RESUMO
MSTO1 encodes a cytosolic mitochondrial fusion protein, misato homolog 1 or MSTO1. While the full genotype-phenotype spectrum remains to be explored, pathogenic variants in MSTO1 have recently been reported in a small number of patients presenting with a phenotype of cerebellar ataxia, congenital muscle involvement with histologic findings ranging from myopathic to dystrophic and pigmentary retinopathy. The proposed underlying pathogenic mechanism of MSTO1-related disease is suggestive of impaired mitochondrial fusion secondary to a loss of function of MSTO1. Disorders of mitochondrial fusion and fission have been shown to also lead to mitochondrial DNA (mtDNA) depletion, linking them to the mtDNA depletion syndromes, a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of cellular mtDNA content. However, the consequences of pathogenic variants in MSTO1 on mtDNA maintenance remain poorly understood. We present extensive phenotypic and genetic data from 12 independent families, including 15 new patients harbouring a broad array of bi-allelic MSTO1 pathogenic variants, and we provide functional characterization from seven MSTO1-related disease patient fibroblasts. Bi-allelic loss-of-function variants in MSTO1 manifest clinically with a remarkably consistent phenotype of childhood-onset muscular dystrophy, corticospinal tract dysfunction and early-onset non-progressive cerebellar atrophy. MSTO1 protein was not detectable in the cultured fibroblasts of all seven patients evaluated, suggesting that pathogenic variants result in a loss of protein expression and/or affect protein stability. Consistent with impaired mitochondrial fusion, mitochondrial networks in fibroblasts were found to be fragmented. Furthermore, all fibroblasts were found to have depletion of mtDNA ranging from 30 to 70% along with alterations to mtDNA nucleoids. Our data corroborate the role of MSTO1 as a mitochondrial fusion protein and highlight a previously unrecognized link to mtDNA regulation. As impaired mitochondrial fusion is a recognized cause of mtDNA depletion syndromes, this novel link to mtDNA depletion in patient fibroblasts suggests that MSTO1-deficiency should also be considered a mtDNA depletion syndrome. Thus, we provide mechanistic insight into the disease pathogenesis associated with MSTO1 mutations and further define the clinical spectrum and the natural history of MSTO1-related disease.
Assuntos
Proteínas de Ciclo Celular/genética , Doenças Cerebelares/genética , Proteínas do Citoesqueleto/genética , DNA Mitocondrial , Doenças Mitocondriais/genética , Distrofias Musculares/genética , Mutação , Adolescente , Adulto , Atrofia , Células Cultivadas , Doenças Cerebelares/diagnóstico por imagem , Doenças Cerebelares/patologia , Doenças Cerebelares/fisiopatologia , Criança , Variações do Número de Cópias de DNA , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais/diagnóstico por imagem , Doenças Mitocondriais/patologia , Doenças Mitocondriais/fisiopatologia , Músculos/patologia , Distrofias Musculares/diagnóstico por imagem , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Fenótipo , Adulto JovemRESUMO
It is unclear how improvements in peripheral motor function in children with spinal muscular atrophy (SMA), treated with nusinersen, translate into clinically significant respiratory/sleep outcomes. A retrospective chart review of SMA children at the Sydney Children's Hospital Network was undertaken looking at 2 years before and after receiving their first dose of nusinersen. Polysomnography (PSG), spirometry and clinical data were collected and analysed using paired and unpaired t-tests for PSG parameters and generalised estimating equations for longitudinal lung function data. Forty-eight children (10 Type 1, 23 Type 2, 15 Type 3) at mean age 6.98 yrs (SD 5.25) for nusinersen initiation were included. There was a statistically significant improvement in oxygen nadir during sleep in individuals post nusinersen (mean of 87.9% to 92.3% (95%CI 1.24 - 7.63, p = 0.01)). Based on clinical and PSG findings, 6/21 patients (5 Type 2, 1 Type 3) ceased nocturnal NIV post nusinersen. Non-significant improvements were demonstrated in mean slope for FVC% predicted, FVC Z-score and mean FVC% predicted. Within 2 years of commencing nusinersen, stabilisation of respiratory outcomes occurred. Whilst some of the SMA type 2/3 cohort ceased NIV, there were no statistically significant improvements lung function and most PSG parameters.
