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The present paper deals with the phlebotomine species captured during the period from January 1998 to June 2000 in 12 caves located in the Serra da Bodoquena, situated in the south central region of Mato Grosso do Sul State, Brazil. Three of the caves are situated further north (in Bodoquena county), seven in the central area (Bonito county) and two in the south (Jardim county). These last two caves and three of those in Bonito are located at the west side of the ridge. Eighteen species of phlebotomines were captured within the caves: Brumptomyia avellari (Costa Lima, 1932), Brumptomyia brumpti (Larrousse, 1920), Brumptomyia cunhai (Mangabeira, 1942), Brumptomyia galindoi (Fairchild & Hertig, 1947), Evandromyia corumbaensis (Galati, Nunes, Oshiro & Rego, 1989), Lutzomyia almerioi Galati & Nunes, 1999, Lutzomyia longipalpis (Lutz & Neiva, 1912), Martinsmyia oliveirai (Martins, Falcão & Silva, 1970), Micropygomyia acanthopharynx (Martins, Falcão & Silva, 1962), Micropygomyia peresi (Mangabeira, 1942), Micropygomyia quinquefer (Dyar, 1929), Nyssomyia whitmani (Antunes & Coutinho, 1939), Psathyromyia campograndensis (Oliveira, Andrade-Filho, Falcão & Brazil, 2001), Psathyromyia punctigeniculata (Floch & Abonnenc, 1944), Psathyromyia shannoni (Dyar, 1929), Pintomyia kuscheli (Le Pont, Martinez, Torrez-Espejo & Dujardin, 1998), Sciopemyia sordellii (Shannon & Del Ponte, 1927) and Sciopemyia sp. A total of 29,599 phlebotomine sandflies was obtained. Lutzomyia almerioi was absolutely predominant (91.5%) over the other species on both sides of the Bodoquena ridge, with the exception of the southern caves in which it was absent. It presents summer predominance, with nocturnal and diurnal activities. The species breeds in the caves and was captured during daytime both in the dark area and in the mouth of the caves. Martinsmyia oliveirai, the second most frequent sandfly, also presents a summer peak and only predominated over the other species in one cave, in which there were human residues.0

OBJETIVO - Avaliar o efeito dos ésteres de fitoesteróis (FE) da dieta, em função dos diferentes genótipos de apo E, sobre os lípides plasmáticos em indivíduos moderadamente hipercolesterolêmicos. MÉTODOS - Pacientes moderadamente hipercolesterolêmicos (20 a 60 anos de idade; 50 mulheres e 10 homens), mantidos em dieta habitual, receberam margarina (20g/dia) enriquecida com fitoesteróis (2,8g/dia=1,68g de FE), ou margarina normal (placebo), por dois períodos de quatro semanas cada, em estudo duplo cego e cruzado. RESULTADOS - Fitoesteróis reduziram significativamente o colesterol total e o LDL-colesterol, respectivamente, em 10% e 12% quando comparado à fase basal de admissão ao programa, e 6% e 8% quando comparado à fase placebo. O HDL-colesterol e a trigliceridemia não se modificaram. CONCLUSÃO - Ésteres de fitoesteróis da dieta reduziram a colesterolemia e a redução do LDL-colesterol foi mais pronunciada nos indivíduos que apresentavam valores maiores de LDL-colesterol, quando admitidos no estudo. Em relação ao sinótipo de apo E não houve diferença significativa entre apo E 3/3 e apo E 3/4.

O presente estudo visou estabelecer a prevalência de Cryptosporidium parvum em crianças abaixo de 5 anos, residentes na zona urbana de Campo Grande, MS, 1996/97, através de exames coprológicos e avaliar epidemiologicamente os casos diagnosticados. Tratou-se de um estudo transversal com inquérito domiciliar, onde foram examinadas 1051 amostras fecais, processadas segundo a técnica de Blagg, utilizando-se a coloração de Ziehl-Neelsen modificada para a pesquisa de oocistos de C. parvum. Concluiu-se que: a prevalência de C. parvum (1,1%) observada não foi estatisticamente significativa; foi relatado diarréia em 58,3% das crianças com diagnóstico positivo, supondo-se associação entre diarréia e a presença do parasita; o C. parvum foi mais freqüente em crianças com idade de 25 a 36 meses (50%), porém sem diferença estatisticamente significativa; o sexo não teve papel diferencial em relação ao parasitismo por C. parvum; entre as 12 crianças com criptosporidiose, 83,3% tiveram contato com animais domésticos (cão e ou gato).

INTRODUÇÃO: Nas Américas, Lutzomyia longipalpis tem sido incriminada como vetora da leishmaniose visceral em, praticamente, todas as áreas de ocorrência dessa parasitose. A notificação de casos humanos a partir de 1980 e a presença de cães com aspecto sugestivo de leishmaniose visceral no Município de Corumbá, Estado de Mato Grosso do Sul, Brasil, levaram a investigações entomológicas na área, com o objetivo de identificar a população de flebotomíneo vetora. MATERIAL E MÉTODO: A pesquisa foi realizada no peri e intradomicílio de três residências urbanas e em ecótopo natural, representado por uma gruta, situado fora do perímetro urbano. As capturas, semanais em sua maioria, foram realizadas com armadilhas automáticas luminosas, no período de 1984 a 1986. Os dados metereológicos desse período foram obtidos junto à Estação Metereológica da cidade e os de 1925 a 1982, de bibliografia. RESULTADOS: A fauna flebotomínea urbana, composta de oito espécies, mostrou-se semelhante à da gruta, porém nesta, a abundância das espécies foi maior. Na área urbana, Lu. cruzi predominou tanto no intra como no peridomicílio: no bairro central, representou 90,3% dos espécimens e nos dois bairros periféricos, os seus percentuais foram menores. Lu. forattinii, também, teve freqüência expressiva em um dos bairros periféricos (39,0 %). Na gruta, Lu. corumbaensis foi a espécie predominante. Comenta-se o impacto das condições climáticas e do inseticida aplicado na área urbana na freqüência das espécies, e da utilização da gruta como criadouro pelos flebotomíneos, com base na evolução da razão entre os sexos ao longo do período. Foram adicionadas informações sobre antropofilia e de coleta com isca canina de Lu. forattinii. CONCLUSÃO: O predomínio de Lu. cruzi na área urbana; a expressiva freqüência de Lu. forattinii na periferia da cidade, bem como a sua antropofilia e o estreito grau de parentesco destas espécies com Lu. longipalpis, a principal vetora da leishmaniose visceral em outras áreas da América, são aspectos que sugerem a participação de ambas na transmissão da doença, em Corumbá.

