RESUMO
The MAP kinase p38α is associated with numerous processes in eukaryotes, and its elevated activity is a prominent feature of inflammatory diseases, allergies, and aging. Since p38α is a nodal component of a complex signaling network, it is difficult to reveal exactly how p38α contributes to disparate outcomes. Identification of p38α -specific effects requires activation of p38α per se in vivo. We generated a transgenic mouse model that meets this requirement by allowing inducible and reversible expression of an intrinsically active p38α molecule (p38αD176A+F327S ). p38α's activation across all murine tissues resulted in a significant loss of body weight and death of about 40% of the mice within 17 weeks of activation, although most tissues were unaffected. Flow cytometric analysis of the lungs and bronchoalveolar lavage fluid detected an accumulation of 'debris' within the airways, suggesting impaired clearance. It also revealed increased numbers of alternatively activated alveolar macrophages and myeloid-derived suppressor cells within the lung, pointing at suppression and resolution of inflammation. Blood count suggested that mice expressing p38αD176A+F327S suffer from hemolytic anemia. Flow cytometry of bone marrow revealed a reduced number of hematopoietic stem cells and abnormalities in the erythroid lineage. Unexpectedly, p38α's substrate MAPKAPK2, mitogen-activated protein kinase-activated protein kinase 2 was downregulated in mice expressing p38αD176A+F327S , suggesting that constitutive activity of p38α may impose pathological phenotypes by downregulating downstream components, perhaps via a feedback inhibition mechanism. In summary, this new mouse model shows that induced p38α activity per se is hazardous to mouse vitality and welfare, although pathological parameters are apparent only in blood count, bone marrow, and lungs.
Assuntos
Anemia/genética , Regulação Enzimológica da Expressão Gênica , Macrófagos/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/genética , Mutação , Células Supressoras Mieloides/metabolismo , Anemia/enzimologia , Animais , Peso Corporal/genética , Citocinas/sangue , Citocinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/classificação , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Células Supressoras Mieloides/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Especificidade por SubstratoRESUMO
Chronic kidney disease (CKD) is one of the most prominent diseases affecting our population today. According to the Factsheet published by Centers for Disease Control and Prevention (CDC), it effects approximately 15% of the total population in the United States in some way, shape, or form. Within the myriad of symptomatology associated with CKD, one of the most prevalent factors in terms of affecting quality of life is anemia. Anemia of CKD cannot be completely attributed to one mechanism or cause, but rather has a multifactorial origin in the pathophysiology of CKD. While briefly summarizing well-documented risk factors, this review, as a hypothesis, aims to explore the possible role of Na-K-ATPase and its signaling function [especially recent identified reactive oxygen species (ROS) amplification function] in the interwoven mechanisms of development of the anemia of CKD.
Assuntos
Anemia/enzimologia , Anemia/etiologia , Insuficiência Renal Crônica/complicações , Transdução de Sinais/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Humanos , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a new class of oral medicines being developed for the treatment of anemia in chronic kidney disease (CKD) patients. This study aimed to compare the efficacy and safety of HIF-PHI vs epoetin and darbepoetin in CKD patients with anemia not undergoing dialysis. The PubMed, Embase, Cochrane Library, Web of Science, and clinicaltrials.gov databases were searched from inception to October 2019 for randomized controlled trials investigating different agents (six HIF-PHIs, epoetin, darbepoetin, and placebo) for treating CKD patients with anemia that did not undergo dialysis. The outcomes included a change in hemoglobin (Hb) levels and all-cause mortality. A total of 19 studies were included. Compared with the placebo, except for vadadustat (mean differences: 1.12, 95 % confidence interval [CI]: â0.11-2.35), the other drugs significantly increased Hb levels, with mean differences of 2.46 (95 % CI: 0.93-3.99) for desidustat, 1.81 (0.87-2.75) for enarodustat, 1.68 (0.64-2.72) for molidustat, 1.66 (0.89-2.44) for epoetin, 1.63 (0.69-2.56) for darbepoetin, 1.61 (0.99-2.22) for roxadustat, and 1.55 (0.74-2.36) for daprodustat. No differences were found in the Hb level elevations among these eight drugs. Compared with the placebo, there also was no significant association between the drugs and all-cause mortality (molidustat of RR, 0.39 [95 % CI, 0.06-2.59]; roxadustat, 0.40 (0.06-2.84); enarodustat, 0.33 (0.01-16.25); desidustat, 0.34 (0.01-17.00); epoetin, 0.50 (0.18-1.42); daprodustat, 0.54 (0.09-3.31); darbepoetin, 1.03 (0.65-1.65); and vadadustat, 1.43 (0.15-13.27)). No differences were observed in the all-cause mortality among the drugs. In conclusion, these HIF-PHIs are effective and relatively tolerant for treating anemia patients with CKD not undergoing dialysis. Further research should consider the limitations of our study to evaluate the value of these HIF-PHIs in clinical settings.
