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.

Transporters involved in glucose and water absorption in the Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) anterior midgut

Little is known about insect intestinal sugar absorption, in spite of the recent findings, and even less has been published regarding water absorption. The aim of this study was to shed light on putative transporters of water and glucose in the insect midgut Glucose and water absorptions by the anterior ventriculus of Dysdercus peruvianus midgut were determined by feeding the insects with a glucose and a non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventricular contents. Glucose absorption decreases glucose/dye ratios and water absorption increases dye concentrations. Water and glucose transports are activated (water 50%, glucose 33%) by 50 mM K(2)SO(4) and are inhibited (water 46%, glucose 82%) by 0.2 mM phloretin, the inhibitor of the facilitative hexose transporter (GLUT) or are inhibited (water 45%, glucose 35%) by 0.1 mM phlorizin, the inhibitor of the Na(+)-glucose cotransporter (SGLT). The results also showed that the putative SGLT transports about two times more water relative to glucose than the putative GLUT. These results mean that D. peruvianus uses a GLUT-like transporter and an SGLT-like transporter (with K(+) instead of Na(+)) to absorb dietary glucose and water. A cDNA library from D. peruvianus midgut was screened and we found one sequence homologous to GLUT1, named DpGLUT, and another to a sodium/solute symporter, named DpSGLT. Semi-quantitative RT-PCR studies revealed that DpGLUT and DpSGLTs mRNA were expressed in the anterior midgut, where glucose and water are absorbed, but not in fat body, salivary gland and Malpighian tubules. This is the first report showing the involvement of putative GLUT and SGLT in both water and glucose midgut absorption in insects. (C) 2010 Elsevier Inc. All rights reserved.

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

Aims Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. Methods and results NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO(2)) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 mu M). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. Conclusion Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Long-term regulation of vacuolar H(+)-ATPase by angiotensin II in proximal tubule cells

Long-term effects of angiotensin II (Ang II) on vacuolar H(+)-ATPase were studied in a SV40-transformed cell line derived from rat proximal tubules (IRPTC). Using pH(i) measurements with the fluorescent dye BCECF, the hormone increased Na(+)-independent pH recovery rate from an NH(4)Cl pulse from 0.066 +/- 0.014 pH U/min (n = 7) to 0.14 +/- 0.021 pH U/min (n = 13; p < 0.05) in 10 h Ang II (10(-9) M)-treated cells. The increased activity of H(+)-ATPase did not involve changes in mRNA or protein abundance of the B2 subunit but increased cell surface expression of the V-ATPase. Inhibition of tyrosine kinase by genistein blocked Ang II-dependent stimulation of H(+)-ATPase. Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortmannin and of p38 mitogen-activated protein kinase (MAPK) by SB 203580 also blocked this effect. Thus, long-term exposure of IRPTC cells to Ang II causes upregulation of H(+)-ATPase activity due, at least in part, to increased B2 cell surface expression. This regulatory pathway is dependent on mechanisms involving tyrosine kinase, p38 MAPK, and PI3K activation.

Soy lecithin supplementation alters macrophage phagocytosis and lymphocyte response to concanavalin A: a study in alloxan-induced diabetic rats

Dietary soy lecithin supplementation decreases hyperlipidemia and influences lipid metabolism. Although this product is used by diabetic patients, there are no data about the effect of soy lecithin supplementation on the immune system. The addition of phosphatidylcholine, the main component of lecithin, to a culture of lymphocytes has been reported to alter their function. If phosphatidylcholine changes lymphocyte functions in vitro as previously shown, then it could also affect immune cells in vivo. In the present study, the effect of dietary soy lecithin oil macrophage phagocytic capacity and on lymphocyte number in response to concanavalin A (ConA) stimulation was investigated in non-diabetic and alloxan-induced diabetic rats. Supplementation was carried Out daily with 2 g kg(-1) b.w. lecithin during 7 days. After that, blood was drawn from fasting rats and peritoneal macrophages and mesenteric lymph node lymphocytes were collected to determine the phospholipid content. Plasma triacylglycerol (TAG), total and HDL cholesterol and glucose levels were also determined. Lymphocytes were stimulated by Conk The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye reduction method and flow cytometry were employed to evaluate lymphocyte metabolism and cell number, respectively. Soy lecithin supplementation significantly increased both macrophage phagocytic capacity (+29%) in non-diabetic rats and the lymphocyte number in diabetic rats (+92%). It is unlikely that plasma lipid levels indirectly affect immune cells, since plasma cholesterol, TAG, or phospholipid content was not modified by lecithin supplementation. In Conclusion, lymphocyte and macrophage function were altered by lecithin supplementation, indicating ail immunomodulatory effect of phosphatidylcholine. Copyright (C) 2008 John Wiley & Sons, Ltd.

