Elevated extracellular inorganic phosphate inhibits ecto-phosphatase activity in breast cancer cells: Regulation by hydrogen peroxide.

For cells to obtain inorganic phosphate, ectoenzymes in the plasma membrane, which contain a catalytic site facing the extracellular environment, hydrolyze phosphorylated molecules. In this study, we show that increased Pi levels in the extracellular environment promote a decrease in ecto-phosphatase activity, which is associated with Pi-induced oxidative stress. High levels of Pi inhibit ecto-phosphatase because Pi generates H2 O2 . Ecto-phosphatase activity is inhibited by H2 O2 , and this inhibition is selective for phospho-tyrosine hydrolysis. Additionally, it is shown that the mechanism of inhibition of ecto-phosphatase activity involves lipid peroxidation. In addition, the inhibition of ecto-phosphatase activity by H2 O2 is irreversible. These findings have new implications for understanding ecto-phosphatase regulation in the tumor microenvironment. H2 O2 stimulated by high Pi inhibits ecto-phosphatase activity to prevent excessive accumulation of extracellular Pi, functioning as a regulatory mechanism of Pi variations in the tumor microenvironment.

Inorganic Phosphate (Pi) in the Breast Cancer Microenvironment: Production, Transport and Signal Transduction as Potential Targets for Anticancer Strategies.

Tumor cells develop a high demand for inorganic phosphate (Pi) due to their high growth rates and energy requirements. Serum Pi concentrations in cancer patients have been found to be two to four times higher than baseline levels in healthy individuals. Twofold Pi accumulation was observed in breast cancer cells in the mouse tumor microenvironment. In the breast tumoral microenvironment, ectonucleotidases and ectophosphatases-presenting catalytic sites facing the extracellular environment-could be involved in the extracellular release of Pi to be internalized by Pi transporters to fuel the high energy requirement typical of cancer cells. Two Pi transporters were characterized in breast cancer cells (Na+-dependent and H+-dependent) with strong associations with tumor processes such as proliferation, migration, adhesion, and epithelium-mesenchymal transition (EMT). Moreover, a high extracellular Pi concentration stimulates ROS production in triple-negative breast cancer cells by Pi transport stimulation. Several compounds show a potent ability to inhibit ectonucleotidases, ectophosphatases, Pi transporters, and Pi-modulated signal pathways in breast cancer cells and regulate proliferation, migration, adhesion, and EMT. This review article aimed to gather the relevant experimental records regarding Pi's effects on the breast cancer microenvironment and points to possible inhibitors for ectonucleotidases, ectophosphatases, Pi transporters, and Pi-modulated signal pathways as potential chemotherapeutic agents or Pi acting as a potent enhancer of classical chemical-induced cytotoxicity in triple-negative breast cancer cells.

Anaerobic ATP synthesis pathways and inorganic phosphate transport and their possible roles in encystment in Acanthamoeba castellanii.

Acanthamoeba castellanii is the etiological agent of amoebic keratitis and is present in the environment in trophozoite or cyst forms. Both forms can infect the vertebrate host and colonize different tissues. The high resistance of cysts to standard drugs used in clinics contributes to the lack of effective treatments. Therefore, in this context, studies have emerged to understand cyst physiology and metabolism. Phosphate transporters are proteins responsible for the uptake of extracellular inorganic phosphate and transport to the cytosol. This work aims to verify the relationship between Pi transport and energetic metabolism in cysts of A. castellanii. The phosphate uptake ratio was higher in cysts compared with trophozoites. Recently, three sequences related to phosphate transporters have been identified in the A. castellanii genome (AcPHS1, AcPHS2, and AcPHS3); the messenger RNA expression levels of which differ depending on the amoeba life form. Pi uptake in cysts displayed peak activity at alkaline pH, whereas Pi transport in trophozoites was not affected in the same pH ranges. Cysts harbor a low-affinity Pi transport system (K0,5 and Vmax values of 1.76 ± 0.26 mM and 104.6 ± 6.3 nmol Pi × h-1 × 106 cells) compared to the trophozoite phosphate transport system. Pi transport seems important for anaerobic adenosine triphosphate synthesis in cysts, which initially occurs through the glycolytic pathway and subsequently through the pyruvate ferredoxin oxidoreductase pathway. Altogether, these results suggest that contrary to that previously postulated, cysts are active metabolic forms, and, as noted in trophozoites, phosphate uptake is important for energetic metabolism.

