Quantitative profiling of CD13 on single acute myeloid leukemia cells by super-resolution imaging and its implication in targeted drug susceptibility assessment.

Quantitative profiling of membrane proteins on the cell surface is of great interest in tumor targeted therapy and single cell biology. However, the existing technologies are either of insufficient resolution, or unable to provide precise information on the localization of individual proteins. Here, we report a new method that combines the use of quantum dot labeling, super-resolution microscopy (structured illumination microscopy, SIM) and software modeling. In this proof-of-principle study, we assessed the biological effects of Bestatin on individual cells from different AML cell lines expressing CD13 proteins, a potential target for tumor targeted therapy. Using the proposed method, we found that the different AML cell lines exhibit different CD13 expression densities, ranging from 0.1 to 1.3 molecules per µm2 cell surface, respectively. Importantly, Bestatin treatment assays shows that its effects on cell growth inhibition, apoptosis and cell cycle change are directly proportional to the density of CD13 on the cell surface of these cell lines. The results suggest that the proposed method advances the quantitative analysis of single cell surface proteins, and that the quantitative profiling information of the target protein on single cells has potential value in targeted drug susceptibility assessment.

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

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

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

Educação permanente em alimentação e nutrição na Estratégia Saúde da Família: encontros e desencontros em municípios brasileiros de grande porte / Ongoing food and nutrition education within the family health strategy: points of agreement and disagreement in major Brazilian cities

O objetivo foi conhecer fatores facilitadores e dificultadores e estratégias utilizadas por 28 municípios brasileiros de grande porte na realização de Educação Permanente em alimentação e nutrição, na Estratégia Saúde da Família. Método qualitativo de múltiplos casos. A técnica de coleta de dados foi a entrevista com coordenadores municipais das ações de alimentação e nutrição, das cinco regiões do país. O estudo foi realizado entre 2009 e 2010. Utilizou-se o software NVivo e a análise de conteúdo foi orientada por categorias temáticas. Foram entrevistados 44 profissionais, sendo 19 nutricionistas. A maioria dos municípios era do Nordeste e Sudeste, 14 eram capitais, 7 metrópoles e 14 tinham Núcleo de Apoio à Saúde da Família. Os fatores facilitadores para Educação Permanente em nutrição mais citados foram as parcerias e a disponibilidade de recursos. Os dificultadores mais frequentes foram a indisponibilidade de agendas e a falta de profissionais na gestão das ações de nutrição. As estratégias mais utilizadas foram a realização de ações educativas no nível local, por grupos e o planejamento e programação. Concluiu-se que são necessários maiores investimentos para que a Educação Permanente em alimentação e nutrição se concretize.

This study sought to ascertain the facilitating and inhibiting factors and strategies used by 28 major Brazilian cities in conducting ongoing food and nutrition education within the Family Health Strategy. It involved a qualitative study of multiple cases. The data collection technique was conducted in interviews with municipal coordinators of food and nutrition campaigns from the five regions of the country. The study was conducted between 2009 and 2010. NVivo 10 software was used and content analysis was divided up into thematic categories. Forty-four professionals were interviewed, 19 of which were nutritionists. Most cities were from the Northeast and Southeast; 14 were capitals, 7 were metropolises and 14 had Family Health Suppor Units. The most frequently mentioned facilitating factors for Ongoing Education in Nutrition were partnerships and the availability of funds. The most frequent inhibiting factors were difficulty in scheduling and a lack of professionals in management of nutrition actions. The strategies most commonly used were conducting training at the local level, in groups and planning and programming. The conclusion drawn is that more investment is needed for Ongoing Education in Feeding and Nutrition to be effectively implemented.

