SENP1 induced protein deSUMO modification increased the chemotherapy sensitivity of endometrial cancer side population cells / 中华肿瘤杂志

Objective: To inhibit the stemness maintenance potential of endometrial cancer and increase the sensitivity of endometrial cancer side population cells to chemotherapy drugs by inducing extensive deSUMOylation modification of proteins. Methods: Flow cytometry was used to sort and culture CD133(+) CD44(+) KLE endometrial cancer cell clone spheres. Protein expression level of small ubiquitin-related modifier 1 (SUMO1) and two stemness maintenance genes of tumor side population cells, octamer binding transcription factor-4 (Oct4) and sex determining region Y-box2 (Sox2), were detected by western blotting method. Lentivirus-mediated Sentrin/SUMO-specific proteases 1 (SENP1) gene was stably transfected into KLE side population cells. Western blotting was used to detect the protein expressions of SENP1, SUMO1, Oct4 and Sox2. The clone formation rate was compared between KLE side population cells with or without SENP1 overexpression. Flow cytometry was applied to detect cell cycle changes. 3-(4, 5-Dimethylthiazole-2)-2, 5-diphenyl-tetrazolium bromide (MTT) experiment and flow cytometry apoptosis method were used to detect the chemosensitivity of the side population of endometrial cancer cells to cisplatin. Tumor-bearing mouse models of endometrial cancer were established to detect the effect of SENP1 overexpression on the chemotherapy sensitivity of cisplatin. Results: Compared with CD133(-)CD44(-) KLE cells, CD133(+) CD44(+) KLE side population cells could form clonal spheres and express higher levels of SUMO1, Oct4 and Sox2 proteins (P<0.05). Compared with KLE side population cells that were not transfected with SENP1 gene, the expression level of SENP1 protein in KLE side population cells overexpressing SUMO1、Oct4 and Sox2 were lower. The clonal sphere formation rate was reduced from (25.67±5.44)% to (7.46±1.42)%, and cell cycle shifted from G(0)/G(1) phase to G(2) phase. IC(50) of cisplatin decreased from (55.46±6.14) μg/ml to (11.55±3.12) μg/ml, and cell apoptosis rate increased from (9.76±2.09)% to (16.79±3.44)%. Overexpression of SENP1 could reduce the tumorigenesis rate of KLE side population cells in vivo and increase their chemotherapy sensitivity to cisplatin (P<0.05). Conclusion: Overexpression of SENP1 can induce protein deSUMOylation modification, inhibit the stemness maintenance potential of endometrial cancer side population cells, and enhance their chemotherapy sensitivity, which provides a new reference for gene therapy of endometrial cancer.

Resultados de dos encuestas dirigidas a inmigrantes y nativos del sureste español: salud, uso de servicios y necesidad de asistencia médica / Results of two surveys of immigrants and natives in Southeast Spain: health, use of services, and need for medical assistance

Objetivo. Comparar la salud, uso de servicios sanitarios y necesidad insatisfecha de atención médica (NIAM) entre inmigrantes y nativos del sureste español. Material y métodos. Estudio transversal de dos muestras representativas de población: inmigrante (n=1150) y nativa (n=1303; Encuesta Nacional de Salud). Se creó una única base de datos con ponderación específica para cada muestra y se estimaron razones de prevalencia (RP) mediante regresión multivariante. Resultados. Marroquíes, ecuatorianos y europeos del este (EE) declararon peor salud que los nativos (RPs [IC95%]: 2.45 [1.91-3.15]; 1.51 [1.28-1.79] y 1.44 [1.08-1.93], respectivamente). Los inmigrantes hicieron mayor uso de las urgencias (excepto EE) y consumieron menos fármacos. Los marroquíes mostraron la mayor diferencia en la frecuencia de NIAM (RP [IC95%]: 12.20 [5.25-28.37]), principalmente por razones laborales (46%). Conclusiones. La salud y el uso de servicios sanitarios difirieron significativamente entre inmigrantes y nativos. Destaca la NIAM alta en marroquíes por causa laboral.