Extracellular Inorganic Phosphate-Induced Release of Reactive Oxygen Species: Roles in Physiological Processes and Disease Development.

Inorganic phosphate (Pi) is an essential nutrient for living organisms and is maintained in equilibrium in the range of 0.8-1.4 mM Pi. Pi is a source of organic constituents for DNA, RNA, and phospholipids and is essential for ATP formation mainly through energy metabolism or cellular signalling modulators. In mitochondria isolated from the brain, liver, and heart, Pi has been shown to induce mitochondrial reactive oxygen species (ROS) release. Therefore, the purpose of this review article was to gather relevant experimental records of the production of Pi-induced reactive species, mainly ROS, to examine their essential roles in physiological processes, such as the development of bone and cartilage and the development of diseases, such as cardiovascular disease, diabetes, muscle atrophy, and male reproductive system impairment. Interestingly, in the presence of different antioxidants or inhibitors of cytoplasmic and mitochondrial Pi transporters, Pi-induced ROS production can be reversed and may be a possible pharmacological target.

A chloride channel blocker prevents the suppression by inorganic phosphate of the cytosolic calcium signals that control muscle contraction.

KEY POINTS: Accumulation of inorganic phosphate (Pi ) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of Pi into SR are fully established.  We showed that Pi promoted Ca2+ release at concentrations below 10 mm and decreased it at higher concentrations. This decrease correlated well with that of [Ca2+ ]SR .  Pre-treatment of permeabilized myofibres with 2 mm Cl- channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of Pi .  The biphasic dependence of Ca2+ release on [Pi ] is explained by a direct effect of Pi acting on the SR Ca2+ release channel, combined with the intra-SR precipitation of Ca2+ salts. The effects of 9AC demonstrate that Pi enters the SR via a Cl- pathway of an as-yet-undefined molecular nature. ABSTRACT: Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (Pi ). Pi diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca2+ salts, thus contributing to the impairment of Ca2+ release. Information on the Pi entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca2+ spark frequency is correlated with the Ca2+ load of the SR and can be used to monitor this variable. We studied the effects of Pi on Ca2+ sparks in permeabilized fibres of the frog. Relative event frequency (f/fref ) rose with increasing [Pi ], reaching 2.54 ± 1.6 at 5 mm, and then decreased monotonically, reaching 0.09 ± 0.03 at [Pi ] = 80 mm. Measurement of [Ca2+ ]SR confirmed a decrease correlated with spark frequency at high [Pi ]. A large [Ca2+ ]SR surge was observed upon Pi removal. Anion channels are a putative path for Pi into the SR. We tested the effect of the chloride channel blocker 9-anthracenecarboxylic acid (9AC) on Pi entrance. 9AC (400 µm) applied to the cytoplasm produced a non-significant increase in spark frequency and reduced the Pi effects on this parameter. Fibre treatment with 2 mm 9AC in the presence of high cytoplasmic Mg2+ suppressed the effects of Pi on [Ca2+ ]SR and spark frequency up to 55 mm [Pi ]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that Pi impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR, thus contributing to myogenic fatigue.

The Roles of Sodium-Independent Inorganic Phosphate Transporters in Inorganic Phosphate Homeostasis and in Cancer and Other Diseases.