The adaptation problems of patients undergoing hemodialysis: socio-economic and clinical aspects / Problemas adaptativos de pacientes em hemodiálise: aspectos socioeconômicos e clínicos / Problemas adaptativos de pacientes en hemodiálisis: aspectos socioeconómicos y clínicos

OBJECTIVES: to identify adaptation problems under Roy's Model in patients undergoing hemodialysis and to correlate them with the socioeconomic and clinical aspects. METHOD: a transversal study, undertaken using a questionnaire. The sample was made up of 178 individuals. The Chi-squared and Mann-Whitney U tests were undertaken. RESULTS: the adaptation problems and the socioeconomic and clinical aspects which presented statistical associations were: Hyperkalemia and age; Edema and income; Impairment of a primary sense: touch and income; Role failure and age; Sexual dysfunction and marital status and sex; Impairment of a primary sense: vision and years of education; Intolerance to activity and years of education; Chronic pain and sex and years of education; Impaired skin integrity and age: Hypocalcemia and access; Potential for injury and age and years of education; Nutrition below the organism's requirements and age; Impairment of a primary sense: hearing and sex and kinetic evaluation of urea; Mobility in gait and/or coordination restricted, and months of hemodialysis; and, Loss of ability for self-care, and months of hemodialysis and months of illness. CONCLUSION: adaptation problems in the clientele undergoing hemodialysis can be influenced by socioeconomic/clinical data. These findings contribute to the development of the profession, fostering the nurse's reflection regarding the care. .

OBJETIVOS: identificar os problemas adaptativos de Roy em pacientes submetidos a hemodiálise e correlacioná-los aos aspectos socioeconômicos e clínicos. MÉTODO: estudo transversal, realizado através de um formulário. A amostra foi de 178 indivíduos. Efetuaram-se os testes qui-quadrado e U de Mann-Whitney. RESULTADOS: os problemas adaptativos e os aspectos socioeconômicos e clínicos que apresentaram associações estatísticas foram: hipercalemia e idade; edema e renda; deficiência de um sentido primário: tátil e renda; falha no papel e idade; disfunção sexual e estado civil e sexo; deficiência de um sentido primário: visão e anos de estudo; intolerância à atividade e anos de estudo; dor crônica e sexo e anos de estudo; integridade da pele prejudicada e idade; hipocalcemia e acesso; potencial para lesão e idade e anos de estudo; nutrição menor que as necessidades do organismo e idade; deficiência de um sentido primário: audição e sexo e avaliação cinética da ureia; mobilidade andar e/ou coordenação restritas e meses de hemodiálise e perda de habilidade de autocuidado e meses de hemodiálise e meses de doença. CONCLUSÃO: problemas adaptativos da clientela hemodialítica podem sofrer influências de dados socioeconômicos/clínicos. Tais achados contribuem para o desenvolvimento da profissão, proporcionando reflexão por parte do enfermeiro acerca do cuidado. .

OBJETIVOS: identificar los problemas adaptativos de Roy en pacientes sometidos a hemodiálisis y correlacionarlos a los aspectos socioeconómicos y clínicos. MÉTODO: estudio transversal, realizado a través de un formulario. La muestra fue de 178 individuos. Se efectuaron las pruebas Chi-cuadrado y U de Mann-Whitney. RESULTADOS: los problemas adaptativos y los aspectos socioeconómicos y clínicos que presentaron asociaciones estadísticas fueron: Hiperkalemia y edad; Edema y renta; Deficiencia de un sentido primario: táctil y renta; Fracaso en el papel y edad; Disfunción sexual y estado civil y sexo; Deficiencia de un sentido primario: visión y años de estudio; Intolerancia a la actividad y años de estudio; Dolor crónico y sexo y años de estudio; Integridad de la piel perjudicada y edad; Hipocalcemia y acceso; Potencial para lesión y edad y años de estudio; Nutrición menor que las necesidades del organismo y edad; Deficiencia de un sentido primario: audición y sexo y evaluación cinética de la urea; Movilidad andar y/o coordinación restringidas y meses de hemodiálisis; y, Pérdida de habilidad de autocuidado y meses de hemodiálisis y meses de enfermedad. CONCLUSIÓN: los problemas adaptativos de la clientela hemodialítica pueden sufrir influencias de datos socioeconómicos/clínicos. Esos hallazgos contribuyen para el desarrollo de la profesión, permitiendo la reflexión del enfermero acerca del cuidado. .

Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles.

