Short-term evaluation of non-absorbable microgranular hydroxyapatite infiltration in the guinea pig subepidermal abdominal region

Non-absorbable microgranular hydroxyapatite was infiltrated into the subepidermal abdominal region of guinea pigs in order to assess the possibility of using this material to correct deficiencies in orbital volume. Microgranular hydroxyapatite (2.0 ml) was subepidermally infiltrated into the abdominal region of 20 guinea pigs. The animals were divided into four experimental groups of 5 animals each, which were killed 7 (G1), 15 (G2), 30 (G3) and 60 (G4) days after infiltration. The area and the largest and smallest diameters of the nodules formed by infiltration were evaluated at the site of infiltration and histological examination was performed. The mean granuloma area was similar in all groups. Histopathological examination showed that the material remained isolated from surrounding tissues by a pseudocapsule that became denser throughout the experiment. A host reaction started with young fibroblastic tissue that evolved to dense tissue until cartilaginous tissue was formed in G4, progressively advancing towards the center of the granuloma from G1 to G4. Non-absorbable microgranular hydroxyapatite is an inert material that was well tolerated by the animals studied, with maintenance of the infiltrated volume, and may perhaps be useful to fill anophthalmic cavities

Short-term evaluation of non-absorbable microgranular hydroxyapatite infiltration in the guinea pig subepidermal abdominal region.

Non-absorbable microgranular hydroxyapatite was infiltrated into the subepidermal abdominal region of guinea pigs in order to assess the possibility of using this material to correct deficiencies in orbital volume. Microgranular hydroxyapatite (2.0 ml) was subepidermally infiltrated into the abdominal region of 20 guinea pigs. The animals were divided into four experimental groups of 5 animals each, which were killed 7 (G1), 15 (G2), 30 (G3) and 60 (G4) days after infiltration. The area and the largest and smallest diameters of the nodules formed by infiltration were evaluated at the site of infiltration and histological examination was performed. The mean granuloma area was similar in all groups. Histopathological examination showed that the material remained isolated from surrounding tissues by a pseudocapsule that became denser throughout the experiment. A host reaction started with young fibroblastic tissue that evolved to dense tissue until cartilaginous tissue was formed in G4, progressively advancing towards the center of the granuloma from G1 to G4. Non-absorbable microgranular hydroxyapatite is an inert material that was well tolerated by the animals studied, with maintenance of the infiltrated volume, and may perhaps be useful to fill anophthalmic cavities.

[Effect of sucrose-containing gum and fluoridated dentifrice on in situ remineralization of artificial caries]. / Efeito da goma de mascar contendo sacarose e do dentifrício fluoretado na remineralização in situ de lesões de cárie artificiais.

The aim of this study was to evaluate the remineralization of incipient carious lesions in bovine enamel in situ. Artificial carious lesions were produced and fixed in removable lower appliances in the region of the lingual surfaces of first molars, in six volunteers with ages between 18 and 22 years, who were subjected to 3 distinct experimental periods of 1 week each. In the first period (control group), patients brushed their teeth with a non-fluoridated dentifrice 4 times a day (after meals), and, in the second period (group I), patients used a dentifrice containing 1,500 ppm of fluorine (in the form of MFP). In the third period (group II) volunteers brushed their teeth with non-fluoridated dentifrice and used chewing gum containing 60% of sucrose during 20 minutes, 4 times a day (after meals). Before and after each treatment, the specimens underwent Vicker's hardness test (200 g of load), and the remineralization percentage (alpha) was calculated. The control group showed 2.78% of demineralization, and groups I and II showed 3.36 and 5.21% of remineralization, respectively. Statistical analysis (with Kruskal-Wallis and Miller's tests) showed significant difference (p < 0.05) between the control and experimental groups (I and II). Group II showed greater alpha than group I, but this difference was not significant. These results suggest that the use of sucrose-containing chewing gum and fluoridated dentifrice has a considerable effect on the remineralization of incipient carious lesions and may be a valuable alternative for their prevention.

Endogenous lectin as a possible regulator of the hydrolysis of physiological substrates by soybean seed acid phosphatase.

