C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2+/phosphate-induced impairment

Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and eventually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidant and neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injury induced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in rat retinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). In SH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, and the reduction was effectively prevented by treatment with C-PC in the low micromolar concentration range. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120 mmHg for 45 min, which was followed by 15 min of reperfusion. This event resulted in a cell density reduction to lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocular pre-treatment with C-PC for 15 min. In the RBM exposed to 3 mM phosphate and/or 100 mu M Ca2+, C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition as assessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygen species levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a strong inhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potential neuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the protection of mitochondria from impairment. (C) 2012 Elsevier Inc. All rights reserved.

Partitioning and extraction of collagenase from Penicillium aurantiogriseum in poly(ethylene glycol)/phosphate aqueous two-phase system

Purification of collagenase produced by Penicillium aurantiogriseum URM4622 was carried using a PEG/phosphate aqueous two-phase system (ATPS). A 2(3)-full experimental design was used to investigate the influence of PEG molar mass, PEG concentration and phosphate concentration on the selected responses, namely partition coefficient, activity yield and purification factor. The ATPS was composed of PEG (molar mass of 550, 1500 and 4000 g/mol) at concentrations of 15.0, 17.5 and 20.0% (w/w) and phosphate at concentrations of 12.5, 15.0 and 17.5% (w/w). The best results of one-step extraction of collagenase from the fermentation broth (partition coefficient of 1.01, activity yield of 242% and purification factor of 23.5) were obtained at pH 6.0 using 20.0% (w/w) PEG 550 and 17.5% (w/w) phosphate. The results of this preliminary study demonstrate that the selected ATPS is satisfactorily selective for the extraction of such a collagenase. (C) 2012 Elsevier B.V. All rights reserved.

Cytocompatibility of Porous Biphasic Calcium Phosphate Granules With Human Mesenchymal Cells by a Multiparametric Assay

This work aims to evaluate the cytocompatibility of injectable and moldable restorative biomaterials based on granules of dense or porous biphasic calcium phosphates (BCPs) with human primary mesenchymal cells, in order to validate them as tools for stem cell-induced bone regeneration. Porous hydroxyapatite (HA) and HA/beta-tricalcium phosphate (beta-TCP) (60: 40) granules were obtained by the addition of wax spheres and pressing at 20 MPa, while dense materials were compacted by pressing at 100 MPa, followed by thermal treatment (1100 degrees C), grinding, and sieving. Extracts were prepared by 24-h incubation of granules on culture media, with subsequent exposition of human primary mesenchymal cells. Three different cell viability parameters were evaluated on the same samples. Scanning electron microscopy analysis of the granules revealed distinct dense and porous surfaces. After cell exposition to extracts, no significant differences on mitochondrial activity (2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) or cell density (Crystal Violet Dye Elution) were observed among groups. However, Neutral Red assay revealed that dense materials extracts induced lower levels of total viable cells to porous HA/beta-TCP (P < 0.01). Calcium ion content was also significantly lower on the extracts of dense samples. Porogenic treatments on BCP composites do not affect cytocompatibility, as measured by three different parameters, indicating that these ceramics are well suited for further studies on future bioengineering applications.

IMPROVEMENT TO THE MARGINAL COPING FIT OF COMMERCIALLY PURE TITANIUM CAST IN PHOSPHATE-BONDED INVESTMENT BY USING A SIMPLE PATTERN COATING TECHNIQUE