Assuntos
Atrofia Muscular Espinal , Atrofias Musculares Espinais da Infância , Humanos , Criança , Estudos Retrospectivos , Atrofia Muscular Espinal/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Atrofias Musculares Espinais da Infância/complicações , Atrofias Musculares Espinais da Infância/tratamento farmacológico , SonoRESUMO
L-selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. To identify mechanisms for upregulation and assess how this influences leukocyte/endothelial cell interactions, cell-surface dimers of L-selectin were induced using the coumermycin-GyrB dimerization strategy for cross-linking L-selectin cytoplasmic domains in L-selectin cDNA-transfected lymphoblastoid cells. Coumermycin- induced L-selectin dimerization resulted in an approximately fourfold increase in binding of phosphomanan monoester core complex (PPME), a natural mimic of an L-selectin ligand, comparable to that observed after leukocyte activation. Moreover, L-selectin dimerization significantly increased (by approximately 700%) the number of lymphocytes rolling on vascular endothelium under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. Therefore, L-selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. Inducible oligomerization may also be a common mechanism for rapidly upregulating the adhesive or ligand-binding function of other cell-surface receptors.
Assuntos
Selectina L/metabolismo , Leucócitos/metabolismo , Células Cultivadas , DNA Girase , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Dimerização , Endotélio Vascular , Humanos , Selectina L/genética , Mananas/metabolismo , Manosefosfatos/metabolismoRESUMO
OBJECTIVE: In the context of increasing numbers of childhood cancer survivors (CCS), this study aimed to enhance understanding of the biophysical basis for long term chemotherapy induced peripheral neuropathy from different chemotherapy agents in CCS. METHODS: Detailed cross-sectional neurophysiological examination, using median nerve axonal excitability studies, alongside clinical assessments, in 103 long term CCS (10.5 ± 0.6 years post-treatment). RESULTS: Cisplatin treated CCS (n = 16) demonstrated multiple sensory axonal excitability changes including increased threshold (P < 0.05), alterations in depolarising and hyperpolarising threshold electrotonus (P < 0.05) and reduction in resting and minimum IV slope (P < 0.01). Vincristine treated CCS (n = 73) were comparable to controls, except for prolonged distal motor latency (P = 0.001). No differences were seen in the non-neurotoxic chemotherapy group (n = 14). Abnormalities were more evident in the cisplatin subgroup with greater clinical neuropathy manifestations. CONCLUSION: Persistent long term changes in axonal biophysical properties vary with different chemotherapy agents, most evident after cisplatin exposure. Longitudinal studies of nerve function during chemotherapy treatment are required to further evaluate these differences and their mechanistic basis. SIGNIFICANCE: This study provides a unique biophysical perspective for persistent cisplatin related neurotoxicity in children, previously under recognised.
Assuntos
Potenciais de Ação/fisiologia , Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Nervo Mediano/fisiopatologia , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Vincristina/efeitos adversos , Adolescente , Sobreviventes de Câncer , Criança , Estudos Transversais , Feminino , Humanos , Masculino , Neoplasias/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/fisiopatologiaRESUMO
Prostacyclin (PGI2) plays an integral role in O2 mediation of pulmonary vasomotor tone in the fetus and newborn. We hypothesized that O2 modulates PGI2 synthesis in vitro in ovine fetal intrapulmonary arteries, with decreasing O2 causing attenuated synthesis. A decline in PO2 from 680 to 40 mmHg caused a 26% fall in basal PGI2 synthesis. PGI2 synthesis maximally stimulated by bradykinin, A23187, and arachidonic acid were also attenuated at low PO2, by 35%, 33%, and 35%, respectively. PGE2 synthesis was equally affected. In contrast, varying O2 did not alter PGI2 synthesis with exogenous PGH2, which is the product of cyclooxygenase and the substrate for prostacyclin synthetase. Prostaglandin-mediated effects of O2 on cAMP production were also examined. Decreasing PO2 to 40 mmHg caused complete inhibition of basal cAMP production, whereas cAMP production stimulated by exogenous PGI2 was not affected. In parallel studies of mesenteric arteries, PGI2 synthesis and cAMP production were enhanced at low O2. Thus, PGI2 synthesis in fetal intrapulmonary arteries is modulated by changes in O2, with decreasing O2 causing attenuated synthesis. This process is due to an effect on cyclooxygenase activity, it causes marked parallel alterations in cAMP production, and it is specific to the pulmonary circulation.
Assuntos
AMP Cíclico/metabolismo , Epoprostenol/biossíntese , Oxigênio/fisiologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Artéria Pulmonar/embriologia , Animais , Calcimicina/farmacologia , Cálcio/fisiologia , Colforsina/farmacologia , Dinoprostona/biossíntese , Isoproterenol/farmacologia , Músculo Liso Vascular/fisiologia , Prostaglandina H2 , Prostaglandinas H/farmacologia , Transdução de Sinais , SuínosRESUMO
Prostacyclin is a critical mediator of structure and function in the pulmonary circulation, causing both the inhibition of vascular smooth muscle growth and vasodilation via the stimulation of adenylate cyclase. To examine the potential role of alterations in prostacyclin production or mechanism of action in chronic hypoxic pulmonary hypertension, we determined the effects of prolonged (7 d) in vivo hypoxia on in vitro prostacyclin synthesis and mediation of adenylate cyclase activity in rat main pulmonary arteries. In control arteries prostacyclin production exceeded that of prostaglandin (PG) E2 by 25-fold, with 42% originating from the endothelium. Studies utilizing indomethacin revealed that endogenous prostaglandins mediate at least 69% of basal adenylate cyclase activity. Prostacyclin-stimulated enzyme activity was enhanced by exogenous GTP, indicating that this is a receptor-mediated process involving G protein amplification. Comparable dose-related responses to prostacyclin and PGE2 suggest that these agents may activate a common receptor. After 7 d of in vivo hypoxia there was a 2.7-fold increase in in vitro prostacyclin production, with equivalent increases in synthesis in the endothelium and vascular smooth muscle. However, despite this increase there was no change in basal adenylate cyclase activity, and this was associated with attenuated sensitivity of the enzyme to prostacyclin stimulation. Concomitant diminution of the response to beta-adrenergic stimulation, with previously-demonstrated beta receptor downregulation and unaltered postreceptor-mediated activity, suggests that the blunted response to prostacyclin is due to receptor downregulation. Parallel studies of the thoracic aorta indicated that these changes are specific to the pulmonary artery. It is postulated that attenuation of the response of adenylate cyclase to prostacyclin may contribute to the structural changes and hypertension observed in the pulmonary vasculature of the rat with chronic hypoxia.
Assuntos
Adenilil Ciclases/metabolismo , Epoprostenol/biossíntese , Artéria Pulmonar/metabolismo , Animais , Endotélio Vascular/metabolismo , Ativação Enzimática , Epoprostenol/fisiologia , Guanosina Trifosfato/farmacologia , Hipertensão Pulmonar/etiologia , Hipóxia/metabolismo , Indometacina/farmacologia , Isoproterenol/farmacologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
Signal transducer and activator of transcription 5 (STAT5) activation occurs frequently in human progenitor B-cell acute lymphoblastic leukemia (B-ALL). To identify gene alterations that cooperate with STAT5 activation to initiate leukemia, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice in which a mutagenic Sleeping Beauty transposon (T2/Onc) was mobilized only in B cells. Stat5b-CA mice typically do not develop B-ALL (<2% penetrance); in contrast, 89% of Stat5b-CA mice in which the T2/Onc transposon had been mobilized died of B-ALL by 3 months of age. High-throughput sequencing approaches were used to identify genes frequently targeted by the T2/Onc transposon; these included Sos1 (74%), Kdm2a (35%), Jak1 (26%), Bmi1 (19%), Prdm14 or Ncoa2 (13%), Cdkn2a (10%), Ikzf1 (8%), Caap1 (6%) and Klf3 (6%). Collectively, these mutations target three major cellular processes: (i) the Janus kinase/STAT5 pathway (ii) progenitor B-cell differentiation and (iii) the CDKN2A tumor-suppressor pathway. Transposon insertions typically resulted in altered expression of these genes, as well as downstream pathways including STAT5, extracellular signal-regulated kinase (Erk) and p38. Importantly, expression of Sos1 and Kdm2a, and activation of p38, correlated with survival, further underscoring the role these genes and associated pathways have in B-ALL.
Assuntos
Linfócitos B/metabolismo , Elementos de DNA Transponíveis/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Fator de Transcrição STAT5/genética , Animais , Western Blotting , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Regulação Leucêmica da Expressão Gênica , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Estimativa de Kaplan-Meier , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese Insercional , Mutação , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína SOS1/genética , Proteína SOS1/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We have previously reported the complete sequence of the cDNAs corresponding to the alleles at the polymorphic loci (DRB1, DRB3, DQA1, DQB1) of the DRw11(5) cell line Swei. We now report the nucleotide and derived amino acid sequence of the alleles at the remaining two polymorphic loci, DPA1 and DPB1, from Swei, which types as DPw4b. Comparison of the DPw4b alpha sequence with previous DP alpha sequences indicates that it is identical to the DP alpha chains of DPw4 and DPw2. However, the DPw4b alpha cDNA encodes a unique 3' untranslated region that corresponds to sequences contained within the last intron of the DP alpha genomic gene. These results indicate that alternative splicing occurs within the Swei DP alpha RNA. Comparison of the DPw4b beta sequence with previously reported DP beta sequences indicates that the first domain is identical to the sequence of the DP beta chain derived from the Burkhardt (Burk) cell line, but that the second domain differs by two amino acids. In addition, a single amino acid difference from DPw2 at position 69 in the first domain of the DPw4b beta chain suggests that this residue is important in determining DP specificity. Furthermore, comparison with other DP beta DNA sequences suggests that variable regions occur at positions 8-11, 55-57, and 84-87.
Assuntos
Antígenos HLA-DP/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Antígenos HLA-D/genética , Cadeias beta de HLA-DP , Homozigoto , Humanos , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
BACKGROUND: Prior histologic studies have examined smooth muscle cell, macrophage and thrombus constituents of atherosclerotic coronary atherectomy specimens. Lymphocytes and mononuclear leukocytes are also detectable in atherosclerotic surgical pathology specimens utilizing immunocytochemical techniques. METHODS: In order to quantify the histological contribution of cytokine receptor-expressing immunocompetent cells to human coronary artery stenoses, 30 directional atherectomy catheter biopsy specimens (wet weight < or = 10 mg) from 16 patients were snap frozen (-70 degrees C) for quantitative immunocytochemical studies. Following computer-assisted quantification of total intimal nuclei per tissue section (mean 297 +/- 177; cell density 7 +/- 5/10(4) microns 2), monoclonal antibody cytochemistry was used to identify the percentage of these cells expressing antigenic clusters of differentiation (CD) characteristic of T-lymphocytes, B-lymphocytes and monocytes. Identification of alpha (low affinity) and beta (intermediate affinity) interleukin-2 receptors on intimal cells was accomplished using a three-step streptavidin-biotin method. RESULTS: A significant percentage of intimal cells were of lymphocytic (11 +/- 13%) or monocytic (12 +/- 14%) origin, with helper T-cells (9 +/- 12%) outnumbering both suppressor T-cells (2 +/- 4%) and B-lymphocytes (1 +/- 2%). Interleukin-2 receptors were noted on 9 +/- 12% of intimal cells, including cells with a vascular smooth muscle phenotype. CONCLUSIONS: These quantitative immunocytochemical data conclusively demonstrate that lymphocytes and monocytes account for over 20% of coronary plaque cells obtained by in-vivo atherectomy, and that helper (CD4) T-cells predominate over suppressor (CD8) T-cells and B-lymphocytes. Variable interleukin-2 receptor subtype expression occurs in mononuclear leukocytes infiltrating chronic human atheroma. By applying these techniques, the therapeutic effects of cytotoxic agents on selectively targeted cytokine receptor-expressing cells may now be evaluated in vivo in small human directional coronary atherectomy specimens.
Assuntos
Aterectomia Coronária , Doença das Coronárias/metabolismo , Leucócitos Mononucleares/metabolismo , Receptores de Citocinas/metabolismo , Adulto , Idoso , Linfócitos B/metabolismo , Corantes , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/cirurgia , Doença das Coronárias/patologia , Doença das Coronárias/cirurgia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Linfócitos T/metabolismoAssuntos
Cumarínicos/farmacologia , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Proteínas Recombinantes de Fusão/química , Aminocumarinas , Animais , Linhagem Celular , DNA Girase , Dimerização , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Novobiocina/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Spinal muscular atrophy (SMA) is a relatively common autosomal recessive neuromuscular disorder characterised by muscle weakness and atrophy due to degeneration of motor neurons of the spinal cord and cranial motor nuclei. The clinical phenotype incorporates a wide spectrum. No effective treatment is currently available and patients may experience severe physical disability which is often life limiting. The most common type of SMA is caused by homozygous disruption of the survival motor neuron 1 (SMN1) gene by deletion, conversion or mutation and results in insufficient levels of survival motor neuron (SMN) protein in motor neurons. While diagnosis is usually achieved by genetic testing, an illustrative clinical case is described that highlights the molecular and diagnostic complexities. While there is an emerging picture concerning the function of the SMN protein and the molecular pathophysiological mechanisms underpinning the disease, a number of substantial issues remain unresolved. The selective vulnerability of the motor neuron and the site and timing of the primary pathogenesis are not yet determined. Utilising the organisation of the SMN genomic region, recent advances have identified a number of potential therapeutic targets. As such, this review incorporates discussion of the clinical manifestations, molecular genetics, diagnosis, mechanisms of disease pathogenesis and development of novel treatment strategies.
Assuntos
Atrofia Muscular Espinal , Proteínas do Complexo SMN/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Processamento Alternativo , Animais , Ensaios Clínicos como Assunto , Diagnóstico Diferencial , Humanos , Neurônios Motores/patologia , Neurônios Motores/fisiologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/fisiopatologia , Guias de Prática Clínica como Assunto , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas do Complexo SMN/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Interferon-gamma (IFN gamma) is an important immunomodulatory cytokine produced by activated T cells and NK cells that plays a pivotal role in promoting host defense. IFN gamma is distinguished from IFN alpha and IFN beta by its ability to regulate a number of immune functions. IFN gamma induces its biologic effects by interacting with a specific IFN gamma receptor expressed at the cell surface. Recently, IFN gamma receptors have been purified from human and murine cells and their cDNAs cloned and expressed. This work has revealed that IFN gamma receptors are 90 kDa, single chain glycoproteins that bind ligand with high affinity in a species specific manner. There appears to be only a single type of IFN gamma receptor that is expressed on nearly all cell types. Whereas this single polypeptide is sufficient to confer ligand binding and processing activity to transfected cells, a second, as yet undefined, component is required to form a functionally active IFN gamma receptor. The identity of this second component is currently being investigated. In addition, recent work has revealed novel structure-function relationships that exist within the IFN gamma receptor's intracellular domain. This work has shown that distinct portions of the intracellular domain are differentially responsible for mediating different biologic activities of the receptor.
Assuntos
Interferon gama , Receptores de Interferon , Animais , Humanos , Camundongos , Receptor de Interferon gamaRESUMO
The last ten years have seen an explosive growth in our understanding of IFN gamma. The cloning of the cDNAs for IFN gamma and its receptor have made available large amounts of highly purified recombinant IFN gamma and soluble IFN gamma receptor. In addition, highly specific neutralizing monoclonal antibodies have been generated to both of these proteins. Using these reagents, IFN gamma and the IFN gamma receptor have been characterized on a molecular basis. Structure-function studies carried out on these proteins have identified key molecular regions that are required for biologic activity. Moreover, a great deal is now known concerning the physiologic role that IFN gamma plays in vivo. In this review we focus on the new developments in the areas of IFN gamma biochemistry and biology and pay particular attention to the IFN gamma receptor and three aspects of IFN gamma biology that are of special interest to immunologists: host defense, inflammation, and autoimmunity.
Assuntos
Interferon gama/fisiologia , Receptores de Interferon/fisiologia , Sequência de Aminoácidos , Animais , Autoimunidade , Humanos , Inflamação/etiologia , Interferon gama/química , Interferon gama/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Receptores de Interferon/química , Receptores de Interferon/genética , Transdução de Sinais , Receptor de Interferon gamaRESUMO
Functionally active human interferon-gamma (IFN gamma) receptors require the presence of at least two polypeptides: the IFN gamma receptor and an accessory molecule encoded by a gene on human chromosome 21. Here we have used a murine L cell line that stably contains human chromosome 21 (SCC16-5) to determine whether the receptor's cytoplasmic domain is important for receptor function. SCC16-5 stably transfected with the full-length human IFN gamma receptor cDNA bound, internalized, and responded to human IFN gamma. In contrast, SCC16-5 expressing human IFN gamma receptors lacking a cytoplasmic domain bound human IFN gamma but did not internalize or respond to it. Using a family of IFN gamma receptor deletion mutants, two functionally important regions within the intracellular domain were identified: (a) a membrane proximal region (residues 256-303) required for ligand processing and biologic responsiveness and (b) the carboxyl-terminal 39 amino acids (residues 434-472) needed exclusively for biologic responses.
Assuntos
Receptores Imunológicos/genética , Animais , Sequência de Bases , Cromossomos Humanos Par 21 , DNA/genética , Citometria de Fluxo , Genes MHC Classe I , Humanos , Interferons/metabolismo , Interferons/farmacologia , Células L , Camundongos , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Testes de Precipitina , Receptores de Interferon , Proteínas Recombinantes/genética , TransfecçãoRESUMO
To better understand the role of prostacyclin [prostaglandin (PG) I2] in oxygen mediation of vasomotor tone in the developing lung, we determined the ontogeny of the direct effects of acute changes in oxygen on in vitro PGI2 synthesis and adenosine 3',5'-cyclic monophosphate (cAMP) production in intrapulmonary arteries from fetal and newborn lambs. In the absence of varying oxygen, PGI2 synthesis increased 6.9-fold from early to late in the third trimester, and it rose an additional 3.2-fold from late gestation to 1 wk of age, and another 2.1-fold from 1 to 4 wk. PGE2 synthesis similarly rose 4.9-fold during the third trimester, but it then fell 69% from late gestation to 1 wk of age and remained unchanged postnatally. Paralleling the developmental increase in PGI2 synthesis, basal cAMP production rose 6.2-fold from the early third trimester to 4 wk of age. In contrast, PGI2-stimulated cAMP production was similar in all age groups. With an acute decline in PO2 in vitro from 680 to 40 mmHg, PGI2 and PGE2 synthesis in fetal arteries fell 33-46 and 39-55%, respectively. In contrast, they were increased by 9-145% and 44-130%, respectively, at lower PO2 in arteries from newborn lambs. Basal cAMP production was altered by decreased oxygen in a similar manner, falling by 35-39% in fetal arteries yet increasing by 21-47% in the newborn. PGI2-stimulated cAMP production, however, was not affected by oxygen at all ages except in the early third trimester. Thus there is a dramatic developmental increase in pulmonary arterial PGI2 synthesis that causes a marked maturational rise in cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Epoprostenol/biossíntese , Feto/metabolismo , Oxigênio/farmacologia , Artéria Pulmonar/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , AMP Cíclico/biossíntese , Dinoprostona/biossíntese , Prostaglandinas/biossíntese , OvinosRESUMO
Herein, we report that overexpression of either human or murine interferon-gamma (IFN-gamma) receptors lacking their entire intracellular domains in cells bearing functionally active IFN gamma receptors ablates responsiveness to homologous ligand in a dominant negative manner. Unresponsiveness could also be induced in murine cells overexpressing murine IFN gamma receptor mutants that either lack 39 COOH-terminal amino acids or contain an alanine substitution for a functionally critical tyrosine. Overexpression of the full-length receptor did not alter cellular responsiveness to IFN gamma. The inhibitory activities of the receptor mutants were dose-dependent, generalizable to a variety of cellular responses, and specific. Cells expressing 100:1 ratios of mutant to wild-type receptor were unresponsive to IFN gamma even at doses 30,000 times greater than that required to induce a maximal response in wild-type cells. These results provide an example of a dominant negative mutation that effects the complete inactivation of a transmembrane receptor lacking a kinase domain and suggest a more general utility for dominant negative mutations in the study of cytokine receptor function.
Assuntos
Interferon gama/farmacologia , Receptores de Interferon/biossíntese , Receptores de Interferon/genética , Animais , Sequência de Bases , Linhagem Celular , Cromossomos Humanos Par 21 , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Genes MHC Classe I/efeitos dos fármacos , Genes MHC da Classe II/efeitos dos fármacos , Humanos , Interferon Tipo I/farmacologia , Células L , Camundongos , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Oligodesoxirribonucleotídeos , Plasmídeos , Proteínas Recombinantes , Transfecção , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimentoRESUMO
To define the role of endothelial nitric oxide (NO) in developmental changes in pulmonary vascular resistance and oxygen responsiveness, we determined the ontogeny of endothelial NO production and of oxygen modulation of that process in pulmonary arteries from fetal and newborn lambs. NO production was assessed by measuring endothelium-dependent arterial guanosine 3',5'-cyclic monophosphate synthesis. Basal NO rose two-fold from late gestation to 1 wk of age and another 1.6-fold from 1 to 4 wk. Acetylcholine-stimulated NO also increased 1.6-fold from 1 to 4 wk. The maturational rise in NO was evident at high Po2 in vitro, and it was not modified by L-arginine. This suggests that the developmental increase may alternatively involve enhanced calcium-calmodulin-mediated mechanisms, increased expression of NO synthase, or greater availability of required cofactor(s). With an acute decline in Po2 in vitro from 680 to 150 or 40 mmHg, there was 50-88% attenuation of basal and acetylcholine-stimulated NO late in the third trimester and in the newborn but not early in the third trimester. Parallel studies of mesenteric endothelium revealed postnatal increases in basal and stimulated NO but no decline in NO at lower Po2. Ontogenic changes in endothelial NO production and in oxygen modulation of that process may be involved in the maturational decrease in vascular resistance and the development of oxygen responsiveness in the pulmonary circulation.
