RESUMEN
BACKGROUND: The literature provides conflicting data on sexual function in women with inflammatory bowel disease (IBD). We aim to describe sexual function at baseline and over time in a prospective inception cohort of adult women with IBD. METHODS: Women age 18 years or older enrolled in the Ocean State Crohn's & Colitis Area Registry (OSCCAR) with 2 years of prospective follow-up were included in the study. All subjects were enrolled within 1 year of IBD diagnosis. Female sexual function was assessed using the Female Sexual Function Index (FSFI). Linear mixed effects models were used to assess changes in FSFI by various demographic and clinical factors. RESULTS: One hundred sixteen of 130 eligible women (89%) were included in the study. Ninety-seven percent of women had sexual dysfunction, defined as an FSFI score of <26.55, with a baseline mean FSFI score (SD) of 16.4 (8.4) overall (15.5 [8.6] in Crohn's disease, 17.4 [8.1] in UC, P = 0.22). Despite improvement in overall disease activity, there was no significant change in the FSFI score or individual domain scores over the entire 2-year study period. Among all women with IBD, older age, nonsingle marital status, lower Short Form Health Survey (SF-36) Physical Component Summary score, and the use of biologics were independent risk factors for sexual dysfunction. CONCLUSIONS: Almost all women experienced sexual dysfunction that did not improve over time despite improvement in overall disease activity. Future studies are warranted to identify underlying mechanisms that explain the associations between demographic and clinical factors and sexual dysfunction among newly diagnosed women.
Asunto(s)
Enfermedades Inflamatorias del Intestino/complicaciones , Sistema de Registros/estadística & datos numéricos , Índice de Severidad de la Enfermedad , Disfunciones Sexuales Fisiológicas/etiología , Disfunciones Sexuales Psicológicas/etiología , Adolescente , Adulto , Anciano , Femenino , Estudios de Seguimiento , Humanos , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/psicología , Estudios Longitudinales , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Factores de Riesgo , Disfunciones Sexuales Fisiológicas/patología , Disfunciones Sexuales Fisiológicas/psicología , Disfunciones Sexuales Psicológicas/patología , Disfunciones Sexuales Psicológicas/psicología , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.
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Perfilación de la Expresión Génica , Sialiltransferasas/química , Animales , Baculoviridae/metabolismo , Cristalografía por Rayos X , Citidina Monofosfato/química , Vectores Genéticos , Glicósido Hidrolasas/química , Glicosilación , Células HEK293 , Humanos , Insectos , Cinética , Proteínas Recombinantes/química , Sulfotransferasas/químicaRESUMEN
BACKGROUND: Traditional cohort studies are important contributors to our understanding of inflammatory bowel diseases, but they are labor intensive and often do not focus on patient-reported outcomes. Internet-based studies provide new opportunities to study patient-reported outcomes and can be efficiently implemented and scaled. If a traditional cohort study was linked to an Internet-based study, both studies could benefit from added synergy. Existing cohort studies provide an opportunity to develop and test processes for cohort linkage. The Crohn's and Colitis Foundation of America's (CCFA) Partners study is an Internet-based cohort of more than 14,000 participants. The Ocean State Crohn's and Colitis Area Registry (OSCCAR) is an inception cohort. The Sinai-Helmsley Alliance for Research Excellence (SHARE) is a multicentered cohort of inflammatory bowel disease patients. Both the later cohorts include medical record abstraction, patient surveys, and biospecimen collection. OBJECTIVE: Given the complementary nature of these existing cohorts, we sought to corecruit and link