Statement of problem. Coatings of zirconite, Y2O3 or ZrO2 on wax patterns before investing in phosphate-bonded investments have been recommended to reduce the reaction layer in titanium castings, but they are not easily obtainable. Spinel-based investments are relatively stable with molten titanium and could be used as coatings to improve the quality of castings made with those investments. Purpose. The purpose of this study was to evaluate the effect of pattern coating with a commercial spinel-based investment before investing in 1 of 3 phosphate-bonded investments on the marginal coping fit and surface roughness of commercially pure titanium castings. Material and methods. Ten square acrylic resin patterns (12 x 12 x 2 mm) per group were invested in the phosphate-bonded investments Rematitan Plus (RP), Rema Exakt (RE), and Castorit Super C (CA) with or without a coating of the spinel-based investment, Rematitan Ultra (RU). After casting, the specimens were cleaned and the surface roughness was measured with a profilometer. Copings for dental implants with conical abutment were invested, eliminated, and cast as previously described. The copings were cleaned and misfit was measured with a profile projector (n=10). For both tests, the difference between the mean value of RU only and each value of the phosphate-bonded investment was calculated, and the data were analyzed by 2-way ANOVA and Tukey's HSD test (alpha=.05). In addition, the investment roughness was measured in bar specimens (30 x 10 x 10 mm), and the data (n=10) were analyzed by 1-way ANOVA and Tukey's HSD post hoc test (alpha=.05). Results. Two-way ANOVA for casting surface roughness was significant because of the investment, the coating technique, and the interaction between variables. One-way ANOVA was performed to prove the interaction term, and Tukey's post hoc test showed that RP with coating had the lowest mean, while RP had the highest. CA with coating was not different from RP with coating or CA without coating. RE with coating was similar to CA, while RE was different from all groups. For coping marginal fit, the 2-way ANOVA was significant for the investment, the coating technique, and the interaction between variables. The interaction was analyzed by1-way ANOVA and Tukey's HSD test that showed no significant difference among the coated groups, which had better marginal fit than the groups without coating. Among the groups without coating, CA had significant lower marginal misfit than RP, while RE was not different from CA and RP. For the investment surface roughness, the 1-way ANOVA was significant. CA and RU were smoother than RE and RP (P<.001). Conclusions. The coating technique improved the quality of castings fabricated with phosphate-bonded investments. (J Prosthet Dent 2012;108:51-57)

The Influence of Osteoblast Differentiation Stage on Bone Formation in Autogenously Implanted Cell-Based Poly(Lactide-Co-Glycolide) and Calcium Phosphate Constructs

We tested the hypothesis that the osteoblast differentiation status of bone marrow stem cells (BMSCs) combined with a three-dimensional (3D) structure modulates bone formation when autogenously implanted. Rat BMSCs were aspirated, expanded, and seeded into a 3D composite of poly(lactide-co-glycolide) and calcium phosphate (PLGA/CaP) to produce a hybrid biomaterial. Calvarial defects were implanted with (1) scaffold without cells (SC/NC), (2) scaffold and BMSCs (SC + BMSC), (3) scaffold and osteoblasts differentiated for 7 days (SC + OB7), and (4) for 14 days (SC + OB14). After 4 weeks, there was more bone formation in groups combining scaffold and cells, SC + BMSC and SC + OB7. A nonsignificant higher amount of bone formation was observed on SC + OB14 compared with SC/NC. Additionally, more blood vessels were counted within all hybrid biomaterials, without differences among them, than into SC/NC. These findings provide evidences that the cell differentiation status affects in vivo bone formation in autogenously implanted cell-based constructs. Undifferentiated BMSCs or osteoblasts in early stage of differentiation combined with PLGA/CaP scaffold favored bone formation compared with plain scaffold and that one associated with more mature osteoblasts.

Kinetics and product distribution of p-nitrophenyl phosphate dianion solvolysis in ternary DMSO/alcohol/water mixtures are compatible with metaphosphate formation

The rate of solvolysis of p-nitrophenyl phosphate (PNPP) dianion in DMSO/water strongly decreases by increasing water concentration. Addition of linear alcohols (methanol, propanol, butanol, pentanol, and hexanol) at constant DMSO/water molar ratio produced an even sharper rate decrease. Alkyl phosphate formation, resulting from PNPP solvolysis in ternary DMSO/water/alcohol mixtures, increased with alcohol concentration and was essentially temperature independent. Methanol and hexanol were the poorest nucleophiles under all conditions. Activation energies and enthalpies for solvolysis in ternary mixtures were similar and entropies varied with alcohol concentration. Taken together these results can be best interpreted in terms of a dissociative mechanism with the intervention of metaphosphate. Copyright (C) 2011 John Wiley & Sons, Ltd.

