from UV-C-induced apoptosis, even in the presence of DNA lesions. (C) 2007 Elsevier B.V. All rights reserved.

The genotoxic effects of DNA lesions induced by artificial UV-radiation and sunlight

Solar radiation sustains and affects all life forms on Earth. The increase in solar UV-radiation at environmental levels, due to depletion of the stratospheric ozone layer, highlights serious issues of social concern. This becomes still more dramatic in tropical and subtropical regions where radiation-intensity is still higher. Thus, there is the need to evaluate the harmful effects of solar UV-radiation on the DNA molecule as a basis for assessing the risks involved for human health, biological productivity and ecosystems. In order to evaluate the profile of DNA damage induced by this form of radiation and its genotoxic effects, plasmid DNA samples were exposed to artificial-UV lamps and directly to sunlight. The induction of cyclobutane pyrimidine dimer photoproducts (CPDs) and oxidative DNA damage in these molecules were evaluated by means of specific DNA repair enzymes. On the other hand, the biological effects of such lesions were determined through the analysis of the DNA inactivation rate and mutation frequency, after replication of the damaged pCMUT vector in an Escherichia coli MBL50 strain. The results indicated the induction of a significant number of CPDs after exposure to increasing doses of UVC, UVB, UVA radiation and sunlight. Interestingly, these photoproducts are those lesions that better correlate with plasmid inactivation as well as mutagenesis, and the oxidative DNA damages induced present very low correlation with these effects. The results indicated that DNA photoproducts play the main role in the induction of genotoxic effects by artificial UV-radiation sources and sunlight. (C) 2010 Elsevier B.V. All rights reserved.

p53 Mutant Human Glioma Cells Are Sensitive to UV-C-Induced Apoptosis Due to Impaired Cyclobutane Pyrimidine Dimer Removal

The p53 protein is a key regulator of cell responses to DNA damage, and it has been shown that It sensitizes glioma cells to the alkylating agent temozolomide by up-regulating the extrinsic apoptotic pathway, whereas it increases the resistance to chloroethylating agents, such as ACNU and BCNU, probably by enhancing the efficiency of DNA repair. However, because these agents induce a wide variety of distinct DNA lesions, the direct Importance of DNA repair is hard to access. Here, it is shown that the Induction of photoproducts by UV light (UV-C) significantly Induces apoptosis In a p53-mutated glioma background. This Is caused by a reduced level of photoproduct repair, resulting In the persistence of DNA lesions in p53-mutated glioma cells. UV-C-Induced apoptosis in p53 mutant glioma cells Is preceded by strong transcription and replication inhibition due to blockage by unrepaired photolesions. Moreover, the results Indicate that UV-C-induced apoptosis of p53 mutant glioma cells Is executed through the intrinsic apoptotic pathway, with Bcl-2 degradation and sustained Bax and Bak up-regulation. Collectively, the data Indicate that unrepaired DNA lesions Induce apoptosis In p53 mutant gliomas despite the resistance of these gliomas to temozolomide, suggesting that efficiency of treatment of p53 mutant gliomas might be higher with agents that Induce the formation of DNA lesions whose global genomic repair is dependent on p53. (Mol Cancer Res 2009;7(2):237-46)

Results of Joint Experiments and other IAEA activities on research using small tokamaks

This paper presents an overview of the results obtained during the Joint Experiments organized in the framework of the IAEA Coordinated Research Project on `Joint Research Using Small Tokamaks` that have been carried out on the tokamaks CASTOR at IPP Prague, Czech Republic (2005), T-10 at RRC `Kurchatov Institute`, Moscow, Russia (2006), and the most recent one at ISTTOK at IST, Lisbon, Portugal, in 2007. Experimental programmes were aimed at diagnosing and characterizing the core and the edge plasma turbulence in a tokamak in order to investigate correlations between the occurrence of transport barriers, improved confinement, electric fields and electrostatic turbulence using advanced diagnostics with high spatial and temporal resolution. On CASTOR and ISTTOK, electric fields were generated by biasing an electrode inserted into the edge plasma and an improvement of the global particle confinement induced by the electrode positive biasing has been observed. Geodesic acoustic modes were studied using heavy ion beam diagnostics on T-10 and ISTTOK and correlation reflectometry on T-10. ISTTOK is equipped with a gallium jet injector and the technical feasibility of gallium jets interacting with plasmas has been investigated in pulsed and ac operation. The first Joint Experiments have clearly demonstrated that small tokamaks are suitable for broad international cooperation to conduct dedicated joint research programmes. Other activities within the IAEA Coordinated Research Project on Joint Research Using Small Tokamaks are also overviewed.

