RESUMO
Although genome-wide association studies have shown that potassium voltage-gated channel subfamily Q member 1 (KCNQ1) is one of the genes that is most significantly associated with type 2 diabetes mellitus (T2DM), functionally annotating disease-associated single nucleotide polymorphisms (SNPs) remains a challenge. Recently, our group described a novel strategy to identify proteins that bind to SNP-containing loci in an allele-specific manner. The present study successfully applied this strategy to investigate rs163184, a T2DM susceptibility SNP located in the intronic region of KCNQ1. Comparative analysis of DNA-binding proteins revealed that the binding activities for the genomic region containing SNP rs163184 differed between alleles for several proteins, including Sp3 and Lsd1/Kdm1a. Sp3 preferentially bound to the non-risk rs163184 allele and stimulated transcriptional activity in an artificial promoter containing this region. Lsd1/Kdm1a was identified to be preferentially recruited to the non-risk allele of the rs163184 region and reduced Sp3-dependent transcriptional activity in the artificial promoter. In addition, expression of the nearby cyclindependent kinase inhibitor 1C (CDKN1C) gene was revealed to be upregulated after SP3 knockdown in cells that possessed non-risk alleles. This suggests that CDKN1C is potentially one of the functional targets of SNP rs163184, which modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a.
Assuntos
Inibidor de Quinase Dependente de Ciclina p57/genética , Diabetes Mellitus Tipo 2/genética , Histona Desmetilases/genética , Canal de Potássio KCNQ1/genética , Fator de Transcrição Sp3/genética , Alelos , Diabetes Mellitus Tipo 2/patologia , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Ligação Proteica/genéticaRESUMO
Recent studies suggest that dedifferentiation of pancreatic ß-cells is involved in compromised ß-cell function in diabetes mellitus. We have previously shown that the promoter activity of MafB, which is expressed in α-cells of adult islets and immature ß-cells in embryonic pancreas but not in mature ß-cells in mice, is increased in compromised ß-cells of diabetic model mice. Here, we investigated a rat ß-cell line of INS1 cells with late-passage numbers, which showed extremely low expression of MafA and insulin, as an in vitro model of compromised ß-cells. In these INS1 cells, the mRNA expression and the promoter activity of MafB were upregulated compared with the early-passage ('conventional') INS1 cells. Analysis of the MafB promoter in these late-passage INS1 cells revealed that specific CpG sites in the MafB promoter were partially demethylated. The reporter assay revealed that the unmethylated promoter activity of the 373âbp region containing these CpG sites was higher than the in vitro methylated promoter activity. These results suggest that the chronic culture of the rat ß-cell line resulted in partial DNA demethylation of the MafB promoter, which may have a role in MafB promoter activation and possible dedifferentiation in our compromised ß-cell model.
Assuntos
Metilação de DNA/genética , Células Secretoras de Insulina/metabolismo , Fator de Transcrição MafB/genética , Proteínas Oncogênicas/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Ilhas de CpG/genética , Genes Reporter/genética , Células HeLa , Humanos , Insulina/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Ratos , Regulação para Cima/genéticaRESUMO
Although recent genome-wide association studies (GWAS) have been extremely successful, it remains a big challenge to functionally annotate diseaseassociated single nucleotide polymorphisms (SNPs), as the majority of these SNPs are located in noncoding regions of the genome. In this study, we described a novel strategy for identifying the proteins that bind to the SNPcontaining locus in an allelespecific manner and successfully applied this method to SNPs in the type 2 diabetes mellitus susceptibility gene, potassium voltagegated channel, KQTlike subfamily Q, member 1 (KCNQ1). DNA fragments encompassing SNPs, and risk or nonrisk alleles were immobilized onto the novel nanobeads and DNAbinding proteins were purified from the nuclear extracts of pancreatic ß cells using these DNAimmobilized nanobeads. Comparative analysis of the allele-specific DNA-binding proteins indicated that the affinities of several proteins for the examined SNPs differed between the alleles. Nuclear transcription factor Y (NFY) specifically bound the nonrisk allele of the SNP rs2074196 region and stimulated the transcriptional activity of an artificial promoter containing SNP rs2074196 in an allelespecific manner. These results suggest that SNP rs2074196 modulates the affinity of the locus for NFY and possibly induces subsequent changes in gene expression. The findings of this study indicate that our comparative method using novel nanobeads is effective for the identification of allelespecific DNAbinding proteins, which may provide important clues for the functional impact of diseaseassociated noncoding SNPs.