Investigation of Acid-Base Catalysis in Halimadienyl Diphosphate Synthase Involved in Mycobacterium tuberculosis Virulence.

The devastating human pathogenMycobacterium tuberculosis (Mtb) is able to parasitize phagosomal compartments within alveolar macrophage cells due, in part, to the activity of its cell-surface lipids. Prominent among these is 1-tuberculosinyl-adenosine (1-TbAd), a derivative of the diterpenoid tuberculosinyl (halima-5,13-dienyl) diphosphate produced by the class II diterpene cyclase encoded by Rv3377c, termed here MtHPS. Given the demonstrated ability of 1-TbAd to act as a virulence factor for Mtb and the necessity for Rv3377c for its production, there is significant interest in MtHPS activity. Class II diterpene cyclases catalyze a general acid-base-mediated carbocation cascade reaction initiated by protonation of the terminal alkene in the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate and terminated by deprotonation of the final cyclized (and sometimes also rearranged) intermediate. Here, structure-guided mutagenesis was applied to characterize the various residues contributing to activation of the enzymatic acid, as well as identify the enzymatic base in MtHPS. Particularly given the ability of conservative substitution for the enzymatic base (Y479F) to generate an alternative product (labda-7,13-dienyl diphosphate) via deprotonation of an earlier unrearranged intermediate, further mutational analysis was carried out to introduce potential alternative catalytic bases. The results were combined with mechanistic molecular modeling to elucidate how these mutations affect the catalytic activity of this important enzyme. This not only provided detailed structure-function insight into MtHPS but also further emphasized the inert nature of the active site of MtHPS and class II diterpene cyclases more generally.

Medicinal plants and natural products as neuroprotective agents in age-related macular degeneration.

The retina may suffer neurodegenerative damages, as other tissues of the central nervous system do, and serious eye diseases may develop. One of them is age-related macular degeneration, which causes progressive loss of vision due to retina degeneration. Treatment of age-related macular degeneration focuses on antioxidant agents and anti-vascular endothelial growth factor compounds, among others, that prevent/diminish oxidative stress and reduce neovascularisation respectively. The phytochemicals, medicinal plants and/or plant-diet supplements might be a useful adjunct in prevention or treatment of age-related macular degeneration owing to their antioxidant and anti-vascular endothelial growth factor properties. This review article presents the most investigated plants and natural products in relation to age-related macular degeneration, such as saffron, ginkgo, bilberry and blueberry, curcuma or turmeric, carotenoids, polyphenols, and vitamins C and E. This study provides up-to-date information on the effects, treatments, safety and efficiency of these phytotherapy products.

Overexpression of IL-10 in C2D macrophages promotes a macrophage phenotypic switch in adipose tissue environments.

Adipose tissue macrophages are a heterogeneous collection of classically activated (M1) and alternatively activated (M2) macrophages. Interleukin 10 (IL-10) is an anti-inflammatory cytokine, secreted by a variety of cell types including M2 macrophages. We generated a macrophage cell line stably overexpressing IL-10 (C2D-IL10) and analyzed the C2D-IL10 cells for several macrophage markers after exposure to adipocytes compared to C2D cells transfected with an empty vector (C2D-vector). C2D-IL10 macrophage cells expressed more CD206 when co-cultured with adipocytes than C2D-vector cells; while the co-cultured cell mixture also expressed higher levels of Il4, Il10, Il1ß and Tnf. Since regular C2D cells traffic to adipose tissue after adoptive transfer, we explored the impact of constitutive IL-10 expression on C2D-IL10 macrophages in adipose tissue in vivo. Adipose tissue-isolated C2D-IL10 cells increased the percentage of CD206(+), CD301(+), CD11c(-)CD206(+) (M2) and CD11c(+)CD206(+) (M1b) on their cell surface, compared to isolated C2D-vector cells. These data suggest that the expression of IL-10 remains stable, alters the C2D-IL10 macrophage cell surface phenotype and may play a role in regulating macrophage interactions with the adipose tissue.

Effect of cooking and germination on phenolic composition and biological properties of dark beans (Phaseolus vulgaris L.).

