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Polymorphisms of HLA genes, which play a crucial role in presenting peptides with diverse sequences in their peptide-binding pockets, are also thought to affect HLA gene expression, as many studies have reported associations between HLA gene polymorphisms and their expression levels. In this study, we devised an ectopic expression assay for the HLA class I genes in the context of the entire gene, and used the assay to show that the HLA-C*03:03:01 and C*04:01:01 polymorphic differences observed in association studies indeed cause different levels of RNA expression. Subsequently, we investigated the C*03:23N null allele, which was previously noted for its reduced expression, attributed to an alternate exon 3 3' splice site generated by G/A polymorphism at position 781 within the exon 3. We conducted a thorough analysis of the splicing patterns of C*03:23N, and revealed multiple aberrant splicing, including the exon 3 alternative splicing, which overshadowed its canonical counterpart. After confirming a significant reduction in RNA levels caused by the G781A alteration in our ectopic assay, we probed the function of the G-rich sequence preceding the canonical exon 3 3' splice site. Substituting the G-rich sequence with a typical pyrimidine-rich 3' splice site sequence on C*03:23N resulted in a marked elevation in RNA levels, likely due to the enhanced preference for the canonical exon 3 3' splice site over the alternate site. However, the same substitution led to a reduction in RNA levels for C*03:03:01. These findings suggested the dual roles of the G-rich sequence in RNA expression, and furthermore, underscore the importance of studying polymorphism effects within the framework of the entire gene, extending beyond conventional mini-gene reporter assays.
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Antígenos HLA-C , Nucleótidos , Antígenos HLA-C/genética , Sitios de Empalme de ARN/genética , Empalme del ARN , Empalme AlternativoRESUMEN
The clustered regularly interspersed palindromic repeats (CRISPR) system is a powerful genome-editing tool to modify genomes, virtually in any species. The CRISPR tool has now been utilized in many areas of medical research, including gene therapy. Although several proof-of-concept studies show the feasibility of in vivo gene therapy applications for correcting disease-causing mutations, and new and improved tools are constantly being developed, there are not many choices of suitable reporter models to evaluate genome editor tools and their delivery methods. Here, we developed and validated reporter mouse models containing a single copy of disrupted EGFP (ΔEGFP) via frameshift mutations. We tested several delivery methods for validation of the reporters, and we demonstrated their utility to assess both non-homologous end-joining (NHEJ) and via homology-directed repair (HDR) processes in embryos and in somatic tissues. With the use of the reporters, we also show that hydrodynamic delivery of ribonucleoprotein (RNP) with Streptococcus pyogenes (Sp)Cas9 protein mixed with synthetic guide RNA (gRNA) elicits better genome-editing efficiencies than the plasmid vector-based system in mouse liver. The reporters can also be used for assessing HDR efficiencies of the Acidaminococcus sp. (As)Cas12a nuclease. The results suggest that the ΔEGFP mouse models serve as valuable tools for evaluation of in vivo genome editing.
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The highly polymorphic human major histocompatibility complex (MHC) also known as the human leukocyte antigen (HLA) encodes class I and II genes that are the cornerstone of the adaptive immune system. Their unique diversity (>25,000 alleles) might affect the outcome of any transplant, infection, and susceptibility to autoimmune diseases. The recent rapid development of new next-generation sequencing (NGS) methods provides the opportunity to study the influence/correlation of this high level of HLA diversity on allele expression levels in health and disease. Here, we describe the NGS capture RNA-Seq method that we developed for genotyping all 12 classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5) and assessing their allelic imbalance by quantifying their allele RNA levels. This is a target enrichment method where total RNA is converted to a sequencing-ready complementary DNA (cDNA) library and hybridized to a complex pool of RNA-specific HLA biotinylated oligonucleotide capture probes, prior to NGS. This method was applied to 161 peripheral blood mononuclear cells and 48 umbilical cord blood cells of healthy donors. The differential allelic expression of 10 HLA loci (except for HLA-DRA and HLA-DPA1) showed strong significant differences (P < 2.1 × 10-15). The results were corroborated by independent methods. This newly developed NGS method could be applied to a wide range of biological and medical questions including graft rejections and HLA-related diseases.
