Elastin remodeling: Does it play a role in priming the malignant phenotype of oral mucosa?

Background: The extracellular matrix (ECM) is a dynamic tissue that provides nutrition and support to overlying epithelium. During tumorigenesis, the tumor microenvironment (TME) dysregulates the ECM. This is reflected by morphological changes seen in collagen and elastic fibers and is thought to facilitate metastasis. Aim: To study the degradation of elastic fibers in different grades of oral squamous cell carcinoma (OSCC) and in oral epithelial dysplasia (OED) using histochemistry and to correlate it to the TNM stage of OSCC. Materials and Methods: Tumor cores from 38 cases of OSCC (well-differentiated[15], moderately differentiated[14], and poorly differentiated[9]) and 15 incisional biopsies of OED were analyzed. Hematoxylin-eosin and Verhoeff's–Van Gieson (VVG) stains were used. The stained sections were assessed for morphological changes in elastic fibers. Statistical Analysis: Data were analyzed using Statistical Package for Social Sciences (SPSS) version 22 software. Fisher's exact, Kruskal–Wallis, one-way ANOVA, and Turkey post hoc tests were used to establish significance (P ? 0.05). Spearman's correlation test was used to correlate elastin fiber degradation with TNM stage of OSCC. Results: All grades of OSCC showed absence of elastic fibers around the tumor islands. Elastic fiber degradation (fragmented and clumped type fibers) increased proportionately with the grade and TNM stage of OSCC. In OED, A significant reduction in the amount of elastic fibers with increasing grade was noted. Conclusion: A positive correlation was noted between elastin degradation and grade and stage of OSCC. Therefore, it may be implicated in tumor progression of OSCC.

Monitoring of urban and rural basins: water quality of Mourão basin / Monitoramento de bacias urbana e rural: análise integrada de qualidade de água da bacia hidrográfica do Rio Mourão

The Mourão River basin is located on the central western region of the Paraná State – Brazil, between coordinates 23º 44’ - 24º 25 South latitude and 52º 12’ - 52º 30’ West longitude, between 270 and 820 m above sea level, and 1,648.21 km2 drainage area. Water quality was evaluated by monitoring physical, chemical and microbiological parameters. Monthly samplings were performed for a year at five sites in the basin for analysis of: pH, temperature, dissolved oxygen, biochemical oxygen demand, total nitrogen, ammoniacal nitrogen, nitrite, nitrate, total phosphorus, turbidity, total solids, volatile solids and fecal coliforms. The results of the evaluated parameters showed higher values than the limits set by CONAMA Resolution 357 from 2005 for Class 2 in some samples. The Water Quality Index (WQI) indicated that 72% of samples had average quality and 28% had good quality for the Mourão River basin. Higher values of WQI were observed after rainfall period with median of 75 compared to the dry period with median of 62. The source of the Mourão River is contaminated with fecal coliforms, evidencing the real need to treat sewage in rural areas.

Resumo A Bacia Hidrográfica do Rio Mourão, localizada na porção centro ocidental do Estado do Paraná – Brasil, entre as coordenadas 23º 44’ - 24º 25’ Latitude Sul e 52º 12’ - 52º 30’ Longitude Oeste, entre 270 e 820 m acima do nível mar, possui área de drenagem de 1.648,21 km2. Nesta bacia foi avaliada a qualidade da água por meio de monitoramento de parâmetros físico-químicos e microbiológicos. Foram realizadas coletas mensais durante um ano em 5 pontos selecionados da bacia, sendo realizadas análises de: potencial Hidrogeniônico, temperatura, oxigênio dissolvido, demanda bioquímica de oxigênio, nitrogênio total, nitrogênio amoniacal, nitrito, nitrato, fósforo total, turbidez, sólidos totais, sólidos voláteis e coliformes fecais. Os resultados dos parâmetros avaliados indicaram valores superiores aos limites preconizados na Resolução CONAMA 357 de 2005 para rios de classe 2 em algumas amostras. Com a análise do Índice de Qualidade das Águas (IQA), obteve-se 72% das amostras com qualidade média e 28% com qualidade boa para a bacia do Rio Mourão. Os maiores valores do IQA foram observados na época de chuvas com mediana de 75 comparados ao período de seca com mediana de 62. Constatou-se que a nascente do rio Mourão está contaminada com coliformes fecais, mostrando a real necessidade de se tratar os esgotos sanitários no meio rural.

