RESUMEN
Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation-deficient hearts from p32-knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD+ ) biosynthetic enzymes-Nmnat3 and Nampt-and NAD+ levels were decreased, suggesting that NAD+ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α-Nmnat3-mediated NAD+ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD+ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD+ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.
Asunto(s)
Lisosomas/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/genética , NAD/metabolismo , Animales , Células Cultivadas , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/deficiencia , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Fosfoglicerato Quinasa/metabolismoRESUMEN
An autism-associated gene Shank3 encodes multiple splicing isoforms, Shank3a-f. We have recently reported that Shank3a/b-knockout mice were more susceptible to kainic acid-induced seizures than wild-type mice at 4 weeks of age. Little is known, however, about how the N-terminal and ankyrin repeat domains (NT-Ank) of Shank3a/b regulate multiple molecular signals in the developing brain. To explore the functional roles of Shank3a/b, we performed a mass spectrometry-based proteomic search for proteins interacting with GFP-tagged NT-Ank. In this study, NT-Ank was predicted to form a variety of complexes with a total of 348 proteins, in which RNA-binding (n = 102), spliceosome (n = 22), and ribosome-associated molecules (n = 9) were significantly enriched. Among them, an X-linked intellectual disability-associated protein, Nono, was identified as a NT-Ank-binding protein. Coimmunoprecipitation assays validated the interaction of Shank3 with Nono in the mouse brain. In agreement with these data, the thalamus of Shank3a/b-knockout mice aberrantly expressed splicing isoforms of autism-associated genes, Nrxn1 and Eif4G1, before and after seizures with kainic acid treatment. These data indicate that Shank3 interacts with multiple RNA-binding proteins in the postnatal brain, thereby regulating the homeostatic expression of splicing isoforms for autism-associated genes after birth.
RESUMEN
The 3243A > G in mtDNA is a representative mutation in mitochondrial diseases. Mitochondrial protein synthesis is impaired due to decoding disorder caused by severe reduction of 5-taurinomethyluridine (τm5U) modification of the mutant mt-tRNALeu(UUR) bearing 3243A > G mutation. The 3243A > G heteroplasmy in peripheral blood reportedly decreases exponentially with age. Here, we found three cases with mild respiratory symptoms despite bearing high rate of 3243A > G mutation (>90%) in blood mtDNA. These patients had the 3290T > C haplotypic mutation in addition to 3243A > G pathogenic mutation in mt-tRNALeu(UUR) gene. We generated cybrid cells of these cases to examine the effects of the 3290T > C mutation on mitochondrial function and found that 3290T > C mutation improved mitochondrial translation, formation of respiratory chain complex, and oxygen consumption rate of pathogenic cells associated with 3243A > G mutation. We measured τm5U frequency of mt-tRNALeu(UUR) with 3243A > G mutation in the cybrids by a primer extension method assisted with chemical derivatization of τm5U, showing that hypomodification of τm5U was significantly restored by the 3290T > C haplotypic mutation. We concluded that the 3290T > C is a haplotypic mutation that suppresses respiratory deficiency of mitochondrial disease by restoring hypomodified τm5U in mt-tRNALeu(UUR) with 3243A > G mutation, implying a potential therapeutic measure for mitochondrial disease associated with pathogenic mutations in mt-tRNAs.
