Structural insights into leishmanolysins encoded on chromosome 10 of Leishmania (Viannia) braziliensis

BACKGROUND Leishmanolysins have been described as important parasite virulence factors because of their roles in the infection of promastigotes and resistance to host's defenses. Leishmania (Viannia) braziliensis contains several leishmanolysin genes in its genome, especially in chromosome 10. However, the functional impact of such diversity is not understood, but may be attributed partially to the lack of structural data for proteins from this parasite. OBJECTIVES This works aims to compare leishmanolysin sequences from L. (V.) braziliensis and to understand how the diversity impacts in their structural and dynamic features. METHODS Leishmanolysin sequences were retrieved from GeneDB. Subsequently, 3D models were built using comparative modeling methods and their dynamical behavior was studied using molecular dynamic simulations. FINDINGS We identified three subgroups of leishmanolysins according to sequence variations. These differences directly affect the electrostatic properties of leishmanolysins and the geometry of their active sites. We identified two levels of structural heterogeneity that might be related to the ability of promastigotes to interact with a broad range of substrates. MAIN CONCLUSION Altogether, the structural plasticity of leishmanolysins may constitute an important evolutionary adaptation rarely explored when considering the virulence of L. (V.) braziliensis parasites.

Enterotoxigenic and non-enterotoxigenic Bacteroides fragilis from fecal microbiota of children

Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.

Contexto social del debate sobre el control natal en Colombia en las décadas de 1960 y 1970: política, medicina y sociedad / The social context of the birth control debate in Colombia in the 1960s and 1970s: politics, medicine and society

El artículo busca presentar el contexto y aproximación preliminares necesarios para comprender y abordar el debate sobre el control natal en Colombia en las décadas de 1960 y 1970. Recoge las principales posturas en conflicto en dicho período, y los discursos y lógicas que permearon la llegada de los programas de planificación norteamericanos a América Latina como forma de control político de los movimientos revolucionarios.

The article seeks to present the necessary context and a preliminary approach to understanding and addressing the birth control debate in Colombia in the 1960s and 1970s. It covers the main conflicting positions during that period and the discourses and logics permeating the arrival of North American family planning programs to Latin America as a form of political control of revolutionary movements.

Posible papel de Bacteroides fragilis enterotoxigénico en la etiología de la vaginitis infecciosa / Possible role of enterotoxigenic Bacteroides fragilis in the etiology of infectious vaginitis

La vaginitis es un trastorno ginecológico frecuente producido por distintas causas, algunas de las cuales permanecen desconocidas. Bacteroides fragilis es el anaerobio más importante en bacteriología clínica. Algunas cepas son enterotoxigénicas y se asocian con síndromes intestinales y extraintestinales. Recientemente han sido aisladas de pacientes con vaginitis. En este trabajo se planteó investigar la posible asociación de B. fragilis enterotoxigénico con la vaginitis infecciosa. Fueron procesadas 265 muestras de exudado vaginal. 202 de mujeres sintomáticas y 63 mujeres sanas. La identificación de los microorganismos se realizó por métodos convencionales. En 31,2% de las pacientes sintomáticas se identificaron: Gardnerella vaginalis, Candida albicans, Mobiluncus, Mycoplasma hominis, Ureaplasma urealyticum y Streptococcus agalactiae. En 27 pacientes sintomáticas y en 5 mujeres sanas se identificó B. fragilis. Estas cepas fueron cultivadas en medio líquido e incubadas durante 48 h a 36° C en anaerobiosis. La toxicidad en los sobrenadantes se ensayó en células HT-29. 18 cepas de B. fragilis aisladas de pacientes sintomáticas fueron enterotoxigénicas, ya que indujeron alteraciones en la monocapa celular y en las células. No se identificó en mujeres sanas (P<0,05). 77,7% de las cepas de B. fragilis enterotoxigénicas no se encontraron asociadas con otros patógenos específicos. Este hecho sugiere que pudiera ser un agente causante de vaginitis, ya que el efecto de la enterotoxina sobre la E-cadherina del epitelio vaginal podría facilitar la invasión y su posible papel patógeno en la vagina. Esta es la primera investigación que asocia a Bacteroides fragilis enterotoxigénico como posible causa de vaginitis infecciosa.

