RESUMO
Degranulation, a fundamental effector response from mast cells (MCs) and platelets, is an example of regulated exocytosis. This process is mediated by SNARE proteins and their regulators. We have previously shown that several of these proteins are essential for exocytosis in MCs and platelets. Here, we assessed the role of the SNARE protein SNAP23 using conditional knockout mice, in which SNAP23 was selectively deleted from either the megakaryocyte/platelet or connective tissue MC lineages. We found that removal of SNAP23 in platelets results in severe defects in degranulation of all three platelet secretory granule types, i.e., alpha, dense, and lysosomal granules. The mutation also induces thrombocytopenia, abnormal platelet morphology and activation, and reduction in the number of alpha granules. Therefore, the degranulation defect might not be secondary to an intrinsic failure of the machinery mediating regulated exocytosis in platelets. When we removed SNAP23 expression in MCs, there was a complete developmental failure in vitro and in vivo. The developmental defects in platelets and MCs and the abnormal translocation of membrane proteins to the surface of platelets indicate that SNAP23 is also involved in constitutive exocytosis in these cells. The MC conditional deletant animals lacked connective tissue MCs, but their mucosal MCs were normal and expanded in response to an antigenic stimulus. We used this mouse to show that connective tissue MCs are required and mucosal MCs are not sufficient for an anaphylactic response.
Assuntos
Anafilaxia/imunologia , Plaquetas/imunologia , Tecido Conjuntivo/imunologia , Mastócitos/imunologia , Proteínas Qb-SNARE/imunologia , Proteínas Qc-SNARE/imunologia , Anafilaxia/genética , Anafilaxia/patologia , Animais , Plaquetas/patologia , Tecido Conjuntivo/patologia , Exocitose/genética , Exocitose/imunologia , Mastócitos/patologia , Camundongos , Camundongos Knockout , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Vesículas Secretórias/genética , Vesículas Secretórias/imunologiaRESUMO
The antioxidant, antimicrobial, antiproliferative, and enzyme inhibitory properties of five extracts from aerial parts of Salvia pachyphylla Epling ex Munz were examined to assess the prospective of this plant as a source of natural products with therapeutic potential. These properties were analyzed by performing a set of standard assays. The extract obtained with dichloromethane showed the most variety of components, as they yielded promising results in all completed assays. Furthermore, the extract obtained with ethyl acetate exhibited the greatest antioxidant activity, as well as the best xanthine oxidase inhibitory activity. Remarkably, both extracts obtained with n-hexane or dichloromethane revealed significant antimicrobial activity against the Gram-positive bacteria; additionally, they showed greater antiproliferative activity against three representative cell lines of the most common types of cancers in women worldwide, and against a cell line that exemplifies cancers that typically develop drug resistance. Despite that, other extracts were less active, such as the methanolic or aqueous; their results are promising for the isolation and identification of novel bioactive molecules.
RESUMO
Tuberculosis (TB) remains as a major public health issue in developing countries. Accurate detection is essential for the proper management of patients with active disease. Here, we present a simple DNAzol-LAMP (loop-mediated isothermal amplification) procedure for the detection of Mycobacterium tuberculosis in sputum specimens. Twenty smear-positive sputum samples were analyzed as follows: (i) Genetic material was extracted by a standard DNAzol protocol, and (ii) mycobacterial DNA was detected by a typical TB-specific loop-mediated isothermal amplification method. Results and diagnostic test performance attests to the suitability of the proposed procedure.