data. METHODS: Eligible OSCCAR and SHARE participants were invited to join the CCFA Partners study and provide consent for data sharing between the 2 cohorts. After informed consent, participants were directed to the CCFA Partners website to complete enrollment and a baseline Web-based survey. Participants were linked across the 2 cohorts by the matching of an email address. We compared demographic and clinical characteristics between OSCCAR and SHARE participants who did and did not enroll in CCFA Partners and the data linkage. RESULTS: Of 408 participants in the OSCCAR cohort, 320 were eligible for participation in the CCFA Partners cohort. Of these participants, 243 consented to participation; however, only 44 enrolled in CCFA Partners and completed the linkage. OSCCAR participants who enrolled in CCFA Partners were better educated (17% with doctoral degrees) than those who did not (3% with doctoral degrees, P=.01). In the SHARE cohort, 436 participants enrolled and linked to the Partners cohort. More women (60% vs 50%) linked and those who linked were predominantly white (96%; P<.01). Crohn's disease patients who linked had lower mean scores on the Harvey-Bradshaw Index (3.6 vs 4.4, P<.01). Ulcerative colitis patients who linked had less extensive disease than those who did not link (45% vs 60%, P<.01). CONCLUSIONS: Linkage of CCFA Partners with cohorts such as OSCCAR and SHARE may be a cost-effective way to expand the infrastructure for clinical outcomes and translational research. Although linkage is feasible from a technical, legal, and regulatory perspective, participant willingness appears to be a limiting factor. Overcoming this barrier will be needed to generate meaningful sample sizes to conduct studies of biomarkers, natural history, and clinical effectiveness using linked data.
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Enfermedades Inflamatorias del Intestino , Internet , Selección de Paciente , Sistema de Registros , Adulto , Estudios de Cohortes , Colitis Ulcerosa , Enfermedad de Crohn , Femenino , Humanos , Almacenamiento y Recuperación de la Información , Masculino , Registros Médicos , Medición de Resultados Informados por el Paciente , Proyectos de Investigación , Encuestas y CuestionariosRESUMEN
BACKGROUND: Studies describing the incidence of Crohn's disease (CD) and ulcerative colitis (UC) are uncommon in the United States. We sought to determine the incidence of CD and UC in the state of Rhode Island. METHODS: The Ocean State Crohn's and Colitis Area Registry is a state-based inception cohort of patients newly diagnosed with inflammatory bowel disease (IBD) in Rhode Island. To confirm a diagnosis of CD, UC, or IBD unclassified (IBDU), the National Institute of Diabetes and Digestive and Kidney Diseases IBD Genetics Consortium criteria were applied in a review of medical records from gastroenterology practices located in the state of Rhode Island and adjacent to the Rhode Island border in Massachusetts and Connecticut. Using population-based data, we determined the statewide incidence of IBD in Rhode Island from 2008 to 2010. RESULTS: A total of 971 Rhode Island residents were diagnosed with IBD, including 444 with CD, 486 with UC, and 41 with IBD unclassified from 2008 to 2010. The overall age- and sex-adjusted IBD incidence was 30.2 (95% confidence interval, 28.3-32.1) per 100,000 persons in this time frame with 13.9, 15.1, and 1.3 per 100,000 diagnosed with CD, UC, and IBD unclassified, respectively. Of the total incident cases in Rhode Island, 30% (n = 291) were enrolled in Ocean State Crohn's and Colitis Area Registry for follow-up. CONCLUSIONS: The incidence of IBD in Rhode Island is higher than that previously reported by other population-based cohorts in the United States. Prospective follow-up of individuals enrolled in the community-based Ocean State Crohn's and Colitis Area Registry cohort is ongoing.