Vaginitis is a common gynecologic disorder. It is due to several causes, some even unknown. Bacteroides fragilis is the most important anaerobe in clinical bacteriology, some strains of this group are notable for being enterotoxigenic and they have been associated with intestinal and extraintestinal syndromes. They have recently been isolated from patients with vaginitis. The purpose of this study was to investigate a possible association of enterotoxigenic B. fragilis with infectious vaginitis. 265 samples of vaginal exudate were processed, 202 from symptomatic patients and 63 healthy women. The identification of the microorganisms was carried out by conventional methods. In 31.2% of symptomatic patients were identified: Gardnerella vaginalis, Mobiluncus, Candida albicans, Mycoplasma hominis, Ureaplasma urealyticum and Streptococcus agalactiae. B. fragilis was identified in 27 symptomatic patients and 5 healthy women. These strains were cultivated in liquid medium and incubated during 48 h at 36°C in anaerobe chambers. Supernatant activity was assayed in HT-29 cells. Eighteen B. fragilis strains isolated from symptomatic patients were enterotoxigenic, because induced alterations in target cell morphology. It was not identified in healthy women (P<0.05). 77.7% of enterotoxigenic B. fragilis strains were not associated with other specific pathogens. This fact suggests that enterotoxigenic B. fragilis could be a cause for vaginitis. The effect of enterotoxin on E-cadherin of vaginal epithelium could facilitate invasion and its possible pathogenic role in the vagina. This is the first report that associates enterotoxigenic Bacteroides fragilis as a possible cause of infectious vaginitis.

Púrpura trombótica trombocitopenica en adultos / Trombocitopenica trombótica purple in adults

Se revisa la presentación de 6 pacientes adultos con púrpura trombótica trombocitopénica (PTT), que fueron atendidos en el Servicio de Clínica Médica del Hospital Escuela de Corrientes durante un período de 11 años. La púrpura trombótica trombocitopénica es una anemia hemolítica microangiopática caracterizada por una pentada clínica típica: trombocitopenia, anemia hemolítica microangiopática, fallo renal, fiebre y manifestaciones neurológicas. Todos los pacientes presentaron trombocitopenia y anemia hemolítica microangiopática. El compromiso neurológico fue la segunda característica más frecuente. Todos los pacientes fueron tratados con plasmaféresis. Tres pacientes se recuperaron completamente y 3 fallecieron. El pilar del tratamiento es la transfusión de plasma y la plasmaféresis. Se hace hincapié en su implementación inmediata dada la alta mortalidad de esta patología.

Ocurrence of blaSPM-1 and blaIMP-1 genes of metallo-beta-lactamases in clinical isolates of Pseudomonas aeruginosa from three universitary hospitals in the city of Porto Alegre, Brazil

We described the ocurrence of metallo-beta-lactamases (MBL) genes blaSPM-1 and blaIMP-1 in clinical isolates of Pseudomonas aeruginosa resistant to imipenem and/or ceftazidime obtained from three universitary hospitals in the city of Porto Alegre, Brazil. The MBL production was screened by phenotypic test and the genes were detected by PCR.

Descrevemos a ocorrência dos genes de metalo-beta-lactamases (MBL) blaSPM-1 e blaIMP-1 em isolados clínicos de Pseudomonas aeruginosa resistentes ao imipenem e/ou ceftazidima obtidos em três hospitais universitários de Porto Alegre, Brasil. A produção de MBL foi observada através de técnica fenotípica e os genes foram detectados pelo método de PCR.

The ubiquitous gp63-like metalloprotease from lower trypanosomatids: in the search for a function

Plant and insect trypanosomatids constitute the "lower trypanosomatids", which have been used routinely as laboratory models for biochemical and molecular studies because they are easily cultured under axenic conditions, and they contain homologues of virulence factors from the classic human trypanosomatid pathogens. Among the molecular factors that contribute to Leishmania spp. virulence and pathogenesis, the major surface protease, alternatively called MSP, PSP, leishmanolysin, EC 3.4.24.36 and gp63, is the most abundant surface protein of Leishmania promastigotes. A myriad of functions have been described for the gp63 from Leishmania spp. when the metacyclic promastigote is inside the mammalian host. However, less is known about the functions performed by this molecule in the invertebrate vector. Intriguingly, gp63 is predominantly expressed in the insect stage of Leishmania, and in all insect and plant trypanosomatids examined so far. The gp63 homologues found in lower trypanosomatids seem to play essential roles in the nutrition as well as in the interaction with the insect epithelial cells. Since excellent reviews were produced in the last decade regarding the roles played by proteases in the vertebrate hosts, we focused in the recent developments in our understanding of the biochemistry and cell biology of gp63-like proteins in lower trypanosomatids.