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Sitios Genéticos , Antígenos HLA/genética , Haplotipos , RNA-Seq , Frecuencia de los Genes , Antígenos HLA/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Polimorfismo de Nucleótido Simple , Valor Predictivo de las Pruebas , Reproducibilidad de los ResultadosRESUMEN
The typical products of enzymatic circularization of DNA, using DNA ligase or recombinase, are covalently closed and mostly relaxed DNA circles. Because they are difficult to analyze on conventional gels, they are often converted to nicked circles prior to electrophoresis. Herein, we present a sensitive and quantitative procedure for directly analyzing ligated closed circle DNA on agarose gels without additional treatments. Specifically, inclusion of GelStar dye in the gel allowed detection of ligated closed circle DNAs, which were likely super-twisted by being intercalated by GelStar, as discrete bands with good separation from linear DNA of the same sizes.
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ADN Circular/química , Electroforesis en Gel de Agar/métodos , ADN Ligasas/metabolismo , Fluorescencia , Conformación de Ácido NucleicoRESUMEN
We have identified 88 interactor candidates for human growth hormone (GH) by the yeast two-hybrid assay. Among those, we focused our efforts on carboxypeptidase E (CPE), which has been thought to play a key role in sorting prohormones, such as pro-opiomelanocortin (POMC), to regulated secretory vesicles. We found that CPE co-localizes with and interacts with GH in AtT20 pituitary cells. Downregulation of CPE led to decreased levels of GH secretion, consistent with involvement of CPE in GH sorting/secretion. Our binding assay in vitro with bacterially expressed proteins suggested that GH directly interacts with CPE but in a manner different from POMC.
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Carboxipeptidasa H/metabolismo , Hormona de Crecimiento Humana/metabolismo , Transporte Biológico , Carboxipeptidasa H/genética , Humanos , TransfecciónRESUMEN
BACKGROUND: Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders that occur owing to the abnormalities in type 1 collagen, and is characterized by increased bone fragility and other extraskeletal manifestations. We report the case of a patient who was diagnosed with OI following subarachnoid hemorrhage (SAH) secondary to a ruptured saccular intracranial aneurysm (IA). CASE PRESENTATION: A 37-year-old woman was referred to our hospital because of sudden headache and vomiting. She was diagnosed with SAH (World Federation of Neurosurgical Society grade 2) owing to an aneurysm of the middle cerebral artery. She then underwent surgical clipping of the aneurysm successfully. She had blue sclerae, a history of several fractures of the extremities, and a family history of bone fragility and blue sclerae in her son. According to these findings, she was diagnosed with OI type 1. We performed genetic analysis for a single nucleotide G/C polymorphism (SNP) of exon 28 of the gene encoding for alpha-2 polypeptide of collagen 1, which is a potential risk factor for IA. However, this SNP was not detected in this patient or in five normal control subjects. Other genetic analyses did not reveal any mutations of the COL1A1 or COL1A2 gene. The cerebrovascular system is less frequently involved in OI. OI is associated with increased vascular weakness owing to collagen deficiency in and around the blood vessels. SAH secondary to a ruptured IA with OI has been reported in only six cases. CONCLUSION: The patient followed a good clinical course after surgery. It remains controversial whether IAs are caused by OI or IAs are coincidentally complicated with OI.