Multitemporal analysis of estimated soil loss for the river Mourão watershed, Paraná – Brazil / Análise multitemporal da perda de solo estimada para a bacia hidrográfica do rio Mourão, Paraná – Brasil

The multitemporal behavior of soil loss by surface water erosion in the hydrographic basin of the river Mourão in the center-western region of the Paraná state, Brazil, is analyzed. Forecast was based on the application of the Universal Soil Loss Equation (USLE) with the data integration and estimates within an Geography Information System (GIS) environment. Results had shown high mean annual rain erosivity (10,000 MJ.mm.ha–1.h–1.year–1), with great concentration in January and December. As a rule, soils have average erodibilities, exception of Dystroferric Red Latisol (low class) and Dystrophic Red Argisol (high class). Although the topographic factor was high (>20), rates lower than 1 were predominant. Main land uses comprise temporal crops and pasture throughout the years. The watershed showed a natural potential for low surface erosion. When related to usage types, yearly soil loss was also low (<50 ton.ha–1.year–1), with more critical scores that reach rates higher than 150 ton.ha–1.year–1. Soil loss over the years did not provide great distinctions in distribution standards, although it becames rather intensified in some sectors, especially in the center-eastern and southwestern sections of the watershed.

Resumo Este estudo visa analisar o comportamento multitemporal da perda de solo por erosão hídrica laminar na bacia hidrográfica do rio Mourão, localizada na região centro-ocidental do Estado do Paraná – Brasil. A predição foi baseada na aplicação da Equação Universal de Perdas de Solo (USLE), com integração dos dados e estimativas realizados em ambiente SIG (Sistema de Informações Geográficas). Os resultados mostram elevada erosividade da chuva média anual (10.000 MJ.mm.ha–1.h–1.ano–1). Os solos apresentam, em geral, média erodibilidade, com exceções do Latossolo Vermelho Distroférrico (classe baixa) e o Argissolo Vermelho Distrófico (classe alta). O fator topográfico atingiu índices elevados (>20), no entanto, predominaram os valores inferiores a 1. Os tipos de usos principais são as lavouras temporárias e pastagem, para todos os anos. A bacia apresentou Potencial Natural a Erosão laminar predominante baixo, quando relacionado aos tipos de usos resultou numa perda de solo anual estimada também baixa (<50 ton.ha–1.ano–1), com pontos mais críticos que atingem valores superiores a 150 ton.ha–1.ano–1. A perda de solo entre os anos não apresentou grandes distinções nos padrões de distribuição, apenas intensificou-se em alguns setores, como nas porções centro-leste e sudoeste da bacia.

Influence of urban area on the water quality of the Campo River basin, Paraná State, Brazil / Influência da área urbana na qualidade das águas na Bacia do Rio do Campo River, estado do Paraná, Brasil

The Campo River basin is located on the third plateau of the Paraná State or trap plateau of Paraná, at the middle portion between the rivers Ivaí and Piquiri, southern Brazil, between the coordinates 23° 53 and 24° 10’ South Latitude and 52° 15’ and 52° 31’ West Longitude. The basin has 384 Km² area, being 247 km2 in the municipality of Campo Mourão and 137 km2 in the municipality of Peabiru, in Paraná State. The Campo River is a left bank tributary of the Mourão River, which flows into the Ivaí River. The objective of this study was to monitor water quality in the Km 119 River and the Campo River, tributaries of the Mourão River, with monthly collection of water samples to determine pH, temperature, turbidity, biochemical oxygen demand, dissolved oxygen, fecal coliforms, total solids, total nitrogen, ammoniacal nitrogen, nitrite, nitrate and total phosphorus. The results obtained were compared with the indices established by the environmental legislation and applied in the determination of the Water Quality Index (WQI) used by the Water Institute of Paraná State, regulating environmental agency. Poor water quality in these rivers presents a worrying scenario for the region, since this river is the main source of water supply for the public system. Results of organic matter, fecal coliforms and total phosphorus were higher than the limits established by Resolution CONAMA 357/2005 to river class 2, specially at downstream of the Km 119 River and the Campo River, due to the significant influence of the urban anthropic activity by the lack of tertiary treatment and also rural by the lack of basic sanitation in this area. Results of WQI of Km 119 River and do Campo River indicated that water quality can be classified as average in 71% and good in 29% of the sites evaluated.