Tripanossomatídeos de insetos e de plantas são informalmente denominados de "tripanossomatídeos inferiores". Estes microrganismos são utilizados rotineiramente como modelos para estudos de bioquímica e de biologia molecular porque são facilmente cultivados sob condições axênicas e porque possuem homólogos aos fatores de virulência encontrados nos tripanossomatídeos que são patógenos humanos clássicos. Dentre os fatores moleculares que contribuem para a virulência e patogênese de Leishmania spp. destaca-se a principal protease de superfície, também conhecida como MSP, PSP, leishmanolisina, EC 3.4.24.36 e gp63, que é a proteína de superfície mais abundante encontrada nas formas promastigotas de Leishmania. Diversas funções foram descritas para a gp63 de Leishmania no hospedeiro vertebrado. Entretanto, pouco é conhecido sobre as funções desempenhadas por essa molécula no inseto vetor. Curiosamente, a gp63 é predominantemente expressa na forma evolutiva de Leishmania encontrada no inseto, e em todos os tripanossomatídeos de insetos e plantas analisados até o presente momento. Os homólogos da gp63 presentes nos tripanossomatídeos inferiores desempenham um papel essencial na nutrição assim como na interação com as células epiteliais do inseto. Uma vez que revisões de excelente qualidade foram produzidas na última década sobre a função de proteases nos hospedeiros vertebrados, nesta revisão nós abordamos os recentes progressos sobre os aspectos bioquímicos e as prováveis funções biológicas desempenhadas pelas proteínas homólogas à gp63 nos tripanossomatídeos inferiores.

Metaloproteasas en la progresión tumoral: revisión / Metalloproteinases and tumor progression: review

Las dos propiedades biológicas que determinan la malignidad en el cáncer son la infiltración y la metástasis, lo cual ha llevado al estudio de todos los mecanismos que tienen que ver tanto con la invasión de las células tumorales como con la relación de estas células con su estroma, con el cual interactúan produciendo factores que provoca la movilización y acumulación de células inflamatorias, formación de nuevos vasos sanguíneos, multiplicación de fibroblastos y síntesis de componentes de la matriz extracelular. La invasión tumoral está mediada por diversas enzimas, en particular las proteasas, que degradan la matriz extracelular y cuyos productos facilitan la progresión tumoral, destacando entre ellas las metaloproteasas (MMP), que al jugar un papel importante en la biología del cáncer, son blanco potencial para usarlas en la terapia contra esta enfermedad. Por ello inhibidores sintéticos de las metaloproteasas ya han sido desarrollados, que si bien han presentando actividad antitumoral al usarse en varios modelos animales, en estudios clínicos hechos en pacientes con cánceres avanzados no se ha logrado, hasta ahora, obtener una respuesta terapéutica efectiva. Dada la importancia y significado biológico y clínico de estas investigaciones, este artículo resume el papel que desempeñan las metaloproteasas en la promoción tumoral, proliferación celular, invasión tumoral y la angiogénesis

High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.

Visión clínica de la ADAMTS13 / Clinical vision of the ADAMTS13

La disfunción ADAMTS13 ha sido implicada en la patogénesis de la púrpura trombocitopénica trombótica (PTT). Este desorden ocurre con mayor frecuencia en mujeres y, en el 13 por ciento de ellas, se asocia al embarazo. Sin embargo, hay poca información sobre el comportamiento de la proteasa en embarazo normal. Estudiamos el factor de de von Willebrand y la actividad ADAMTS13 en mujeres sanas no embarazadas, embarazadas y puerperio. Se incluyeron a cincuenta y cinco mujeres no embarazadas, donantes normales del banco de sangre, que no tomaban píldoras anticonceptivas, como controles. Se incluyeron 270 embarazadas y puérperas normales. La actividad ADAMTS13 disminuyó progresivamente a partir del período de 12-16 semanas hasta el final del puerperio temprano (media el 52 por ciento, rango 22-89, p < 0,0001), con un aumento leve posterior. Los niveles fueron levemente menores en nulíparas que en mujeres con paridad previa (65 por ciento vs 83 por ciento, p = 0,0003), y primíparas que en multiparas entre 6-11 semanas hasta 17-23 semanas del embarazo (69 por ciento vs. 80 por ciento, p = 0,005). Aunque en todas las mujeres los níveles de proteasa fueron iguales en los diferentes grupos sanguíneos, las mujeres no embarazadas del grupo sanguíneo O demostraron una media mayor de actividad de ADAMTS13 que el no-O (78 por ciento vs 69 por ciento, p = 0,064). Nuestros resultados sugieren que los cambios de actividad de ADAMTS13, durante el embarazo y puerperio, podrían hacer que el final del embarazo fuera un período más vulnerable para el desarrollo de microangiopatía trombótica.

