Papel das proteases na erosão dentinária / The role of proteases on dental erosion

Na dentina, a matriz orgânica desmineralizada tem um papel protetor contra desafios erosivos subsequentes. Porém, essa camada pode ser degradada por proteases, como as metaloproteinases da matriz (MMPs) e cisteína catepsinas (CCs). Recentemente, o uso de inibidores de proteases da matriz surgiu como uma importante ferramenta preventiva contra a erosão dentinária. Entretanto, o(s) mecanismo(s) exato(s) pelo(s) qual(is) os inibidores de proteases podem prevenir a erosão dentinária, bem como os tipos de proteases mais envolvidas neste processo ainda não são completamente conhecidos. O projeto foi desenvolvido em 2 subprojetos, com os seguintes objetivos: A)Subprojeto 1:Avaliar o papel das proteases na progressão da erosão dentária; B)subprojeto 2: Testar o potencial inibitório do NaF em CCs dentinárias. Para cumprir esses objetivos, foram utilizadas dentina de terceiros molares humanos para a preparação dos espécimes. A)Subprojeto1:Blocos de dentina (4 X 4 x 2 mm) (n=119) foram obtidos de raízes. Os espécimes foram divididos em 7 grupos de acordo com o seu tratamento (E-64, inibidor especifico II de catepsinas B, clorexidina, galardina NaF, placebo) ou sem tratamento, géis foram aplicados uma única vez sobre a superfície e feito o desafio erosivo (90s, 4x por dia por 5 dias) e feita analise perfilométrica. Os espécimes foram incubadas em solução contendo colagenase de Clostridium histolyticum tipo VII por 96hrs e então feita uma segunda analise perfilometrica para se determinar a espessura da MOD. Dois espécimes foram separados para análise de microscopia eletrônica de varredura. B)Subprojeto 2: Palitos de dentina (6 mm X 2 mm X 1 mm) (n=60) foram cortados da porção médio coronária dos dentes e completamente desmineralizados por imersão em EDTA 0,5 M (pH7,4) por 30 dias e lavados em água deionizada sob constante agitação a 4ºC por 72 h. Os espécimes foram divididos em 6 grupos (E-64, NaF e controle negativo, pH 5,5 ou 7,2) e incubados em saliva artificial contendo seus respectivos inibidores por 24 h 7 dias e 21 dias; ao termino de cada período, os espécimes eram pesados para avaliar a perda de massa e analisada a presença de CTX. A)Subprojeto 1: a perda de tecido desmineralizado (m, média± SD) foi: CHX 8,4±1,7b, Gala 8,6±1,9b, IECB 9,6±1,4a, E64 9,9±1,3a, NaF 9,9±1,7a, P 10,9±2,2a, ST 11,0±1,5a. A perda de tecido mineralizado foi: CHX 15,4±2,2b, Gala 16,0±1,8b, IECB 17,6±2,4a, E64 17,6±2,0a, NaF 17,3±2,8a, P 19,1±2,1a, ST 18,9±2,4a. Os inibidores de MMP reduziu significativamente a perda de matriz orgânica e tecido mineralizado em comparação com os outros grupos (p<0,05). Não foi achada diferença significante entre a espessura da matriz orgânica desmineralizada remanescente (p=0,845). B)Subprojeto 2: Na perda de massa houve diferença significante em relação ao inibidor (F=20,047, p<0,0001) e tempo de incubação (F=222,462, p<0,0001) com significante interação entre esses critérios, nos período de menor tempo de incubação, a perda foi similar para todos os grupos testados, no período de maior tempo de incubação, o grupo contendo NaF demostrou os melhores resultados. Na analise de CTX, houve diferença significante em relação aos inibidores (F46,543, p<0,0001), pH (F=14,836, p<0,0004) e tempo de incubação (F=161,438, p<0,0001) com significante interação entre esses critérios, como ocorrido na perda de massa, não houve diferença estatística nos períodos de menor incubação. No período de maior tempo de incubação, mais uma vez o grupo NaF mostrou os melhores resultados. No valor acumulado de CTX, os grupos E64 e controle negativo tiveram os maiores valores de CTX acumulado, o grupo NaF, independente do pH mostrou redução significante em relação aos demais grupos. Após analise dos resultados dos dois subprojetos, podemos indicar que as MMPs são as proteases de maior importância na progressão da erosão dentinária, assim, sua inibição é de maior importância para a redução desta patologia. Mesmo as CCs não exercendo papel direto para a progressão da erosão, elas são efetivas na cascata da ativação de outras proteases, como as próprias MMPs. Com isso, sua inibição também pode ser importante para a redução indireta da progressão da erosão. Neste presente estudo, pudemos comprovar que o NaF tem potencial inibitório sobre as CCs dentinárias, assim, sugerindo um novo inbidor de CCs. Com os resultados deste estudo, podemos afirmar que as MMPs são as principais proteases na progressão da erosão dentinária e que o NaF tem potencial inibitório nas CCs dentinárias.(AU)