Resumo A Bacia Hidrográfica Rio do Campo está situada no terceiro planalto paranaense, na porção média entre os rios Ivaí e Piquiri, Sul do Brasil, entre as coordenadas 23° 53’ e 24°10’ de Latitude Sul e 52°15’ e 52°31’ de Longitude Oeste. A bacia ocupa área de 384 Km2, sendo 247 km2 no município de Campo Mourão e 137 km2 no município de Peabiru, no estado do Paraná. O Rio do Campo é um afluente da margem esquerda do Rio Mourão, que deságua no Rio Ivaí. O objetivo deste trabalho foi monitorar a qualidade da água do rio Km 119 e rio do Campo, afluentes do Rio Mourão, com coletas mensais de amostras de água para determinação do pH, temperatura, turbidez, demanda bioquímica de oxigênio, oxigênio dissolvido, coliformes fecais, sólidos totais, nitrogênio total, nitrogênio amoniacal, nitrito, nitrato e fósforo total. Os resultados obtidos foram comparados aos limites estabelecidos na legislação ambiental vigente para poluentes na água e aplicados na determinação do Índice de Qualidade das Águas (IQA) usado pelo Instituto das Águas do Paraná, órgão ambiental fiscalizador. A má qualidade observada nestes rios é preocupante para a região que usufruiu destas águas como principal fonte de abastecimento público. Resultados de matéria orgânica, coliformes totais e fósforo total extrapolaram os padrões estabelecidos na Resolução CONAMA 357/2005 para rio classe 2, especialmente à jusante do rio Km 119 e do Rio do Campo, devido à influência significativa da atividade antrópica urbana pela falta de tratamento terciário e rural pela falta de saneamento básico nesta área. Os resultados do IQA do Rio Km 119 e Rio do Campo indicam que as águas podem ser classificadas com qualidade média em 71% e boa em 29% nos pontos avaliados.

Influence of urban area on the water quality of the Campo River basin, Paraná State, Brazil

The Campo River basin is located on the third plateau of the Paraná State or trap plateau of Paraná, at the middle portion between the rivers Ivaí and Piquiri, southern Brazil, between the coordinates 23° 53 and 24° 10 South Latitude and 52° 15 and 52° 31 West Longitude. The basin has 384 Km² area, being 247 km2 in the municipality of Campo Mourão and 137 km2 in the municipality of Peabiru, in Paraná State. The Campo River is a left bank tributary of the Mourão River, which flows into the Ivaí River. The objective of this study was to monitor water quality in the Km 119 River and the Campo River, tributaries of the Mourão River, with monthly collection of water samples to determine pH, temperature, turbidity, biochemical oxygen demand, dissolved oxygen, fecal coliforms, total solids, total nitrogen, ammoniacal nitrogen, nitrite, nitrate and total phosphorus. The results obtained were compared with the indices established by the environmental legislation and applied in the determination of the Water Quality Index (WQI) used by the Water Institute of Paraná State, regulating environmental agency. Poor water quality in these rivers presents a worrying scenario for the region, since this river is the main source of water supply for the public system. Results of organic matter, fecal coliforms and total phosphorus were higher than the limits established by Resolution CONAMA 357/2005 to river class 2, specially at downstream of the Km 119 River and the Campo River, due to the significant influence of the urban anthropic activity by the lack of tertiary treatment and also rural by the lack of basic sanitation in this area. Results of WQI of Km 119 River and do Campo River indicated that water quality can be classified as average in 71% and good in 29% of the sites evaluated.