The effects of two lectins concanavalin A (conA) and soybean agglutinin, on soybean seed acid phosphatase activity were investigated using p-nitrophenylphosphate (pNPP), pyrophosphate (PPi) and phosphoenolpyruvate (PEP) as substrates. Of the four acid phosphatase isoforms (AP1, AP2, AP3A and AP3B) purified from soybean seeds, only AP1 was activated 40 and 60% by conA and soybean agglutinin, respectively. Both lectins affected some of the kinetic parameters of AP1. The activation by lectins was not affected by 1 mM Ca2+ or Mn2+ but glucose and methylmannopyranoside (100 mM) prevented activation by conA. Under the same conditions, galactose had no effect. These results suggest that plant acid phosphatases may be regulated by lectins, the effects vary according to the substrate used.

Effectiveness of surface protection of resin modified glass ionomer cements evaluated spectrophotometrically.

The effectiveness of four surface protectors for resin-modified glass ionomer cements was evaluated by spectrophotometrically determining dye uptake. Ninety specimens, 3.0 mm in diameter and 1.0 mm in height, were made with Photac-Fil, Fuji II LC and Vitremer and divided into six groups for each material. Positive and negative controls were not protected while experimental specimens were protected with proprietary glaze, nail varnish, flowable resin and glaze. The discs were immersed in 0.1% methylene blue solution for 10 minutes after mixing, except for those negative control specimens that were immersed in deionized-water. After 24 hours, the specimens were washed and the protectors trimmed with Sof-Lex discs. The specimens were then removed from the matrixes and individually placed in 1.5 mL of 65% nitric acid for five hours. The absorbance was determined spectrophotometrically at 590 nm. Dye uptake was expressed in microgram dye/specimen. The data were analyzed by two-way ANOVA and Tukey-Kramer tests. All surface protectors tested were effective. For Fuji II LC and Vitremer no differences were observed among tested protections. For Photac-Fil, nail varnish showed better performance than the proprietary glaze.

Rabbit bone marrow response to bovine osteoinductive proteins and anorganic bovine bone.

The effects caused by the implantation of bioabsorbable hydroxyapatite (HA) bound to a pool of bone morphogenetic proteins (BMPs) and other bone noncollagenous hydrophobic proteins mixed with anorganic bovine bone inside rabbit bone marrow were assessed. Within the interior of hollow cylindric titanium prototypes, the following biomaterials were inserted: (1) test group: HA containing a pool of BMPs and noncollagenous hydrophobic proteins mixed with anorganic bovine bone; (2) control group: HA without any protein mixed with anorganic bovine bone; and (3) negative control group: blood clot. The cylinders were placed surgically into the medial portion of the tibiae of 7 rabbits in a manner that allowed the biomaterials to contact just the bone marrow. Morphometric analysis showed that: (1) the biomaterials containing the protein mixture resulted in significantly less new bone than the biomaterials without such a mixture; (2) the group without the protein pool formed larger amounts of bone within the cylinder when compared to the negative control (blood clot only); and (3) the biomaterials containing the protein pool did not show any difference in relation to the negative control. It was concluded that a pool of BMPs and other bone noncollagenous hydrophobic proteins had an inhibitory effect on osteogenesis, and that the biomaterials without a protein pool formed a favorable substrate to bone formation.

Inhibition of acid phosphatase isoforms purified from mature soybean (Glycine max) seeds.

The four soybean seed acid phosphatase isoforms AP1, AP2, AP3A and AP3B were competitively inhibited by phosphate, vanadate, fluoride and molybdate, using p-nitrophenylphosphate as substrate. The four isoforms were not significantly affected by compounds that can interact with SH residues or by pyridoxal phosphate. These results indicated that cysteine and lysine residues are not present in the active site of the four soybean seed acid phosphatase isoforms. The inhibition constant values for phosphate, vanadate, fluoride and molybdate at pH 5.0 were respectively: API (250, 12.8, 1.7, 0.05 microM). AP2 (800, 10, 500, 0.025 microM), AP3A (250, 24.2,250, 0.032 microM ), AP3B (2400 36.9, 750, 0.05 microM).

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.

Soybean seed acid phosphatases: unusual optimum temperature and thermal stability studies.

In contrast to other acid phosphatases, four cytoplasmic isoforms (AP1, AP2, AP3A, and AP3B) purified from mature soybean seeds presented high activities at temperatures above 80 degrees C, when p-nitrophenylphosphate (p-NPP) was utilized as substrate. However, with tyrosine phosphate and inorganic pyrophosphate as substrates, maximum activities were observed at temperature of 60 degrees C during 10 min reaction. In the absence of substrate, enzymes lost only 20% activity after 60 min at 60 degrees C; the isoforms AP3A and AP3B retained 30% of activity at 70 degrees C after 60 min and all the isoforms were inactivated at 80 degrees C, after 5 min. Thermal inactivation studies indicated that the soybean enzymes showed different temperature dependences in relation to most plant acid phosphatases. A best protective effect was observed when the isoforms were preincubated, at 70 degrees C, with phosphate (10 mM) and p-nitrophenol (10 mM) which indicates that the enzyme inactivation was prevented only in the presence of both reaction products.