Desenvolveram-se estudos sobre flebotomíneos em área de leishmaniose tegumentar, fazenda Boa Sorte, Município de Corguinho, Mato Grosso do Sul, Brasil, com vistas a incriminar vetor dessa parasitose. No início dos estudos, encontravam-se bem preservados vários tipos da cobertura vegetal primitiva, com predomínio de cerrado e cerradão, denominado localmente de "croa". Decorridos quatro meses, parte significativa da "croa" e do cerrado foi queimada para transformação em áreas de pastagens. Durante julho/1991 a junho/93, realizaram-se coletas semanais das 18:00 às 6:00 horas, com armadilha CDC (Center on Disease Control), em floresta-galeria, floresta de encostas, cerrado, "croa", peridomicílio (chiqueiro e poleiro) e no interior de uma tulha; coletas mensais com armadilha de Shannon das 18:00 às 24:00 horas em floresta-galeria e "croa". De junho/91 a setembro de 1992, capturas mensais com isca humana, por 24 horas, em floresta-galeria. Investigou-se infecção natural por flagelados em flebotomíneos coletados com armadilha de Shannon e isca humana. As coletas com CDC resultaram 24 espécies de Lutzomyia e duas de Brumptomyia. A "croa" foi o ambiente que mais contribuiu com espécimens e que apresentou a maior diversidade, juntamente com a floresta de encostas. Nas coletas com CDC, L. whitmani revelou-se a mais abundante, índice de abundância padronizado = 0,991; porém, esteve muito pouco representada no interior do anexo domiciliar; apresentou prevalência de 96,0% nas armadilhas de Shannon e isca humana, respectivamente com 3.265 e 516 espécimens. Sua maior freqüência deu-se em épocas frias e secas. Dotada de atividade quase que exclusivamente noturna, exibiu pico de ocorrência das 18:00 às 19:00 horas. A taxa de infecção natural por flagelados, em 680 fêmeas de flebotomíneos dissecadas, foi de 0,15% e, entre 613 fêmeas de L. whitmani, de 0,16%. Com base em seu comportamento, L. whitmani foi incriminada como provável vetora da leishmaniose tegumentar na área. A segunda espécie mais abundante, L. lenti, não demonstrou antropofilia. Apresenta-se também a fauna flebotomínica por ambientes.

In the Amazon region the phlebotomine fauna is considered one of the most diverse in the world. The use of Shannon traps may provide information on the anthropophily of the species and improve the traps’ performance in terms of diversity and quantity of insects collected when white and black colored traps are used together. This study sought to verify the attractiveness of the traps to the phlebotomine species of the Brazilian Amazon basin using Shannon traps under these conditions. The insects were collected using two Shannon traps installed side by side, one white and the other black, in a primary forest area of the municipality of Xapuri, Acre, Brazil. Samples were collected once a month during the period August 2013 to July 2015. A sample of females was dissected to test for natural infection by flagellates. A total of 6,309 (864 males and 5,445 females) specimens (36 species) were collected. Psychodopygus carrerai carrerai (42%), Nyssomyia shawi (36%), and Psychodopygus davisi (13%), together represented 90% of the insects collected. Nyssomyia shawi and Psychodopygus davisi were more attracted by the white color. Specimens of Nyssomyia shawi, Nyssomyia whitmani, and Psychodopygus hirsutus hirsutus were found naturally infected by flagellates in the mid and hindgut. This is the first study in Acre state using and comparing both black and white Shannon traps, demonstrating the richness, diversity, and anthropophilic behavior of the phlebotomine species and identifying proven and putative vectors of the etiological agents of leishmaniasis.

The phlebotomine fauna of Campo Grande city, capital of Mato Grosso do Sul state in Brazil, an endemic area for visceral leishmaniasis, has been thoroughly investigated, but all the insect collections were undertaken with automatic light traps. The present study sought to investigate the fauna in this city using Shannon and Disney traps, having human beings and hamsters, respectively, as bait. Both types of traps were installed in forest fragment and peridomiciliary areas in the period from 2007 to 2009. The phlebotomine females were analyzed by PCR for Leishmania identification. Lutzomyia longipalpis was the only species collected in the peridomiciles and rendered a total of 574 specimens with a 5.2:1 male:female ratio. A total of eight species were attracted to the two traps (one of each type) installed in the forest fragment, including: Bichromomyia flaviscutellata, Evandromyia bourrouli, Evandromyia lenti, Lutzomyia longipalpis, Nyssomyia whitmani, Pintomyia christenseni, Psathyromyia bigeniculata, and Sciopemyia sordellii. A total of 143 specimens were collected, Bi. flaviscutellata accounting for 81% and Lu. longipalpis for 1.4% of them. In one female of Lu. longipalpis collected in a Disney trap installed in a peridomicile, Leishmania (Leishmania) infantum DNA was found, thus strengthening the hypothesis that the transmission of leishmaniasis is in fact occurring in the anthropic environment.

Abstract Background Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7alpha- hydroxycholesterol and 7beta- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. Methods Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. Results In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. Conclusion In macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus.

Relata-se um caso de raiva em morcego insetívoro, Lasyurus borealis, na região urbana, em bairro residencial, no Município de Jundiaí, SP (Brasil).

Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. FAM3B, a secreted protein, has been extensively studied in various types of tumors. However, its function in breast cancer remains poorly understood., Methods: We analyzed FAM3B expression data from breast cancer patients available at TCGA database and overall survival was analyzed by using the Kaplan-Meier plotter. MDA-MB-231 TNBC tumor cell line and hormone-responsive MCF-7 cell lines were transfected to overexpress FAM3B. We assessed cell death, tumorigenicity, and invasiveness in vitro through MTT analysis, flow cytometry assays, anchorage-independent tumor growth, and wound healing assays, respectively. We performed in vivo evaluation by tumor xenograft in nude mice., Results: In silico analysis revealed that FAM3B expression was lower in all breast tumors. However, TNBC patients with high FAM3B expression had a poor prognosis. FAM3B overexpression protected MDA-MB-231 cells from cell death, with increased expression of Bcl-2 and Bcl-xL, and reduced caspase-3 activity. MDA-MB-231 cells overexpressing FAM3B also exhibited increased tumorigenicity and migration rates in vitro, displaying increased tumor growth and reduced survival rates in xenotransplanted nude mice. This phenotype is accompanied by the upregulation of EMT-related genes Slug, Snail, TGFBR2, vimentin, N-cadherin, MMP-2, MMP-9, and MMP-14. However, these effects were not observed in the MCF-7 cells overexpressing FAM3B., Conclusion: FAM3B overexpression contributes to tumor growth, promotion of metastasis, and, consequently, leads to a poor prognosis in the most aggressive forms of breast cancer. Future clinical research is necessary to validate FAM3B as both a diagnostic and a therapeutic strategy for TNBC., Competing Interests: Disclosure The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Currently, there is a great need for the development of three-dimensional (3D) in vitro lung models. Particularly, the production of a suitable 3D model of pulmonary epithelium for understanding the pathophysiology of diseases such as the COVID-19 must consider the tissue architecture and presence, for example, of the angiotensin-converting enzyme-2 (ACE-2) in the cells. Different polymeric membranes are being used to support cell culturing, especially of lung cells, however, there is still no information about the culture of these cells onto bacterial nanocellulose (BNC) matrices. We have used the BNC matrix CellFate® as a support for the assembly of a 3D in vitro model of lung epithelium, composed of human lung fibroblasts (HLF) and lung adenocarcinoma cells (CALU-3). CellFate® matrices were made from bacterial fermentation resulting in a natural and biocompatible biopolymer. Cells were cultured onto CellFate® and maintained in a 5% CO 2 humidified atmosphere at 37°C. Cell viability was assessed by the resazurin method The samples were, then, exposed to the air-liquid interface (ALI), and histologically analyzed. ACE-2 activity was verified on the hydrolyze of the fluorogenic substrate Mca-APK(Dnp)-OH, and its presence was evaluated by flow cytometry. The expression of the anionic transporter SLCO3A1 was evaluated by qPCR. Cell viability analysis indicates that CellFate® was not toxic to these cells. By flow cytometry, the presence of the ACE-2 was identified in the CALU-3 cells surface corroborating the results obtained from enzymatic activity analysis. The SLCO3A1 transporter expression was identified in cells cultured onto CellFate®, but not in cells cultured onto the transwell (control). CALU-3 cells cultivated onto CellFate® resulted in a pseudostratified organization, a typical morphology of the human respiratory tract epithelium. The current model opens perspectives for studies involving physiological characterization, improving its relevance for the understanding of the pathophysiology of diseases as well as the response to drugs., (© 2023 Wiley Periodicals LLC.)

Mucosal vaccination appears to be suitable to protect against SARS-CoV-2 infection. In this study, we tested an intranasal mucosal vaccine candidate for COVID-19 that consisted of a cationic liposome containing a trimeric SARS-CoV-2 spike protein and CpG-ODNs, a Toll-like receptor 9 agonist, as an adjuvant. In vitro and in vivo experiments indicated the absence of toxicity following the intranasal administration of this vaccine formulation. First, we found that subcutaneous or intranasal vaccination protected hACE-2 transgenic mice from infection with the wild-type (Wuhan) SARS-CoV-2 strain, as shown by weight loss and mortality indicators. However, when compared with subcutaneous administration, the intranasal route was more effective in the pulmonary clearance of the virus and induced higher neutralizing antibodies and anti-S IgA titers. In addition, the intranasal vaccination afforded protection against gamma, delta, and omicron virus variants of concern. Furthermore, the intranasal vaccine formulation was superior to intramuscular vaccination with a recombinant, replication-deficient chimpanzee adenovirus vector encoding the SARS-CoV-2 spike glycoprotein (Oxford/AstraZeneca) in terms of virus lung clearance and production of neutralizing antibodies in serum and bronchial alveolar lavage (BAL). Finally, the intranasal liposomal formulation boosted heterologous immunity induced by previous intramuscular vaccination with the Oxford/AstraZeneca vaccine, which was more robust than homologous immunity.

Background: Type 2 diabetes mellitus (T2DM) is frequently associated with dyslipidemia, which corresponds to the increase in the triglycerides and fatty acid concentrations in tissues, such as the skeletal muscle. Also, T2DM molecular mechanism involves increasing in reactive oxygen species (ROS) production and oxidative stress. The use of herbal medicines such as Uncaria tomentosa (Ut) has been proposed as an auxiliary treatment for patients with T2DM. In this study, it was evaluated the effect of Ut aqueous extract on cell viability and ROS production, in skeletal myoblasts from C2C12 lineage exposed to the free fatty acid palmitate (PA)., Methods: Cells were incubated with PA in different concentrations ranging from 10 to 1000 μM, for 24 or 48 h, for cytotoxicity assay. Cell death, DNA fragmentation and ROS production assays were performed in cell cultures incubated with PA for 24 h, in the pre (preventive condition) or post treatment (therapeutic condition) with 250 μg/ml Ut aqueous extract, for 2 or 6 h. Cell death was evaluated by MTT method or flow cytometry. ROS generation was measured by fluorescence spectroscopy using the DCFDA probe., Results: Cell viability was reduced to approximately 44% after the incubation with PA for 24 h from the concentration of 500 µM. In the incubation of cells with 500 μM PA and Ut extract for 6 h, in both conditions (preventive or therapeutic), it was observed an increase of 27 and 70% in cell viability respectively, in comparison to the cultures incubated with only PA. Also, the incubation of cultures with 500 μM PA, for 24 h, increased 20-fold the ROS formation, while the treatment with Ut extract, for 6 h, both in the preventive or therapeutic conditions, promoted decrease of 21 and 55%, respectively., Conclusion: The Ut extract was efficient in promoting cell protection against PA lipotoxicity and ROS generation, potentially preventing oxidative stress in C2C12 skeletal muscle cells. Since T2DM molecular mechanism involves oxidative stress condition and it is often associated with dyslipidemia and fatty acid accumulation in muscle tissue, these results open perspectives for the use of Ut as an auxiliary strategy for T2DM management., (© 2023. The Author(s).)

Cultivated meat (CM) technology has the potential to disrupt the food industry-indeed, it is already an inevitable reality. This new technology is an alternative to solve the environmental, health and ethical issues associated with the demand for meat products. The global market longs for biotechnological improvements for the CM production chain. CM, also known as cultured, cell-based, lab-grown, in vitro or clean meat, is obtained through cellular agriculture, which is based on applying tissue engineering principles. In practice, it is first necessary to choose the best cell source and type, and then to furnish the necessary nutrients, growth factors and signalling molecules via cultivation media. This procedure occurs in a controlled environment that provides the surfaces necessary for anchor-dependent cells and offers microcarriers and scaffolds that favour the three-dimensional (3D) organisation of multiple cell types. In this review, we discuss relevant information to CM production, including the cultivation process, cell sources, medium requirements, the main obstacles to CM production (consumer acceptance, scalability, safety and reproducibility), the technological aspects of 3D models (biomaterials, microcarriers and scaffolds) and assembly methods (cell layering, spinning and 3D bioprinting). We also provide an outlook on the global CM market. Our review brings a broad overview of the CM field, providing an update for everyone interested in the topic, which is especially important because CM is a multidisciplinary technology.

