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1.
Am J Transplant ; 13(9): 2308-21, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23855618

RESUMEN

To ensure safety tolerance induction protocols are accompanied by conventional immunosuppressive drugs (IS). But IS such as calcineurin inhibitors (CNI), for example, cyclosporin A (CsA), can interfere with tolerance induction. We investigated the effect of an additional transient CsA treatment on anti-CD4mAb-induced tolerance induction upon rat kidney transplantation. Additional CsA treatment induced deteriorated graft function, resulting in chronic rejection characterized by glomerulosclerosis, interstitial fibrosis, tubular atrophy and vascular changes. Microarray analysis revealed enhanced intragraft expression of the B cell attracting chemokine CXCL13 early during CsA treatment. Increase in CXCL13 expression is accompanied by enhanced B cell infiltration with local and systemic IgG production and C3d deposition as early as 5 days upon CsA withdrawal. Adding different CNIs to cultures of primary mesangial cells isolated from glomeruli resulted in a concentration-dependent increase in CXCL13 transcription. CsA in synergy with TNF-α can enhance the B cell attracting and activating potential of mesangial cells. Transient B cell depletion or transfer of splenocytes from tolerant recipients 3 weeks after transplantation could rescue tolerance induction and did inhibit intragraft B cell accumulation, alloantibody production and ameliorate chronic rejection.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Linfocitos T CD4-Positivos/inmunología , Inhibidores de la Calcineurina , Tolerancia Inmunológica/inmunología , Inmunosupresores/farmacología , Trasplante de Riñón , Animales , Linfocitos B/inmunología , Calcineurina/farmacología , Quimiocina CXCL13/biosíntesis , Ciclosporina/farmacología , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Riñón/metabolismo , Activación de Linfocitos , Masculino , Ratas , Ratas Endogámicas Lew
2.
Am J Transplant ; 9(8): 1742-51, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19563340

RESUMEN

The frequency of delayed function of kidney transplants varies greatly and is associated with quality of graft, donor age and the duration of cold ischemia time. Furthermore, body weight differences between donor and recipient can affect primary graft function, but the underlying mechanism is poorly understood. We transplanted kidney grafts from commensurate body weight (L-WD) or reduced body weight (H-WD) donor rats into syngeneic or allogeneic recipients. Twenty-four hours posttransplantation, serum creatinine levels in H-WD recipients were significantly higher compared to L-WD recipients indicating impaired primary graft function. This was accompanied by upregulation of IL-6 transcription and increased tubular destruction in grafts from H-WD recipients. Using DNA microarray analysis, we detected decreased expression of genes associated with kidney function and an upregulation of other genes such as Cyp3a13, FosL and Trib3. A single application of IL-6 into L-WD recipients is sufficient to impair primary graft function and cause tubular damage, whereas immediate neutralization of IL-6 receptor signaling in H-WD recipients rescued primary graft function with well-preserved kidney graft architecture and a normalized gene expression profile. These findings have strong clinical implication as anti-IL6R treatment of patients receiving grafts from lower-weight donors could be used to improve primary graft function.


Asunto(s)
Peso Corporal/fisiología , Interleucina-6/fisiología , Trasplante de Riñón/fisiología , Transducción de Señal/fisiología , Animales , Apoptosis/fisiología , Creatinina/sangre , Hemo-Oxigenasa 1/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Trasplante de Riñón/patología , Túbulos Renales/patología , Masculino , Modelos Animales , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas
3.
Brief Bioinform ; 2(4): 329-40, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11808745

RESUMEN

During recent years, DNA microarrays have become the method of choice to monitor the expression level of a large number of genes. Depending on the focus of the study and the method of microarray fabrication, a number of different strategies for probe selection may be most appropriate. One consideration concerns the length of the probe, ranging from some 25 residues used for oligonucleotide arrays to complete cDNAs. Unless resources are truly unlimited, an important decision to be made is the amount of effort to be put into the selection of genes and gene fragments. While high-throughput cDNA arraying projects usually will select from a collection of existing cDNA clones, smaller projects focusing on a number of selected genes can afford to selectively amplify fragments optimised for that purpose. This paper discusses the full scope of probe selection strategies, highlighting the problems that may be encountered in the various systems.


Asunto(s)
Sondas de ADN , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Empalme Alternativo/genética , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Poli A/metabolismo , Regiones Promotoras Genéticas
4.
J Biol Chem ; 275(8): 5710-7, 2000 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-10681556