Resumo A Bacia Hidrográfica Rio do Campo está situada no terceiro planalto paranaense, na porção média entre os rios Ivaí e Piquiri, Sul do Brasil, entre as coordenadas 23° 53 e 24°10 de Latitude Sul e 52°15 e 52°31 de Longitude Oeste. A bacia ocupa área de 384 Km2, sendo 247 km2 no município de Campo Mourão e 137 km2 no município de Peabiru, no estado do Paraná. O Rio do Campo é um afluente da margem esquerda do Rio Mourão, que deságua no Rio Ivaí. O objetivo deste trabalho foi monitorar a qualidade da água do rio Km 119 e rio do Campo, afluentes do Rio Mourão, com coletas mensais de amostras de água para determinação do pH, temperatura, turbidez, demanda bioquímica de oxigênio, oxigênio dissolvido, coliformes fecais, sólidos totais, nitrogênio total, nitrogênio amoniacal, nitrito, nitrato e fósforo total. Os resultados obtidos foram comparados aos limites estabelecidos na legislação ambiental vigente para poluentes na água e aplicados na determinação do Índice de Qualidade das Águas (IQA) usado pelo Instituto das Águas do Paraná, órgão ambiental fiscalizador. A má qualidade observada nestes rios é preocupante para a região que usufruiu destas águas como principal fonte de abastecimento público. Resultados de matéria orgânica, coliformes totais e fósforo total extrapolaram os padrões estabelecidos na Resolução CONAMA 357/2005 para rio classe 2, especialmente à jusante do rio Km 119 e do Rio do Campo, devido à influência significativa da atividade antrópica urbana pela falta de tratamento terciário e rural pela falta de saneamento básico nesta área. Os resultados do IQA do Rio Km 119 e Rio do Campo indicam que as águas podem ser classificadas com qualidade média em 71% e boa em 29% nos pontos avaliados.

Multitemporal analysis of estimated soil loss for the river Mourão watershed, Paraná Brazil

The multitemporal behavior of soil loss by surface water erosion in the hydrographic basin of the river Mourão in the center-western region of the Paraná state, Brazil, is analyzed. Forecast was based on the application of the Universal Soil Loss Equation (USLE) with the data integration and estimates within an Geography Information System (GIS) environment. Results had shown high mean annual rain erosivity (10,000 MJ.mm.ha1.h1.year1), with great concentration in January and December. As a rule, soils have average erodibilities, exception of Dystroferric Red Latisol (low class) and Dystrophic Red Argisol (high class). Although the topographic factor was high (>20), rates lower than 1 were predominant. Main land uses comprise temporal crops and pasture throughout the years. The watershed showed a natural potential for low surface erosion. When related to usage types, yearly soil loss was also low ( 50 ton.ha1.year1), with more critical scores that reach rates higher than 150 ton.ha1.year1. Soil loss over the years did not provide great distinctions in distribution standards, although it becames rather intensified in some sectors, especially in the center-eastern and southwestern sections of the watershed.

Resumo Este estudo visa analisar o comportamento multitemporal da perda de solo por erosão hídrica laminar na bacia hidrográfica do rio Mourão, localizada na região centro-ocidental do Estado do Paraná Brasil. A predição foi baseada na aplicação da Equação Universal de Perdas de Solo (USLE), com integração dos dados e estimativas realizados em ambiente SIG (Sistema de Informações Geográficas). Os resultados mostram elevada erosividade da chuva média anual (10.000 MJ.mm.ha1.h1.ano1). Os solos apresentam, em geral, média erodibilidade, com exceções do Latossolo Vermelho Distroférrico (classe baixa) e o Argissolo Vermelho Distrófico (classe alta). O fator topográfico atingiu índices elevados (>20), no entanto, predominaram os valores inferiores a 1. Os tipos de usos principais são as lavouras temporárias e pastagem, para todos os anos. A bacia apresentou Potencial Natural a Erosão laminar predominante baixo, quando relacionado aos tipos de usos resultou numa perda de solo anual estimada também baixa ( 50 ton.ha1.ano1), com pontos mais críticos que atingem valores superiores a 150 ton.ha1.ano1. A perda de solo entre os anos não apresentou grandes distinções nos padrões de distribuição, apenas intensificou-se em alguns setores, como nas porções centro-leste e sudoeste da bacia.

Monitoring of urban and rural basins: water quality of Mourão basin

The Mourão River basin is located on the central western region of the Paraná State Brazil, between coordinates 23º 44 - 24º 25 South latitude and 52º 12 - 52º 30 West longitude, between 270 and 820 m above sea level, and 1,648.21 km2 drainage area. Water quality was evaluated by monitoring physical, chemical and microbiological parameters. Monthly samplings were performed for a year at five sites in the basin for analysis of: pH, temperature, dissolved oxygen, biochemical oxygen demand, total nitrogen, ammoniacal nitrogen, nitrite, nitrate, total phosphorus, turbidity, total solids, volatile solids and fecal coliforms. The results of the evaluated parameters showed higher values than the limits set by CONAMA Resolution 357 from 2005 for Class 2 in some samples. The Water Quality Index (WQI) indicated that 72% of samples had average quality and 28% had good quality for the Mourão River basin. Higher values of WQI were observed after rainfall period with median of 75 compared to the dry period with median of 62. The source of the Mourão River is contaminated with fecal coliforms, evidencing the real need to treat sewage in rural areas.