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 2/genética , Canal de Potássio KCNQ1/genética , Nanopartículas de Magnetita , Animais , Linhagem Celular Tumoral , Predisposição Genética para Doença , Células HEK293 , Células HeLa , Humanos , Células Secretoras de Insulina/metabolismo , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Ratos , Ativação Transcricional/genéticaRESUMO
BACKGROUND: Yersinia pseudotuberculosis generally infects the gastrointestinal tract and causes enteropathy symptoms suggesting infection. Y. pseudotuberculosis infections are often complicated with intraceliac lymphoadenopathy mimicking malignant lymphoma. This is a first case of Yersinia pseudotuberculosis enteropathy mimicking enteropathic γδ T-cell lymphoma. This case highlighted the γδ T-cell reaction to Yersinia enterocolitis sometimes mimicking malignant lymphoma clinically. CASE PRESENTATION: A 72-year-old female was referred to our institute due to abdominal pain with skin rush, fever and diarrhea. Computed tomography (CT) scanning revealed mucosal swelling of the cecum with enlargement of regional lymph nodes. Laboratory data showed elevated CRP (7.74 mg/dL), an increased level of soluble interleukin-2 receptor (sIL-2R 3095 IU/mL), and CD3+ γδ T-cell circulation in peripheral blood and bone marrow (10.9% and 3.9%, respectively). Increased proportions of γδ T-cells supported the diagnosis of malignant lymphoma. Colonoscopy demonstrated hemorrhagic mucosal erosion with partial ulceration, and the subsequent pathological findings at the inflammation site suggested malignant lymphoma histopathology in the colon. These objective findings were entirely consistent with enteropathic γδ T-cell lymphoma. Thereafter, however, the microbiological results of the patient's stool at admission showed Yersinia pseudotuberculosis, and she was diagnosed as having Yersinia enterocolitis. All abnormal findings including subjective symptoms were in remission or mitigated within 2 weeks after her onset. Even the γδ T-cell circulation disappeared (0.04% in peripheral blood), and we speculate that those cells were a reaction to the Yersinia infection. CONCLUSION: In this case, a differential diagnosis included infectious enterocolitis from other immunogenic or malignant diseases. Although a measurement of sIL-2R is critical in differentiating malignant lymphoma in patients suffering with lymph adenopathy, that is not confirmative. This patient's case indicates that T cells expressing the γδ T-cell receptor might be associated with the acute and late phase reactions, in which T cells play a role in the construction of granulomas and the establishment of sequelae.
Assuntos
Enterocolite/diagnóstico , Linfoma de Células T Associado a Enteropatia/diagnóstico , Yersiniose/diagnóstico , Yersinia pseudotuberculosis/isolamento & purificação , Idoso , Colo/patologia , Colonoscopia , Diagnóstico Diferencial , Enterocolite/microbiologia , Enterocolite/patologia , Feminino , Humanos , Mucosa Intestinal/patologia , Linfonodos/patologia , Linfoma , Linfoma de Células T , Linfócitos T , Yersiniose/microbiologia , Yersiniose/patologiaRESUMO
Organ regenerative therapy aims to reproduce fully functional organs to replace organs that have been lost or damaged as a result of disease, injury, or aging. For the fully functional regeneration of ectodermal organs, a concept has been proposed in which a bioengineered organ is developed by reproducing the embryonic processes of organogenesis. Here, we show that a bioengineered hair follicle germ, which was reconstituted with embryonic skin-derived epithelial and mesenchymal cells and ectopically transplanted, was able to develop histologically correct hair follicles. The bioengineered hair follicles properly connected to the host skin epithelium by intracutaneous transplantation and reproduced the stem cell niche and hair cycles. The bioengineered hair follicles also autonomously connected with nerves and the arrector pili muscle at the permanent region and exhibited piloerection ability. Our findings indicate that the bioengineered hair follicles could restore physiological hair functions and could be applicable to surgical treatments for alopecia.