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Colitis Ulcerosa/epidemiología , Enfermedad de Crohn/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Colitis Ulcerosa/patología , Enfermedad de Crohn/patología , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Sistema de Registros , Rhode Island/epidemiología , Adulto JovenRESUMEN
BACKGROUND: Systemic corticosteroids (CS) are a mainstay of treatment for patients with newly diagnosed inflammatory bowel disease (IBD). Previous population-based studies report CS exposure rates range from 39 to 75 % within the first year of diagnosis with surgical resection rates as high as 13-18 % in the same time frame. These reports represent an older cohort of patients enrolled over prolonged periods of time and do not necessarily reflect current treatment approaches. We examine CS use during the first year of IBD diagnosis in a community-based, inception cohort. METHODS: Data were derived from the Ocean State Crohn's and Colitis Area Registry (OSCCAR), a prospective inception cohort of IBD patients who are residents of Rhode Island. RESULTS: A total of 272 patients were included in the current analyses. Overall, 60 % of Crohn's disease and 57 % of ulcerative colitis patients were exposed to at least one course of CS during year 1 of study enrollment. Most notably, only 2 % of patients (n = 5) required a surgical resection. CONCLUSIONS: In this community-based cohort, 59 % of patients were exposed to at least one course of CS during their first year of enrollment. In contrast to previous studies, OSCCAR represents a more modern cohort of patients. While steroid exposure rates were similar or slightly higher than those in previous reports, we observed a low rate of surgical resection. As our cohort ages, future analysis will focus on the role more contemporary agents may play on the low rates of surgery we observed.
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Corticoesteroides/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Enfermedad de Crohn/tratamiento farmacológico , Adolescente , Adulto , Niño , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
PURPOSE: Patients with inflammatory bowel disease (IBD) may be exposed to high doses of diagnostic radiation. The purpose of this study is to identify subsets of this population at risk for significant radiation exposure. METHODS: This HIPAA compliant, IRB approved study consists of 336 patients (237 adult and 99 pediatric) within the Ocean State Crohn's & Colitis Area Registry (OSCCAR). All were newly diagnosed with IBD and prospectively enrolled between 1/2008 and 12/2012. Comprehensive chart review was performed. RESULTS: 207 (61.6%) patients were diagnosed with Crohn's disease (CD), 120 (35.7%) with ulcerative colitis (UC), and 9 (2.7%) with inflammatory bowel disease, type unspecified (IBDU). 192 (57.1%) patients were exposed to GI-specific radiation. Average GI-specific radiation dose for adult IBD patients was 14.1 mSV and was significantly greater among adult CD than adult UC patients (p = 0.01). Pediatric patients underwent fewer CT scans (p < 0.0001). Risk factors for increased radiation exposure include: GI surgery (p = 0.003), biologic therapy (p = 0.01), pain-predominant symptoms (as compared to diarrhea-predominant symptoms; p < 0.05), and isolated ileal disease (p = 0.02). Patients with stricturing or penetrating disease received higher radiation doses than patients with non-stricturing, non-penetrating disease (p < 0.0001). CONCLUSIONS: A variety of risk factors are associated with increased exposure to ionizing radiation after diagnosis of IBD. Knowledge of these risk factors can help physicians prospectively identify patients at risk for elevated radiation exposure and consider low-dose or radiation-free imaging.
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Enfermedades Inflamatorias del Intestino/diagnóstico por imagen , Exposición a la Radiación , Tomografía Computarizada por Rayos X , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Estudios Prospectivos , Sistema de Registros , Factores de RiesgoRESUMEN
BACKGROUND: Crohn's disease (CD) is a form of inflammatory bowel disease with different described behaviors, including stricture. At present, there are no laboratory studies that can differentiate stricturing CD from other phenotypes of inflammatory bowel disease. We performed a pilot study to examine differences in the proteome among patients with stricturing CD, nonstricturing CD, and ulcerative colitis. METHODS: Serum samples were selected from the Ocean State Crohn's and Colitis Area Registry, an established cohort of patients with inflammatory bowel disease. Patients with CD with surgically resected stricture were matched with similar patients with CD without known stricture and with ulcerative colitis. Serum samples from each patient were digested and analyzed using liquid chromatography-mass spectrometry to characterize the proteome. Statistical analyses were performed to identify peptides and proteins that can differentiate CD with stricture. RESULTS: Samples from 9 patients in each group (27 total patients) were analyzed. Baseline demographic characteristics were similar among the 3 groups. We quantified 7668 peptides and 897 proteins for analysis. Receiver operating characteristic analysis identified a subset of peptides with an area under the curve greater than 0.9, indicating greater separation potential. Partial least squares discriminant analysis was able to distinguish among the three groups with up to 70% accuracy by peptides and up to 80% accuracy by proteins. We identified the significantly different proteins and peptides and determined their function based on previously published literature. CONCLUSIONS: The serum of patients with stricturing CD, nonstricturing CD, and ulcerative colitis is distinguishable through proteomic analysis. Some of the proteins that differentiate the stricturing phenotype have been implicated in complement activation, fibrinolytic pathways, and lymphocyte adhesion.
