A Cell-Free Multi-enzyme Cascade Reaction for the Synthesis of CDP-Glycerol.

CDP-glycerol is a nucleotide-diphosphate-activated version of glycerol. In nature, it is required for the biosynthesis of teichoic acid in Gram-positive bacteria, which is an appealing target epitope for the development of new vaccines. Here, a cell-free multi-enzyme cascade was developed to synthetize nucleotide-activated glycerol from the inexpensive and readily available substrates cytidine and glycerol. The cascade comprises five recombinant enzymes expressed in Escherichia coli that were purified by immobilized metal affinity chromatography. As part of the cascade, ATP is regenerated in situ from polyphosphate to reduce synthesis costs. The enzymatic cascade was characterized at the laboratory scale, and the products were analyzed by high-performance anion-exchange chromatography (HPAEC)-UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After the successful synthesis had been confirmed, a design-of-experiments approach was used to screen for optimal operation conditions (temperature, pH value and MgCl2 concentration). Overall, a substrate conversion of 89 % was achieved with respect to the substrate cytidine.

Efecto de la exposición a bajas dosis de ozono en la expresión de la interleucina 17A durante el proceso de neurodegeneración progresiva en el hipocampo de ratas / Effect of exposure to low doses of ozone on interleukin 17A expression during progressive neurodegeneration in the rat hippocampus

Introducción: La exposición crónica a bajas dosis de ozono causa un estado de estrés oxidativo y pérdida de la regulación de la respuesta inflamatoria, lo cual lleva a un proceso de neurodegeneración progresiva.ObjetivoEstudiar el efecto de la exposición crónica a bajas dosis de ozono sobre la concentración de IL-17A y su expresión en neuronas, microglía, astrocitos y células T en hipocampo de ratas.MétodosSe utilizaron 72 ratas Wistar, divididas en 6 grupos (n = 12): control (sin ozono) y expuestos a ozono (0,25 ppm, 4 h diarias) durante 7, 15, 30, 60 y 90 días, respectivamente. Seis sujetos de cada grupo fueron procesados para cuantificar IL-17A por ELISA y los 6 restantes para inmunohistoquímica (frente a IL-17A y GFAP, Iba1, NeuN o CD3).ResultadosLos datos obtenidos por el ELISA mostraron un incremento significativo en las concentraciones de IL-17A en los grupos de 7, 15, 30 y 60 días de exposición, comparados con el control (p < 0,05). Los resultados muestran que las neuronas del hipocampo son las células con una mayor inmunorreactividad frente a IL-17A entre los 60 y 90 días de exposición a ozono; además, se observó un aumento de astrocitos activados en los grupos de 30 y 60 días de exposición.ConclusiónLa exposición a ozono induce un incremento en la expresión de la IL-17A, principalmente en las neuronas hipocampales, acompañado de una activación de astrocitos en el hipocampo de ratas durante el proceso de neurodegeneración progresiva, similar a lo que ocurre en la enfermedad de Alzheimer en humanos. (AU)

Introduction: Chronic exposure to low doses of ozone causes oxidative stress and loss of regulation of the inflammatory response, leading to progressive neurodegeneration.ObjectiveWe studied the effect of chronic exposure to low doses of ozone on IL-17A concentration and expression in neurons, microglia, astrocytes, and T cells in the rat hippocampus.MethodsWe used 72 Wistar rats, divided into 6 groups (n = 12): a control group (no ozone exposure) and 5 groups exposed to ozone (0.25 ppm, 4 h daily) for 7, 15, 30, 60, and 90 days. We processed 6 rats from each group to quantify IL-17A by ELISA; the remaining 6 were processed for immunohistochemistry (against IL-17A and GFAP, Iba1, NeuN, and CD3).ResultsThe ELISA study data showed a significant increase in IL-17A concentrations in the 7-, 15-, 30-, and 60-day exposure groups, with regard to the control group (P < .05). Furthermore, they indicate that hippocampal neurons were the cells showing greatest immunoreactivity against IL-17A between 60 and 90 days of exposure to ozone; we also observed an increase in activated astrocytes in the 30- and 60-day exposure groups.ConclusionExposure to ozone in rats induces an increase in IL-17A expression, mainly in hippocampal neurons, accompanied by hippocampal astrocyte activation during chronic neurodegeneration, similar to that observed in Alzheimer disease in humans. (AU)

