RESUMO
The human concentrative nucleoside transporter, CNT3 (SLC28A3), plays an important role in mediating the cellular entry of a broad array of physiological nucleosides and synthetic anticancer nucleoside analog drugs. As a first step toward understanding the genetic basis for interindividual differences in the disposition and response to antileukemic nucleoside analogs, we examined the genetic and functional diversity of CNT3. In all, 56 variable sites in the exons and flanking intronic region of SLC28A3 were identified in a collection of 270 DNA samples from US populations (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). Of the 16 coding region variants, 12 had not been previously reported. Also, 10 resulted in amino-acid changes and three of these had total allele frequencies of >/=1%. Nucleotide diversity (pi) at nonsynonymous and synonymous sites was estimated to be 1.81 x 10(4) and 18.13 x 10(4), respectively, suggesting that SLC28A3 is under negative selection. All nonsynonymous variants, constructed by site-directed mutagenesis and expressed in Xenopus laevis oocytes, transported purine and pyrimidine model substrates, except for c. 1099G>A (p. Gly367Arg). This rare variant alters an evolutionarily conserved site in the putative substrate recognition domain of CNT3. The presence of three additional evolutionarily conserved glycine residues in the vicinity of p. Gly367Arg that are also conserved in human paralogs suggest that these glycine residues are critical in the function of the concentrative nucleoside transporter family. The genetic analysis and functional characterization of CNT3 variants suggest that this transporter does not tolerate nonsynonymous changes and is important for human fitness.
Assuntos
Proteínas de Membrana Transportadoras/genética , Vidarabina/análogos & derivados , Adenosina/metabolismo , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Proteínas de Transporte de Cátions , Cladribina/metabolismo , Sequência Conservada , DNA/genética , Etnicidade , Variação Genética , Haplótipos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Vidarabina/metabolismo , Xenopus laevisRESUMO
Two mouse models are widely used for Down syndrome (DS) research. The Ts65Dn mouse carries a small chromosome derived primarily from mouse chromosome 16, causing dosage imbalance for approximately half of human chromosome 21 orthologs. These mice have cerebellar pathology with direct parallels to DS. The Ts1Cje mouse, containing a translocated chromosome 16, is at dosage imbalance for 67% of the genes triplicated in Ts65Dn. We quantified cerebellar volume and granule cell and Purkinje cell density in Ts1Cje. Cerebellar volume was significantly affected to the same degree in Ts1Cje and Ts65Dn, despite that Ts1Cje has fewer triplicated genes. However, dosage imbalance in Ts1Cje had little effect on granule cell and Purkinje cell density. Several mice with dosage imbalance for the segment of the Ts65Dn chromosome not triplicated in Ts1Cje had phenotypes that contrasted with those in Ts1Cje. These observations do not readily differentiate between two prevalent hypotheses for gene action in DS.
Assuntos
Cerebelo/patologia , Modelos Animais de Doenças , Síndrome de Down/genética , Síndrome de Down/patologia , Fenótipo , Animais , Cerebelo/diagnóstico por imagem , Cruzamentos Genéticos , Feminino , Marcadores Genéticos , Granulócitos/patologia , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Biossíntese de Proteínas , Células de Purkinje/patologia , Deleção de Sequência , Trissomia , UltrassonografiaRESUMO
The phenotype of NK1R-/- mice was compared with that of acute pharmacological blockade of the tachykinin NK1 receptor on sensorimotor function and in assays relevant to depressive illness and anxiety. The dose range for L-760735 and GR205171 that was associated with functional blockade of central NK1 receptors in the target species was established by antagonism of the behavioural effects of intracerebroventricular NK1 agonist challenge in gerbils, mice and rats. The caudal grooming and scratching response to GR73632 was absent in NK1R-/- mice, confirming that the receptor had been genetically ablated. There was no evidence of sedation or motor impairment in NK1R-/- mice or following administration of L-760735 to gerbils, even at doses in excess of those required for central NK1 receptor occupancy. In the resident-intruder and forced swim test, the behaviour of NK1R-/- mice, or animals treated acutely with L-760735 or GR205171, resembled that seen with the clinically used antidepressant drug fluoxetine. However, the effects of GR205171 were not clearly enantioselective in mice. In contrast, although NK1R-/- mice also exhibited an increase in the duration of struggle behaviour in the tail suspension test, this was not observed following pharmacological blockade with L-760735 in gerbils or GR205171 in mice, suggesting that this may reflect a developmental alteration in the knockout mouse. There was no effect of NK1 receptor blockade with L-760735 in guinea-pigs or GR205171 in rats, or deletion of the NK1 receptor in mice, on behaviour in the elevated plus-maze test for anxiolytic activity. These findings extend previous observations on the phenotype of the NK1R-/- mouse and establish a broadly similar profile following acute pharmacological blockade of the receptor. These studies also serve to underscore the limitations of currently available antagonists that are suitable for use in rat and mouse behavioural assays.
