RESUMEN

Structural proteomics techniques are useful for the determination of protein interaction interfaces. Each technique provides orthogonal structural information on the structure and the location of protein interaction sites. Here, we have characterized a monoclonal antibody epitope for a protein antigen by a combination of differential photoreactive surface modification (SM), cross-linking (CL), differential hydrogen-deuterium exchange (HDX), and epitope extraction/excision. We found that experimental data from different approaches agree with each other in determining the epitope of the monoclonal antibody on the protein antigens using the HIV-1 p24-mAb E complex as an illustrative example. A combination of these multiple structural proteomics approaches results in a detailed picture of the interaction of the proteins and increases confidence in the determination of the final structure of the protein interaction interface. Data are available via ProteomeXchange with identifier PXD040902.

Asunto(s)

Anticuerpos Monoclonales , Proteómica , Epítopos/química , Anticuerpos Monoclonales/química , Mapeo Epitopo/métodos , Espectrometría de Masas/métodos

RESUMEN

In the Amazon, the leaching from soil left unprotected by deforestation increases the entry of iron, among other elements, in aquatic ecosystems, which can cause cyanobacterial blooms. This study aimed to investigate the physiological response of a strain of Microcystis panniformis to iron variation. The strain was isolated from a reservoir located in the Western Amazon and produces microcystin-LR. After a period of iron deprivation, the cultures were submitted to three conditions: control (223 µgFe.L-1), treatment with 23 µgFe.L-1, and absence of iron. At regular intervals for eight days, the cell density, levels of chlorophyll a and microcystins were determined. On the second and fourth day, transcription of genes responsive to iron limitation was quantified. Starting on the fourth day of the experiment, the different iron concentrations affected growth, and on the eighth day in the iron-free condition cell density was 90% lower than in control. Chlorophyll cell quota in 23 µgFe.L-1 and control presented similar values, while without iron the cells became chlorotic as of the fourth day Toxin concentration in cells grow in 0 µgFe.L-1 in relation to the control. Higher transcription levels of the feo and fut genes were observed in the 0 µgFe.L-1 and 23 µgFe.L-1 treatments, indicating that the cells were activating high-affinity capture systems to reestablish an adequate concentration of intracellular iron. The increasing deforestation in the Jamari River Basin (Amazon region), can contribute to the occurrence of toxic cyanobacterial blooms due to the greater entrance of iron in water bodies.

Asunto(s)

Microcistinas , Microcystis , Clorofila A , Ecosistema , Hierro , Microcystis/genética

RESUMEN

The energy transfer in the aquatic food chain is an important way for mercury (Hg) to enter other trophic levels. The objective of this work was to evaluate the Hg concentrations in plankton upstream and downstream of the Samuel Hydroelectric Reservoir, Rondônia, Brazil. Phytoplankton and zooplankton samples were collected with 20-µm and 68-µm nylon nets. An aliquot was removed for taxonomic analysis and another for total mercury determination, performed by cold vapor atomic absorption spectroscopy. Water physical-chemical parameters were also measured. The Hg concentrations in total plankton (phytoplankton and zooplankton samples) obtained at the three sampling upstream stations showed the same behavior, with the highest values registered in June 2005 (232 µg kg-1, 118 µg kg-1, 128 µg kg-1). The lowest values at stations J1 and M1 were recorded in November 2005 (4 µg kg-1 and 22 µg kg-1, respectively), while the lowest values at stations M4 and M8 were recorded in October 2005 (22 µg kg-1 and 5 µg kg-1, respectively). The Hg results found in the plankton in this study corroborate the results of other recent studies in the same region. The statistical analyses revealed that Hg concentrations in plankton do not explain the distribution of these organisms at the four sampling stations of Samuel Reservoir. Graphical Abstract.