Inorganic phosphate (Pi) is an essential nutrient for the maintenance of cells. In healthy mammals, extracellular Pi is maintained within a narrow concentration range of 0.70 to 1.55 mM. Mammalian cells depend on Na+/Pi cotransporters for Pi absorption, which have been well studied. However, a new type of sodium-independent Pi transporter has been identified. This transporter assists in the absorption of Pi by intestinal cells and renal proximal tubule cells and in the reabsorption of Pi by osteoclasts and capillaries of the blood-brain barrier (BBB). Hyperphosphatemia is a risk factor for mineral deposition, the development of diseases such as osteoarthritis, and vascular calcifications (VCs). Na+-independent Pi transporters have been identified and biochemically characterized in vascular smooth muscle cells (VSMCs), chondrocytes, and matrix vesicles, and their involvement in mineral deposition in the extracellular microenvironment has been suggested. According to the growth rate hypothesis, cancer cells require more phosphate than healthy cells due to their rapid growth rates. Recently, it was demonstrated that breast cancer cells (MDA-MB-231) respond to high Pi concentration (2 mM) by decreasing Na+-dependent Pi transport activity concomitant with an increase in Na+-independent (H+-dependent) Pi transport. This Pi H+-dependent transport has a fundamental role in the proliferation and migratory capacity of MDA-MB-231 cells. The purpose of this review is to discuss experimental findings regarding Na+-independent inorganic phosphate transporters and summarize their roles in Pi homeostasis, cancers and other diseases, such as osteoarthritis, and in processes such as VC.

Acanthamoeba castellanii phosphate transporter (AcPHS) is important to maintain inorganic phosphate influx and is related to trophozoite metabolic processes.

Acanthamoeba castellanii is a free-living amoeba and the etiological agent of granulomatous amoebic encephalitis and amoebic keratitis. A. castellanii can be present as trophozoites or cysts. The trophozoite is the vegetative form of the cell and has great infective capacity compared to the cysts, which are the dormant form that protect the cell from environmental changes. Phosphate transporters are a group of proteins that are able to internalize inorganic phosphate from the extracellular to intracellular medium. Plasma membrane phosphate transporters are responsible for maintaining phosphate homeostasis, and in some organisms, regulating cellular growth. The aim of this work was to biochemically characterize the plasma membrane phosphate transporter in A. castellanii and its role in cellular growth and metabolism. To measure inorganic phosphate (Pi) uptake, trophozoites were grown in liquid PYG medium at 28 °C for 2 days. The phosphate uptake was measured by the rapid filtration of intact cells incubated with 0.5 µCi of 32Pi for 1 h. The Pi transport was linear as a function of time and exhibited Michaelis-Menten kinetics with a Km = 88.78 ± 6.86 µM Pi and Vmax = 547.5 ± 16.9 Pi × h-1 × 10-6 cells. A. castellanii presented linear phosphate uptake up to 1 h with a cell density ranging from 1 × 105 to 2 × 106 amoeba × ml-1. The Pi uptake was higher in the acidic pH range than in the alkaline range. The oxygen consumption of living trophozoites increased according to Pi addition to the extracellular medium. When the cells were treated with FCCP, no effect from Pi on the oxygen flow was observed. The addition of increasing Pi concentrations not only increased oxygen consumption but also increased the intracellular ATP pool. These phenomena were abolished when the cells were treated with FCCP or exposed to hypoxia. Together, these results reinforce the hypothesis that Pi is a key nutrient for Acanthamoeba castellanii metabolism.

Effect of Graded Levels of an Enhanced E coli Phytase with Step-Wise Reduction of Supplemental Inorganic Phosphate on Growth Performance of Broilers Fed Corn-Soy Diet

This study evaluated the potential of incremental doses of an enhanced Escherichia coli-derived phytase to support step-wise reduction of supplemental inorganic phosphate in an all-vegetable broiler diet. Corn-soybean meal-based diets containing 0.40/0.80%, 0.35/0.70%, and 0.30/0.60% avP and Ca, respectively from 0-10, 10-25, and 26-42 days posthatch served as experimental control (PC). Three test diets were formulated with 500, 1000, and 1500 FTU/kg of phytase assigned respectively an avP matrix of 0.15, 0.19, and 0.23% and a fixed Ca matrix of 0.15%. An additional diet (PC++) containing extra avP and Ca (+0.05% avP/+0.1% Ca) to that of PC was included to test if avP and Ca were not limiting in PC. Each diet was offered to 16 replicates of straight-run broilers kept in floor pens (30 birds per pen). PC++ had lower (p 0.05; 10 and 25 d) or similar (42 d) BW and toe ash compared with PC confirming the avP and Ca set in PC were sufficient to support optimal growth and bone mineralization. Compared to the PC, diets containing 1000 and 1500 FTU phytase had higher BW (p 0.05) at 10 and 25 d. For the overall period of 0-42 d, FI, BW and FCR did not differ across treatments. Percentage-, but not the absolute-, toe ash at phytase treated groups was significantly (p 0.05) low compared with the PC. The experiment demonstrated that 1500 FTU/kg of enhance E. coli phytase supports optimal BW and FCR of broilers fed corn-soy diet largely void of supplemental inorganic phosphate.(AU)