Objective. To compare the self-perceived health, use of health services and unmet need for health care (UNHC) among immigrants and native populations of Southeast Spain. Materials and methods. Cross-sectional study of two representative samples of 1150 immigrants, and 1303 native participants from the National Health Survey. A single database was created with specific weights for each sample, and prevalence ratios (PR) were estimated by multivariate regression. Results. Moroccans, Ecuadorians and Eastern Europeans (EE) reported poorer health than the native population (PRs [CI95%]: 2.45 [1.91-3.15]; 1.51 [1.28-1.79] and 1.44 [1.08-1.93], respectively). Immigrants made greater use of emergencies that natives (except for EE) and had lower use of medication. Moroccan showed the greatest difference in the frequency of UNHC (PR [CI95%]:12.20 [5.25 - 28.37]), mainly because of working limitations (46%). Conclusions. The health status and use of health services among immigrants differ significantly from those of natives. Results highlight the higher frequency of UNHC among immigrants, especially high in Moroccans.

Estratégias para o enfrentamento do absenteísmo em consultas odontológicas nas Unidades de Saúde da Família de um município de grande porte: uma pesquisa-ação / Strategies for tackling absenteeism in dental appointments in the Family Health Units of a large municipality: action research

O objetivo deste trabalho foi analisar os motivos das faltas às consultas odontológicas em Unidades de Saúde da Família (USF) e implementar estratégias para sua redução por meio da pesquisa-ação. O estudo foi realizado em 12 USF de Piracicaba/SP, de 01 de janeiro a 31 de dezembro de 2010. A amostra se consistiu de 385 usuários, entrevistados por telefone, sobre os motivos das faltas, além de 12 cirurgiões-dentistas e 12 enfermeiras. Realizaram-se duas oficinas com os profissionais: uma para problematização dos dados coletados nas entrevistas e elaboração de estratégias; e outra após 4 meses, para avaliação. O maior motivo de faltas foi a coincidência do horário de funcionamento das unidades com o de trabalho dos usuários. Dentre as estratégias ressaltou-se a realização de palestras sobre saúde bucal, educação permanente nas reuniões de equipe, capacitação dos Agentes Comunitários de Saúde, participação em grupos terapêuticos e parcerias entre Equipe de Saúde Bucal e equipamentos sociais da comunidade. A adoção de prontuário único foi a estratégia desafiadora encontrada pelos profissionais. Concluiu-se que as estratégias implementadas levaram à diminuição das faltas em 66,6% e o caráter motivador das oficinas possibilitou a reflexão crítica para o redirecionamento da prática em saúde.

The aim of this study was to analyze the reasons for missed appointments in dental Family Health Units (FHU) and implement strategies to reduce same through action research. This is a study conducted in 12 FHUs in Piracicaba in the State of São Paulo from January, 1 to December, 31 2010. The sample was composed of 385 users of these health units who were interviewed over the phone and asked about the reasons for missing dental appointments, as well as 12 dentists and 12 nurses. Two workshops were staged with professionals: the first to assess the data collected in interviews and develop strategy, and the second for evaluation after 4 months. The primary cause for missed appointments was the opening hours of the units coinciding with the work schedule of the users. Among the strategies suggested were lectures on oral health, ongoing education in team meetings, training of Community Health Agents, participation in therapeutic groups and partnerships between Oral Health Teams and the social infrastructure of the community. The adoption of the single medical record was the strategy proposed by professionals. The strategies implemented led to a 66.6% reduction in missed appointments by the units and the motivating nature of the workshops elicited critical reflection to redirect health practices.

Listeriosis en México: importancia clínica y epidemiológica / Listeriosis in Mexico: Clinical and epidemiological importance

La listeriosis es una enfermedad transmitida por alimentos (ETA) y ocasionada por Listeria monocytogenes. La importancia de ésta se debe a su impacto clínico, la alta tasa de mortalidad y el efecto económico derivado de los brotes asociados con el consumo de alimentos. En México, las fallas en los sistemas de vigilancia epidemiológicos son causa de información imprecisa sobre la incidencia de la listeriosis y sobre su caracterización como ETA. En este trabajo se presentan datos referentes a la presencia de la bacteria en alimentos, reportes de casos de la enfermedad y patologías relacionadas con infección por L. monocytogenes. La falta de datos exactos sobre la importancia de esta bacteria plantea la necesidad de concientizar a las instancias correspondientes para definir estrategias de búsqueda intencionada de L. monocytogenes en alimentos y de la recopilación de información clínica precisa que permita conocer la importancia clínica y epidemiológica de la listeriosis en México.