Asunto(s)

Mercurio/análisis , Contaminantes Químicos del Agua/análisis , Animales , Brasil , Monitoreo del Ambiente , Peces , Cadena Alimentaria , Plancton

RESUMEN

Hepatocytes are replenished gradually during homeostasis and robustly after liver injury1, 2. In adults, new hepatocytes originate from the existing hepatocyte pool3-8, but the cellular source of renewing hepatocytes remains unclear. Telomerase is expressed in many stem cell populations, and mutations in telomerase pathway genes have been linked to liver diseases9-11. Here we identify a subset of hepatocytes that expresses high levels of telomerase and show that this hepatocyte subset repopulates the liver during homeostasis and injury. Using lineage tracing from the telomerase reverse transcriptase (Tert) locus in mice, we demonstrate that rare hepatocytes with high telomerase expression (TERTHigh hepatocytes) are distributed throughout the liver lobule. During homeostasis, these cells regenerate hepatocytes in all lobular zones, and both self-renew and differentiate to yield expanding hepatocyte clones that eventually dominate the liver. In response to injury, the repopulating activity of TERTHigh hepatocytes is accelerated and their progeny cross zonal boundaries. RNA sequencing shows that metabolic genes are downregulated in TERTHigh hepatocytes, indicating that metabolic activity and repopulating activity may be segregated within the hepatocyte lineage. Genetic ablation of TERTHigh hepatocytes combined with chemical injury causes a marked increase in stellate cell activation and fibrosis. These results provide support for a 'distributed model' of hepatocyte renewal in which a subset of hepatocytes dispersed throughout the lobule clonally expands to maintain liver mass.

Asunto(s)

Hepatocitos/citología , Hepatocitos/enzimología , Homeostasis , Regeneración Hepática , Hígado/citología , Hígado/lesiones , Telomerasa/metabolismo , Animales , Linaje de la Célula/genética , Autorrenovación de las Células/genética , Femenino , Hepatocitos/metabolismo , Homeostasis/genética , Hígado/metabolismo , Hígado/patología , Regeneración Hepática/genética , Masculino , Ratones , Análisis de Secuencia de ARN , Telomerasa/genética

RESUMEN

CONTEXT: In clinical practice, the importance of screening for anxiety and depression in patients with medical illness is highlighted. In many cases, the suicidal ideation makes up the framework of mental disorders, which may be exacerbated in these individuals. OBJECTIVE: To investigate the role of symptoms of mental disorders in the presence of suicidal ideation. METHODS: A total of 103 patients with diagnosis of coronary artery disease in cardiac treatment were interviewed for symptoms of anxiety and depression using the Beck Depression Inventory and Hospital Anxiety and Depression Scale. All patients were also analyzed for presence of suicidal ideation, wishes, attitude and suicidal plans using the Beck Suicidal Ideation Scale. The relationship between social and demographic variables and mental disturbances and the presence of suicidal ideation was assessed using chi-square test and coefficient of sperm. Logistic regression analysis was used to explain the change in the role of each of the variables in suicidal ideation. RESULTS: The results showed that predictors for suicidal ideation were isolated anxiety (B=0.29; Wald 4.77; p=0.03) with an odds ratio of 1.34 (CI 1.03-1.75) and isolated depression (B=0.33; Wald 5.35; p=0.02) with an odds ratio of 1.39 (CI 1.05-1.85). Frequencies of interaction depression and anxiety were higher among patients who were single, widowed and divorced. Chi-square test and the coefficient of sperm showed an association between marital status and suicidal ideation (χ(2)(2)=9.17; p=0.01). CONCLUSION: Anxiety and depression are risk factors for isolated patients with suicidal ideation. Early clinical identification of mental disorders in patients with medical illness contributed to preventing the risk of suicide.

Asunto(s)

Trastornos de Ansiedad/epidemiología , Ansiedad/epidemiología , Depresión/epidemiología , Trastorno Depresivo/epidemiología , Ideación Suicida , Adulto , Anciano , Enfermedad de la Arteria Coronaria/psicología , Femenino , Humanos , Masculino , Trastornos Mentales/epidemiología , Persona de Mediana Edad , Escalas de Valoración Psiquiátrica , Factores de Riesgo , Suicidio