Introduction: In skin traumas, such as burns, epidermal homeostasis is affected, often requiring clinical approaches. Different therapeutic strategies can be used including transplantation, besides the use of synthetic or natural materials with allogeneic cells. In this context, tissue engineering is an essential tool for skin regeneration, and using mesenchymal stem cells (MSC) from the umbilical cord appears to be a promising strategy in regenerative medicine due to its renewal and differentiation potential and hypo immunogenicity. We evaluated the transdifferentiation of MSC from umbilical cord into keratinocytes in three-dimensional (3D) in vitro skin models, using dermal equivalents composed by type I collagen with dermal fibroblasts and a commercial porcine skin decellularized matrix, both cultured at air-liquid interface (ALI). Methods: The expression of epidermal proteins cytokeratins (CK) 5, 14 and 10, involucrin and filaggrin was investigated by real-time PCR and immunofluorescence, in addition to the activity of epidermal kallikreins (KLK) on the hydrolysis of fluorogenic substrates. Results and discussion: The cultivation of MSCs with differentiation medium on these dermal supports resulted in organotypic cultures characterized by the expression of the epidermal markers CK5, CK14, CK10 and involucrin, mainly on the 7 th day of culture, and filaggrin at 10 th day in ALI. Also, there was a 3-fold increase in the KLK activity in the epidermal equivalents composed by MSC induced to differentiate into keratinocytes compared to the control (MSC cultivated in the proliferation medium). Specifically, the use of collagen and fibroblasts resulted in a more organized MSC-based organotypic culture in comparison to the decellularized matrix. Despite the non-typical epithelium structure formed by MSC onto dermal equivalents, the expression of important epidermal markers in addition to the paracrine effects of these cells in skin may indicate its potential use to produce skin-based substitutes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dos Santos, Freitas-Marchi, Reigado, de Assis, Maria Engler, Chambergo Alcalde and Nunes.)

Purpose: To analyze the cytotoxicity and cell in porcine-derived decellularized skin matrix., Methods: We analyzed the effect of multiple decellularization processes by histological analysis, DNA quantification, and flow cytometry. Subsequently, we analyzed the most appropriate hydrogel concentration to minimize cytotoxicity on fibroblast culture and to maximize cell proliferation., Results: After the fourth decellularization, the DNA quantification showed the lowest DNA concentration (< 50 ng/mg). Histological analysis showed no cell components in the hydrogel. Moreover, hematoxylin and eosin showed a heterogeneous structure of collagen fibers. The best hydrogel concentration ranged from 3 to 25%, and there was no significant difference between the 24 hours and seven days., Conclusions: The process of hydrogel production was effective for removing cells and DNA elements. The best hydrogel concentration ranged from 3 to 25%.

Trichoderma reesei superoxide dismutase (TrSOD) is a well-characterized enzyme being stable between 30 and 90°C for 1 h with activity at pH between 2.6 and 9.0. This work aimed to clone, express, purify, and evaluate the protective effect antioxidant of this enzyme on skin cells when fused to transactivator of transcription (TAT) protein transduction domain of HIV-1 and abalone (Ab) peptides to allow cell penetration. TrSOD, TAT-TrSOD-Yfp (fused to yellow fluorescent protein), and Ab-TrSOD were expressed in E. coli and purified as soluble proteins. The cytotoxicity of the enzymes, at the concentrations of 1, 3, and 6 μmol/L, was evaluated for a period of 24 and 48 h of incubation, with no cytotoxic effect on 3T3 fibroblasts. The 3T3 cells were exposed to the oxidant agent tert-butyl hydroperoxide and evaluated for reactive oxygen species (ROS) generation, in the presence or not of the recombinant enzymes. TAT-TrSOD-Yfp was able to decrease the generation of ROS by 15% when used in the concentrations of 3 and 6 μmol/L in comparison to the control, but there was no difference in relation to the effect of TrSOD. Ab-TrSOD, when compared to TrSOD, promoted a decrease in the formation of ROS of 19% and 14% at the concentrations of 1 and 6 μmol/L, respectively, indicating that this recombinant form was more effective in reducing oxidative stress compared to SOD without the cell-penetrating peptide (CPP). Together, these results indicate that the fusion of SOD with these CPP increased the antioxidant capacity of fibroblasts, identified by the reduction in the generation of ROS. In addition, such molecules, in the concentrations initially used, were not toxic to the cells, opening perspectives for the development of products for antioxidant protection of the skin that may have therapeutic and cosmetic application., (© 2022 International Union of Biochemistry and Molecular Biology, Inc.)