Assuntos
Bioengenharia/métodos , Folículo Piloso/transplante , Cabelo/fisiologia , Regeneração , Alopecia/cirurgia , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cabelo/citologia , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Transplante de Pele/métodos , Células-Tronco/citologia , Células-Tronco/metabolismo , Engenharia Tecidual/métodosRESUMO
Organ replacement regenerative therapy is purported to enable the replacement of organs damaged by disease, injury or aging in the foreseeable future. Here we demonstrate fully functional hair organ regeneration via the intracutaneous transplantation of a bioengineered pelage and vibrissa follicle germ. The pelage and vibrissae are reconstituted with embryonic skin-derived cells and adult vibrissa stem cell region-derived cells, respectively. The bioengineered hair follicle develops the correct structures and forms proper connections with surrounding host tissues such as the epidermis, arrector pili muscle and nerve fibres. The bioengineered follicles also show restored hair cycles and piloerection through the rearrangement of follicular stem cells and their niches. This study thus reveals the potential applications of adult tissue-derived follicular stem cells as a bioengineered organ replacement therapy.
Assuntos
Células-Tronco Adultas/citologia , Folículo Piloso/citologia , Adulto , Células-Tronco Adultas/transplante , Animais , Bioengenharia , Diferenciação Celular , Proliferação de Células , Folículo Piloso/transplante , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Vibrissas/citologia , Vibrissas/transplanteRESUMO
The autologous transplantation of hair follicles that have been separated into single follicular units is an accepted treatment for androgenetic alopecia. Recent studies demonstrate that the multiple stem cell populations and surrounding cutaneous tissues coordinately regulate the hair follicle functions and skin homeostasis. Therefore, the critical issues for consideration regarding functional hair restoration therapy are reproduction the correct connectivity and cooperation with host cutaneous tissues, including the arrector pili muscle (APM) and nerve system. We report successful establishment of mouse single follicular transplantation model and autonomous restoration of transplanted hair follicle piloerection in mouse skin. Transplanted hair follicles were responsive to the neurotransmitter acetylcholine and formed proper connections with surrounding host tissues such as APM and nerve fibers, which in turn connect with not only the hair follicle bulge region but also the APM. These results demonstrate that the piloerection ability of transplanted hair follicles can be estimated quantitatively. This study makes a substantial contribution towards the development of transplantation therapy that will facilitate future functional regeneration therapy for skin and skin appendages.
Assuntos
Alopecia/terapia , Folículo Piloso/transplante , Piloereção/fisiologia , Acetilcolina/farmacologia , Animais , Folículo Piloso/citologia , Folículo Piloso/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologiaRESUMO
BACKGROUND: Markedly activated neutrophils or higher plasma levels of neutrophil elastase are involved in the poor response to intravenous immunoglobulin (IVIG) and the formation of coronary artery lesions (CAL) in patients with acute Kawasaki disease. We hypothesized that ulinastatin (UTI), by both direct and indirect suppression of neutrophils, would reduce the occurrence of CAL. METHODS AND RESULTS: We retrospectively analyzed the clinical records of patients with Kawasaki disease between 1998 and 2009. Three hundred sixty-nine patients were treated with a combination of UTI, aspirin, and IVIG as an initial treatment (UTI group), and 1178 were treated with a conventional initial treatment, and IVIG with aspirin (control group). The baseline characteristics did not demonstrate notable differences between the two groups. The occurrence of CAL was significantly lower in the UTI group than in the control group (3% versus 7%; crude odds ratio [OR], 0.46; 95% confidence interval [CI], 0.25-0.86; P=0.01). The OR adjusted for sex, Gunma score (the predictive score for IVIG unresponsiveness), and dosage of initial IVIG (1 or 2 g/kg) was 0.32 (95% CI, 0.17-0.60; P<0.001). In addition, most CAL occurred in patients requiring additional rescue treatment and the proportion of those patients was significantly lower in the UTI group than in the control group (13% versus 22%; crude OR, 0.52; 95% CI, 0.38-0.73; P<0.001). The adjusted OR was 0.30 (95% CI, 0.20-0.44; P<0.001). CONCLUSIONS: UTI was associated with fewer patients requiring additional rescue treatment and reduction of CAL in this retrospective study.