Assuntos
Animais Recém-Nascidos/metabolismo , Desenvolvimento Embrionário e Fetal , Endotélio Vascular/metabolismo , Feto/metabolismo , Óxido Nítrico/biossíntese , Artéria Pulmonar/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Arginina/análogos & derivados , GMP Cíclico/metabolismo , Endotélio Vascular/embriologia , Endotélio Vascular/crescimento & desenvolvimento , Técnicas In Vitro , Oxigênio/fisiologia , Artéria Pulmonar/embriologia , Artéria Pulmonar/crescimento & desenvolvimento , OvinosRESUMO
We have previously shown that the intracellular domain of the interferon-gamma (IFN-gamma) receptor plays an obligate role in receptor-mediated signal transduction. Moreover, we have specifically identified two regions within the human IFN-gamma receptor's intracellular domain required for functional activity: the membrane-proximal 48 amino acids required for both functional activity and receptor-mediated ligand internalization and the C-terminal 39 amino acids required exclusively for biologic response induction. Herein we report the identification of the 3 amino acids within the C-terminal region of the receptor that are obligatorily required for receptor function. By using a set of overlapping truncation mutants, the minimal functional sequence within the C-terminal region was localized to residues 434-444 (APTSFGYD-KPH). By mutating each individual residue within this sequence to alanine, three residues (Tyr-440, Asp-441, and His-444) were identified as being critical for IFN-gamma-dependent (i) upregulation of major histocompatibility complex class I proteins, (ii) activation of the IFN regulatory factor 1 gene, and (iii) stimulation of cells to produce nitric oxide. The more conservative Tyr-440-->Phe substitution also resulted in a nonfunctional receptor. Subsequent mutational analysis of all five of the IFN-gamma receptor's intracellular tyrosine residues revealed that Tyr-440 was the sole tyrosine required for receptor activity. These results thus identify a unique sequence in the IFN-gamma receptor that is required for initiation of IFN-gamma-dependent biologic responses and highlight the importance of the hydroxyl side chain of Tyr-440 in this process.
Assuntos
Interferon gama/fisiologia , Receptores de Interferon/química , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Genes MHC Classe I , Humanos , Fator Regulador 1 de Interferon , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Óxido Nítrico/metabolismo , Fosfoproteínas/genética , Proteínas Recombinantes , Transdução de Sinais , Tirosina/químicaRESUMO
Prostacyclin (PGI2) is an important local mediator of vasomotor tone in the fetus and newborn, acting via stimulation of cAMP production by vascular smooth muscle (VSM) adenylate cyclase. In this investigation PGI2 and prostaglandin (PG) E2 synthesis and stimulation of cAMP production were compared in vitro in ovine fetal pulmonary versus systemic (mesenteric) arteries. PGI2 synthesis was similar in the pulmonary and systemic arteries (2.4 +/- 0.2 and 2.6 +/- 0.3 ng/mg protein.h, respectively), as was PGE2 synthesis (1.9 +/- 0.2 and 1.5 +/- 0.2 ng/mg protein.h, respectively); synthesis was greater for PGI2 versus PGE2 in both artery types. 65-71% of PGI2 synthesis and 51-59% of PGE2 synthesis occurred in the endothelium. Basal (nonstimulated) cAMP production was fully attenuated by indomethacin, indicating that it is mediated exclusively by endogenous PG. Basal cAMP production was 3.8-fold less in pulmonary versus systemic arteries, and this was related to a 9.7-fold difference in responsiveness to PG. A 14.7-fold difference in the response to forskolin indicates that underlying mechanism may be a disparity in the quantity and/or function of the adenylate cyclase enzyme complex. Thus, prostacyclin and PGE2 synthesis are comparable in ovine fetal pulmonary versus systemic arteries, but the cAMP response to the prostanoids is markedly greater in the latter artery type due to differences in the activation of adenylate cyclase. This heterogeneity in VSM intracellular signalling may contribute to differential vasomotor tone and responses in the fetal pulmonary and systemic circulation.