Various approaches have been explored to study skin biology, including the use of stem cells. Mesenchymal stem cells (MSCs) from the umbilical cord can be safely and easily obtained; however, a simple strategy to monitor their differentiation is essential. Involucrin is a marker of keratinocyte differentiation, and its promoter (pINV) directs stratum-specific expression of this protein. We designed a reporter system containing EGFP under the control of pINV to assess MSC transdifferentiation into keratinocytes. The functional sequence of pINV was inserted into a lentiviral vector, producing LeGO-GpINV. MSCs were transduced with LeGO-GpINV and induced to transdifferentiate into keratinocytes under cultivation with keratinocyte serum-free medium. MSC transdifferentiation was confirmed by morphological changes and by the expression of epidermal markers by flow cytometry, quantitative PCR, Western blot and the activity of epidermal kallikreins 5, 6 and 7. After 14 days of transdifferentiation, MSCs transduced with LeGO-GpINV showed an increase in EGFP fluorescence and expressed CK10, CK14, involucrin and filaggrin. There was also an increase in kallikrein activity. This reporter system allowed us to temporally assess epidermal differentiation, simultaneously with involucrin expression, opening possibilities for the in vivo study of skin biology and in regenerative medicine., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Eczema is a skin condition characterized by itchy and inflammatory patches. The accumulation of neutrophils and the imbalance between enzymes and their inhibitors appears to be related to this condition. We proposed a neutrophil elastase (NE)-based eczema model in mice in order to verify histopathological features as well as the expression and activity of proteases and inhibitors. Mice skins were topically administered with human NE (0-2 pmol/cm 2 ) for 24-168 h. It was observed thickening of epidermis, parakeratosis, spongiosis and leukocyte infiltration. Also, NE-treated skins presented high activity of epidermal kallikreins 5 and 7, and cathepsin B on synthetic substrates, and expression evaluated by RT-qPCR. The proteolytic activity was inhibited by soybean trypsin inhibitor, CA074 and Caesalpinia echinata kallikrein inhibitor (CeKI). The topic application of CeKI reversed eczema phenotype in NE-treated skins. Elafin expression was shown to be increased in NE-treated skins. These results suggest that the NE may trigger morphological and biochemical changes in skin similar to those observed in eczematous diseases. In addition to the establishment of this in vivo model, this work opens perspectives for the use of protease inhibitor-based drugs for the management of this skin condition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Abstract: Santos, WDNd, Vieira, CA, Bottaro, M, Nunes, VA, Ramirez-Campillo, R, Steele, J, Fisher, JP, and Gentil, P. Resistance training performed to failure or not to failure results in similar total volume, but with different fatigue and discomfort levels. J Strength Cond Res 35(5): 1372-1379, 2021-The purpose of this study was to compare the acute response to 4 sets of high velocity parallel squats performed to momentary failure (MF) or not to momentary failure (NF). Twelve women (24.93 ± 5.04 years) performed MF and NF protocols, in a randomized order with 2-3 interday rest. The protocol involved 4 sets of parallel squats executed at high velocity at 10RM load, with 2 minutes of rest interval between sets. During the NF protocol, the sets were interrupted when the subject lost more than 20% of mean propulsive velocity. The analysis involved the number of repetitions performed per set, total number of repetitions, movement velocity loss, power output loss, rating of perceived exertion (RPE), rating of perceived discomfort (RPD), and session rating of perceived exertion (sRPE). Compared with NF, MF resulted in a higher number of repetitions in the first set (11.58 ± 1.83 vs. 7.58 ± 1.72, p < 0.05), but a lower in the last set (3.58 ± 1.08 vs. 5.41 ± 1.08, p < 0.05). Total number of repetitions was similar between the protocols (MF 26.25 ± 3.47 vs. NF 24.5 ± 3.65, p > 0.05). In both protocols, there were significant decreases in maximum and mean movement velocity loss and power output loss, but higher decreases were observed in MF than NF (p < 0.05). Values for RPE, sRPE, and RPD were higher during MF than NF (p < 0.05). Controlling the movement velocity in NF protocol enabled performance of a similar total volume of repetitions with lower movement velocity and power output losses, RPE, sRPE, and RPD than during an MF protocol., (Copyright © 2019 National Strength and Conditioning Association.)

Protease inhibitors have been widely used in several therapeutic applications such as in the treatment of bleeding disorders, hypertension, cancer and pulmonary diseases. In a previous work, we demonstrated that a Kunitz-type serine protease inhibitor isolated from the seeds of Caesalpinia echinata (CeEI) exhibits pharmacological potential in lung inflammatory diseases in which neutrophil elastase plays a crucial role. However, an important challenge in the use of natural products is to ensure a commercially viable production. In this work, we report the cloning, expression and purification of two recombinant CeEI isoinhibitors with 700 base pairs encoding two proteins with 181 amino acid residues (rCeEI-4 and rCeEI-5). After the expression, each yielding 22 mg/L of active protein, both isoinhibitors presented a molecular mass of about 23.0 kDa, evaluated by SDS-PAGE. The inhibition constants for human neutrophil elastase (HNE) were 0.67 nM (rCeEI-4) and 0.57 nM (rCeEI-5), i.e., similar to the native inhibitor (1.90 nM). Furthermore, rCeEI-4 was used as a template to design smaller functional peptides flanking the inhibitor reactive site: rCeEI-36, delimited between the amino acid residues N 36 and S 88 containing a disulfide bond in the reactive-site loop, and rCeEI-46, delimited between S 46 and L 75 without the disulfide bond. The yields were 18 mg/L (rCeEI-36) and 12 mg/L (rCeEI-46). Both peptides inhibit HNE in the nanomolar range (K i 0.30 ± 0.01 and 8.80 ± 0.23, respectively). Considering their size and the inhibitory efficiency, these peptides may be considered in strategies for the development of drugs targeting pulmonary disorders where elastase is involved., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Gestational diabetes (GD) is a condition defined as carbohydrate intolerance and hyperglycemia beginning in the second trimester of pregnancy, which overlaps with the progesterone exponential increase. Progesterone has been shown to cause pancreatic β -cell death by a mechanism dependent on the generation of reactive oxygen species and oxidative stress. Herein, we studied the effect of this hormone on the expression of 84 genes related to oxidative stress and oxidant defense in pancreatic RINm5F cell lineage. Cells were incubated with 0.1, 1.0, or 100  μ M progesterone for 6 or 24 h, in the presence or absence of the vitamins E and C. Among the investigated genes, five of them had their expression increased, at least 2-fold, in two different concentrations independently of the time of incubation, or at the same concentration at the different time points, including those that encode for stearoyl-CoA desaturase 1 ( Scd1 ), dual oxidase 1 ( Duox1 ), glutathione peroxidase 6 ( GPx6 ), heme oxygenase 1 ( Hmox1 ), and heat shock protein a1a ( Hspa1a ). Vitamins E and C were able to increase, in progesterone-treated cells, the expression of genes with antioxidant function such as Hmox1 , but decreased Scd1 expression, a gene with prooxidant function. At cytoplasmic level, progesterone positively modulated Hmox1 and Hspa1a content. These results suggest that the protein encoded by these genes might protect cells against progesterone induced-oxidative damage, opening perspectives to elucidate the molecular mechanism involved in progesterone action in GD, as well as for the development of antioxidant strategies for the prevention and treatment of this disease., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Nathália Ruder Borçari et al.)

Polymeric scaffolds incorporating plant-derived compounds, produced by electrospinning, have attracted attention in the field of skin tissue engineering. This study evaluates the sustained antioxidant activity of polycaprolactone (PCL)/gelatin nanofibers prepared by electrospinning and incorporating loaded liposomes of epigallocatechin-3-gallate (EGCG), a strong antibacterial and antioxidant molecule found in green tea, that significantly accelerates the wound-healing process. The morphology and the structural properties of the membranes were characterized by scanning electron microscopy (SEM) and FTIR spectroscopy. Results revealed that the EGCG released from PCL+gelatin nanofibers scavenges the toxic ROS species generated by exposure to either H 2 O 2 or UV radiation and slows down the oxidation events associated with damage. This study provides the basis for development of promising nanofiber formulations containing EGCG that might enhance repair/regeneration of skin tissue.

Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions. Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period. Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology., (© 2019 Wiley Periodicals, Inc.)