Effect of exposure to low doses of ozone on interleukin 17A expression during progressive neurodegeneration in the rat hippocampus.

INTRODUCTION: Chronic exposure to low doses of ozone causes oxidative stress and loss of regulation of the inflammatory response, leading to progressive neurodegeneration. OBJECTIVE: We studied the effect of chronic exposure to low doses of ozone on IL-17A concentration and expression in neurons, microglia, astrocytes, and T cells in the rat hippocampus. METHODS: We used 72 Wistar rats, divided into 6 groups (n=12): a control group (no ozone exposure) and 5 groups exposed to ozone (0.25ppm, 4h daily) for 7, 15, 30, 60, and 90 days. We processed 6 rats from each group to quantify IL-17A by ELISA; the remaining 6 were processed for immunohistochemistry (against IL-17A and GFAP, Iba1, NeuN, and CD3). RESULTS: The ELISA study data showed a significant increase in IL-17A concentrations in the 7-, 15-, 30-, and 60-day exposure groups, with regard to the control group (P<.05). Furthermore, they indicate that hippocampal neurons were the cells showing greatest immunoreactivity against IL-17A between 60 and 90 days of exposure to ozone; we also observed an increase in activated astrocytes in the 30- and 60-day exposure groups. CONCLUSION: Exposure to ozone in rats induces an increase in IL-17A expression, mainly in hippocampal neurons, accompanied by hippocampal astrocyte activation during chronic neurodegeneration, similar to that observed in Alzheimer disease in humans.

Mix-and-Match System for the Enzymatic Synthesis of Enantiopure Glycerol-3-Phosphate-Containing Capsule Polymer Backbones from Actinobacillus pleuropneumoniae, Neisseria meningitidis, and Bibersteinia trehalosi.

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.

Effect of chronic unpredictable stress on the progression of experimental apical periodontitis in rats.

AIM: To establish an experimental model combining chronic stress and apical periodontitis by assessing the development of periapical lesions in rats in three different time points. METHODOLOGY: Forty-eight male Wistar rats were randomly assigned into two equal groups: Apical periodontitis (AP) and AP + Stress (AP + S). The animals of the AP group were not exposed to stressful conditions whereas the AP + S group were exposed to a variety of stressors on a daily basis until the end of the experiment. After three weeks of chronic unpredictable stress, apical periodontitis was induced in both groups by exposing the pulpal tissue of the mandibular first molar to the oral environment. Each group was further subdivided into three subgroups according to the euthanasia period: 14, 21 and 28 days after pulp exposure. The animals were weighed, and the blood was collected for corticosterone serum dosage by radioimmunoassay. The mandibles were removed and submitted to histopathological and microtomography analyses to assess the inflammatory response and the progression of periapical lesions. Comparisons between the AP and AP + S groups were performed using Student's t-test and Mann-Whitney U-test for parametric and nonparametric data, respectively. The one-way anova test followed by Tukey's test (parametric data) and Kruskal-Wallis followed by Dunn's test (nonparametric data) were used for comparisons between the three time points within the same group (P < 0.05). RESULTS: The AP + S group had a significantly lower average percentage of weight gain at 14 days and 21 days after AP induction (P < 0.05). Significantly higher levels of corticosterone were found in the AP + S group at 21 days (P < 0.05). The AP + S group had a significantly greater intensity and extension of inflammatory infiltrate with larger areas of bone loss compared to the AP groups at 21 days (P < 0.05). The volume of the periapical lesions in the AP + S group was significantly larger than that of the AP group 21 days following pulp exposure (P < 0.05). CONCLUSIONS: The chronic unpredictable stress model applied for 6 weeks exacerbated the inflammatory response and increased bone loss associated with AP, especially 21 days after its induction. This model appears to be suitable for investigating the bidirectional relationship between apical periodontitis and chronic stress.