Cell coupling in mouse pancreatic beta-cells measured in intact islets of Langerhans

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified beta-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent beta-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between beta-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell ( due to the. ring of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of beta-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to -55 mV) gave rise to a slow capacitive current indicative of coupling between beta-cells, but not in non-beta-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial beta-cell in an islet is in electrical contact with six to seven other beta-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell K(ATP) channel conductance (G(K,ATP)) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC(50) of approximately 4 mM. Theoretical considerations indicate that the coupling between beta-cells within the islet is sufficient to allow propagation of [Ca(2+)](i) waves to spread with a speed of approximately 80 mu m s(-1), similar to that observed experimentally in confocal [Ca(2+)](i) imaging.

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.

The allergic inflammatory reaction in neonatal streptozotocininduced diabetic rats: evidence of insulin resistance and microvascular dysfunction

To investigate the allergic reaction in neonatal streptozotocin (nSTZ)-induced diabetes mellitus. Male newborn Wistar rats were made diabetic by the injection of streptozotocin (160 mg/kg, i. p.) and used 8 weeks thereafter. Animals were sensitized against ovalbumin (OA, 50 mu g and Al(OH)3, 5 mg, s. c.) and challenged 14 or 21 days thereafter. OA-induced airway inflammation and OA-induced pleurisy models were used to investigate leukocyte migration (total and differential leukocyte counts) and lung vascular permeability (Evans blue dye extravasation). nSTZ-diabetic rats presented glucose intolerance and insulin resistance. Relative to controls, nSTZ rats exhibited a 30% to 50% reduction in lung vascular permeability. Leukocyte infiltration in both models of allergen-induced inflammation, and number of pleural mast cells did not differ between groups. Data suggest that the reduction of allergic inflammatory reactions in nSTZ rats is restricted to microvascular dysfunctions and associated, probably, with insulin resistance in lung microvascular endothelium.

Synthesis, characterization and catalytic evaluation of cubic ordered mesoporous iron-silicon oxides

Iron was successfully incorporated in FDU-1 type cubic ordered mesoporous silica by a simple direct synthesis route. The (Fe/FDU-1) samples were characterized by Rutherford back-scattering spectrometry (RBS), small angle X-ray scattering (SAXS). N(2) sorption isotherm, X-ray diffraction (XRD) and X-ray absorption spectroscopy (XAS). The resulting material presented an iron content of about 5%. Prepared at the usual acid pH of -0.3, the composite was mostly formed by amorphous silica and hematite with a quantity of Fe(2+) present in the structure. The samples prepared with adjusted pH values (2 and 3.5) were amorphous. The samples` average pore diameter was around 12.0 nm and BET specific surface area was of 680 m(2) g(-1). Although the iron-incorporated material presented larger lattice parameter, about 25 nm compared to pure FDU-1, the Fe/FDU-1 composite still maintained its cubic ordered fcc mesoporous structure before and after the template removal at 540 degrees C. The catalytic performance of Fe/FDU-1 was investigated in the catalytic oxidation of Black Remazol B dye using a catalytic ozonation process. The results indicated that Fe/FDU-1 prepared at the usual acid pH exhibited high catalytic activity in the mineralization of this pollutant when compared to the pure FDU-1. Fe(2)O(3) and Fe/FDU-1 prepared with higher pH of 2 and 3.5. (C) 2010 Elsevier B.V. All rights reserved.