In the dentine, the demineralized organic matrix has a protector part against the following erosive challenges. Nevertheless, this layer can be degraded by proteases, like the matrix metalloproteinases (MMPS) and cystein cathepsins (CCs). Recently, the use of proteases of the matrix´s inhibitors, emerged as an important preventive tool against the dentinária erosion. However, the exact mechanisms from which the inhibitors of the proteases may prevent the dentin erosion, as much as the kinds of proteases more involved in this process are not completely known yet. Therefore, the general objective of this project was to investigate the part of the two main proteases of the matrix (MMPs and CCs) in the dental erosion. The project was developed in 2 subprojects, with the following objectives: A)Subproject 1: Evaluate the part of the proteases in the progression of the dental erosion; B)subproject 2: To test the NaF inhibitory potencial in the dentin CCs. To accomplish these objectives, human third molar dentin were used for the preparation of the specimens, obtained in the surgery and urgency clinics of FOB-USP (subproject 1) or granted by the University of Oulu (subproject 2). A) Subproject 1: Dentine blocks 4 X 4 X 2 mm) (n=119) were obtained from the roots of the obtained teeth. The specimens were divided in 7 groups according with their treatment. Gels containing inhibitors (E-64, specific cathepsin B inhibitor II, chlorhexidine, galardin NaF, placebo), or without treatment, were produced, applied only one time over the surface and made the erosive challenge (90s, 4x a day for 5 days) and made profilometric analysis. The specimens were incubated in a solution containing collagenase of Clostridium histolyticum type VII for 96 hours and then a second profilometric analysis was made to determine the thickness of the MOD. Two specimens were separated for the electronic microscopy scan analysis. B) Subproject 2: Dentine sticks (6 mm X 2 mm X 1 mm) (n=60) were cut from the medium coronary portion of the teeth and completely demineralized by immersion in EDTA 0,5 M (pH7,4) ifor 30 days and washed in deionized water under constant agitation in 4º C for 72 hours. The specimens were divided in 6 groups (divided by inhibitors: E-64, NaF and negative control, pH 5,5 or 7,2) and incubated in artificial saliva containing their respective inhibitors for 24 hours, 7 days and 21 days; by the end of each period, the specimens were weighted to evaluate the loss of mass and analised the presence of CTX. A)Subproject 1: the loss of demineralized tissue (m, média± SD) was : CHX 8,4±1,7b, Gala 8,6±1,9b, IECB 9,6±1,4a, E64 9,9±1,3a, NaF 9,9±1,7a, P 10,9±2,2a, ST 11,0±1,5a. The loss of demineralized tissue was: CHX 15,4±2,2b, Gala 16,0±1,8b, IECB 17,6±2,4a, E64 17,6±2,0a, NaF 17,3±2,8a, P 19,1±2,1a, ST 18,9±2,4a. The MMP inhibitors reduced significantly the loss of organic matrix and demineralized tissue in comparison with other groups (p<0,05). There was no significant difference found between the thickness of the remaining demineralized organic matrix.(p=0,845). B)Subproject: In the loss of mass, there was a significant difference in relation to the inhibitor (F=20,047, p<0,0001) and incubation time (F=222,462, p<0,0001) with significant interaction between these criteria, in the periods of lesser time of incubation, the loss was similar for all the tested groups, in the period of higher time of incubation, the group containing NaF demonstrated the best results. In the analysis of CTX, there was significant difference in relation the inhibitors (F46,543, p<0,0001), pH (F=14,836, p<0,0004) and time of incubation (F=161,438, p<0,0001)with significant interaction between these criteria, as occurred in the mass loss, there was no statistic difference in the period of lesser incubation. In the period of higher time of incubation, once again, the NaF group demonstrated the best results. The CTX accumulated value, the E64 groups and negative control had the greater accumulated values of CTX, the NaF group, regardlessof the pH, demonstrated significant reduction in relation to the other groups. After the analysisof the results of both subprojects, we can indicate that the MMPs are the proteases of greater importance in the progression of the dentin erosion, thus, its inhibition is of graeter importance for the reduction of this pathology. Even the CCs don´t playing the part directly for the progression of erosion, they are effective in the cascade of the activation of other proteases, like the MMPs themselves. In this manner, its inhibition can also be important for the indirect reduction of the progression of the erosion. In this present study, we can prove that the NaF has inhibiting potential over the dentin CCs, thus, suggesting a new inhibitor of CCs. With the results of this study, we can affirm that the MMPs are the main proteases in the progression of the dentin erosion and that the NaF has inhibiting potential in the dentin CCs.(AU)

