José Mauro Granjeiro

Purification and characterization of multiple forms of soybean seed acid phosphatases

Abstract Four isoforms of acid phosphatase (EC 3.1.3.2), AP1, AP2, AP3A and AP3B, have been detected and partially purified from soybean seed ( Glycine max ) through DEAE- and SP-Sephadex chromatographies. Specific activity values of 822, 163, 14 and 66 nkat·mg −1 were obtained for AP1 (903-fold purification), AP2 (180-fold), AP3A (15-fold), and AP3B (73-fold), respectively, using p -nitrophenylphosphate as substrate. Relative native molecular mass values for AP1. AP2, AP3A and AP3B, determined by gel filtration on calibrated SW-300 Waters Protein Glass column, were found to be 51 000, 58 000, 52 000 and 30 000, respectively. All four acid phosphatase isoforms presented a carbohydrate moiety in their structures and revealed only single phosphatase activity bands following nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. AP1 and AP2 exhibited greater substrate specificity than AP3A and AP3B. The K m values were determined for p -nitrophenylphosphate, tyrosinephosphate and inorganic pyrophosphate, at pH 5.0 and 37 °C. The acid phosphatases presented the following apparent K m values: AP1 ( p NPP — 0.49, PPi — 0.21 and TyrP — 1.14 mM); AP2 ( p NPP — 0.38, PPi — 1.33 and TyrP — 1.14 mM); AP3A ( p NPP -0.20, PPi — 0.16 and TyrP — 0.19 mM) and AP3B ( p NPP — 0.086, PPi — 0.17 and TyrP — 0.17 mM). All four isoforms were inhibited by inorganic phosphate, fluoride, vanadate, molybdate, Cu 2+ and Zn 2+ . The soybean seed acid phosphatases did not catalyze the transphosphorylation reaction since no stimulation was observed with inorganic phosphate acceptors, such as glycerol, methanol and ethanol.

Bovine kidney low molecular weight acid phosphatase: FMN‐dependent kinetics

A low molecular weight bovine kidney acid phosphatase, electrophoretically homogeneous and with a relative molecular mass of 17.8 kDa, was used in this work. Among the various substrates tested, FMN was found to be the most effective, at pH 7.0. Distinct activation energy values were obtained for p‐nitrophenyl phosphate‐ (45.44 kJ mol‐1) and flavin mononucleotide‐ (28.60 kJ mol‐1) hydrolysis reactions. The FMN hydrolysis was strongly inhibited by Cu2+ and pCMB, but activated by guanosine. Pyridoxal‐phosphate and vanadate were competitive inhibitors for the FMN‐dependent reaction.

Effect of homologous series of n-alkyl sulfates and n-alkyl trimethylammonium bromides on low molecular mass protein tyrosine phosphatase activity

The effect of anionic and cationic surfactants on acid phosphatase denaturation has been extensively studied. Low molecular mass (LMr) protein tyrosine phosphatase (PTP), a key regulatory enzyme involved in many different processes in the cell, was distinctly affected by anionic (homologous series of n-alkyl sulfates (C8-C14)) and cationic (n-alkyl trimethylammonium bromides (C12-C16)) surfactants. At concentrations 10-fold lower critical micellar concentration (cmc) values, the enzyme was completely inactivated in the presence of anionic surfactants, in a process independent of the pH, and dependent on the chain length of the surfactants. Under the same conditions, the effect of cationic surfactants on the enzyme activity was pH-dependent and only at pH 7.0 full inactivation was observed at concentrations 10-fold higher cmc values. In contrast to cationic surfactants the effect of anionic surfactants on the enzyme activity was irreversible and was not affected by the presence of NaCl. Inorganic phosphate, a known competitive inhibitor of PTP, protected the enzyme against inactivation by the surfactants. Our results suggest that the inactivation of the LMr PTP by anionic and cationic surfactants involved both electrostatic and hydrophobic interactions, and that the interactions enzyme-surfactants probably occurred at or near the active site. (Mol Cell Biochem 265: 133–140, 2004)

Kinetic Characterization of Bovine Lung Low-Molecular-Weight Protein Tyrosine Phosphatase

Protein tyrosine phosphatase is an important class of enzymes that plays an essential role in the cellular proliferation, differentiation, and oncogenesis. In this paper we report characterization of a low-molecular-weight protein tyrosine phosphatase purified from bovine lung. The enzyme activity was essentially independent of metal ions and sensitive to sulfhydryl reagents. Both vanadate and inorganic phosphate are competitive inhibitors, with Ki values of 0.38 microM and 0.28 mM, respectively. Besides p-nitrophenyl phosphate, the enzyme was also able to efficiently hydrolyze tyrosine phosphate, beta-naphthyl phosphate, and flavine mononucleotide.