Listeriosis is caused by Listeria monocytogenes, an important food-borne disease due to its clinical forms, high mortality rate, and the economic impact in both clinical and food production industries. In Mexico, the lack of epidemiological surveillance systems leads to the need of accurate data on the incidence of listeriosis and its association with food-borne disease. In this paper, we present data about the presence of this bacterium in food, reports related to clinical cases of listeriosis, and information of diseases in which L. monocytogenes may be involved. However, in most of these cases the etiology was not established. Given this, there's a need to inform and warn the appropriate entities, to define strategies for the mandatory search of L. monocytogenes through the whole food production chain and clinical suspects, for the epidemiological importance and control of listeriosis in Mexico.

La Escuela de Salud Pública de México: innovación educativa y tecnológica en el nuevo milenio / School of Public Health of Mexico: Educational and technological innovation in the new millennium

Este artículo fue concebido para analizar la función de la Escuela de Salud Pública de México (ESPM) desde el año 2000 hasta el presente. Uno de sus puntos centrales es el análisis del proceso de reorientación de la labor educativa de la escuela con la finalidad de responder a los retos en materia de salud y educación surgidos a finales del siglo XX. Para exponer cómo ha evolucionado dicho proceso, retomamos tres ejes rectores que caracterizan la labor de la escuela en la actualidad: el cambio de modelo pedagógico, la incorporación de las tecnologías de la información y las comunicaciones, y la profesionalización de la docencia. Con la exposición de este tema, y a través del contraste entre el pasado y el presente, buscamos completar la historia de trabajo ininterrumpido de la Escuela durante sus 92 años de existencia, que ha trascendido los confines del país.

This article was conceived to analyze the work of the School of Public Health of Mexico (ESPM for is acronym in Spanish) from the year 2000 to the present day. One of the highlights that we will examine is the reorientation of the educational work of the school in order to meet the challenges in health and education that emerged during the end of the twentieth century. In order to explain the evolution of this process, we will describe the three main guiding principles that characterize the present work of the school: the pedagogical model's change, the incorporation of the information and communication technologies, and the professionalization in teaching. The purpose of this work is to define those guiding principles, and to expose, through the contrast between past and present, the complete history of uninterrupted work of the School of Public Health of Mexico during its ninety-two years of existence, that has gone beyond the boundaries of the country.

O programa de assistência pré-natal nos Cuidados de Saúde Primários em Portugal: uma reflexão / The prenatal care program in Primary Care in Portugal: a reflection / El programa de vigilancia prenatal en la Atención Primaria en Portugal: una reflexión

A melhoria da qualidade dos cuidados prestados à grávida e ao recém-nascido é uma das áreas de intervenção prioritária do Plano Nacional de Saúde. Embora se reconheça que as medidas introduzidas nos últimos anos têm contribuído para diminuir os valores da mortalidade materna e perinatal, é necessário referir, também, que continuam a ocorrer gravidezes não planeadas que, não raras vezes, resultam do início tardio, ou mesmo da ausência, da assistência pré-natal. Neste artigo, procuramos refletir sobre a assistência pré-natal no contexto de saúde reprodutiva, de forma a constituir um contributo para os enfermeiros que prestam uma assistência integral e humanizada às grávidas e às suas famílias. Concluímos que a assistência pré-natal engloba um conjunto de cuidados específicos dirigidos a um grupo vulnerável, constituindo uma área muito importante na avaliação dos cuidados de saúde primários.

The quality improvement of care provided to the pregnant women and newborn is one of the priority areas for intervention of the National Health Plan. While acknowledging that the measures introduced in recent years have contributed to lower the values of maternal and perinatal mortality, it should also be mentioned that unplanned pregnancies continue to occur, and that they often result in a delayed or absent prenatal surveillance. In this paper, we seek to reflect on the prenatal surveillance program under Primary Health Care relating to quality of health care provided in the context of reproductive health. We concluded that prenatal surveillance includes a set of specific care services targeted at a vulnerable group, constituting an important and susceptible area of evaluation in primary care.