Legumes are the basés diet in several countries. They hold a high nutritional value, but other properties related to human health are nowadays being studied. The aim of this work was to study the influence of processes (boiling or germination) on the phenolic composition of dark beans (Phaseolus vulgaris L. c.v. Tolosana) and their effect on their antioxidant, neuroprotective and anticancer ability. Phenolic composition of raw and processed dark beans was analysed by HPLC-PAD and HPLC-ESI/MS. The antioxidant activity was evaluated by ORAC. Astrocytes cultures (U-373) have been used to test their neuroprotective effect. Anticancer activities were evaluated on three different cell lines (renal adenocarcinoma (TK-10), breast adenocarcinoma (MCF-7) and melanoma (UACC-62)) by sulphorhodamine B method. Qualitative and quantitative differences in phenolic composition have been observed between raw and processed dark beans that influence the antioxidant activity, mainly for germinated samples which show a decrease of antioxidant capacity. Although every assayed extracts decreased reactive oxygen species release and exhibited cytotoxicity activities on cancer cell lines, raw beans proved to be the most active in neuroprotective and antitumoral effects; this sample is especially rich in phenolic compounds, mainly anthocyanins. This study further demonstrated that phenolic composition of dark beans is related with cooking process and so with their neuroprotective and anticancer activity; cooking of dark beans improves their digestion and absorption at intestinal level, while maintaining its protective ability on oxidative process at cellular level.

Tratamiento de la sobrecarga de volumen en IC refractaria / Treatment of volume overload in refractory heart failure / Tratamento da sobrecarga de volume na insuficiência cardíaca refratária

La insuficiencia cardíaca es un problema de salud creciente debido al envejecimiento de la población y a la mejoría en el pronóstico de los pacientes con cardiopatía establecida. En los últimos años, han ido apareciendo nuevos tratamientos que intentan mejorar la situación clínica, la calidad de vida y la supervivencia de aquellos pacientes que desarrollan refractariedad a los diuréticos. El empleo de estos nuevos fármacos podría tener un impacto positivo en las situaciones agudas, pero no parece que aporten beneficios adicionales para los enfermos con insuficiencia cardíaca congestiva crónica. En estos casos, las técnicas de ultrafiltración extracorpórea y, sobre todo, la diálisis peritoneal parecen demostrar mejoría de la clase funcional, reducción en las tasas de hospitalización, mejoría en la calidad de vida y reducción de la mortalidad, siendo además coste-eficiente.

Heart failure is an increasing health problem due to the aging population and the improvement in the prognosis of patients with established heart disease. In recent years new treatments have emerged that seek to improve the clinical situation, the quality of life and survival of patients who develop refractoriness to diuretics. The use of these new drugs could have a positive impact on the acute but it would not provide additional benefits for patients with chronic congestive heart failure. In these cases, extracorporeal ultrafiltration techniques and especially peritoneal dialysis seem to show improvement in functional class, reduction in hospitalization rates, improved quality of life and reduction in mortality, besides being cost-efficient.

A insuficiência cardíaca é um problema crescente de saúde devido ao envelhecimento da população e a melhora do prognóstico dos pacientes com doença cardíaca estabelecida. Nos últimos anos, novos tratamentos surgiram que procuram melhorar a situação clínica, a qualidade de vida e sobrevida dos pacientes que desenvolvem refratariedade ao uso de diuréticos. O uso desses novos fármacos poderia ter um impacto positivo sobre os efeitos agudos, mas não oferecem benefícios adicionais para os pacientes com insuficiência cardíaca congestiva crônica. Nestes casos, as técnicas de ultrafiltração extracorpórea e, especialmente, a diálise peritoneal parecem mostrar melhoria da classe funcional, redução das taxas de hospitalização, melhoria da qualidade de vida e a redução da mortalidade, e tambémé custo-eficiente.

Cationic liposomes bearing IL-2 on their external surface induced mice leukocytes to kill human cervical cancer cells in vitro, and significantly reduced tumor burden in immunodepressed mice.