Clinical influence of calcium hydroxide and N-acetylcysteine on the levels of resolvins E1 and D2 in apical periodontitis.

AIM: To investigate the presence of resolvins E1 (RvE1) and D2 (RvD2) in teeth with primary endodontic infections and apical periodontitis, and to assess the influence of calcium hydroxide medication [Ca(OH)2 ], in association with 2% chlorhexidine gel (2% CHX gel), and N-acetylcysteine (NAC) on the levels of RvE1 and RvD2 in periapical tissues. METHODOLOGY: Thirty-six single-rooted teeth with primary endodontic infections and apical periodontitis were selected and randomly divided into three groups according to the medication: [Ca(OH)2 ] + saline solution (SSL) [Ca(OH)2  + SSL group] (n = 12), Ca(OH)2  + 2% chlorhexidine gel [Ca(OH)2  + 2% CHX gel group] (n = 12) and NAC [NAC group] (n = 12). Samples were collected from the periapical interstitial fluid at two different sampling times: before (S1) and after 14 days of intracanal medications (S2). Resolvins were measured using the enzyme-linked immunosorbent assay. Data were analysed using paired t-test, Wilcoxon test and Kruskal-Wallis test, followed by Dunn's post hoc test; all statistical tests were performed at a significance level of 5%. RESULTS: RvE1 and RvD2 were detected in 100% of the samples (36/36) at S1 and S2. Ca(OH)2 medication did not increase the levels of RvE1 or RvD2 (both P > 0.05); however, NAC significantly increased the levels of RvE1 and RvD2 after 14 days of treatment (P < 0.05). CONCLUSIONS: RvE1 and RvD2 were detected in periapical tissues from teeth with root canal infections. Moreover, calcium hydroxide medication did not increase the levels of resolvins in apical periodontitis. In contrast, the use of NAC intracanal medication significantly increased the levels of RvE1 and RvD2 after 14 days of treatment.

Effect of exposure to low doses of ozone on interleukin 17A expression during progressive neurodegeneration in the rat hippocampus. / Efecto de la exposición a bajas dosis de ozono en la expresión de la interleucina 17A durante el proceso de neurodegeneración progresiva en el hipocampo de ratas.

INTRODUCTION: Chronic exposure to low doses of ozone causes oxidative stress and loss of regulation of the inflammatory response, leading to progressive neurodegeneration. OBJECTIVE: We studied the effect of chronic exposure to low doses of ozone on IL-17A concentration and expression in neurons, microglia, astrocytes, and T cells in the rat hippocampus. METHODS: We used 72 Wistar rats, divided into 6 groups (n=12): a control group (no ozone exposure) and 5 groups exposed to ozone (0.25ppm, 4h daily) for 7, 15, 30, 60, and 90 days. We processed 6 rats from each group to quantify IL-17A by ELISA; the remaining 6 were processed for immunohistochemistry (against IL-17A and GFAP, Iba1, NeuN, and CD3). RESULTS: The ELISA study data showed a significant increase in IL-17A concentrations in the 7-, 15-, 30-, and 60-day exposure groups, with regard to the control group (P<.05). Furthermore, they indicate that hippocampal neurons were the cells showing greatest immunoreactivity against IL-17A between 60 and 90 days of exposure to ozone; we also observed an increase in activated astrocytes in the 30- and 60-day exposure groups. CONCLUSION: Exposure to ozone in rats induces an increase in IL-17A expression, mainly in hippocampal neurons, accompanied by hippocampal astrocyte activation during chronic neurodegeneration, similar to that observed in Alzheimer disease in humans.