Mejorar la calidad de la atención a embarazada y recién nacido es una de las áreas prioritarias de intervención del plan nacional de salud. Aunque se reconoce que las medidas adoptadas en los últimos años han contribuido a reducir los valores de la mortalidad materna y perinatal, es necesario mencionar, también, que embarazos no planificados siguen produciéndose a menudo resultado de la aparición, o incluso ausencia, de vigilancia prenatal. En este artículo, reflexionamos sobre el programa de vigilancia prenatal en el marco de la atención primaria de salud, vinculándola con la calidad de la atención de la salud en el contexto de la salud reproductiva. Concluimos que la vigilancia prenatal comprende un conjunto de cuidados específicos dirigidos a un grupo vulnerable, lo que constituye un área sensible y evaluación importante en atención primaria.

Determinação da relevância da cruzipaína na interação de Trypanosoma cruzi com Rhodnius prolixus / Determination of cruzipain relevance in the interaction of Trypanosoma cruzi with Rhodnius prolixus

A cruzipaína é a cisteína peptidase mais abundante do Trypanossoma cruzi, o qual é o agente etiológico da doença de Chagas. A cruzipaína é um importante fator de virulência do T. cruzi envolvida em várias etapas cruciais na interação com células de mamíferos. Essa enzima é expressa em níveis variáveis em todas as formas evolutivas e cepas do parasito, sendo abundantemente expressa nas formas epimastigotas, encontradas apenas no inseto vetor. Esse dado nos levou a investigar se a cruzipaína poderia estar envolvida na interação do T. cruzi com células do hospedeiro invertebrado. Para tal, foram analisados os efeitos do pré-tratamento do T. cruzi com um painel de diferentes inibidores de cisteína peptidases ou anticorpos anti-cruzipaína na adesão do parasito ao epitélio intestinal médio posterior dissecado de Rhodnius prolixus. Paralelamente, foi analisado o índice de adesão ao eptélio do T. cruzi que superexpressa a chagasina (pCHAG), um inibidor endógeno da cruzipaína. A taxa de adesão dos parasitos tratados com os inibidores de cisteína peptidase (iodoacetamida, leupeptina, antipaína ou E-64 a 10 (Miu)M, ou cistatina a 1 (Miu)g/mL) foi em média 70por cento inferior em comparação aos parasitos não tratados, com exceção do Ca074me (um inibidor de catepsina B) que não mostrou alteração significativa. O tratamento de parasitos com a cistatina apresentou um efeito dose-dependente sobre a taxa de adesão em realçao aos parasitos não tratados. Além disso, o tratamento de epimastigotas com anticorpos anti-cruzipaína (1:1000)induziu uma redução significativa de 64por cento na adesão.Os parasitos pCHAG apresentaram baixa capacidade de ligação ao epitélio intestinal dissecado de R. prolixus, enquanto o pTEX (plasmídeo sem o gene inserido) não apresentou mudanças significativas na taxa de adesão em relação ao controle. Nós também comparamos a habilidade de vários isolados de T. cruzi na adesão ao intestino do R. prolixus. A cepa G, que naturalmente apresenta...

Arginine and glutamate levels in the gingival crevicular fluid from patients with chronic periodontitis

The objectives of this study were to determine arginine and glutamate levels in the gingival crevicular fluid (GCF) of adult chronic periodontitis patients versus periodontally healthy controls, and to compare two kinds of microdialysis probes: normal and U-shaped probes. The analysis of GCF components was developed to improve the diagnosis of periodontal disease (PD). Proteolysis in the periodontal tissues increases the concentration of amino acids (aa) in the GCF and the levels of these aa may reveal PD features and stages. GCF samples were collected by microdialysis in situ from 5 periodontally affected sites (probing depth >5 mm, clinical attachment loss >3 mm) in 14 adult chronic periodontitis patients and from 14 adult periodontally healthy controls. Capillary zone electrophoresis coupled to laser induced fluorescence detection was used to measure concentration of arginine and glutamate in the GCF. Data were analyzed statistically by ANOVA and Tukey's post-hoc tests (?=0.05). Arginine concentration was increased (p<0.001) and glutamate concentration was decreased (p<0.001) in chronic periodontitis patients as compared to controls. There were no significant differences (p=0.069) between the normal and U-shaped probes. In conclusion, the increase of arginine and decrease of glutamate concentration in GCF were associated to the presence of periodontitis, and might be used as markers to recognize periodontally susceptible subjects as well as to evaluate the treatment course.