Effect of Graded Levels of an Enhanced E coli Phytase with Step-Wise Reduction of Supplemental Inorganic Phosphate on Growth Performance of Broilers Fed Corn-Soy Diet

This study evaluated the potential of incremental doses of an enhanced Escherichia coli-derived phytase to support step-wise reduction of supplemental inorganic phosphate in an all-vegetable broiler diet. Corn-soybean meal-based diets containing 0.40/0.80%, 0.35/0.70%, and 0.30/0.60% avP and Ca, respectively from 0-10, 10-25, and 26-42 days posthatch served as experimental control (PC). Three test diets were formulated with 500, 1000, and 1500 FTU/kg of phytase assigned respectively an avP matrix of 0.15, 0.19, and 0.23% and a fixed Ca matrix of 0.15%. An additional diet (PC++) containing extra avP and Ca (+0.05% avP/+0.1% Ca) to that of PC was included to test if avP and Ca were not limiting in PC. Each diet was offered to 16 replicates of straight-run broilers kept in floor pens (30 birds per pen). PC++ had lower (p 0.05; 10 and 25 d) or similar (42 d) BW and toe ash compared with PC confirming the avP and Ca set in PC were sufficient to support optimal growth and bone mineralization. Compared to the PC, diets containing 1000 and 1500 FTU phytase had higher BW (p 0.05) at 10 and 25 d. For the overall period of 0-42 d, FI, BW and FCR did not differ across treatments. Percentage-, but not the absolute-, toe ash at phytase treated groups was significantly (p 0.05) low compared with the PC. The experiment demonstrated that 1500 FTU/kg of enhance E. coli phytase supports optimal BW and FCR of broilers fed corn-soy diet largely void of supplemental inorganic phosphate.

H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment.

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H+-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H+-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H+-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of Km = 1.387 ±â€¯0.1674 mM Pi and Vmax = 198.6 ±â€¯10.23 Pi × h-1 × mg protein-1 hence reflecting a low affinity Pi transport. H+-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H+-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H+-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H+-dependent Pi transport was five-fold higher than the Na+-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H+-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na+-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.

On the Roles of Wheat Endosperm ADP-Glucose Pyrophosphorylase Subunits.

The ADP-glucose pyrophosphorylase from wheat endosperm controls starch synthesis in seeds and has unique regulatory properties compared to others from this family. It comprises two types of subunits, but despite its importance little is known about their roles. Here, we synthesized de novo the wheat endosperm ADP-glucose pyrophosphorylase small (S) and large (L) subunit genes, heterologously expressed them in Escherichia coli, and kinetically characterized the recombinant proteins. To understand their distinct roles, we co-expressed them with well characterized subunits from the potato tuber enzyme to obtain hybrids with one S subunit from one source and an L subunit from the other. After kinetic analyses of these hybrids, we concluded that the unusual insensitivity to activation of the wheat endosperm enzyme is caused by a pre-activation of the L subunit. In addition, the heat stability and sensitivity to phosphate are given by the S subunit.

Inorganic phosphate transporters in cancer: Functions, molecular mechanisms and possible clinical applications.