RESUMO
OBJECTIVE: Presently, non-LTR retrotransposons are the most active mobile elements in the human genome. Among these, Alu elements are highly represented in the modern population. Worldwide, distribution of Alu polymorphisms (insertion/deletion; I/D) shows variability between different populations. Two Alu insertion loci, ACE and PR, are significant biomarkers that have served in several genotype-phenotype association studies. In Mexico, studies concerning the frequency of these biomarkers have been conducted mainly in subpopulations from central and southern regions. Here, we screened a population sample of the northwestern region to gain further knowledge regarding the prevalence of Alu polymorphisms within ACE and PR loci. RESULTS: For ACE locus, the observed genotype frequencies were 26.5, 51.0 and 22.5% for II, ID, and DD, respectively; and allelic frequencies for I and D were 52 and 48%. Whereas respective genotype frequencies for PR locus were 2.7, 26.5 and 70.8%, and the corresponding allele frequencies were 16 and 84%. Furthermore, the insertion frequency within ACE locus was similar between central, western and northwestern subpopulations, and rather higher in southeastern subpopulation (p < 0.05). Although the occurrence of Alu polymorphisms within PR locus has not been widely examined, the insertion frequency was higher in northwestern subpopulation, as compared with western and southeastern subpopulations (p < 0.05). Based on the frequency of Alu insertions found in ACE and PR loci, subpopulations from the northwestern, western and central regions share a common genetic origin, but apparently not with the subpopulation from the southeastern region, in accordance with the notion that assumes the existence of a broad genomic diversity in the Mexican population. In addition, the high prevalence of Alu insertions reveals their potential application as biomarkers with prognostic value for the associated diseases; e.g., as part of the standard protocols for clinical diagnosis.
Assuntos
Elementos Alu/genética , Biomarcadores , Loci Gênicos/genética , Genótipo , Polimorfismo Genético/genética , Humanos , MéxicoRESUMO
Human amebiasis, caused by the parasitic protozoan Entamoeba histolytica, remains as a significant public health issue in developing countries. The life cycle of the parasite compromises two main stages, trophozoite and cyst, linked by two major events: encystation and excystation. Interestingly, the cyst stage has a chitin wall that helps the parasite to withstand harsh environmental conditions. Since the amebic chitinase, EhCHT1, has been recognized as a key player in both encystation and excystation, it is plausible to consider that specific inhibition could arrest the life cycle of the parasite and, thus, stop the infection. However, to selectively target EhCHT1 it is important to recognize its unique biochemical features to have the ability to control its cellular function. Hence, to gain further insights into the structure-function relationship, we conducted an experimental approach to examine the effects of pH, temperature, and denaturant concentration on the enzymatic activity and protein stability. Additionally, dependence on in vivo oxidative folding was further studied using a bacterial model. Our results attest the potential of EhCHT1 as a target for the design and development of new or improved anti-amebic therapeutics. Likewise, the potential of the oxidoreductase EhPDI, involved in oxidative folding of amebic proteins, was also confirmed.
Assuntos
Quitinases/química , Quitinases/metabolismo , Entamoeba histolytica/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Quitinases/genética , Entamoeba histolytica/genética , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Cinética , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Protein disulfide isomerases (PDI) are eukaryotic oxidoreductases that catalyze the formation and rearrangement of disulfide bonds during folding of substrate proteins. Structurally, PDI enzymes share as a common feature the presence of at least one active thioredoxin-like domain. PDI enzymes are also involved in holding, refolding, and degradation of unfolded or misfolded proteins during stressful conditions. The EhPDI enzyme (a 38 kDa polypeptide with two active thioredoxin-like domains) has been used as a model to gain insights into protein folding and disulfide bond formation in E. histolytica. Here, we performed a functional complementation assay, using a ΔdsbC mutant of E. coli, to test whether EhPDI exhibits isomerase activity in vivo. Our preliminary results showed that EhPDI exhibits isomerase activity; however, further mutagenic analysis revealed significant differences in the functional role of each thioredoxin-like domain. Additional studies confirmed that EhPDI protects heat-labile enzymes against thermal inactivation, extending our knowledge about its chaperone-like activity. The characterization of EhPDI, as an oxidative folding catalyst with chaperone-like function, represents the initial step to dissect the molecular mechanisms involved in protein folding in E. histolytica.