RESUMEN

Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin, an essential lipid constituent of the plasma membrane, lysosomal membranes, endoplasmic reticulum, and the Golgi membrane stacks of mammalian cells. In this study, we report the biochemical and functional characterization and subcellular localization of magnesium-dependent nSMase1 from overexpressing human embryonic kidney (HEK293) cells. Site-directed mutagenesis of conserved residues probably involved in the enzymatic sphingomyelin cleavage as well as the removal of one or both putative transmembrane domains lead to the complete loss of enzymatic activity of human nSMase1 expressed in HEK293 cells. Polyclonal antibodies raised against recombinant mammalian nSMase1 immunoprecipitated and inactivated the enzyme in membrane extracts of overexpressing HEK293 cells and different murine tissues. Cell fractionation combined with immunoprecipitation studies localized the nSMase1 protein predominantly in the microsomal fraction. The enzyme colocalized with marker proteins of the endoplasmic reticulum and the Golgi apparatus in immunocytochemistry. Anti-nSMase1 antibodies did not affect the nSMase activity in the plasma membrane fraction and membrane extracts from murine brain. Our study leads to the conclusion that nSMase1 is one of at least two mammalian neutral sphingomyelinases with different subcellular localization, tissue specificity, and enzymatic properties.


Asunto(s)
Magnesio/metabolismo , Esfingomielina Fosfodiesterasa/química , Animales , Northern Blotting , Encéfalo/enzimología , Línea Celular , Membrana Celular/enzimología , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico Liso/enzimología , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Ratones , Microscopía Fluorescente , Microsomas Hepáticos/enzimología , Mutagénesis Sitio-Dirigida , Mutación Puntual , Pruebas de Precipitina , Procesamiento Proteico-Postraduccional , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielina Fosfodiesterasa/metabolismo , Distribución Tisular , Transfección
5.
Proc Natl Acad Sci U S A ; 97(11): 5895-900, 2000 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-10823942

RESUMEN

The enzymatic breakdown of sphingomyelin by sphingomyelinases is considered the major source of the second messenger ceramide. Studies on the contribution of the various described acidic and neutral sphingomyelinases to the signaling pool of ceramide have been hampered by the lack of molecular data on the neutral sphingomyelinases (nSMases). We recently identified a mammalian nSMase, an integral membrane protein with remote similarity to bacterial sphingomyelinases. However, its ubiquitous expression pattern is in contrast to previous findings that sphingomyelinase activity is found mainly in brain tissues. By using an improved database search method, combined with phylogenetic analysis, we identified a second mammalian nSMase (nSMase2) with predominant expression in the brain. The sphingomyelinase activity of nSMase2 has a neutral pH optimum, depends on Mg(2+) ions, and is activated by unsaturated fatty acids and phosphatidylserine. Immunofluorescence reveals a neuron-specific punctate perinuclear staining, which colocalizes with a Golgi marker in a number of cell lines. The likely identity of nSMase2 with cca1, a rat protein involved in contact inhibition of 3Y1 fibroblasts, suggests a role for this enzyme in cell cycle arrest. Both mammalian nSMases are members of a superfamily of Mg(2+)-dependent phosphohydrolases, which also contains nucleases, inositol phosphatases, and bacterial toxins.


Asunto(s)
Encéfalo/enzimología , Magnesio/metabolismo , Proteínas del Tejido Nervioso/genética , Esfingomielina Fosfodiesterasa/genética , Secuencia de Aminoácidos , Animales , Ciclo Celular/fisiología , Células Cultivadas , Clonación Molecular , Inhibición de Contacto/fisiología , ADN Complementario/genética , Activación Enzimática , Ácidos Grasos Insaturados/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas del Tejido Nervioso/metabolismo , Células PC12 , Fosfatidilserinas/metabolismo , Isoformas de Proteínas/química , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielina Fosfodiesterasa/fisiología , Fracciones Subcelulares/metabolismo
6.
Proc Natl Acad Sci U S A ; 95(7): 3638-43, 1998 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-9520418

RESUMEN

Sphingomyelin is an abundant constituent of the plasma membranes of mammalian cells. Ceramide, its primary catabolic intermediate, is released by either acid sphingomyelinase or neutral sphingomyelinase (nSMase) and has emerged as a potential lipid signaling molecule. nSMase is regarded as a key enzyme in the regulated activation of the "sphingomyelin cycle" and cell signaling. We report here the cloning, identification, and functional characterization of murine and human nSMase, a ubiquitously expressed integral membrane protein, which displays all established properties of the Mg2+-dependent nSMase of the plasma membrane. Stably nSMase-overexpressing U937 and human embryonic kidney cell lines have been generated for the study of the role of nSMase in signal transduction pathways. Their stimulation by tumor necrosis factor alpha leads only to a moderately elevated ceramide concentration. Activation of Jun kinase and NFkappaB and poly(ADP-ribose) polymerase cleavage are identical in mock- and nSMase-transfected cells. Tumor necrosis factor alpha triggers the ERK1 pathway in none of the cell lines. The cloned nSMase will facilitate further controlled experiments aiming at the definition of a possible role of ceramide as signal transduction molecule.


Asunto(s)
Transducción de Señal , Esfingolípidos/metabolismo , Esfingomielina Fosfodiesterasa/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , ADN Complementario/análisis , ADN Complementario/genética , Técnicas de Transferencia de Gen , Humanos , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia , Esfingomielina Fosfodiesterasa/metabolismo
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