Análisis de expresión global del cáncer cérvico uterino: rutas metabólicas y genes alterados / Global expression analysis in uterine cervical cancer: Metabolic pathways and altered genes

High risk human papillomavirus (HPV) infection is considered to be the most important etiological factor of Cervical Uterine Cancer. In order to determine the global expression pattern and to identify possible molecular markers of cervical cancer, cDNA arrays with probe sets complementary to 8,000 human genes were used to examine the expression profiles among 5 cell lines derived from human cervical cancer, three HPV16(+) tumor samples and three normal cervical tissues HPV(-). The levels of expression of different cellular processes were identified. Hierarchical clustering was performed and the gene expression using RT-PCR was confirmed. Two genes were found to be consistently overexpressed in invasive cervical cancer biopsies; one of them, IL-6 was previously reported to be overexpressed in cervical cancer and one novel gene, MMP10, previously not known to be related to cervical cancer. Hierarchical clustering of the expression data revealed that samples with common HPV type infection grouped together, maybe this could mean that differences between HPV types could be indirectly determined by expression profiles.

La infección por virus de papiloma de alto riesgo (VPH) es considerada como el factor etiológico más importante del cáncer cérvico uterino (CaCU). Con el fin de determinar el patrón de expresión global e identificar algunos posibles genes marcadores del CaCU, se utilizaron microhileras de DNA que contenían 8,000 secuencias que codificaban para transcritos diferentes, para estudiar los perfiles de expresión de cinco líneas celulares derivadas de CaCU, tres muestras tumorales conteniendo VPH 16 y tres muestras normales negativas para la presencia de VPH. Se identificaron los niveles de expresión de genes relacionados con diferentes rutas metabólicas. Se llevó a cabo el análisis de agrupamiento jerárquico y posteriormente se confirmó la sobrexpresión de dos genes mediante RT-PCR. Estos dos genes se encontraron sobrexpresados en biopsias tumorales cervicales. Uno de ellos, el gen de IL6, que ha sido previamente reportado en relación con CaCU, así como el gen de la matriz-metaloproteasa 10 (MMP10) por primera vez relacionado con esta neoplasia. El análisis de agrupamiento jerárquico, además, reveló que las muestras que contienen el mismo tipo viral están asociadas, sugiriendo posibles diferencias en expresión entre tipos virales.

Expresión MMP-13 en enfermedad periodontal / MMP-13 expression in periodontal disease

Objetivo: determinar si existe diferencia en la expresión y localización de MMP-13 en periodonto sano, agrandamiento gingival inducido por placa bacteriana, periodontitis crónica moderada/avanzada y periodontitis agresiva, mediatne inmunohistoquímica para evidenciar un posible papel de esta colagenaa en la progresión del daño tisular en patologías infecciosas del periodonto. Métodos: el tipo de diseño seguido en este estudio fue observacional comparativo. Se tomaron biopsias de 22 pacientes que fueron diagnosticados como 5 sanos (S), 6 periodontitis crónica (PC), 6 periodontitis agresia (PA) y 5 agrandamiento gingival inducido por placa bacteriana (AGIPB), analizándose 4 estratos tisulares en cada biopsia: epitelio oral (EO), epitelio de unión (EU), conectivo adyacente al epitelio de unión (CAEU) y conectivo profundo (CP). El tipo de muestreo fue intencional, ya que los investigadores conocían las características de los sujetos que se escogieron para el estudio. Las muestras se colocaron en formalina al 10 por ciento y se incluyeron en bloques de parafina, para posteriormente realizar la técnica de inmunohistoquímica. Se hizo valoración semicuantitativa determinada por conteo celular al microscopio de luz (40X). Resultados: la mayor expresión de MMP-13 se da en la periodontitis crónica (PC) seguida por el agrandamiento gingival inducido por placada bacteriana (AGIPB), periodontitis agresiva (PA) y finalmente los pacientes sanos que no mostraron expresión alguna de esta colagenasa. El estrato tisular que más expresión mostró fue el epitelio oral a nivel del estrato granuloso. Conclusión: la MMP-13 no se expresa en biopsias de tejido sano. La expresión se presenta en tejidos que se encuentran en procesos de destrucción o de remodelación