Microarray Analysis of Differentially Expressed Genes between Cysts and Trophozoites of Acanthamoeba castellanii

Acanthamoeba infection is difficult to treat because of the resistance property of Acanthamoeba cyst against the host immune system, diverse antibiotics, and therapeutic agents. To identify encystation mediating factors of Acanthamoeba, we compared the transcription profile between cysts and trophozoites using microarray analysis. The DNA chip was composed of 12,544 genes based on expressed sequence tag (EST) from an Acanthamoeba ESTs database (DB) constructed in our laboratory, genetic information of Acanthamoeba from TBest DB, and all of Acanthamoeba related genes registered in the NCBI. Microarray analysis indicated that 701 genes showed higher expression than 2 folds in cysts than in trophozoites, and 859 genes were less expressed in cysts than in trophozoites. The results of real-time PCR analysis of randomly selected 9 genes of which expression was increased during cyst formation were coincided well with the microarray results. Eukaryotic orthologous groups (KOG) analysis showed an increment in T article (signal transduction mechanisms) and O article (posttranslational modification, protein turnover, and chaperones) whereas significant decrement of C article (energy production and conversion) during cyst formation. Especially, cystein proteinases showed high expression changes (282 folds) with significant increases in real-time PCR, suggesting a pivotal role of this proteinase in the cyst formation of Acanthamoeba. The present study provides important clues for the identification and characterization of encystation mediating factors of Acanthamoeba.

Quantitative determination of S-allyl-L-cysteine in extraction of garlic by LC-MS / 复旦学报（医学版）

Objective To develop a liquid chromatography-mass spectrometry (LC-MS) method for the determination of S-allyl-L-cystein in extraction of garlic. Methods A LC-MS method was established on a ZORBAX Eclipse XDB-C8(4.6 mm×250 mm, 5 μm)column with the mobile phase consisting of 1‰ formic acid water-methanol (95:5), flow rate of 0.8 mL/min and detection with post-column splitting. The split ratio was 2:1, column temperature was set at 25 ℃. The mass spectrometer equipped with ESI and the ion source was operated in negative mode. The dry gas flow was 10.0 L/min, the nebulizer pressure was 30 psig, and the vaporizer temperature was 350 ℃. SIM detector, S-allyl-L-cystein m/z 160 and S-allyl-L-cystein sulfoxide m/z 176. Results The calibration curves showed good linearity in the range of 0.062 5-2 μg/mL of S-allyl-L-cystein. The detection limit was 0.01 μg/mL. The within-day RSD was 4.11% and the day-to-day RSD was 4.49%.The average recovery for S-allyl-L-cystein was 101.63%. Conclusions The method is simple and accurate. It is adapted to determine and analyze S-allyl-L-cystein in extraction of garlic and the average content of S-allyl-L-cystein is 0.514 μg/mg.