Assuntos
Ansiolíticos/farmacologia , Antidepressivos/farmacologia , Ansiedade/genética , Nível de Alerta/genética , Depressão/genética , Mutação/genética , Fenótipo , Receptores da Neurocinina-1/genética , Animais , Nível de Alerta/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Gerbillinae , Cobaias , Desamparo Aprendido , Masculino , Camundongos , Morfolinas/farmacologia , Motivação , Antagonistas dos Receptores de Neurocinina-1 , Piperidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Tetrazóis/farmacologiaRESUMO
1-(5-[[(2R,3S)-2-([(1R)-1-[3,5-Bis(trifluoromethyl)phenyl]ethyl]oxy)-3-(4-fluorophenyl)morpholin-4-yl]methyl]-2H-1,2,3-triazol-4-yl)-N,N-dimethylmethanamine hydrochloride 3 is a high affinity, orally active, h-NK(1) receptor antagonist with a long central duration of action and a solubility in water of >100 mg/mL. The construction of the 5-dimethylaminomethyl 1,2,3-triazol-4-yl unit, which incorporates the solubilizing group of 3, was accomplished by thermal rearrangement of a propargylic azide in the presence of dimethylamine. Compound 3 is highly effective in pre-clinical tests that are relevant to clinical efficacy in emesis and depression.
Assuntos
Antidepressivos/síntese química , Antieméticos/síntese química , Morfolinas/síntese química , Antagonistas dos Receptores de Neurocinina-1 , Triazóis/síntese química , Administração Oral , Aminas/síntese química , Aminas/química , Aminas/farmacologia , Animais , Animais Recém-Nascidos , Antidepressivos/química , Antidepressivos/farmacologia , Antieméticos/química , Antieméticos/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cães , Furões , Gerbillinae , Cobaias , Técnicas In Vitro , Injeções Intravenosas , Macaca mulatta , Morfolinas/química , Morfolinas/farmacologia , Ensaio Radioligante , Ratos , Solubilidade , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologia , Vocalização Animal/efeitos dos fármacosRESUMO
Mn superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, has been shown to be essential for animal survival. MnSOD mutant mice (Sod2-/- mice) on the CD1 background develop severe dilated cardiomyopathy and usually die within 10 d after birth. To characterize better the phenotype and understand the mechanism of superoxide-mediated tissue damage in Sod2-/- mice, congenic Sod2-/- mice on inbred backgrounds were generated to ensure genetic homogeneity. When generated on a C57BL/6J background (B6
Assuntos
Acidose/metabolismo , Cardiomiopatia Dilatada/enzimologia , Morte Fetal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Acidose/genética , Aconitato Hidratase/genética , Aconitato Hidratase/metabolismo , Animais , Cardiomiopatia Dilatada/genética , Catalase/genética , Catalase/metabolismo , Morte Fetal/genética , Genótipo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Rim/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Mitocôndrias/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Fenótipo , Superóxido Dismutase/deficiência , Regulação para CimaRESUMO
Misfolded proteins in the endoplasmic reticulum (ER) are degraded by N-terminal threonine proteases within the 26S proteasome. Each protease is formed by an activated beta subunit, beta5/X, beta1/Y, or beta2/Z, that exhibits chymotrypsin-like, peptidylglutamyl-peptide hydrolyzing, or trypsin-like activity, respectively. Little is known about the relative contribution of specific beta subunits in the degradation of endogenous protein substrates. Using active site proteasome inhibitors and a reconstituted degradation system, we now show that all three active beta subunits can independently contribute to ER-associated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR). Complete inactivation (>99.5%) of the beta5/X subunit decreased the rate of ATP-dependent conversion of CFTR to trichloroacetic acid soluble fragments by only 40%. Similarly, proteasomes containing only active beta1/Y or beta2/Z subunits degraded CFTR at approximately 50% of the rate observed for fully functional proteasomes. Simultaneous inhibition (>93%) of all three beta subunits blocked CFTR degradation by approximately 90%, and inhibition of both protease and ATPase activities was required to completely prevent generation of small peptide fragments. Our results demonstrate both a conserved hierarchy (ChT-L > PGPH > or = T-L) as well as a redundancy of beta subunit function and provide insight into the mechanism by which active site proteasome inhibitors influence degradation of endogenous protein substrates at the ER membrane.