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Aneurisma Intracraneal/complicaciones , Osteogénesis Imperfecta/complicaciones , Hemorragia Subaracnoidea/complicaciones , Adulto , Aneurisma Roto/complicaciones , Femenino , HumanosRESUMEN
BACKGROUND: Ursodeoxycholic acid (UDCA) partly exerts choleretic effects by modifying the function of the bile salt export pump (Bsep, ABCB11). UDCA induces insertion of Bsep into the canalicular membrane of hepatocytes; however, underlying mechanisms remain unknown. We aimed to elucidate molecular mechanisms behind UDCA-induced Bsep activation. METHODS: We established MDCK II cells stably expressing both Bsep and Na(+)-taurocholate cotransporting polypeptide, and investigated the effect of UDCA on activity and protein expression of Bsep using these cells. We performed inhibitor study to know the molecules involved in UDCA-induced Bsep activation, and also tested the influence of UDCA on Bsep having a disease-associated mutation. RESULTS: UDCA activated Bsep in a dose-dependent manner. UDCA did not affect Bsep protein expression in whole cell lysates but increased its apical surface expression by extending the half-life from 2.4 to 5.0 h. This effect was specific to Bsep because UDCA did not affect other apical and basolateral proteins, and was independent of protein kinase A, adenylate cyclase, p38(MAPK), phosphatidylinositide 3-kinase, Ca(2+), and microtubules. NorUDCA activated Bsep similar to UDCA; however, cholic acid, taurocholic acid, and tauroUDCA had no effect. UDCA significantly increased the activity of Bsep with a benign recurrent intrahepatic cholestasis 2 mutation (A570T) but did not affect Bsep with a progressive familial intrahepatic cholestasis 2 mutation (G982R or D482G). CONCLUSIONS: We demonstrated that UDCA stabilizes Bsep protein in the apical membrane and increases its activity in MDCK II cells, presumably by retarding the endocytotic process.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Membrana Celular/metabolismo , Hepatocitos/metabolismo , Ácido Ursodesoxicólico/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Animales , Colestasis Intrahepática/genética , Perros , Endocitosis/fisiología , Células de Riñón Canino Madin Darby , Mutación , Ratas , Ácido Taurocólico/metabolismo , Ácido Ursodesoxicólico/administración & dosificaciónRESUMEN
Sick building syndrome (SBS) is a set of several clinically recognizable symptoms reported by occupants of a building without a clear cause. Neuropathy target esterase (NTE) is a membrane bound serine esterase and its reaction with organophosphates (OPs) can lead to OP-induced delayed neuropathy (OPIDN) and nerve axon degeneration. The aim of our study was to determine whether there was a difference in NTE activity in the peripheral blood mononuclear cells (PBMCs) of Japanese patients with SBS and healthy controls and whether PNPLA6 (alias NTE) gene polymorphisms were associated with SBS. We found that the enzymatic activity of NTE was significantly higher (P < 0.0005) in SBS patients compared with controls. Moreover, population with an AA genotype of a single nucleotide polymorphism (SNP), rs480208, in intron 21 of the PNPLA6 gene strongly reduced the activity of NTE. Fifty-eight SNP markers within the PNPLA6 gene were tested for association in a case-control study of 188 affected individuals and 401 age-matched controls. Only one SNP, rs480208, was statistically different in genotype distribution (P = 0.005) and allele frequency (P = 0.006) between the cases and controls (uncorrected for testing multiple SNP sites), but these were not significant by multiple corrections. The findings of the association between the enzymatic activity of NTE and SBS in Japanese show for the first time that NTE activity might be involved with SBS.
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Hidrolasas de Éster Carboxílico/metabolismo , Fosfolipasas/metabolismo , Polimorfismo de Nucleótido Simple , Síndrome del Edificio Enfermo/enzimología , Síndrome del Edificio Enfermo/genética , Adulto , Pueblo Asiatico/genética , Hidrolasas de Éster Carboxílico/genética , Estudios de Casos y Controles , Femenino , Humanos , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/metabolismo , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Fosfolipasas/genética , Síndrome del Edificio Enfermo/metabolismo , Adulto JovenRESUMEN
There have been several reports of temozolomide (TMZ) treatment of pituitary carcinomas and atypical adenomas. O(6)-methyl-guanine-DNA methyltransferase is not the sole molecule determining the sensitivity to TMZ in pituitary carcinomas and atypical adenomas. The Japan Society of Hypothalamic and Pituitary Tumors study suggests that MSH6, one of mismatch repair pathway enzyme, fulfills a contributory role to the efficacy of TMZ treatment for pituitary carcinomas and atypical adenomas. The preserved MSH6 function might be essential for the responsiveness to TMZ treatment in pituitary carcinomas and atypical adenomas.