Assuntos
Anemia/tratamento farmacológico , Darbepoetina alfa/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Epoetina alfa/uso terapêutico , Hematínicos/uso terapêutico , Hemoglobinas/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Insuficiência Renal Crônica/complicações , Idoso , Idoso de 80 Anos ou mais , Anemia/enzimologia , Anemia/etiologia , Anemia/mortalidade , Biomarcadores/sangue , Darbepoetina alfa/efeitos adversos , Inibidores Enzimáticos/efeitos adversos , Epoetina alfa/efeitos adversos , Feminino , Hematínicos/efeitos adversos , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Masculino , Pessoa de Meia-Idade , Metanálise em Rede , Ensaios Clínicos Controlados Aleatórios como Assunto , Insuficiência Renal Crônica/mortalidade , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: Blood-related hereditary diseases are widespread in Eastern and SouthWestern regions of Saudi Arabia until recently. In this study, we used Na+, K+ATPase as an enzymatic indicator for the diagnosis of the diseases. MATERIALS AND METHODS: Individuals with different blood diseases (iron deficiency (n=13), anemia (n=14), thalassemia (n=16) and sickle cell anemia (n=12) were studied for Na+, K+-ATPase activity in the plasma membrane of red blood cell and compared with those of the healthy ones (n=20) of the same age and gender living in Jeddah, Saudi Arabia. RESULTS: There was a significant elevation in the specific activity of Na+, K+ATPase in individuals with anemia compared with those of control (0.0094 + 0.001 nmol / mg protein/min versus 0.0061 ± 0.001). On the other hand, there was a significant reduction in enzyme activity in thalassemia (0.0028 ± 0.002 nmol / mg protein/min) and sickle cell anemia cases (0.0042 ±0.001 nmol / mg protein/min) compared to the control group. The cut off value for Na+, K+ATPase activity is 0.005 µmol Pi/min-showing 94% sensitivity and 93% specificity for the differentiation of blood abnormality. CONCLUSION: It can be recommended that the activity of Na+, K+-ATPase can be used for the diagnosis of individuals with blood diseases/disorders.