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Síndrome MELAS , Enfermedades Mitocondriales , Humanos , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , ARN de Transferencia de Leucina/metabolismo , Taurina , Haplotipos , Mutación , ADN Mitocondrial/genética , Enfermedades Mitocondriales/genéticaRESUMEN
Heart failure (HF) is a leading cause of death and repeated hospitalizations and often involves cardiac mitochondrial dysfunction. However, the underlying mechanisms largely remain elusive. Here, using a mouse model in which myocardial infarction (MI) was induced by coronary artery ligation, we show the metabolic basis of mitochondrial dysfunction in chronic HF. Four weeks after ligation, MI mice showed a significant decrease in myocardial succinyl-CoA levels, and this decrease impaired the mitochondrial oxidative phosphorylation (OXPHOS) capacity. Heme synthesis and ketolysis, and protein levels of several enzymes consuming succinyl-CoA in these events, were increased in MI mice, while enzymes synthesizing succinyl-CoA from α-ketoglutarate and glutamate were also increased. Furthermore, the ADP-specific subunit of succinyl-CoA synthase was reduced, while its GDP-specific subunit was almost unchanged. Administration of 5-aminolevulinic acid, an intermediate in the pathway from succinyl-CoA to heme synthesis, appreciably restored succinyl-CoA levels and OXPHOS capacity and prevented HF progression in MI mice. Previous reports also suggested the presence of succinyl-CoA metabolism abnormalities in cardiac muscles of HF patients. Our results identified that changes in succinyl-CoA usage in different metabolisms of the mitochondrial energy production system is characteristic to chronic HF, and although similar alterations are known to occur in healthy conditions, such as during strenuous exercise, they may often occur irreversibly in chronic HF leading to a decrease in succinyl-CoA. Consequently, nutritional interventions compensating the succinyl-CoA consumption are expected to be promising strategies to treat HF.
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Insuficiencia Cardíaca , Infarto del Miocardio , Acilcoenzima A , Adenosina Difosfato/metabolismo , Ácido Aminolevulínico , Metabolismo Energético , Glutamatos/metabolismo , Insuficiencia Cardíaca/metabolismo , Hemo/metabolismo , Humanos , Ácidos Cetoglutáricos , Fosforilación OxidativaRESUMEN
OBJECTIVES: To evaluate the long-term impact of immunosuppressive therapeutic agents on antibody response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) mRNA vaccination in patients with autoimmune rheumatic diseases (AIRD) in order to propose a strategy for annual vaccination. METHODS: This prospective multicentre cohort study evaluated the humoral response to second and third BNT162b2 and/or mRNA-1273 vaccines in 382 Japanese AIRD patients classified into 12 different medication groups and in 326 healthy controls (HCs). The third vaccination was administered six months after the second vaccination. Antibody titres were measured using the Elecsys Anti-SARS-CoV-2 S assay. RESULTS: The seroconversion rate and antibody titres were lower in AIRD patients than in HCs 3-6 weeks after the second vaccination and 3-6 weeks after the third vaccination. Seroconversion rates were <90% after the third vaccination in patients receiving mycophenolate mofetil and rituximab. Antibody levels after the third vaccination were significantly lower in the groups prescribed TNF inhibitor with or without methotrexate, abatacept and rituximab or cyclophosphamide than those of HCs in a multivariate analysis adjusting for age, sex, and glucocorticoid dosage. The third vaccination induced an adequate humoral response in patients treated with sulfasalazine, bucillamine, methotrexate monotherapy, iguratimod, interleukin-6 inhibitors or calcineurin inhibitors including tacrolimus. CONCLUSIONS: Repeated vaccinations in many immunosuppressed patients produced antibody responses similar to those observed in HCs. In contrast, annual vaccination in patients receiving TNF inhibitors, abatacept, mycophenolate mofetil and rituximab may require caution.
