RESUMEN
Telomere-led rapid chromosome movements (RPMs) are a conserved characteristic of chromosome dynamics in meiosis. RPMs have been suggested to influence critical meiotic functions such as DNA repair and the association of the homologous chromosomes. Here, we describe a method using 3D time-lapse fluorescence imaging to monitor RPMs in Hoechst-stained mouse seminiferous tubules explants. We supplement visualization with customized quantitative motion analysis and in silico simulation. The ability to carry out live imaging, combined with quantitative image analysis, offers a sensitive tool to investigate the regulation of RPMs, chromosome reorganizations that precede dynamic mid-prophase events, and their contribution to faithful transmission of genetic information.
Asunto(s)
Meiosis , Animales , Ratones , Masculino , Imagen de Lapso de Tiempo/métodos , Telómero/genética , Telómero/metabolismo , Túbulos Seminíferos/citología , Túbulos Seminíferos/metabolismo , Cromosomas/genéticaRESUMEN
Chromatin undergoes extensive remodeling during meiosis, leading to specific patterns of gene expression and chromosome organization, which ultimately controls fundamental meiotic processes such as recombination and homologous chromosome associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies we adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin-binding sites by ChIP-seq. High-quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin-binding sites.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Cromatina , Espermatocitos , Animales , Espermatocitos/metabolismo , Espermatocitos/citología , Ratones , Masculino , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Meiosis/genética , Inmunoprecipitación de Cromatina/métodos , Sitios de UniónRESUMEN
Gametogenesis in mammals encompasses highly regulated developmental transitions. These are associated with changes in transcription that cause characteristic patterns of gene expression observed during distinct stages of gamete development, which include specific activities with critical meiotic functions. SWI/SNF chromatin remodelers are recognized regulators of gene transcription and DNA repair, but their composition and functions in meiosis are poorly understood. We have generated gamete-specific conditional knockout mice for ARID2, a specific regulatory subunit of PBAF, and have compared its phenotype with BRG1 knockouts, the catalytic subunit of PBAF/BAF complexes. While Brg1Δ/Δ knockout acts at an early stage of meiosis and causes cell arrest at pachynema, ARID2 activity is apparently required at the end of prophase I. Striking defects in spindle assembly and chromosome-spindle attachment observed in Arid2Δ/Δ knockouts are attributed to an increase in aurora B kinase, a master regulator of chromosome segregation, at centromeres. Further genetic and biochemical analyses suggest the formation of a canonical PBAF and a BRG1-independent complex containing ARID2 and PBRM1 as core components. The data support a model in which different PBAF complexes regulate different stages of meiosis and gametogenesis.
Asunto(s)
Cromatina , Proteínas Cromosómicas no Histona , Factores de Transcripción , Animales , Aurora Quinasa B/genética , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Meiosis/genética , Ratones , Factores de Transcripción/metabolismoRESUMEN
Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of Chd4 and Chd3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of chd4 early in gametogenesis, but not chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that Chd4 controls expression of dmrt1 and its downstream target plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of Chd4 in mammalian gametogenesis pointing to functions in gene expression early in the process.
Asunto(s)
ADN Helicasas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Espermatogonias , Animales , Diferenciación Celular , Gónadas , Masculino , Mamíferos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ratones , Factores de Transcripción/genéticaAsunto(s)
Enfermedades del Pene , Humanos , Masculino , Enfermedades del Pene/cirugía , Pene/cirugíaRESUMEN
Mineral exploration areas are recognized for negatively affecting site environmental quality. The recent contaminations in the cities of Brumadinho, Mariana, Santo Antônio do Grama (Minas Gerais), and Barcarena (Pará) point to the seriousness of this issue in Brazil. However, studies on the influence of mining tailings from the extraction of semiprecious rocks on the quality of the sediments of water systems are rare. The aim of this study is to evaluate the influence of mining activities (amethyst, quartz, agate, calcite, and gypsum) on the quality of the sediments of Rio de Várzea, southern Brazil, the biggest region of amethyst rock extraction in the world. The concentrations of the chemical species Al2O3, SiO2, P2O5, K2O, CaO, TiO2, Fe2O3, Cr, Mn, Co, Cu, Zn, Zr, Ba, Cd, and Pb were determined by the technique energy-dispersive X-ray emission spectrometry (EDXRF). In the study, moderate contamination of the sediments of the Várzea River was demonstrated by means of background strategies (contamination factor, enrichment factor, and geoaccumulation index). Statistical analysis with the use of ANOVA, Tukey test, and principal component analysis revealed significant differences of concentrations of the chemical species of the sediments at the exit of the mining zone in relation to the other study areas.