Assuntos
Anemia/enzimologia , Membrana Celular/enzimologia , Membrana Eritrocítica/enzimologia , Eritrócitos/enzimologia , ATPase Trocadora de Sódio-Potássio/sangue , Anemia Ferropriva , Anemia Falciforme , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Arábia Saudita , TalassemiaRESUMO
Ruxolitinib is a potent Janus kinase (JAK)1/JAK2 inhibitor that has demonstrated rapid reductions in splenomegaly and marked improvement in disease-related symptoms and quality of life in patients with myelofibrosis (MF). The present analysis reports the 3-year follow-up (median, 151 weeks) of the efficacy and safety of Controlled Myelofibrosis Study With Oral Janus-associated Kinase (JAK) Inhibitor Treatment-II (the COMFORT-II Trial), comparing ruxolitinib with the best available therapy (BAT) in 219 patients with intermediate-2 and high-risk MF. In the ruxolitinib arm, with continued therapy, spleen volume reductions of ≥35% by magnetic resonance imaging (equivalent to approximately 50% reduction by palpation) were sustained for at least 144 weeks, with the probability of 50% (95% confidence interval [CI], 36-63) among patients achieving such degree of response. At the time of this analysis, 45% of the patients randomized to ruxolitinib remained on treatment. Ruxolitinib continues to be well tolerated. Anemia and thrombocytopenia were the main toxicities, but they were generally manageable, improved over time, and rarely led to treatment discontinuation (1% and 3.6% of patients, respectively). No single nonhematologic adverse event led to definitive ruxolitinib discontinuation in more than 1 patient. Additionally, patients randomized to ruxolitinib showed longer overall survival than those randomized to BAT (hazard ratio, 0.48; 95% CI, 0.28-0.85; log-rank test, P = .009).
Assuntos
Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/mortalidade , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Anemia/induzido quimicamente , Anemia/tratamento farmacológico , Anemia/enzimologia , Anemia/mortalidade , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Masculino , Nitrilas , Mielofibrose Primária/enzimologia , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis/efeitos adversos , Pirimidinas , Taxa de Sobrevida , Trombocitopenia/induzido quimicamente , Trombocitopenia/tratamento farmacológico , Trombocitopenia/enzimologia , Trombocitopenia/mortalidade , Fatores de TempoRESUMO
The aim of this study was to evaluate the ecto-adenosine deaminase (E-ADA) activity in erythrocytes of lambs experimentally infected with Haemonchus contortus, correlating it with the degrees of anemia of the experimental animals. A total of 14 healthy lambs, with negative fecal exam for parasites, were to carry out the present study. They were divided into two groups, composed by seven animals: Group A represented the healthy animals (uninfected), while in Group B the animals were infected with 15,000 larvae of H. contortus. Blood was drawn on the days 15, 45 and 75 post-infection (PI) in order to perform the hematological analysis, as well as the mensuration of E-ADA activity in erythrocytes. Parasitological stool exam were performed on the same days mentioned above to follow up the evolution of the infection, as well to determine the number of eggs per gram of feces (EPG). On day 15PI, the animals presented negative EPG and there was not significant (P>0.05) difference between groups in relation to E-ADA activity and hematologic parameters. Animals in Group B had positive EPG for helminths on days 45 and 75 PI, accompanied by varying degrees of anemia, when compared to Group A. At the same periods E-ADA activity was significantly (P<0.05) increased in the erythrocytes of animals of Group B when compared with the not-infected ones. Statistically, there was a negative correlation (P<0.01) between activity E-ADA in erythrocytes and hematocrit on days 45 (r = -0.76) and 75 (r = -0.85)PI. Based on these results and in the scientific literature, it is possible to conclude that the E-ADA may participate on mechanisms related with the pathogenesis and host response against anemia caused by H. contortus.
