ATP pulse and calcium homeostasis in cells from hepatopancreas of Dilocarcinus pagei, a freshwater crab

Calcium (Ca) is critical for crustaceans due to their molting cycle and its presence in the carapace as calcium carbonate, apart from the usual functions of Ca, such as cell signalling. Ca transport in Dilocarcinus pagei, a freshwater crab, was studied in isolated cells from hepatopancreas to further characterize Ca transport mechanisms in these crabs. Cells were isolated and loaded with Fluo-3, a calcium fluorescent dye. Three different cell treatments were performed: Group 1 cells were Ca free during cell dissociation, and calcium was present (at 1mM) for fluorescence cell loading and transport experiments (FC); Group 2 cells were calcium free during cell dissociation and for transport experiments, but not during cell loading (LC); and Group 3 cells were Ca free during cell dissociation, cell loading and transport experiments (WC). Intracellular Ca was recorded through time after ATP was added to the cells and ATP caused an increase in Ca efflux within 30s in all cells. WC cells showed the smallest Ca efflux compared to the other cells, probably because it was intracellularly Ca ""depleted"". Vanadate and amiloride decreased the Ca efflux when ATP was added to the cells, while verapamil did not cause any effect in Ca efflux, confirming the presence of a Ca(2+)-ATPase sensitive to vanadate in hepatopancreas of D. pagei. In a different set of experiments, cells were also exposed to a Ca pulse of 1 and 10mM during 180s. 10mM Ca increased intracellular Ca compared to 1mM, and the increase was not recovered during the experimental time. Additionally, Ca influx was reduced by verapamil and amiloride, but not completely. The results suggest that Ca influx probably occurs through an undefined exchanger, apart from Ca channels (verapamil sensitive) and electrogenic 1Na(+)(1H(+))/1 Ca(2+) exchanger (amiloride-sensitive). Similarities between freshwater and seawater crabs, lobsters and crayfish in relation to plasma membrane Ca transporters, although the environment where they live is quite diverse, suggest that universal mechanisms for Ca homeostasis are widespread among crustaceans. (C) 2010 Elsevier Inc. All rights reserved.

Taurine Supplementation Increases K(atp) Channel Protein Content, Improving Ca2+ Handling And Insulin Secretion In Islets From Malnourished Mice Fed On A High-fat Diet.

Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.

The Analgesic Effect Of Dipyrone In Peripheral Tissue Involves Two Different Mechanisms: Neuronal K(atp) Channel Opening And Cb(1) Receptor Activation.

Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100ng/50µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.

Pulse train fluorescence technique for measuring triplet state dynamics

We report on a method to study the dynamics of triplet formation based on the fluorescence signal produced by a pulse train. Basically, the pulse train acts as sequential pump-probe pulses that precisely map the excited-state dynamics in the long time scale. This allows characterizing those processes that affect the population evolution of the first excited singlet state, whose decay gives rise to the fluorescence. The technique was proven to be valuable to measure parameters of triplet formation in organic molecules. Additionally, this single beam technique has the advantages of simplicity, low noise and background-free signal detection. (C) 2011 Optical Society of America

Selective Decrease of Components of the Creatine Kinase System and ATP Synthase Complex in Chronic Chagas Disease Cardiomyopathy

Background: Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC) and ischemic (IC) cardiomyopathies. Methodology/Principal Findings: Myocardium homogenates from CCC (N = 5), IC (N = 5) and IDC (N = 5) patients, as well as from heart donors (N = 5) were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit) and muscular creatine kinase (CKM) and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. Conclusions/Significance: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

Influence of pulse repetition rate on temperature rise and working time during composite filling removal with the Er : YAG laser