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COVID-19 , Enfermedades Reumáticas , Humanos , Vacunas contra la COVID-19 , Rituximab , Abatacept , Vacuna BNT162 , Estudios de Cohortes , Metotrexato/uso terapéutico , Ácido Micofenólico , Estudios Prospectivos , COVID-19/prevención & control , SARS-CoV-2 , Terapia de Inmunosupresión , Inmunosupresores/uso terapéutico , Enfermedades Reumáticas/tratamiento farmacológico , Vacunación , AnticuerposRESUMEN
OBJECTIVES: To determine the optimal management for early-onset thrombophilia (EOT), the genetic and clinical features of protein C (PC)-, protein S (PS)-, or antithrombin (AT)-deficient patients of ≤20 years of age were studied in Japan. METHODS/RESULTS: Clinical and genetic information of all genetically diagnosed cases was collected through the prospective, retrospective study, and literature review. One-hundred-one patients had PC (n = 55), PS (n = 29), or AT deficiency (n = 18). One overlapping case had PC- and PS-monoallelic variant. Fifty-five PC-deficient patients (54%) had 26 monoallelic or 29 biallelic variant(s), and 29 (29%) PS-deficient patients had 20 monoallelic or nine biallelic variant(s). None of the patients had AT-biallelic variants. The frequent low-risk allele p.K193del (PC-Tottori) was found in five patients with monoallelic (19%) but not 29 with biallelic variant(s). The most common low-risk allele p.K196E (PS-Tokushima) was found in five with monoallelic (25%) and six with biallelic variant(s) (67%). One exceptional de novo PC variant was found in 32 families with EOT. Only five parents had a history of thromboembolism. Thrombosis concurrently developed in three mother-newborn pairs (two PC deficiency and one AT deficiency). The prospective cohort revealed the outcomes of 35 patients: three deaths with PC deficiency and 20 complication-free survivors. Neurological complications were more frequently found in patients with PC-biallelic variants than those with PC-, PS-, or AT-monoallelic variants (73% vs. 24%, p = .019). CONCLUSIONS: We demonstrate the need for elective screening for EOT targeting PC deficiency in Japan. Early prenatal diagnosis of PC deficiency in mother-infant pairs may prevent perinatal thrombosis in them.
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Deficiencia de Antitrombina III , Deficiencia de Proteína C , Deficiencia de Proteína S , Trombofilia , Trombosis , Recién Nacido , Femenino , Embarazo , Humanos , Estudios Retrospectivos , Estudios Prospectivos , Japón/epidemiología , Deficiencia de Proteína S/complicaciones , Deficiencia de Proteína S/diagnóstico , Deficiencia de Proteína S/genética , Trombofilia/complicaciones , Trombosis/etiología , Trombosis/genética , Deficiencia de Proteína C/genética , Deficiencia de Proteína C/complicaciones , Proteína C/genética , Anticoagulantes , Antitrombina III , AntitrombinasRESUMEN
Mitochondrial tRNAs are indispensable for the intra-mitochondrial translation of genes related to respiratory subunits, and mutations in mitochondrial tRNA genes have been identified in various disease patients. However, the molecular mechanism underlying pathogenesis remains unclear due to the lack of animal models. Here, we established a mouse model, designated 'mito-mice tRNALeu(UUR)2748', that carries a pathogenic A2748G mutation in the tRNALeu(UUR) gene of mitochondrial DNA (mtDNA). The A2748G mutation is orthologous to the human A3302G mutation found in patients with mitochondrial diseases and diabetes. A2748G mtDNA was maternally inherited, equally distributed among tissues in individual mice, and its abundance did not change with age. At the molecular level, A2748G mutation is associated with aberrant processing of precursor mRNA containing tRNALeu(UUR) and mt-ND1, leading to a marked decrease in the steady-levels of ND1 protein and Complex I activity in tissues. Mito-mice tRNALeu(UUR)2748 with ≥50% A2748G mtDNA exhibited age-dependent metabolic defects including hyperglycemia, insulin insensitivity, and hepatic steatosis, resembling symptoms of patients carrying the A3302G mutation. This work demonstrates a valuable mouse model with an inheritable pathological A2748G mutation in mt-tRNALeu(UUR) that shows metabolic syndrome-like phenotypes at high heteroplasmy level. Furthermore, our findings provide molecular basis for understanding A3302G mutation-mediated mitochondrial disorders.
