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1.
Cell ; 157(3): 726-39, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24746791

RESUMO

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.


Assuntos
Neuroimagem/métodos , Animais , Encéfalo/citologia , Callithrix , Indicadores e Reagentes/química , Camundongos , Microscopia/métodos
2.
Cell ; 154(6): 1314-25, 2013 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-24034253

RESUMO

G-protein-coupled receptors (GPCRs) are known to possess two different conformations, active and inactive, and they spontaneously alternate between the two in the absence of ligands. Here, we analyzed the agonist-independent GPCR activity for its possible role in receptor-instructed axonal projection. We generated transgenic mice expressing activity mutants of the ß2-adrenergic receptor, a well-characterized GPCR with the highest homology to odorant receptors (ORs). We found that mutants with altered agonist-independent activity changed the transcription levels of axon-targeting molecules--e.g., Neuropilin-1 and Plexin-A1--but not of glomerular segregation molecules--e.g., Kirrel2 and Kirrel3--thus causing shifts in glomerular locations along the anterior-posterior (A-P) axis. Knockout and in vitro experiments demonstrated that Gs, but not Golf, is responsible for mediating the agonist-independent GPCR activity. We conclude that the equilibrium of conformational transitions set by each OR is the major determinant of expression levels of A-P-targeting molecules.


Assuntos
Axônios/metabolismo , Condutos Olfatórios/embriologia , Receptores Odorantes/metabolismo , Células Receptoras Sensoriais/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Animais , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Condutos Olfatórios/citologia , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Odorantes/genética
3.
Nature ; 594(7864): 547-552, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34108685

RESUMO

Tissue stem cells are generated from a population of embryonic progenitors through organ-specific morphogenetic events1,2. Although tissue stem cells are central to organ homeostasis and regeneration, it remains unclear how they are induced during development, mainly because of the lack of markers that exclusively label prospective stem cells. Here we combine marker-independent long-term 3D live imaging and single-cell transcriptomics to capture a dynamic lineage progression and transcriptome changes in the entire epithelium of the mouse hair follicle as it develops. We found that the precursors of different epithelial lineages were aligned in a 2D concentric manner in the basal layer of the hair placode. Each concentric ring acquired unique transcriptomes and extended to form longitudinally aligned, 3D cylindrical compartments. Prospective bulge stem cells were derived from the peripheral ring of the placode basal layer, but not from suprabasal cells (as was previously suggested3). The fate of placode cells is determined by the cell position, rather than by the orientation of cell division. We also identified 13 gene clusters: the ensemble expression dynamics of these clusters drew the entire transcriptional landscape of epithelial lineage diversification, consistent with cell lineage data. Combining these findings with previous work on the development of appendages in insects4,5, we describe the 'telescope model', a generalized model for the development of ectodermal organs in which 2D concentric zones in the placode telescope out to form 3D longitudinally aligned cylindrical compartments.


Assuntos
Linhagem da Célula , Folículo Piloso/citologia , Células-Tronco/citologia , Animais , Rastreamento de Células , Ectoderma , Embrião de Mamíferos , Células Epiteliais/citologia , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Transgênicos , Família Multigênica , RNA-Seq , Análise de Célula Única , Pele , Técnicas de Cultura de Tecidos , Transcriptoma , Vibrissas
4.
Genes Cells ; 29(10): 808-819, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39044347

RESUMO

Mammalian oocytes undergo a long-term meiotic arrest that can last for almost the entire reproductive lifespan. This arrest occurs after DNA replication and is prolonged with age, which poses a challenge to oocytes in maintaining replication-dependent chromosomal proteins required for the completion of meiosis. In this study, we show that chromosomal histones are reduced with age in mouse oocytes. Both types of histone H3 variants, replication-dependent H3.1/H3.2 and replication-independent H3.3, decrease with age. Aging-associated histone reduction is associated with transcriptomic features that are caused by genetic depletion of histone H3.3. Neither the genetic reduction of chromosomal H3.1/H3.2 nor H3.3 accelerates the aging-associated increase in premature chromosome separation that causes meiotic segregation errors. We suggest that aging-associated reduction of chromosomal histones is linked to several transcriptomic abnormalities but does not significantly contribute to errors in meiotic chromosome segregation during the reproductive lifespan of mice.