Objective: The purpose of this study was to assess the efficacy of Er:YAG laser energy for composite resin removal and the influence of pulse repetition rate on the thermal alterations occurring during laser ablation. Materials and Methods: Composite resin filling was placed in cavities (1.0 mm deep) prepared in bovine teeth and the specimens were randomly assigned to five groups according to the technique used for composite filling removal. In group I (controls), the restorations were removed using a high-speed diamond bur. In the other groups, the composite fillings were removed using an Er: YAG laser with different pulse repetition rates: group 2-2 Hz; group 3-4 Hz; group 4-6 Hz; and group 5-10 Hz. The time required for complete removal of the restorative material and the temperature changes were recorded. Results: Temperature rise during composite resin removal with the Er: YAG laser occurred in the substrate underneath the restoration and was directly proportional to the increase in pulse repetition rate. None of the groups had a temperature increase during composite filling removal of more than 5.6 degrees C, which is considered the critical point above which irreversible thermal damage to the pulp may result. Regarding the time for composite filling removal, all the laser-ablated groups (except for group 5 [10 Hz]) required more time than the control group for complete elimination of the material from the cavity walls. Conclusion: Under the tested conditions, Er: YAG laser irradiation was efficient for composite resin ablation and did not cause a temperature increase above the limit considered safe for the pulp. Among the tested pulse repetition rates, 6 Hz produced minimal temperature change compared to the control group (high-speed bur), and allowed composite filling removal within a time period that is acceptable for clinical conditions.

Expression of an activating mutation in the gene encoding the K(ATP) channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K(+) (K(ATP)) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of K(ATP) channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of K(ATP) channels, closed K(ATP) channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of K(ATP) channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Effectiveness of 980-mm Diode and 1064-nm Extra-Long-Pulse Neodymium-Doped Yttrium Aluminum Garnet Lasers in Implant Disinfection

Objective: To evaluate the potential of 980-nm gallium aluminum arsenide (GaAlAs) and 1064-nm neodymium-doped yttrium aluminum garnet (Nd:YAG) lasers to reduce bacteria after irradiation of implant surfaces contaminated with Enterococcus faecalis and Porphyromonas gingivalis and on irradiated implant surface morphology. Background: Despite the frequency of implant success, some implant loss is related to peri-implantitis because of difficulty in eliminating the biofilm. Methods: Implants (3.75 x 13 mm) with machined surfaces, surfaces sand blasted with titanium oxide (TiO(2)), and sand-blasted and acid-etched surfaces were exposed to P. gingivalis and E. faecalis cultures and irradiated with 980-nm GaAlAs or 1064-nm Nd: YAG lasers. After laser treatments, the number of remaining colony-forming units and implant surface morphology were analyzed using scanning electron microscopy (SEM). Results: The Nd: YAG laser was able to promote a total contamination reduction on all implants irradiated. The results with the GaAlAs laser showed 100% bacteria reduction on the implants irradiated with 3 W. Irradiation with 2.5 W and 3 W achieved 100% of bacteria reduction on P. gingivalis-contaminated implants. Decontamination was not complete for the sand-blasted TiO(2) (78.6%) and acid-etched surfaces (49.4%) contaminated with E. faecalis and irradiated with 2.5 W. SEM showed no implant surface changes. Conclusion: The wavelengths used in this research provided bacteria reduction without damaging implant surfaces. New clinical research should be encouraged for the use of this technology in the treatment of peri-implantitis.

A high voltage pulse power supply for metal plasma immersion ion implantation and deposition

We describe the design and implementation of a high voltage pulse power supply (pulser) that supports the operation of a repetitively pulsed filtered vacuum arc plasma deposition facility in plasma immersion ion implantation and deposition (Mepiiid) mode. Negative pulses (micropulses) of up to 20 kV in magnitude and 20 A peak current are provided in gated pulse packets (macropulses) over a broad range of possible pulse width and duty cycle. Application of the system consisting of filtered vacuum arc and high voltage pulser is demonstrated by forming diamond-like carbon (DLC) thin films with and without substrate bias provided by the pulser. Significantly enhanced film/substrate adhesion is observed when the pulser is used to induce interface mixing between the DLC film and the underlying Si substrate. (C) 2010 American Institute of Physics. [doi:10.1063/1.3518969]