Polarization and solvatochromic shift of ortho-betaine in water

Betaine dyes are known to show very large transition energy shifts in different solvents. The ortho-betaine molecule - a simple two-ring prototype of the E-T(30) Reichardt dye - has been investigated theoretically from a combined statistical and quantum mechanics approach. Using sequential Monte Carlo (MC) simulations and MP2/cc-pVDZ calculations the in-water dipole moment of ortho-betaine is obtained as 12.30 +/- 0.05 D. This result shows a considerable increase of 75% compared to the in-vacuum dipole moment. For comparison, the use of a polarizable continuum model using the same MP2/cc-pVDZ leads to an in-water dipole moment of 11.6 D, in good agreement. This large polarization is incorporated in the classical potential for another MC simulation to generate solute-solvent configurations and to obtain the contribution of the polarization effect in the solvatochromic shift. Using statistically uncorrelated configurations and supermolecular INDO/CIS calculations, including the solute and, explicitly, 230 solvent water molecules, the statistically converged calculated shift is obtained here as 6360 cm(-1), in good agreement with the experimental result of 7550 cm(-1). (c) 2007 Elsevier B.V. All rights reserved.

Ultrafast dynamics of bis (n-butylimido) perylene thin films excited by two-photon absorption

We report a pump-probe study of the two-photon induced reflectivity changes in bis (n-butylimido) perylene thin films. To enhance the two-photon excitation we deposited bis (n-butylimido) perylene films on top of gold nanoislands. The observed transient response in the reflectivity spectrum of bis (n-butylimido) perylene is due to a depletion of the molecule`s ground state and excited state absorption.

A combination of techniques to evaluate photodynamic efficiency of photosensitizers

Photodynamic therapy, used mainly for cancer treatment and microorganisms inaction, is based on production of reactive oxygen species by light irradiation of a sensitizer. Hematoporphyrin derivatives as Photofrin (R) (PF) Photogem (R) (PG) and Photosan (R) (PF), and chlorin-c6-derivatives as Photodithazine (R)(PZ), have suitable sensitizing properties. The present study provides a way to make a fast previous evaluation of photosensitizers efficacy by a combination of techniques: a) use of brovine serum albumin and uric acid as chemical dosimeters; b) photo-hemolysis of red blood cells used as a cell membrane interaction model, and c) octanol/phosphate buffer partition to assess the relative lipophilicity of the compounds. The results suggest the photodynamic efficient rankings PZ > PG >= PF > PS. These results agree with the cytotoxicity of the photosensitizers as well as to chromatographic separation of the HpDs, both performed in our group, showing that the more lipophilic is the dye, the more acute is the damage to the RBC membrane and the oxidation of indol, which is immersed in the hydrophobic region of albumin.

Optical birefringence induced by two-photon absorption in polythiophene bearing an azochromophore

We carried out experiments of induced birefringence via two-photon absorption in spin-coated films of the conjugated polymer poly[2-[ethyl-[4-(4-nitro-phenylazo)-phenyl] -amino]-ethane (3-thienyl)ethanoate], PAzT, at 680 and 775 nm. This process allows recording in the bulk because of the spatial confinement of the bireffingence provided by the two-photon absorption. The induced birefringence is associated with molecular reorientation caused by the two-photon induced isomerization of the azochromophores attached to the polymer backbone. In addition, the two-photon absorption spectrum of PAzT was measured to help selecting the excitation wavelength for two-photon absorption induced birefringence. (c) 2008 Elsevier Ltd. All rights reserved.

Influence from the sulfonate group (RSO(3)(-)) on the conversion process and emission efficiency of poly(p-phenylene vinylene)

The discovery of an alternative route to convert poly(xylyliden tetrahydrothiophenium chloride) (PTHT) into poly(p-phenylene vinylene) (PPV) using dodecylbenzenesulfonate (DBS) has allowed the formation of ultrathin films with unprecedented control of architecture and emission properties. In this work, we show that this route may be performed with several sufonated compounds where RSO(3)(-) replaces the counter-ion (Cl(-)) of PTHT, some of which are even more efficient than DBS. Spin-coating films were produced from PTHT and azo-dye molecules, an azo-polymer and organic salts as counter-ions of PTHT. The effects of the thermal annealing step of PTHT/RSO(3)(-) films at 110 and 230 degrees C were monitored by measuring the absorption and emission spectra. The results indicate that the exchange of the counterion Cl(-) of PTHT by a linear long chain with RSO(3)(-) group is a general procedure to obtain PPV polymer at lower conversion temperature (ca. 110 degrees C) with significant increase in the emission efficiency, regardless of the chemical position and the number of sulfonate groups. With the enhanced emission caused by Congo Red and Tinopal as counter-ions, it is demonstrated that the new synthetic route is entirely generic, which may allow accurate control of conversion and emission properties. (C) 2010 Elsevier B.V. All rights reserved.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.