RESUMEN

The aim of this study was to conduct a systematic review of the literature regarding the prevalence of sexual dysfunction in patients with cardiovascular diseases. An article search of the ISI Web of Science and PubMed databases using the search terms "sexual dysfunction", "cardiovascular diseases", "coronary artery disease", "myocardial infarct" and "prevalence" was performed. In total, 893 references were found. Non-English-language and repeated references were excluded. After an abstract analysis, 91 references were included for full-text reading, and 24 articles that evaluated sexual function using validated instruments were selected for this review. This research was conducted in October 2012, and no time restrictions were placed on any of the database searches. Reviews and theoretical articles were excluded; only clinical trials and epidemiological studies were selected for this review. The studies were mostly cross-sectional, observational and case-control in nature; other studies used prospective cohort or randomized clinical designs. In women, all domains of sexual function (desire, arousal, vaginal lubrication, orgasm, sexual dissatisfaction and pain) were affected. The domains prevalent in men included erectile dysfunction and premature ejaculation and orgasm. Sexual dysfunction was related to the severity of cardiovascular disease. When they resumed sexual activity, patients with heart disease reported significant difficulty, including a lack of interest in sex, sexual dissatisfaction and a decrease in the frequency of sexual activity.

Asunto(s)

Enfermedades Cardiovasculares/epidemiología , Disfunciones Sexuales Fisiológicas/epidemiología , Enfermedades Cardiovasculares/complicaciones , Estudios Epidemiológicos , Femenino , Humanos , Masculino , Prevalencia , Ensayos Clínicos Controlados Aleatorios como Asunto , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factores Sexuales , Disfunciones Sexuales Fisiológicas/etiología

RESUMEN

The aim of this study was to conduct a systematic review of the literature regarding the prevalence of sexual dysfunction in patients with cardiovascular diseases. An article search of the ISI Web of Science and PubMed databases using the search terms "sexual dysfunction”, “cardiovascular diseases”, “coronary artery disease", “myocardial infarct" and “prevalence” was performed. In total, 893 references were found. Non-English-language and repeated references were excluded. After an abstract analysis, 91 references were included for full-text reading, and 24 articles that evaluated sexual function using validated instruments were selected for this review. This research was conducted in October 2012, and no time restrictions were placed on any of the database searches. Reviews and theoretical articles were excluded; only clinical trials and epidemiological studies were selected for this review. The studies were mostly cross-sectional, observational and case-control in nature; other studies used prospective cohort or randomized clinical designs. In women, all domains of sexual function (desire, arousal, vaginal lubrication, orgasm, sexual dissatisfaction and pain) were affected. The domains prevalent in men included erectile dysfunction and premature ejaculation and orgasm. Sexual dysfunction was related to the severity of cardiovascular disease. When they resumed sexual activity, patients with heart disease reported significant difficulty, including a lack of interest in sex, sexual dissatisfaction and a decrease in the frequency of sexual activity. .

Asunto(s)

Femenino , Humanos , Masculino , Enfermedades Cardiovasculares/epidemiología , Disfunciones Sexuales Fisiológicas/epidemiología , Enfermedades Cardiovasculares/complicaciones , Estudios Epidemiológicos , Prevalencia , Ensayos Clínicos Controlados Aleatorios como Asunto , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factores Sexuales , Disfunciones Sexuales Fisiológicas/etiología

RESUMEN

A produção do maracujá amarelo cresce no Brasil e o descarte inadequado das cascas representa tanto umproblema para o meio ambiente, como a perda de ingrediente rico em fibras. O seu uso é fonte de rendapara o produtor rural. Esta revisão traz os estudos que têm sido realizados sobre o aproveitamento da cascado maracujá, considerando-se os aspectos nutricionais e, também, uma alternativa segura, em termostoxicológicos, para o aproveitamento desse resíduo. Muitos autores avaliaram a casca do maracujá edetectaram teores de fibra alimentar de 35 a 90 %; e os benefícios do seu consumo foram identificados pormeio de testes in vivo em ratos e em humanos. Em função de interesse, incentivou-se o desenvolvimentode produtos enriquecidos em fibra alimentar utilizando esse resíduo como componente da formulação. Hámuito tempo foram identificados glicosídeos cianogênicos no maracujá, exceto nas sementes. Os efeitostóxicos de alimentos com cianogênicos são observados em países africanos, onde é comum o consumoda mandioca brava sem o tratamento adequado. O processo de extrusão termoplástica tem sido estudadocomo ferramenta útil na redução de substâncias tóxicas e sua utilização será importante para processar ascascas e reduzir cianogênicos aos níveis aceitáveis pela legislação.