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Enfermedades Mitocondriales , ARN de Transferencia de Leucina , Humanos , Animales , Ratones , ARN de Transferencia de Leucina/metabolismo , Enfermedades Mitocondriales/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Mutación , Procesamiento Postranscripcional del ARNRESUMEN
Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising â¼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Replicación del ADN , ADN de Hongos/genética , Inestabilidad Genómica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteína 9 Asociada a CRISPR/química , Variaciones en el Número de Copia de ADN , ADN de Hongos/metabolismo , ADN de Cadena Simple/genética , Edición Génica , Genes Fúngicos , Metalotioneína/genética , Mutagénesis , Niacinamida/farmacología , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Secuencias Repetidas en TándemRESUMEN
OBJECTIVES: To evaluate the impact of medication on antibody response to severe acute respiratory syndrome coronavirus-2 mRNA vaccines in Japanese patients with rheumatic diseases. METHODS: This prospective multicentre cohort study evaluated the humoral response in 12 different medication groups. Antibody levels before the first vaccination and 3-6 weeks after the second vaccination were measured using the Elecsys Anti-SARS-CoV-2 S assay. Statistical analysis included comparing antibody titres among the different medication groups using the Kruskal-Wallis test followed by the Bonferroni-Dunn test and multiple linear regression analysis. RESULTS: 295 patients were analysed. The seroconversion rate was 92.2% and the median antibody titre was 255 U/ml (interquartile range, 34.1-685) after the second mRNA vaccination. Antibody levels were significantly lower in the groups treated with Tumour necrosis factor inhibitor with methotrexate, abatacept, mycophenolate mofetil (MMF), MMF or mizoribine combined with calcineurin inhibitor, and rituximab or cyclophosphamide compared with those treated with sulfasalazine and/or bucillamine or calcineurin inhibitor (p < 0.01). The correlation between antibody titre and treatment was significant after adjusting for age, gender, and glucocorticoid dose (p < 0.01). CONCLUSIONS: Additional early vaccination is required in patients treated with Tumour necrosis factor inhibitor and methotrexate, abatacept, MMF, MMF or mizoribine combined with calcineurin inhibitor and rituximab or cyclophosphamide.
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COVID-19 , Enfermedades Reumáticas , Humanos , Inmunosupresores/uso terapéutico , Rituximab , Metotrexato/uso terapéutico , Abatacept , Inhibidores de la Calcineurina , Japón , Formación de Anticuerpos , Vacunas contra la COVID-19/uso terapéutico , Estudios Prospectivos , Estudios de Cohortes , Inhibidores del Factor de Necrosis Tumoral/uso terapéutico , COVID-19/prevención & control , SARS-CoV-2 , Ácido Micofenólico/uso terapéutico , Ciclofosfamida , Enfermedades Reumáticas/tratamiento farmacológicoRESUMEN
Early detection and long-term monitoring are important for urothelial carcinoma of the bladder (UCB). Urine cytology and existing markers have insufficient diagnostic performance. Here, we examined medium-sized extracellular vesicles (EVs) in urine to identify specific markers for UCB and evaluated their usefulness as diagnostic material. To identify specific markers in urinary EVs derived from UCB, we undertook shotgun proteomics using urine from four UCB patients and four healthy subjects. Next, 29 healthy specimens, 18 noncancer specimens, and 33 UCB specimens, all from men, were analyzed for urinary EVs by flow cytometry to evaluate the diagnostic performance of UCB-specific EVs. Nanoparticle-tracking analysis indicated that the size of EVs extracted from urine was mostly <400 nm. By shotgun proteomics, we detected several proteins characteristic of UCB and found that carcinoembryonic antigen-related adhesion molecule (CEACAM) proteins were increased in patients. Flow cytometric analysis revealed that the degree of expression of CEACAM1, CEACAM5, and CEACAM6 proteins on the surface of EVs varied among patients. Extracellular vesicles expressing CEACAM proteins also expressed mucin 1, suggesting that they were derived from tumorigenic uroepithelial cells. The number of EVs expressing CEACAM1, 5, and 6 proteins was significantly increased in UCB (mean ± SD, 8.6 ± 13%) compared to non-UCB (0.69 ± 0.46) and healthy (0.46 ± 0.34) by flow cytometry. The results of receiver operating characteristic (ROC) analysis showed a good score of area under the ROC curve of 0.907. We identified EVs that specifically express CEACAM proteins in urine and have potential for diagnostic applications. These EVs are potential targets in a new liquid biopsy test for UCB patients.