Tumor cells are known to modify their surroundings in order to escape immunologic detection, and IL-2, a killer cell activator, is one of the factors known to overcome this escape mechanism. In this regard, when we cocultured cells from the human cervical cancer cell line INBL with mice blood leukocytes, no inhibition of tumor cell growth was observed, but when a similar coculture was done in the presence of cationic liposomes bearing IL-2 on their external surface (CL-IL-2), all the INBL cells were killed. In order to evaluate whether this in vitro property of CL-IL-2 to overcome tumor cell detection by lymphocytes could also be reproduced in vivo, INBL cells were intraperitoneally (i.p.) inoculated into immunodepressed mice to produce solid tumors. We observed that the subsequent i.p. delivery of CL-IL-2 rendered the tumor masses significantly smaller. The presence of a large number of infiltrating lymphocytes on those tumors, and the fact that many had a cytotoxic CD8(+) phenotype suggests that these lymphocytes were responsible for the observed antitumor effect. Finally, the possible formation of a bridge between the IL-2R receptors on both, the lymphocytes and the INBL cells, mediated by the IL-2-bearing liposomes, and its possible effect on the activation of antitumor cytotoxic lymphocytes is discussed.

Papel de la diálisis peritoneal en el tratamiento de la insuficiencia cardíaca / Role of peritoneal dialysis in the treatment of heart failure / Papel da diálise peritoneal no tratamento da insuficiência cardíaca

Introducción. La insuficiencia cardíaca (IC) es una patología progresiva incluso con tratamiento adecuado. La eliminación de líquido ayuda en el manejo de estos pacientes. Evaluamos la eficacia de la diálisis peritoneal (DP) para tratar la IC refractaria. Pacientes y métodos. Estudio prospectivo no randomizado en pacientes con IC refractaria a dosis máximas toleradas de fármacos. Todos fueron tratados con DP. Se analizaron datos clínicos y estado funcional. Para determinar su eficacia se comparó el estado de salud percibido en los pacientes con DP respecto a los reportes de aquéllos que utilizaban tratamientos conservadores. Finalmente, se realizó un estudio de coste-utilidad. Resultados. Fueron incluidos 25 pacientes (68% varones, 66±10 años) y 16 siguieron en DP a la finalización del período de seguimiento (20±9 meses). Realizaron un solo intercambio nocturno 21 pacientes; el resto 2 ó 3 en relación al grado de insuficiencia renal. Todos mejoraron su estado funcional (4% tres grados, 64% dos y 32% uno; P<0,001); se redujo la presión sistólica en la arteria pulmonar (46±12 vs 25±9 mm Hg; P=0,007). Las tasas de hospitalización disminuyeron de forma importante (de 65±16 a 9±5 días/paciente/año; P=0,002). La expectativa de vida fue del 84% al año de tratamiento y del 72% y 58% tras 18 y 24 meses, respectivamente. La DP se asoció a mejor percepción del estado de salud que los tratamientos conservadores (0,6978 vs 0,4104; P<0,01). La DP es coste-efectiva comparada con los regímenes diuréticos. Conclusiones. La DP es una buena opción para el manejo del paciente con IC refractaria; mejora el grado funcional y la calidad de vida y reduce la hospitalización, mortalidad y los costes sanitarios.

Background. Heart failure (HF) is a progressive disorder even with adequate treatment. Fluid removal may aid in the management of these patients. We evaluated the efficacy of peritoneal dialysis (PD) in the treatment of refractory HF. Patients and methods. Prospective, non-randomized study involving patients with congestive HF refractory to maximum tolerable drug treatment. All of them were treated with PD. We analysed clinical data and functional status. To determine efficacy we compared the perceived state of health to PD patients respect to those reported with conservative therapies. Finally, we carried out a cost-utility evaluation. Results. Twenty-five patients (68% men, 66±10 years) were included and 16 were still undergoing PD at the end of the follow-up period (20±9 months). Twenty-one patients underwent only one daily nocturnal exchange; the rest, 2 or 3 exchanges due to different degrees of renal failure. All of them improved their NYHA functional status, (4% three classes, 64% two, 32% one; P<0.001), with a reduction in their pulmonary artery systolic pressure (46±12 vs 25±9 mm Hg; P=0.007). Hospitalization rates underwent a dramatic reduction (from 65±16 to 9±5 days/patient/year; P=0.002). PD life expectancy was 84% after 12 months of treatment, and 72% and 58% after 18 and 24 months. PD was associated with a higher perception state of health than the conservative therapy, (0.6978 vs 0.4104; P<0.01). PD is cost-effective compared with the diuretic regimens. Conclusions. PD is a good option for patients with refractory HF; it improves the functional status and quality of life, reduces morbidity, mortality and health care costs.