Inflammatory effects of snake venom metalloproteinases

Metalloproteinases are abundant enzymes in crotaline and viperine snake venoms. They are relevant in the pathophysiology of envenomation, being responsible for local and systemic hemorrhage frequently observed in the victims. Snake venom metalloproteinases (SVMP) are zinc-dependent enzymes of varying molecular weights having multidomain organization. Some SVMP comprise only the proteinase domain, whereas others also contain a disintegrin-like domain, cysteine-rich, and lectin domains. They have strong structural similarities with both mammalian matrix metalloproteinases (MMP) and members of ADAMs (a disintegrin and metalloproteinase) group. Besides hemorrhage, snake venom metalloproteinase induce local myonecrosis, skin damage, and inflammatory reaction in experimental models. Local inflammation is an important characteristic of snakebite envenomations inflicted by viperine and crotaline snake species. Thus, in the recent years there is a growing effort to understand the mechanisms responsible for SVMP-induced inflammatory reaction and the structural determinants of this effect. This short review focuses the inflammatory effects evoked by SVMP.

Macrophage elastase (MMP-12): a pro-inflammatory mediator?

As many metalloproteinases (MMPs), macrophage elastase (MMP-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes. Studies using animal models of acute and chronic pulmonary inflammatory diseases, such as pulmonary fibrosis and chronic obstrutive pulmonary disease (COPD), have given evidences that MMP-12 is an important mediator of the pathogenesis of these diseases. However, as very few data regarding the direct involvement of MMP-12 in inflammatory process in the airways were available, we have instilled a recombinant form of human MMP-12 (rhMMP-12) in mouse airways. Hence, we have demonstrated that this instillation induced a severe inflammatory cell recruitment characterized by an early accumulation of neutrophils correlated with an increase in proinflammatory cytokines and in gelatinases and then by a relatively stable recruitment of macrophages in the lungs over a period of ten days. Another recent study suggests that resident alveolar macrophages and recruited neutrophils are not involved in the delayed macrophage recruitment. However, epithelial cells could be one of the main targets of rhMMP-12 in our model. We have also reported that a corticoid, dexamethasone, phosphodiesterase 4 inhibitor, rolipram and a non-selective MMP inhibitor, marimastat could reverse some of these inflammatory events. These data indicate that our rhMMP-12 model could mimic some of the inflammatory features observed in COPD patients and could be used for the pharmacological evaluation of new anti-inflammatory treatment. In this review, data demonstrating the involvement of MMP-12 in the pathogenesis of pulmonary fibrosis and COPD as well as our data showing a pro-inflammatory role for MMP-12 in mouse airways will be summarized.

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina / Factores de virulencia de Vibrio cholerae no-O1 no-O139 aislados en Córdoba, Argentina

V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no-clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and “in vivo” studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.

En este trabajo se analizó la presencia y expresión de genes de virulencia en V. cholerae no-O1 no-O139 de origen clínico y ambiental, aislados en Córdoba, Argentina. La mayoría de las cepas estudiadas contiene los genes toxR y hlyA, pero no ctxA, zot, ace, tcpA y stn. Se analizó la actividad hemolítica y citotóxica de estas cepas en los sobrenadantes de cultivo, así como su potencial enterotóxico en ensayos de asa ileal ligada de conejo. Además, los aislamientos fueron comparados por sus perfiles genéticos en PFGE. Las cepas del medio ambiente mostraron variación en su fenotipo de virulencia y no mostraron relación clonal. Las cepas clínicas fueron muy enterotóxicas, hemolíticas, proteolíticas y mostraron perfiles indistinguibles de PFGE, aunque mostraron diferencias en su actividad citotóxica. En este trabajo se describen por primera vez, utilizando ensayos de cultivo celular e “in vivo”, propiedades de virulencia de V. cholerae no-O1 no-O139 aislados en Argentina.