Qualitative analysis of governance trends after health system reforms in Latin America: lessons from Mexico.

OBJECTIVE: Health policies in Latin America are centered on the democratization of health. Since 2003, during the last generation of reforms, health systems in this region have promoted governance strategies for better agreements between governments, institutions, and civil society. In this context, we develop an evaluative research to identify trends and evidence of governance after health care reforms in six regions of Mexico. METHODS: Evaluative research was developed with a retrospective design based on qualitative analysis. Primary data were obtained from 189 semi-structured interviews with purposively selected health care professionals and key informants. Secondary data were extracted from a selection of 95 official documents on results of the reform project at the national level, national health policies, and lines of action for good governance. Data processing and analysis were performed using ATLAS.ti and PolicyMaker. RESULTS: A list of main strengths and weaknesses is presented as evidence of health system governance. Accountability at the federal level remains prescriptive; in the regions, a system of accountability and transparency in the allocation of resources and in terms of health democratization strategies is still absent. CONCLUSION: Social protection and decentralization schemes are strategies that have allowed for improvements with a proactive role of users and civil society. Regarding challenges, there are still low levels of governance and difficulties in the effective conduct of programs and reform strategies together with a lack of precision in the rules and roles of the different actors of the health system.

Peripheral, functional and postural asymmetries related to the preferred chewing side in adults with natural dentition.

The aim of this cross-sectional study was to determine the preferred chewing side and whether chewing side preference is related to peripheral, functional or postural lateral preferences. One hundred and forty-six adults with natural dentition performed three masticatory assays, each consisting of five trials of chewing three pieces of silicon placed into a latex bag for 20 cycles, either freestyle or unilaterally on the right- or left-hand side. Occlusal contact area in the intercuspal position, maximum bite force, masticatory performance and cycle duration were measured and the lateral asymmetry of these variables was calculated. Laterality tests were performed to determine handedness, footedness, earedness and eyedness as functional preferences, and hand-clasping, arm-folding and leg-crossing as postural lateral preferences. The preferred chewing side was determined using three different methods: assessment of the first chewing cycle for each trial, calculation of the asymmetry index from all cycles and application of a visual analogue scale. Bivariate relationship and multiple linear regression analyses were performed. Among unilateral chewers, 77% of them preferred the right side for chewing. The factors most closely related to the preferred chewing side were asymmetry of bite force, asymmetry of masticatory performance and earedness, which explained up to 16% of the variance. Although several functional or postural lateral preferences seem to be related to the preferred chewing side, peripheral factors such as asymmetry of bite force and of masticatory performance are the most closely related to the preferred chewing side in adults with natural dentition.

Effectiveness of daily versus non-daily granulocyte colony-stimulating factors in patients with solid tumours undergoing chemotherapy: a multivariate analysis of data from current practice.

We conducted a multicentre, retrospective, observational study including patients with solid tumours (excluding breast cancer) that received granulocyte colony-stimulating factors (G-CSF) and chemotherapy. We investigated the effectiveness of daily vs. non-daily G-CSFs (pegfilgrastim) adjusting by potential confounders. The study included 391 patients (211 daily G-CSF; 180 pegfilgrastim), from whom 47.3% received primary prophylaxis (PP) (57.8% pegfilgrastim), 26.3% secondary prophylaxis (SP: initiation after cycle 1 and no reactive treatment in any cycle) (51.5% pegfilgrastim) and 26.3% reactive treatment (19.4% pegfilgrastim). Only 42.2% of patients with daily G-CSF and 46.2% with pegfilgrastim initiated prophylaxis within 72 h after chemotherapy, and only 10.5% of patients with daily G-CSF received it for ≥ 7 days. In the multivariate models, daily G-CSF was associated with higher risk of grade 3-4 neutropenia (G3-4N) vs. pegfilgrastim [odds ratio (OR): 1.73, 95% confidence interval (CI): 1.004-2.97]. Relative to SP, PP protected against G3-4N (OR for SP vs. PP: 6.0, 95%CI: 3.2-11.4) and febrile neutropenia (OR: 3.1, 95%CI: 1.1-8.8), and was associated to less chemotherapy dose delays and reductions (OR for relative dose intensity <85% for SP vs. PP: 3.1, 95%CI: 1.7-5.4) and higher response rate (OR: 2.1, 95%CI: 1.2-3.7). Data suggest that pegfilgrastim, compared with a daily G-CSF, and PP, compared with SP, could be more effective in preventing neutropenia and its related events in the clinical practice.

Entamoeba histolytica: the over expression of a mutated EhRabB protein produces a decrease of in vitro and in vivo virulence.

Vesicular trafficking, which is implicated in secretion of cytolytic molecules as well as in phagocytosis, plays an important role in the pathogenic mechanism of Entamoeba histolytica, the protozoan parasite causative of human amoebiasis. Thus, Rab GTPases, that are key regulators of vesicle trafficking, should be considered as molecules involved in the parasite virulence. EhRabB is a Rab protein located in cytoplasmic vesicles that are translocated to phagocytic mouths during ingestion of target cells, suggesting that this Rab protein is involved in phagocytosis. To prove this hypothesis, we over expressed the wild type EhrabB gene and a mutant gene encoding for a protein (RabBN118I) unable to bind guanine nucleotides and therefore constitutively inactive. The over expression of the mutated protein in E. histolytica trophozoites provoked a dominant negative effect, reflected in a significant decrease of both phagocytosis and cytopathic effect as well as in a failure to produce hepatic abscesses in hamsters. These results confirm that EhRabB is involved in phagocytosis and virulence of E. histolytica.

Alpha-1-acid glycoprotein, its local production and immunopathological participation in experimental pulmonary tuberculosis.

Alpha-1-acid glycoprotein (AGP) is one of the major acute-phase proteins (APPs). Hepatic production and serum concentrations increase in response to systemic injury, inflammation, or infection. We reported previously that expression of the AGP gene is induced in the liver during experimental pulmonary tuberculosis. Since AGP may also be produced at the infection site and has some immunomodulatory properties, we used a model of progressive pulmonary tuberculosis in Balb/c mice to study the kinetics of AGP production in the lung and its influence on immunopathology. We found that AGP was produced in the lung during experimental tuberculosis. Alveolar macrophages and type II pneumocytes were the most important cellular sources during early infection (days 1-14). From day 21 postinfection, during the progressive phase of the infection, foamy macrophages located in pneumonic areas were the most important source of AGP and 10-fold higher concentrations were found on day 60. In a second part of the study, AGP was inactivated during the progressive phase by the administration of specific blocking antibodies. In comparison with control infected animals, tuberculous mice treated with blocking AGP antibodies showed higher expression of interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in association with significantly reduced bacillary loads and tissue damage. Thus, AGP is produced in the lung during experimental pulmonary tuberculosis and it has immunomodulatory activities, suppressing cell-mediated immunity and facilitating growth of bacilli and disease progression.

Concurrencia de síndromes paraneoplásicos neurológicos, en paciente afecta de cáncer de mama y metástasis cerebral: revisión de la literatura / Concurrence of neurologic paraneroplastic syndromes in a woman with brain metastatic breast cancer: review of the literature

