Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR

OBJECTIVE: The aim of this study was to evaluate, by PCR-RFLP and Real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature. MATERIAL AND METHODS: A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by DNA extraction. The concentration, purity and integrity of the DNA were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 A/G (rs 4444903) polymorphism by PCR-RFLP and for IRF6 polymorphism (rs 17015215) using Real-Time PCR. RESULTS: There was no significant difference of DNA yield (p=0.75) and purity (p=0.86) among the three different incubation times. DNA obtained from different incubation times presented high-molecular weight. The PCR-RFLP and Real time PCR reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by Real-Time PCR presented C allele for IRF6 gene polymorphism (homozygous: CC; heterozygous: CT) and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques. CONCLUSION: We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and PCR amplified after storage in mouthwash solution at room temperature

Purification and kinetic proper ties of a castor bean seed acid phosphatase containing sulfhydryl groups

An acid phosphatase (EC 3.1.3.2) has been identified and purified from castor bean (Ricinus communis L., IAC-80) seed through sulphopropyl (SP)-Sephadex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A-Sepharose chromatography. The enzyme was purified 2000-fold to homogeneity, with a final specific activity of 3.8 mu kat mg(-1) protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was found to be 60 kDa, The acid phosphatase had a pH optimum of 5.5 and an apparent K-m value for p-nitrophenylphosphate of 0.52 mM, The enzyme-catalyzed reaction was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p-chloromercuribenzoate (pCMB), Cu2+ and Zn2+, The strong inhibition by pCMB, Cu2+ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate and inorganic pyrophosphate (PPi) as substrate. The highest specificity constant (V-max/K-m) was observed with PPi, making it a potential physiological substrate.107215115

Purification and characterization of multiple forms of soybean seed acid phosphatases

Four isoforms of acid phosphatase (EC 3.1.3.2), API, 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 API (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 API, 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, tyrosine-phosphate and inorganic pyrophosphate, at pH 5.0 and 37 degrees C. The acid phosphatases presented the following apparent K-m values: API (pNPP - 0.49, PPi - 0.21 and TyrP - 1.14 mM); AP2 (pNPP - 0.38, PPi - 1.33 and TyrP - 1.14 mM); AP3A (pNPP1 - 0.20, PPi - 0.16 and TyrP - 0.19 mM) and AP3B (pNPP - 0.086, PPI 0.17 and TyrP 0.17 mM). All four isoforms were inhibited by inorganic phosphate, fluoride, vanadate, molybdate, Cu2+ and Zn2+. The soybean seed acid phosphatases did not catalyze the transphosphorylation reaction since no stimulation was observed with inorganic phosphate accepters, such as glycerol, methanol and ethanol. (C) Elsevier, Paris.36748749

Inhibition of bovine kidney low molecular mass phosphotyrosine protein phosphatase by uric acid

Uric acid inhibited 50% of the activity of bovine kidney low molecular mass phosphotyrosine protein phosphatase at concentrations of 1.0, 0.4, 1.3, and 0.2 mM, respectively for p -nitrophenyl phosphate (p -NPP), flavine mononucleotide, beta-naphthyl phosphate and tyrosine phosphate (Tyr-P) as substrates. The mixed type inhibition of p -NPP hydrolysis was fully reversible, with K-ic and K-iu values of 0.4 and 1.1 mM, respectively; the inhibition by uric acid shifted the pH optimum from 5.0 to 6.5. When Tyr-P was the substrate, competitive inhibition was observed with a K-i value of 0.05 mM. Inhibition studies by uric acid in the presence of thiol compounds, and preincubation studies in the presence of inorganic phosphate suggest that the interaction of uric acid with the enzyme occurred at the active site, but did not involve SH residues, and that the mechanism of inhibition depended on the structure of the substrates.17534535

Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei

Procyclic forms of Trypanosoma brucei possess a phosphatase activity on their external cell surface. This activity, while it dephosphorylates [P-32]phosphocasein, is inhibited weakly by NaF and tartrate but strongly by vanadate. In this work, we describe the presence of an external phosphatase activity in intact bloodstream forms of T brucei. With p-nitrophenyl phosphate (pNPP) as substrate, these intact cells produced 3-5 nmol pNP min(-1) mg(-1), linearly for up to at least 30 min. The activity was not significantly increased by Mg2+, Mn2+, Ca2+ and Co2+, but was inhibited by vanadate, NaF, p-chloromercuribenzoate and Zn2+ and was insensitive to okadaic acid. Membrane-enriched fractions of parasites contained an acid phosphatase activity, with a pH optimum in the range of 4.5-5.5. This activity hydrolyzed phosphotyrosine (40 nmol phosphate min(-1) mg(-1)) better than phosphothreonine or phosphoserine. Partial purification of this phosphatase yielded a single activity band following gel electrophoresis, a K-m value of 0.29 mM with pNPP and was insensitive to the Fe2+/H2O2/ascorbate system. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.220219720

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 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.4161201120

Use of chemically modified silica with beta-diketoamine groups for separation of alpha-lactoalbumin from bovine milk whey by affinity chromatography

Silica gel surface was chemically modified with beta-diketoamine groups by reacting the silanol from the silica surface with 3-aminopropyl-triethoxysilane and 3-bromopentanedione, With this material, copper ions were adsorbed from aqueous solutions, The chemical analysis of the silica-gel-immobilized acetylacetone provided a quantity of 0.67 mmol g(-1) of organic groups attached to the support and 0.63 mmol g(-1) of copper, This material was used as a stationary phase in IMAC (immobilized metal affinity chromatography), to separate alpha-lactoalbumin from bovine milk whey, The results showed an efficient separation in the chromatographic column, The possibility of reutilization of the stationary phase was also investigated. (C) 1997 Academic Press185231331

Kinetic characterization of bovine lung low-molecular-weight protein tyrosine phosphatase

Protein tyrosine phosphatase is an important Glass 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 mu M 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.24326927

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 (C-8-C-14)) and cationic (n-alkyl trimethylammonium bromides (C-12-C-16)) 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.2654167113314

Análise histológica, radiográfica e do perfil de imunoglobulinas após implantação de enxerto de osso esponjoso bovino desmineralizado em bloco em músculo de ratos Histologic, radiographic and imunoglobuline profile analysis after implantation blocks of demineralized bovine cancellous bone graft in muscle of rats

O objetivo deste trabalho foi avaliar a biocompatibilidade de blocos de enxerto de osso bovino esponjoso acelular e desmineralizado (Gen-Ox®, Baumer S.A.). Um bloco cilíndrico (5x12mm) de material de enxerto foi implantado em músculo abdutor da coxa de 30 ratos, sendo os animais sacrificados 3, 7, 14, 21 e 28 dias (n=6) após as cirurgias. Após a tomada das radiografias, as peças foram removidas para o processamento histológico. A análise histológica mostrou que nos períodos de 3 e 7 dias foi evidenciado um processo inflamatório agudo, caracterizado pela presença de neutrófilos, reabsorção do coágulo sanguíneo e angiogênese. Entre 14 e 21 dias, verificou-se a reabsorção da matriz implantada por células mononucleadas, raras células gigantes e sua substituição por tecido conjuntivo fibroso rico em vasos e células. Aos 28 dias, na maioria dos casos, observou-se apenas pequenos fragmentos de matriz implantada envolto por tecido conjuntivo característico da região. Radiograficamente, não se notou evidências de mineralização. Com base nos resultados obtidos concluímos que o enxerto de matriz de osso esponjoso bovino desmineralizado em bloco é biocompatível quando implantado em tecido conjuntivo intramuscular de ratos, sendo absorvido e substituído por tecido conjuntivo característico da região, sem qualquer indício de ocorrência de osteogênese ectópica.<br>The aim of this study was to evaluate the biocompatibility of blacks of organic bovine cancellous bone graft material (Gen-OxTM, Baumer) in ectopic sites. A cylinder of black of graft material measuring 5mm in diameter and 12mm in length was implanted in abdutor muscle of 30 Wistar rats. After 3, 7, 14, 21e 28 days, (6 animals/period) were killed, radiographies were taken and the tissues and blood were collected to histological and imunoglobuline G and M profile analysis, respectively. The analysis of the sections revelead an acute inflammatory process at 3 and 7 days, characterized by the presence of neutrophils. Absorption of the implanted matrix by mononuclear cells and scarce giant cells and their replacement by fibrous connective tissue rich in vessels and cells, was suggested. At 28 days, in most cases just remnants of the implanted matrix involved by the typical connective tissue of the perimisium were found. There was no radiographic evidence of mineral content or change in the IgG and IgM profile. On the basis in results described here, it could be concluded that the material is biocompatible and absorbable but, with no signs of osteoindutive capacity was observed