Assuntos
Doença da Artéria Coronariana/prevenção & controle , Glicoproteínas/administração & dosagem , Síndrome de Linfonodos Mucocutâneos/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Inibidores da Tripsina/administração & dosagem , Doença Aguda , Anti-Inflamatórios não Esteroides/administração & dosagem , Aspirina/administração & dosagem , Pré-Escolar , Terapia Combinada , Doença da Artéria Coronariana/imunologia , Doença da Artéria Coronariana/terapia , Quimioterapia Combinada , Feminino , Glicoproteínas/efeitos adversos , Humanos , Imunoglobulinas Intravenosas/administração & dosagem , Lactente , Masculino , Síndrome de Linfonodos Mucocutâneos/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Estudos Retrospectivos , Resultado do Tratamento , Inibidores da Tripsina/efeitos adversosRESUMO
BACKGROUND: The effect of carvedilol on heart failure (HF) in patients with a functionally univentricular heart (UVH) remains unclear. METHODS AND RESULTS: Carvedilol was used to treat HF in 51 patients with a UVH, classified into 3 groups: after the Fontan operation (F), after the bidirectional Glenn operation (G), and patients who had not undergone Fontan or Glenn operation (NF). Carvedilol therapy was started at a mean age of 10 ± 12 years (range: 1 month to 34 years). The initial and maximum doses of carvedilol were 0.04 ± 0.03 and 0.42 ± 0.29 mg · kg(-1) · day(-1), respectively. After a mean follow-up of 11 months, the cardiothoracic ratio improved from 60 ± 8 to 58 ± 8% (P<0.01), and the dosage of furosemide was reduced from 1.4 ± 0.9 to 0.7 ± 0.7 mg · kg(-1) · day(-1) (P < 0.01). The ejection fraction also improved from 35 ± 12 to 40 ± 11% (P < 0.05), and this improvement was prominent in the F group (from 35 ± 15 to 45 ± 9%; P < 0.05). Clinical signs, symptoms, and New York Heart Association functional class also improved. CONCLUSIONS: Carvedilol may play an important role in treating HF associated with a UVH.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 1/uso terapêutico , Carbazóis/uso terapêutico , Cardiopatias Congênitas/tratamento farmacológico , Insuficiência Cardíaca/tratamento farmacológico , Propanolaminas/uso terapêutico , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Direita/efeitos dos fármacos , Adolescente , Adulto , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Antiarrítmicos/uso terapêutico , Carvedilol , Criança , Pré-Escolar , Quimioterapia Combinada , Feminino , Técnica de Fontan/efeitos adversos , Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/fisiopatologia , Cardiopatias Congênitas/cirurgia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Lactente , Japão , Masculino , Recuperação de Função Fisiológica , Estudos Retrospectivos , Volume Sistólico/efeitos dos fármacos , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Sperm-specific phospholipase C, PLCzeta, is a candidate for the Ca(2+) oscillation-inducing factor that is introduced into the ooplasm upon sperm-egg fusion. In addition to the 647-residue full-length PLCzeta, s-PLCzeta lacking the N-terminal 110 amino acids is known to be present in the mouse testis. In this study, we attempted to obtain full-term offspring from s-PLCzeta-activated eggs by round spermatid injection. Metaphase II-arrested eggs injected with a high RNA concentration of s-PLCzeta RNA normally developed to blastocysts. When the round spermatid nucleus was injected into telophase II-stage eggs previously activated by s-PLCzeta RNA, three live offspring were successfully obtained by transfer of the developed 4-cell embryos to pseudopregnant mice. These three offspring all grew to be normal adults and reproduced healthy second-generation mice.