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Carcinoma de Células Transicionales , Vesículas Extracelulares , Neoplasias de la Vejiga Urinaria , Antígeno Carcinoembrionario/metabolismo , Carcinoma de Células Transicionales/metabolismo , Vesículas Extracelulares/metabolismo , Citometría de Flujo , Humanos , Masculino , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
BACKGROUND: A patient repeatedly developed bacteraemia despite the continuous use of antibiotics. We obtained two Klebsiella pneumoniae isolates from the patient's blood on Days 72 and 105 after hospitalization. Each of the two isolates belonged to ST45, but while the first isolate was susceptible to most antibiotics, the second one was resistant to multiple drugs including carbapenems. OBJECTIVES: To identify the genetic differences between the two isolates and uncover alterations formed by the within-host bacterial evolution leading to the antimicrobial resistance. METHODS: Whole-genome comparison of the two isolates was carried out to identify their genetic differences. We then profiled their outer membrane proteins related to membrane permeability to drugs. To characterize a ramR gene mutation found in the MDR isolate, its WT and mutant genes were cloned and expressed in the MDR isolate. RESULTS: The two isolates showed only three genomic differences, located in mdoH, ramR and upstream of ompK36. In the MDR isolate, a single nucleotide substitution in the ompK36 upstream region attenuated OmpK36 expression. A single amino acid residue insertion in RamR in the MDR isolate impaired its function, leading to the down-regulation of OmpK35 and the subsequent up-regulation of the AcrAB-TolC transporter, which may contribute to the MDR. CONCLUSIONS: We identified very limited genomic changes in the second K. pneumoniae clone during within-host evolution, but two of the three identified mutations conferred the MDR phenotype on the clone by modulating drug permeability.
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Infecciones por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Clonales/metabolismo , Resistencia a Múltiples Medicamentos , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Proteínas de Transporte de Membrana , Pruebas de Sensibilidad Microbiana , Mutación , beta-Lactamasas/genéticaRESUMEN
OBJECTIVE: To clarify the incidence and genetic risk of neonatal-thromboembolism, we conducted a nationwide study exploring the impact of thrombophilia on neonatal-thromboembolism in Japan. STUDY DESIGN: A questionnaire survey was conducted for perinatal centers in Japan, focusing on the clinical expression, genotype, treatment, and outcome of patients who developed thromboembolism within 28 days of birth from 2014 to 2018. RESULTS: The estimated incidence of neonatal-thromboembolism was 0.39 cases per 10 000 live births. Intracranial lesions and purpura fulminans occurred in 66 and 5 of 77 patients, respectively. Fifty-eight (75.3%) infants presented within 3 days after birth. Four (5.2%) died, and 14 (18.2%) survived with disability. At the diagnosis, <20% plasma activity of protein C was noted in 16 infants, protein S (in 2), and antithrombin (in 1). Thirteen genetic tests identified 4 biallelic and 5 monoallelic protein C-variants but no protein S- or antithrombin-variants. Protein C-variants had purpura fulminans (P < .01), ocular bleeding (P < .01), positive-family history (P = .01), and death or disability (P = .03) more frequently than others. Protein C-variants were independently associated with disability (OR 5.74, 95% CI 1.16-28.4, P = .03) but not death. Four biallelic variants had serious thrombotic complications of neurologic disability, blindness, and/or amputation. Three monoallelic variants survived without complications. The only protein C-variant death was an extremely preterm heterozygote infant. CONCLUSIONS: Monoallelic protein C-variants had a higher incidence of neonatal-thromboembolism than biallelic variants. Thrombophilia genetic testing should be performed in the setting of neonatal-thromboembolism and low protein C to identify the underlying genetic defect.