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Biomarcadores/sangre , Constricción Patológica/sangre , Enfermedad de Crohn/sangre , Proteoma/análisis , Proteómica/métodos , Adolescente , Adulto , Anciano , Niño , Cromatografía Líquida de Alta Presión , Constricción Patológica/diagnóstico , Enfermedad de Crohn/diagnóstico , Análisis Discriminante , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Proyectos Piloto , Pronóstico , Curva ROC , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes.
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Heparina/química , Heparina/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Transporte de Electrón , Humanos , Radical Hidroxilo , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/genética , Unión Proteica , Huella de Proteína , Dominios y Motivos de Interacción de Proteínas , Receptores Inmunológicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteínas RoundaboutRESUMEN
BACKGROUND: Despite the fact that the inflammatory bowel diseases (IBD) and their treatments may affect physical appearance, the effect of IBD on body image is poorly understood. The aims of this study were to determine whether body image dissatisfaction (BID) changes over time in patients with IBD and to examine the demographic and disease-related variables associated with decreased body image. METHODS: Adults aged 18 and above in the Ocean State Crohn's and Colitis Area Registry with at least 2 years of follow-up were eligible for this study. All patients were enrolled within 6 months of IBD diagnosis and followed prospectively. BID was assessed using a modified version of the Adapted Satisfaction With Appearance questionnaire. Total Adapted Satisfaction With Appearance scores and 2 subscores were calculated. To assess for changes over time, general linear models for correlated data were used for continuous outcomes, and generalized estimating equations were used for discrete outcomes. RESULTS: Two hundred seventy-four patients were studied. BID was found to be stable over time among men and women with IBD despite overall improvements in disease activity. No differences were found in BID according to IBD subtype. Female gender, greater disease activity, higher symptom burden, longer duration of steroid use, dermatologic and musculoskeletal manifestations of IBD, and ileocolonic disease location among patients with Crohn's disease were associated with greater BID. Greater BID was associated with lower health-related quality of life. CONCLUSIONS: BID remains stable in an incident cohort of IBD despite improved disease activity and is associated with lower health-related quality of life.
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Imagen Corporal/psicología , Enfermedades Inflamatorias del Intestino/psicología , Satisfacción del Paciente/estadística & datos numéricos , Calidad de Vida , Adolescente , Adulto , Anciano , Estudios de Cohortes , Emociones , Femenino , Estudios de Seguimiento , Humanos , Enfermedades Inflamatorias del Intestino/fisiopatología , Masculino , Persona de Mediana Edad , Pronóstico , Sistema de Registros , Encuestas y Cuestionarios , Adulto JovenRESUMEN
BACKGROUND: The incidence of Clostridium difficile infection (CDI) in inflammatory bowel disease (IBD) is increasing, and CDI has a negative impact on IBD outcomes with both increased morbidity and mortality. Data are lacking regarding the rate of appropriate testing for CDI at the time of diagnosis. METHODS: We sought to determine the rate of CDI testing and CDI positivity at diagnosis of IBD using data collected through the Ocean State Crohn's and Colitis Area Registry (OSCCAR), a prospective cohort of patients with newly diagnosed IBD. CDI testing and CDI positivity were determined by reviewing the medical records of patients enrolled into the registry and diagnosed with IBD between January 2008 and July 2011. RESULTS: Of 320 enrolled patients, 227 (70.9%) reported diarrhea, and CDI testing was performed for 113 (49.8%) of the 227 patients. CDI testing was not recorded as being performed for the remaining 114 patients who reported having diarrhea. An additional 24 patients were tested for CDI but did not report having diarrhea. Seven (5.1%) of the 137 patients tested for CDI were positive. CONCLUSIONS: Testing for CDI is significantly lower than expected at diagnosis of IBD. Although the prevalence of CDI among tested patients is approximately 5%, a low testing rate suggests a significant quality issue in the diagnosis of IBD, with the potential for delayed diagnosis of CDI.