Introdução. A insuficiência cardíaca (IC) é uma patologia progressiva, mesmo com tratamento adequado. A remoção do líquido pode auxiliar no tratamento desses pacientes. Nós avaliamos a eficácia da diálise peritoneal (PD) no tratamento da insuficiência cardíaca refratária. Pacientes e métodos. Estudo prospectivo, não randomizado envolvendo pacientes com insuficiência cardíaca congestiva refratária ao tratamento medicamentoso máximo tolerado. Todos foram tratados com DP. Nós analisamos dados clínicos e estado funcional. Nós determinamos a eficácia, comparando o estado de saúde em PD em comparação com relação aos relatados com tratamento conservador. Finalmente, foi realizada uma avaliação de custo-utilidade. Resultados. Foram incluídos 25 pacientes (68% homens, 66±10 anos) e 16 estavam ainda em fase de PD no final do período de follow-up (20±9 meses). Vinte e um pacientes foram submetidos a apenas uma troca diária noturna, o resto, as trocas de 2 ou 3 devido a diferentes graus de insuficiência renal. Todos melhoraram seu status funcional da NYHA (4% três graus, 64% dois y 32% um; P<0,001); com uma redução da sua pressão sistólica da artéria pulmonar (46±12 vs 25±9 mm Hg; P=0,007). As taxas de hospitalização decresceram significativamente (de 65±16 a 9±5 dias/paciente/ano; P=0,002). A expectativa de vida era de 84% por ano de tratamento e 72% e 58% após 18 e 24 meses, respectivamente. PD foi associada com um estado de maior percepção de saúde do que o tratamento conservador (0,6978 vs. 0,4104; P<0,01). O DP é custo-utilidade em comparação com os regimes de diuréticos. Conclusàµes. O DP é uma boa opção para o manejo de pacientes com IC refratária, que melhora o estado funcional e qualidade de vida, reduz a internação, mortalidade e custos de saúde.

Study of red wine neuroprotection on astrocytes.

Phenolic composition of wine depends not only on the grape variety from which it is made, but on some external factors such as winemaking technology. Red wine possesses the most antioxidant effect because of its high polyphenolic content. The aim of this work is to study for the first time, the neuroprotective activity of four monovarietal Spanish red wines (Merlot (ME), Tempranillo (T), Garnacha (G) and Cabernet-Sauvignon (CS)) through its antioxidant ability, and to relate this neuroprotection to its polyphenolic composition, if possible. The wine effect on neuroprotection was studied through its effect as free radical scavenger against FeSO4, H2O2 and FeSO4 + H2O2. Effect on cell survival was determined by 3(4,5-dimethyltiazol-2-il)-2,5-diphenyltetrazolium reduction assay (MTT) and lactate dehydrogenase (LDH) release assay on astrocytes cultures. Results showed that most of the studied wine varieties induced neuroprotection through their antioxidant ability in astrocytes, Merlot being the most active; this variety is especially rich in phenolic compounds, mainly catechins and oligomeric proanthocyanidins. Our results show that red wine exerts a protection against oxidative stress generated by different toxic agents and that the observed neuroprotective activity is related to their polyphenolic content.

Identification of epitopes and immunodominant regions on the MICA protein defined by alloantibodies from kidney transplant patients.