Autophagy is a degradation pathway that recycles cell materials upon encountering stress conditions or during specific developmental processes. To better understand the physiological roles of autophagy, proper monitoring methods are very important. In mammals and yeast, monitoring of autophagy is often performed with a green fluorescent protein (GFP)-ATG8 fusion protein or with acidotropic dyes such as monodansylcadaverine (MDC) and LysoTracker Red (LTR). To evaluate these monitoring methods, here we examined these systems by inducing autophagy in Arabidopsis thaliana roots as a model for monitoring autophagy in planta. Under carbon- and nitrogen-starved conditions, the number and size of vesicles labeled by GFP-ATG8 was increased for several hours and then gradually decreased to a level higher than that observed before the start of the experiment. We also observed the disappearance of GFP-ATG8-labeled vesicles after treatment with wortmannin, a phosphatidylinositol 3-kinase inhibitor known as an autophagy inhibitor, showing that the GFP-ATG8 transgenic line constitutes an excellent method for monitoring autophagy. These data were compared with plants stained with MDC and LTR. There was no appreciable MDC/LTR staining of small organelles in the root under the induction of autophagy. Some vesicles were eventually observed in the root tip only, but co-localization experiments, as well as experiments with autophagy-deficient atg mutants, provided the evidence that these structures were located in the vacuole and were not manifestly autophagosomes and/or autolysosomes. Extreme caution should therefore be used when monitoring autophagy with the aid of MDC/LTR. Additionally, our observations strongly suggest that autophagosomes fuse directly to vacuoles in Arabidopsis roots.

Judicialização da saúde na garantia do acesso ao medicamento / The judicialisation of health as a means ensuring access to medicines / La Judicialización de la salud como garantía de acceso a medicamentos

Objetivo Visando conhecer o impacto das demandas judiciais sobre a organização dos serviços públicos de saúde, realizou-se uma revisáo sistemática com enfoque na "judicialização da saúde" para fornecimento de medicamentos. Métodos Foram analisados artigos originais publicados no período de 2007 a 2011, na literatura nacional e internacional, resultando no total de 49239 artigos disponíveis nas bases de dados Science Direct e BIREME. Resultados A pesquisa indicou predominância da bibliografia proveniente do Brasil, principalmente do sudeste, bem como de estudo realizado na Colômbia. Discursáo Dentre os pleitos, configuraram-se como principais agravos relatados as doenças crônicas, podendo-se citar: diabetes, hipertensáo, cânceres e artrite reumatóide. Por serem afecções parte de programas específicos do Sistema Único de Saúde, a dificuldade de acesso a esses fármacos e consequente judicialização da saúde demonstrou a fragilidade das políticas públicas existentes. Conclusão Por fim, conclui-se que a via judicial, apesar de ser uma estratégia para garantir o acesso ao medicamento, apresenta inabilidade para lidar com o julgamento das ações e gera, dessa forma, distorções no fluxo dos sistemas públicos.

Objective A systematic review, focusing on the judicialisation of health regarding gaining access to medicines, was aimed at understanding the impact of lawsuits on the organisation of public health services. Method Original articles published between 2007 and 2011 in the pertinent national and international literature were analysed, resulting in 49,239 articles being found in Science Direct and BIREME databases. Results The survey indicated a predominance of literature from Brazil, mainly the southeast, as well as a study from Colombia. Discussion The aforementioned chronic disease-related claims involved diabetes, high blood pressure, cancer and rheumatoid arthritis. Forming part of specific Unified Healthcare System programmes highlighted the difficulty in gaining access to the appropriate medicine and consequent health judicialisation demonstrated the fragility of existing public policy. Conclusion It was concluded that the courts (despite being a strategy for ensuring access to medicine) were unable to deal with the current spate of lawsuits, thereby leading to disruption regarding the flow of public systems.

Objetivo El estudio tiene como objetivo evaluar el impacto de las demandas judiciales sobre la organización de los servicios públicos de salud, mediante la realización de una revisión sistemática centrada en el uso de los tribunales para el suministro de medicamentos. Método Fueron identificados 49239 artículos en las bases de datos Science Direct e BIREME. Resultado El estudio indicó que la mayor parte de la bibliografía es de Brasil, con uno estudio en Colombia. Discusión Aparecen como los principales trastornos de salud relatados a las enfermedades crónicas, se pueden citar: la diabetes, la hipertensión, el cáncer y la artritis reumatoide. Debido a que son parte de los programas específicos de lo sistema de salud, la dificultad de acceso a estos fármacos y la consiguiente judicialización de la salud de manifiesto la fragilidad de las políticas públicas existentes. Conclusiones Por último, está la conclusión de que los tribunales, a pesar de ser una estrategia para garantizar el acceso a la medicina, presenta incapacidad para hacer frente al juicio de las acciones y por lo tanto genera distorsiones en el flujo de los sistemas públicos.