Resumo A Bacia Hidrográfica do Rio Mourão, localizada na porção centro ocidental do Estado do Paraná Brasil, entre as coordenadas 23º 44 - 24º 25 Latitude Sul e 52º 12 - 52º 30 Longitude Oeste, entre 270 e 820 m acima do nível mar, possui área de drenagem de 1.648,21 km2. Nesta bacia foi avaliada a qualidade da água por meio de monitoramento de parâmetros físico-químicos e microbiológicos. Foram realizadas coletas mensais durante um ano em 5 pontos selecionados da bacia, sendo realizadas análises de: potencial Hidrogeniônico, temperatura, oxigênio dissolvido, demanda bioquímica de oxigênio, nitrogênio total, nitrogênio amoniacal, nitrito, nitrato, fósforo total, turbidez, sólidos totais, sólidos voláteis e coliformes fecais. Os resultados dos parâmetros avaliados indicaram valores superiores aos limites preconizados na Resolução CONAMA 357 de 2005 para rios de classe 2 em algumas amostras. Com a análise do Índice de Qualidade das Águas (IQA), obteve-se 72% das amostras com qualidade média e 28% com qualidade boa para a bacia do Rio Mourão. Os maiores valores do IQA foram observados na época de chuvas com mediana de 75 comparados ao período de seca com mediana de 62. Constatou-se que a nascente do rio Mourão está contaminada com coliformes fecais, mostrando a real necessidade de se tratar os esgotos sanitários no meio rural.

Staphylococcus aureus e estafilococos coagulase negativosresistentes à vancomicina em um Hospital Universitário Brasileiro / Vancomycin-resistant Staphylococcus aureus and coagulase-negative staphylococci in a Brazilian University Hospital

Infecções hospitalares por Methicillin-Resistant S. aureus (MRSA) e Methicillin-Resistant Coagulase-Negative Staphylococci (MRCoNS) estão entre as mais frequentes mundialmente, justificando um aumento significativo no uso de vancomicina. Com o objetivo de avaliar a presença de estafilococos resistentes aos glicopeptídeos em pacientes em uso terapêutico desse antimicrobiano, internados no Hospital de Clínicas da Universidade Federal de Uberlândia, Uberlândia MG, foi realizado um estudo longitudinal prospectivo incluindo 41 pacientes, 21 adultos e 20 crianças, entre dezembro de 2000 e março de 2002. O monitoramento microbiológico foi realizado por meio de swabs coletados a partir da cavidade bucal e reto e cultivo primário em Ágar Manitol Salgado acrescido de 6 mg/mL de oxacilina. Amostras selecionadas foram testadas quanto à sensibilidade aos glicopeptídeos pelas técnicas de gel difusão e diluição em ágar e a análise de heterorresistência, pela semeadura utilizando inóculo correspondente à escala 0,5 de McFarland (108 UFC/mL) e análise do perfil populacional. Um único paciente, nefropata em programa de hemodiálise apresentou-se colonizado com uma amostra do fenótipo Vancomycin-Intermediate S. aureus (VISA) (CIM = 8 miug/mL) e em doze, foram isoladas amostras de estafilococos heterorresistentes, correspondendo oito hVISA e quatro hVICoNS. O estudo do perfil populacional, confirmou a presença de subpopulações de células resistentes, sendo seis hVISA e duas hVICoNS. A presença de amostras heterorresistentes à vancomicina pode representar um risco potencial no futuro.

Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-resistant Coagulase-negative Staphylococci (MRCoNS) are among the most frequent hospital infections around the world, and are associated with a significant rise in the use of vancomycin. In order to assess the presence of staphylococci resistant to glycopeptides in patients taking this antibiotic in Uberlândia Federal University Hospital, Uberlândia (MG, Brazil), a prospective longitudinal study of 41 patients (21 adults and 20 children) was performed between December 2000 and March 2002. Microbiological monitoring was carried out by means of swabs collected from the oral cavity and rectum, screened by culturing in Salt Mannitol Agar plus 6 ?g/mL oxacillin. Selected samples were tested for susceptibility to glycopeptides, by the techniques of gel diffusion and dilution in agar, and for heteroresistance, by seeding with an inoculum at a density of 0.5 on the McFarland scale (108 CFU/mL) and performing a population analysis profile (PAP). A single nephropathy patient was colonized with a strain of vancomycin intermediate S. aureus (VISA) phenotype (MIC = 8 miug/mL). In twelve patients, heteroresistant staphylococci were isolated, corresponding to eight hVISA and four hVICoNS. The PAP study confirmed the presence of six hVISA and two hVICoNS. The presence of heteroresistant vancomycin samples may pose a potential risk in the future.

Hospital surface contamination in wards occupied by patients infected with MRSA or MSSA in a Brazilian university hospital

The hospital environment, especially surfaces, represents a secondary reservoir for pathogens such as Staphylococcus aureus. The present study was carried out at the Hospital de Clínicas da Universidade Federal de Uberlândia (HC-UFU) in Brazil, from January to August 2004, with the aim of qualitatively and quantitatively assessing the presence of S. aureus on surfaces touched by hands (bed rail, bedside table and door handle), on the floor and in the air, in wards where the patients were infected or not infected with this organism. Twenty-six wards with at least one infected patient and 26 wards with uninfected patients were investigated. Surfaces and air were sampled during bedmaking, by means of sterile 10cm2 adhesive tapes and 90mm-diameter exposed culture plates, respectively. Cultures were carried out on Egg Yolk Salt Agar, containing 7.5% NaCl and 1% egg yolk, and colonies identified by lecithinase and coagulase tests and Gramstain. About 50% of the wards were S. aureus contaminated, without significant differences between the groups (46.1% in the infected patient wards versus 53.8% in controls); the correlation between counts of the pathogen on the surfaces and in the air was 66.7%. Inspite of the fact that these bacteria were frequently present in the assessed wards, the microbial density waslow (less than 1 CFU/cm2). The association between environmental contamination and the epidemiology of S. aureus nosocomial infections is complex and thus further investigations are needed to reach a better understanding of this relationship.

Behavioral and electroencephalographic analysis of seizures induced by intrahippocampal injection of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

In this study, the behavioral and electroencephalographic (EEG) analysis of seizures induced by the intrahippocampal injection in rats of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera, was determined. The first alterations occurred during microinjection of granulitoxin (8 µg) into the dorsal hippocampus and consisted of seizure activity that began in the hippocampus and spread rapidly to the occipital cortex. This activity lasted 20-30 s, and during this period the rats presented immobility. During the first 40-50 min after its administration, three to four other similar short EEG seizure periods occurred and the rats presented the following behavioral alterations: akinesia, facial automatisms, head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements. Within 40-50 min, the status epilepticus was established and lasted 8-12 h. These results are similar to those observed in the acute phase of the pilocarpine model of temporal lobe epilepsy and suggest that granulitoxin may be a useful tool not only to study the sodium channels, but also to develop a new experimental model of status epilepticus.

A liver metalloendopeptidase which degrades the circulating hypotensive peptide hormones bradykinin and atrial natriuretic peptide

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human liver using successive steps of chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-200. The purified enzyme hydrolyzed the Pro7-Phe8 bond of bradykinin and the Ser25-Tyr26 bond of atrial natriuretic peptide. No cleavage was produced in other peptide hormones such as vasopressin, oxytocin or Met- and Leu-enkephalin. This enzyme activity was inhibited by 1 mM divalent cation chelators such as EDTA, EGTA and o-phenanthroline and was insensitive to 1 µM phosphoramidon and captopril, specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. With Mr 85 kDa, the enzyme exhibits optimal activity at pH 7.5. The high affinity of this endopeptidase for bradykinin (Km = 10 µM) and for atrial natriuretic peptide (Km = 5 µM) suggests that it may play a physiological role in the inactivation of these circulating hypotensive peptide hormones

Partial sequence and toxic effects of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