Las proteínas alergénicas: un novedoso blanco para el desarrollo de estudios en proteomica funcional / The allergenic proteins: a novel target for the studies' development in functional proteomica

En la patogénesis de las enfermedades alérgicas están involucrados el ambiente, la carga genética y la inmunocompetencia del individuo. Continuamente nuestro sistema inmune está expuesto a numerosas proteínas, sin embargo, solo unas pocas inducen una respuesta inmune alérgica. El potencial intrínsico de una proteína alergénica para inducir sensibilización solo se manifiesta en individuos susceptibles, genéticamente condicionados a presentar respuestas atópicas. Muchas de estas proteínas alergénicas comparten alguna homología en su secuencia de aminoácidos. Estos alérgenos poseen un amplio rango de características moleculares, ninguna de las cuales es única solo para estas proteínas alergénicas. A pesar de esto, algunas de estas características son más comunes entre algunos alérgenos que con otras proteínas. Se ha demostrado que algunas proteínas con actividad enzimática inducen reacciones alérgicas y que esta propiedad biológica está asociada con su actividad catalítica. En la presente revisión se describen las principales características moleculares de las proteínas alergénicas, y se hace énfasis en la cistein proteasas de los ácaros intradomiciliarios, en razón a que ellas son un factor de riesgo en el desarrollo de una respuesta inmune alérgica en individuos susceptibles y se constituyen en factores desencadenantes de respuestas inflamatorias en la fisiopatología de las enfermedades alérgicas respiratorias...

The components involved in the pathogenesis of disorder allergic include the environment, the genetics and the immune competence. Continuously we are exposed to thousands of different proteins, but a single few of them induce an allergic immune response. The unique potential of an allergic protein to induce sensitization, only produce a response in susceptible individuals. Many of those allergic proteins share some homology in their sequence of amino acids, nevertheless, allergens have an ample rank of characteristic, no of which is unique for allergenic proteins. In spite of this, some characteristics are more common between allergens than in other proteins. It has been demonstrated that some proteins with enzymatic activity have a predisposition to induce allergic reactions and that its biological characteristics is associated with its catalytic activity. Those characteristic could be contribute directly to the allergenicity of this protein. In the present review we presented/displayed the main intrinsic characteristics of allergenic proteins associated with an allergic immune response, making emphasis in their Properties as activity cystein protease how and this biological faculty determining a factor in the development of the inflammatory cascade as an allergic immune response in susceptible individuals...

Antioxidant Effects of Sulfur-Containing Amino Acids

Sulfur is an essential element for the entire biological kingdom because of its incorporation into amino acids, proteins and other biomolecules. Sulfur atoms are also important in the iron-containing flavoenzymes. Unlike humans, plants can use inorganic sulfur to synthesize sulfur-containing amino acids. Therefore, plants are an important source of sulfur for humans. Sulfur-containing compounds are found in all body cells and are indispensable for life. Some of sulfur-containing antioxidant compounds are, cysteine, methionine, taurine, glutathione, lipoic acid, mercaptopropionylglycine, N-acetylcysteine, and the three major organosulfur compounds of garlic oil, diallylsulfide, diallyldisulfide and diallyltrisulfide. In a comparison of the structure-function relationship among these sulfur-containing antioxidant compounds, dihydrolipoic acid (the reduced form of LA) is the most effective antioxidant. Dihydrolipoic acid contains two sulfhydryl groups and can undergo further oxidation reaction to form lipoic acid. The antioxidative activities of sulfur-containing compounds follow a general trend, the more highly reduced forms are stronger antioxidants and the number of sulfur atoms determine, at least in part, their modulatory activites on the glutathione related antioxidant enzymes. In this article, the antioxidant effects and the antioxidative activities, of sulfur-containing amino acids, are reviewed. In addition, the general antioxidant effects and the structure-function relationship of some sulfur-containing compounds are also reviewed.