Repair of the anophthalmic cavity of rats with synthetic hydroxyapatite

We placed spheres of synthetic hydroxyapatite (calcium chloride combined with sodium phosphate) in the eviscerated or enucleated orbital cavity of rats in order to evaluate the biocompatibility of this material with the orbital cavity. The study was conducted on 50 albino rats, 25 of which were submitted to enucleation and 25 to evisceration of one eye. The animals were sacrificed 7, 15, 21, 30 and 60 days after surgery and the orbital content was submitted to histopathological examination. A reaction of the young granulation tissue type was observed first. The hydroxyapatite was gradually surrounded by a granulomatous macrophage inflammatory response and covered with dense connective tissue that formed a sort of "mesh" septating and supporting progressively smaller blocks of the substance. The same type of reaction was observed in the enucleated and eviscerated cavities. We conclude that synthetic hydroxyapatite is an inert nonallergenic material which is appropriate for volume replacement in the anophthalmic cavity.

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.

Purification and characterization of a low-molecular-weight bovine kidney acid phosphatase.

A relative low molecular mass bovine kidney acid phosphatase was purified 1,640-fold to homogeneity, with 7% recovery. The purified enzyme (specific activity 100 mumol min-1 mg-1) was electrophoretically homogeneous with a relative molecular mass of 17.8 kDa, as determined by SDS-polyacrylamide gel electrophoresis. A broad pH optimum of 4.0-5.5 and a maximal enzyme activity at 60 degrees C were determined for the p-nitrophenyl phosphate hydrolysis. Apparent Km values of 0.14 mM, 0.4 mM, 0.3 mM and 7.9 mM were obtained, at 37 degrees C and pH 5.0, for the best substrates p-nitrophenyl phosphate, beta-naphtyl-phosphate, flavin mononucleotide and tyrosine-phosphate, respectively. The enzyme activity was enhanced by guanosine but inhibited by ZnCl2 and CuSO4, p-cloromercuribenzoate and ammonium molybdate. Vanadate (Ki 0.47 microM), pyridoxal 5'-phosphate (Ki 2.2 microM), inorganic phosphate (Ki 0.77 microM) are competitive inhibitors. Both glycerol and methanol increased significantly the acid phosphatase activity, acting as good phosphate acceptors in the transphosphorylation reaction.

Repair of the anophthalmic cavity of rats with synthetic hydroxyapatite.

We placed spheres of synthetic hydroxyapatite (calcium chloride combined with sodium phosphate) in the eviscerated or enucleated orbital cavity of rats in order to evaluate the biocompatibility of this material with the orbital cavity. The study was conducted on 50 albino rats, 25 of which were submitted to enucleation and 25 to evisceration of one eye. The animals were sacrificed 7, 15, 21, 30 and 60 days after surgery and the orbital content was submitted to histopathological examination. A reaction of the young granulation tissue type was observed first. The hydroxyapatite was gradually surrounded by a granulomatous macrophage inflammatory response and covered with dense connective tissue that formed a sort of "mesh" septating and supporting progressively smaller blocks of the substance. The same type of reaction was observed in the enucleated and eviscerated cavities. We conclude that synthetic hydroxyapatite is an inert nonallergenic material which is appropriate for volume replacement in the anophthalmic cavity.

Effect of nail varnishes and petroleum jelly combinations on glass ionomer dye uptake.

PURPOSE: To evaluate the effectiveness of different brands of nail varnish alone or associated with petroleum jelly as surface protectors for glass ionomer cements by determining dye uptake spectrophotometrically. MATERIALS AND METHODS: Three hundred thirty six specimens, 3.0 mm in diameter and 1.0 mm thick, were made with Chelon-Fil (CF) and ChemFil II (CII) and divided into 14 groups for each material. Positive control (A) and negative control (B) specimens were not protected, while experimental specimens were protected with six brands of nail varnish used with and without petroleum jelly. The specimens were immersed in 0.05% methylene blue solution 10 minutes after mixing except for negative control specimens that were immersed in deionized water. RESULTS: Dye uptake (microgram dye/restoration) for CF was: A = 11.3 +/- 3.1; B = 0.0 +/- 0.0 with varnish groups ranging from 0.6 to 2.5 and for CII: A = 12.4 +/- 2.5; B = 0.0 +/- 0.0 with varnish groups ranging from 0.4 to 2.4. The data were analyzed by ANOVA. The dye uptake among the groups was not significantly different (P < 0.01), except for the control group (unprotected cements).