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Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/diagnóstico , Infecciones por Clostridium/microbiología , Pruebas Diagnósticas de Rutina/métodos , Heces/microbiología , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Infecciones por Clostridium/epidemiología , Femenino , Humanos , Enfermedades Inflamatorias del Intestino/epidemiología , Masculino , Persona de Mediana Edad , New York/epidemiología , Prevalencia , Pronóstico , Estudios Prospectivos , Características de la Residencia , Factores de Riesgo , Adulto JovenRESUMEN
The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.
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Glicoproteínas/biosíntesis , Proteínas Recombinantes/biosíntesis , Transfección/métodos , Animales , Cromatografía de Afinidad , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Células HEK293 , Humanos , Procesamiento Proteico-Postraduccional , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Numerous in vivo functional studies have indicated that the dimeric extracellular domain (ECD) of the CaSR plays a crucial role in regulating Ca(2+) homeostasis by sensing Ca(2+) and l-Phe. However, direct interaction of Ca(2+) and Phe with the ECD of the receptor and the resultant impact on its structure and associated conformational changes have been hampered by the large size of the ECD, its high degree of glycosylation, and the lack of biophysical methods to monitor weak interactions in solution. In the present study, we purified the glycosylated extracellular domain of calcium-sensing receptor (CaSR) (ECD) (residues 20-612), containing either complex or high mannose N-glycan structures depending on the host cell line employed for recombinant expression. Both glycosylated forms of the CaSR ECD were purified as dimers and exhibit similar secondary structures with â¼ 50% α-helix, â¼ 20% ß-sheet content, and a well buried Trp environment. Using various spectroscopic methods, we have shown that both protein variants bind Ca(2+) with a Kd of 3.0-5.0 mm. The local conformational changes of the proteins induced by their interactions with Ca(2+) were visualized by NMR with specific (15)N Phe-labeled forms of the ECD. Saturation transfer difference NMR approaches demonstrated for the first time a direct interaction between the CaSR ECD and l-Phe. We further demonstrated that l-Phe increases the binding affinity of the CaSR ECD for Ca(2+). Our findings provide new insights into the mechanisms by which Ca(2+) and amino acids regulate the CaSR and may pave the way for exploration of the structural properties of CaSR and other members of family C of the GPCR superfamily.
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Calcio/química , Multimerización de Proteína , Receptores Sensibles al Calcio/química , Calcio/metabolismo , Glicosilación , Células HEK293 , Humanos , Ligandos , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismo , Relación Estructura-ActividadRESUMEN
Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far-reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally because of collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis; however, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10-L (IRX10-L) and ESKIMO1/TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O-acetyltransferase, we have resolved two long-standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways used by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties.
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Proteínas de Arabidopsis/metabolismo , Xilanos/biosíntesis , Acetilación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Green fluorescent proteins (GFPs) and their derivatives are widely used as markers to visualize cells, protein localizations in in vitro and in vivo studies. The use of GFP fusion protein for visualization is generally thought to have negligible effects on cellular function. However, a number of reports suggest that the use of GFP may impact the biological activity of these proteins. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins mediating diverse patho-physiological processes. In the heparin-based interactome studies, heparin-binding proteins are often prepared as GFP fusion proteins. In this report, we use surface plasmon resonance (SPR) spectroscopy to study the impact of the GFP tagging on the binding interaction between heparin and a heparin-binding protein, the Roundabout homolog 1 (Robo1). SPR reveals that heparin binds with higher affinity to Robo1 than GFP-tagged Robo1 and through a different kinetic mechanism. A conformational change is observed in the heparin-Robo1 interaction, but not in the heparin-Robo1-GFP interaction. Furthermore the GFP-tagged Robo1 requires a shorter (hexasaccharide) than the tag-free Robo1 (octadecasaccharide). These data demonstrate that GFP tagging can reduce the binding affinity of Robo1 to heparin and hinder heparin binding-induced Robo1 conformation change.