Hyper-Rayleigh scattering with picosecond pulse trains

We propose a method for measuring hyper-Rayleigh scattering employing pulse trains produced by a Q-switched and mode-locked Nd:YAG laser. The use of the entire pulse train under the Q-switch envelope avoids the need of any device to scan the irradiance, as is usually done with nanosecond and femtosecond single-pulse lasers. To verify the feasibility of the technique, we performed measurements in different solutions of para-nitroaniline and compared the results with those obtained with nanosecond pulses. In both cases, the agreement with the hyperpolarizability values reported in the literature is about the same, but the measurements carried out with pulse trains are at least 20 times faster. Besides the advantage of acquisition speed, the use of pulse trains also allows the instantaneous inspection of slow luminescence contributions arising from multiphoton absorption. (C) 2008 Optical Society of America.

Potent Cardioprotective Effect of the 4-Anilinoquinazoline Derivative PD153035: Involvement of Mitochondrial K(ATP) Channel Activation

Background: The aim of the present study was to evaluate the protective effects of the 4-anilinoquinazoline derivative PD153035 on cardiac ischemia/reperfusion and mitochondrial function. Methodology/Principal Findings: Perfused rat hearts and cardiac HL-1 cells were used to determine cardioprotective effects of PD153035. Isolated rat heart mitochondria were studied to uncover mechanisms of cardioprotection. Nanomolar doses of PD153035 strongly protect against heart and cardiomyocyte damage induced by ischemia/reperfusion and cyanide/aglycemia. PD153035 did not alter oxidative phosphorylation, nor directly prevent Ca(2+) induced mitochondrial membrane permeability transition. The protective effect of PD153035 on HL-1 cells was also independent of AKT phosphorylation state. Interestingly, PD153035 activated K(+) transport in isolated mitochondria, in a manner prevented by ATP and 5-hydroxydecanoate, inhibitors of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)). 5-Hydroxydecanoate also inhibited the cardioprotective effect of PD153035 in cardiac HL-1 cells, demonstrating that this protection is dependent on mitoK(ATP) activation. Conclusions/Significance: We conclude that PD153035 is a potent cardioprotective compound and acts in a mechanism involving mitoK(ATP) activation.

A Family of Autoconnected Transformers for 12-and 18-Pulse Converters-Generalization for Delta and Wye Topologies

Multipulse rectifier topologies based on autoconnections are increasingly applied as interface stages between mains and power electronics converters. These topologies are attractive and cost-effective solutions for meeting the requirements of low total harmonic distortion of line current and high power factor. Furthermore, as only a small fraction of the total power required by the load is processed in the magnetic core, the overall resulting volume and weight are reduced. This paper proposes a mathematical analysis based on phasor diagrams that results in a single and general expression capable of unifying all delta and wye step-up or step-down autotransformer connections for 12-and 18-pulse ac-dc converters. The expression obtained allows the choice of a wide range of input/output voltage ratio for step-up or step-down autotransformer, and this general expression is also presented in a graphical form for each converter. Moreover, it simplifies the procedure for determining turn ratios and polarities for all windings of the autotransformer. A routine for easy and fast calculations is developed and validated by a design example. Finally, experimental results are presented along with comments on a 6-kW 220-V line voltage, 400-V rectified voltage, and 18-pulse delta-autoconnected prototype.

A numerical investigation on the visco-plastic response of structures to different pulse loading shapes

The time varying intensity character of a load applied to a structure poses many difficulties in analysis. A remedy to this situation is to substitute a complex pulse shape by a rectangular equivalent one. It has been shown by others that this procedure works well for perfectly plastic elementary structures. This paper applies the concept of equivalent pulse to more complex structures. Special attention is given to the material behavior, which is allowed to be strain rate and strain hardening sensitive. Thanks to the explicit finite element solution, it is shown in this article that blast loads applied to complex structures made of real materials can be substituted by equivalent rectangular loads with both responses being practically the same. (c) 2007 Elsevier Ltd. All rights reserved.