Assuntos
Envelhecimento , Segregação de Cromossomos , Histonas , Meiose , Oócitos , Animais , Oócitos/metabolismo , Histonas/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Camundongos , Feminino , Meiose/genética , Replicação do DNA , Camundongos Endogâmicos C57BL
5.
Mol Cell ; 65(1): 176-190, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-28017587

RESUMO

To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1-/-:Cry2-/- background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.


Assuntos
Relógios Circadianos , Ritmo Circadiano , Criptocromos/metabolismo , Células-Tronco Embrionárias/metabolismo , Animais , Comportamento Animal , Criptocromos/química , Criptocromos/deficiência , Criptocromos/genética , Genótipo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Atividade Motora , Mutação , Células NIH 3T3 , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fenótipo , Fosforilação , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Tempo , Transfecção
6.
Dev Biol ; 497: 26-32, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36868446

RESUMO

Reptiles are important model organisms in developmental and evolutionary biology, but are used less widely than other amniotes such as mouse and chicken. One of the main reasons for this is that has proven difficult to conduct CRISPR/Cas9-mediated genome editing in many reptile species despite the widespread use of this technology in other taxa. Certain features of reptile reproductive systems make it difficult to access one-cell or early-stage zygotes, which represents a key impediment to gene editing techniques. Recently, Rasys and colleagues reported a genome editing method using oocyte microinjection that allowed them to produce genome-edited Anolis lizards. This method opened a new avenue to reverse genetics studies in reptiles. In the present article, we report the development of a related method for genome editing in the Madagascar ground gecko (Paroedura picta), a well-established experimental model, and describe the generation of Tyr and Fgf10 gene-knockout geckos in the F0 generation.


Assuntos
Sistemas CRISPR-Cas , Lagartos , Animais , Camundongos , Sistemas CRISPR-Cas/genética , Lagartos/genética , Microinjeções , Genética Reversa , Edição de Genes/métodos , Oócitos
7.
Biochem Biophys Res Commun ; 704: 149713, 2024 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-38428304

RESUMO

As life expectancy continues to increase, age-related kidney diseases are becoming more prevalent. Chronic kidney disease (CKD) is not only a consequence of aging but also a potential accelerator of aging process. Here we report the pivotal role of podocyte ERCC1, a DNA repair factor, in maintaining glomerular integrity and a potential effect on multiple organs. Podocyte-specific ERCC1-knockout mice developed severe proteinuria, glomerulosclerosis, and renal failure, accompanied by a significant increase in glomerular DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). ERCC1 gene transfer experiment in the knockout mice attenuated proteinuria and glomerulosclerosis with reduced DNA damage. Notably, CD44+CD8+ memory T cells, indicative of T-cell senescence, were already elevated in the peripheral blood of knockout mice at 10 weeks old. Additionally, levels of senescence-associated secretory phenotype (SASP) factors were significantly increased in both the circulation and multiple organs of the knockout mice. In older mice and human patients, we observed an accumulation of DSBs and an even greater buildup of SSBs in glomeruli, despite no significant reduction in ERCC1 expression with age in mice. Collectively, our findings highlight the crucial role of ERCC1 in repairing podocyte DNA damage, with potential implications for inflammation in various organs.


Assuntos
Nefropatias , Podócitos , Humanos , Camundongos , Animais , Podócitos/metabolismo , Glomérulos Renais/metabolismo , Nefropatias/metabolismo , Camundongos Knockout , Proteinúria/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo
8.
Jpn J Clin Oncol ; 54(4): 489-497, 2024 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-38157885

RESUMO

OBJECTIVE: The companion diagnosis for olaparib, a poly (ADP-ribose) polymerase inhibitor for prostate cancer, aims to detect BRCA1/2 gene variants. In clinical practice, the frequency of germline BRCA1/2 variants in patients receiving castration-resistant prostate cancer treatment is unknown. We aimed to evaluate the prevalence of germline BRCA1/2 variants and their relationship to prognosis and treatment efficacy in castration-resistant prostate cancer. METHODS: Between June 2021 and 2023, 92 patients receiving castration-resistant prostate cancer treatment were examined for germline BRCA1/2 variants using BRACAnalysis CDx®. Furthermore, the associations between BRCA1/2 pathogenic variants and clinical outcomes were assessed. RESULTS: Of the 92 patients referred for genetic testing, 6 (6.5%) carried germline pathogenic variants in BRCA1/2. The BRCA2 variant was the most frequent (n = 5), followed by BRCA1 variant (n = 1). Among the five variants in BRCA2, the p.Asp427Thrfs*3 variant was identified for the first time in prostate cancer. Overall survival from castration-resistant prostate cancer for patients with BRCA1/2 variants was significantly shorter than for patients without BRCA1/2 variants (P = 0.043). Progression-free survival of androgen receptor signaling inhibitors for patients with BRCA1/2 variants was significantly shorter than for those without (P = 0.003). Progression-free survival of taxane chemotherapy was significantly shorter in patients with BRCA1/2 variants than in those without (P = 0.0149). CONCLUSIONS: In clinical practice, 6.5% of patients treated with castration-resistant prostate cancer carried germline BRCA1/2 pathogenic variants. Japanese castration-resistant prostate cancer patients with germline BRCA1/2 mutants have a poor prognosis and may be less responsive to treatment with androgen receptor signaling inhibitors and taxane-based chemotherapy for castration-resistant prostate cancer.