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Deficiencia de Proteína C/complicaciones , Tromboembolia/epidemiología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Recién Nacido , Japón , Masculino , Deficiencia de Proteína C/genética , Estudios Retrospectivos , Factores de Riesgo , Encuestas y Cuestionarios , Tromboembolia/genéticaRESUMEN
Developmental and epileptic encephalopathy (DEE) represents a group of neurodevelopmental disorders characterized by infantile-onset intractable seizures and unfavorable prognosis of psychomotor development. To date, hundreds of genes have been linked to the onset of DEE. GNAO1 is a DEE-associated gene encoding the alpha-O1 subunit of guanine nucleotide-binding protein (GαO ). Despite the increasing number of reported children with GNAO1 encephalopathy, the molecular mechanisms underlying their neurodevelopmental phenotypes remain elusive. We herein present that co-immunoprecipitation and mass spectrometry analyses identified another DEE-associated protein, SPTAN1, as an interacting partner of GαO . Silencing of endogenous Gnao1 attenuated the neurite outgrowth and calcium-dependent signaling. Inactivation of GNAO1 in human-induced pluripotent stem cells gave rise to anomalous brain organoids that only weakly expressed SPTAN1 and Ankyrin-G. Furthermore, GNAO1-deficient organoids failed to conduct synchronized firing to adjacent neurons. These data indicate that GαO and other DEE-associated proteins organize the cytoskeletal remodeling and functional polarity of neurons in the developing brain.
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Citoesqueleto/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Animales , Encéfalo/metabolismo , Encefalopatías/metabolismo , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Trastornos del Neurodesarrollo/metabolismo , Neuronas/metabolismo , FenotipoRESUMEN
OBJECTIVE: To investigate the somatic mutation profiles of testicular germ cell tumors in Japanese men. METHODS: We analyzed the somatic missense mutation profile of testicular germ cell tumors among 21 Japanese men with seminoma (n = 14), pure embryonic carcinoma (n = 3) and mixed testicular germ cell tumor (n = 4) by targeted next-generation sequencing of 409 cancer-related genes covering 1.23 Mb of the genome. RESULTS: We identified a total of 22 missense mutations in 21 primary testicular germ cell tumor samples (0.89 mutations/Mb), of which seven mutations were confirmed to be absent from the germline. KIT:p.Asn822Tyr, KIT:p.Leu576Pro, PIK3CA:p.Glu542Lys and FBXW7:p.Arg505His were statistically and functionally potential. A total of 18 missense mutations were previously unknown in testicular germ cell tumors. PDGFRA amplification from one patient with seminoma was detected. KIT, BCR,PIK3CG, PIK3CA and PDGFRA mutations involved in aberrant signaling of the KIT-PI3K-AKT pathway was detected in 27.3% of detected mutations. CONCLUSIONS: The present investigation identified a low mutation rate in testicular germ cell tumors among Asian patients, 18 novel mutations and PDGFRA amplification. Limitations of the present study are the small sample and missing normal DNA for some testicular germ cell tumors.
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Neoplasias de Células Germinales y Embrionarias , Neoplasias Testiculares , Exones , Genómica , Humanos , Japón , Masculino , Mutación , Neoplasias de Células Germinales y Embrionarias/genética , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-kit/genética , Neoplasias Testiculares/genéticaRESUMEN
Mitochondrial cardiomyopathy can be described as a condition characterized by abnormal heart-muscle structure and/or function, secondary to mutations in nuclear or mitochondrial DNA. Its severity can range from subclinical to critical conditions. We presented three cases of mitochondrial cardiomyopathy with m.3243A > G mutation and compared the clinical manifestations with the histological findings for each of these cases. All cases showed cardiac hypertrophy, juvenile-onset diabetes mellitus, and hearing loss. Case 1 (43-year-old male) showed less cardiac involvement and shorter duration of mitochondrial disease-related symptoms than case 2 (67-year-old female) and case 3 (51-year-old male), who showed the most advanced cardiac condition and longest duration from the manifestation of heart failure. The histological findings revealed that cardiomyocytes from case 1 showed no hypertrophy and mitochondrial degeneration in electron microscopy. Alternatively, cases 2 and 3 showed hypertrophy in their cardiomyocytes, and mitochondrial degeneration (e.g. onion-like lesions, swollen cristae, and lamellar bodies) was most apparent in case 3. These results suggested that mitochondrial degeneration, as evaluated by electron microscopy, might be correlated with impaired heart function in patients with mitochondrial cardiomyopathy.