In Vivo Evaluation of Strontium-Containing Nanostructured Carbonated Hydroxyapatite

Bone tissue is a composite material that has hydroxyapatite (HA) as its main inorganic phase component. The biological apatites have low crystallinity and contain cationic and anionic substitutions in their structure, which differ from the available synthetic ceramics. The purpose of this study is to evaluate the biocompatibility of nanostructured carbonated hydroxyapatite microspheres containing 5 wt% strontium (SrcHA) compared with the biocompatibility of carbonated hydroxyapatite (cHA), both synthesized at 37°C and non-sintered, used to control stoichiometric HA microspheres in subcutaneous tissue of mice. The biomaterials (BM) were characterized using X-ray Diffraction (XRD), Vibrational Spectroscopy in an Infrared Fourier Transform (VSIRFT) and Scanning Electron Microscopy (SEM). Forty five balb-C mice were randomly divided into four groups of 15 animals each: SrcHA, cHA, HA, and without material implantation (Sham group). All samples were histologically processed for descriptive evaluation of the biological effect. At each experimental period (1, 3 and 9 weeks), there was a higher biosorption of the tested biomaterials observed in contrast with the HA. The cHA group was the only group completely phagocytosed by macrophages and giant cells after 9 weeks. All biomaterials proved to be biocompatible, and the cHA and SrcHA 3% groups exhibited a faster bioabsorption in comparison with the control group. The doping of strontium did not cause a greater biological response after the 3 experimental periods.

Evaluation of Cytocompatibility of Bioglass-Niobium Granules with Human Primary Osteoblasts: A Multiparametric Approach

The pursuit for an ideal bone substitute remains the main focus of many tissue engineering researchers. Among the myriad types of grafts available, synthetic bone grafts are of special importance, because it is available in large amounts, reduce the surgical trauma and eliminate the risk of diseases’ transmission. In this context, bioactive glasses have received attention mostly due to its described biocompatibility and rapid rate of surface reactivity when compared with other materials, allowing for faster interactions with the local tissue. The addition of niobium to this material has been shown as increasing the chemical resistance of the compound and providing greater stability. However, alterations on the chemical composition of biomaterials may impact on its biocompatibility. Therefore, the aim of this study was to evaluate the in vitro biocompatibility of bioglass-Niobium (BgNb) granules, in comparison with standard commercial bioglass (Biogran®) throughout an interesting multiparametrical approach, employing Phenol 2% and dense polystyrene beads as positive and negative controls, respectively. Extracts from each material were prepared by 24 hours incubation in culture medium (DMEM). Human primary osteoblasts were then exposed for 24 hours to each extract and cell viability was evaluated by three parameters: mitochondrial activity (XTT method), membrane integrity (neutral red dye uptake) and cell density (crystal violet dye exclusion test). BgNb extracts were highly compatible, since the levels of viable cells were similar to the control group (unexposed cells), on all parameters studied. The mean cell density on the Biogran® group was slightly lower than BgNb, even though this material was also non-cytotoxic. The excellent in vitro response for BgNb granules indicates the suitability of this material to future studies on its biological and physical properties when applied in vivo.

Radiopacidade de novos biomateriais usados em cirurgia parendodôntica

Resumen pt: Objetivo: Comparar a radiopacidade dos materiais retrobturadores: Cimento Portland (CP), Super EBA (SEBA), MTA-Angelus com Sulfato de Bario (MTA-SB), MTA...