Assuntos
Antineoplásicos , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Proteína BRCA1/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Proteína BRCA2/genética , Receptores Androgênicos/uso terapêutico , Prevalência , Japão/epidemiologia , Antineoplásicos/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Taxoides/uso terapêutico , Células Germinativas
9.
Biol Pharm Bull ; 47(2): 394-398, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38325828

RESUMO

Midbrain dopaminergic neurons respond to rewards and have a crucial role in positive motivation and pleasure. Electrical stimulation of dopaminergic neurons and/or their axonal fibers and arborization has been often used to motivate animals to perform cognitive tasks. Still, the electrical stimulation is incompatible with electrophysiological recordings. In this light, optical stimulation following artificial expression of channelrhodopsin-2 (ChR2) in the cell membrane has been also used, but the expression level of ChR2 varies among researchers. Thus, we attempted to stably express ChR2 fused with a red fluorescence protein, mCherry, in dopaminergic neurons. Since dopamine transporter (DAT) gene is known as a marker for dopaminergic neurons, we inserted ChR2-mCherry into the downstream of the DAT gene locus of the rat genome by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) genome editing and created DAT-ChR2-mCherry knock-in rats. Immunohistochemistry showed that ChR2-mCherry was expressed in dopaminergic neurons in homozygote knock-in rats, whereas whole-cell recordings revealed that ChR2-mCherry-positive neurons did not fire action potentials upon blue light stimulation, indicating that ChR2 was not functional for optogenetics. Nevertheless, fluorescent labeling of dopaminergic neurons mediated by mCherry could help characterize them physiologically and histologically.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Ratos , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Proteína Vermelha Fluorescente , Neurônios Dopaminérgicos/metabolismo
10.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33893234

RESUMO

The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca2+- and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (∼60 min) Ca2+ elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca2+ elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis.


Assuntos
Morte Celular/fisiologia , Células Epidérmicas/metabolismo , Queratinócitos/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Diferenciação Celular , Epiderme/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Queratinócitos/fisiologia , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp/métodos , Pele
11.
J Phys Ther Sci ; 36(3): 117-122, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38434990

RESUMO

[Purpose] This study investigated the changes in caregiving risk and motor function among older adults participating in community gatherings ("Kayoinoba") in Koshigaya. [Participants and Methods] A total of 257 older participants who engaged in the Kayoinoba program for 6 months from its inception were included in the analysis. Caregiving risk and motor function were assessed twice-once at the beginning of the Kayoinoba (first assessment) and again 6 months later (second assessment). The Kihon Checklist was used to evaluate caregiving risk, and the timed up-and-go, one-leg standing, and 30-s chair-stand tests were done to evaluate motor functioning. Participants were divided into pre-frail and healthy groups, and the first and second assessments were compared. [Results] The Kihon Checklist score of the pre-frail group significantly improved from the first to the second assessment. The pre-frail group had lower composite scores for physical function, outdoor activities, and depression mood items based on the Kihon Checklist; the healthy group showed no such differences. Performance on the 30-s chair-stand test was significantly better in the second assessment than in the first assessment in both groups. [Conclusion] The findings of this study emphasize the benefits of participating in Kayoinoba among high-risk older adults and provide the knowledge for developing a healthier community-based symbiotic society.