Os objetivos deste estudo foram determinar os níveis de arginina e glutamato no fluido gengival crevicular (FGC) em pacientes com periodontite crônica contra controles saudáveis e comparar dois tipos de cânulas de microdiálise: normais e em forma de U. A análise dos componentes do FGC desenvolveu-se para melhorar o diagnóstico da doença periodontal (DP). A proteólise dos tecidos periodontais aumenta a concentração de aminoácidos (aa) no FGC e os níveis destes aa podem revelar as características e estágios da DP. Amostras de FGC foram obtidas pela técnica de microdiálise in situ de cinco zonas com o periodonto afetado (profundidade de sondagem >5 mm, perda da inserção clínica >3 mm) em 14 pacientes adultos com periodontite crônica e 14 controles saudáveis. Para medir a concentração de arginina e glutamato no GFC, usou-se a técnica de eletroforese capilar com detecção de fluorescência induzida por laser. Nos pacientes com periodontite crônica, a concentração de arginina aumentou significantemente (p<0.001), enquanto a de glutamato diminuiu significantemente (p<0.001) em comparação aos controles. Não houve diferenças significantes (p=0.069) entre as cânulas normais e as cânulas em forma de U. Conclui-se que o aumento da concentração de arginina e diminuição de glutamato no FGC estavam associados à presença de periodontite, e podem ser usados como marcadores para identificar pacientes suscetíveis à periodontite bem como avaliar a evolução do tratamento.

Partial Purification and Characterization of a Cysteine Protease Inhibitor from the Plerocercoid of Spirometra erinacei

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.

Cysteine proteinases of Trypanosoma cruzi: from digestive enzymes to programmed cell death mediators

Trypanosoma cruzi, the parasite causing Chagas disease, contains a number of proteolytic enzymes. The recent completion of the genome sequence of the T. cruzi CL Brener clone suggests the presence of 70 cysteine peptidases, 40 serine peptidases (none of them from the chymotrypsin family), about 250 metallopeptidases (most leishmanolysin homologues), 25 threonine peptidases, and only two aspartyl peptidases, none of them from the pepsin family. The cysteine peptidases belong to 7 families of Clan CA, 3 families of Clan CD, and one each of Clans CE and CF In Clan CA, the C1 family is represented by cruzipains 1 and 2, biochemically well characterized, as well as cathepsin B and two other cathepsins. There are a number of homologues to calpains (family C2), probably non-functional, lacking the Ca-binding domain. Family C54 includes the Atg4 proteinases (autophagins), which seem to be involved in the autophagic process. Clan CD includes family C14, the metacaspases. We have expressed the metacaspases TcMCA3 and TcMCA5, and obtained indirect evidence of their participation in programmed cell death induced by fresh human serum in the parasite. More experiments are required to better define their role in apoptosis.

Behavior of Araujiain, a new cysteine phytoprotease, in organic media with low water content

In this paper we studied the effect of different organic solvents (1-octanol, trichloroethylene, ethanol, ethyl acetate, tetrahydrofuran, cyclohexane, propanone, acetonitrile, dichloromethane, chlorobenzene, N,N-dimethylformamide, acetophenone, diethyl ether, methanol, ethylene glycol and toluene) with low and constant water content on substrate preferences, thermostability and stability (caseinolytic activity retention after 4 h) of proteases of Araujia hortorum Fourn. (Asclepiadaceae). The stability of araujiain was high in N,N-dimethylformamide and ethanol at 40 ºC, but decreased at higher temperature. Araujiain substrates preferences in buffer Tris-HCl (pH 8), ethylene glycol and N,N-dimethylformamide exhibited different patterns, but the enzyme showed a high preference by glutamine derivative in all cases. According to FTIR spectroscopy studies, araujiain changed its secondary structure and as a consequence, it also changed its substrate preferences. This enzyme showed lower beta-helical character and greater beta-sheet folding in buffer than in organic media. A larger amount of antiparallel beta-sheet residues indicates the formation of tighter intermolecular hydrogen bonds and enzymatic aggregates. These facts could explain the higher esterolytic activities, the greater stability and good hydrolytic potential of araujiain in some organic media such as N,N-dimethylformamide.

