RESUMEN
Organ donors are sources of physiologically healthy organs and tissues for life-saving transplantation, and have been recently used for human immunology studies which are typically confined to the sampling of peripheral blood. Donors comprise a diverse population with different causes of death and clinical outcomes during hospitalization, and the effects of such variations on immune parameters in blood and tissues are not known. We present here a coordinate analysis of innate and adaptive immune components in blood, lymphoid (bone marrow, spleen, lymph nodes), and mucosal (lungs, intestines) sites from a population of brain-dead organ donors (2 months-93 years; n = 291) across eight clinical parameters. Overall, the blood of donors exhibited similar monocyte and lymphocyte content and low serum levels of pro-inflammatory cytokines as healthy controls; however, donor blood had increased neutrophils and serum levels of IL-8, IL-6, and MCP-1 which varied with cause of death. In tissues, the frequency and composition of monocytes, neutrophils, B lymphocytes and T cell subsets in lymphoid or mucosal sites did not vary with clinical state, and was similar in donors independent of the extent of clinical complications. Our results reveal that organ donors maintain tissue homeostasis, and are a valuable resource for fundamental studies in human immunology.
Asunto(s)
Muerte Encefálica/inmunología , Linfocitos/inmunología , Células Mieloides/inmunología , Trasplante de Órganos , Donantes de Tejidos , Obtención de Tejidos y Órganos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Muerte Encefálica/patología , Estudios de Casos y Controles , Niño , Preescolar , Citocinas/sangre , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Persona de Mediana Edad , Pronóstico , Subgrupos de Linfocitos T/inmunología , Adulto JovenRESUMEN
Successful strategies for transplantation of neural precursor cells for replacement of lost or dysfunctional CNS cells require long-term survival of grafted cells and integration with the host system, potentially for the life of the recipient. It is also important to demonstrate that transplants do not result in adverse outcomes. Few studies have examined the long-term properties of transplanted neural precursor cells in the CNS, particularly in non-neurogenic regions of the adult. The aim of the present study was to extensively characterize the fate of defined populations of neural precursor cells following transplantation into the developing and adult CNS (brain and spinal cord) for up to 15 months, including integration of graft-derived neurons with the host. Specifically, we employed neuronal-restricted precursors and glial-restricted precursors, which represent neural precursor cells with lineage restrictions for neuronal and glial fate, respectively. Transplanted cells were prepared from embryonic day-13.5 fetal spinal cord of transgenic donor rats that express the marker gene human placental alkaline phosphatase to achieve stable and reliable graft tracking. We found that in both developing and adult CNS grafted cells showed long-term survival, morphological maturation, extensive distribution and differentiation into all mature CNS cell types (neurons, astrocytes and oligodendrocytes). Graft-derived neurons also formed synapses, as identified by electron microscopy, suggesting that transplanted neural precursor cells integrated with adult CNS. Furthermore, grafts did not result in any apparent deleterious outcomes. We did not detect tumor formation, cells did not localize to unwanted locations and no pronounced immune response was present at the graft sites. The long-term stability of neuronal-restricted precursors and glial-restricted precursors and the lack of adverse effects suggest that transplantation of lineage-restricted neural precursor cells can serve as an effective and safe replacement therapy for CNS injury and degeneration.