Asunto(s)

Aprovechamiento Integral de los Alimentos , Fibras de la Dieta , Glicósidos , Passiflora , Producción de Alimentos

RESUMEN

Setd8/PR-Set7/KMT5a-dependent mono-methylation of histone H4 at lysine 20 is essential for mitosis of cultured cells; yet, the functional roles of Setd8 in complex mammalian tissues are unknown. We use skin as a model system to explore how Setd8 may regulate cell division in vivo. Deletion of Setd8 in undifferentiated layers of the mouse epidermis impaired both proliferation and differentiation processes. Long-lived epidermal progenitor cells are lost in the absence of Setd8, leading to an irreversible loss of sebaceous glands and interfollicular epidermis. We show that Setd8 is a transcriptional target of c-Myc and an essential mediator of Myc-induced epidermal differentiation. Deletion of Setd8 in c-Myc-overexpressing skin blocks proliferation and differentiation and causes apoptosis. Increased apoptosis may be explained by our discovery that p63, an essential transcription factor for epidermal commitment is lost, while p53 is gained upon removal of Setd8. Both overexpression of p63 and deletion of p53 rescue Setd8-induced apoptosis. Thus, Setd8 is a crucial inhibitor of apoptosis in skin and its activity is essential for epidermal stem cell survival, proliferation and differentiation.

Asunto(s)

N-Metiltransferasa de Histona-Lisina/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Fenómenos Fisiológicos de la Piel , Animales , Apoptosis , Secuencia de Bases , Diferenciación Celular , Proliferación Celular , Cartilla de ADN , Ratones , Reacción en Cadena de la Polimerasa , Unión Proteica , Piel/metabolismo

RESUMEN

How the proto-oncogene c-Myc balances the processes of stem-cell self-renewal, proliferation and differentiation in adult tissues is largely unknown. We explored c-Myc's transcriptional roles at the epidermal differentiation complex, a locus essential for skin maturation. Binding of c-Myc can simultaneously recruit (Klf4, Ovol-1) and displace (Cebpa, Mxi1 and Sin3a) specific sets of differentiation-specific transcriptional regulators to epidermal differentiation complex genes. We found that Sin3a causes deacetylation of c-Myc protein to directly repress c-Myc activity. In the absence of Sin3a, genomic recruitment of c-Myc to the epidermal differentiation complex is enhanced, and re-activation of c-Myc-target genes drives aberrant epidermal proliferation and differentiation. Simultaneous deletion of c-Myc and Sin3a reverts the skin phenotype to normal. Our results identify how the balance of two transcriptional key regulators can maintain tissue homeostasis through a negative feedback loop.

Asunto(s)

Epidermis/fisiología , Retroalimentación Fisiológica/fisiología , Homeostasis/genética , Queratinocitos/fisiología , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Represoras/fisiología , Transcripción Genética/fisiología , Animales , Células Epidérmicas , Femenino , Queratinocitos/citología , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Represoras/genética , Complejo Correpresor Histona Desacetilasa y Sin3

RESUMEN

Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex 1 (PRC1)-associated protein, maintains human epidermal stem cells as slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its antisenescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors such as Ezh2, Dnmt1, and Bmi1 to prevent the active stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation.

Asunto(s)

Células Madre Adultas/metabolismo , Proliferación Celular , Queratinocitos/metabolismo , Proteínas Represoras/metabolismo , Proteína SUMO-1/metabolismo , Adulto , Células Madre Adultas/patología , Diferenciación Celular/genética , Células Cultivadas , Senescencia Celular/genética , Ensamble y Desensamble de Cromatina , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Epidermis/patología , Prepucio/patología , Perfilación de la Expresión Génica , Humanos , Recién Nacido , Queratinocitos/patología , Ligasas , Masculino , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/genética , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas

RESUMEN

Myc-induced SUN domain-containing protein (Misu or NSun2) is a nucleolar RNA methyltransferase important for c-Myc-induced proliferation in skin, but the mechanisms by which Misu contributes to cell cycle progression are unknown. In this study, we demonstrate that Misu translocates from the nucleoli in interphase to the spindle in mitosis as an RNA-protein complex that includes 18S ribosomal RNA. Functionally, depletion of Misu caused multiple mitotic defects, including formation of unstructured spindles, multipolar spindles, and chromosome missegregation, leading to aneuploidy and cell death. The presence of both RNA and Misu is required for correct spindle assembly, and this process is independent of active translation. Misu might mediate its function at the spindle by recruiting nucleolar and spindle-associated protein (NuSAP), an essential microtubule-stabilizing and bundling protein. We further identify NuSAP as a novel direct target gene of c-Myc. Collectively, our results suggest a novel mechanism by which c-Myc promotes proliferation by stabilizing the mitotic spindle in fast-dividing cells via Misu and NuSAP.