Los Síndromes paraneoplásicos neurológicos sonentidades complejas, de difícil diagnóstico, y quepueden afectar a cualquier parte del sistemanervioso. El descubrimiento de nuevos antígenosonconeuronales y su relación con las alteracionesinmunológicas asociadas están ayudando a undiagnóstico precoz. La asociación con el carcinomade mama es infrecuente. Presentamos el caso de unapaciente de 62 años, diagnosticada de carcinoma demama izquierda, mastectomizada y tratada conquimioterapia adyuvante. Presentó recidiva, conmetástasis cerebelosa resecada quirúrgicamente ytratada con radioterapia. Se complicó con cuadromiasteniforme de tipo Lambert-Eaton, disfunciónautonómica y posterior encefalítis límbica con comairreversible. La aplicación de los nuevos criteriosdiagnósticos, los descubrimientos moleculares y larealización de ensayos clínicos terapeúticos,podrían mejorar las expectativas en estas pacientes

Neurological paraneoplastic syndromes arecomplex entities of difficult diagnosis. They canlocate in any part of the nervous system producingimportant neurological dysfunctions. The discoveryof new specific anti-neuronal antibodies and theirrelation with the associated immunologicalalterations help to make an early diagnosis.Association with breast cancer is not frequent. Wepresent the case of a 62-year old patient with leftbreast carcinoma that had been treated bymastectomy and adjuvant chemotherapy one yearbefore. She relapsed with cerebellum metastasesand was treated by surgery and radiotherapy. Thepicture was complicated with a Lambert-Eaton-likemyasthenic syndrome, neuroplastic sensoryneuronopathy, limbic encephalitis and irreversiblecoma. The application of new diagnostic criteria,the molecular discoveries and the performance oftherapeutic clinical trials could better the lifeexpectancy of these patients

Entamoeba histolytica: structural and functional analysis of the Ehadh112 gene promoter.

The Entamoeba histolytica Ehcp112 and Ehadh112 genes that encode the EhCPADH complex are separated by a non-coding 188pb region. Their proximity suggests a coordinated expression regulation for both genes. Here, we studied the structure and function of 996 bp (p996CAT) upstream the ATG start codon of the Ehadh112 gene. The p996CAT plasmid drove CAT transcription with a 78% of the activity showed by actin promoter. Deletion of 330 bp at the 5' end of p966CAT to produce the p776CAT plasmid, decreased activity to 40% in relation to actin promoter and to 50% of p996CAT, suggesting the presence of a silencer in this region. Interestingly, deletion of other 297 bp to the p776CAT to generate the p469CAT plasmid, augmented activity in 2.5-fold compared with p776CAT construction, showing the presence of a proximal enhancer promoter. Transcription initiation sites (-69 and -150 bp), TATA like box, GAAC, and Inr elements, as well as putative DNA binding motifs, were mapped in the -1 to -469 bp core promoter region.

The Entamoeba histolytica Ehcp112 gene has a distal and weak promoter.

Ehcp112 encodes the Entamoeba histolytica EhCP112 cysteine protease that is part of the EhCPADH complex. By in silico analysis we identified putative transcription factor-binding sites along 837 bp upstream the Ehcp112 gene ATG codon. A TATA-like motif (TATATAAA) was located at -36 to -29 bp, a GAAC box (GAACC) was found at -10 to -14 bp and an Inr sequence (TTCAAC) at -8 to -2 bp. These tripartite promoter elements are in non-canonical positions, downstream the transcription initiation site (-280 bp). We cloned four Ehcp112 promoter fragments in pBSCAT-ACT plasmid to obtain pI (355 bp), pII (681 bp), pIII (833 bp), and pIV (554 bp) constructs. In transfected trophozoites, only pIII drove CAT activity with 44% efficiency in relation to actin promoter activity. Our results showed the presence of a distal and weak promoter in the Ehcp112 gene. The active DNA region is inside the open reading frame of the Ehrab B gene, suggesting that expression of both genes could be coordinately regulated.

Functional characterization of EhADH112: an Entamoeba histolytica Bro1 domain-containing protein.