Assuntos
Adenosina Desaminase/sangue , Anemia/veterinária , Eritrócitos/enzimologia , Hemoncose/veterinária , Haemonchus , Doenças dos Ovinos/parasitologia , Adenosina Desaminase/fisiologia , Anemia/enzimologia , Anemia/parasitologia , Animais , Fezes/parasitologia , Hemoncose/enzimologia , Hematócrito/veterinária , Hemoglobinas/análise , Masculino , Contagem de Ovos de Parasitas/veterinária , Ovinos/parasitologia , Doenças dos Ovinos/enzimologiaRESUMO
AIM: To investigate and clarify, for the first time, the role of inosine triphosphate pyrophosphatase (ITPA) polymorphism in Egyptian chronic hepatitis C virus (HCV) patients. METHODS: The human genomic DNA of all patients was extracted from peripheral blood cells in order to determine the single nucleotide polymorphism (SNP) of ITPA (rs1127354). SNP genotyping was performed by real time polymerase chain reaction (PCR, ABI TaqMan allelic discrimination kit) for 102 treatment-naive Egyptian patients with chronic HCV. All patients had no evidence of cardiovascular or renal diseases. They received a combination treatment of pegylated interferon α (PEG-IFNα) as a weekly subcutaneous dose plus an oral weight-adjusted dose of ribavirin (RBV). The majority received PEG-IFNα2a (70.6%) while 29.4% received PEG-IFNα2b. The planned duration of treatment was 24-48 wk according to the viral kinetics throughout the course of treatment. Pre-treatment liver biopsy was done for each patient for evaluation of fibrosis stage and liver disease activity. The basal viral load level was detected quantitatively by real time PCR while viral load throughout the treatment course was performed qualitatively by COBAS TaqMan assay. RESULTS: Ninety-three patients (91.2%) had ITPA SNP CC genotype and 9 (8.8%) had non-CC genotype (CA and AA). The percentage of hemoglobin (Hb) decline was higher for CC patients than for non-CC patients, particularly at weeks 4 and 8 (P = 0.047 and 0.034, respectively). During the first 12 wk of treatment, CC patients had significantly more Hb decline > 3 g/dL than non-CC patients: 64.5% vs 22.2% at weeks 8 and 12, respectively, (P = 0.024 and 0.038). Reduction of the amount of the planned RBV dose was significantly higher for CC patients than non-CC patients during the first 12 wk (18% ± 12.1% vs 8.5% ± 10.2%, P = 0.021). The percentage of CC patients with RBV dose reduction was significantly greater than that of non-CC patients (77.4% vs 44.4%, P = 0.044). Multivariate analysis identified only the percentage of RBV dose as a predictor for Hb decline. Platelet decline was significantly higher in non-CC patients than CC patients at weeks 12, 24 and 48 (P = 0.018, 0.009 and 0.026, respectively). CONCLUSION: Rs1127354 ITPA polymorphism plays a decisive role in protecting against treatment-induced anemia and the need for RBV dose reduction in Egyptian HCV patients.
Assuntos
Anemia/prevenção & controle , Antivirais/efeitos adversos , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/efeitos adversos , Polietilenoglicóis/efeitos adversos , Polimorfismo de Nucleotídeo Único , Pirofosfatases/genética , Ribavirina/efeitos adversos , Adulto , Anemia/sangue , Anemia/induzido quimicamente , Anemia/enzimologia , Anemia/genética , Biomarcadores/sangue , Quimioterapia Combinada , Egito/epidemiologia , Feminino , Frequência do Gene , Predisposição Genética para Doença , Hemoglobinas/metabolismo , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/enzimologia , Hepatite C Crônica/epidemiologia , Hepatite C Crônica/genética , Humanos , Interferon alfa-2 , Modelos Logísticos , Masculino , Análise Multivariada , Razão de Chances , Fenótipo , Proteínas Recombinantes/efeitos adversos , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: Acute pancreatitis is a significant potential complication with double-balloon enteroscopy. Hyperamylasemia is frequently observed after both double-balloon enteroscopy and single-balloon enteroscopy but often without associated pancreatitis. Whether the same phenomenon occurs with spiral enteroscopy is currently unknown. AIMS: To determine the incidence of pancreatitis and hyperamylasemia following spiral enteroscopy. METHODS: A prospective cohort study of consecutive patients undergoing proximal spiral enteroscopy was conducted. Serum amylase levels were measured immediately before and following the procedure, combined with observation for clinical signs of pancreatitis. RESULTS: A total of 32 patients underwent proximal spiral enteroscopy, with a mean total procedure time of 51 min (range 30 min to 100 min) and mean depth of insertion of 240 cm (range 50 cm to 350 cm). The diagnostic yield was 50%, with 31% of all procedures being therapeutic. While no patients exhibited signs that raised suspicion of pancreatitis, hyperamylasemia was common (20%). Hyperamylasemia was not significantly associated with procedure duration or depth of insertion but was linked to patients with Peutz-Jeghers syndrome and with the use of propofol sedation, suggesting that it may be more common in difficult cases. CONCLUSIONS: Postprocedural hyperamylasemia occurred frequently with proximal spiral enteroscopy, while no associated pancreatitis was observed. This finding suggests that hyperamylasemia may not necessarily reflect pancreatic injury nor portend a risk for pancreatitis.