Asunto(s)

Nucléolo Celular/enzimología , Metiltransferasas/metabolismo , Huso Acromático/enzimología , Animales , Apoptosis , Línea Celular Tumoral , Humanos , Inmunoprecipitación , Interfase , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos , Microtúbulos/enzimología , Mitosis , Necrosis , Unión Proteica , Biosíntesis de Proteínas , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Ribosómico 18S/metabolismo , Tubulina (Proteína)/metabolismo

RESUMEN

Lrig1 is a marker of human interfollicular epidermal stem cells and helps maintain stem cell quiescence. We show that, in mouse epidermis, Lrig1 defines the hair follicle junctional zone adjacent to the sebaceous glands and infundibulum. Lrig1 is a Myc target gene; loss of Lrig1 increases the proliferative capacity of stem cells in culture and results in epidermal hyperproliferation in vivo. Lrig1-expressing cells can give rise to all of the adult epidermal lineages in skin reconstitution assays. However, during homeostasis and on retinoic acid stimulation, they are bipotent, contributing to the sebaceous gland and interfollicular epidermis. beta-catenin activation increases the size of the junctional zone compartment, and loss of Lrig1 causes a selective increase in beta-catenin-induced ectopic hair follicle formation in the interfollicular epidermis. Our results suggest that Lrig1-positive cells constitute a previously unidentified reservoir of adult mouse interfollicular epidermal stem cells.

Asunto(s)

Células Epidérmicas , Glicoproteínas de Membrana/metabolismo , Células Madre Multipotentes/citología , Proteínas del Tejido Nervioso/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Epidermis/metabolismo , Folículo Piloso/citología , Folículo Piloso/metabolismo , Mamíferos , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Células Madre Multipotentes/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , beta Catenina/metabolismo

RESUMEN

Dielectrophoresis is a method that has demonstrated great potential in cell discrimination and isolation. In this study, the dielectrophoretic sorting of normal and Babesia bovis infected erythrocytes was performed using a microfabricated flow cytometer. Separation was possible through exploitation of the dielectric differences between normal and infected erythrocytes, essentially due to the higher ionic membrane permeability of B. bovis infected cells. Sorting experiments were performed inside a microchip made from Pt microelectrodes and SU-8 channels patterned on a glass substrate. Optimum cell separation was achieved at 4 MHz using an in vitro culture of B. bovis suspended in 63 mS/m phosphate buffer and applying a sinusoidal voltage of 15 V peak-to-peak. Normal erythrocytes experienced stronger positive dielectrophoresis (pDEP) than B. bovis infected cells, moving them closer to the microelectrodes. Under these conditions it was possible to enrich the fraction of infected cells from 7 to 50% without the need of extensive sample preparation or labelling. Throughout the experiments very few microliters of sample were used, suggesting that this system may be considered suitable for integration in a low-cost automated device to be used in the in situ diagnostic of babesiosis.

Asunto(s)

Babesia bovis , Separación Celular/métodos , Electroforesis/métodos , Eritrocitos/parasitología , Citometría de Flujo/métodos , Animales , Bovinos , Electrodos , Recuento de Eritrocitos , Teoría Cuántica , Coloración y Etiquetado

RESUMEN

We present a particle-sorting device based on the opposition of dielectrophoretic forces. The forces are generated by an array of electrode chambers located in both sidewalls of a main flow channel. Particles with different dielectric response perceive different force magnitudes and are therefore continuously focused to different streamlines in the flow channel. We relate the particles' dielectric response to their output position in the downstream channel. We demonstrate the performance of the device by separating a mixed yeast cell population into pure fractions of viable and nonviable cells. Finally, we use the device to enrich red blood cells infected with Babesia bovis, a major pathogen in cattle and simultaneously confirm the hypothesis that infection with B. bovis causes significant changes in the dielectric response of red blood cells.