In Vivo and In Vitro Biocompatibility Study of Nanostructured Carbonate-Apatite

The objective of this study was to investigate the in vitro and in vivo biological responses to carbonate apatite (cHA) in comparison to hydroxyapatite (HA). Spheres (400500 μm) of both materials were synthesized under 5°C (cHA) and 90°C (HA) and not sintered. The in vitro cytocompatibility was determined by the XTT assay, according to ISO 10993-5:2009, after exposure of MC3T3-E1 cells to the materials extracts. Ethics Commission on Teaching and Research in Animals approved this project (CEPA/NAL 193/10) and, subsequently, the biomaterials were grafted in the subcutaneous tissues of mice (n=15). After 1 and 3 weeks, five animals of each group were killed for samples removal containing biomaterials and surrounding tissues for histological examination. Semi-serial (5-μm thick) sections were cut and stained with Hematoxylin and Eosin (HE) and the presence of inflammatory infiltrates and biomaterials resorption were evaluated. The experimental group of 3 weeks didn’t show the presence of spheres of both biomaterials and few spheres were observed after 1 week. Histological analysis showed the granulation tissue around the biomaterials with the presence of multinucleated giant cells. After 3 weeks it was observed the presence of fibrous tissue around biomaterials and few inflammatory cells. No signals of tissue necrosis were observed in both groups in all experimental studied periods. Nanostructured carbonate apatite spheres are cytocompatible, biocompatible and present initial biosorption on the subcutaneous comparable to stoichiometric HA, indicating its suitability for further studies on regenerative medicine.

Cytocompatibility and Structural Arrangement of the Collagen Fibers: An In Vitro and In Vivo Evaluation of 5% Zinc Containing Hydroxyapatite Granules

Hydroxyapatite (HA) is one of the most employed materials for bone therapy due to its structural similarity with bone, its biocompatibility and physicochemical properties. Additionally, HA performance may be improved by ionic substitution of calcium with divalent bioactive metallic cations such as zinc. In this context, zinc incorporation into HA have been well studied, in spite of conflicting results regarding its biocompatibility: while previous reports on in vitro cytocompatibility have described 5% zinc containing HA (ZnHA) as slightly cytotoxic, this material presented an excellent response on in vivo studies. In order to bring more information on ZnHA biocompatibility, we performed a multiparametric assay evaluating sequentially on the same cells three different viability parameters: mitochondrial activity (XTT), membrane integrity (Neutral Red) and cell density (Crystal Violet Dye Exclusion test). Additionally, we intended to complement the existing data on ZnHA in vivo performance, by assessing its ability to affect the arrangement of collagen fibers on the grafted area, an important indicative of bone maturation. MC3T3-E1 cells were exposed to 24-hours extracts of ZnHA or stoichiometric HA on culture medium (DMEM) and cell viability was assayed. ZnHA was very cytocompatible, since the levels of viable cells on all 3 tests were similar to the HA and polystyrene (negative control) extracts, but significantly higher than cells treated with 4% phenol (positive control). For the in vivo studies, critical size defects in rats calvaria were filled with HA or ZnHA granules. The histological evaluation after 30 and 180 days revealed an increase along time. Event tough ZnHA is cytocompatible the presence of Zn was unable to alter the interaction between collagen fibers and the mineral bone phase, as compared to stoichiometric HA.

Biocompatibility of Carbonated Hydroxyapatite Nanoparticles with Different Crystallinities

Carbonated apatite (CHA) is commonly considered a promising synthetic material for biomedical applications in orthopedic and dental surgery due to its biocompatibility, bioresorption and bioactivity. CHA5, CHA37 and CHA90 powders were synthesized from wet method and the DRX patterns showed that the crystallinity and particle size of CHA samples increased proportionally with the synthesis temperature. Powder extracts medium were obtained from each sample to interact with MC3T3-E1 osteoblastics cells. It was evaluated morphology, citotoxicity, pH and Ca2+ concentration. Citotoxicity assays showed high metabolic activity on all samples when compared to control. The polygonal shaped and the confluent monolayer observed in control cells progressively changed according to the crystallinity increase of samples. Cells under mitosis and spindle-like shaped where the main alterations observed. In addition the cell viability could be sensitive to the acid reactivity and crystallinity of carbonated apatite samples.