Asunto(s)
Diferenciación Celular/fisiología , Sistema Nervioso Central/fisiología , Neuronas/fisiología , Trasplante de Células Madre/métodos , Células Madre/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Células Cultivadas , Sistema Nervioso Central/citología , Sistema Nervioso Central/cirugía , Embrión de Mamíferos , Femenino , Gangliósidos/metabolismo , Supervivencia de Injerto/efectos de los fármacos , Inmunohistoquímica/métodos , Inmunosupresores/farmacología , Microscopía Electrónica de Transmisión/métodos , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuroglía/fisiología , Neuroglía/ultraestructura , Neuronas/ultraestructura , Embarazo , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de TiempoRESUMEN
Successful strategies for transplantation of neural precursor cells for replacement of lost or dysfunctional CNS cells require long-term survival of grafted cells and integration with the host system, potentially for the life of the recipient. It is also important to demonstrate that transplants do not result in adverse outcomes. Few studies have examined the long-term properties of transplanted neural precursor cells in the CNS, particularly in non-neurogenic regions of the adult. The aim of the present study was to extensively characterize the fate of defined populations of neural precursor cells following transplantation into the developing and adult CNS (brain and spinal cord) for up to 15 months, including integration of graft-derived neurons with the host. Specifically, we employed neuronal-restricted precursors and glial-restricted precursors, which represent neural precursor cells with lineage restrictions for neuronal and glial fate, respectively. Transplanted cells were prepared from embryonic day-13.5 fetal spinal cord of transgenic donor rats that express the marker gene human placental alkaline phosphatase to achieve stable and reliable graft tracking. We found that in both developing and adult CNS grafted cells showed long-term survival, morphological maturation, extensive distribution and differentiation into all mature CNS cell types (neurons, astrocytes and oligodendrocytes). Graft-derived neurons also formed synapses, as identified by electron microscopy, suggesting that transplanted neural precursor cells integrated with adult CNS. Furthermore, grafts did not result in any apparent deleterious outcomes. We did not detect tumor formation, cells did not localize to unwanted locations and no pronounced immune response was present at the graft sites. The long-term stability of neuronal-restricted precursors and glial-restricted precursors and the lack of adverse effects suggest that transplantation of lineage-restricted neural precursor cells can serve as an effective and safe replacement therapy for CNS injury and degeneration.
Asunto(s)
Trasplante de Células/métodos , Sistema Nervioso Central/fisiología , Neuronas/fisiología , Células Madre/fisiología , Factores de Edad , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Animales Modificados Genéticamente , Células Cultivadas , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/ultraestructura , Técnicas de Cocultivo/métodos , Embrión de Mamíferos , Femenino , Gangliósidos/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunohistoquímica/métodos , Proteínas de Filamentos Intermediarios/metabolismo , Microscopía Inmunoelectrónica/métodos , Proteínas del Tejido Nervioso/metabolismo , Nestina , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuroglía/fisiología , Neuronas/ultraestructura , Fosfopiruvato Hidratasa/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Tiempo , TrasplantesRESUMEN
Although prostate cancer is the most common non-cutaneous malignancy diagnosed in men in the United States, little is known about inherited factors that influence its genetic predisposition. Here we report that germline mutations in the gene encoding 2'-5'-oligoadenylate(2-5A)-dependent RNase L (RNASEL) segregate in prostate cancer families that show linkage to the HPC1 (hereditary prostate cancer 1) region at 1q24-25 (ref. 9). We identified RNASEL by a positional cloning/candidate gene method, and show that a nonsense mutation and a mutation in an initiation codon of RNASEL segregate independently in two HPC1-linked families. Inactive RNASEL alleles are present at a low frequency in the general population. RNASEL regulates cell proliferation and apoptosis through the interferon-regulated 2-5A pathway and has been suggested to be a candidate tumor suppressor gene. We found that microdissected tumors with a germline mutation showed loss of heterozygosity and loss of RNase L protein, and that RNASEL activity was reduced in lymphoblasts from heterozyogous individuals compared with family members who were homozygous with respect to the wildtype allele. Thus, germline mutations in RNASEL may be of diagnostic value, and the 2-5A pathway might provide opportunities for developing therapies for those with prostate cancer.