Partial purification and characterisation of cysteine protease in wheat germ.

BACKGROUND: Proteases have become an essential part of the modern food and feed industry, being incorporated in a large and diversified range of products for human and animal consumption. The objective of this study was to purify and characterise a protease from wheat germ. RESULTS: After purification a single protease of molecular weight 61-63 kDa (determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) was obtained. The purified protease had optimal activity at 50 °C and maintained its activity completely after incubation at 30 °C for 30 min, while over 47% of the activity was lost after incubation at 80 °C for 30 min. The purified protease had optimal activity and maintained maximum stability at pH 5.5, while the activity decreased after incubation for 30 min at other pH values. The protease was inhibited by Mg(2+), Mn(2+), Ba(2+) and iodoacetic acid and stimulated by Li(+), Ca(2+), Cu(2+), ß-mercaptoethanol and dithiothreitol, while Zn(2+), L-cysteine and glutathione had no significant effect on its activity. At pH 5.5 the enzyme had a K(m) of 0.562 mg mL(-1) with casein as substrate and showed higher affinity to casein than to bovine serum albumin, ovalbumin and gelatin. CONCLUSION: The purified enzyme from wheat germ was identified as a cysteine protease.

Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro.

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock.

Entamoeba histolytica: correlation of assessment methods to measure erythrocyte digestion, and effect of cysteine proteinases inhibitors in HM-1:IMSS and HK-9:NIH strains.

Entamoeba histolytica trophozoites are able to degrade human erythrocytes; the loss of erythrocyte cellular matrix and degradation of plasma membrane were observed, along with the decrease in the average size of digestive vacuoles. Ninety-six percent of hemoglobin ingested was hydrolyzed by trophozoites within 3h, as evidenced by electrophoresis. Accordingly, X-ray spectroscopy revealed the presence of iron inside vacuoles after erythrophagocytosis, the concentration of which decreased to control levels in a similar period. Quantification of erythrocyte digestion at the early and late periods was determined by a spectrophotometric procedure, with t(1/2)=1.67 h and 35-min for HM-1:IMSS and HK-9:NIH trophozoites, respectively. In the latter, activity was due to the combined action of intracellular enzymatic activity and exocytosis. E-64c and leupeptin totally inhibited erythrocyte digestion within a 3-h period, thereafter hydrolysis took place at lower rate. Our results suggest that erythrocyte digestion in E. histolytica proceeds in different ways in these two amebic strains, and can be blocked by proteinase inhibitors.

Assessment of cell-signaling pathways in the regulation of mammalian target of rapamycin (mTOR) by amino acids in rat adipocytes.

Enhanced phosphorylation of the ribosomal protein s6 kinase, p70(s6k), and the translational repressor, 4E-BP1, are associated with either insulin-induced or amino acid-induced protein synthesis. Hyperphosphorylation of p70(s6k) and 4E-BP1 in response to insulin or amino acids is mediated through the mammalian target of rapamycin (mTOR). In several cell lines, mTOR or its downstream targets can be regulated by phosphatidylinositol (PI) 3-kinase; protein kinases A, B, and C; heterotrimeric G-proteins; a PD98059-sensitive kinase or calcium; as well as by amino acids. Regulation by amino acids appears to involve detection of levels of charged t-RNA or t-RNA synthetase activity and is sensitive to inhibition by amino acid alcohols. In the present article, however, we show that the rapamycin-sensitive regulation of 4E-BP1 and p70(s6k) in freshly isolated rat adipocytes is not inhibited by either L-leucinol or L-histidinol. This finding is in agreement with other recent studies from our laboratory suggesting that the mechanism by which amino acids regulate mTOR in freshly isolated adipocytes may be different than the mechanism found in a number of cell lines. Therefore we investigated the possible role of growth factor-regulated and G-protein-regulated signaling pathways in the rapamycin-sensitive, amino acid alcohol-insensitive actions of amino acids on 4E-BP1 phosphorylation. We found, in contrast to previously published results using 3T3-L1 adipocytes or other cell lines, that the increase in 4E-BP1 phosphorylation promoted by amino acids was insensitive to agents that regulate protein kinase A, mobilize calcium, or inhibit protein kinase C. Furthermore, amino acid-induced 4E-BP1 phosphorylation was not blocked by pertussis toxin nor was it mimicked by the G-protein agonists fluoroaluminate or MAS-7. However, amino acids failed to activate either PI 3-kinase, protein kinase B, or mitogen-activated protein kinase and failed to promote tyrosine phosphorylation of cellular proteins, similar to observations made using cell lines. In summary, amino acids appear to use an amino acid alcohol-insensitive mechanism to regulate mTOR in freshly isolated adipocytes. This mechanism is independent of cell-signaling pathways implicated in the regulation of mTOR or its downstream targets in other cells. Overall, our study emphasizes the need for caution when extending results obtained using established cell lines to the differentiated nondividing cells found in most tissues.