Cellular and tissue effects induced by photogemA (R) and red LED in photodynamic therapy

In order to consider the photodynamic therapy (PDT) as a clinical treatment for candidosis, it is necessary to know its cytotoxic effect on normal cells and tissues. Therefore, this study evaluated the toxicity of PDT with PhotogemA (R) associated with red light-emitting diode (LED) on L929 and MDPC-23 cell cultures and healthy rat palatal mucosa. In the in vitro experiment, the cells (30000 cells/cm(2)) were seeded in 24-well plates for 48 h, incubated with PhotogemA (R) (50, 100, or 150 mg/l) and either irradiated or not with a red LED source (630 +/- 3 nm; 75 or 100 J/cm(2); 22 mW/cm(2)). Cell metabolism was evaluated by the MTT assay (ANOVA and Dunnet`s post hoc tests; p < 0.05) and cell morphology was examined by scanning electron microscopy. In the in vivo evaluation, PhotogemA (R) (500 mg/l) was applied to the palatal mucosa of Wistar rats during 30 min and exposed to red LED (630 nm) during 20 min (306 J/cm(2)). The palatal mucosa was photographed for macroscopic analysis at 0, 1, 3, and 7 days posttreatment and subjected to histological analysis after sacrifice of the rats. For both cell lines, there was a statistically significant decrease of the mitochondrial activity (90-97%) for all PhotogemA (R) concentrations associated with red LED regardless of the energy density. However, in the in vivo evaluation, the PDT-treated groups presented intact mucosa with normal characteristics both macroscopically and histologically. From these results, it may be concluded that the association of PhotogemA (R) and red LED caused severe toxic effects on normal cell cultures, characterized by the reduction of mitochondrial activity and morphological alterations, but did not cause damage to the rat palatal mucosa in vivo.

Alteration of purinergic P2X(4) and P2X(7) receptor expression in rats with temporal-lobe epilepsy induced by pilocarpine

Although ATP and P2X receptor activity have been lately associated with epilepsy, little is known regarding their exact roles in epileptogenesis. Temporal-lobe epilepsy (TLE) in rat was induced by pilocarpine in order to study changes of hippocampal P2X(2), P2X(4) and P2X(7) receptor expression during acute, latent or chronic phases of epilepsy. During acute and chronic phases increased P2X(7) receptor expression was principally observed in glial cells and glutamatergic nerve terminals, suggesting participation of this receptor in the activation of inflammatory and excitotoxic processes during epileptogenesis. No significant alterations of hippocampal P2X(2) and P2X(4) receptor expression was noted during the acute or latent phase when compared to the control group, indicating that these receptors are not directly involved with the initiation of epilepsy. However, the reduction of hippocampal P2X(4) receptor immunostaining in the chronic phase could reflect neuronal toss or decreased GABAergic signaling. (C) 2008 Elsevier B.V. All rights reserved.

Chlorella vulgaris up-modulation of myelossupression induced by lead: The role of stromal cells

In this study, Chlorella vulgaris (CV) was examined for its chelating effects on the ability of bone marrow stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice, using the long-term bone marrow culture (LTBMC). In addition, the levels of interleukin (IL)-6, an important hematopoietic stimulator, as well as the numbers of adherent and non-adherent cells were also investigated. Mice were gavage treated daily with a single 50 mg/kg dose of CV for 10 days, concomitant to continuous offering of 1300 ppm lead acetate in drinking water. We found that CV up-modulates the reduced ability of stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice and restores both the reduced number of non-adherent cells and the ability of stromal cells from these mice to produce IL-6. Monitoring of lead poisoning demonstrated that CV treatment significantly reduced lead levels in blood and tissues, completely restored the normal hepatic ALA levels, decreased the abnormally high plasma ALA and partly recovered the liver capacity to produce porphyrins. These findings provide evidence for a beneficial use of CV for combination or alternative chelating therapy to protect the host from the damage induced by lead poisoning. (C) 2008 Elsevier Ltd. All rights reserved.