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Endorribonucleasas/genética , Mutación de Línea Germinal , Oncogenes , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Nucleótidos de Adenina/metabolismo , Secuencia de Bases , Estudios de Casos y Controles , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Femenino , Ligamiento Genético , Heterocigoto , Homocigoto , Humanos , Pérdida de Heterocigocidad , Linfocitos/enzimología , Masculino , Oligorribonucleótidos/metabolismo , LinajeRESUMEN
Polymer-directed enzyme prodrug therapy (PDEPT) is a novel two-step antitumour approach using a combination of a polymeric prodrug and polymer-enzyme conjugate to generate cytotoxic drug selectively at the tumour site. In this study the polymeric prodrug N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-Gly-Phe-Leu-Gly-doxorubicin conjugate PK1 (currently under Phase II clinical evaluation) was selected as the model prodrug, and HPMA copolymer-cathepsin B as a model for the activating enzyme conjugate. Following polymer conjugation (yield of 30-35%) HPMA copolymer-cathepsin B retained approximately 20-25% enzymatic activity in vitro. To investigate pharmacokinetics in vivo,(125)I-labelled HPMA copolymer-cathepsin B was administered intravenously (i.v.) to B16F10 tumour-bearing mice. HPMA copolymer-cathespin B exhibited a longer plasma half-life (free cathepsin B t(1/2alpha)= 2.8 h; bound cathepsin B t(1/2alpha)= 3.2 h) and a 4.2-fold increase in tumour accumulation compared to the free enzyme. When PK1 (10 mg kg(-1)dox-equiv.) was injected i.v. into C57 mice bearing subcutaneously (s.c.) palpable B16F10 tumours followed after 5 h by HPMA copolymer-cathepsin B there was a rapid increase in the rate of dox release within the tumour (3.6-fold increase in the AUC compared to that seen for PK1 alone). When PK1 and the PDEPT combination were used to treat established B16F10 melanoma tumour (single dose; 10 mg kg(-1)dox-equiv.), the antitumour activity (T/C%) seen for the combination PDEPT was 168% compared to 152% seen for PK1 alone, and 144% for free dox. Also, the PDEPT combination showed activity against a COR-L23 xenograft whereas PK1 did not. PDEPT has certain advantages compared to ADEPT and GDEPT. The relatively short plasma residence time of the polymeric prodrug allows subsequent administration of polymer-enzyme without fear of prodrug activation in the circulation and polymer-enzyme conjugates have reduced immunogenicity. This study proves the concept of PDEPT and further optimisation is warranted.
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Catepsina B/farmacología , Ácidos Polimetacrílicos/farmacología , Profármacos , Animales , Área Bajo la Curva , Catepsina B/química , Catepsina B/farmacocinética , Terapia Combinada , Activación Enzimática , Semivida , Infusiones Intravenosas , Masculino , Melanoma/tratamiento farmacológico , Melanoma/veterinaria , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales , Ácidos Polimetacrílicos/química , Ácidos Polimetacrílicos/farmacocinética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/veterinariaRESUMEN
Mice lacking expression of the p66 isoform of the ShcA adaptor protein (p66(ShcA)) are less susceptible to oxidative stress and have an extended life span. Specifically, phosphorylation of p66(ShcA) at serine 36 is critical for the cell death response elicited by oxidative damage. We sought to identify the kinase(s) responsible for this phosphorylation. Utilizing the SH-SY5Y human neuroblastoma cell model, it is demonstrated that p66(ShcA) is phosphorylated on serine/threonine residues in response to UV irradiation. Both c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinases are activated by UV irradiation, and we show that both are capable of phosphorylating serine 36 of p66(ShcA) in vitro. However, treatment of cells with a multiple lineage kinase inhibitor, CEP-1347, that blocks UV-induced JNK activation, but not p38, phosphatidylinositol 3-kinase, or MEK1 inhibitors, prevented p66(ShcA) phosphorylation in SH-SY5Y cells. Consistent with this finding, transfected activated JNK1, but not the kinase-dead JNK1, leads to phosphorylation of serine 36 of p66(ShcA) in Chinese hamster ovary cells. In conclusion, JNKs are the kinases that phosphorylate serine 36 of p66(ShcA) in response to UV irradiation in SH-SY5Y cells, and blocking p66(ShcA) phosphorylation by intervening in the JNK pathway may prevent cellular damage due to light-induced oxidative stress.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Proteínas Quinasas Activadas por Mitógenos/efectos de la radiación , Proteínas/metabolismo , Serina/metabolismo , Rayos Ultravioleta , Proteína Adaptadora GRB2 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas/química , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