Colágeno na cartilagem osteoartrótica / Collagen in osteoarthrotic cartilage

A cartilagem articular é um tecido altamente especializado, composto por células, os condrócitos, e um conjunto de macromoléculas, como o colágeno e os proteoglicanos. O colágeno é uma proteína fibrilar que garante resistência ao tecido, enquanto os proteoglicanos têm a função de mola biológica, sendo responsáveis pela compressibilidade da cartilagem. A complexa interação entre duas proteínas garante a elasticidade. Estas características específicas da cartilagem são essenciais para amortecer as grandes forças de impacto a que as articulações diartrodiais estão submetidas, sem muito gasto de energia, visto tratar-se de um tecido avascular. Em processos artrósicos ocorre um desequilíbrio entre a produção de componentes da matriz extracelular e destruição pelas metaloproteases, levando à degradação e perda do tecido cartiloginoso. A fase inicial da osteoartrose é marcada por perda de fragmentos de proteoglicanos para o líquido sinovial, aumento dos colágenos tipo II e tipo IV, aparecimento dos colágenos I e III, não típicos da cartilagem, e diminuição do colágeno tipo IX, que é importante para manter a integridade da matriz extracelular, além do entumescimento da cartilagem. Como conseqüência, a cartilagem perde suas características específicas, levando a alterações na função articular. A evolução da doença promove dimimuição significativa das proteínas, até mesmo do colágeno tipo XI, que tem localização mais interna na estrutura da fibrila heterotípica, e, portanto levando até a exposição do osso. Até o momento, o tratamento da osteoartrose está baseado principlamente no controle da dor e/ou inflamação, não diminuindo ou impedindo a degradação da cartilagem articular. Neste aspecto a perspectiva de tratamento futuro da osteoartrose estaria na utilização de inibidores das metaloproteases associadas a condroprotetores interferindo no "turnover" da cartilagem e impedindo, deste modo, o processo de degradação

The calcium-dependent protease of Loxosceles gaucho venom acts preferentially upon red cell band 3 transmembrane protein

Eighty micrograms red blood cell (RBC) ghosts from patients who had previously exhibited the cutaneous form of loxoscelism (presenting localized dermonecrosis) and the viscerocutaneous form of loxoscelism (presenting dermonecrosis, hemoglobinuria, hematuria, and jaundice) and from controls were incubated with 2.5 æg crude Loxosceles gaucho venom in 5 mM phosphate buffer, pH 7.4, at 37ºC. Among all membrane proteins, quantitative proteolysis of the important integral transmembrane protein 3 increased with venom dose and with incubation time from 30 to 120 min, as demonstrated by gel densitometry. Similar quantitative data were obtained for RBC ghosts from patients and from control subjects, a fact that argues against the possibility of genetic factors favoring the hemolytic viscerocutaneous form. These data suggest that the clinical forms may be different types of the same disease, with the viscerocutaneous form being the result of large amounts of intravascularly injected venom and the superficial form being the result of in situ venom action. Since protein 3 is a housekeeping integral membrane protein, whose genetic deficiency leads to hemolytic anemia, it is reasonable to relate it to the hemolysis which occurs in the viscerocutaneous form of loxoscelism. The venom protease responsible for the process was not inhibited after 120-min incubation by 0.2 mM paramethylsulfonyl fluoride or by 0.2 mM N-ethylmaleimide but was inhibited by 25 mM ethylenediaminetetraacetic acid (a calcium-chelating agent) in 5 mM phosphate buffer at pH 7.4, which suggests that the enzyme is a calcium-dependent metalloprotease

Von Willebrand factor cleaving protease activity in the physiopathology of microangiopathic disorders

The von Willebrand factor cleaving protease (VWFCP) modulates the von Willebrand factor (VWF) multimeric size in normal plasma. VWFCP activity levels are decreased in different physiological and pathologic situations. Different techniques have been developed to unfold the purified VWF (perfusion at high shear rate, dialysis against urea in nitrocellulose filters), to detect the VWFCP activity on it (multimeric analysis of VWF, collagen binding to VWF assay) and to use the patient plasma both as the source of the enzyme and substrate. In this paper we compared the above mentioned methods with new ones: normal plasma dialyzed on membranes instead of purified VWF, dialysis of the samples against urea in tubing instead of nitrocellulose filters, and sonicated plasma to remove the endogenous VWF. The perfusion assay and detection by multimeric analysis showed a limit of detection (25%) of VWFCP activity. Dialysis against urea in both supports and detection by multimeric analysis, showed a better limit of detection (3%), but the recovery of the samples was not as efficient in nitrocellulose filters as it was in tubing. The detection by collagen binding to VWF has more advantages because it allows to analyze more samples than the multimeric analysis does in the same assay. The dialysis of plasma by membranes to obtain the source of exogenous VWF requires no complex equipment. The method, which uses patient plasma as the source of the enzyme and substrate, was inapplicable in our experience because the values could not be interpolated in the reference curve