A neurotoxic peptide, granulitoxin (GRX), was isolated from the sea anemone Bunodosoma granulifera. The N-terminal amino acid sequence of GRX is AKTGILDSDGPTVAGNSLSGT and its molecular mass is 4958 Da by electrospray mass spectrometry. This sequence presents a partial degree of homology with other toxins from sea anemones such as Bunodosoma caissarum, Anthopleura fuscoviridis and Anemonia sulcata. However, important differences were found: the first six amino acids of the sequence are different, Arg-14 was replaced by Ala and no cysteine residues were present in the partial sequence, while two cysteine residues were present in the first 21 amino acids of other toxins described above. Purified GRX injected ip (800 µg/kg) into mice produced severe neurotoxic effects such as circular movements, aggressive behavior, dyspnea, tonic-clonic convulsion and death. The 2-h LD50 of GRX was 400 ñ 83 µg/kg

A new brain metalloendopeptidase which degrades the Alzheimer beta-amyloid 1-40 peptide producing soluble fragments without neurotoxic effects

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human brain using successive steps of chromatography on DEAE-Trisacryl, hydroxylapatite and Sephacryl S-200. The purified enzyme cleaved the Gly(33)-Leu(34) bond of the 25-35 neurotoxic sequence of the Alzheimer Beta-amyloid 1-40 peptide producing soluble fragments without neurotoxic effects. This enzyme activity was only inhibited by divalent cation chelators such as EDTA, AGTA and o-phenanthroline (1 mM) and was insensitive to phosphoramidon and captopril (1 muM concentration), specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. The high affinity of this human brain endopeptidase for Beta-amyloid 1-40 peptide (Km = 5 muM) suggests that it may play a physiological role in the degradation of this substance produced by normal cellular metabolism. It may also be hypothesized that the abnormal accumulation of the amyloid Beta-protein in Alzheimer´s disease may be initiated by a defect or an inactivation of this enzyme.

Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Two intramolecularly quenched fluorogenic peptides containing oaminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid rsidues, Abz-Darg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were Km = 2.8muM, Kcat = 5.3 min-1, Kcat/Km = 2 min-1 muM-1 and Km = 5.0 muM, Kcat = 7.0 min-1, Kcat/Km = 1.4 min-1 muM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE;EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by alpha-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations.

Metabolism of diglycine and triglycine by non-filtering kidneys

We have studied the metabolism of diglycine and triglycine in the isolated non-filtering rat kidney. Kidneys from adult male Wistar Kyoto rats weighing 250-350 g were perfused with Krebs-Henseleit solution containing either 1 mM diglycine or triglycine. The analysis of the peptide residues and their components was performed using an amino acid microanalyzer utilizing ion exchange chromatography. Diglycine was degraded to a final concentration of 0.09 mM after 120 min (91 per cent); this degradation occurred predominantly during the first hour, with a 56 per cent reduction of the initial concentration. The metabolism of triglycine occurred similarly, with a final concentration of 0.18 mM (82 per cent); during the first hour there was a 67 per cent reduction of the initial concentration of the tripeptide. Both peptides produced glycine in increasing concentrations, but there was a slightly lower recovery of glycine, suggesting its utilization by the kidney as fuel. The hydrolysis of triglycine also produced diglycine, which was also hydrolyzed to glycine. The results of the present study show the existence of functional endothelial or contraluminal membrane peptidases which may be important during parenteral nutrition.

A new fluorometric assay for neutral endopeptidase (EC 3.4.24.11)

An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, Abz-GGdFLRRV-EDDnp, was selectively hydrolyzed at the R-V bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 µM,Kcat = 127 / min and Kcat / Km = 42 / min µM) similar to those of Leu-enkephalin. The specificity of the assay for NEP was demostrated by incubating Abz-GGdFLRRV-EDDnp with a kidney homogenate and with crude membrane preparations of brain and lung. For all three homogenates the complementary fragments Abz-GGdFLRRnp accounted for more than 95 percent of the products wich were totally inhibited by 1 µM thiorphan, a highly specific NEP inhibitor. A continuous fluorometric assay for only 5 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified NEP or the equivalent enzymatic activity in crude tissue preparations.