The aim of this study was to develop a saturated transcript map of the region encompassing the HPC1 locus to identify the susceptibility genes involved in hereditary prostate cancer (OMIM 176807) and hyperparathyroidism-jaw tumor syndrome (OMIM 145001). We previously reported the generation of a 6-Mb BAC/PAC contig of the candidate region and employed various strategies, such as database searching, exon-trapping, direct cDNA hybridization, and sample sequencing of BACs, to identify all potential transcripts. These efforts led to the identification and precise localization on the BAC contig of 59 transcripts representing 22 known genes and 37 potential transcripts represented by ESTs and exon traps. Here we report the detailed characterization of these ESTs into full-length transcript sequences, their expression pattern in various tissues, their genomic organization, and their homology to known genes. We have also identified an Alu insertion polymorphism in the intron of one of the transcripts. Overall, data on 13 novel transcripts and the human RGS8 gene (homologue of the rat RGS8 gene) are presented in this paper. Ten of the 13 novel transcripts are expressed in prostate tissue and represent positional candidates for HPC1.
Asunto(s)
Cromosomas Humanos Par 1 , Síndromes Neoplásicos Hereditarios/genética , Neoplasias de la Próstata/genética , Proteínas RGS/genética , ARNt Metiltransferasas/genética , Secuencia de Aminoácidos , Animales , Mapeo Contig , ADN Complementario , Etiquetas de Secuencia Expresada , Expresión Génica , Perfilación de la Expresión Génica/métodos , Predisposición Genética a la Enfermedad , Genoma Humano , Humanos , Hiperparatiroidismo/genética , Neoplasias Maxilomandibulares/genética , Masculino , Datos de Secuencia Molecular , Mutación , Neoplasias de las Paratiroides/genética , Ratas , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC(50) values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.
Asunto(s)
Carbazoles/farmacología , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas , Animales , Células CHO , Muerte Celular , Cricetinae , Activación Enzimática , Proteínas Quinasas JNK Activadas por Mitógenos , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Químicos , Células PC12 , Ratas , Proteina Quinasa Quinasa Quinasa 11 Activada por MitógenoRESUMEN
PURPOSE: To evaluate tumor, normal tissue, and plasma pharmacokinetics of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA). The study aimed to determine the pharmacokinetics of carbon-11-labeled DACA ([11C]DACA) and evaluate the effect of pharmacologic doses of DACA on radiotracer kinetics. PATIENTS AND METHODS: [11C]DACA (at 1/1,000 phase I starting dose) was administered to 24 patients with advanced cancer (pre-phase I) or during a phase I trial of DACA in five patients. Positron emission tomography (PET) was performed to assess pharmacokinetics and tumor blood flow. Plasma samples were analyzed for metabolite profile of [11C]DACA. RESULTS: There was rapid systemic clearance of [11C]DACA over 60 minutes (1.57 and 1.46 L x min(-1) x m(-2) in pre-phase I and phase I studies, respectively) with the production of several radiolabeled plasma metabolites. Tumor, brain, myocardium, vertebra, spleen, liver, lung, and kidneys showed appreciable uptake of 11C radioactivity. The area under the time-versus-radioactivity curves (AUC) showed the highest variability in tumors. Of interest to potential toxicity, maximum radiotracer concentrations (Cmax) in brain and vertebra were low (0.67 and 0.54 m(2) x mL(-1), respectively) compared with other tissues. A moderate but significant correlation was observed for tumor blood flow with AUC (r = 0.76; P =.02) and standardized uptake value (SUV) at 55 minutes (r = 0.79; P =.01). A decrease in myocardial AUC ( P =.03) and splenic and myocardial SUV ( P =.01 and.004, respectively) was seen in phase I studies. Significantly higher AUC, SUV, and Cmax were observed in tumors in phase I studies. CONCLUSION: The distribution of [11C]DACA and its radiolabeled metabolites was observed in a variety of tumors and normal tissues. In the presence of unlabeled DACA, pharmacokinetics were altered in myocardium, spleen, and tumors. These data have implications for predicting activity and toxicity of DACA and support the use of PET early in drug development.