Asunto(s)

Babesia bovis/aislamiento & purificación , Separación Celular/instrumentación , Separación Celular/métodos , Eritrocitos/citología , Técnicas Analíticas Microfluídicas/instrumentación , Saccharomyces cerevisiae/citología , Animales , Babesiosis/parasitología , Calibración , Supervivencia Celular , Electrodos , Electroforesis/instrumentación , Electroforesis/métodos , Eritrocitos/parasitología , Humanos , Técnicas Analíticas Microfluídicas/métodos , Sensibilidad y Especificidad

RESUMEN

Impedance spectroscopy is a powerful tool for label-free analysis and characterisation of living cells. In this work, we achieved the detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer. The cellular modifications caused by the intracellular parasite result in a shift in impedance which can be measured dielectrically. Thus, a rapid cell-by-cell detection with microliter amounts of reagents is possible. Unlike other diagnostic tests, this method does not depend on extensive sample pre-treatment or expensive chemicals and equipment.

Asunto(s)

Babesia bovis/aislamiento & purificación , Babesiosis/veterinaria , Enfermedades de los Bovinos/diagnóstico , Eritrocitos/parasitología , Citometría de Flujo/instrumentación , Análisis Espectral/métodos , Animales , Babesiosis/diagnóstico , Babesiosis/parasitología , Bovinos , Enfermedades de los Bovinos/parasitología , Impedancia Eléctrica , Parasitología/métodos

RESUMEN

A new pathogenic Theileria species transmitted by Haemaphysalis qinghaiensis was identified in the Northwestern part of China and was shown to be highly pathogenic for small ruminants. The present article aimed at identifying merozoite antigens that might be suitable for developing diagnostic methods and designing a potential vaccine. Absence of other theilerial or babesial infections was confirmed by reverse line blot in all antigen samples used. Extensive Western blot analyses using serum from infected and noninfected animals led to the identification of four potential merozoite immunoreactive proteins at different molecular weights. Further protein characterization using peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) followed by database searching resulted in two significant hits that identified two proteins of parasite origin, one homologous to a possible MO25-family protein from Cryptosporidium parvum and the other with an HSP70 from Theileria annulata. Another protein was also identified as a parasite protein but without significant homology. Immunization of rabbits with selected proteins produced antisera that reacted specifically on Western blots with merozoite antigens of the corresponding sizes. This article represents the first identification and characterization of potential antigenic proteins of Theileria sp. (China) for veterinary purposes.

Asunto(s)

Antígenos de Protozoos/análisis , Rumiantes/parasitología , Theileria/inmunología , Theileria/patogenicidad , Theileriosis/inmunología , Animales , Western Blotting/veterinaria , China , Electroforesis en Gel de Poliacrilamida/veterinaria , Sueros Inmunes/inmunología , Ixodidae/parasitología , Merozoítos/inmunología , Peso Molecular , Proteínas Protozoarias/análisis , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Conejos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/veterinaria

RESUMEN

OBJECTIVE: To establish optimal conditions for long-term culture of the erythrocytic stage of Theileria uilenbergi. SAMPLE POPULATION: Red blood cells from 3 splenectomized sheep experimentally infected with a blood stabilate of T uilenbergi. PROCEDURES: Cultures of T uilenbergi were initiated by use of blood from experimentally infected sheep collected when parasites were detected in Giemsa-stained thin blood smears. Different culture conditions were tested to optimize in vitro growth of the organisms. Subcultures were performed at a ratio of 1:2, 1:4, and 1:8 when the percentage of parasitized erythrocytes (PPE) was at least 1% or when the initial PPE was doubled. RESULTS: The optimal culture medium was HL-1 medium (a complete chemically defined medium) supplemented with 20% sheep serum and 0.75% chemically defined lipids. Optimal culture conditions included incubation in a humidified 2% O(2), 5% CO(2), and 93% N(2) atmosphere at 37 degrees C. Cultures of the merozoite stage of the parasite were continuously propagated in vitro for > 1 year. The PPE reached values of up to 3%. CONCLUSIONS AND CLINICAL RELEVANCE: Optimization of culture conditions to reach a high PPE seems worthwhile. The continuous propagation of T uilenbergi in culture allows the production of parasite material without infecting animals and provides a continuous laboratory source of parasites for further studies.