Assuntos
Cisteína Endopeptidases/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Complexos Multienzimáticos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Sistema Livre de Células , Retículo Endoplasmático/efeitos dos fármacos , Glicerol/farmacologia , Glicosilação , Hemina/farmacologia , Humanos , Técnicas In Vitro , Membranas Intracelulares/efeitos dos fármacos , Lactonas/farmacologia , Leupeptinas/farmacologia , Bicamadas Lipídicas/metabolismo , Complexo de Endopeptidases do Proteassoma , Subunidades Proteicas , Coelhos , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Tripsina/farmacologia , Ubiquitinas/metabolismoRESUMO
Manganese superoxide dismutase (MnSOD) is essential in protecting mitochondria against the damaging effects of superoxide radicals (O(2)(*-)), and increased expression of MnSOD protects cells and transgenic animals from various forms of oxidative stress. In addition, increased levels of MnSOD have been shown to slow down cell growth and induce differentiation. To study the effects of high MnSOD levels in vivo, we generated a series of transgenic mice using a mouse genomic sequence under control of the endogenous promoter. Four transgenic lines produced by pronuclear DNA injection exhibited up to 2-fold elevated MnSOD levels in brain and heart. However, using an embryonic stem cell approach, a line having 10-fold elevated MnSOD levels in the brain and 6- to 7-fold elevated levels in the heart and kidney was generated. Surprisingly, the genetic background of this transgenic line influenced the expression level of the transgene, with DBA/2 (D2) and C57BL/6 (B6) mice exhibiting low- and high-level transgene expression, respectively. This difference was the result of an increased transcription rate of the transgene. High-level MnSOD expression in B6 animals was associated with small size, male infertility, and decreased female fertility. These features are absent on the D2 background and indicate that high levels of MnSOD activity may interfere with normal growth and fertility.
Assuntos
Retardo do Crescimento Fetal/genética , Infertilidade/genética , Superóxido Dismutase/genética , Transcrição Gênica/genética , Transgenes/genética , Regulação para Cima/genética , Animais , Células da Medula Óssea/metabolismo , Encéfalo/metabolismo , Catalase/metabolismo , Feminino , Fibroblastos/metabolismo , Glutationa Redutase/metabolismo , Infertilidade/patologia , Células Intersticiais do Testículo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Miocárdio/metabolismo , Especificidade da Espécie , Superóxido Dismutase/metabolismo , Transferência Intratubária do Zigoto/métodosRESUMO
Mice inheriting both copies of MMU12 either maternally or paternally demonstrate imprinting effects. Whereas maternal uniparental disomy 12 (matUPD12) fetuses are growth retarded and die perinatally, paternal UPD12 (patUPD12) fetuses die during late gestation and exhibit placentomegaly and skeletal muscle maturation defects. To examine further the developmental consequences of UPD12, we intercrossed mouse stocks heterozygous for Robertsonian translocation chromosomes (8.12) and (10.12). We report that at 13.5-14.5 dg patUPD12 hearts exhibit increased ventricular diameter, thinner, less compact myocardium, and deep intertrabecular recesses when compared to controls. These data provide evidence for cardiac failure, a lethal condition, and suggest a role for an imprinted gene(s) in normal heart development.