Expressão de metaloproteinases no adenoma pleomórfico. Análise através de zimografia e de imunofluorescência in vitro e de imunohistoquímica in vivo / Metalloproteinases expression in pleomorphic adenoma. Zimography and imunofluorescence in vitro analysis and imunohistochemistry in vivo analysis

O adenoma pleomórfico é um tumor benigno de origem na glândula salivar caracterizado por um crescimento lento e indolor. Histologicamente apresenta parênquima epitelial de arranjos variáveis, nos quais são vistas células luminais, mioepiteliais e plasmocitóides e um componente estromal rico, cuja gama de macromolélulas constituintes é extensa. Apesar da matriz extracelular tão rica, poucos estudos são realizados para elucidar seu padrão de invasão baseado na proteólise dessa matriz. Além disso, locais de erosão capsular do adenoma pleomórfico por provável proteólise e expressões variáveis em locais distintos de N-CAM nesta neoplasia, foram fatos que nos fizeram supor que o adenoma pleomórfico secreta metaloproteinases e estas devem contribuir para sua invasão. Por isso, realizamos estudo em cultura de células originadas de um adenoma pleomórfico de glândula submandibular, células AP5, e estudo in vivo através de biópsias de adenoma pleomórfico emblocados em parafina. In vitro estudamos os meios condicionados por 24 horas pelas células AP5 através do gel de gelatina em zimografia. Também em cultura dessas células foram feitas reações de imunofluorescência para as MMPs 1, 2, 3 e 9. No material emblocado também realizamos reações de imunohistoquímica para as MMPs 1, 2, 3 e 9. Como resultados verificamos através da zimografia que as células AP5 secretam as MMPs 9 latente e ativa e 2 latente. Na imunofluorescência as células AP5 foram positivas para MMPs 9, 2 e 1. Em imunohistoquímica o tumor foi positivo para MMPs 1, 2, 3 e 9, porém em expressões e padrões variáveis. Apesar da expressão de metaloproteinases no adenoma pleomórfico in vivo e in vitro, é cedo concluir que estas proteases são as principais responsáveis pelo padrão de invasão desta neoplasia, já que a invasão neoplásica é um processo complexo e dinâmico, o qual envolve múltiplas etapas.

Estresse oxidativo e ruptura da placa aterosclerótica / Oxidative stress and atherosclerotic plaque rupture

Inflamação, estresse oxidativo e disfunção do endotélio vascular constituem importantes fatores ligados à aterogênese, intermediando a relação entre fatores de risco e indução do ateroma. A inflamação, particularmente, é o fenômeno básico que determina a instabilização da placa, que culmina com ruptura do ateroma, trombose oclusiva e eventos clínicos agudos. Eventos inflamatórios na placa aterosclerótica incluem: infiltração de leucócitos mononucleares, aumento da expressão e atividade de metaloproteinases de matriz, aumento da expressão de fatores pró-trombóticos e morte celular programada (apoptose) de células musculares lisas e endoteliais. Todos esses fatores podem ser favorecidos ou mediados por estresse oxidativo, que é a produção excessiva ou não compensada de espécies reativas de oxigênio. O estresse oxidativo reduz a biodisponibilidade do óxido nítrico, está envolvido na oxidação da lipoproteína de baixa densidade e constitui segundo mensageiro intracelular de fatores de crescimento indutores de proliferação, secreção de matriz extracelular, diferenciação e senescência. O estresse oxidativo é um importante indutor do fator de transcrição NF-kappaB, que induz a transcrição de um programa gênico relacionado à inflamação. Recentemente vem sendo reconheci da a importância da via de sinalização CD40/CD40L no controle de todos esses processos. É interessante notar que muitos desses fenômenos são modulados por espécies reativas de oxigênio, sugerindo a participação do estresse oxidativo na ruptura da placa aterosclerótica...