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Acridinas/farmacocinética , Antineoplásicos/farmacocinética , Tomografía Computarizada de Emisión , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Radioisótopos de Carbono , Femenino , Humanos , Cinética , Masculino , Persona de Mediana Edad , Neoplasias/tratamiento farmacológico , Distribución TisularRESUMEN
The 2.5-kilobase pair poly(purine.pyrimidine) (poly(R.Y)) tract present in intron 21 of the polycystic kidney disease 1 (PKD1) gene has been proposed to contribute to the high mutation frequency of the gene. To evaluate this hypothesis, we investigated the growth rates of 11 Escherichia coli strains, with mutations in the nucleotide excision repair, SOS, and topoisomerase I and/or gyrase genes, harboring plasmids containing the full-length tract, six 5'-truncations of the tract, and a control plasmid (pSPL3). The full-length poly(R.Y) tract induced dramatic losses of cell viability during the first few hours of growth and lengthened the doubling times of the populations in strains with an inducible SOS response. The extent of cell loss was correlated with the length of the poly(R.Y) tract and the levels of negative supercoiling as modulated by the genotype of the strains or drugs that specifically inhibited DNA gyrase or bound to DNA directly, thereby affecting conformations at specific loci. We conclude that the unusual DNA conformations formed by the PKD1 poly(R.Y) tract under the influence of negative supercoiling induced the SOS response pathway, and they were recognized as lesions by the nucleotide excision repair system and were cleaved, causing delays in cell division and loss of the plasmid. These data support a role for this sequence in the mutation of the PKD1 gene by stimulating repair and/or recombination functions.
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Reparación del ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Proteínas/genética , ADN Bacteriano/química , Humanos , Mutación , Conformación de Ácido Nucleico , Recombinación Genética , Canales Catiónicos TRPPRESUMEN
beta-Amyloid (A beta) has been strongly implicated in the pathophysiology of Alzheimer's disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c-Jun N-terminal kinases (JNKs) and of their substrate, c-Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated A beta. The activities of JNK family members increased in neuronal PC12 cells within 2 h of A beta treatment and reached 3--4-fold elevation by 6 h. To test the role of these changes in death caused by A beta, we examined the effects of CEP-1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP-1347 (100--300 nM) in the culture medium effectively blocked the increases in cellular JNK activity caused by A beta and, at similar concentrations, protected both PC12 cells and sympathetic neurons from A beta-evoked-death. Effective protection required addition of CEP-1347 within 2 h of A beta treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant-negative c-Jun construct also conferred protection from A beta-evoked death, supporting a model in which JNK activation contributes to death via activation of c-Jun. Finally, CEP-1347 blocked A beta-stimulated activation of caspase-2 and -3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by A beta and hence identify it as a potential therapeutic target in AD.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Apoptosis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/enzimología , Fragmentos de Péptidos/farmacología , Enfermedad de Alzheimer/enzimología , Péptidos beta-Amiloides/metabolismo , Animales , Carbazoles/farmacología , Caspasa 2 , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genes Dominantes , Indoles/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Células PC12/citología , Células PC12/efectos de los fármacos , Células PC12/enzimología , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Pruebas de Precipitina , Ratas , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/enzimología , TransfecciónAsunto(s)
Vacuna Antisarampión , Sarampión , Programas de Inmunización , Brotes de Enfermedades , Burkina FasoRESUMEN