Assuntos
Anemia/terapia , Enteroscopia de Duplo Balão/efeitos adversos , Hiperamilassemia/epidemiologia , Pancreatite/epidemiologia , Síndrome de Peutz-Jeghers/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Amilases/sangue , Anemia/enzimologia , Anemia/patologia , Proteína C-Reativa/metabolismo , Estudos de Coortes , Feminino , Humanos , Hiperamilassemia/diagnóstico , Incidência , Masculino , Pessoa de Meia-Idade , Pancreatite/diagnóstico , Síndrome de Peutz-Jeghers/enzimologia , Síndrome de Peutz-Jeghers/patologiaRESUMO
In red cell development, the differentiation program directed by the transcriptional regulator GATA1 requires signaling by the cytokine erythropoietin, but the mechanistic basis for this signaling requirement has remained unknown. Here we show that erythropoietin regulates GATA1 through protein kinase D activation, promoting histone deacetylase 5 (HDAC5) dissociation from GATA1, and subsequent GATA1 acetylation. Mice deficient for HDAC5 show resistance to anemic challenge and altered marrow responsiveness to erythropoietin injections. In ex vivo studies, HDAC5(-/-) progenitors display enhanced entry into and passage through the erythroid lineage, as well as evidence of erythropoietin-independent differentiation. These results reveal a molecular pathway that contributes to cytokine regulation of hematopoietic differentiation and offer a potential mechanism for fine tuning of lineage-restricted transcription factors by lineage-specific cytokines.
Assuntos
Eritropoese/fisiologia , Fator de Transcrição GATA1/fisiologia , Histona Desacetilases/fisiologia , Proteína Quinase C/fisiologia , Acetilação , Anemia/enzimologia , Anemia/genética , Anemia/patologia , Animais , Carbazóis/farmacologia , Linhagem da Célula , Citocinas/fisiologia , Ativação Enzimática , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/enzimologia , Eritropoese/efeitos dos fármacos , Eritropoetina/farmacologia , Eritropoetina/uso terapêutico , Humanos , Indóis/farmacologia , Maleimidas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transdução de SinaisRESUMO
Carotenoids and their metabolites are widespread and exert key biological functions in living organisms. In vertebrates, the carotenoid oxygenase BCMO1 converts carotenoids such as ß,ß-carotene to retinoids, which are required for embryonic pattern formation and cell differentiation. Vertebrate genomes encode a structurally related protein named BCDO2 but its physiological function remains undefined. Here, we show that BCDO2 is expressed as an oxidative stress-regulated protein during zebrafish development. Targeted knockdown of this mitochondrial enzyme resulted in anemia at larval stages. Marker gene analysis and staining for hemoglobin revealed that erythropoiesis was not impaired but that erythrocytes underwent apoptosis in BCDO2-deficient larvae. To define the mechanism of this defect, we have analyzed the role of BCDO2 in human cell lines. We found that carotenoids caused oxidative stress in mitochondria that eventually led to cytochrome c release, proteolytic activation of caspase 3 and PARP1, and execution of the apoptotic pathway. Moreover, BCDO2 prevented this induction of the apoptotic pathway by carotenoids. Thus, our study identifying BCDO2 as a crucial protective component against oxidative stress establishes this enzyme as mitochondrial carotenoid scavenger and a gatekeeper of the intrinsic apoptotic pathway.