Assuntos
Entamoeba histolytica/metabolismo , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Catálise , Escherichia coli/metabolismo , Oxirredução , Oxirredutases/metabolismo , Ligação Proteica/fisiologia , Dobramento de Proteína , Estrutura Terciária de Proteína/fisiologia , Tiorredoxinas/metabolismoRESUMO
From 1966 to 1983, Argentina underwent a period of political radicalization as fascist regimes used terror to control its citizens and leftist guerrillas resorted to violence to spark revolution. During this politically volatile period, psychiatry transformed from an apolitical clinical specialty into an ideological tool used for both leftist resistance and military oppression. The largest psychiatric organization at the time, the Federación Argentina de Psiquiatras (FAP), became the center for a new politically committed brand of psychiatry in Argentina that united psychoanalysis and community psychiatry with Marxist theory. Though the military targeted and eventually dismantled the FAP and its leftist brand of psychoanalysis and community psychiatry, sectors of the government also paradoxically appropriated and reframed community-based psychiatric perspectives to pathologize leftist subversion and advance their own conservative ideology.
Assuntos
Distúrbios Civis/história , Transtornos Mentais/classificação , Política , Psiquiatria/história , Argentina , História do Século XXRESUMO
Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy.
Assuntos
Entamoeba histolytica/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Antibacterianos/farmacologia , Bacitracina/farmacologia , Entamoeba histolytica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Insulina/metabolismo , Chaperonas Moleculares/metabolismo , Muramidase/química , Muramidase/metabolismo , Oxirredutases/metabolismo , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/química , Dobramento de Proteína , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismoRESUMO
Specific information about the population pharmacogenetics can be the starting point to study the inheritance of these traits, to design individual drug therapy, and to develop new drugs rationally. Pharmacogenetic studies have been performed in some regions of Mexico, such as Central and Northeast, but this kind of study has not been conducted in the Northwest region so far. Here, we report the distribution of NAT2, TPMT, and MTHFR gene polymorphisms in Baja California, Mexico. We found that our population sample exhibits allele and genotype frequencies that are highly similar to those observed in Caucasian populations, although it should be noted that there are slight similarities with those determined in other populations. As allelic variants of drug-metabolizing enzymes are prevalent in our population, it is important to consider pharmacogenetic testing as part of the standard diagnostic protocols before medication.
Assuntos
Arilamina N-Acetiltransferase/genética , Etnicidade/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metiltransferases/genética , Farmacogenética/métodos , Polimorfismo Genético/genética , Adolescente , Adulto , Arilamina N-Acetiltransferase/metabolismo , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Metiltransferases/metabolismo , México , Adulto JovemRESUMO
Patients with fragile X syndrome present a variable phenotype, which contributes to the underdiagnosing of this condition. The use of clinical checklists in individuals with intellectual disability can help in selecting patients to be given priority in the molecular investigation of the fragile X mutation in the FMR1 gene. Some features included in checklists are better predictors than others, but they can vary among different populations and with patient age. In the present study, we evaluated 20 features listed in four clinical checklists from the literature, using a sample of 192 Brazilian male patients presenting with intellectual disability (30 positive and 162 negative for fragile X mutation). After statistical analysis, 12 out of the 20 items analyzed showed significant differences in their distributions between the two groups. These features were grouped in a new checklist that can help clinicians in their referral for fragile X testing in patients with developmental delay.
Assuntos
Países em Desenvolvimento , Síndrome do Cromossomo X Frágil/diagnóstico , Deficiência Intelectual/diagnóstico , Programas de Rastreamento , Adolescente , Adulto , Brasil , Criança , Pré-Escolar , Estudos Transversais , Síndrome do Cromossomo X Frágil/epidemiologia , Humanos , Deficiência Intelectual/epidemiologia , Masculino , Adulto JovemRESUMO
Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze the correct formation of disulfide bonds during protein folding. Structurally they are characterized by the presence of functional thioredoxin-like (Trx) domains. For the protozoan parasite causative of the human amebiasis (Entamoeba histolytica), the correct formation of disulfide bonds is important for an accurate folding of its proteins, including some virulence factors. However, little is known about the enzymes involved in this mechanism. We undertook a post-genomic approach to identify the PDI family of this parasite. The genome database survey revealed a set of 11 PDI-encoding sequences with predictable protein thiol/disulfide oxidoreductase activities.