Inflammation is an essential process in many pulmonary diseases in which kinins are generated by protease action on kininogen, a phenomenon that is blocked by protease inhibitors. We evaluated kinin release in an in vivo lung inflammation model in rats, in the presence or absence of CeKI ( C. echinata kallikrein inhibitor), a plasma kallikrein, cathepsin G, and proteinase-3 inhibitor, and rCeEI (recombinant C. echinata elastase inhibitor), which inhibits these proteases and also neutrophil elastase. Wistar rats were intravenously treated with buffer (negative control) or inhibitors and, subsequently, lipopolysaccharide was injected into their lungs. Blood, bronchoalveolar lavage fluid (BALF), and lung tissue were collected. In plasma, kinin release was higher in the LPS-treated animals in comparison to CeKI or rCeEI groups. rCeEI-treated animals presented less kinin than CeKI-treated group. Our data suggest that kinins play a pivotal role in lung inflammation and may be generated by different enzymes; however, neutrophil elastase seems to be the most important in the lung tissue context. These results open perspectives for a better understanding of biological process where neutrophil enzymes participate and indicate these plant inhibitors and their recombinant correlates for therapeutic trials involving pulmonary diseases., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Progesterone has been associated with the development of gestational diabetes (GD) due to the enhancement of insulin resistance. As β-cell apoptosis participates in type 1 and type 2 diabetes pathophysiology, we proposed the hypothesis that progesterone might contribute to the development of GD through a mechanism that also involves β-cell death. To address this question, RINm5F insulin-producing cells were incubated with progesterone (25-100 μM), in the presence or absence of α-tocopherol (40 μM). After 24 or 48 h, membrane integrity and DNA fragmentation were analyzed by flow cytometry. Caspase activity was used to identify the mode of cell death. The involvement of endoplasmic reticulum stress in the action of progesterone was investigated by western blotting. Oxidative stress was measured by 2',7'-dichlorofluorescein diacetate (DCFDA) oxidation. Isolated rat islets were used in similar experiments in order to confirm the effect of progesterone in primary β-cells. Incubation of RINm5F cells with progesterone increased the number of cells with loss of membrane integrity and DNA fragmentation. Progesterone induced generation of reactive species. Pre-incubation with α-tocopherol attenuated progesterone-induced apoptosis. Western blot analyses revealed increased expression of CREB2 and CHOP in progesterone-treated cells. Progesterone caused apoptotic death of rat islet cells and enhanced generation of reactive species. Our results show that progesterone can be toxic to pancreatic β-cells through an oxidative-stress-dependent mechanism that induces apoptosis. This effect may contribute to the development of GD during pregnancy, particularly under conditions that require administration of pharmacological doses of this hormone.

Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. For this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEI is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K(i) 3.7 μM), leading to hydrolysis of specific synthetic substrates. In a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. In experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Zn(+2), HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PK binding to cell- or ECM-bound-HK and PK activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn(2+). Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Although numerous inflammation pathways have been implicated in Alzheimer's disease, the involvement of the kallikrein-kinin system is still under investigation. We anatomically localized and quantified the density of kinin B(1) and B(2) receptors binding sites in the rat brain after the infusion of amyloid-beta (Abeta) peptide in the right lateral brain ventricle for 5 weeks. The conditioned avoidance test showed a significant reduction of memory consolidation in rats infused with Abeta (68.6+/-20.9%, P<0.05) when compared to control group (90.8+/-4.1%; infused with vehicle). Autoradiographic studies performed in brain samples of both groups using [(125)I]HPP-[des-Arg(10)]-Hoe-140 (150pM, 90min, 25 degrees C) showed a significant increase in density of B(1) receptor binding sites in the ventral hippocampal commissure (1.23+/-0.07fmol/mg), fimbria (1.31+/-0.05fmol/mg), CA1 and CA3 hippocampal areas (1.05+/-0.03 and 1.24+/-0.02fmol/mg, respectively), habenular nuclei (1.30+/-0.04fmol/mg), optical tract (1.30+/-0.05fmol/mg) and internal capsule (1.26+/-0.05fmol/mg) in Abeta group. For B(2) receptors ([(125)I]HPP-Hoe-140, 200pM, 90min, 25 degrees C), a significant increase in density of binding sites was observed in optical tract (2.04+/-0.08fmol/mg), basal nucleus of Meynert (1.84+/-0.18fmol/mg), lateral septal nucleus - dorsal and intermediary portions (1.66+/-0.29fmol/mg), internal capsule (1.74+/-0.19fmol/mg) and habenular nuclei (1.68+/-0.11fmol/mg). In control group, none of these nuclei showed [(125)I]HPP-Hoe-140 labeling. This significant increase in densities of kinin B(1) and B(2) receptors in animals submitted to Abeta infusion was observed mainly in brain regions related to cognitive behavior, suggesting the involvement of the kallikrein-kinin system in Alzheimer's disease in vivo.

Background Information: DMD (Duchenne muscular dystrophy) is a devastating X-linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose-derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X-linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown., Results: We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co-cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)-positive ASCs and DAPI (4',6-diamidino-2-phenylindole)-stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion., Conclusions: These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.

Background Information: Duchenne muscular dystrophy is a disease characterized by progressive and irreversible muscle degeneration for which there is no therapy. HUCB (human umbilical cord blood) has been considered as an important source of haematopoietic and mesenchymal stem cells, each having been shown to differentiate into distinct cell types. However, it remains unclear if these cells are able to differentiate into muscle cells., Results: We have showed that stem cells from HUCB did not differentiate into myotubes or express dystrophin when cultured in muscle-conditioned medium or with human muscle cells. However, delivery of GFP (green fluorescent protein)-transduced mononucleated cells from HUCB, which comprises both haematopoietic and mesenchymal populations, into quadriceps muscle of mdx (mouse dystrophy X-chromosome linked) mice resulted in the expression of human myogenic markers. After recovery of these cells from mdx muscle and in vitro cultivation, they were able to fuse and form GFP-positive myotubes that expressed dystrophin., Conclusions: These results indicate that chemical factors and cell-to-cell contact provided by in vitro conditions were not enough to trigger the differentiation of stem cells into muscle cells. Nevertheless, we showed that the HUCB-derived stem cells were capable of acquiring a muscle phenotype after exposure to an in vivo muscle environment, which was required to activate the differentiation programme.

The kallikrein-kinin system is involved in a variety of physiological and pathological processes. Components of this system, identified in rat and human brains, can be altered in neurodegenerative processes such as Alzheimer's disease. Here, we studied kinin release and its inactivation in rats submitted to chronic cerebroventricular infusion of beta-amyloid (Abeta) peptide. Neurodegeneration was confirmed by histological analysis of brain samples. In cerebrospinal fluid of animals infused with Abeta, bradykinin concentration was increased, as determined by radioimmunoassay. However, in the brain of Abeta group, we only detected the tripeptide Arg-Pro-Pro, purified by reversed-phase chromatography and characterized by liquid chromatography-electrospray ionization mass spectrometry. This fragment of bradykinin indicated the possible participation of kinin-processing enzymes in the brain such as a prolyl oligopeptidase.