Asunto(s)
Monitoreo del Ambiente , Sedimentos Geológicos/química , Minería , Contaminantes Químicos del Agua/análisis , Brasil , Ciudades , Estudios Longitudinales , Metales Pesados/análisis , Ríos/química , Dióxido de Silicio/análisisRESUMEN
Chromosome segregation errors during meiosis result in the formation of aneuploid gametes and are the leading cause of pregnancy loss and birth defects in humans. Proper chromosome segregation requires pairwise associations of maternal and paternal homologous chromosomes. Chiasmata, which are the cytological manifestations of crossovers (COs), provide a physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Although CO-promoting activities ensure a balanced number and position of COs, their identity and mechanism of action in mammals remain understudied. Previous work in yeast and Arabidopsis has shown that Zip2 and Shoc1 are ortholog proteins with an important role in promoting the formation of COs. Our work is the first study in mammals showing the in vivo and in vitro function of mouse and human SHOC1. We show that purified recombinant human SHOC1, an XPF/MUS81 family member, preferentially binds branched DNA molecules but apparently lacks in vitro endonuclease activity, despite its conserved ERCC4-(HhH)2 core structure. Cytological observations suggest that initial steps of recombination are normal in a majority of spermatocytes from SHOC1 hypomorphic mice. However, late stages of recombination appear abnormal, as chromosomal localization of MLH1 is reduced. In agreement, chiasma formation is reduced, and cells arrest at metaphase I with a few lagging chromosomes and subsequent apoptosis. This analysis of SHOC1-deficient mice and the selective localization of SHOC1 to a subset of recombination sites show that SHOC1 acts at key mid-stage steps of the CO formation process. The formation of chromosome axial elements and homologous pairing are apparently normal, but synapsis is altered with SYCP1 frequently failing to extend the full length of the chromosome axes. Finally, we describe that SHOC1 interacts with TEX11, another protein important for the formation of COs, connecting SHOC1 to chromosome axis and structure.
Asunto(s)
Intercambio Genético , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Meiosis/genética , Animales , Emparejamiento Cromosómico/genética , Segregación Cromosómica/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Recombinación Genética , Espermatocitos/metabolismoRESUMEN
The efficiency and type of pathway chosen to repair DNA double-strand breaks (DSBs) are critically influenced by the nucleosome packaging and the chromatin architecture surrounding the DSBs. The Swi/Snf (PBAF and BAF) chromatin-remodeling complexes contribute to DNA damage-induced nucleosome remodeling, but the mechanism by which it contributes to this function is poorly understood. Herein, we report how the Baf200 (Arid2) PBAF-defining subunit regulates DSB repair. We used cytological and biochemical approaches to show that Baf200 plays an important function by facilitating homologous recombination-dependent processes, such as recruitment of Rad51 (a key component of homologous recombination) to DSBs, homology-directed repair, and cell survival after DNA damage. Furthermore, we observed that Baf200 and Rad51 are present in the same complex and that this interaction is mediated by C-terminal sequences in both proteins. It has been recognized previously that the interplay between distinct forms of Swi/Snf has profound functional consequences, but we understand little about the composition of complexes formed by PBAF protein subunits. Our biochemical analyses reveal that Baf200 forms at least two distinct complexes. One is a canonical form of PBAF including the Swi/Snf-associated Brg1 catalytic subunit, and the other contains Baf180 but not Brg1. This distinction of PBAF complexes based on their unique composition provides the foundation for future studies on the specific contributions of the PBAF forms to the regulation of DNA repair.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación/fisiología , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , Humanos , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Recombinasa Rad51/genética , Factores de Transcripción/genéticaRESUMEN
Brosimum gaudichaudii Trécul., a plant that belongs to Moraceae family, is found throughout the Brazilian Cerrado. The antimicrobial activities of ethanolic bark and leaf extracts of B. gaudichaudii were tested against multiresistant bacteria isolated from diabetic foot infections (DFIs). Antimicrobial activity of the extracts was evaluated by agar disc diffusion (DD) and broth dilution (BD) methods. By BD method, bark (53.85, 45.83%) and leaf (42.31, 50.00%) extracts contained antimicrobial activity against both gram-negative and gram-positive bacteria. Increased antimicrobial activity was observed when bark and leaf extracts were tested against Staphylococcus aureus (63.64%) and Pseudomonas aeruginosa (66.67%). Statistical analyses of bark and leaf extract demonstrated antimicrobial activity against both gram-positive (p = 0.000) and gram-negative bacteria (p = 0.012). Extract of bark (p = 0.075) or leaf (p = 0.005) associated with ACA antibiotic showed antimicrobial activity against gram-positive bacteria. Our study suggests that the bark and leaf extracts contain bioactive compounds with antimicrobial activity against multidrug resistant strains.