Assuntos
Apoptose/fisiologia , Carotenoides/metabolismo , Mitocôndrias/metabolismo , Oxigenases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Acetilcisteína/farmacologia , Anemia/enzimologia , Anemia/genética , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Carotenoides/farmacologia , Linhagem Celular , Dioxigenases , Eritropoese/genética , Eritropoese/fisiologia , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Fenretinida/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Estresse Oxidativo , Oxigenases/antagonistas & inibidores , Oxigenases/genética , RNA Interferente Pequeno/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
INTRODUCTION: The hypoxia-inducible factor (HIF) system mediates the body's response to hypoxia, locally, inducing angiogenesis and a shift to anaerobic metabolism, and systemically, increasing red cell mass in anemia. HIF prolyl hydroxylases (HIF-PH) modify HIF, decreasing its activity. Increasing HIF activity through inhibition of HIF-PH may provide an alternative treatment for anemia and may protect against damage related to ischemia-reperfusion. AREAS COVERED: The review discusses the basic science underpinnings of the HIF system and the clinical effects of the HIF system and its pharmacologic manipulation. EXPERT OPINION: Manipulation of the HIF system may improve outcomes in anemia by bypassing the effective iron deficiency found in anemia of chronic disease and by increasing red cell mass without supraphysiologic increases in erythropoietin. HIF-PH may also find a clinical use in the prevention of ischemia-reperfusion damage in strokes, cardiac ischemia, ischemic renal failure, etc.
Assuntos
Anemia/tratamento farmacológico , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Anemia/enzimologia , Animais , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Isquemia Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Pró-Colágeno-Prolina Dioxigenase/metabolismoRESUMO
AMP-activated protein kinase (AMPK) is an αßγ heterotrimer conserved throughout evolution and important for energy sensing in all eukaryote cells. AMPK controls metabolism and various cellular events in response to both hormones and changes in cellular energy status. The γ subunit senses intracellular energy status through the competitive binding of AMP and ATP. We show here that targeted disruption of the mouse AMPKγ1 gene (Prkag1) causes regenerative hemolytic anemia by increasing the sequestration of abnormal erythrocytes. Prkag1(-/-) mice displayed splenomegaly and iron accumulation due to compensatory splenic erythropoiesis and erythrophagocytosis. Moreover, AMPKγ1-deficient erythrocytes were highly resistant to osmotic hemolysis and poorly deformable in response to increasing shear stress, consistent with greater membrane rigidity. No change in cytoskeletal protein composition was observed; however, the phosphorylation level of adducin, a protein promoting the binding of spectrin to actin, was higher in AMPKγ1-deficient erythrocytes. Together, these results demonstrate that AMPKγ1 subunit is required for the maintenance of erythrocyte membrane elasticity.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Anemia/enzimologia , Membrana Eritrocítica/metabolismo , Esplenomegalia/enzimologia , Proteínas Quinases Ativadas por AMP/genética , Anemia/sangue , Anemia/genética , Anemia Hemolítica/enzimologia , Anemia Hemolítica/genética , Animais , Western Blotting , Elasticidade , Eritroblastos/metabolismo , Eritroblastos/patologia , Contagem de Eritrócitos , Deformação Eritrocítica , Feminino , Hiperplasia , Ferro/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/metabolismo , Baço/patologia , Esplenomegalia/sangue , Esplenomegalia/genéticaRESUMO
AMP-activated protein kinase (AMPK) plays a pivotal role in regulating cellular energy metabolism. We previously showed that AMPKalpha1-/- mice develop moderate anemia associated with splenomegaly and high reticulocytosis. Here, we report that splenectomy of AMPKalpha1-/- mice worsened anemia supporting evidence that AMPKalpha1-/- mice developed a compensatory response through extramedullary erythropoiesis in the spleen. Transplantation of bone marrow from AMPKalpha1-/- mice into wild-type recipients recapitulated the hematologic phenotype. Further, AMPKalpha1-/- red blood cells (RBC) showed less deformability in response to shear stress limiting their membrane flexibility. Thus, our results highlight the crucial role of AMPK to preserve RBC integrity.