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Adenoma/tratamiento farmacológico , Biomarcadores de Tumor/metabolismo , Dacarbazina/análogos & derivados , Neoplasias Hipofisarias/tratamiento farmacológico , Adenoma/genética , Adenoma/metabolismo , Antineoplásicos Alquilantes/uso terapéutico , Biomarcadores de Tumor/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dacarbazina/uso terapéutico , Humanos , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismo , Temozolomida , Resultado del TratamientoRESUMEN
Skull base metastasis from differentiated thyroid carcinoma including follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC) is a rare clinical entity. Eighteen FTC cases and 10 PTC cases showing skull base metastasis have been reported. The most common symptom of skull base metastasis from FTC and PTC is cranial nerve dysfunction. Bone destruction and local invasion to the surrounding soft tissues are common on radiological imaging. Skull base metastases can be the initial clinical presentation of FTC and PTC in the presence of silent primary sites. The possibility of skull base metastasis from FTC and PTC should be considered in patients with the clinical symptoms of cranial nerve dysfunction and radiological findings of bone destruction. A variety of genetic alterations in thyroid tumors have been identified to have a fundamental role in their tumorigenesis. Molecular histochemical studies are useful for elucidating the histopathological features of thyroid carcinoma. Recent molecular findings may provide novel molecular-based treatment strategies for thyroid carcinoma.
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We modified and tested scaffold/matrix attachment region (S/MAR) episomal vectors. The new vectors would be useful in obtaining cells stably expressing fluorescent protein-tagged transgenes with small, mostly within 10-fold cell-to-cell fluctuations. In the vectors, the same transcript directs episomal replication and expression of transgene/antibiotic marker, and only antibiotic selection without any other extra steps was sufficient to obtain desired stable cells, including those expressing two different proteins simultaneously. Furthermore, the two test cases (expression of human growth hormone in AtT20 and four protein kinase C isoforms in HEK293) would prove to be useful in visualizing and analyzing regulatory processes involving these proteins.
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Expresión Génica , Genes Reporteros , Vectores Genéticos , Regiones de Fijación a la Matriz/genética , Plásmidos , Animales , Biomarcadores/metabolismo , Corticotrofos/citología , Corticotrofos/metabolismo , Replicación del ADN , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Células HEK293 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , TransgenesRESUMEN
Adult growth hormone (GH) deficiency (AGHD) in Japan is diagnosed based on peak GH concentrations during GH provocative tests such as GHRP-2 stimulation test. In this study, we aimed to evaluate the ability of serum insulin-like growth factor-1 (sIGF-1) and urinary GH (uGH) at the time of awakening to diagnose AGHD. Fifty-nine patients with pituitary disease (32 men and 27 women; age 20-85 y (57.5 ± 15.5, mean ± SD) underwent GHRP-2 stimulation and sIGF-1 testing. Thirty-six and 23 patients were diagnosed with and without severe AGHD, respectively based on a peak GH response of <9 ng/mL to GHRP-2 stimulation. Serum IGF-1 was evaluated as a standard deviation score (IGF-1 SDS) based on age and sex. We determined whether uGH levels in urine samples from 42 of the 59 patients at awakening were above or below the sensitivity limit. We evaluated IGF-1 SDS and uGH levels in a control group of 15 healthy volunteers. Values for IGF-1 SDS were significantly lower in patients with, than without (-2.07 ± 1.77 vs.-0.03 ± 0.92, mean ± SD; p < 0.001) AGHD whereas the range of IGF-1 SDS substantially overlapped at > -1.4. IGF-1 SDS discriminated AGHD more effectively in patients aged ≤60 years. The χ2 test revealed a statistical relationship between uGH and AGHD (test statistic: 7.0104 ≥ χ2 (1; 0.01) = 6.6349). When IGF-1 SDS is < -1.4 or uGH is below the sensitivity limit, AGHD can be detected with high sensitivity.
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Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/orina , Factor I del Crecimiento Similar a la Insulina/análisis , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oligopéptidos , Enfermedades de la Hipófisis/complicacionesRESUMEN
Combined in situ hybridization (ISH) and immunohistochemistry (IHC) under electron microscopy (EM-ISH & IHC) has sufficient ultrastructural resolution to provide two-dimensional images of subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (Quantum dots; Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH & IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationship between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin revealed that both rab3B and SNARE system proteins play an important role and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. An experimental pituitary cell line, the GH3 cell, in which growth hormone (GH) is linked to enhanced yellow fluorescein protein (EYFP), has been developed. This stable GH3 cell secretes GH linked to EYFP upon being stimulated by Ca(2+) influx or Ca(2+) release from storage. This GH3 cell is useful for real-time visualization of the intracellular transport and secretion of GH. These three methods enable us to visualize consecutively the processes of transcription, translation, transport, and secretion of pituitary hormone.