Inorganic phosphate is one of the most essential nutrients for the maintenance of cell life. Because of its essential role in nutrient supplementation, the study of plasma membrane inorganic phosphate transporters in cancer biology has received much attention in recent years. Several studies suggest that these transporters are up-regulated in tumor cells and thus have been considered to be important promoters of tumor progression. Altered expression levels of inorganic phosphate transporters, such as NaPi-IIb (SLC34A2) and PiT-1 (SLC20A1), have been demonstrated. The purpose of this review article was to gather the relevant experimental records on inorganic phosphate transporters in tumors and to demonstrate the importance of these proteins in clinical applications. In this work, we demonstrate that for decades, the potential use of the inorganic phosphate transporter as an antigen for the diagnosis of tumor subtypes remained unknown. With the advancement in molecular biology techniques, phosphate transporters have been identified as being associated with cancer. In addition to their altered expression in cancer, several studies have demonstrated other functions of inorganic phosphate transporters, such as transceptors, rearrangements with oncogenes and modifications in the expression of ABC transporters, aiding in the process of proliferation and drug resistance.

RSL4 Takes Control: Multiple Signals, One Transcription Factor.

Root hair growth dramatically expands the root surface area, thus facilitating water and nutrient uptake. Until recently, the molecular mechanism underlying root hair growth was unknown. Recent studies have revealed that the transcription factor ROOT HAIR DEFECTIVE 6 LIKE 4 (RSL4) coordinates hormonal, environmental, and developmental factors to trigger polar growth.

Mitochondrial involvement in memory impairment induced by scopolamine in rats.

OBJECTIVE: Scopolamine (SCO) administration to rats induces molecular features of AD and other dementias, including impaired cognition, increased oxidative stress, and imbalanced cholinergic transmission. Although mitochondrial dysfunction is involved in different types of dementias, its role in cognitive impairment induced by SCO has not been well elucidated. The aim of this work was to evaluate the in vivo effect of SCO on different brain mitochondrial parameters in rats to explore its neurotoxic mechanisms of action. METHODS: Saline (Control) or SCO (1 mg/kg) was administered intraperitoneally 30 min prior to neurobehavioral and biochemical evaluations. Novel object recognition and Y-maze paradigms were used to evaluate the impact on memory, while redox profiles in different brain regions and the acetylcholinesterase (AChE) activity of the whole brain were assessed to elucidate the amnesic mechanism of SCO. Finally, the effects of SCO on brain mitochondria were evaluated both ex vivo and in vitro, the latter to determine whether SCO could directly interfere with mitochondrial function. RESULTS: SCO administration induced memory deficit, increased oxidative stress, and increased AChE activities in the hippocampus and prefrontal cortex. Isolated brain mitochondria from rats administered with SCO were more vulnerable to mitochondrial swelling, membrane potential dissipation, H2O2 generation and calcium efflux, all likely resulting from oxidative damage. The in vitro mitochondrial assays suggest that SCO did not affect the organelle function directly. CONCLUSION: In conclusion, the present results indicate that SCO induced cognitive dysfunction and oxidative stress may involve brain mitochondrial impairment, an important target for new neuroprotective compounds against AD and other dementias.

H+-dependent inorganic phosphate uptake in Trypanosoma brucei is influenced by myo-inositol transporter.

Trypanosoma brucei is an extracellular protozoan parasite that causes human African trypanosomiasis or "sleeping sickness". During the different phases of its life cycle, T. brucei depends on exogenous inorganic phosphate (Pi), but little is known about the transport of Pi in this organism. In the present study, we showed that the transport of 32Pi across the plasma membrane follows Michaelis-Menten kinetics and is modulated by pH variation, with higher activity at acidic pH. Bloodstream forms presented lower Pi transport in comparison to procyclic forms, that displayed an apparent K0.5 = 0.093 ± 0.008 mM. Additionally, FCCP (H+-ionophore), valinomycin (K+-ionophore) and SCH28080 (H+, K+-ATPase inhibitor) inhibited the Pi transport. Gene Tb11.02.3020, previously described to encode the parasite H+:myo-inositol transporter (TbHMIT), was hypothesized to be potentially involved in the H+:Pi cotransport because of its similarity with the Pho84 transporter described in S. cerevisiae and other trypanosomatids. Indeed, the RNAi mediated knockdown remarkably reduced TbHMIT gene expression, compromised cell growth and decreased Pi transport by half. In addition, Pi transport was inhibited when parasites were incubated in the presence of concentrations of myo-inositol that are above 300 µM. However, when expressed in Xenopus laevis oocytes, two-electrode voltage clamp experiments provided direct electrophysiological evidence that the protein encoded by TbHMIT is definitely a myo-inositol transporter that may be only marginally affected by the presence of Pi. These results confirmed the presence of a Pi carrier in T. brucei, similar to the H+-dependent inorganic phosphate system described in S. cerevisiae and other trypanosomatids. This transport system contributes to the acquisition of Pi and may be involved in the growth and survival of procyclic forms. In summary, this work presents the first description of a Pi transport system in T. brucei.