The interplay of different proteases and glycosaminoglycans is able to modulate the activity of the enzymes and to affect their structures. Human plasma kallikrein (huPK) is a proteolytic enzyme involved in intrinsic blood clotting, the kallikrein-kinin system and fibrinolysis. We investigated the effect of heparin on the action, inhibition and secondary structure of huPK. The catalytic efficiency for the hydrolysis of substrates by huPK was determined by Michaelis-Menten kinetic plots: 5.12x10(4) M-1 s-1 for acetyl-Phe-Arg-p-nitroanilide, 1.40x10(5) M-1 s-1 for H-D-Pro-Phe-Arg-p-nitroanilide, 2.25x10(4) M-1 s-1 for Abz-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-EDDnp, 4.24x10(2)M-1 s-1 for factor XII and 5.58x10(2) M-1 s-1 for plasminogen. Heparin reduced the hydrolysis of synthetic substrates (by 2.0-fold), but enhanced factor XII and plasminogen hydrolysis (7.7- and 1.4-fold, respectively). The second-order rate constants for inhibition of huPK by antithrombin and C1-inhibitor were 2.40x10(2) M-1 s-1 and 1.70x10(4) M-1 s-1, respectively. Heparin improved the inhibition of huPK by these inhibitors (3.4- and 1.4-fold). Despite the fact that huPK was able to bind to a heparin-Sepharose matrix, its secondary structure was not modified by heparin, as monitored by circular dichroism. These actions may have a function in the control or maintenance of some pathophysiological processes in which huPK participates.

Apoptosis and necrosis are two forms of cell death that can occur in response to various agents and oxidative damage. In addition to necrosis, apoptosis contributes to muscle fiber loss in various muscular dystrophies as well participates in the exudative diathesis in chicken, pathology caused by dietary deficiency of vitamin E and selenium, which affects muscle tissue. We have used chicken skeletal muscle cells and bovine fibroblasts to study molecular events involved in the cell death induced by oxidative stress and apoptotic agents. The effect of vitamin E on cell death induced by oxidants was also investigated. Treatment of cells with anti-Fas antibody (50 to 400 ng/mL), staurosporine (0.1 to 100 microM) and TNF-alpha (10 and 50 ng/mL) resulted in a little loss of Trypan blue exclusion ability. Those stimuli conducted cells to apoptosis detected by an enhancement in caspase activity upon fluorogenic substrates but this activity was not fully blocked by the caspase inhibitor Z-VAD-fmk. Oxidative stress induced by menadione (10, 100 and 250 muM) promoted a significant reduction in cell viability (10%, 20% and 35% for fibroblasts; 20%, 30% and 75% for muscle cells, respectively) and caused an increase in caspase activity and DNA fragmentation. H2O2 also promoted apoptosis verified by caspase activation and DNA fragmentation, but in higher doses induced necrosis. Vitamin E protected cells from death induced by low doses of oxidants. Although it was ineffective in reducing caspase activity in fibroblasts, this vitamin diminished the enzyme activity in muscle cells. These data suggested that oxidative stress could activate apoptotic mechanisms; however the mode of cell death will depend on the intensity and duration of the stimulus, and on the antioxidant status of the cells.

Several IAP (inhibitor of apoptosis) proteins regulate cell fate decisions, and the X-linked IAP (XIAP) does so in part by inhibiting caspases, proteases that execute the apoptotic pathway. A tissue-specific homologue of XIAP, known as ILP2 (IAP-like protein 2), has previously been implicated in the control of apoptosis in the testis by direct inhibition of caspase 9. In examining this protein we found that the putative caspase 9 interaction domain is a surprisingly weak inhibitor and is also conformationally unstable. Comparison with the equivalent domain in XIAP demonstrated that the instability is due to the lack of a linker segment N-terminal to the inhibitory BIR (baculovirus IAP repeat) domain. Fusion of a 9-residue linker from XIAP to the N-terminus of ILP2 restored tight caspase 9 inhibition, dramatically increased conformational stability and allowed crystallization of the ILP2 BIR domain in a form strikingly similar to the XIAP third BIR domain. We conclude that ILP2 is an unstable protein, and cannot inhibit caspase 9 in a physiological way on its own. We speculate that ILP2 requires assistance from unidentified cellular factors to be an effective inhibitor of apoptosis in vivo.

The relationship between thyroid, gonads and bone morphology was investigated in 5 month-old Wistar rats, castrated and kept in hyperthyroid or euthyroid state for periods of 30, 60 and 90 days. A non-castrated control group was maintained in the same condition. At the end of each period, plasma concentrations of free T4, progesterone and estradiol were measured. The bones from each group were submitted to radiological and histological analysis. Hyperthyroidism in intact rats caused variation in bone morphology throughout the experiment, leading to loss of trabecular bone tissue at day 60, due to increased bone resorption. At day 90 there was no bone loss, because the increase in bone resorption was accompanied by higher bone apposition. In castrated euthyroid rats reduction in progesterone levels inhibited the bone apposition, causing a slight loss of trabecular bone tissue in vertebrae and alveolar bone at day 30, which was intensified at days 60 and 90, affecting cortical bone tissue. The administration of thyroxine to castrated rats reversed osteopenia at days 30 and 60, which did not occur at day 90 when bone loss was more extensive. In conclusion, hypoprogesteronism and hypoestrogenism alter bone metabolism and the bone response to hyperthyroidism depends on the plasma profile of sex steroids, time of exposure and bone tissue configuration.

Caesalpinia echinata is a tree belonging to the Leguminosae family. The red color of the trunk, looking like burning wood ('brasa' in Portuguese), is the origin of the name Brazil. Seeds of leguminous plants contain high amounts of serine proteinase inhibitors that can affect different biological processes. Here we show that a protein isolated from seeds of C. echinata is able to inhibit enzymes that participate in blood coagulation and fibrinolysis. This inhibitor (CeKI) was purified to homogeneity by ion exchange and reversed-phase chromatography. SDS-PAGE indicated a single polypeptide chain with a molecular mass of 20 kDa. CeKI inhibits human plasma kallikrein ( K i =3.1 nM), plasmin ( K i =0.18 nM), factor XIIa ( K i =0.18 nM), trypsin ( K i =21.5 nM) and factor Xa ( K i =0.49 mM). CeKI inhibited kinin release from highmolecular- mass kininogen by kallikrein in vitro . The N-terminal sequence, determined by automatic Edman degradation, identified the inhibitor as a member of the Kunitz family. The secondary structure, determined by circular dichroism, is mainly a random coil followed by beta-sheet structure. The action of CeKI on enzymes of the blood-clotting intrinsic pathway was confirmed by prolongation of the activated partial thromboplastin time.