EhADH112 is part of the EhCPADH complex, a protein involved in key events of the Entamoeba histolytica host invasion. EhADH112 participates in trophozoite adherence to target cells and in phagocytosis. We report here the finding of two EhADH112 homologues in the E. histolytica genome (EhADH112-like proteins). EhADH112 and its relatives have a Bro1 domain at their amino-terminus and a consensus context for phosphorylation by Src-tyrosine kinases, both involved in signal transduction processes in other organisms. Our findings associate EhADH112 to supplementary functions related to those reported for the Alix/AIP1 family. To elucidate the precise function of EhADH112, we studied the phenotypes displayed by trophozoites transfected with the Ehadh112 full gene. Transfected trophozoites overexpressed a 78 kDa protein, which was mainly targeted to the EhCPADH complex. Moreover, these trophozoites exhibited enhanced phagocytic rates, providing further evidence of EhADH112 contribution to adhesion and phagocytosis activities.

Structural characterization of SmMn2GeO7 single microcrystals by electron microscopy.

Single microcrystals of the new compound samarium dimanganese germanium oxide, SmMn2GeO7, were grown using the flux method in a double spherical mirror furnace (DSMF). The micrometric crystals were observed and chemically analysed with scanning electron microscopy (SEM) and X-ray energy dispersive spectroscopy (EDX). The structural characterization and chemical analysis of these crystals were also carried out using transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM), together with electron-energy-loss spectroscopy (EELS). We found that the new quaternary compound crystallizes in the orthorhombic system with the point group mmm (D2h), space group Immm (No. 71) and cell parameters a=8.30 (10), b=8.18 (10), c=8.22 (10) A and V=558.76 A3.

HER2 y carcinoma de próstata: ¿una relación de utilidad clínica? / HER2 and prostate carcinoma: is there any relation with clinical utility?

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Ehp53, an Entamoeba histolytica protein, ancestor of the mammalian tumour suppressor p53.

This paper reports the identification of Ehp53, a p53-like Entamoeba histolytica protein, which binds to the human p53 DNA consensus sequence (oli-p53). Monoclonal antibodies against p53 (Ab-1 and Ab-2) recognized a single 53 kDa spot in two-dimensional gels and inhibited the formation of complexes produced by E. histolytica nuclear extracts and oli-p53. Additionally, E. histolytica gene promoter sequences with high homology to oli-p53 formed complexes with nuclear proteins that were abolished by oli-p53. Ehp53 protein levels increased in UV-irradiated trophozoites. This protein was also detected in Entamoeba moshkovskii and Entamoeba invadens. By confocal microscopy, Ehp53 was located in the nuclei, EhkO organelles and cytoplasm. The Ehp53-encoding gene was cloned and its predicted amino acid sequence showed 30-54 % and 50-57 % homology with important domains of the human and the Drosophila melanogaster p53 proteins, respectively. This homology included the tetramerization domain, the nuclear export signal and a nuclear localization signal. Ehp53 also contains seven of the eight DNA-binding residues and two of the four Zn(2+)-binding sites described for p53. A recombinant Ehp53 was recognized by Ab-2. Ehp53 is believed to be the first p53-like protein found in protozoa and may be the evolutionary ancestor of the mammalian p53.

Multidrug resistance in the protozoan parasite Entamoeba histolytica.

In this review we discuss the mechanisms and molecules involved in the multidrug resistance (MDR) of the protozoan parasite Entamoeba histolytica. Drug resistant mutants exhibited the main characteristics presented by the MDR mammalian cells. They showed cross-resistance to several unrelated drugs that is reverted by calcium channel blockers. MDR phenotype in E. histolytica is regulated at a transcriptional level by the EhPgp1 gene, which is constitutively expressed and by the EhPgp5 gene, whose expression is induced in the presence of the drug. Transcription factors participate in the expression regulation of these genes. After over transcription, the EhPgp genes are amplified, cooperating to produce the MDR phenotype. Post-transcriptional mechanisms such as mRNA stability seem to be involved in this phenomenon. As for other mdr gene products, the EhPGP5 protein functions as a chloride current inductor or as a regulator of cellular regulatory volume decrease.