Asunto(s)
Antibacterianos/aislamiento & purificación , Moraceae/química , Antibacterianos/química , Antibacterianos/farmacología , Brasil , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Corteza de la Planta/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Hojas de la Planta/químicaRESUMEN
Nef is an HIV-1 accessory protein that promotes viral replication and pathogenesis. A key function of Nef is to ensure sustained depletion of CD4 and MHC-I molecules in infected cells by inducing targeting of these proteins to multivesicular bodies (MVBs), and ultimately to lysosomes for degradation. Nef also affects cellular secretory routes promoting its own secretion via exosomes. To better understand the effects of Nef on the exocytic pathway, we investigated whether this viral factor modifies the composition of exosomes released by T lymphocytes. We showed that both CD4 and MHC-I molecules are secreted in exosomes from T cells and that the expression of Nef reduces the amount of these proteins in exosomes. To investigate the functional role for this novel activity of Nef, we performed in vitro HIV-1 infection assays in the presence of distinct populations of exosomes. We demonstrated that exosomes released by CD4+ T cells, but not CD4- T cells, efficiently inhibit HIV-1 infection in vitro. Because CD4 is the main receptor for HIV-1 infection, these results suggest that CD4 molecules displayed on the surface of exosomes can bind to envelope proteins of HIV-1 hindering virus interaction with target cells and infection. Importantly, CD4-depleted exosomes released by CD4+ T cells expressing Nef have a reduced capacity to inhibit HIV-1 infection in vitro. These results provide evidence that Nef promotes HIV-1 infection by reducing the expression of CD4 in exosomes from infected cells, besides the original role of Nef in reducing the CD4 levels at the cell surface.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Exosomas/inmunología , Productos del Gen nef/inmunología , Infecciones por VIH/inmunología , Línea Celular , Regulación hacia Abajo , Células HEK293 , VIH-1 , Humanos , Complejo Mayor de Histocompatibilidad/inmunología , Microscopía FluorescenteRESUMEN
Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef.
Asunto(s)
Antígenos CD4/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Lisosomas/enzimología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Antígenos CD4/genética , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Endosomas/genética , Endosomas/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Humanos , Lisosomas/genética , Unión Proteica , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: Despite advances, inappropriate analgesic treatment for burn patients is still seen. The objective of this review was to collect data on pain management in burn patients. CONTENT: We reviewed the mechanisms of pain, burn patient assessment, as well as pharmacological and non-pharmacological treatment. CONCLUSION: Pain management in burn patients is still a challenge for the multidisciplinary team. Frequent and continuous evaluation of the patient's response is very important due to the various stages that the hospitalized burn patient goes through, as well as a combination therapy with analgesic and non-pharmacological measures. Understanding the complexity of the pathophysiological, psychological, and biochemical changes a burn patient presents is the first step to achieve success in analgesic management.
Asunto(s)
Analgésicos/uso terapéutico , Quemaduras/complicaciones , Manejo del Dolor , Dolor/etiología , HumanosRESUMEN
BACKGROUND AND OBJECTIVES: Despite advances, inappropriate analgesic treatment for burn patients is still seen. The objective of this review was to collect data on pain management in burn patients. CONTENT: We reviewed the mechanisms of pain, burn patient assessment, as well as pharmacological and non-pharmacological treatment. CONCLUSION: Pain management in burn patients is still a challenge for the multidisciplinary team. Frequent and continuous evaluation of the patient's response is very important due to the various stages that the hospitalized burn patient goes through, as well as a combination therapy with analgesic and non-pharmacological measures. Understanding the complexity of the pathophysiological, psychological, and biochemical changes a burn patient presents is the first step to achieve success in analgesic management.
Asunto(s)
Quemaduras/complicaciones , Manejo del Dolor/métodos , Quemaduras/terapia , Humanos , Dolor/tratamiento farmacológico , Dolor/etiología , Grupo de Atención al PacienteRESUMEN
Activation of the high affinity IgE-binding receptor (FcεRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the COOH terminus of the protein-tyrosine kinase Syk. Synthetic peptides representing the last 10 amino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2, and Grb2-related adaptor downstream of Shc (GADS) and the protein phosphatases SHIP-1 and TULA-2 (also known as UBASH3B or STS-1). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far Western blots showed direct binding of the phosphorylated peptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2, and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcεRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-γ2. There was parallel enhancement of the receptor-induced degranulation and activation of nuclear factor for T cells or nuclear factor κB, indicating that TULA-2, like SHIP-1, functions as a negative regulator of FcεRI signaling in mast cells. Therefore, once phosphorylated, the terminal tyrosines of Syk bind complexes of proteins that are positive and negative regulators of signaling in mast cells.