Assuntos
Proteínas Quinases Ativadas por AMP/sangue , Deformação Eritrocítica/fisiologia , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Anemia/sangue , Anemia/enzimologia , Anemia/genética , Animais , Transplante de Medula Óssea , Deformação Eritrocítica/genética , Eritropoese , Hematopoese Extramedular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragilidade Osmótica , Esplenectomia , Esplenomegalia/sangue , Esplenomegalia/enzimologia , Esplenomegalia/genéticaRESUMO
Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock-out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic-transformed fibroblasts, results in a senescence-like G(1) cell cycle arrest, accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21(Cip). Notably, concomitant genetic inactivation of p53 or p21(Cip) indicates that Hdac1 and Hdac2 regulate p53-p21(Cip)-independent pathways critical for maintaining cell cycle progression. In vivo, we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte-megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism-based toxicities observed in patients treated with HDAC inhibitors.
Assuntos
Ciclo Celular , Hematopoese , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Anemia/enzimologia , Animais , Apoptose , Biocatálise , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Histona Desacetilase 1/deficiência , Histona Desacetilase 2/deficiência , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Trombocitopenia/enzimologia , Trombocitopenia/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 --> Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency.
Assuntos
Glucose-6-Fosfato Isomerase/metabolismo , Fosfolipídeos/biossíntese , Triglicerídeos/biossíntese , Anemia/enzimologia , Anemia/genética , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Células CHO , Cricetinae , Cricetulus , Glucose-6-Fosfato Isomerase/genética , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Doenças Neuromusculares/enzimologia , Doenças Neuromusculares/genética , Proteínas Associadas a Pancreatite , Fosfolipídeos/genética , Mutação Puntual , Triglicerídeos/genéticaAssuntos
Anemia/genética , Células-Tronco Hematopoéticas/imunologia , Integrases/genética , Receptores de Estrogênio/genética , Recombinação Genética/imunologia , Ativação Transcricional , Anemia/enzimologia , Anemia/imunologia , Animais , Técnicas de Introdução de Genes , Células-Tronco Hematopoéticas/metabolismo , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas/genética , RNA não Traduzido , Receptores de Estrogênio/metabolismo , Tamoxifeno/administração & dosagemRESUMO
The CreER(T2) for conditional gene inactivation has become increasingly used in reverse mouse genetics, which enables temporal regulation of Cre activity using a mutant estrogen binding domain (ER(T2)) to keep Cre inactive until the administration of tamoxifen. In this study, we present the severe toxicity of ubiquitously expressed CreER(T2) in adult mice and embryos. The toxicity of Cre recombinase or CreER(T2) in vitro or in vivo organisms are still less sufficiently recognized considering the common use of Cre/loxP system, though the toxicity might compromise the phenotypic analysis of the gene of interest. We analyzed two independent lines in which CreER(T2) is knocked-in into the Rosa26 locus (R26CreER(T2) mice), and both lines showed thymus atrophy, severe anemia, and illegitimate chromosomal rearrangement in hematopoietic cells after the administration of tamoxifen, and demonstrated complete recovery of hematological toxicity in adult mice. In the hematopoietic tissues in R26CreER(T2) mice, reduced proliferation and increased apoptosis was observed after the administration of tamoxifen. Flow cytometric analysis revealed that CreER(T2) toxicity affected several hematopoietic lineages, and that immature cells in these lineages tend to be more sensitive to the toxicity. In vitro culturing of hematopoietic cells from these mice further demonstrated the direct toxicity of CreER(T2) on growth and differentiation of hematopoietic cells. We further demonstrated the cleavage of the putative cryptic/pseudo loxP site in the genome after the activation of CreER(T2) in vivo. We discussed how to avoid the misinterpretation of the experimental results from potential toxic effects due to the activated CreER(T2).