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Hormona del Crecimiento , Imagenología Tridimensional/métodos , Proteínas Qa-SNARE , ARN Mensajero/metabolismo , Proteína 25 Asociada a Sinaptosomas , Proteínas de Unión al GTP rab3 , Animales , Proteínas Bacterianas , Transporte Biológico/fisiología , Línea Celular , Exocitosis/fisiología , Hormona del Crecimiento/metabolismo , Humanos , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Proteínas Luminiscentes , Microscopía Confocal/métodos , Microscopía Electrónica/métodos , Hipófisis/metabolismo , Hipófisis/ultraestructura , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/ultraestructura , Puntos Cuánticos , Ratas , Proteína 25 Asociada a Sinaptosomas/metabolismo , Proteína 25 Asociada a Sinaptosomas/ultraestructura , Proteínas de Unión al GTP rab3/metabolismo , Proteínas de Unión al GTP rab3/ultraestructuraRESUMEN
In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of pituitary hormone synthesis on the rough endoplasmic reticulum. A combined ISH and immunohistochemistry (IHC) under EM (EM-ISH&IHC) approach has sufficient ultrastructural resolution, and provides two-dimensional images of the subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationships between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin has revealed that both rab3B and SNARE system proteins play important roles and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulation by Ca²+ influx or Ca²+ release from storage. This GH3 cell line is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods from conventional immunohistochemistry and fluorescein imaging allow us to consecutively visualize the process of transcription, translation, transport and secretion of anterior pituitary hormone.
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Inmunohistoquímica/métodos , Hipófisis/citología , Hipófisis/metabolismo , Animales , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Humanos , Hibridación in Situ/métodos , Microscopía ElectrónicaRESUMEN
BACKGROUND AND IMPORTANCE: The case presented here describes the clinical evolution of a pituitary carcinoma from an atypical prolactinoma after temozolomide (TMZ) treatment. The mechanism of acquisition of TMZ resistance was analyzed. CLINICAL PRESENTATION: A 60-year-old woman with atypical prolactinoma had been treated for 7 years with multiple therapies, including dopamine agonists, surgical intervention (5 times), conventional radiotherapy, and radiosurgery. The patient deteriorated as a result of tumor enlargement. Ten cycles of TMZ therapy, 200 mg/m for 5 days every 4 weeks, improved the patient's performance status and caused tumor shrinkage. Six months after discontinuation of TMZ, the tumor progressed into pituitary carcinoma with tumor regrowth and intraventricular dissemination. TMZ therapy was ineffective this time. A sixth surgery and salvage chemotherapy failed to improve the patient's condition, and she died 9 years after the first diagnosis. Throughout the treatment course, O6-methyl-guanine-DNA methyltransferase (MGMT) was immunonegative in the tumor specimens, including the TMZ-refractory pituitary carcinoma. Mutation of p53 was identified in both the atypical prolactinoma and pituitary carcinoma. In contrast, major differences were noted for mismatch repair protein MSH6 immunostaining: Although MSH6 was diffusely immunopositive in the atypical adenoma, it became immunonegative when the tumor evolved into TMZ-refractory pituitary carcinoma. CONCLUSION: Loss of MSH6 occurred during the progression from an atypical prolactinoma to a pituitary carcinoma, which may have caused resistance to TMZ treatment. This case suggests that preserving MSH6 function is essential for responsiveness to TMZ treatment in MGMT-negative and p53-mutated atypical pituitary adenoma or pituitary carcinoma.
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Antineoplásicos Alquilantes/uso terapéutico , Carcinoma/genética , Transformación Celular Neoplásica/genética , Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos/genética , Neoplasias Hipofisarias/genética , Prolactinoma/genética , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Metilasas de Modificación del ADN/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Dacarbazina/uso terapéutico , Resultado Fatal , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Mutación , Neoplasias Hipofisarias/tratamiento farmacológico , Neoplasias Hipofisarias/patología , Prolactina/metabolismo , Prolactinoma/tratamiento farmacológico , Prolactinoma/patología , Temozolomida , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo 'gain-of-function' and 'loss-of-function' studies.
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Marcación de Gen/métodos , Ratones Transgénicos , Transgenes , Animales , Núcleo Celular , Células Madre Embrionarias/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Inyecciones , Integrasas/metabolismo , Ratones , Óvulo/metabolismo , Recombinación Genética , Eliminación de SecuenciaRESUMEN
BACKGROUND: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)gamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5)P(2), and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. CONCLUSIONS/SIGNIFICANCE: Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.
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Lisina/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Sirtuina 1/metabolismo , Tirotropina/metabolismo , Acetilación , Animales , Western Blotting , Línea Celular , Células Cultivadas , Cromatografía Liquida , Electroporación , Genotipo , Humanos , Inmunoprecipitación , Masculino , Espectrometría de Masas , Ratones , Ratones Noqueados , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Unión Proteica , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 1/genéticaRESUMEN
Sick building syndrome (SBS) is a chronic disorder caused by exposure to diverse indoor environmental or chemical pollutants. This study examined the association between seven detoxification genes (CYP1A1, CYP2E1, EPHX1, GSTM1, GSTT1, GSTP1, and NAT2) and SBS in the Japanese population. One hundred eighty patients with SBS and 401 healthy controls were enrolled in this study. We examined the prevalence for total of eleven genetic polymorphisms of detoxification genes. However, no statistically significant differences in allele and genotype frequency distributions of eleven genetic polymorphisms of these detoxification genes were found between patients and controls. On this basis, we conclude that the polymorphisms that we assessed for the detoxification genes do not contribute to the etiology of SBS.
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UNLABELLED: It is unclear how hepatic adiponectin resistance and sensitivity mediated by the adiponectin receptor, AdipoR2, contributes to the progression of nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the roles of hepatic AdipoR2 in NASH, using an animal model. We fed C57BL/6 mice a methionine-deficient and choline-deficient (MCD) diet for up to 8 weeks and analyzed changes in liver pathology caused by either an AdipoR2 short hairpin RNA-expressing adenovirus or an AdipoR2-overexpressing adenovirus. Inhibition of hepatic AdipoR2 expression aggravated the pathological state of NASH at all stages: fatty changes, inflammation, and fibrosis. In contrast, enhancement of AdipoR2 expression in the liver improved NASH at every stage, from the early stage to the progression of fibrosis. Inhibition of AdipoR2 signaling in the liver diminished hepatic peroxisome proliferator activated receptor (PPAR)-alpha signaling, with decreased expression of acyl-CoA oxidase (ACO) and catalase, leading to an increase in lipid peroxidation. Hepatic AdipoR2 overexpression had the opposite effect. Reactive oxygen species (ROS) accumulation in liver increases hepatic production of transforming growth factor (TGF)-beta1 at all stages of NASH; adiponectin/AdipoR2 signaling ameliorated TGF-beta-induced ROS accumulation in primary cultured hepatocytes, by enhancing PPAR-alpha activity and catalase expression. CONCLUSION: The adiponectin resistance and sensitivity mediated by AdipoR2 in hepatocytes regulated steatohepatitis progression by changing PPAR-alpha activity and ROS accumulation, a process in which TGF-beta signaling is implicated. Thus, the liver AdipoR2 signaling pathway could be a promising target in treating NASH.
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Hígado Graso/metabolismo , Hígado Graso/patología , Hígado/metabolismo , Receptores de Adiponectina/metabolismo , Transducción de Señal , Animales , Catalasa/metabolismo , Células Cultivadas , Deficiencia de Colina/complicaciones , Dieta , Progresión de la Enfermedad , Activación Enzimática , Hígado Graso/etiología , Técnicas de Transferencia de Gen , Hepatocitos/metabolismo , Masculino , Metionina/deficiencia , Ratones , Ratones Endogámicos C57BL , PPAR alfa/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Interferon beta 6 million units per week was administered to a patient with an aggressive astrocytoma in the tectum that was resistant to cisplatin, etoposide, vinblastine, and the oral alkylating agent temozolomide. The tumor was immunopositive for O6-methylguanine-DNA methyltransferase (MGMT). Interferon beta caused the disappearance of the gadolinium-enhanced lesion in the tectum. Interferons have apoptotic and antiangiogenic effects on tumor cells, and the lesion's disappearance may have been induced by complexes of these effects. Administration of interferon beta might have a favorable effect on tectal gliomas that are immunopositive for MGMT and resistant to chemoradiotherapy including temozolomide.