Asunto(s)
Degranulación de la Célula/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mastocitos/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Degranulación de la Célula/efectos de los fármacos , Línea Celular , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Mastocitos/inmunología , Ratones , FN-kappa B/genética , FN-kappa B/inmunología , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/inmunología , Factores de Transcripción NFATC/metabolismo , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/inmunología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/farmacología , Receptores de IgE/genética , Receptores de IgE/inmunología , Receptores de IgE/metabolismo , Transducción de Señal/efectos de los fármacos , Quinasa SykRESUMEN
The protein tyrosine kinase Syk plays a critical role in FcεRI signaling in mast cells. Binding of Syk to phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) of the receptor subunits results in conformational changes and tyrosine phosphorylation at multiple sites that leads to activation of Syk. The phosphorylated tyrosines throughout the molecule play an important role in the regulation of Syk-mediated signaling. Reconstitution of receptor-mediated signaling in Syk(-/-) cells by wild-type Syk or mutants which have substitution of these tyrosines with phenylalanine together with in vitro assays has been useful strategies to understand the regulation and function of Syk.
RESUMEN
The aggregation by antigen of the IgE bound to its high affinity receptor on mast cells initiates a complex series of biochemical events that result in the release of inflammatory mediators. The essential role of the protein tyrosine kinase Syk has been appreciated for some time, and newer results have defined the mechanism of its activation. The use of siRNA has defined the relative contribution of Syk, Fyn and Gab2 to signaling and has made possible a screening study to identify previously unrecognized molecules that are involved in these pathways.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mastocitos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Animales , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Mastocitos/enzimología , Proteínas Tirosina Quinasas/genética , ARN Interferente Pequeño , Quinasa SykRESUMEN
The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as FcepsilonRI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased FcepsilonRI-induced degranulation, nuclear factor for T cell activation and NFkappaB activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Tirosina/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Péptidos y Proteínas de Señalización Intracelular/fisiología , Mastocitos/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Tirosina Quinasas/fisiología , Ratas , Quinasa SykRESUMEN
BACKGROUND: Pelvic organ prolapse (POP) is a downward descent of pelvic organs that results in protrusions of the vagina, the uterus, or both. The cause of this disorder is likely to be multifactorial, attributable to a combination of risk factors, especially connective tissue disorders. Our objective was to characterize and quantify a component of the extracellular matrix (ECM)-sulfated glycosaminoglycan (GAG)-in the parametrium and vaginal apex of women with and without uterine prolapse. METHODS: Parametrium and vaginal apex tissue was obtained from 42 women who underwent surgery. Patients underwent a physical examination and were divided into groups according to the type of genital prolapse. Standard biopsies were taken during surgery and were assessed by biochemical methods. GAGs were obtained by proteolysis. The relative concentration of GAGs was determined by densitometry. Data were compared using an independent sample t-test or chi(2) test. RESULTS: In both groups (with and without prolapse) and in both types of tissue, dermatan sulfate (DS) was the most predominant glycosaminoglycan, followed by chondroitin sulfate (CS) and heparan sulfate (HS). We did not observe significant differences in the total amounts of GAGs, DS, CS, or HS. CONCLUSIONS: This study did not show altered biochemical characteristics in the ECM of parametrium and vaginal apex tissue of women either with or without uterine prolapse.
Asunto(s)
Glicosaminoglicanos/análisis , Prolapso Uterino/patología , Vagina/química , Anciano , Biopsia , Femenino , Humanos , Persona de Mediana Edad , Examen Físico , PosmenopausiaRESUMEN
Gangliosides are complex glycosphingolipids that are important in many biological processes. The present study investigated the role of gangliosides in the organization of lipid rafts in RBL-2H3 mast cells and in the modulation of mast cell degranulation via FcvarepsilonRI. The role of gangliosides was examined using two ganglioside deficient cell lines (B6A4A2III-E5 and B6A4C1III-D1) as well as the parent cell line (RBL-2H3). All three cell lines examined express FcvarepsilonRI, Lyn, Syk and LAT. However, only in RBL-2H3 cells were FcvarepsilonRI, LAT and alpha-galactosyl derivatives of ganglioside GD(1b) mobilized to lipid raft domains following FcvarepsilonRI stimulation. The inhibition of glycosphingolipid synthesis in RBL-2H3 cells also resulted in a decrease in the release of beta-hexosaminidase activity after FcvarepsilonRI activation. The two mutant cell lines have a reduced release of beta-hexosaminidase activity after FcvarepsilonRI stimulation, but not after exposure to calcium ionophore. These results indicate that the alpha-galactosyl derivatives of ganglioside GD(1b) are important in the initial events of FcvarepsilonRI signaling upstream of Ca2+ influx. Since the initial signaling events occur in lipid rafts and in the mutant cell lines the rafts are disorganized, these results also suggest that these gangliosides contribute to the correct assembly of lipid rafts and are essential for mast cell activation via FcvarepsilonRI.