Isolation and properties of a multicatalytic proteinase complex from Xenopus laevis skin secretion

A multicatalytic proteinase complex present in the skin secretion of Xenopus laevis was purified and its enzymatic activity towards natural and synthetic peptides was investigated. We identified three activities: i) a C-terminal deamidation enzyme activity which exhibited selectivity for the Asp-Phe-NH2 and Phe-Leu-NH2 motifs of cerulein, minigastrin Leu-enkephalinamide, (des-Tyr1)Leu-enkephalinamide and diaminobenzylthiocyanate-DVDERDVRGFASFL NH2 (DABTCDR8kermit); ii) an endopeptidase activity that cleaves peptide bonds on the carboxyl side of hydrophobic amino acid residues such as Tyr-Gly of LHRH, Ile-Ala of PGLa and Leu-Ala of buccalin; iii) an enzyme activity that cleaves peptide bonds at the dibasic sites of peptides of the dynorphin family. The molecular weight determined by Sephacryl S-400 molecular sieve filtration indicated an Mr about 600 kDa. The activities characterized here exhibit and optimal pH of about 7.4. The activities of the multicatalytic complex were differentially inhibited by the classical inhibitors of proteases

Human neuroblastoma cells express a novel metallo-endopeptidase activity able to inactivate atrial natriuretic factor: inhibition during retinoic acid-induced differentiation

A new metallo-endopeptidase which hydrolyzes atrium natriuretic factor (ANF) has been isolated from human neuroblastoma NB-OK-1 cells. In the present study we show that this metallo-endopeptidase is also present in several other human neuroblastoma cell lines, which include CHP 100, SH-SY5Y, SK-N-BE(2), BE(2)-C and BE(2)M-17. Additionally, we show that this endopeptidase activity is reduced to about 20 per cent of the control during retinoic acid (RA)-induced neuronal differentiation in the RA-sensitive SK-N-BE(2) cells, but not in the RA-resistant BE(2)-M17 cells. This suggests that the inhibition is related to neuronal differentiation and not to a direct effect of 5 microM RA on the enzyme activity. This new enzyme is clearly distinct from neutral endopeptidase (NEP, EC 3.4.24.11) and angiotensin-converting enzyme (ACE,EC 3.4.15.1), since specific inhibitors for these endopeptidases (10 microM phosphoramidon and 1 mM captopril, respectively) had no effect on their activity. However, this enzyme was inhibited 100 per cent by 10 mM o-phenanthroline showing an inhibitory spectrum similar to that of another novel metallo-endopeptidase recently isolated in our laboratory from Xenopus laevis skin secretion. Although the physiological function of this new enzyme in human neuroblastoma cells is not known at the present time, we suggest that it may participate in inactivation of neuropeptides such as atrium natriuretic factor (ANF), substance P, somatostatin-14 and bradykinin in vivo

Metabolism of dipeptides by isolated and perfused nonfiltering kidneys

Rehal metabolism of Glycyl-glycine (Gly-gly), Glycyl-proline (Gly-pro) and Prolyl-glycine (Pro-gly) was studied in the non-filtering, isolated perfused rat kidney. Gly-gly is metabolized by more than 90% after 120 min of perfusion. Gly-pro is more resistant to degradation and about 75% of the original peptide can be found intact in the perfusate at the end of perfusion. For Pro-gly only 25% reamins intact at the end of the experiment. Glycine was also monitored as another marker for dipepptide degratation and ins production increased throughout the perfusion time. In some experiments we also determined the production of proline. We conclude from these experiments that the basolateral membrane, or perhaps the kidney blood vessels, posses an efficient apparatus for the hydrolysis of Gly-gly and Pro-gly. This mechanism is less efficient in the case of Gly-pro. This confirms an earlier hypothesis that dipeptide metabolism does not occur solely in the brush-border membranes

Causas de excisao de globos oculares pos-ferimento perfurante. / Causes of ocular excision following penetrating injuries

Os autores apresentam as causas de excisao de 39 olhos de 768 pacientes com ferimento perfurante ocular. Lembram que nenhuma excisao ocorreu por receio de oftalmia simpatica. A excisao em l8 casos foi determinada pela ocorrencia de FPO com prolapso irremediavel do material intra-ocular, em 17 casos por endoftalmite nao controlavel clinicamente, em 3 por "phthisis bulbi" e em 1 por hemoftalmo e CEIO. Ressaltam que a conduta inicial deve ser conservadora, destacando que mesmo olhos severamente lesados podem ser recuperados atraves de cirurgias combinadas