Degranulation of human eosinophils induced by Paragonimus westermani-secreted protease

Eosinophil degranulation is considered to be a key effector function for the killing of helminthic worms and tissue inflammation at worm-infected lesion sites. However, relatively little data are available with regard to eosinophil response after stimulation with worm-secreted products which contain a large quantity of cysteine proteases. In this study, we attempted to determine whether the degranulation of human eosinophils could be induced by the direct stimulation of the excretory-secretory products (ESP) of Paragonimus westermani, which causes pulmonary paragonimiasis in human beings. Incubation of eosinophils for 3 hr with Paragonimus-secreted products resulted in marked degranulation, as evidenced by the release of eosinophil-derived neurotoxin (EDN) in the culture supernatants. Moreover, superoxide anion was produced by eosinophils after stimulation of the ESP. The ESP-induced EDN release was found to be significantly inhibited when the ESP was pretreated with protease inhibitor cocktail or the cysteine protease inhibitor, E-64. These findings suggest that human eosinophils become degranulated in response to P. westermani-secreted proteases, which may contribute to in vivo tissue inflammation around the worms.

Contact of Entamoeba histolytica with baby hamster kidney-21 (BHK-21) cell line on cysteine proteinase activity.

BACKGROUND & OBJECTIVES: Entamoeba histolytica, the causative agent of amoebiasis and amoebic liver abscess, lyses host cells by direct contact using surface lectins and releases cysteine proteinase (CP). Virulence of E. histolytica is directly related to activity of its CP. The relationship of CP activity and cytotoxicity has not been established. The present study was carried out to explore the events following contact of E. histolytica with target cells. METHODS: Protease activity of E. histolytica was measured by azocaseine and haemoglobin assays, and cysteine proteinase activity was assessed by substrate gel electrophoresis. Target cell lysis was measured by chromium release assay. RESULTS: Protease activity of E. histolytica was increased 2.5-fold following contact with BHK-21 cell line. CP activity of trophozoites alone was visualized at position 56, 35 and 29 kDa in substrate gel electrophoresis. Contact of trophozoites with target cells augmented the cytotoxic activity of amoebic CP. The increase in CP activity seen by substrate gel electrophoresis and cytotoxicity assay was blocked by pretreatment with E 64, a specific CP inhibitor and GalNAc, a contact inhibitor. INTERPRETATION & CONCLUSION: The present data showed the involvement of amoebic CP in cytotoxicity and that the CP activity was enhanced on lectin-mediated contact of E. histolytica to the target cells. Further studies need to be done to understand the mechanism at the molecular level.

Pro domain peptide of HGCP-Iv cysteine proteinase inhibits nematode cysteine proteinases

Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides. A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form. The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region. HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs. Recombinant propeptides of HGCP-Iv, expressed in E. coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems. Cysteine proteinases from other species produced no noticeable activity.

Cysteine proteases in nodulation and nitrogen fixation.

The cysteine proteinases or cysteine endopeptidases (EC 3.4.22) are known to occur widely in plant cells. They are involved in almost all aspects of plant growth and development including germination, circadian rhythms, senescence and programmed cell death. They are also involved in mediating plant cell responses to environmental stress (such as water stress, salinity, low temperature, wounding, ethylene, and oxidative conditions) and plant-microbe interactions (including nodulation). In the development and function of legume root nodules, cysteine proteases could be involved in several important processes:-(i) a defence response to root invasion by microorganisms; (ii) protein turnover required during the formation of new tissue; (iii) cellular homeostasis and metabolism; (iv) adaptation of host cells to physiological stresses; (v) control of nodule senescence. Because of their central importance to plant physiology, cysteine proteases could serve as important targets for the study of nodule development and functioning at the molecular level. Because of their widespread occurrence in nodulating plants they could also serve as candidate genes for targeted plant breeding programmes.