Assuntos
Anemia/genética , Aberrações Cromossômicas , Cromossomos/genética , Inibidores do Crescimento/genética , Células-Tronco Hematopoéticas/patologia , Integrases/genética , Receptores de Estrogênio/genética , Recombinação Genética , Anemia/enzimologia , Anemia/patologia , Animais , Técnicas de Cocultura , Proteínas da Matriz Extracelular/genética , Feminino , Técnicas de Introdução de Genes , Inibidores do Crescimento/toxicidade , Células-Tronco Hematopoéticas/imunologia , Integrases/metabolismo , Integrases/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese Insercional , Proteína-Lisina 6-Oxidase/genética , Receptores de Estrogênio/metabolismo , Células Estromais/patologiaRESUMO
Evaluation of two patients with transfusion dependent anemia revealed RBC pyruvate kinase to be 33% and 41% of the mean normal value, with normal or high values of other RBC enzymes. Parental PK activities were just below normal in three of four of the parents. Subsequent DNA analysis revealed both patients to be compound heterozygotes for PKLR gene mutations, two of which are previously undescribed. Borderline low pyruvate kinase activities with increased in other RBC enzyme activities should prompt consideration of measurement of parental enzyme activities, and confirmation by DNA analysis if available.
Assuntos
Anemia/enzimologia , Eritrócitos/enzimologia , Piruvato Quinase/deficiência , Piruvato Quinase/genética , Anemia/genética , DNA/genética , Feminino , Heterozigoto , Humanos , Lactente , Masculino , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: To evaluate the status and diagnostic utility of antioxidant paraoxonase 1 (PON1) in gastroesophageal cancers. DESIGN AND METHODS: PON1's arylesterase/paraoxonase activities and phenotype were determined in 82 cancers and 57 controls, and related to clinicopathological features, anemia and cachexia coexistence, cholesterol levels, liver function panel, and inflammatory and angiogenic indices. RESULTS: PON1's activities were decreased in cancer. Arylesterase activity correlated with cancer T and N stages, being an N1-independent predictor. Both activities correlated with transferrin, albumin, CRP and inflammation-based Glasgow Prognostic Score, and arylesterase activity also with interleukin-6 and midkine. Phenotype A (Q192 homozygotes) was associated with lower transferrin and higher TNF-alpha concentrations. PON1's arylesterase activity reflected anemia severity, being correlated with hemoglobin, hematocrit, and iron. PON1 arylesterase activity negatively correlated with alkaline phosphatase and gamma-glutamyl transferase, but not with bilirubin, aminotransferases, HDL or LDL cholesterol. PON1 moderately indicated cancer presence and regional metastasis. CONCLUSIONS: PON1 activity decreases in gastroesophageal cancers and corresponds to inflammation severity and cancer-related anemia. PON1 decrease indicates lymph node metastasis, but its moderate predictive power does not recommend PON1 determination alone for clinical application.
Assuntos
Arildialquilfosfatase/sangue , Neoplasias Esofágicas/complicações , Neoplasias Esofágicas/enzimologia , Síndromes Paraneoplásicas/enzimologia , Síndromes Paraneoplásicas/etiologia , Neoplasias Gástricas/complicações , Neoplasias Gástricas/enzimologia , Adenocarcinoma/complicações , Adenocarcinoma/enzimologia , Adenocarcinoma/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia/sangue , Anemia/enzimologia , Anemia/etiologia , Carcinoma de Células Escamosas/complicações , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/fisiopatologia , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/fisiopatologia , Feminino , Humanos , Inflamação/enzimologia , Inflamação/etiologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Síndromes Paraneoplásicas/patologia , Síndromes Paraneoplásicas/fisiopatologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/fisiopatologiaRESUMO
Objetivo: destacar a infecção por parvovirus B19 como uma das causas de anemia em lactentes com doença hemolítica de base...
Objective: to highlight B19 parvovirus infection as a cause of anemia in infants with underlying hemolytic disease...