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Cardiomiopatías/genética , ADN Mitocondrial/genética , Sordera/genética , Diabetes Mellitus/genética , Mitocondrias/patología , Enfermedades Mitocondriales/genética , Mutación , Adulto , Anciano , Cardiomiopatías/diagnóstico , Cardiomiopatías/patología , Sordera/diagnóstico , Sordera/patología , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/patología , Femenino , Humanos , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Mitocondrias/ultraestructura , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/patología , SíndromeRESUMEN
Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp-/- mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp-/- MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia.
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Cardiomiopatías/genética , Proteínas Portadoras/genética , Transporte de Electrón/fisiología , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Mutación , Adulto , Edad de Inicio , Anciano , Alelos , Secuencia de Aminoácidos , Animales , Cardiomiopatías/complicaciones , Cardiomiopatías/patología , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Células Cultivadas , Preescolar , Estudios de Cohortes , ADN Mitocondrial , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Recién Nacido , Masculino , Ratones , Persona de Mediana Edad , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/patología , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa , Linaje , Conformación Proteica , Homología de Secuencia , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Oxidative damage results in protein modification and is observed in many diseases, such as heart failure and renal insufficiency. Human serum albumin is an index of oxidative change and is conventionally measured using high-performance liquid chromatography (HPLC). Although this method is more sensitive than the colorimetric method, it is time-consuming for clinical practice and the sera must be stored at -80°C before analysis. To overcome these limitations, in the present study we developed a new reagent for a more rapid and convenient quantification of oxidative stress, involving determination of the ratio of human nonmercaptalbumin to total albumin using a colorimetric method with bromocresol purple. The clinical utility of the developed reagent was confirmed by demonstrating the consistently higher oxidative stress levels in dialysis patients than in healthy control subjects, matching the results of the conventional HPLC method. This novel approach could be a valuable tool for immediate estimation of the state of oxidative stress during the course of disease and treatment, and could aid clinical treatment decisions.
RESUMEN
Evidence of the molecular epidemiology of thrombophilia is growing, and the clinical management of adult thromboembolism patients has recently made significant progress. On the other hand, there is little or no evidence concerning the genetic variation, treatment, and prophylaxis of thromboembolism development in the early life stage. The clinical presentation of early-onset thrombosis/thrombophilia, which mostly occurs in newborns and adolescents, differs from that in cases of adult-onset. Recurrent purpura fulminans and/or intracranial hemorrhage/infarction leads to dangerous lifelong complications. As in the setting of cancer genomic medicine, germline variants require determination for the individualized control of early-onset thrombophilia. The genetic predisposition to thrombosis varies among ethnicities. In the Japanese population, the protein S variant (PS-Tokushima, K196E) has attracted attention as the cause of a common and low-risk prothrombotic predisposition in adults, while protein C deficiency greatly impacts the onset of pediatric thrombosis. In 2020, 3 years after the registration of idiopathic thrombosis as a designated intractable disease, genetic tests have been promulgated for health insurance portability. Disease-specific therapy for early-onset thrombophilia is crucial. Here, we review the genetic heterogeneity, prophylaxis, and treatment strategy of the rare subgroups of severe heritable thrombosis conditions in Japan.