Microtubule-associated protein 1B is a major constituent of the neuronal cytoskeleton during the early stages of development. This protein and its phosphorylated isoform, microtubule-associated protein 1B-P, defined by the monoclonal antibody 1B-P [Boyne L. J. et al. (1995) J. Neurosci. Res. 40, 439-450], are present in growing axons and concentrated in the distal end near the growth cone. In most regions of the central nervous system, microtubule-associated protein 1B and microtubule-associated protein 1B-P are developmentally down-regulated. They remain, however, at relatively high levels in the adult peripheral nervous system, where microtubule-associated protein 1B-P is localized exclusively in axons. The aim of this study was to examine the levels of microtubule-associated protein 1B and its phosphorylated isoform during regenerative growth of peripheral axons. Following transection and re-apposition of the sciatic nerve at midthigh, the levels of total microtubule-associated protein 1B, microtubule-associated protein 1B-P and microtubule-associated protein 1B messenger RNA were analysed in dorsal root ganglion neurons and sciatic nerve axons using western blots and RNase protection assays. After the lesion, there was a small decrease in the levels of microtubule-associated protein 1B and its messenger RNA in dorsal root ganglion neurons. The proximal axonal stump showed a similar decrease in the levels of microtubule-associated protein 1B 30days after lesion and returned to normal 60-90days post-lesion. In the distal stump of the sciatic nerve, the levels of microtubule-associated protein 1B increased dramatically and rapidly between three and 14days, but the protein was localized mainly in activated Schwann cells and myelin-like structures, and not in axons [Ma D. et al. (1999) Brain Res. 823, 141-153]. With the regeneration of axons into the distal stump, an intense expression of microtubule-associated protein 1B was observed in these axons. Microtubule-associated protein 1B-P, however, disappeared from the degenerated distal axonal stump as early as three days post-operation, and was absent in the regenerating axons and in Schwann cells between three and 14days. The levels of microtubule-associated protein 1B-P recovered slowly and did not reach the normal levels even after 90days post-operation. In contrast to the response following transection, the levels of microtubule-associated protein 1B and microtubule-associated protein 1B-P were much less affected after nerve crush. We propose that the relatively high levels of microtubule-associated protein 1B and its messenger RNA in adult dorsal root ganglions support peripheral neuron regeneration. The presence of microtubule-associated protein 1B in the regenerating axons suggests that microtubule-associated protein 1B is involved in axonal growth during peripheral nerve regeneration. However, the phosphorylated microtubule-associated protein 1B-P isoform, associated with growing axons during development, is not present in the regenerating axons after transection, presumably because of changes in the activities of kinases and phosphatases associated with the injury. These observations underscore the difference between axonal development and regeneration and the importance of injury-related effects that occur locally.
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Axones/metabolismo , Ganglios Espinales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Regeneración Nerviosa/fisiología , Nervio Ciático/fisiología , Animales , Axotomía , Femenino , Neuronas/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Nervio Ciático/lesionesRESUMEN
Ral GDP dissociation stimulator (RalGDS) and its family members RGL, RLF and RGL2 are involved in Ras and Ral signaling pathways as downstream effector proteins. Here we report the precise localization and cloning of two forms of human RGL gene differing at the amino terminus. Transcript A, cloned from liver cDNA libraries has the same amino terminus as the mouse RGL, whereas transcript B cloned from brain has a substitution of 45 amino acids for the first nine amino acids. At the genomic level, exon 1 of transcript A is replaced by two alternative exons (1B1 and 1B2) in transcript B. Both forms share exons 2 through 18. The human RGL protein shares 94% amino acid identity with the mouse protein. Northern blot analysis shows that human RGL is expressed in a wide variety of tissues with strong expression being seen in the heart, brain, kidney, spleen and testis.