Leishmania amazonensis: characterization of an ecto-pyrophosphatase activity.

Several ecto-enzymatic activities have been described in the plasma membrane of the protozoan Leishmania amazonensis, which is the major etiological agent of diffuse cutaneous leishmaniasis in South America. These enzymes, including ecto-phosphatases, contribute to the survival of the parasite by participating in phosphate metabolism. This work identifies and characterizes the extracellular hydrolysis of inorganic pyrophosphate related to an ecto-pyrophosphatase activity of the promastigote form of L. amazonensis. This ecto-pyrophosphatase activity is insensitive to MnCl2 but is strongly stimulated by MgCl2. This stimulation was not observed during the hydrolysis of p-nitrophenyl phosphate (p-NPP) or ß-glycerophosphate, two substrates for different ecto-phosphatases present in the L. amazonensis plasma membrane. Furthermore, extracellular PPi hydrolysis is more efficient at alkaline pHs, while p-NPP hydrolysis occurs mainly at acidic pHs. These results led us to conclude that extracellular PPi is hydrolyzed not by non-specific ecto-phosphatases but rather by a genuine ecto-pyrophosphatase. In the presence of 5mM MgCl2, the ecto-pyrophosphatase activity from L. amazonensis is sensitive to micromolar concentrations of NaF and millimolar concentrations of CaCl2. Moreover, this activity is significantly higher during the first days of L. amazonensis culture, which suggests a possible role for this enzyme in parasite growth.

Disturbance of brain energy and redox homeostasis provoked by sulfite and thiosulfate: potential pathomechanisms involved in the neuropathology of sulfite oxidase deficiency.

Sulfite oxidase (SO) deficiency is biochemically characterized by tissue accumulation and high urinary excretion of sulfite, thiosulfate and S-sulfocysteine. Affected patients present severe neurological symptoms and cortical atrophy, whose pathophysiology is still poorly established. Therefore, in the present work we investigated the in vitro effects of sulfite and thiosulfate on important parameters of energy metabolism in the brain of young rats. We verified that sulfite moderately inhibited the activity of complex IV, whereas thiosulfate did not alter any of the activities of the respiratory chain complexes. It was also found that sulfite and thiosulfate markedly reduced the activity of total creatine kinase (CK) and its mitochondrial and cytosolic isoforms, suggesting that these metabolites impair brain cellular energy buffering and transfer. In contrast, the activity of synaptic Na(+),K(+)-ATPase was not altered by sulfite or thiosulfate. We also observed that the inhibitory effect of sulfite and thiosulfate on CK activity was prevented by melatonin, reduced glutathione and the combination of both antioxidants, as well as by the nitric oxide synthase N(ω)-nitro-l-arginine methyl ester, indicating the involvement of reactive oxygen and nitrogen species in these effects. Sulfite and thiosulfate also increased 2',7'-dichlorofluorescin oxidation and hydrogen peroxide production and decreased the activity of the redox sensor aconitase enzyme, reinforcing a role for oxidative damage in the effects elicited by these metabolites. It may be presumed that the disturbance of cellular energy and redox homeostasis provoked by sulfite and thiosulfate contributes to the neurological symptoms and abnormalities found in patients affected by SO deficiency.

Trypanosoma rangeli: an alkaline ecto-phosphatase activity is involved with survival and growth of the parasite.

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using ß-glycerophosphate (ß-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of ß-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of ß-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. ß-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when ß-GP was the sole source of Pi and stopped it in the absence of ß-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.

On the effect of the injection of potassium phosphate in vivo inducing the precipitation of serum calcium with inorganic phosphate.