Assuntos
Cardiomiopatias/embriologia , Cardiomiopatias/genética , Aberrações Cromossômicas/genética , Impressão Genômica/genética , Miocárdio/patologia , Animais , Aberrações Cromossômicas/embriologia , Cruzamentos Genéticos , Feminino , Morte Fetal/genética , Coração/embriologia , Heterozigoto , Cariotipagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Translocação Genética/genéticaRESUMO
Heart mitochondria from heterozygous (Sod2(-/+)) knockout mice have a 50% reduction in manganese superoxide dismutase (MnSOD) activity. The decrease in MnSOD activity was associated with increased mitochondrial oxidative damage as demonstrated by a decrease in the activities of iron sulfhydryl proteins sensitive to oxygen stress (aconitase and reduced nicotinamide adenine dinucleotide-oxidoreductase). Mitochondrial function was altered in the Sod2(-/+) mice, as shown by decreased respiration by complex I and an increase in the sensitivity of the permeability transition to induction by calcium and t-butylhydroperoxide. The increased induction of the permeability transition in heart mitochondria from Sod2(-/+.)mice was associated with increased release of cytochrome c and an increase in DNA fragmentation. Cardiomyocytes isolated from neonatal Sod2(-/+) and Sod2(-/-) mice were more sensitive to cell death than cardiomyocytes from Sod2(+/+) mice after t-butylhydroperoxide treatment, and this increased sensitivity was prevented by inhibiting the permeability transition with cyclosporin A. These experiments demonstrate that MnSOD may play an important role in the induction of the mitochondrial pathway of apoptosis in the heart, and this appears to occur primarily through the permeability transition.
Assuntos
Apoptose , Heterozigoto , Mitocôndrias Cardíacas/enzimologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Aconitato Hidratase/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citosol/enzimologia , Ativação Enzimática/fisiologia , Glutamato-Amônia Ligase/metabolismo , Glutationa/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/efeitos dos fármacos , Miocárdio/citologia , Miocárdio/enzimologia , Oxidantes/farmacologia , Estresse Oxidativo/fisiologia , Consumo de Oxigênio/fisiologia , Permeabilidade/efeitos dos fármacos , Superóxido Dismutase/deficiência , terc-Butil Hidroperóxido/farmacologiaRESUMO
Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive decline in Alzheimer's disease and Down's syndrome. With aging, the partial trisomy 16 (Ts65Dn) mouse model of Down's syndrome exhibited reductions in BFCN size and number and regressive changes in the hippocampal terminal fields of these neurons with respect to diploid controls. The changes were associated with significantly impaired retrograde transport of nerve growth factor (NGF) from the hippocampus to the basal forebrain. Intracerebroventricular NGF infusion reversed well established abnormalities in BFCN size and number and restored the deficit in cholinergic innervation. The findings are evidence that even BFCNs chronically deprived of endogenous NGF respond to an intervention that compensates for defective retrograde transport. We suggest that age-related cholinergic neurodegeneration may be a treatable disorder of failed retrograde NGF signaling.