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Factores de Intercambio de Guanina Nucleótido/genética , Secuencia de Aminoácidos , Northern Blotting , Encéfalo/metabolismo , Clonación Molecular , ADN Complementario/genética , Expresión Génica , Humanos , Hígado/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteínas ras/metabolismoRESUMEN
Several hereditary disease loci have been genetically mapped to the chromosome 1q24-q31 interval, including the hereditary prostate cancer 1 (HPC1) locus. Here, we report the construction of a 20-Mb yeast artificial chromosome contig and a high-resolution 6-Mb sequence-ready bacterial artificial chromosome (BAC)/P1-derived artificial chromosome (PAC) contig of 1q25 by sequence and computational analysis, STS content mapping, and chromosome walking. One hundred thirty-six new STSs, including 10 novel simple sequence repeat polymorphisms that are being used for genetic refinement of multiple disease loci, have been generated from this contig and are shown to map to the 1q25 interval. The integrity of the 6-Mb BAC/PAC contig has been confirmed by restriction fingerprinting, and this contig is being used as a template for human chromosome 1 genome sequencing. A transcription mapping effort has resulted in the precise localization of 18 known genes and 31 ESTs by database searching, exon trapping, direct cDNA hybridization, and sample sequencing of BACs from the 1q25 contig. An additional 11 known genes and ESTs have been placed within the larger 1q24-q31 interval. These transcription units represent candidate genes for multiple hereditary diseases, including HPC1.
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Cromosomas Humanos Par 1 , Mapeo Físico de Cromosoma , Neoplasias de la Próstata/genética , Secuencia de Bases , Cromosomas Artificiales de Levadura , Mapeo Contig , Dermatoglifia del ADN/métodos , ADN Complementario , Predisposición Genética a la Enfermedad , Humanos , Masculino , Datos de Secuencia Molecular , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
Spinal cord injury in adult mammals causes atrophy or loss of axotomized neurons. We have previously found that the product of the antiapoptotic gene Bcl-2, delivered by intraspinal injection of a DNA plasmid, reduces atrophy and loss of axotomized Clarke's nucleus neurons in adult rats. Here we studied whether the same treatment protects axotomized red nucleus (RN) neurons. Two months after the right dorsolateral funiculus was ablated in adult Sprague-Dawley rats by C3/C4 subtotal hemisection, there was approximately 48% loss of RN neurons in the magnocellular portion of the RN contralateral to the lesion and atrophy of many surviving neurons. When a DNA plasmid encoding the human Bcl-2 gene and the bacterial reporter gene LacZ, complexed with cationic lipids, was injected just rostral to the subtotal hemisection site, 87% of RN neurons survived, and there was partial, but robust, protection from atrophy. These and our previous results indicated that intraspinal administration of the Bcl-2 gene can prevent retrograde cell loss and reduce atrophy of axotomized RN and Clarke's nucleus neurons in adult rats and provide an effective means to rescue neurons whose survival depends on different growth factors.
Asunto(s)
ADN/genética , Genes bcl-2/genética , Terapia Genética , Neuronas/patología , Plásmidos/genética , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/terapia , Animales , Atrofia , Axotomía , Recuento de Células , Tamaño de la Célula , ADN/administración & dosificación , Femenino , Histocitoquímica , Operón Lac , Plásmidos/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
The Bcl-2 proto-oncogene regulates apoptosis and prevents cell death. We studied the effect of Bcl-2 gene over-expression on the survival of axotomized red nucleus (RN) neurons after unilateral hemisection at cervical segment 4/5 (C4/5) in mice. Seventy-five percent of RN neurons survived in Bcl-2 over-expressing mice 1 or 2 months after surgery compared with only 55% of RN neurons in wild-type mice. However, Bcl-2 gene over-expression does not prevent lesion-induced shrinkage of RN neurons.