Effects of carbon dioxide feeding rate and light intensity on the fed-batch pulse-feeding cultivation of Spirulina platensis in helical photobioreactor

The behavior of S. platensis was investigated in this study through fed-batch pulse-feeding cultures performed at different carbon dioxide feeding rates (F = 0.44-1.03 g L-1 d(-1)) and photosynthetic photon flux density (PPFD = 80-250 mu mol photons m(-2) s(-1)) in a bench-scale helical photobioreactor. To achieve this purpose, an inorganic medium lacking the carbon source was enriched by gaseous carbon dioxide from a cylinder. The maximum cell concentration achieved was 12.8 g L-1 at PPFD = 166 mu mol photons m(-2) s(-1) and F= 0.44 g L-1 d(-1) of CO2. At PPFD = 80 and 125 mu mol photons m(-2) s(-1), the carbon utilization efficiency (CUE) reached maximum values of 50 and 69%, respectively, after about 20 days, and then it decreased, thus highlighting a photolimitation effect. At PPFD = 166 mu mol photons m(-2) s(-1), CUE was >= 90% between 20 and 50 days. The photosynthetic efficiency reached its maximum value (9.4%) at PPFD = 125 mu mol photons m(-2) s(-1). The photoinhibition threshold appeared to strongly depend on the feeding rate: at high PPFD, an increase in the amount of fed CO2 delayed the inhibitory effect on biomass growth, whereas at low PPFD, excess CO2 addition caused the microalga to stop growing. (c) 2007 Elsevier B.V. All rights reserved.

ATP-induced apoptosis involves a Ca(2+)-independent phospholipase A(2) and 5-lipoxygenase in macrophages

Macrophages express P2X(7) and other nucleotide (P2) receptors, and display the phenomena of extracellular ATP (ATP(e))-induced P2X(7)-dependent membrane permeabilization and cell death by apoptosis and necrosis. P2X7 receptors also cooperate with toll-like receptors (TLRs) to induce inflammasome activation and IL-1 beta secretion. We investigated signaling pathways involved in the induction of cell death by ATP, in intraperitoneal murine macrophages. Apoptosis (hypodiploid nuclei) and necrosis (LDH release) were detected 6 h after an induction period of 20 min in the presence of ATP Apoptosis was blocked by caspase 3 and caspase 9 inhibitors and by cyclosporin A. The MAPK inhibitors PD-98059, SB-203580 and SB-202190 provoked no significant effect oil apoptosis, but SB-203580 blocked LDH release. Neither apoptosis nor necrosis was inhibited when both intra- and extracellular Ca(2+) were chelated during the induction period. Mepacrine, a generic PLA(2) inhibitor and BEL, an inhibitor of Ca(2+)-independent PLA(2) (iPLA(2)) blocked apoptosis, while pBPB and AACOOPF(3). inhibitors of secretory and Ca(2+)-dependent PLA(2) respectively, had no significant effect. Cycloxygenase inhibitors had no effect on apoptosis, while the inhibitors of lipoxygenase (LOX) and leukotriene biosynthesis nordihydroguaiaretic acid (NDGA), zileuton, AA-861, and MK-886 significantly decreased apoptosis. Neither NDGA nor MK-886 blocked apoptosis of 5-LOX(-/-) macrophages. CP-105696 and MK-571, antagonists of leukotriene receptors, had no significant effect on apoptosis. None of the inhibitors of PLA(2) and LOX/leukotriene pathway had a significant inhibitory effect on LDH release. Our results indicate that a Ca(2+) -independent step involving an iPLA(2) and 5-LOX are involved in the triggering of apoptosis but not necrosis by P2X7 in macrophages. (C) 2008 Elsevier Inc. All rights reserved.