Assuntos
Motivos EF Hand/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Parto/efeitos dos fármacos , Fosfoinositídeo Fosfolipase C/farmacologia , Fosfolipases Tipo C/farmacologia , Animais , Animais Recém-Nascidos , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Feminino , Isoenzimas/química , Isoenzimas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Oogênese/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Fosfoinositídeo Fosfolipase C/química , Gravidez , Telófase/efeitos dos fármacos , Fosfolipases Tipo C/químicaRESUMO
We report a case of a patient with severe heart failure after Fontan procedure in whom carvedilol was very effective. A 27-year-old man had intractable congestive heart failure due to severe ventricular dysfunction after Fontan operation. Central venous pressure was elevated to 29 mmHg. A right-to-left shunt was noted across a large collateral vessel between the innominate vein and the pulmonary vein. He was administered carvedilol (initial dose, 2 mg/day; maximum dose, 30 mg/day). Cardiac catheterization performed 1 year after carvedilol administration revealed a decrease in atrial pressure and improvement of ventricular function. He underwent a conversion operation to total cavopulmonary connection (TCPC) and ligation of a collateral vein communicating with the innominate and pulmonary veins. Carvedilol may be a legitimate treatment before TCPC conversion or heart transplantation for the high-risk group of patients with a failed Fontan circulation.
Assuntos
Antagonistas Adrenérgicos beta/uso terapêutico , Carbazóis/uso terapêutico , Técnica de Fontan/efeitos adversos , Insuficiência Cardíaca/tratamento farmacológico , Propanolaminas/uso terapêutico , Adulto , Carvedilol , Embolização Terapêutica , Derivação Cardíaca Direita , Insuficiência Cardíaca/etiologia , Comunicação Interventricular/cirurgia , Humanos , Masculino , Valva Mitral/anormalidades , Valva Mitral/cirurgia , Estenose da Valva Pulmonar/cirurgia , Reoperação , Transposição dos Grandes Vasos/cirurgia , Disfunção Ventricular/complicações , Disfunção Ventricular/terapiaRESUMO
Cytoplasmic polyadenylation of mRNAs is involved in post-transcriptional regulation of genes, including translational activation. In addition to yeast Cid1 and Cid13 and mouse TPAP, GLD-2 has been recently identified as a cytoplasmic poly(A) polymerase in Caenorhabditis elegans and Xenopus oocytes. In this study, we have characterized mouse GLD-2, mGLD-2, in adult tissues, meiotically maturing oocytes, and NIH3T3 cultured cells. mGLD-2 was ubiquitously present in all tissues and cells tested. mGLD-2 was localized in the nucleus as well as in the cytoplasm of somatic, testicular, and cultured cells. Transfection of expression plasmids encoding mGLD-2 and the mutant proteins into NIH3T3 cells revealed that a 17-residue sequence in the N-terminal region of mGLD-2 probably acts as a localization signal required for the transport into the nucleus. Analysis of reverse transcriptase-polymerase chain reaction indicated the presence of mGLD-2 mRNA in the oocytes throughout meiotic maturation. However, 54-kDa mGLD-2 was found in the oocytes only at the metaphases I and II after germinal vesicle breakdown, presumably due to translational control. When mGLD-2 synthesis was artificially inhibited and enhanced by injection of double-stranded and polyadenylated RNAs into the germinal vesicle-stage oocytes, respectively, oocyte maturation was significantly arrested at the metaphase-I stage. These results suggest that mGLD-2 may act in the ooplasm on the progression of metaphase I to metaphase II during oocyte maturation.