Asunto(s)
Axotomía , Neuronas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Núcleo Rojo/patología , Animales , Recuento de Células , Muerte Celular/fisiología , Tamaño de la Célula/fisiología , Genotipo , Histocitoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/fisiología , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Núcleo Rojo/citología , Médula Espinal/citología , Médula Espinal/fisiologíaRESUMEN
Preclinical toxicology studies are performed prior to phase I trials with novel cancer therapeutics to identify a safe clinical starting dose and potential human toxicities. The primary aim of this study was to evaluate the ability of rodent-only toxicology studies to identify a safe phase I trial starting dose. In addition, the ability of murine studies to predict the quantitative and qualitative human toxicology of cancer therapeutics was studied. Data for 25 cancer drugs were collated for which the preclinical and clinical routes and schedules of administration were either the same (22/25), or closely matched. The maximum tolerated dose/dose lethal to 10% of mice (MTD/LD10) was identified for 24 drugs, and in patients the maximum administered dose (MAD) was associated with dose-limiting toxicity (DLT) in initial clinical trials with 20 compounds. In addition, for 13 agents, the toxicity of the drug at one-tenth the mouse MTD/LD10 was also investigated in rats, following repeated administration (20 doses). A phase I trial starting dose of one-tenth the mouse MTD/LD10 (mg m(-2)) was, or would have been, safe for all 25 compounds. With the exception of nausea and vomiting, which cannot be assessed in rodents, other common DLTs were accurately predicted by the murine studies (i.e. 7/7 haematological and 3/3 neurological DLTs). For two of the 13 drugs studied in rats, repeated administration of one-tenth the mouse MTD/LD10 was toxic, leading to a reduction in the phase I trial starting dose; however, one-tenth the mouse MTD/LD10 was subsequently tolerated in patients. For the 20 drugs where clinical DLT was reached, the median ratio of the human MAD to the mouse MTD/LD10 was 2.6 (range 0.2-16) and the median ratio of the clinical starting dose to the MAD was 35 (range 2.3-160). In contrast, in 13 subsequent phase I trials with 11 of the initial 25 drugs, the median ratio of the clinical starting dose to the MAD was 2.8 (range 1.6-56), emphasizing the value of early clinical data in rapidly defining the dose range for therapeutic studies. For all 25 drugs studied, rodent-only toxicology provided a safe and rapid means of identifying the phase I trial starting dose and predicting commonly encountered DLTs. This study has shown that the routine use of a non-rodent species in preclinical toxicology studies prior to initial clinical trials with cancer therapeutics is not necessary.
Asunto(s)
Antineoplásicos/toxicidad , Ensayos de Selección de Medicamentos Antitumorales/métodos , Pruebas de Toxicidad/métodos , Animales , Antineoplásicos/efectos adversos , Ensayos Clínicos Fase II como Asunto/métodos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Dosis Máxima Tolerada , Ratones , Valor Predictivo de las Pruebas , Ratas , Estudios RetrospectivosRESUMEN
Microtubule-associated protein 1B (MAP1B) is expressed at high levels during development of the nervous system and is localized primarily in neurons while specific phosphorylated isoforms of MAP1B are localized exclusively in growing axons. The levels of MAP1B are down regulated in most regions of the adult CNS, but remain high in neurons and axons of the PNS. This study demonstrates that the expression of MAP1B is induced in adult Schwann cells following sciatic nerve lesion and regeneration. High levels of both mRNA and the MAP1B protein were detected in Schwann cells associated with the axotomized distal stump. Expression of MAP1B was also observed in cultured primary Schwann cells from neonatal rats. The properties of the MAP1B protein in cultured Schwann cells were further characterized by Western blot analysis using specific antibodies that recognize the N-terminal, middle and C-terminal domains of MAP1B. All of these antibodies detected a protein of 320-340 kDa demonstrating that MAP1B expressed by Schwann cells is very similar, or identical, to MAP1B expressed by neurons. The phosphorylation of MAP1B in Schwann cells was also studied using monoclonal antibodies (mAb) that recognize specific phosphorylation epitopes. The results indicated that the expression of MAP1B in Schwann cells exhibited a differential phosphorylation state that was recognized by mAb 1B6 but not by other mAbs, including 1B-P, 150 and RT97, that recognize phosphorylated MAP1B in growing axons. We therefore conclude that MAP1B is expressed in Schwann cells during both development and axonal regeneration, suggesting that the developmental pattern of MAP1B in these cells is recapitulated in adult Schwann cells during the early stages of regeneration and remyelination of injured peripheral axons. The presence of MAP1B in Schwann cells may support morphological changes of these cells, particularly the formation of processes prior to their differentiation into myelin forming Schwann cells.