The Effects of Prostaglandin E1 and N-Acetyl-L-Cysteine for Ischemic Renal Reperfusion Injury in the Rat / 대한이식학회지

BACKGROUND: Some studies has showed that even though reperfusion is essential for the recovery of renal function, it, itself, might be caused of renal injury through liberation of oxygen free radical due to surplus oxygen following reperfusion. If it is so, it can be presumed that oxygen free radical scavenger during reperfusion might be useful drugs to protect renal injury during reperfusion. On the base of this hypothesis, we selected prostaglandin E1 (PGE1) which is vasodilator and cellular protector which act through decreased demand of ATP and N-acetyl-L-cystein (NAC) which is oxygen radical scavenger for the purpose to see oxygen free radical scavenger related drugs will work on protection from renal injury during reperfusion. MATERIALS AND METHODS: To define the efficacy of these drugs, It was evaluated by a serial check of the serum creatinine level, and pathologic change on the light microscopy. And also to investigate the role of HSP 70 and HSP 72 on reperfusion injury, immunohistochemical study and Western blot analysis were performed on the experiment rats. The experimental ischemic renal model was made by clamping hilum of both kidney for 60 minutes on Wistar male rats. After declamping, PGE1, NAC and saline through a tail vein: PGE1 35 microgram/kg/hr, NAC 6 mM/kg/hr and 0.9% saline 1 ml/kg/hr were infused for 60 minutes and then blood samples were obtained from IVC at 1st hour, 2nd hour, 1st day hour, 2nd day and 5th day after reprefusion for creatinine. RESULTS: The serum creatinine was normalized in group receiving PGE1 at 5th day after reperfusion. But creatinine was not normalized in group receiving saline and NAC at 5th day after reperfusion: serum creatinine at 5th day after reperfusion were 0.58 0.08 mg/dl in group receiving PGE1, 2.85 0.65 mg/dl in group receiving saline and 2.96 0.51 mg/dl in group receiving NAC. There was expression of HSP 72 in group receiving PGE1 at 5th day after reperfusion. But not in group receiving NAC and saline at 5th day after reperfusion. It was noted to have prominent expression of HSP 70 in renal cortex in group receiving PGE1 at 2nd and 5th day. On contrary, there were prominent expression of HSP 70 in renal cortex in group receiving NAC at 1st and 2nd hour. CONCLUSION: It is clear that PGE1 has a protective effect for ischemic renal reperfusion injury by showing early regeneration of renal tubule, persistent expression of HSP 72 and HSP 70 and normalization of serum creatinine at 5th day after reperfusion.

EFFECTS OF Cu~(2+) ON ISOLATED SUPERIOR CERVICAL SYMPATHETIC GANGLION OF RABBITS / 中国药理学通报

The effects of Cu2+ on superior cervical sympathetic ganglion of rabbits were studied by the sucrose-gap method. Cu2+ at cumula- tive concentration inhibited ganglion action potentials. IC50 of Cu2+ was 0.26 mmol/L. The effects of Cu2+ were antagonized by neostig-miae and L-cystein, while enhanced under 1/2[Ca2+] and reduced under 3[Ca2+] .Cu2+ inhibited ganglionic N wave and P wave,without affecting LN wave.

EFFECTS OF Zn~(2+) ON ISOLATED SUPERIOR CERVICAL SYMPATHETIC GANGLION OF RABBITS / 西安交通大学学报(医学版)

The effects of Zn~(2+) on the isolated superior cervical sympathetic ganglion of rabbit were stud ied with sucrese-gap method. It was found thatZn~(2+) significantly inhibited ganglienic action poten-tial with a manner of concentration-dependence.The IC-50 value was 1. 48 mmol/L. The effects ofZn~(2+) were antagonized by neostigmine and 1-cys-tein, reduced under high concentration of Ca~(2+) andenhanced under low concentration of Ca~(2+). It wasalso found that Zn~(2+) inhibited ganglionic N wave,less affected p wave and without any effect on LNwave.

EFFECTS OF Zn~(2+) ON THE NEUROMUSCULAR TRANSMISSION / 西安交通大学学报(医学版)

Experiments were carried out on the isolatedrat phrenic nerve diaphragm preparations and chickbiventer cervicis preparations. Zn~(2+) at cumulativeconcentration inhibited muscle contractile responseto indirect stimulation without affecting the con-ductivity of nerve. The inhibiting. action of Zn~(2+) onthe muscle contractile response to indirect stimula-tion was stronger than that on contractile responseto direct stimulation. Zn~(2+) did not affect the Achsensitivity of the chick biventer cervicis muscleThe effects of Zn~(2+) on neuromuscular transmis-sion were antagonized by Ca~(2+) and L-cystein.