Asunto(s)
Deficiencia de Proteína C , Trombofilia , Trombosis , Adolescente , Edad de Inicio , Niño , Genotipo , Humanos , Recién Nacido , Japón , Factores de Riesgo , Trombofilia/diagnóstico , Trombofilia/tratamiento farmacológico , Trombofilia/genética , Trombosis/diagnóstico , Trombosis/etiologíaRESUMEN
BACKGROUND: Linoleic acid is the major fatty acid moiety of cardiolipin, which is central to the assembly of components involved in mitochondrial oxidative phosphorylation (OXPHOS). Although linoleic acid is an essential nutrient, its excess intake is harmful to health. On the other hand, linoleic acid has been shown to prevent the reduction in cardiolipin content and to improve mitochondrial function in aged rats with spontaneous hypertensive heart failure (HF). In this study, we found that lower dietary intake of linoleic acid in HF patients statistically correlates with greater severity of HF, and we investigated the mechanisms therein involved. METHODS: HF patients, who were classified as New York Heart Association (NYHA) functional class I (n = 45), II (n = 93), and III (n = 15), were analyzed regarding their dietary intakes of different fatty acids during the one month prior to the study. Then, using a mouse model of HF, we confirmed reduced cardiolipin levels in their cardiac myocytes, and then analyzed the mechanisms by which dietary supplementation of linoleic acid improves cardiac malfunction of mitochondria. RESULTS: The dietary intake of linoleic acid was significantly lower in NYHA III patients, as compared to NYHA II patients. In HF model mice, both CI-based and CII-based OXPHOS activities were affected together with reduced cardiolipin levels. Silencing of CRLS1, which encodes cardiolipin synthetase, in cultured cardiomyocytes phenocopied these events. Feeding HF mice with linoleic acid improved both CI-based and CII-based respiration as well as left ventricular function, together with an increase in cardiolipin levels. However, although assembly of the respirasome (i.e., CI/CIII2/CIV complex), as well as assembly of CII subunits and the CIII2/CIV complex statistically correlated with cardiolipin levels in cultured cardiomyocytes, respirasome assembly was not notably restored by dietary linoleic acid in HF mice. Therefore, although linoleic acid may significantly improve both CI-based and CII-based respiration of cardiomyocytes, respirasomes impaired by HF were not easily repaired by the dietary intake of linoleic acid. CONCLUSIONS: Dietary supplement of linoleic acid is beneficial for improving cardiac malfunction in HF, but is unable to completely cure HF.
Asunto(s)
Complejo III de Transporte de Electrones/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Insuficiencia Cardíaca/metabolismo , Ácido Linoleico/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Anciano , Animales , Cardiolipinas/metabolismo , Complejo II de Transporte de Electrones/química , Femenino , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Ácido Linoleico/metabolismo , Masculino , Ratones , Mitocondrias Cardíacas/metabolismo , Subunidades de Proteína/metabolismoRESUMEN
MDCK-induced amino acid (AA) mutation, such as D151G/N in the neuraminidase (NA) of influenza A/H3N2 viruses, is of concern. MDCK-SIAT1 cells, modified derivatives with an increased expression of α2,6-linked sialic acid receptors are increasingly used due to their superiority in a viral recovery. However, MDCK-SIAT1 induced AA mutations have not been fully examined. In this study, we compared NA and hemagglutinin (HA) genes of recent circulating influenza viruses isolated after an MDCK-SIAT1 passage with those directly obtained from the original samples. A total of 22 samples collected during the 2016-17 seasons included 9 A/H3N2, 5 H1N1pdm, and 8 B viruses. None of the deduced AA mutations in the NA or HA segments were detected after an MDCK-SIAT1 passage, except for one AA mutation in the NA of an influenza B virus sample. NA D151G/N changes were not seen in any of the MDCK-SIAT1 passaged A/H3N2 viruses, even in the small variants analysis conducted using deep sequencing. AA mutations induced by an MDCK-SIAT1 passage are currently rare, although careful observation is needed in the future.