Assuntos
Síndrome de Down/metabolismo , Fator de Crescimento Neural/metabolismo , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Transporte Biológico Ativo , Contagem de Células , Células , Fibras Colinérgicas/efeitos dos fármacos , Fibras Colinérgicas/metabolismo , Fibras Colinérgicas/patologia , Modelos Animais de Doenças , Síndrome de Down/tratamento farmacológico , Síndrome de Down/patologia , Hipocampo/metabolismo , Humanos , Infusões Parenterais , Camundongos , Camundongos Mutantes , Degeneração Neural/tratamento farmacológico , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Fator de Crescimento Neural/administração & dosagem , Fenótipo , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/metabolismo , Prosencéfalo/patologia , TrissomiaRESUMO
Mucinous colorectal cancers exhibit a characteristic set of molecular genetic alterations and may be derived from progenitor cells committed to the goblet cell lineage. Previously, we demonstrated that the MUC2 mucin gene promoter drives transgene reporter expression with high specificity in small intestinal goblet cells of transgenic mice. On the basis of these experiments, we reasoned that the MUC2 promoter could be used to drive SV40 T antigen (Tag) expression in the same cell type, decoupling them from their normal antiproliferative controls. A line of mice was established (MUCTag6) that expressed Tag in intestinal goblet cells as determined by RNA blot and immunohistochemical analysis. These goblet cells were markedly involuted however, most notably in the villi. Endogenous intestinal MUC2 message levels were reduced to about one third the normal level in these mice. However, absorptive cell lineage markers were comparable with nontransgenics. Bromodeoxyuridine-positive S-phase cells are limited to crypts in nontransgenic intestine but are present in both crypts and villi in MUCTag6. In contrast, mitotic cells were not present in the villi, indicating that MUCTag6 villi goblet cells do not progress into M phase. Apoptotic cells positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling were increased more than fourfold in MUCTag6 villi (P < 0.0001), and apoptotic goblet cells were evident. Electron microscopic examination of MUCTag6 intestinal villi revealed the presence of degraded cell remnants containing mucin goblets together with other cell debris, further indicating apoptosis of the goblet cell lineage. Thus, the expression of Tag in intestinal goblet cells releases them from normal antiproliferative controls, causing their inappropriate entry into S phase even after they transverse the crypt/villus junction. They do not, however, progress to M phase. Instead, they undergo apoptosis with a high degree of efficiency in S or G(2) phase. These experiments demonstrate that apoptosis effectively blocks inappropriate goblet cell proliferation in the intestine, supporting its proposed role as an antineoplastic mechanism.
Assuntos
Antígenos Transformantes de Poliomavirus/biossíntese , Apoptose/fisiologia , Movimento Celular/fisiologia , Células Caliciformes/citologia , Intestino Delgado/citologia , Mucinas/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Feminino , Células Caliciformes/imunologia , Células Caliciformes/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microvilosidades/fisiologia , Mucina-2 , Oncogenes , Regiões Promotoras Genéticas , Fase S/fisiologiaRESUMO
There is substantial evidence implicating mitochondrial dysfunction and free radical generation as major mechanisms of neuronal death in neurodegenerative diseases. The major free radical scavenging enzyme in mitochondria is manganese superoxide dismutase (SOD2). In the present study we investigated the susceptibility of mice with a partial deficiency of SOD2 to the neurotoxins 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), 3-nitropropionic acid (3-NP), and malonate, which are commonly used animal models of Parkinson's and Huntington's disease. Heterozygous SOD2 knockout (SOD2(+/-)) mice showed no evidence of neuropathological or behavioral abnormalities at 2-4 months of age. Compared to littermate wild-type mice, mice with partial SOD2 deficiency showed increased vulnerability to dopamine depletion after systemic MPTP treatment and significantly larger striatal lesions produced by both 3-NP and malonate. SOD2(+/-) mice also showed an increased production of "hydroxyl" radicals after malonate injection measured with the salicylate hydroxyl radical trapping method. These results provide further evidence that reactive oxygen species play an important role in the neurotoxicity of MPTP, malonate, and 3-NP. These findings show that a subclinical deficiency in a free radical scavenging enzyme may act in concert with environmental toxins to produce selective neurodegeneration.