Asunto(s)

Técnicas de Cultivo de Célula , Enfermedades de las Ovejas/parasitología , Theileria/crecimiento & desarrollo , Theileriosis/parasitología , Animales , Criopreservación/veterinaria , Medio de Cultivo Libre de Suero , Cartilla de ADN , Eritrocitos/parasitología , Reacción en Cadena de la Polimerasa/veterinaria , Ovinos , Enfermedades de las Ovejas/sangre , Theileriosis/sangre

RESUMEN

This work presents quality control results on the mercury concentrations in different environmental (river sediments, forest soils, river suspended matter and fish) and human samples from the lower Madeira River, Amazon sampled between 2001 and 2003, about 15-20 years after the nearly cessation of gold mining activities in the region, which reached its peak in the late 1980s. The study aimed to compare mercury concentrations in these environmental samples with those reported by other authors during the gold rush of the Madeira River Basin. Today, in the Madeira River the releases of mercury register a sudden reduction due the gold price fall in the international trade. However, about 100 t of Hg were released to the atmosphere and to aquatic systems in the region during the gold rush. The present survey shows that notwithstanding the reduction of Hg emissions to the Madeira River Basin from gold mining proper, concentrations in fish and humans are similar to those measured during the gold rush. Reduction in Hg concentrations is restricted to areas close to old point sources and only for abiotic compartments (air and sediments). Remobilization of Hg from bottom sediments plus re-emission from soils due to land use changes are probably responsible for keeping high Hg concentrations in biological samples.

Asunto(s)

Mercurio/análisis , Contaminantes del Suelo/análisis , Contaminantes Químicos del Agua/análisis , Animales , Brasil , Monitoreo del Ambiente , Peces , Contaminación de Alimentos , Sedimentos Geológicos/análisis , Cabello/química , Humanos , Ríos

RESUMEN

A reforma do sistema de saúde tem sido marcada por intensos debates sobre os limites dos setores público e privado na provisão de assistência médica. A demanda à assistência médica tem sido um grande problema para a política da saúde no Brasil. As empresas visando amenizar essa situação buscam firmar convênios junto às operadoras de assistência médica privada, estabelecendo um processo de assistência suplementar à saúde aos seus funcionários. Este estudo, objetivando estudar a atenção médica suplementar focalizando sua atuação no Estado do Paraná, pesquisa os convênios firmados por duas indústrias de Ponta Grossa (Paraná) com a operadora de assistência médica suplementar - Unimed, visto que Ponta Grossa é a 3° Regional de Saúde do Estado e polo de atendimento de diversos municípios da região, e, portanto, se qualificando como importante amostra para o estudo em tela. Por outro lado, a Unimed como uma das operadoras de maior atuação no Estado do Paraná permite melhor compreender a complexidade da assistência suplementar. Assim, o desenvolvimento do estudo se verificou pela revisão das políticas de saúde e assistência médica suplementar, focalizando o Paraná e particularizando a cidade de Ponta Grossa. A caracterização das conjunturas e das estruturas políticas de atenção à saúde permitiu a formulação crítica dos convênios mencionados, à luz da Lei 9.656/98 e a atuação da ANS para a regulamentação desses convênios. A pesquisa, além de buscar nos referenciais teóricos subsídios para interpretar os procedimentos da operadora, realizou pesquisas de campo, através do acesso a documentos e entrevistas com os usuários e contratantes, possibilitando assim a obtenção de dados que deram sustentabilidade às críticas e sugestões estabelecidas na conclusão. Entre as conclusões principais destacam-se: o desconhecimento generalizado entre gerentes, trabalhadores e sindicatos das empresas sobre o modus operandi da Unimed tanto quanto sobre os reais custos e limites dos planos oferecidos, bem como a não adesão da operadora às normas da ANS, com os subseqüentes prejuízos para os usários. Finalmente, salienta-se que o estudo de caso mostrou ser possível introduzir mudanças nesses âmbitos desde que haja transparência nas transações e informações e sejam processadas mediante negociações e consensos entre todos os interesses envolvidos.

Asunto(s)

Planes de Asistencia Médica para Empleados , Seguro de Salud , Cobertura de Servicios Privados de Salud