BACKGROUND: Several reports showed a contribution of anti-MICA (major histocompatibility complex class I chain-related molecule A) antibodies (Abs) to the development of acute and chronic rejection. Identification of the epitopes to which the Abs bind may help to determine immunoreactive regions essential for the major histocompatibility complex compatibility between donor and recipients, leading to the best outcome of the transplant. METHODS: Sera from 284 kidney transplant patients were screened for anti-MICA Abs by Luminex assay. MICA allele typing of the recipients was determined. The epitopes of MICA were mapped by screening a synthesized library of overlapping peptides from the extracellular domains of the protein against the sera from kidney transplant patients with anti-MICA Abs. RESULTS: Antibodies against MICA were detected in 50 of 284 patients (17.6%) and correlated with the development of acute rejection. Nine antigenic regions were immunoreactive with anti-MICA Abs in the sera samples. Four of these continuous epitopes mapped to polymorphic amino acids (aa). Five antigenic regions were shared epitopes found in all the MICA alleles. The polymorphic residues, 173 (E/K), 175 (S/G), and 181 (R/T), had determined allele-specific epitopes (reactivity patterns 1 and 2). In contrast, the aa 208Y and 213T were implicated in the cross-reactivity among alleles. CONCLUSIONS: The presence of anti-MICA Abs could be an important marker for diagnosis because of their contribution to the outcome of the graft, regardless of presence of anti-HLA Abs. Additionally, the identification of epitopes revealed the in vivo antigens of the transplant and is spurring the development of new matching strategies to reduce the incidence of acute and chronic rejection.

Characterization of cationic liposomes having IL-2 expressed on their external surface, and their affinity to cervical cancer cells expressing the IL-2 receptor.

Anionic, cationic, and neutral liposomes were constructed to contain IL-2 in order to evaluate their affinity to a cervical cancer cell line (INBL) and to determine whether they can present IL-2 on their external surface. When these liposomes were co-cultured with INBL, the anionic liposomes were the only ones found to be cytotoxic. When neutral and cationic liposomes were constructed in the presence of IL-2, IL-2 was detected only on the surface of cationic liposomes by using a fluorescent anti-IL-2 antibody. By co-culturing INBL with IL-2-containing cationic liposomes, and by using fluorescent anti-IL-2 antibody, we found a strong IL-2 presence on the cell membranes thus suggesting a high affinity of the liposomes to the INBL cells.

Estudio de la avicultura de traspatio en el municipio de Dzununcán, Yucatán / A study of the poultry backyard production in the community of Dzununcan, Yucatan, Mexico

Con el fin de realizar un inventario ganadero para determinar algunos indicadores productivos, así como las causas de mortalidad en paquetes familiares de gallinas de doble propósito se llevó a cabo un censo (septiembre a diciembre de 1992) y un estudio de seguimiento (enero a diciembre de 1993) en el municipio de Dzununcán, Yucatán. Mediante el censo, se registró información acerca del número de animales de traspatio, animales de producción y sobre enfermedades más frecuentes. Para el seguimiento, se entregaron paquetes de 10 o 12 pollos de las razas Plymouth Rock barrada, Rhode Island Rojas y cruzas de éstas con criollo a 24 familias distribuidas en tres grupos; posteriormente se llevaron a cabo de 3 a 4 visitas domiciliarias por semana durante un año. Del censo se obtuvo que el 79 por ciento de las familias explotan animales de traspatio. Las tres especies en orden de importancia fueron: gallinas, pavos y cerdo. El número promedio de gallinas por familia fue de 11.5. Las principales causas de mortalidad según los encuestados fueron: La "mortandad" (21 por ciento), catarro (17 por ciento) y viruela (7 por ciento). El seguimiento muestra que los principales padecimientos fueron: Marek (21 por ciento), coccidiosis (22 por ciento) y afecciones respiratorias (13 por ciento). Los pollos alcanzaron un promedio de peso de 1.42 kg a las 21 semanas de edad, rompieron postura a las 38.5 semanas y produjeron un promedio de 39 huevos por gallina durante 24 semanas de producción. La finalidad principal de la producción de huevo fue el autoconsumo. No se encontraron diferencias (P> 0.05) entre genotipos para ninguna de las variables estudiadas