In vivo assessment of mast cell functional alteration induced by L-leucine methyl ester, using the passive cutaneous anaphylaxis technique.

We have recently reported that in vitro, mast cells were sensitive to the action of L-leucine methyl ester (Leu-OMe), a lysosomotropic compound. We now report the in vivo effect of Leu-OMe on mast cells, qualitatively assessed by using the passive cutaneous anaphylaxis (PCA) reaction. The L- but not the D-stereoisomer of Leu-OMe (25 mM) injected together with Dactylis glomerata, pollen-specific, IgE-containing serum inhibited the PCA reaction in rat skin triggered by a subsequent challenge with the corresponding allergen. When specific IgE antibodies were injected in the rat skin 3 days after the L-Leu-OMe, subsequent challenge with the antigen displayed a recovery of the PCA reaction. Thus, an in vivo L-Leu-OMe treatment, at a concentration which did not lead to any macroscopic tissue injury, elicited either an alteration of mast cell mediator release or an inhibition of allergen-induced vasoactive mediator release through a functional deactivation and/or a depletion of mast cells.

An investigation of the natriuretic, antidiuretic and oxytocic actions of neurohypophysial hormones and related peptides: delineation of separate mechanisms of action and assessment of molecular requirements.

The substitution of the 4-glutamine of oxytocin by a lipophilic aliphatic amino acid leucine yields [4-Leu] oxytocin which possesses natriuretic-diuretic anti-arginine-vasopressin (anti-ADH) activities. Alkyl substitutions of the beta-carbon of the 1 half-cystine of oxytocin yield a series of antioxytocin analogs which inhibit the uterotonic response to oxytocin. In this paper, the results of our further investigations on the molecular requirements for natriuretic, anti-ADH and antioxytocic activities of these peptides are reported. A total of 12 analogs of oxytocin and lysine-vasopressin (LVP) with leucine and/or beta-carbon alkyl substitutions were studied. Our findings reveal that the effect of 4-leucine substitution may not be to enhance the natriuretic activity but rather to abolish the antidiuretic activity of oxytocin. The lack of antidiuretic activity of these 4-leucine analogs makes it possible to unmask the intrinsic natriuretic activity of these peptides at the high dose level. Structure-activity correlations suggest that the oxytocin molecule may be the optimal requirement for natriuretic activity of these peptides. Substitution of 4-glutamine by lipophilic aromatic phenylalanine yields [4-Phe] oxytocin which possesses anti-ADH activity with little or no natriuretic activity. The "hybrid" antioxytocin and anti-ADH molecules, beta-carbon alkyl and 4-leucine substituted analogs did not possess enhanced antihormone activity. Although they had antioxytocic and antipressor activities, they were less potent than their respective singly alkyl substituted analogs. Furthermore, they had no demonstrable anti-ADH activity. The single alkyl substituted oxytocin and LVP also had no anti-ADH activity. It therefore appears that beta-carbon alkyl substitution had different effects on activities depending on the morphological features and the functions of the target cell. In target cells of contractile smooth muscles (uterus and vascular), the alkyl substituted analogs had no intrinsic activity but retained a relatively high receptor affinity to become effective antagonists to the natural hormone. On the other hand, in target cells of the renal tubule which are noncontractile epithelial cells, both intrinsic activity and receptor affinity were reduced or abolished. Thus none of these alkyl substituted analogs possessed more than very slight antidiuretic activity, and none had any natriuretic or anti-ADH activity.