High concentrations of inorganic phosphate (Pi) resulted from the hydrolysis of ATP is strongly associated to the weakness of the contractile mechanism of muscles due to its attractiveness to calcium. The majority of the experiments to study such effect are conducted in vitro. This work investigates the effects of different concentrations of Pi, induced by the injection of potassium phosphate in live animals, in the precipitation with serum calcium and the generation of calcium phosphate composites. The experiments were also designed to find out the ideal amount of potassium phosphate to induce an effective reaction. Potassium phosphate was injected in Wistar rats, randomly separated and distributed into seven groups. Group I was injected with 0.5 ml of saline solution (control) and groups II through VII were injected with 0.5, 1.5, 2.5, 5.0, 7.5 and 10.0 mg/kg of potassium phosphate, respectively. Blood collected from the inferior vena cava was submitted to biochemical analyses to measure the concentrations of calcium, Pi, urea and creatinine. The results showed that Pi, induced by the injection of potassium phosphate in live animals, causes precipitation with serum calcium, with statistically significant differences between the control and the treatment groups for doses up to 5.0 mg/kg. No statistically significant differences were found between the different doses and the concentration of urea and creatinine in the plasma. We conclude that potassium phosphate can be used to induce serum calcium precipitation in-vivo, with minor effects on other physiological variables, and the ideal dose to do so is 5.0 mg/kg.

Effect of different 1 percent chlorhexidine varnish regimens on biochemical composition of the dental biofilm / Efeito de diferentes posologias do verniz de clorexidina a 1 por cento na composição bioquímica do biofilme dentário

PURPOSE: This investigation evaluated in a randomized controlled study the effect of different 1 percent chlorhexidine varnish (1 percentCHX-V) regimens on biochemical composition of the dental biofilm. METHODS: Subjects with mutans streptococci > 10(5) CFU/mL saliva, were allocated into 4 groups: A - single 1 percent CHX-V application; B - 1 percent CHX-V was applied once daily on 3 consecutive days; C - 1 percent CHX-V was applied 3 times with an interval of 4 days between each application; and D - placebo varnish was applied once daily on 3 consecutive days. Dental biofilm samples were collected at baseline and 1, 4, and 8 weeks after the final varnish application and evaluated for inorganic phosphate and insoluble polysaccharide concentrations. RESULTS: No difference was observed between all groups during the different experimental periods in relation to biochemical composition. After 1 week, a significant increase was observed in inorganic phosphate concentration, returning to baseline values after 4 weeks. All groups showed a significant decrease in insoluble polysaccharide concentration for more than 8 weeks after treatment. CONCLUSION: Varnish application resulted in modification of the dental biofilm toward a less cariogenic one.

OBJETIVO: Este estudo avaliou o efeito de diferentes posologias do verniz de clorexidina a 1 por cento (V-CHX1 por cento) na composição bioquímica do biofilme dental. METODOLOGIA: Voluntários com níveis de Streptococcus mutans > 10(5) UFC/mL saliva, foram divididos em 4 grupos: A - uma única aplicação do V-CHX1 por cento; B - aplicações de 1x/dia do V-CHX1 por cento durante 3 dias consecutivos; C - 3 aplicações de 1x/dia do V-CHX1 por cento com intervalos de 4 dias entre elas; D - aplicações de 1x/dia do verniz placebo durante 3 dias consecutivos. Amostras de biofilme dental foram coletadas, no início e após 1, 4 e 8 semanas das aplicações dos vernizes, e analisadas quanto às concentrações de fosfato inorgânico (Pi) e polissacarídeos insolúveis (PI). RESULTADOS: Não foram observadas diferenças, em relação à composição bioquímica, entre os grupos durante os diferentes períodos experimentais. Foi observado, após 1 semana, um aumento significativo nas concentrações de Pi, retornando aos valores iniciais após 4 semanas. Todos os grupos apresentaram uma redução significativa na concentração de PI por mais de 8 semanas após os tratamentos. CONCLUSÃO: A aplicação do verniz resultou em uma modificação do biofilme dentário tornando-o menos cariogênico.