Hypertonic saline solution reduces the inflammatory response in endotoxemic rats

OBJECTIVE: Volume replacement in septic patients improves hemodynamic stability. This effect can reduce the inflammatory response. The objective of this study was to evaluate the effect of 7.5% hypertonic saline solution versus 0.9% normal saline solution for volume replacement during an inflammatory response in endotoxemic rats. METHODS: We measured cytokines (serum and gut), nitrite, and lipid peroxidation (TBARS) as indicators of oxidative stress in the gut. Rats were divided into four groups: control group (C) that did not receive lipopolysaccharide; lipopolysaccharide injection without treatment (LPS); lipopolysaccharide injection with saline treatment (LPS + S); and lipopolysaccharide injection with hypertonic saline treatment (LPS + H). Serum and intestine were collected. Measurements were taken at 1.5, 8, and 24 h after lipopolysaccharide administration. RESULTS: Of the four groups, the LPS + H group had the highest survival rate. Hypertonic saline solution treatment led to lower levels of IL-6, IL-10, nitric oxide, and thiobarbituric acid reactive substances compared to 0.9% normal saline. In addition, hypertonic saline treatment resulted in a lower mortality compared to 0.9% normal saline treatment in endotoxemic rats. Volume replacement reduced levels of inflammatory mediators in the plasma and gut. CONCLUSION: Hypertonic saline treatment reduced mortality and lowered levels of inflammatory mediators in endotoxemic rats. Hypertonic saline also has the advantage of requiring less volume replacement.

Intravenous Hypertonic Saline Solution (7.5%) and Oral Electrolytes to Treat of Calves with Noninfectious Diarrhea and Metabolic Acidosis

Objective The aim of this study was to compare the efficacy of treating osmotic diarrhea and dehydration in calves with hypertonic saline solution (HSS) IV, isotonic electrolyte solution (IES) PO, and a combination of these 2 solutions (HSS + IES). Experimental Design Eighteen male calves 830 days of age were used to evaluate the efficacy of 3 methods of fluid therapy after induction of osmotic diarrhea and dehydration. The diarrhea and dehydration were induced by administration of saccharose, spironolactone, and hydrochlorothiazide for 48 hours. The animals were randomly divided into 3 experimental groups: Group 1: 7.2% hypertonic saline solution-HSS (5 mL/kg IV); Group 2: oral isotonic electrolyte solution IES (60 mL/kg PO); or Group 3: HSS+IES. Clinical signs and laboratory finding observed 48 hours post-induction (Time 0) included diarrhea, dehydration, lethargy, and metabolic acidosis. Results Calves treated with HSS + IES experienced decreases in hematocrit, total protein concentration, albumin concentration, urea nitrogen concentration, and plasma volume as well as increases in blood pH, blood bicarbonate concentration, and central venous pressure between 1 and 3 hours post-treatment. These findings also were observed in animals treated with IES, however, at a slower rate than in the HSS + IES-treated animals. Animals treated with HSS continued to display signs of dehydration, lethargy, and metabolic acidosis 24 hours post-treatment. Conclusion Treatment with a combination of HSS and IES produced rapid and sustainable correction of hypovolemia and metabolic acidosis in calves with noninfections diarrhea and dehydration.

Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism

Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.

The Pst system of Streptococcus mutans is important for phosphate transport and adhesion to abiotic surfaces

The Pst system is a high-affinity inorganic phosphate transporter found in many bacterial species. Streptococcus mutans, the etiological agent of tooth decay, carries a single copy of the pst operon composed of six cistrons (pstS, pstC1, pstC, pstB, smu.1134 and phoU). Here, we show that deletion of pstS, encoding the phosphate-binding protein, reduces phosphate uptake and impairs cell growth, which can be restored upon enrichment of the medium with high concentrations of inorganic phosphate. The relevance of Pst for growth was also demonstrated in the wild-type strain treated with an anti-PstS antibody. Nevertheless, a reduced ability to bind to saliva-coated surfaces was observed, along with the reduction of extracellular polysaccharide production, although no difference on pH acidification was observed between mutant and wild-type strains. Taken together, the present data indicate that the S.similar to mutans Pst system participates in phosphate uptake, cell growth and expression of virulence-associated traits.

Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Early Amyloid-like Oligomers and Their Implication in alpha-Synuclein Aggregation

Lewy bodies and Lewy neurites, neuropathological hallmarks of several neurological diseases, are mainly made of filamentous assemblies of alpha-synuclein. However, other macromolecules including Tau, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and glycosaminoglycans are routinely found associated with these amyloid deposits. Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that can form fibrillar aggregates in the presence of acidic membranes, but its role in Parkinson disease is still unknown. In this work, the ability of heparin to trigger the amyloid aggregation of this protein at physiological conditions of pH and temperature is demonstrated by infrared and fluorescence spectroscopy, dynamic light scattering, small angle x-ray scattering, circular dichroism, and fluorescence microscopy. Aggregation proceeds through the formation of short rod-like oligomers, which elongates in one dimension. Heparan sulfate was also capable of inducing glyceraldehyde-3-phosphate dehydrogenase aggregation, but chondroitin sulfates A, B, and C together with dextran sulfate had a negligible effect. Aided with molecular docking simulations, a putative binding site on the protein is proposed providing a rational explanation for the structural specificity of heparin and heparan sulfate. Finally, it is demonstrated that in vitro the early oligomers present in the glyceraldehyde-3-phosphate dehydrogenase fibrillation pathway promote alpha-synuclein aggregation. Taking into account the toxicity of alpha-synuclein prefibrillar species, the heparin-induced glyceraldehyde-3-phosphate dehydrogenase early oligomers might come in useful as a novel therapeutic strategy in Parkinson disease and other synucleinopathies.

CTAB, Triton X-100 and freezing-thawing treatments of Candida guilliermondii: Effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase, xylose reductase and xylitol dehydrogenase enzymes

Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30 degrees C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4 +/- 15.7 U/g(cells)) > freezing-thawing, , (54.3 +/- 1.9 U/g(cells)) > Triton X-100 (23.5 +/- 0.0 U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 +/- 0.1 U/g(cells).

Patient with toxoplasmosis and glucose-6-phosphate dehydrogenase deficiency: a case report

Abstract Introduction Toxoplasmosis, a zoonotic protozoal disease caused by toxoplasma gondii, is prevalent throughout the world, affecting a large proportion of persons who usually have no symptoms. Glucose 6 phosphate dehydrogenase deficiency, an X-linked inherited disorder, is present in over 400 million people world wide. It is more common in tropical and subtropical countries and is one of the important causes of hemolytic anemia. Case presentation This case report relates the occurrence of the two diseases simultaneously in a child of five years old. Conclusion Patients with glucose-6-phosphate dehydrogenase deficiency are more susceptible to toxoplasmosis and this case report, reinforce the findings of this propensity and alert us for such possibility, what it is important, therefore, the treatment of toxoplasmosis can cause serious hemolysis in these patients.

Small volume of hypertonic saline as the initial fluid replacement in experimental hypodynamic sepsis

Abstract Introduction We conducted the present study to examine the effects of hypertonic saline solution (7.5%) on cardiovascular function and splanchnic perfusion in experimental sepsis. Methods Anesthetized and mechanically ventilated mongrel dogs received an intravenous infusion of live Escherichia coli over 30 minutes. After 30 minutes, they were randomized to receive lactated Ringer's solution 32 ml/kg (LR; n = 7) over 30 minutes or 7.5% hypertonic saline solution 4 ml/kg (HS; n = 8) over 5 minutes. They were observed without additional interventions for 120 minutes. Cardiac output (CO), mean arterial pressure (MAP), portal and renal blood flow (PBF and RBF, respectively), gastric partial pressure of CO2 (pCO2; gas tonometry), blood gases and lactate levels were assessed. Results E. coli infusion promoted significant reductions in CO, MAP, PBF and RBF (approximately 45%, 12%, 45% and 25%, respectively) accompanied by an increase in lactate levels and systemic and mesenteric oxygen extraction (sO2ER and mO2ER). Widening of venous-arterial (approximately 15 mmHg), portal-arterial (approximately 18 mmHg) and gastric mucosal-arterial (approximately 55 mmHg) pCO2 gradients were also observed. LR and HS infusion transiently improved systemic and regional blood flow. However, HS infusion was associated with a significant and sustained reduction of systemic (18 ± 2.6 versus 38 ± 5.9%) and mesenteric oxygen extraction (18.5 ± 1.9 versus 36.5 ± 5.4%), without worsening other perfusional markers. Conclusion A large volume of LR or a small volume of HS promoted similar transient hemodynamic benefits in this sepsis model. However, a single bolus of HS did promote sustained reduction of systemic and mesenteric oxygen extraction, suggesting that hypertonic saline solution could be used as a salutary intervention during fluid resuscitation in septic patients.