[Effect of final washing in a small volume of water on processing of periapical radiographs]. / Influência da lavagem final em pequeno volume de água, no processamento de radiografias periapicais.

The main goal this research was to find out the effectiveness of the final wash in periapical X-ray films. Spectrophotometry determined the residues of sodium hyposulphite taken as a positive sign for the effectiveness of wash on developed films. Three different kinds of film, two Kodak and one Agfa were used. Films were different sensibility and underwent tests. Two containers, one for the intermediate and other for the final wash were used in the first test; in the second, only one container was used for both washes. A residue of 0.4 mg of sodium hyposulphite was considered acceptable. On the three films used, the acorent of sodium hyposulphite detected was inferior to 0.4 mg in the first test, after 5 minutes exposure, nevertheless the small volume of water used. The second test showed a with range of variation in results and so it not recommend.

Threonyl-tRNA synthetase from bovine liver. Purification and kinetic properties in the ATP-pyrophosphate exchange reaction.

Threonyl-tRNA synthetase (E.C. 6.1.1.3) from bovine liver has been purified to near homogeneity approximately 500 fold with a recovery of 48%. Two bands of molecular weight 90,000 and 82,000 respectively, were obtained by sodium dodecyl sulfate gel electrophoresis. The enzyme has an isoelectric point of 5.2 by polyacrylamide containing ampholine gel electrophoresis. Optimum assay conditions and apparent Km values have been determined in the ATP-PPi exchange reaction. Effects of divalent cations and diamine in substitution to Mg2+ and effects of sulfhydryl reagents on ATP-PPi exchange activity have also been observed.

Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond.

A peptide N-glycosidase that catalyzes the hydrolysis of N-linked oligosaccharide chains from glycopeptides and glycoproteins has been purified to homogeneity from almond emulsin and from almond meal. Purification from almond emulsin using ion-exchange chromatography, gel filtration chromatography, and preparative polyacrylamide gel electrophoresis gave an enzyme which was purified more than 700-fold and with a yield of 63%. An alternative procedure, more suitable for efficient large scale purification, used ion-exchange, affinity, and gel filtration chromatography. When purification began with almond emulsin, the enzyme was purified 1200-fold with a 37% yield, while when purification began with almond powder, the enzyme was purified 9000-fold with a yield of 45%. The homogeneous enzyme is stable at 4 degrees C for several months in 10 mM sodium acetate, pH 5.0, buffer. The peptide N-glycosidase is itself shown to be a glycoprotein consisting of a single polypeptide chain with a molecular weight of 66 800 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Circular dichroism spectra of the native molecule indicate the presence of a high (approximately 80%) alpha-helix content. The amino acid and carbohydrate contents of the enzyme are presented. When a convenient new assay with a turkey ovomucoid glycopeptide as a substrate is used, the enzyme preparation exhibits a broad pH optimum centered between pH 4 and pH 6. The enzyme is readily inactivated by SDS and guanidine hydrochloride, but it is stable in the presence of moderate concentrations of several other protein denaturants.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the structural basis of the heterogeneity of human prostatic and seminal acid phosphatases.

We studied possible causes of the electrophoretic heterogeneity of the acid phosphatase (EC 3.1.3.2) purified by affinity chromatography from human prostate and human seminal fluid. The isoelectric focusing pattern in polyacrylamide gel shows numerous bands in the pH range 4.0-5.2 and 5.5-5.9. Treatment with neuraminidase under conditions shown to cause complete removal of sialic acid does not abolish the observed heterogeneity. Although there is a change of the more acidic forms to ones having more basic pI values, at least 4 distinct bands remain. Structural differences at the amino terminal end can be ruled out as the cause of the remaining electrophoretic heterogeneity. Lysine is shown to be the amino terminal amino acid for both the prostatic and seminal fluid enzymes. The sequences of the first 23 amino acids are shown to be identical for the prostatic and seminal fluid acid phosphatases. The functional enzyme contains no metal ion but it can be stoichiometrically inactivated by cupric ion.