Assuntos
Entamoeba histolytica/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
One of the most fascinating aspects of the Entamoeba histolytica trophozoite ultrastructure is the lack of a typical secretory pathway, particularly of rough endoplasmic reticulum and Golgi system, in a cell with such a high secretory activity. Here, we describe the isolation of amoeba cell structures containing ER-typical activities. Following isopycnic centrifugation of plasma membrane-free extracts, microsomes enriched in enzymatic activities such as dolichol-P-mannose synthase (DPMS; EC 2.4.1.83), UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (NAGPT; EC 2.7.8.15), and UDP-D-GlcNAc:dolichol-PP GlcNAc (NAGT; EC 2.4.1.141) were resolved from phagolysosomal fractions. Sec61alpha-subunit, an ER-marker involved in the translocation of nascent proteins to the ER, was found to co-fractionate with DPMS activity indicating that they are contained in microsomes with a similar density. Further, we optimized conditions for trophozoite homogenization and differential centrifugation that resulted in the separation of a 57,000 g-sedimenting microsomal fraction containing EhSec61alpha-subunit, EhDPMS, and EhPDI (protein disulfide isomerase, a soluble marker of the lumen of the ER). A relevant observation was the lack of ER markers associated to the nuclear fraction. Large macromolecular structures such as Ehproteasome were sedimented at a higher speed. Our knowledge of the molecular machinery involved in the biosynthesis of dolichol-linked oligosaccharide was enriched with the identification of putative genes related to the stepwise assembly of the dolichol-PP-GlcNAc(2)Man(5) core. No evidence of genes supporting further assembly steps was obtained at this time.
Assuntos
Entamoeba histolytica/ultraestrutura , Microssomos/enzimologia , Proteínas de Protozoários/metabolismo , Acetilglucosaminidase/análise , Fosfatase Ácida/análise , Animais , Western Blotting , Centrifugação com Gradiente de Concentração , Dolicóis/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/fisiologia , Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Entamoeba histolytica/fisiologia , Glucosiltransferases/análise , Glicosilação , Manosiltransferases/análise , Manosiltransferases/genética , Proteínas de Membrana/análise , Microssomos/fisiologia , Microssomos/ultraestrutura , Oligossacarídeos/biossíntese , Complexo de Endopeptidases do Proteassoma/análise , Isomerases de Dissulfetos de Proteínas/análise , Canais de Translocação SECAssuntos
Entamoeba histolytica/genética , Genoma de Protozoário , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Entamoeba histolytica/classificação , Entamoeba histolytica/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Filogenia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/classificação , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Se presentan 142 casos de infarto del miocardio en jóvenes (IMJ) que corresponden al 5.6 por ciento de un total de 2525 infartos estudiados en los últimos cinco años en el Hospital de Especialidades del Centro Médico La Raza, IMSS. De ellos, 124 (87 por ciento) fueron del sexo masculino y 18 (13 por ciento) del femenino. La edad osciló entre 24 y 40 años, promedio 35 años para ambos sexos. Ocupación laboral: 56 pacientes (39.5 por ciento) eran obreros, 27 (19 por ciento) empleados, 22 (15.5 por ciento) profesionistas, 20 (14 por ciento) taxistas, 15 (10.5 por ciento) comerciantes y 2 (1.5 por ciento) estudiantes. Antecedentes cardiológicos personales: 42 (30 por ciento) tuvieron antecedentes de angor y 5 (3.5 por ciento) de infarto del miocardio. Factores de riesgo coronario: 106 pacientes (75 por