Assuntos
Predisposição Genética para Doença , Gentisatos , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso , Doenças Neurodegenerativas/metabolismo , Neurotoxinas/metabolismo , Superóxido Dismutase/deficiência , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Proteínas de Transporte/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Feminino , Predisposição Genética para Doença/genética , Heterozigoto , Ácido Homovanílico/metabolismo , Hidroxibenzoatos/metabolismo , Radical Hidroxila/metabolismo , Masculino , Malonatos , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Neurotoxinas/toxicidade , Nitrocompostos , Propionatos , Ácido Salicílico/metabolismo , Superóxido Dismutase/genéticaRESUMO
Hereditary non-X-linked nephrogenic diabetes insipidus (NDI) is caused by mutations in the aquaporin-2 (AQP2) water channel. In transfected cells, the human disease-causing mutant AQP2-T126M is retained at the endoplasmic reticulum (ER) where it is functional and targetable to the plasma membrane with chemical chaperones. A mouse knock-in model of NDI was generated by targeted gene replacement using a Cre-loxP strategy. Along with T126M, mutations H122S, N124S, and A125T were introduced to preserve the consensus sequence for N-linked glycosylation found in human AQP2. Breeding of heterozygous mice yielded the expected Mendelian distribution with 26 homozygous mutant offspring of 99 live births. The mutant mice appeared normal at 2-3 days after birth but failed to thrive and generally died by day 6 if not given supplemental fluid. Urine/serum analysis showed a urinary concentrating defect with serum hyperosmolality and low urine osmolality that was not increased by a V2 vasopressin agonist. Northern blot analysis showed up-regulated AQP2-T126M transcripts of identical size to wild-type AQP2. Immunoblots showed complex glycosylation of wild-type AQP2 but mainly endoglycosidase H-sensitive core glycosylation of AQP2-T126M indicating ER-retention. Biochemical analysis revealed that the AQP2-T126M protein was resistant to detergent solubilization. Kidneys from mutant mice showed collecting duct dilatation, papillary atrophy, and unexpectedly, some plasma membrane AQP2 staining. The severe phenotype of the AQP2 mutant mice compared with that of mice lacking kidney water channels AQP1, AQP3, and AQP4 indicates a critical role for AQP2 in neonatal renal function in mice. Our results establish a mouse model of human autosomal NDI and provide the first in vivo biochemical data on a disease-causing AQP2 mutant.
Assuntos
Aquaporinas/genética , Diabetes Insípido Nefrogênico/genética , Modelos Animais de Doenças , Genes Recessivos , Camundongos Transgênicos , Animais , Animais Recém-Nascidos , Aquaporina 2 , Aquaporina 6 , Aquaporinas/biossíntese , Diabetes Insípido Nefrogênico/mortalidade , Células Epiteliais/patologia , Túbulos Renais Coletores/patologia , CamundongosRESUMO
Expression of wild type HFE reduces the ferritin levels of cells in culture. In this report we demonstrate that the predominant hereditary hemochromatosis mutation, C282Y(2) HFE, does not reduce ferritin expression. However, the second mutation, H63D HFE, reduces ferritin expression to a level indistinguishable from cells expressing wild type HFE. Further, two HFE cytoplasmic domain mutations engineered to disrupt potential signal transduction, S335M and Y342C, were functionally indistinguishable from wild type HFE in this assay, as was soluble HFE. These results implicate a role for the interaction of HFE with the transferrin receptor in lowering cellular ferritin levels.
Assuntos
Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Ferro/metabolismo , Proteínas de Membrana , Receptores da Transferrina/metabolismo , Linhagem Celular , Antígenos HLA/química , Células HeLa , Hemocromatose/genética , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/química , Homeostase , Humanos , Receptores da Transferrina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Two animal models of Down syndrome (human trisomy 21) with segmental trisomy for all (Ts65Dn) or part (Ts1Cje) of human chromosome 21-homologous region of mouse chromosome 16 have cognitive and behavioral abnormalities. To compare these trisomies directly and to assess the phenotypic contribution of the region of difference between them, Ts65Dn, Ts1Cje, and a new segmental trisomic (Ms1Ts65) for the region of difference (APP: to Sod1) have been generated as littermates and tested in parallel. Although the performance of Ts1Cje mice in the Morris water maze is similar to that of Ts65Dn mice, the reverse probe tests indicate that Ts65Dn is more severely affected. By contrast, the deficits of Ms1Ts65 mice are significantly less severe than those of Ts65Dn. Therefore, whereas triplication of Sod1 to Mx1 plays the major role in causing the abnormalities of Ts65Dn in the Morris water maze, imbalance of APP: to Sod1 also contributes to the poor performance. Ts65Dn mice are hyperactive and Ts1Cje mice are hypoactive; the activity of Ms1Ts65 mice is not significantly above normal. These findings indicate that genes in the Ms1Ts65 trisomic region must interact with others in the Ts1Cje region to produce hyperactivity in Ts65Dn mice.
Assuntos
Comportamento Animal , Síndrome de Down/genética , Síndrome de Down/psicologia , Animais , Transtornos Cognitivos/genética , Modelos Animais de Doenças , Feminino , Humanos , Cariotipagem , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Mutantes , Atividade Motora/genética , Fenótipo , Mapeamento Físico do Cromossomo , TrissomiaRESUMO
OBJECTIVE: The lymphocyte is a principal mediator of the inflammatory response, and lymphocyte depletion via apoptosis may be an important mechanism of modulating inflammation. Increased oxygen consumption occurs during sepsis and results in the generation of reactive oxygen species. Although reactive oxygen species initiate apoptosis in many biological systems, their role in controlling lymphocyte apoptosis during sepsis is unclear. The objective of this study was to better characterize the role of oxidative stress in precipitating lymphocyte apoptosis during sepsis and to specifically define the role of the CuZn superoxide dismutase (SOD) enzyme complex, a major antioxidant defense, in modulating this process. DESIGN: Prospective, randomized, controlled study. SETTING: Research laboratory at an academic medical center. SUBJECTS: Mice that were either genetically normal or that were deficient in or overexpressed the enzyme CuZn SOD. INTERVENTIONS: Mice from each genetic group were randomized to no manipulation (control), sham surgery, or cecal ligation and puncture. Mice were killed 18-24 hrs after study entry, and the thymi and spleen were removed for analysis of apoptosis. MEASUREMENTS AND MAIN RESULTS: Lymphocyte apoptosis was assessed by three independent methods: light microscopy, fluorescent terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, and DNA gel electrophoresis. Comparisons were performed using standard parametric statistical tests. Lymphocyte apoptosis was present in mice after CLP but not in control mice or in mice after sham surgery (p < .05). Mice completely lacking CuZn SOD developed significantly more lymphocyte apoptosis than did either partially CuZn SOD-deficient or genetically normal mice (p < .05). This apoptosis was more pronounced in the thymus than the spleen and, within the thymus, more prominent in the cortex than medulla (p < .05 for all). In contrast, mice that overexpressed CuZn SOD did not differ in the amount of apoptosis after CLP compared with genetically normal mice (p = NS for all). CONCLUSIONS: Oxidative stress occurs in sepsis and appears to be one stimulus for the development of lymphocyte apoptosis, a process that is partly regulated by CuZn SOD. However, we were unable to demonstrate that overexpression of this enzyme suppressed lymphocyte apoptosis, suggesting that either other antioxidant defenses or other pathways independent of oxidative stress may mediate lymphocyte elimination in this syndrome.
Assuntos
Apoptose/fisiologia , Proteínas de Caenorhabditis elegans , Isoenzimas/fisiologia , Linfócitos/enzimologia , Sepse/enzimologia , Superóxido Dismutase/fisiologia , Animais , DNA/análise , Marcação In Situ das Extremidades Cortadas , Isoenzimas/genética , Camundongos , Distribuição Aleatória , Superóxido Dismutase/genéticaRESUMO
Trisomy 21 (Down syndrome) is the most common chromosomal abnormality associated with mental retardation in humans. Sim2, a human homologue of Drosophila sim gene, which acts as a master regulator of the early development of the fly central nervous system midline, is located on chromosome 21, in the Down syndrome critical region, and might therefore be involved in the pathogenesis of some of the morphological features and brain anomalies observed in Down syndrome. We report here the detailed expression pattern of murine mSim2 gene in Ts1Cje mice fetuses, a segmental trisomy 16 mouse model for trisomy 21, and its overexpression in the zona limitans of the diencephalon using a new quantitative method based on the whole-mount RNA hybridization technique.
Assuntos
Proteínas de Ligação a DNA/genética , Diencéfalo/fisiologia , Síndrome de Down/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Nucleares/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diencéfalo/embriologia , Modelos Animais de Doenças , Proteínas de Drosophila , Desenvolvimento Embrionário e Fetal/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hibridização de Ácido Nucleico , Regiões Promotoras Genéticas/fisiologiaRESUMO
Aquaporin-3 (AQP3) is a water channel expressed at the basolateral plasma membrane of kidney collecting-duct epithelial cells. The mouse AQP3 cDNA was isolated and encodes a 292-amino acid water/glycerol-transporting glycoprotein expressed in kidney, large airways, eye, urinary bladder, skin, and gastrointestinal tract. The mouse AQP3 gene was analyzed, and AQP3 null mice were generated by targeted gene disruption. The growth and phenotype of AQP3 null mice were grossly normal except for polyuria. AQP3 deletion had little effect on AQP1 or AQP4 protein expression but decreased AQP2 protein expression particularly in renal cortex. Fluid consumption in AQP3 null mice was more than 10-fold greater than that in wild-type litter mates, and urine osmolality (<275 milliosmol) was much lower than in wild-type mice (>1,200 milliosmol). After 1-desamino-8-d-arginine-vasopressin administration or water deprivation, the AQP3 null mice were able to concentrate their urine partially to approximately 30% of that in wild-type mice. Osmotic water permeability of cortical collecting-duct basolateral membrane, measured by a spatial filtering optics method, was >3-fold reduced by AQP3 deletion. To test the hypothesis that the residual concentrating ability of AQP3 null mice was due to the inner medullary collecting-duct water channel AQP4, AQP3/AQP4 double-knockout mice were generated. The double-knockout mice had greater impairment of urinary-concentrating ability than did the AQP3 single-knockout mice. Our findings establish a form of nephrogenic diabetes insipidus produced by impaired water permeability in collecting-duct basolateral membrane. Basolateral membrane aquaporins may thus provide blood-accessible targets for drug discovery of aquaretic inhibitors.
Assuntos
Aquaporinas/genética , Diabetes Insípido Nefrogênico/genética , Sequência de Aminoácidos , Animais , Aquaporina 3 , Aquaporinas/química , DNA Complementar , Desamino Arginina Vasopressina/administração & dosagem , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Oxidative damage has been implicated in the aging process and in a number of degenerative diseases. To investigate the role of oxygen radicals in the aging process in mammals, the life spans of transgenic mice on a CD-1 background expressing increased levels of CuZn superoxide dismutase (CuZnSOD), the enzyme that metabolizes superoxide radicals, were determined. Homozygous transgenic mice with a two- to five-fold elevation of CuZnSOD in various tissues showed a slight reduction of life span, whereas hemizygous mice with a 15- to 3-fold increase of CuZnSOD showed no difference in life span from that of the nontransgenic littermate controls. The results suggest that constitutive and ubiquitous overexpression of CuZnSOD alone is not sufficient to extend the life spans of transgenic mice.
Assuntos
Envelhecimento/fisiologia , Sequestradores de Radicais Livres/metabolismo , Superóxido Dismutase/metabolismo , Envelhecimento/genética , Análise de Variância , Animais , Distribuição de Qui-Quadrado , Longevidade , Camundongos , Camundongos Transgênicos , Modelos de Riscos ProporcionaisRESUMO
Long-term potentiation (LTP) and depression (LTD) were investigated in hippocampus of a genetic model of Down syndrome, the segmental trisomy (Ts65Dn) mouse. Field excitatory postsynaptic potentials were recorded from hippocampal slices and LTP and LTD evoked sequentially. LTP decreased whereas LTD increased significantly in Ts65Dn compared with control hippocampus.