The effect of hypothyroidism on bone metabolism and the parathyroids in states of deficiency or sufficiency of sex steroids was studied in 32 two-months-old female Wistar rats distributed in 4 groups of 8 animals each: intact euthyroid (IE), castrated euthyroid (CE), intact hypothyroid (IH) and castrated hypothyroid (CH). After 120 days of treatment, animals were sacrificed and plasma taken to assess free T4. Hyperplasia or hypertrophy of all parathyroids were evident only in IH and CE groups. Of all groups, IH rats presented the most extensive osteopenia, reaching lumbar vertebrae, dental alveolae (jaw and mandible) and long bones. In this group osteopenia resulted from the reduced bone growth, inhibition of bone apposition and return of bone resorption. Although osteopenia in the CH group was almost always more intense in relation to osteopenia presented by CE rats, its intensity was variable when compared to IH rats and dependent on the region studied. Even though it also caused necrosis of higher metabolism bones, the association hypothyroidism-castration did not potentialize the resultant osteopenia of the isolated action of hypothyroidism or castration until 120 days of treatment.

The relationship between thyroid-gonads on the blood profile was investigated in adult Wistar female rats. These animals were either castrated or intact and were kept under hypothyroidism, induced by daily administration of propylthiouracil (PTU) during 120 days. Two groups (castrated and intact) were kept in an euthyroid condition and used as controls. It was collected plasma for free T4 dosage and blood for hematological analysis. The significant low values of free T4 in the treated rats confirmed their hypothyroid state. The hypothyroidism caused anemia in the rats with functional gonads. The castration reverted the effects of thyroxine deficit in the erythrogram. Both the hypothyroidism and the hypogonadism showed just a discret effect on the leucogram.

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 micro/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 microM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 microM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 microM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P'1, P'3 and P'4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P'4 position is important for enzyme action.

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambda exc = 320 nm and lambda em = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.

Human plasma kallikrein (huPK) is a serine proteinase involved in many biological processes including those of the kallikrein-kinin system. The action of huPK on kininogen results in bradykinin (BK) release, a potent mediator of inflammatory responses. BK generation may be influenced by several agents, and the aim of this work was to investigate the effect of glycosaminoglycans (GAGs) on human high-molecular-weight kininogen (HK) hydrolysis by huPK and on inflammation. huPK was pre-incubated in the absence and presence of different GAGs, followed by the addition of kininogen. Bradykinin released at different times was measured by radioimmunoassay, and KM and kcat were calculated. Tuna and bovine dermatan sulfates, the most potent GAGs studied, reduced by 80% and 68%, respectively, the catalytic efficiency of huPK (control = 4. x 10(4) M(-1) s(-1) in BK release. The effect of bovine dermatan sulfate (BDS) on inflammatory response was studied in rat paw edema induced by carrageenin and hourly determined (1-4 h) by plethysmography. BDS significantly reduced the inflammatory response in the first and second hours of measurements (24% and 28%, respectively), p < 0.05. GAGs were shown to reduce bradykinin release "in vitro" and in an inflammation model. This reduction may play a role in the control or maintenance of some pathological and physiological processes.

Blood serine protease inhibitors are becoming better understood and increasingly applied in blood clotting, cancer and other diseases. Reptiles are suitable models for blood coagulation and related processes, moreover, caiman is a good comparative model of a non-poisonous reptile. Recently, we reported the purification of a kininogen, the presence of proteases involved in blood clotting, and a serine protease inhibitor in Caiman crocodilus yacare plasma. In this paper, we described the partial sequence of an inhibitor (CcTI). The inhibitor is an 80-kDa protein, and it inactivates trypsin and chymotrypsin the hydrolysis of specific chromogenic substrates and in the degradation of gelatin. The inhibitor is member of Kazal-type inhibitor family and consists of several domains, its putative reactive site is Arg-His.

In order to study the morphology and morphometry and to characterize and quantify the nucleolar organizer regions (NORs) of bovine thyroids containing methylthiouracil (MTU) residues, five animals were orally treated with a suspension of MTU (5 g/animal/day) for 20 days (group A). This treatment protocol was interrupted 5 days before the the animals were slaughtered. Six animals receiving placebos composed group B. A third group (group C) was composed of normal thyroids obtained from a slaughterhouse. All glands were previously assessed for detection of antithyroid residues by chromatography, and only those glands from MTU-treated animals were positive. Follicles of glands from group A showed wide variation in size and shape. There was a predominance of small follicles covered by multiple layers of columnar cells, sometimes forming papillary projections into the lumen, characterizing severe interfollicular and intrafollicular adenomatosis. Many follicles had vacuolated cells with nuclei showing karyolysis or pyknosis and reduced amounts of a low-density and very excavated colloid. They also showed higher follicular epithelia and larger proportions of their structural components when compared with glands of groups B and C. In the thyroids from group A, the argyrophilic nucleolar organizer region-associated proteins (AgNORs) were greater in number, with small ones scattered all over the nucleus. Although the size of AgNORs in thyroids from groups B and C was variable, these AgNORs were fewer and larger than were those in glands from group A. In conclusion, the MTU induces proliferation and regressive changes in follicular cells, and the AgNOR technique is efficient to distinguish different degrees of thyroid hyperplasia.

Young rabbits on high (0.57%) or low (0.24%) calcium were given an aqueous extract of Solanum malacoxylon (S.m.) leaves (20 g dried leaves/200 ml distilled water) intragastrically at 0, 12 and 36 hours. On bothe diets S.m. induced progressive hypophosphatasemia but serum calcium and phosphorus underwent only minor changes. In rabbits necropsied at 0, 12, 36, 60, 84 and 108 hours, S.m. was shown to have a negative effect on the resorbing osteocytes. With retarded osteocytic osteolysis, osteopetrosis resulted. Further regressive changes in the osteocytes resulted in osteonecrosis which was observed within 12 hours after administration of S.m. extract. The osteonecrosis, combined with retarded apposition, later resulted in osteopenia. It was concluded that the recommended dietary calcium for growing rabbits--about 0.6%--is too high. Whereas the histologic appearance of bone in rabbits fed low calcium was normal, bones from rabbits on high calcium showed retarded resorption and the rabbits had a relative hypophosphatasemia.