ciento) presentaron tensión emocional por personalidad tipo "A", 101 (71 por ciento) tabaquismo, 59 (42 por ciento) hipertensión arterial sistémica, 35 (25 por ciento) diabetes mellitus, 35 (25 por ciento) hiperlipidemias y 28 (20 por ciento) obesidad. Treinta y cuatro casos (24 por ciento) no presentaron factor de riesgo alguno. Localización del IM: 48 (34 por ciento) anteroseptal, 37 (26 por ciento) anterolateral, 36 (25 por ciento) posterioinferior y 21 (15 por ciento) inferior. Solamente 83 sujetos fueron cateterizados: 34 pacientes (41 por ciento) tuvieron lesión de la coronaria izquierda y 18 (22 por ciento) de la coronaria derecha; de ellos, 33 (40 por ciento) con enfermedad de un vaso, 15 (18 por ciento) de dos vasos y 4 (5 por ciento) de tres vasos. En 31 (37 por ciento), no se demostró lesión coronaria. Complicaciones: 87 (61 por ciento) trastornos del ritmo, 29 (20.5 por ciento) trastornos de la conducción, 14 (10 por ciento) insuficiencia cardíaca, 14(10 por ciento) extensión al centrículo derecho, 10 (7 por ciento) aneurisma del ventrículo izquierdo (VI), 8 (6 por ciento) trombo en VI, 7 (5 por ciento) pericarditis, 6 (4 por ciento) fenómenos embólicos periféricos, 4 (3 por ciento) bloqueo aurículo-ventricular de tercer grado; requiriendo marcapaso definitivo solamente 2 de ellos (1.5 por ciento). Cuatro enfermos (3 por ciento) presentaron choque cardiogénico, los cuatro fallecieron y representan la mortalidad intrahospitalaria de este estudio. Se concluye que el infarto miocárdico en jóvenes parece tener un incremento en su incidencia en nuestro medio. Es más frecuente en varones con historia familiar importante, así como con tensión emocional por personalidad tipo "A" y fumadores; más comunmente con enfermedad de un vaso, principalmente la coronaria izquieda e IM de cara anterior. Un porcentaje elevado presenta coronariografía normal. Su pronóstico intrahospitalario en general es bueno
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Doença das Coronárias/fisiopatologia , Ecocardiografia , Estresse Psicológico/complicações , Hipertensão/etiologia , Infarto do Miocárdio/fisiopatologiaRESUMO
Estudiamos 40 pacientes con infarto del miocardio (24 de la cara anterior y 16 de la inferior) en evolución, (IME): 37.5 por ciento con bloqueo AV de segundo y tercer graso, 30 por ciento con bloqueos de la rama derecha y de la subdivisión anterior izquierda, 12.5 por ciento con bloqueos de rama derecha y de l asubdivisión posterior izquierda y 20 por ciento con bloqueo troncular de la rama izquierda. Con el fin de precisar el nivel anatómico del bloqueo, a los enfermos se les practicó estudio electrofisiológico. En los pacientes con infarto del miocardio inferior y bloqueo AV, se encontró buena correlación entre el electrocardiograma y el electrograma de His, localizando el trastorno de conducción a nivel del nod AV (suprahisiano). En cambio en los pacientes con IME anteroseptal, en los que el ECG sólo mostraba bloqueo de rama troncular o en alguno de los fascículos distales, el electrograma de His registró bloqueos en múltiples niveles: el 70 por ciento con bloqueos a nivel intra e infrahisianos, que evolucionaron súbitamente hacia bloqueos AV avanzados. Puede ser que el mecanismo de estos bloqueos sea la disociación funcional longitudinal en la conducción, debida más a una lesión isquémica del tronco del haz de His, que a un bloqueo súbito y simultáneo de todos sus fascículos. Concluimos que el estudio eletrofisiológico es un procedimiento de gran utilidad para identificar a los pacientes con múltiples trastornos de conducción, cuyo pronóstico es menor favorable que para aquellos pacientes que sólo presentaron bloqueos suplrahisianos