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Proteínas Fluorescentes Verdes/metabolismo , Heparina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Proteínas del Tejido Nervioso/genética , Unión Proteica , Receptores Inmunológicos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas RoundaboutRESUMEN
BACKGROUND: The effect of the inflammatory bowel diseases (IBD) on menstrual function is largely unknown. The aims of this study were to determine whether changes in menstrual function occur in the year before IBD diagnosis or in the initial years after diagnosis. METHODS: Women aged 18 years and older in the Ocean State Crohn's and Colitis Area Registry with at least 2 years of follow-up were eligible for this study. All patients were enrolled within 6 months of IBD diagnosis and followed prospectively. Menstrual cycle characteristics were retrospectively assessed. To assess for changes over time, general linear models for correlated data were used for continuous outcomes, and generalized estimating equations were used for discrete outcomes. RESULTS: One hundred twenty-one patients were studied. Twenty-five percent of patients experienced a change in cycle interval in the year before IBD diagnosis and 21% experienced a change in the duration of flow. Among women with dysmenorrhea, 40% experienced a change in the intensity of their menstrual pain and 31% experienced a change in its duration. Overall cycle regularity increased over time. Quality of life was significantly lower in women without regular cycles across all time points. CONCLUSIONS: Changes in menstrual function occur frequently in the year before IBD diagnosis; therefore, screening for menstrual irregularities should be considered in women with newly diagnosed IBD. Patients can be reassured that cycles typically become more regular over time.
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Colitis Ulcerosa/fisiopatología , Enfermedad de Crohn/fisiopatología , Ciclo Menstrual/fisiología , Sistema de Registros/estadística & datos numéricos , Adulto , Femenino , Estudios de Seguimiento , Humanos , Océanos y Mares , Pronóstico , Estudios ProspectivosRESUMEN
Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development.
Asunto(s)
Heparitina Sulfato/deficiencia , Hernias Diafragmáticas Congénitas , Neovascularización Fisiológica , Sulfotransferasas/genética , Animales , Apoptosis , Hipoxia de la Célula , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Diafragma/anomalías , Diafragma/irrigación sanguínea , Diafragma/enzimología , Células Endoteliales/enzimología , Femenino , Estudios de Asociación Genética , Hernia Diafragmática/enzimología , Hernia Diafragmática/genética , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Penetrancia , Receptores de Superficie Celular , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Transducción de Señal , Sulfotransferasas/deficiencia , Tendones/anomalías , Tendones/patología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.
Asunto(s)
Cristalografía por Rayos X , Polisacáridos/química , Ácidos Siálicos/metabolismo , Sialiltransferasas/química , Animales , Bacterias/enzimología , Bacterias/genética , Sitios de Unión , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Polisacáridos/biosíntesis , Conformación Proteica , Ratas , Ácidos Siálicos/química , Sialiltransferasas/metabolismo , Relación Estructura-Actividad , Porcinos/genética , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2-Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand-receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ~650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1-heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1-heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.
Asunto(s)
Glicosaminoglicanos/química , Proteínas del Tejido Nervioso/química , Receptores Inmunológicos/química , Glicosaminoglicanos/metabolismo , Heparina/química , Heparina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oligosacáridos/metabolismo , Receptores Inmunológicos/metabolismo , Resonancia por Plasmón de Superficie , Proteínas RoundaboutRESUMEN
Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled.