RESUMEN
INTRODUCTION: Lysosomal glucosidase beta acid (GBA) deficiency is inherent to Gaucher disease, Parkinsonism and Lewy-body dementia. Increased GBA expression has never been associated with human disease. We describe increased GBA expression and activity in placenta from preeclamptic pregnancies. METHODS: 112 placenta biopsies were available for qPCR, analysis of GBA gene expression and activity. Microanalysis was performed on 20 placenta samples. Alternatively spliced placental GBA transcripts were cloned, expressed in HEK293 cells and analyzed by Western blot and activity assay. RESULTS: GBA is expressed in the syncytiotrophoblast layer of human placenta already at 5 weeks of gestation. We identified five novel GBA transcripts in placenta that enzymatically inactive when expressed in HEK293 cells. Both GBA RNA expression and enzymatic activity are upregulated in preeclamptic placenta. Microarray analysis of 20 placenta tissues identified 158 genes co-regulating with GBA expression and gene enrichment analysis highlights lysosomal function. In our micro-array data GBA expression does not correlate with FLT1 expression, currently the most powerful marker for preeclampsia. There are 89 transcripts that are negatively correlated with GBA expression of which BMP4 and TFEB are interesting as they are essential to early placenta function. DISCUSSION: Although very speculative, we hypothesize that increased GBA expression might relate to placentation through decreased BMP4 signaling or vascularization through downregulation of TFEB. Ceramide, the product of hydrolysis of glucosylceramide by GBA and involved in the regulation of cell differentiation, survival and apoptosis, is another putative candidate linking increased GBA activity to preeclampsia. Both pathways merit further investigation.
Asunto(s)
Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Placenta/metabolismo , Preeclampsia/enzimología , Preeclampsia/genética , Ceramidas/metabolismo , Activación Enzimática , Femenino , Regulación Enzimológica de la Expresión Génica , Glucosilceramidas/metabolismo , Células HEK293 , Humanos , Recién Nacido , Masculino , Placenta/enzimología , Preeclampsia/metabolismo , Embarazo , Regulación hacia Arriba/genéticaRESUMEN
Ocular adnexal marginal zone B-cell lymphomas (OAMZLs) arise in the connective tissues of the orbit or in the mucosa-associated lymphoid tissue of the conjunctiva. Here, we present the immunological and genetic analyses of 20 primary Chlamydia psittaci (Cp)-negative OAMZLs. Analysis of the immunoglobulin variable heavy chain (IgV(H)) gene usage demonstrated a significant preference for V(H)4-34. A combined analysis across all previously published OAMZLs confirmed that this is a general feature of OAMZL, in particular of the Cp-negative group. Our series of OAMZLs did not express the characteristic rheumatoid factor V(H)DJ(H) rearrangements that were previously found in salivary gland- and gastric-marginal zone B-cell lymphomas (MZBCLs). We did not detect the MZBCL-specific chromosomal translocations, t(11;18) API2-MALT1 (mucosa-associated lymphoid tissue1) and t(14;18) IgH/MALT1. Two cases contained a premature stop codon in the A20 gene (TNFAIP3) and one case harbored the activating MYD88 hotspot mutation L265P. Variable nuclear expression of BCL10, NFκB1 (p50) and NFκB2 (p52) suggests that other additional genetic abnormalities affecting the NFκB pathway exist within this group of lymphomas. OAMZL showed variable expression of the chemokine receptor CXCR3 and integrin α4ß7 by the tumor B cells, and low interferon-γ and interlukin-4 mRNA levels in the tissue, indicative of an inflammatory environment with features in between those previously found in cutaneous and other extranodal MZBCL. The strongly biased usage of V(H)4-34 in Cp-negative OAMZLs suggests involvement of a particular stimulatory (auto-) antigen in their development.
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Reordenamiento Génico de Cadena Pesada de Linfocito B , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Inflamación/metabolismo , Linfoma de Células B de la Zona Marginal/genética , Linfoma de Células B de la Zona Marginal/inmunología , Western Blotting , Núcleo Celular/metabolismo , Chlamydophila psittaci/genética , Chlamydophila psittaci/aislamiento & purificación , ADN Bacteriano/genética , Humanos , Técnicas para Inmunoenzimas , Inflamación/genética , Inflamación/inmunología , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Linfoma de Células B de la Zona Marginal/microbiología , Mutación/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Pronóstico , Psitacosis/genética , Psitacosis/inmunología , Psitacosis/microbiología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Translocación GenéticaRESUMEN
DNA double-strand breaks (DSBs) can efficiently kill cancer cells, but they can also produce unwanted chromosome rearrangements when DNA ends from different DSBs are erroneously joined. Movement of DSB-containing chromatin domains might facilitate these DSB interactions and promote the formation of chromosome rearrangements. Therefore, we analyzed the mobility of chromatin domains containing DSBs, marked by the fluorescently tagged DSB marker 53BP1, in living mammalian cells and compared it with the mobility of undamaged chromatin on a time-scale relevant for DSB repair. We found that chromatin domains containing DSBs are substantially more mobile than intact chromatin, and are capable of roaming a more than twofold larger area of the cell nucleus. Moreover, this increased DSB mobility, but not the mobility of undamaged chromatin, can be reduced by agents that affect higher-order chromatin organization.
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Núcleo Celular/metabolismo , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/efectos de la radiación , Cromatina/efectos de los fármacos , Cromatina/genética , Cromatina/efectos de la radiación , Aberraciones Cromosómicas/efectos de los fármacos , Aberraciones Cromosómicas/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Daño del ADN , Etopósido/farmacología , Fluorescencia , Rayos gamma , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Movimiento (Física) , Plásmidos , Coloración y Etiquetado , Imagen de Lapso de Tiempo , Transfección , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
In overt diabetic nephropathy (DNP), an increase in the permeability of the glomerular basement membrane (GBM) has been associated with a loss of negatively charged heparan sulfates (HS) in the GBM. Heparanase (HPSE), an endo-beta(1-4)-D-glucuronidase, can cleave HS and could be a potential candidate for the degradation of glomerular HS, leading to the development of proteinuria. We analyzed whether changes in HS expression are associated with HPSE expression in overt DNP. Immunofluorescence staining was performed to analyze HS, HPSE, and agrin core protein expression in kidney biopsies from patients with overt DNP and from rats and mice with streptozotocin (STZ)-induced diabetes. We also investigated the effect of transgenic HPSE overexpression in mice on glomerular HS and agrin expression. We demonstrate that the loss of GBM HS (-50%) and tubular HS (-60%) is associated with a four-fold increased HPSE expression in overt DNP. In addition, glomerular HPSE expression is upregulated in rats (messenger RNA (mRNA) 2.5-fold, protein three-fold) and mice (mRNA seven-fold, protein 1.5-fold) with STZ-induced diabetes. Furthermore, transgenic HPSE overexpression results in disappearance of HS, whereas expression of the core protein agrin remains unaltered. Our observations suggest that HPSE is involved in the pathogenesis of proteinuria in overt DNP by degradation of HS.
Asunto(s)
Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/fisiopatología , Glucuronidasa/genética , Glucuronidasa/metabolismo , Glomérulos Renales/enzimología , Anciano , Animales , Especificidad de Anticuerpos , Membrana Basal/enzimología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Enzimológica de la Expresión Génica , Glucuronidasa/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Proteinuria/metabolismo , Proteinuria/fisiopatología , Ratas , Ratas WistarRESUMEN
For efficient repair of DNA double strand breaks (DSBs) cells rely on a process that involves the Mre11/Rad50/Nbs1 complex, which may help to protect non-repaired DNA ends from separating until they can be rejoined by DNA repair proteins. It has been observed that as a secondary effect, this process can lead to unintended clustering of multiple, initially separate, DSB-containing chromosome domains. This work demonstrates that neither inactivation of the major repair proteins XRCC3 and the DNA-dependent protein kinase (DNA-PK) nor inhibition of DNA-PK by vanillin influences the aggregation of DSB-containing chromosome domains.
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Cromosomas/fisiología , Cromosomas/efectos de la radiación , Daño del ADN/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Animales , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Modelos Biológicos , Dosis de RadiaciónRESUMEN
Type B Niemann-Pick disease (NPD) is a nonneuronopathic lysosomal storage disorder which is characterized by accumulation of sphingomyelin-laden macrophages. The availability of plasma markers for storage cells may be of great value in facilitating therapeutic decisions. Given the similarity of the storage cells in NPD and Gaucher disease, we studied Gaucher plasma markers (chitotriosidase and CCL18) in two siblings homozygous for the R228C mutation in acid sphingomyelinase (ASM) and a type B course of NPD. The older sibling, first examined at the age of 9 months, showed marked hepatosplenomegaly and pulmonary involvement. The younger sibling has mild asymptomatic hepatosplenomgaly at the age of 5 months. Analysis of plasma specimens revealed markedly increased levels of chitotriosidase and CCL18 in the older sibling. In the younger child also, plasma chitotriosidase and CCL18 were clearly elevated above normal values almost immediately after birth and rapidly increased further. Histochemistry confirmed production of CCL18 by foam cells. In conclusion, plasma chitotriosidase and CCL18 may also serve as markers for the formation of pathological lipid-laden macrophages in type B NPD, in analogy to Gaucher disease. The availability of sensitive plasma surrogate markers may be of great value for monitoring the efficacy of enzyme supplementation therapy that is currently being developed.
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Quimiocinas CC/biosíntesis , Hexosaminidasas/sangre , Enfermedades de Niemann-Pick/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Salud de la Familia , Femenino , Células Espumosas/metabolismo , Enfermedad de Gaucher/sangre , Hexosaminidasas/metabolismo , Homocigoto , Humanos , Inmunohistoquímica , Lactante , Metabolismo de los Lípidos , Mutación , Esfingomielina Fosfodiesterasa/genéticaRESUMEN
PURPOSE: A combined treatment of cells with 5-bromo-2'-deoxyurine (BrdU), Hoechst 33 258 and ultraviolet A (UVA) light was used to introduce chromosomal aberrations in cells for the study of bystander effects in non-labelled cells. MATERIALS AND METHODS: Mixtures of BrdU-labelled and non-labelled Chinese hamster cells (V79) in S phase were exposed to Hoechst 33 258 and/or UVA light. Metaphase cells were collected and analysed for chromosomal aberrations by Giemsa staining. BrdU immunostaining was performed to verify BrdU incorporation in the cells. RESULTS: Combined treatment with BrdU, Hoechst dye and UVA light induced reduced cell survival and increased chromosomal aberrations, whereas treatment with Hoechst 33 258 and/or UVA light had no effect on cells. Elevated frequencies of chromosomal aberrations were found in non-labelled cells mixed with BrdU-labelled cells and exposed to Hoechst dye and UVA light, suggesting the induction of bystander effects by damaged BrdU-labelled cells. These bystander clastogenic effects were also observed in non-labelled cells mixed with dying cells, indicating a contribution of dying cells in the induction of the bystander effects. CONCLUSIONS: The combined treatment with BrdU, Hoechst 33 258 and UVA light is a valid method for the study of bystander effects as it enables both induction of DNA damage and discrimination of targeted cells and bystander cells.
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Bisbenzimidazol/farmacología , Bromodesoxiuridina/farmacología , Rayos Ultravioleta , Animales , Efecto Espectador , Línea Celular , Supervivencia Celular , Aberraciones Cromosómicas , Cricetinae , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Colorantes Fluorescentes/farmacología , Metafase/efectos de la radiación , Microscopía Fluorescente , Fármacos Sensibilizantes a Radiaciones/farmacologíaRESUMEN
PURPOSE: It is generally accepted that chromosome exchanges in irradiated cells are formed through interactions between separate DNA double-strand breaks (DSB). Here we tested whether non-irradiated DNA participates in the formation of chromosome aberrations when complex DNA DSB are induced elsewhere in the nucleus. MATERIALS AND METHODS: Synchronized Chinese hamster cells containing an X chromosome with a late replicating q arm (X(q) domain) were labelled with 125I-iododeoxyuridine (125IdUrd) in a period of S-phase when the vast majority of the X(q) domain was not replicating. DNA damage from 125I decay was accumulated at the G1/S border while the cells were stored in liquid nitrogen. Decay of 125I induced DSB in the immediate vicinity of the 125I atom. Chromosome aberrations involving what is essentially the 125I-free X domain were scored at the first mitosis after cell thawing. As a positive control, cells were treated with 125IdUrd at a later period in S-phase when the X(q) domain replicates, yielding a labelled X(q) domain. RESULTS: The 125I-free X(q) domain exhibited chromosome aberrations (exchanges and fragments). The frequency of these aberrations was linearly dependent on the number of 125I decays elsewhere in the cell nucleus. The efficiency of formation of chromosome aberrations by the 125I-free X(q) domain was approximately half of that observed in the 125I-labelled X(q) domain. CONCLUSIONS: The involvement of the 125I-free X(q) domain in chromosome aberrations suggests that DNA not damaged by the decay of incorporated 125I can interact with damaged DNA, indicating the existence of an alternative pathway for the formation of chromosome aberrations.
Asunto(s)
Núcleo Celular/genética , Núcleo Celular/efectos de la radiación , Cromatina/genética , Aberraciones Cromosómicas/efectos de la radiación , Animales , Línea Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cricetinae , Daño del ADN , Idoxuridina/metabolismo , Hibridación Fluorescente in Situ , Radioisótopos de Yodo , Modelos Genéticos , Cromosoma X/genética , Cromosoma X/metabolismo , Cromosoma X/efectos de la radiaciónRESUMEN
BACKGROUND: IgG subclasses of autoantibodies differ in their potential to induce an inflammatory response as they interact differentially with complement and Fcgamma receptors. METHODS: The IgG subclass distribution of anti-nucleohistone and anti-dsDNA antibodies was analysed longitudinally in patients with systemic lupus erythematosus before and at the moment of an extra-renal (n=23) or a renal relapse (n=l7). Kidney biopsy specimens of patients with a renal relapse were analysed for IgG subclass deposition. RESULTS: IgG1 anti-nucleohistone and IgG1 anti-dsDNA antibodies were present in plasma of 39 out of 40 patients. At the moment of a relapse, IgG2 and IgG3 anti-nucleohistone and IgG2 anti-dsDNA antibodies were more frequently present in patients with renal disease compared with those with extra-renal disease. Increases in levels of IgG1 anti-dsDNA were observed in 10 out of 11 patients prior to a renal relapse but only 10 out of 22 patients with an extra-renal relapse (91 vs 45%, P=0.02). Rises in IgG2 anti-dsDNA occurred at an equally low rate prior to both renal and extra-renal relapses. A rise in IgG2 anti-nucleohistone antibodies preceded a renal relapse in eight of 11 patients and an extra-renal relapse in only four out of 22 patients (73 vs 18%, P=0.006). In kidney biopsies all IgG subclasses could be detected. IgG1 and IgG2 subclass antibodies to nucleohistone and to dsDNA are the predominant subclasses found in plasma of lupus patients with renal disease. CONCLUSIONS: The frequent occurrence of a rise in IgG2 anti-nucleohistone and IgG1 anti-dsDNA in patients prior to a renal relapse suggests that, besides IgG1 subclass autoantibodies, IgG2 subclass antibodies to nucleohistone have a particular pathophysiological role in lupus nephritis.
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Autoanticuerpos/análisis , Inmunoglobulina G/análisis , Lupus Eritematoso Sistémico/inmunología , Nefritis Lúpica/inmunología , Adulto , Anticuerpos Antinucleares/análisis , Biopsia con Aguja , Estudios Transversales , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Estudios Longitudinales , Lupus Eritematoso Sistémico/patología , Nefritis Lúpica/patología , Masculino , Probabilidad , Pronóstico , Sensibilidad y Especificidad , Estadísticas no ParamétricasRESUMEN
Brown-Norway (BN) and Dorus Zadel Black (DZB) rats develop a T-cell-dependent membranous glomerulopathy (MGP) with high proteinuria and antiglomerular basement membrane (GBM) autoreactive antibodies (Abs), upon exposure to mercuric chloride (HgCl2). Laminin is an important autoantigenic target of the anti-GBM Abs, absorbing approximately 30% of the anti-GBM reactivity. Although many anti-GBM Abs have undergone isotype switching, it is currently unclear whether affinity maturation occurs during the HgCl2-induced autoimmune response. To address this question we analysed the rearranged immunoglobulin heavy chain variable-region genes (VHDJH regions) of 15 mAbs that were previously obtained from HgCl2-treated rats. Seven of these mAbs exhibit reactivity towards laminin. Our study showed that the VH-gene usage of antilaminin mAbs is largely restricted to the PC7183 VH-gene family (six out of seven). In addition, we demonstrated that at least three out of six laminin reactive and five out of six non-laminin-binding mAbs are encoded by germline VH genes (a total of eight out of 12 mAbs). Of the eight mAbs that are encoded by germline VH genes, seven are of a non-immunoglobulin M (IgM) isotype, indicating that isotype switching has occurred in these mAbs in the absence of somatic mutations. The mutations observed in the VH genes of the four remaining mAbs do not provide strong evidence for antigenic selection. The data support the notion that B cells in this model of MGP are not subjected to affinity maturation and probably result from polyclonal B-cell activation.
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Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/inmunología , Genes de Inmunoglobulinas/inmunología , Glomerulonefritis Membranosa/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/genética , Autoinmunidad , Secuencia de Bases , Regiones Determinantes de Complementariedad/genética , ADN Complementario/genética , Genes de Inmunoglobulinas/genética , Glomerulonefritis Membranosa/inducido químicamente , Glomerulonefritis Membranosa/genética , Cambio de Clase de Inmunoglobulina , Laminina/inmunología , Cloruro de Mercurio , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas BNRESUMEN
BACKGROUND: Connective tissue growth factor (CTGF) is a profibrotic growth factor, which is upregulated in wound healing and renal fibrosis, including anti-Thy-1.1 nephritis. The kinetics of CTGF mRNA expression in anti-Thy-1.1 nephritis suggested that CTGF regulation might contribute to glomerular response to injury downstream of transforming growth factor-beta (TGFbeta). In anti-Thy-1.1 nephritis the initial damage is followed by mesangial repair and limited sclerosis, which involves mesangial cell (MC) activation (alpha-smooth-muscle actin (alphaSMA) expression), proliferation, migration, and extracellular matrix production. The present in vitro study addresses the possible role of CTGF in these different aspects of mesangial response to injury, and how CTGF activity might relate to effects of TGFbeta and platelet-derived growth factor-BB (PDGF-BB). METHODS AND RESULTS: Immunostaining and ELISA showed that alphaSMA expression and transformation of MC into myofibroblast-like cells was induced by TGFbeta, but not affected by PDGF-BB, CTGF, or neutralizing anti-CTGF antibodies. [(3)H]thymidine incorporation and Ki67 staining demonstrated that, unlike PDGF-BB, neither CTGF nor TGFbeta induced the proliferation of MC. In contrast, both CTGF and TGFbeta induced MC migration, as evidenced by approximation of wound edges in scrape-wounded, non-proliferating rat MC monolayers. In addition, fibronectin expression was upregulated by both CTGF and TGFbeta, as measured by dot-blot analysis. Anti-CTGF completely blocked the effect of added CTGF. Moreover, anti-CTGF significantly reduced TGFbeta-induced increase in fibronectin. CONCLUSION: It thus appears that CTGF is specifically involved in a subset of the adaptive changes of MC involved in mesangial repair and sclerosis, which makes it an interesting candidate target for future intervention strategies.
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Regulación de la Expresión Génica/fisiología , Mesangio Glomerular/lesiones , Mesangio Glomerular/fisiología , Sustancias de Crecimiento/fisiología , Proteínas Inmediatas-Precoces/fisiología , Péptidos y Proteínas de Señalización Intercelular , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factor de Crecimiento Transformador beta/genética , Actinas/genética , Animales , Anticuerpos/farmacología , Becaplermina , División Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Factor de Crecimiento del Tejido Conjuntivo , Fibronectinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Mesangio Glomerular/citología , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/farmacología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/farmacología , Antígeno Ki-67/análisis , Cinética , Proteínas Proto-Oncogénicas c-sis , Ratas , Proteínas Recombinantes/farmacología , Timidina/metabolismoAsunto(s)
Inmunoglobulina A/análisis , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Adulto , Niño , Progresión de la Enfermedad , Endotelio/inmunología , Endotelio/patología , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina M/sangre , Fallo Renal Crónico/inmunología , Fallo Renal Crónico/fisiopatología , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Nefritis Lúpica/fisiopatología , ProteinuriaRESUMEN
Exposure of Brown Norway rats to mercuric chloride induces systemic autoimmunity, involving T- and B-lymphocyte activation, (auto-)antibody production and multiorgan inflammation. Several divalent metal ions, such as Mg2+ and Mn2+, can activate binding of integrins to their ligands, thus causing lymphocyte adhesion. To test the hypothesis that Hg2+ acts in a similar way, we studied the effect of HgCl2 on integrin-mediated T-cell adhesion. HgCl2 induced cell-cell aggregation of human T lymphoblasts. Exposure of a human T-cell clone to HgCl2 for 1 hr enhanced, in a dose-dependent way, cell binding to fibronectin (FN) and to intercellular adhesion molecules (ICAM) -1, -2 and -3. Furthermore, HgCl2 induced strong binding of Jurkat T cells to FN. These effects of HgCl2 were of similar magnitude as the effects of phorbol 12-myristate 13-acetate (PMA) or MnCl2. Studies using blocking antibodies indicated the involvement of CD11a in binding to ICAMs, and of CD49d, CD49e, and CD29 in binding to FN. Adhesion to FN induced by HgCl2 or by PMA, but not by MnCl2, was dependent on temperature and on extracellular Ca2+ or Mg2+. Addition of cytochalasin B enhanced synergistically the FN adhesion induced by MnCl2, whereas the effects of PMA and HgCl2 were not modified. These results indicate that Hg2+ is a potent activator of T-cell adhesion, mediated by several integrins and ligands. In contrast to the effect of MnCl2, HgCl2-induced cell adhesion probably involves an intracellular pathway. Activation of integrins by HgCl2 may play an important role in activation and migration of leucocytes involved in HgCl2-induced immune dysregulation in vivo.
Asunto(s)
Antiinfecciosos Locales/farmacología , Desinfectantes/farmacología , Integrinas/fisiología , Cloruro de Mercurio/farmacología , Linfocitos T/efectos de los fármacos , Actinas/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Agregación Celular/efectos de los fármacos , Agregación Celular/inmunología , Técnicas de Cultivo de Célula , Citoesqueleto/inmunología , Humanos , Integrinas/metabolismo , Células Jurkat , Cloruro de Magnesio/farmacología , Linfocitos T/fisiologíaRESUMEN
Susceptibility to chemically induced lung tumorigenesis has previously been mapped to a genomic interval of 27 kb in the MHC class III region of the mouse using two H2 (a/b) intra- H2 recombinants, B10.A(1R) and B10.A(2R). Three genes are located within this interval, G7e (encoding a viral envelope protein), G7a/ Vars2 (encoding valyl-tRNA synthetase), and G7c (a gene with unknown function). A 70 kb contig, spanning the 27 kb region and extending 20 kb either side, was constructed from lambda phage libraries with genomic inserts derived from mouse strains B10.A(1R) and B10.A(2R). The region was analyzed for single-nucleotide polymorphisms, which would facilitate further fine mapping of the interval. Analysis of the expression levels of the candidate genes did not reveal any difference between B10.A(1R) and B10.A(2R). In addition, no differences were found at the sequence level in the 27 kb interval except for an A to T transition in intron 7 of G7c. A database comparison of the sequence surrounding this polymorphism did not identify any DNA-binding or enhancer consensus sequence. In conclusion, the previously observed phenotype could not be associated with or assigned to any of the candidate genes G7e, G7a/ Vars2, or G7c, nor could any of the other susceptibility loci, which have been reported to map to this region ( Cps1, Acp, Orch1, and Igis1).
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Antígenos HLA/genética , Neoplasias Pulmonares/genética , Complejo Mayor de Histocompatibilidad/genética , Recombinación Genética/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Regulación de la Expresión Génica , Genotipo , Hibridación in Situ , Neoplasias Pulmonares/inducido químicamente , Ratones , Datos de Secuencia Molecular , Fenotipo , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The methods covered in this unit include flow cytometry of metaphase chromosomes, chromosome dissection, and the DOP-PCR amplification methods for reverse chromosome painting. Successful application in these areas requires care and attention to methodological details, and this unit is particularly comprehensive.
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Citogenética/métodos , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Colorantes Fluorescentes/análisis , Hibridación Fluorescente in Situ/métodos , Sondas de Oligonucleótidos/análisis , Animales , Cromosomas/ultraestructura , Humanos , MetafaseRESUMEN
We developed a method for partial irradiation of cell nuclei and for highlighting the irradiated chromatin domain(s) in both interphase nuclei and metaphase chromosomes. The method involves the use of the replication program of chromosomes and consists of three major steps: I) selection of a suitable chromatin domain, II) damage induction by 125I, and III) visualization of the domain. Here, the first step of the method, applied to Chinese hamster HA-1 cells, is described. Using pulse labelling with the replication marker IUdR, it was shown that Xq does not replicate at early S-phase and that the replication timing of Xq can be highly effectively synchronized with hydroxyurea in a whole cell population. Thus, the replication timing of Xq may be used to exclude or to incorporate 125I into the Xq. Other chromatin can be selected and targeted with 125I in a similar way. Examples of possible applications of the method are given.
Asunto(s)
Núcleo Celular/genética , Cromatina/efectos de la radiación , Daño del ADN/efectos de la radiación , Radioisótopos de Yodo/farmacología , Animales , Técnicas de Cultivo de Célula , Ciclo Celular , Cricetinae , Daño del ADN/genética , Hidroxiurea/administración & dosificación , Idoxuridina/administración & dosificación , Inmunohistoquímica , Radioisótopos de Yodo/administración & dosificación , Inhibidores de la Síntesis del Ácido Nucleico/administración & dosificación , Radioterapia/métodos , Radioterapia/tendenciasRESUMEN
BACKGROUND & AIMS: Patients with progressive familial intrahepatic cholestasis (PFIC) type 3 have a mutation in the MDR3 gene, encoding the hepatocanalicular phospholipid translocator. In general, liver failure develops within the first decade of life in these patients. Previous studies have shown that in the mdr2-knockout mouse, the animal model for this disease, the absence of phospholipids in bile causes chronic bile salt-induced damage to hepatocytes. We aimed to test the efficacy of hepatocyte transplantation and liver repopulation in this disease model. METHODS: Transgenic MDR3-expressing hepatocytes as well as normal mdr2(+/+) hepatocytes were transplanted in mdr2(-/-) mice, and liver repopulation was assessed by immunohistochemistry and measurement of biliary lipid secretion. RESULTS: Transplanted hepatocytes partially repopulated the liver, restored phospholipid secretion, and diminished liver pathology. Repopulation was stronger when hepatocellular damage was enhanced by a bile salt-supplemented diet. After 1 year, however, these animals developed multiple hepatic tumors, and biliary phospholipid secretion decreased. In transplanted animals receiving a control diet, repopulation was slower but eventually remained stable at 21%, while liver pathology was completely abrogated and tumor formation was prevented. CONCLUSIONS: These results suggest that moderate liver pathology is a safe condition for the induction of effective hepatocyte repopulation and that this therapy is potentially applicable to patients with PFIC type 3.
Asunto(s)
Colestasis/genética , Colestasis/cirugía , Hepatocitos/trasplante , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/genética , Animales , Colatos/administración & dosificación , Colatos/farmacología , Colestasis/metabolismo , Colestasis/patología , Dieta , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/prevención & control , Masculino , Ratones , Ratones Noqueados/genética , Ratones Transgénicos/genética , Fosfolípidos/metabolismoRESUMEN
Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase alpha was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.
Asunto(s)
Replicación del ADN , ADN/metabolismo , Animales , Bromodesoxiuridina/metabolismo , Células CHO , Núcleo Celular/ultraestructura , Cricetinae , ADN Polimerasa I/metabolismo , Halógenos , Hibridación in Situ/métodos , Nucleótidos , Factores de TiempoRESUMEN
Although the aetiology of the hypopigmentary disorder vitiligo is ill understood, it is clear that pigment producing cells are absent from vitiliginous lesional skin. The present study was designed to investigate the possible role of melanocyte-expressed apoptosis regulatory molecules in melanocyte disappearance. Flow cytometric evaluation of p53, p21, Bcl-2 and Bax revealed no differences in in vitro expression levels between normal control and non-lesional melanocytes. Moreover, no in situ immunohistological differences were observed in melanocytes present in control, non-lesional and perilesional skin. However, an enhanced number of p53+ nuclei, in the absence of detectable p21 expression, was detected in involved areas. The observed p53 expression pattern did not involve melanocytes and could be the result of ultraviolet (UV) A irradiation. Further, we showed that UVB is capable of modulating melanocyte-expressed apoptosis regulatory molecules. Consequently, a lethal dose of UVB was given to two groups of cultured normal control and non-lesional melanocytes. No significant differences were found when comparing the percentages and kinetics of UVB-induced apoptosis in these groups. In conclusion, our results indicate that the relative apoptosis susceptibility of melanocytes in vitiligo is comparable with that of normal control cells. It is therefore unlikely that vitiligo is causally related to dysregulation of apoptosis regulatory molecules.
Asunto(s)
Apoptosis/fisiología , Melanocitos/metabolismo , Proteínas/metabolismo , Vitíligo/metabolismo , Apoptosis/efectos de la radiación , Western Blotting , Estudios de Casos y Controles , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Citometría de Flujo , Humanos , Melanocitos/fisiología , Melanocitos/efectos de la radiación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Rayos Ultravioleta , Vitíligo/fisiopatología , Proteína X Asociada a bcl-2RESUMEN
Chromosome territories need to be well defined at high resolution before functional aspects of chromosome organization in interphase can be explored. To visualize chromosomes by electron microscopy (EM), the DNA of Chinese hamster fibroblasts was labeled in vivo with thymidine analogue BrdU. Labeled chromosomes were then segregated during several cell cycles to obtain nuclei containing only 2 to 3 labeled chromosomes. Subsequent immunocytochemical detection of BrdU allowed analysis by EM of chromosome territories and subchromosomal domains in well preserved nuclei. Our results provide the first high resolution visualization of chromosomes in interphase nuclei. We show that chromosome domains are either separated from one another by interchromatin space or are in close contact with no or little intermingling of their DNA. This demonstrates that, while chromosomes form discrete territories, chromatin of adjacent chromosomes may be in contact in limited regions, thus implying chromosome-chromosome interactions. Chromosomes are organized as condensed chromatin with dispersed chromatin extending into the interchromatin space that is largely devoid of DNA. The interchromatin space, which is known to be involved in various nuclear functions, forms interconnecting channels running through and around chromosome territories. Functional implications of this organization are discussed.