Phosphoric acid containing proanthocyanidin enhances bond stability of resin/dentin interface

Abstract Proanthocyanidin (PA) is a promising dentin biomodifier due to its ability to stabilize collagen fibrils against degradation by matrix metalloproteinases (MMPs); however, the most effective protocol to incorporate PA into bonding procedures is still unclear. This study evaluated the effect of dentin biomodification with a PA acid etchant on MMP activity, adhesive interface morphology and resin-dentin microtensile bond strength. Sound extracted human molars were flattened to expose dentin and acid-etched for 15 s according to the groups: EXP - experimental phosphoric acid; EXP+PA - experimental phosphoric acid 10% PA; TE - total-etching system; SE - self-etching system. Samples were restored with composite resin and stored in distilled water (37ºC). MMP activity and interface morphology were analyzed after 24 h by in situ zymography (n=6) and scanning electron microscopy (n=3), respectively. The resin-dentin microtensile bond strength (μTBS) was evaluated after 24 h and 6 months storage (n=6). Significantly higher MMP activity was detected in etched dentin compared with untreated dentin (p<0.05), but no difference among acid groups was found. Resin tags and microtags, indicative of proper adhesive system penetration in dentinal tubules and microtubules, were observed along the hybrid layer in all groups. There was no difference in μTBS between 24 h and 6 months for EXP+PA; moreover, it showed higher long-term μTBS compared with TE and EXP (p<0.05). The results suggest that 15 s of biomodification was not sufficient to significantly reduce MMP activity; nonetheless, EXP+PA was still able to improve resin-dentin bond stability compared with total- and self-etching commercial systems.

Resumo A proantocianidina (PA) é um biomodificador dentinário promissor devido a sua capacidade de estabilizar as fibrilas colágenas contra a degradação por metaloproteinases da matriz (MMPs); no entanto, o protocolo mais eficaz para a incorporação de PA em procedimentos adesivos ainda não está claro. Este estudo avaliou o efeito da biomodificação da dentina com um condicionador ácido contendo PA na atividade de MMPs, morfologia da interface adesiva e resistência à microtração resina-dentina. Molares humanos extraídos foram lixados para exposição da dentina e condicionados com ácido por 15 s de acordo com os grupos: EXP - ácido fosfórico experimental; EXP+PA - ácido fosfórico experimental com 10% PA; TE - sistema total-etch; SE - sistema self-etch. As amostras foram restauradas com resina composta e armazenadas em água destilada (37ºC). A atividade de MMP e morfologia da interface foram analisadas após 24 h por zimografia in situ (n=6) e microscopia eletrônica de varredura (n=3), respectivamente. A resistência à microtração resina-dentina (μTBS) foi avaliada após 24 horas e 6 meses de armazenamento (n=6). Atividade de MMP detectada na dentina condicionada foi significativamente maior em comparação com a dentina não tratada (p <0,05), mas não houve diferenças entre os diferentes ácidos. Tags e microtags de resina, indicativos de uma penetração adequada do sistema adesivo nos túbulos e microtúbulos dentinários, foram observadas ao longo da camada híbrida em todos os grupos. Não houve diferença entre os valores de μTBS de 24 h e 6 meses para EXP+PA; além disso, EXP+PA apresentou maiores valores de μTBS após 6 meses em comparação com TE e EXP (p <0,05). Os resultados sugerem que a biomodificação por 15 s não foi suficiente para reduzir significativamente a atividade de MMP; apesar disso, EXP + PA foi capaz de melhorar a estabilidade da interface resina-dentina em comparação com sistemas total- e self-etch comerciais.

Extraction, Partial Purification and Characterization of Bromelain from Pineapple (Ananas Comosus) Crown, Core and Peel Waste

Abstract Ananas Comosus (also known as pineapple) is a part of Bromeliaceae family and it is consumed as food as well as folk medicine for the treatment of various diseases. It is reported that pineapple is a rich source of bromelain, a cysteine protease and it is considered as an important enzyme in different industries due to its significant therapeutic and industrial applications such as anticancer, anti-inflammatory and meat tenderizing. Bromelain is mostly present in fruit and stem of pineapple, but it is reported that crown, core, and peels, which constitute the waste of the pineapple plant, also contain bromelain but limited data is available. Therefore, the proposed study aimed at utilizing pineapple waste for the extraction and characterization of bromelain. Firstly, crude bromelain was extracted with phosphate buffer (pH 7), then it was subjected to partial purification using different fractions of ammonium sulphate (NH4)2SO4 such as 30, 40, 50 and 60% followed by desalting and concentration. Enzyme activity was calculated by using casein digesting unit (CDU) method. The results demonstrated that the crown bromelain showed highest purification of 4.34-fold at 30% (NH4)2SO4 saturation, whereas core and peel bromelain showed highest purification of 2.75 and 2.59-fold at 40% (NH4)2SO4 saturation. The molecular weight of crude and partially purified bromelain was determined by SDS-PAGE analysis and found to be 26 KDa. The pH and thermal stability of all the parts of pineapple showed maximum stability at pH 7 and at 35oC temperature.

Comparison of Single-Step Methods to Enrich Lipase Concentrations in Bacterial Cell Lysates

Abstract Lipases are currently used in food technology for the modification of fats and oils. The thermal stability of lipase is an essential characteristic for this application. This study compares four individual single-step methods (heat treatment, ethanol precipitation, ammonium sulfate precipitation, and size-exclusion chromatography) to enrich lipase concentrations from thermophilic bacterial (Geobacillius stearothermophilus and Anoxybacillus flavithermus) cell lysates. SDS-PAGE and size exclusion chromatography were used to determine the molecular weights of the lipases and the enrichment efficiencies were determined using specific enzyme activities. The molecular weight of G. stearothermophilus lipase was approximately 42 kDa, and approximately 33 kDa for A. flavithermus lipase. For each organism, ethanol precipitation resulted in the highest enrichment fold, followed by ammonium sulfate precipitation, gel filtration and heat treatment respectively. The highest yields for G. stearothermophilus lipase were obtained with ammonium sulfate precipitation, followed by get filtration, and ethanol precipitation respectively. The highest yields for A. flavithermus lipase were obtained from heat treatment followed by ammonium sulfate precipitation, gel filtration and ethanol precipitation respectively. Ethanol precipitation and heat treatment are simple methods for enzyme enrichment from cell lysates and can result in high enzyme yields with moderate enrichment folds compared to complex multi-step purification methods.

Matrix metalloproteinases 2 and 9 in rabbits with doxorubicin-induced cardiomyopathy / Metaloproteinases de matriz 2 e 9 em coelhos com cardiomiopatia induzida pela doxorrubicina

Some studies have shown the role played by matrix metalloproteinases and their inhibitors in doxorubicin cardiotoxicity. In this study, we sought to investigate how plasma and myocardial MMP 2 and 9 perform in rabbits with doxorubicin-induced cardiomyopathy, searching for a correlation between the activity of these collagenases and cardiac remodeling. Cardiomyopathy was induced by doxorubicin given intravenously twice a week for six consecutive weeks. Plasma MMP activity and the echocardiogram were assessed at baseline, and at 15 and 45 days after first injection of doxorubicin. The myocardial activity of these enzymes was solely evaluated in nine rabbits at 45 days, and results were compared with nine healthy controls. We only identified the full-length forms of both MMP 2 and 9 throughout the study. The plasma pro-MMP 2 reduced along the deterioration of cardiac function, while the pro-MMP 9 increased significantly at T45 as compared to baseline and T15. A negative significant correlation was found to exist between the plasma activity of pro-MMP 2 and mitral E-to-mitral septal annular early diastolic velocity ratio, which is an estimate of mean left atrial pressure and congestion. Only pro-MMP 2 was found in myocardial samples, and mean activity of such enzyme was statistically lower than that recorded for healthy controls. Although no active form was documented for either collagenase, the duration of the treatment with doxorubicin played a role in the alteration of plasma pro-forms activity. However, these changes could not be associated with most echocardiographic parameters that are supportive of cardiac remodeling.(AU)

Alguns estudos já demonstraram o papel exercido pelas metaloproteinases de matriz e seus inibidores na cardiotoxicidade promovida pela doxorrubicina. Assim, este estudo teve como objetivo investigar o comportamento das MMPs 2 e 9 plasmáticas e miocárdicas em coelhos com cardiomiopatia induzida pela doxorrubicina, buscando determinar se há correlação entre a atividade dessas colagenases e o remodelamento cardíaco. A cardiomiopatia foi induzida pela doxorrubicina aplicada por via intravenosa duas vezes por semana ao longo de seis semanas consecutivas. A atividade plasmática das MMPs e o ecocardiograma foram avaliados no momento basal e aos 15 e 45 dias após a primeira aplicação da doxorrubicina. A atividade miocárdica dessas enzimas foi quantificada em apenas nove coelhos aos 45 dias e os resultados comparados com outros nove controles saudáveis. Foram identificadas apenas as formas inativas das MMPs 2 e 9 durante todo o estudo. A pro-MMP 2 plasmática reduziu à medida que a função cardiaca se deteriorou, enquanto a pro-MMP 9 aumentou significativamente em T45 quando comparada aos momentos basal e T15. Houve correlação negativa significativa entre a atividade plasmática da pro-MMP 2 e a relação entre E mitral e a velocidade anular mitral no início da diástole, um parâmetro que permite estimar a pressão atrial esquerda média e a congestão. Apenas a pro-MMP 2 foi documentada nas amostras miocárdicas dos coelhos com cardiomiopatia e atividade media dessa enzima foi estatisticamente menor que aquela observada nos controles saudáveis. Embora a forma ativa de ambas as colagenases não tenha sido identificada, o tempo de tratamento com doxorrubicina interferiu na atividade das formas inativas plasmáticas. Contudo, essas alterações não se associaram com a maioria dos parâmetros ecocardiográficos que indicam remodelamento cardíaco.(AU)

Bee venom induces apoptosis and suppresses matrix metaloprotease-2 expression in human glioblastoma cells

Abstract Glioblastoma is the most common malignant brain tumor representing with poor prognosis, therapy resistance and high metastasis rate. Increased expression and activity of matrix metalloproteinase-2, a member of matrix metalloproteinase family proteins, has been reported in many cancers including glioblastoma. Inhibition of matrix metalloproteinase-2 expression has resulted in reduced aggression of glioblastoma tumors in several reports. In the present study, we evaluated effect of bee venom on expression and activity of matrix metalloproteinase-2 as well as potential toxicity and apoptogenic properties of bee venom on glioblastoma cells. Human A172 glioblastoma cells were treated with increasing concentrations of bee venom. Then, cell viability, apoptosis, matrix metalloproteinase-2 expression, and matrix metalloproteinase-2 activity were measured using MMT assay, propidium iodide staining, real time-PCR, and zymography, respectively. The IC50 value of bee venom was 28.5 µg/ml in which it leads to decrease of cell viability and induction of apoptosis. Incubation with bee venom also decreased the expression of matrix metalloproteinase-2 in this cell line (p < 0.05). In zymography, there was a reverse correlation between bee venom concentration and total matrix metalloproteinase-2 activity. Induction of apoptosis as well as inhibition of matrix metalloproteinase-2 activity and expression can be suggested as molecular mechanisms involved in cytotoxic and antimetastatic effects of bee venom against glioblastoma cells.

Generation of antibodies against disintegrin and cysteine-rich domains by DNA immunization: An approach to neutralize snake venom-induced haemorrhage

Objective To explore whether a DNA immunization approach targeting the major haemorrhage molecule of a prothrombin activator-like metalloproteinase from Echis ocellatus (E. ocellatus) venom could be conceived to inspire antibodies with more prominent specificity and equal adequacy to current conventional antivenoms systems. Methods The isolated DNA EoMP-6 was used as the template for PCR amplification using the EoDC-2-specific forward and reverse primers. A PCR product of approximately 700 bp was obtained and cloned into pSecTag-B expression vector where anti-EoDC-2 antibodies were generated and analysed for their efficacy to neutralise local haemorrhage in vitro and in vivo. Results Our results suggest that the generated anti-EoDC-2 showed a remarkable efficacy by (a) interfering with the interaction of the recombinant disintegrin “EoDC-2” isolated from the E. ocellatus as well as other viper species to the α

Evaluation of matrix metalloproteinase and cysteine cathepsin activity in dentin hybrid layer by gelatin zymography.

Aim: The aim of this study was to comparatively assess the gelatinolytic activity of matrix metalloproteinases(MMPs) and Cysteine Cathepsins (CCs) in the adhesive interface using etch and rinse adhesive at different time intervals using zymographic technique. Methodology: Twenty freshly extracted non-carious human third molars were used in this study. Occlusal surfaces were ground flat and 1mm thick horizontal dentin slabs were obtained from each tooth using a diamond disc. The dentin surface was polished with 600-grit silicon-carbide paper. Five out of 20 samples were directly pulverized. In the remaining fifteen samples, the dentin was etched and adhesive was applied and light cured according to the manufacturer’s instructions. A 1mm thick flowable composite was build up and light cured. Bonded specimens were cut vertically into 3 to 4 dentin slabs by means of diamond disc to expose the adhesive/ dentin interfaces. These were then ground down to 500 μm thick resin-dentin interface using a hard tissue microtome. These sections were then pulverised into powder. Following this, every five samples were subjected to zymographic analysis after 1 day, 7 days and 21 days. Results: Zymograms showed clear, thicker bands on all three isoforms in the etched samples compared to control samples at 1st and 7th day intervals and became inactive at 21st day for all three isoforms. MMP 9 activity was relatively higher when compared to CCs and MMP 2. Conclusion: Etch and rinse adhesive activated MMPs and CCs within the hybrid layer that remained active till 7th day and no gelatinolytic activity was found on 21st day and MMPs are more active compared to CCs and MMP-2.

Diagnóstico clínico en saliva. Revisión

El odontólogo está llamado a convertirse en un elemento importante en el desarrollo de estudios de fenotipado de poblaciones para impulsar la aplicación de la medicina personalizada y la razón se debe a la saliva, que además de resultar un fluido imprescindible para la conservación de las funciones orales, se perfila con cualidades importantes para el diagnóstico clínico con base genética. Con el desarrollo de potentes técnicas de laboratorio, como el inmunoensayo, electroforesis, fluorescencia, cromatografía (HPLC y CG), espectrometría de masa (EM), la reacción en cadena de la polimerasa (PCR) y pruebas genéticas de fenotipado, se ha podido correlacionar la presencia de ciertos biomarcadores de daño tisular en la saliva con los niveles de esas especies en sangre. Estos biomarcadores constituyen señales del daño tisular o de respuestas del organismo a esas injurias, cuando aun no se pueden observar las evidencias clínicas del mismo. Esta aplicación de la saliva le confiere una importancia especial en lo referente al diagnóstico clínico. Al estudio de los biomarcadores orales se ha unido, de forma más reciente, el desarrollo de protocolos para la obtención de ADN genómico de la saliva que permite, por la viabilidad en la colección de muestras, su conservación y facilidades en su traslado, realizar estudios poblacionales para conocer la funcionalidad de ciertos genes que se relacionan con la biotransformación de los medicamentos, una causa importante de las variaciones interindividuales a los tratamientos farmacológicos. El conocimiento de polimorfismos en genes que expresan las enzimas que metabolizan fármacos específicos, permite realizar cambios en los principios activos o ajustes en las dosis de tratamientos individuales, que es el objetivo de la medicina personalizada o farmacogenética. Estos estudios también permiten conocer la predisposición genética de poblaciones al desarrollo de ciertas enfermedades, tanto orales como sistémicas, lo que propicia el establecimiento de nuevas políticas en la profilaxis y medicina preventiva. El uso de la saliva con estos fines abre nuevas perspectivas en la atención odontológica, y requiere de esfuerzos interdisciplinarios y cooperación en el equipo de salud.

The dentist is called to become an important element in the development of studies phenotyping of populations to advance the implementation of personalized medicine and the reason is because saliva, which besides being a prerequisite for the preservation of oral functions fluid , is emerging with important qualities for clinical diagnosis with genetic basis. With the development of powerful laboratory techniques, such as immunoassay, electrophoresis, fluorescence, chromatography (HPLC and GC), mass spectrometry (MS), polymerase chain reaction (PCR) and genetic testing phenotyping, it has been correlate the presence of certain biomarkers of tissue damage in saliva levels of these species in blood. These biomarkers are signs of tissue damage or agency responses to these injuries, when not even be seen clinical evidence of it. This application saliva attaches special importance with regard to clinical diagnosis. The study of oral biomarkers has joined, more recently, the development of protocols for obtaining genomic DNA from saliva that allows for viability in sample collection, preservation of the same and facilities during transport, conduct population studies to determine the function of certain genes that are related to biotransformation of drugs, a major cause of differences among individuals to drug treatments. Knowledge of polymorphisms in genes that express enzymes that metabolize specific drugs can make changes or adjustments active principles in doses of individual treatments, which is the goal of personalized medicine or pharmacogenetics. These studies also provide insight into the genetic predisposition of populations to the development of certain diseases, both oral and systemic, which favors the establishment of new policies in the prophylaxis and preventive medicine. The use of saliva for this purpose opens new perspectives in care dental and requires interdisciplinary efforts and cooperation in the health team.

Análise comparativa da expressão e atividade das metaloproteinases 2 e 9 e de seus inibidores teciduais nas lesões cutâneas das variantes poiquilodérmica e clássica da micose fungoide / A comparative analysis of the expression and activity of metalloproteinases 2 and 9 and their tissue inhibitors in cutaneous lesions of poikilodermatous and classic variants of mycosis fungoides

Micose fungoide poiquilodérmica (MFp) é uma variante clínica de micose fungoide (MF). É mais indolente e caracterizada pela presença da poiquilodermia. As metaloproteinases (MMP) e seus inibidores específicos TIMP (Tissue Inhibitors of Metaloproteinases) estão envolvidos na oncogênese. Especificamente as MMP2 e MMP9 e seus inibidores, TIMP-2 e TIMP-1, respectivamente, foram relacionados ao prognóstico em tumores. Poucos trabalhos estudaram MMP e nenhum estudou a ação dos TIMP na MF. Objetivos: avaliar a relação entre MMP2 e MMP9 e seus inibidores TIMP2 e TIMP1 e a agressividade da MF e descrever a casuística de micose fungoide poiquilodérmica no ambulatório de linfomas cutâneos da Divisão de Clínica Dermatológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Métodos: análise retrospectiva de 54 casos de MFp, sendo 25 de MFp localizada 14 de MFp generalizada e 15 de MFp mista. Para análise das MMP e TIMP, os grupos de MFp foram comparados com 7 amostras de pele normal (PN), 10 casos de MF clássica inicial (MFi), 9 casos de MF tumoral não-transformada (MFT nt) e 10 de MF tumoral transformada (MFT t). Resultados: A proporção de mulheres: homens foi 2,44. MFp apresentou maior tempo entre os primeiros sintomas e o diagnóstico. MFpG apresentou maior prevalência de lesões do tipo pitiríase liquenoide crônica (PLC) (79%). Houve alta prevalência de MF hipocromiante (62%) no grupo MFp mista. A histologia da MFp apresentou características típicas de MF e, adicionalmente, atrofia, telangectasias e derrame pigmentar, específicos da forma poiquilodérmica. Na imuno-histoquímica predominou o fenótipo CD3+, CD4+, CD7-, CD8- em todos os grupos, e MFp apresentou significantemente menor predomínio do fenótipo CD8+ que o grupo MFi. O grupo MFpG apresentou baixa positividade para pesquisa de clonalidade T da pele (12,5%). A MMP2 esteve mais presente na epiderme em MFi e MFp relativamente a MFT. Na derme superficial, os grupos MFi e MFp...

Poikilodermatous mycosis fungoides (pMF) is a clinical variant of mycosis fungoides (MF). It is more indolent than classic MF and is characterized by the presence of poikiloderma. The matrix metalloproteinases (MMPs) and their specific inhibitors TIMP (Tissue Inhibitors of Metalloproteinases) are involved in oncogenesis. Specifically, MMP2 and MMP9 and their inhibitors, TIMP-2 and TIMP-1, respectively, have been related to prognosis in tumors. There are few studies on MMP and none on the role of TIMPs in MF. Objectives: To evaluate if there is a relationship between the presence and activity of MMP2 and MMP9 and their inhibitors TIMP2 and TIMP1, and the aggressiveness of MF. To describe a casuistic of poikilodermatous mycosis fungoides in an outpatient clinic in the Dermatological Division of Hospital das Clinicas of University of Sao Paulo Medical School. Methods: Retrospective analysis of 54 cases of pMF, this included 25 localized pMF (LpMF), 14 generalized pMF (GpMF) and 15 mixed pMF. For the analysis of MMPs and TIMPs, the pMF groups were compared with 7 normal skin samples (NS), 10 cases of initial classical MF (cMF), 9 cases of non-transformed tumor MF (nt MFT) and 10 transformed tumor MF (t MFT). Results: The proportion of women : men was 2.44. The pMFs groups showed a longer period of time from the first symptoms to the diagnosis than the cMF group. The GpMF group had a higher incidence of pityriasis lichenoides chronica-like lesions (PLC) (79%) than the other groups. There was a high incidence of hypopigmented MF (62%) in the mixed pMF group. Histology showed typical characteristics of MF and, additionally, atrophy, telangiectasia and pigmentary alterations compatible with pMF. At immunohistochemistry the cases were predominantly CD3+, CD4+, CD7-, CD8- phenotype in all groups, and the pMF groups had a significantly lower prevalence of CD8+ phenotype than the cMF group. The GPMF group showed low positivity for clonality of the T-cell...

Identification Method for Lumbrokinase by Fibrin Zymography / 中国药师

Objective:To establish a fibrin zymography method for identifying the active proteins in lumbrokinase,and investigate the production difference and batch consistency of 5 different manufacturers. Methods:Fibrin zymography was used with the final con-centration of fibrinogen of 5 × 10 - 4 g·ml - 1 ,renature time of 30 min in 2. 5% Triton-x-100,incubation time of 30 min in PBS buffer (pH = 7. 4)at 37℃ and the protein concentration in the sample of 5. 0-37. 5 μg. Results:The sensitivity of the method was high,and the molecular weight distribution of active protein bands for the samples from five manufacturers was between 15KD and 40KD with 6 common active protein bands. The zymography of the samples from the five manufacturers had slight difference,while various batches of the samples from the same manufacturer showed no difference. Conclusion:The method is special. It can reflect molecular weight distribution and species of active protein,batch consistency and production process stability. It is easy to be standardized and suitable for the identification of lumbrokinase,which can lay foundation for the quality consistency evaluation of marketed products.

Partial Purification and Characterization of β-glucosidase from Monascus sanguineus

The aim of the present work was to study the production and characterization of β-glucosidase from Monascus sanguineus. Agro-waste residues were screened to obtain the maximum yield of enzyme. Jack fruit seed was the best substrate for enzyme production. Studies on the optimization of pH and temperature showed acidic pH favorable for enzymatic activity, whereas the optimum temperature was 60°C. Enzyme kinetics studies with different concentration of pNPG showed the calculated value of Km approximately 0.89 mM with the non-linear regression and 0.98 mM with the linear regression techniques. The enzyme was predominantly inhibited by KCl (69.8%) and moderately inhibited by CaCl2 (14.8%). Studies on the sensitivity for glucose showed that after 100 mM concentration of glucose, inhibition in pNPG hydrolysis took place. The molecular weight of the protein was estimated as 116 and 66 kDa with SDS- PAGE and zymography was carried out to verify the specific activity.

Zymographic Detection of Aspartic Proteinase Activities in Porcine Ovarian Extracts.

Aims: Tightly regulated proteolytic activity is essential in the mammalian ovary to maintain follicular and luteal functions. Studies conducted on ovarian aspartic proteinases (APs) are limited. Previously it has been noted that the AP activity increases towards the latter part of luteal phase. The aim of this study was to isolate AP from porcine ovarian extract and to identify whether multiple AP activities are found in the extracts. Place and Duration of Study: Department of Biochemistry, between December 2009 and February 2012. Methodology: Porcine ovaries (n= 100) were collected and ovarian extracts were prepared. APs were fractionated, using anion exchange chromatography at pH 8.5, gel permeation chromatography and affinity chromatography. AP activity (U/ml) of the fractions were measured in the presence and absence of Pepstatin A. AP specific activities (U/mg) were calculated after measuring total protein concentrations (mg/ml) of the fractions. Fractions were analyzed using polyacrylamide gel electrophoresis conducted under denaturing (SDS-PAGE) and native (PAGE) conditions. PAGE was followed by zymography. Results: With anion exchange chromatography, AP was recovered during column washing as an unbound fraction (~67%) and during elution as a bound fraction (~33%). Bound AP was recovered around 0.23 M NaCl with 0-1 M NaCl gradient used during elution. Both AP fractions had an apparent molecular mass of 40 kDa. AP activity was completely inhibited in the presence of 1 μM Pepstatin. Specific activity of AP increased with fractionation from 1 in the ovarian extract to 747 and 511 U/mg with unbound and bound AP respectively. SDS-PAGE showed elimination of impurities with the progress of fractionation. PAGE and zymography showed the presence of at least three AP activities in porcine ovaries. Conclusion: Proteinases fractionated using three chromatography procedures were APs. Results showed the presence of multiple AP activities in porcine ovary.

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatus larvae / Avaliação e purificação parcial dos inibidores da serina protease de larvas do Rhipicephalus (Boophilus) annulatus

Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

Carrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.

Zimography is an effective method for detection of matrix metalloproteinase 2 (MMP-2) activity in cultured human fibroblasts

PURPOSE: To describe a method to characterize the gelatinase activity of cultured human periodontal fibroblasts stimulated with Pam3Cys and E. coli LPS, ligands of TLR2 and TLR4 respectively, and by centrifugation of the cultures, simulating an orthodontic force. METHODS: To study MMP-2 activity, primary cultures of human periodontal fibroblasts were stimulated with the addition of TLRs 2 and 4 ligands and the application of mechanical force by centrifugation at 141 x g for 30 min. Supernatant media was collected 24 hours later to perform protein quantification and zymography. RESULTS: MMP-2 activity suffered an increase in cultures co-stimulated with TLRs 2 and 4 ligands alone or with the presence of mechanical force application compared to basal levels. CONCLUSION: Zymography, one of the several methods to study MMPs activities, is a simple, qualitative and efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate.

Expression of matrix metalloproteases-2 and -9 in horse hoof laminae after intestinal obstruction, with or without Hydrocortisone treatment / Expressão de metaloproteinases 2 e 9 no tecido laminar do casco de equinos após obstrução intestinal e tratamento com hidrocortisona

Twenty horses were used in the experiment, for composed control group, (Cg) instrumented group, (Ig;without intestinal obstruction), treated group (Tg;submitted to intestinal obstruction and hydrocortisone treatment) and non-treated group (Ntg;submitted to intestinal obstruction without treatment). Immunohistochemistry and zymography techniques were used for researches on MMPs 2 and 9 in horse hoof laminae. There was an increase in the expression of MMP-2 in animals of Tg and Ntg. MMP-9 increased on animals from groups Ntg and Ig, however there was no rise of this MMP on the Tg when compared to the other groups in the immunohistochemistry analysis. Based on the results, it was observed that the intestinal injury caused by enterotomy and intestinal obstruction raise the quantities of MMPs in the hoof laminae.

Vinte cavalos foram usados no experimento: para compor o grupo controle (Cg), grupo instrumentado, Ig (sem obstrução intestinal), grupo tratado, Tg (submetidos à obstrução intestinal e tratamento com hidrocortisona) e grupo não tratado, Ntg (submetidos à obstrução intestinal, sem tratamento. Técnicas de zimografia e imunoistoquímica foram utilizadas para pesquisa de MMP-2 e MMP-9 no tecido laminar do casco dos equinos. Houve um aumento na expressão de MMP-2 nos animais dos grupos Tg e Ntg. A MMP-9 aumentou nos animais dos grupos Ig e Ntg. Houve aumento desta MMP no Tg quando comparado aos demais grupos na análise por zimografia. Observou-se que a injúria intestinal, causada pela enterotomia e obstrução intestinal, eleva a quantidade de MMPs no tecido laminar do casco.

Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

Inhibition of Gelatinases by Vegetable Extracts of the Species Tapirira guianensis (Stick Pigeon).

Tapirira guianensis (Stick pigeon), a widely-used herbal medicine, has been reported to possess various biological activities. The aim of this study was the phytochemical analysis of the fractions of extracts of T. guianensis and the investigation of the action of these extracts on the activity of gelatinases using zymography. Matrix metalloproteinases (gelatinases) have prognostic influences in human cancers, where higher expressions of these enzymes are associated with increased aggressiveness and biological behavior of tumors. Many natural products have been tested on several stages of carcinogenesis to demonstrate their effectiveness in the inhibition or activation of molecules that are important for tumor progression. This study identified the fractions obtained from the crude extract of T. guianensis (Stick pigeon), which efficiently inhibited gelatinases.

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4.

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40°C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu­­­2+, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

Ethanol Extract of Bupleurum falcatum Improves Functional Recovery by Inhibiting Matrix Metalloproteinases-2 and -9 Activation and Inflammation after Spinal Cord Injury

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade the extracellular matrix and other extracellular proteins. Upregulation of MMPs activity is known to be required for the inflammatory cell infiltration after spinal cord injury (SCI) and most likely contributes to early blood spinal barrier disruption and inflammation, thereby leading to the impairment of functional recovery. Here, we examined the effect of ethanol extract of Bupleurum falcatum (BF) on functional recovery by inhibiting MMP-2 and -9 activation and inflammation after SCI. Rats received a moderate, weight-drop contusion injury to spinal cord were administered orally with BF at a dose of 100 mg/kg for 14 d and functional recovery was measured by Basso-Beattie-Bresnahan locomotor open field behavioral rating test, inclined plane test and foot print analysis. To examine the neuroprotective effect of BF, TUNEL staining and counting were also performed. In addition, the expression and/or activation of MMP-2, MMP-9 and inflammatory mediators such as TNF-alpha, IL-1beta, COX-2, and iNOS were examined by RT-PCR and gelatin zymography using spinal cord tissue from 1 d after injury. Our data showed that BF significantly inhibited the expression and activation of both MMP-2 and MMP-9 after SCI. The mRNA expressions of TNF-alpha, IL-1beta, COX-2, and iNOS were also significantly attenuated by BF. Furthermore, BF reduced apoptotic cell death at 1 d after injury, thereby significantly reduced lesion volume and improved functional recovery. Taken together, these results suggest that BF can be used as a potential therapeutic agent for treating acute spinal injury.

Clinical significance of matrix metalloproteinase 2 and 9 in breast cancer.

Background: Invasion and metastasis are the most strenuous problems in the management of breast cancer. These events require diverse proteolytic enzymes, among which MMP-2 and MMP-9 play a significant role in degradation of type IV collagen, the major component of the basement membrane. Therefore, the major objective of the study is to evaluate the clinical usefulness of MMP-2 and MMP-9 with respect to malignant tumor growth, invasion, and metastasis in breast cancer. Materials and Methods: Gelatin zymography was performed on 157 tissue extracts of malignant and adjacent normal breast tissues as well as negative and positive lymph nodes from 49 breast cancer patients. Statistical analysis was carried out using SPSS statistical software (version 10). Results: ProMMP-2 levels were significantly higher in adjacent normal tissues. Active MMP-2 and MMP-9 levels were higher in malignant breast tissues. Activation ratios of MMP-2 and MMP-9 were significantly higher in malignant breast tissues and in patients with lymph node metastasis. ProMMP-2, active MMP-2, and active MMP-9 could significantly discriminate between malignant and adjacent normal breast tissues. The MMP-2 activation ratio showed significant discriminatory efficacy between patients with and without lymph node metastasis and significant association with increased risk of lymph node metastasis in node-negative patients. Conclusion: The results indicate significant clinical utility of these proteolytic enzymes in malignant tumor growth, invasion, and metastasis in breast cancer.