The Levels of MDM2 Protein Are Decreased by a Proteasome-Mediated Proteolysis Prior to Caspase-3-Dependent pRb and PARP Cleavages

MDM2 is a substrate of caspase-3 in p53-mediated apoptosis. In addition, MDM2 mediates its own ubiquitination in a RING finger-dependent manner. Thus, we investigated whether MDM2 is degraded through a ubiquitin-dependent proteasome pathway in the absence of p53. When HL-60 cells, p53 null, were treated with etoposide, MDM2 was markedly decreased prior to caspase-3-dependent retinoblastoma tumor suppressor protein (pRb) and poly (ADP- ribose) polymerase (PARP) cleavages. Moreover, down-regulation of MDM2 level was not coupled with its mRNA down-regulation. However, the level of MDM2 was partially restored by proteasome inhibitors such as LLnL and lactacystin, even in the presence of etoposide. Our results suggest that, in the p53 null status, MDM2 protein level is decreased by proteasome-mediated proteolysis prior to caspase-3-dependent PARP and pRb cleavages.

Specific and reversible inhibition of entamoeba histolytica cysteine proteinase activities by Zn²+: Implications for adhersion and cell damage1

Background. Cysteine-proteinases are thought to play an important role in E. histolytica pathogenicity. Although effective blockage of proteolytic activities can be obtained with sereveral known inhibitors, the high cellular toxicity of most of the inhibitors precludes experimentation with live cells or animal models. Specific cysteine-proteinase inhibitors that could be utilized in studies of virulence are of great need in the field of amebiasis. Methods. Cysteine-proteinase activities were determined in trophozoit lysates by azocasein degradation and after PAGE and gelatin zymograms. Inhibition of the activities was assessed in the presence of 0.01-2.5 mM concentrations of fivalent cations of the IIB and VIII series such as Zn, Cd, Hg, Ni, and Co. Reversibility was induced with 25 mM L-cysteine or 50 mM L-histidine and by metal chelation with 5 mM phenantroline. The inhibitory effect of ZnCI2 was tested with live cells in fibronectin-biding and cytotoxicity assays. Results. ZnCI2 specifically inhibited cysteine-proteinase activities in trophozoite lysates in a concentration-dependent manner. Additionally, 1.0-2.5 mM ZnCI2 bloked proteolysis in more than 70 percent. This inhibition was completely reverted by L-cysteine, L-histidine, or phenantroline. Similar results were obtained by analyzing indivual cysteine-proteinase activities separated in gelatin zymograms. At these concentrations, ZnCI2 significanty interfered with trophozoit adhesion, thus making amebas deficient in substrate degradation and cell damage. Conclusions. ZnCI2 is effective inhibitor of amebic cysteine-proteinases. Its low toxicity at relative high concentrations, high solubility, and low cost make it adequate for live cell experimentation and animal models of amebic virulence

Enhanced expression of cathepsin L in metastatic bone tumors

Cathepsin L is a kind of cystein proteases which are known to facilitate the invasion and metastasis of tumor cells by degrading the components of basement membrane and extracellular matrix. This study was undertaken to investigate the expression of cathepsin L by Northern blot analysis with radiolabeled cDNA specific for cathepsin L in six normal tissues, two osteosarcoma cell lines, MG-63 and Saos-2, six primary bone tumors and six metastatic bone tumors. In six normal tissues, the highest level of cathepsin L was expressed in liver with the descending order of liver > lung > thymus > ovary > kidney > esophagus. One of the two osteosarcoma cell lines established from the primary sites expressed a high level of cathepsin L mRNA. Out of six primary bone tumors, three (50%) expressed cathepsin L mRNA, while all (100%) of six metastatic bone tumors expressed the mRNA. These results demonstrating the higher frequency of expression of cathepsin L in metastatic bone tumors suggest that cathepsin L may participate in tumor invasion and metastasis.

Effect of heat-shock & nutritional stress on the expression of a neutral thiol protease in Leishmania donovani promastigotes.

The soluble intracellular protease was partially purified from L. donovani promastigotes. The activity of this enzyme increased with increase in temperature from 25 degrees C to 37 degrees C and was active optimally at 70 degrees C. This protease activity appeared to be decreased due to heat-shock of the promastigotes for 4 h at 37 degrees C and increased due to nutrient starvation. Inhibition of the protease by p-chloromercuribenzoate and iodoacetamide suggested that this enzyme could be a thiol protease.