12.
J Immunol ; 206(2): 366-375, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33310872

RESUMO

ADP-ribosylation factor (Arf) family consisting of six family members, Arf1-Arf6, belongs to Ras superfamily and orchestrates vesicle trafficking under the control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. It is well established that brefeldin A, a potent inhibitor of ArfGEFs, blocks cytokine secretion from activated T cells, suggesting that the Arf pathway plays important roles in T cell functions. In this study, because Arf1 and Arf6 are the best-characterized members among Arf family, we established T lineage-specific Arf1-deficient, Arf6-deficient, and Arf1/6 double-deficient mice to understand physiological roles of the Arf pathway in the immune system. Contrary to our expectation, Arf deficiency had little or no impact on cytokine secretion from the activated T cells. In contrast, the lack of both Arf1 and Arf6, but neither Arf1 nor Arf6 deficiency alone, rendered naive T cells susceptible to apoptosis upon TCR stimulation because of imbalanced expression of Bcl-2 family members. We further demonstrate that Arf1/6 deficiency in T cells alleviates autoimmune diseases like colitis and experimental autoimmune encephalomyelitis, whereas Ab response under Th2-polarizing conditions is seemingly normal. Our findings reveal an unexpected role for the Arf pathway in the survival of T cells during TCR-induced activation and its potential as a therapeutic target in the autoimmune diseases.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Colite/imunologia , Encefalomielite Autoimune Experimental/imunologia , Linfócitos T/imunologia , Fator 1 de Ribosilação do ADP/genética , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Animais , Apoptose , Sobrevivência Celular , Células Cultivadas , Imunoterapia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais
13.
Gastroenterology ; 161(6): 1907-1923.e26, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34391772

RESUMO

BACKGROUND & AIMS: Metaplasia and dysplasia in the corpus are reportedly derived from de-differentiation of chief cells. However, the cellular origin of metaplasia and cancer remained uncertain. Therefore, we investigated whether pepsinogen C (PGC) transcript-expressing cells represent the cellular origin of metaplasia and cancer using a novel Pgc-specific CreERT2 recombinase mouse model. METHODS: We generated a Pgc-mCherry-IRES-CreERT2 (Pgc-CreERT2) knock-in mouse model. Pgc-CreERT2/+ and Rosa-EYFP mice were crossed to generate Pgc-CreERT2/Rosa-EYFP (Pgc-CreERT2/YFP) mice. Gastric tissues were collected, followed by lineage-tracing experiments and histologic and immunofluorescence staining. We further established Pgc-CreERT2;KrasG12D/+ mice and investigated whether PGC transcript-expressing cells are responsible for the precancerous state in gastric glands. To investigate cancer development from PGC transcript-expressing cells with activated Kras, inactivated Apc, and Trp53 signaling pathways, we crossed Pgc-CreERT2/+ mice with conditional KrasG12D, Apcflox, Trp53flox mice. RESULTS: Expectedly, mCherry mainly labeled chief cells in the Pgc-CreERT2 mice. However, mCherry was also detected throughout the neck cell and isthmal stem/progenitor regions, albeit at lower levels. In the Pgc-CreERT2;KrasG12D/+ mice, PGC transcript-expressing cells with KrasG12D/+ mutation presented pseudopyloric metaplasia. The early induction of proliferation at the isthmus may reflect the ability of isthmal progenitors to react rapidly to Pgc-driven KrasG12D/+ oncogenic mutation. Furthermore, Pgc-CreERT2;KrasG12D/+;Apcflox/flox mice presented intramucosal dysplasia/carcinoma and Pgc-CreERT2;KrasG12D/+;Apcflox/flox;Trp53flox/flox mice presented invasive and metastatic gastric carcinoma. CONCLUSIONS: The Pgc-CreERT2 knock-in mouse is an invaluable tool to study the effects of successive oncogenic activation in the mouse corpus. Time-course observations can be made regarding the responses of isthmal and chief cells to oncogenic insults. We can observe stomach-specific tumorigenesis from the beginning to metastatic development.


Assuntos
Proliferação de Células , Transformação Celular Neoplásica/genética , Celulas Principais Gástricas/enzimologia , Integrases/genética , Pepsinogênio C/genética , Lesões Pré-Cancerosas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Gástricas/genética , Ativação Transcricional , Animais , Desdiferenciação Celular , Linhagem da Célula , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Celulas Principais Gástricas/patologia , Regulação Neoplásica da Expressão Gênica , Genes APC , Predisposição Genética para Doença , Integrases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metaplasia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Pepsinogênio C/metabolismo , Fenótipo , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Vermelha Fluorescente
14.
Biochem Biophys Res Commun ; 628: 91-97, 2022 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-36084556

RESUMO

The adaptor protein GAREM has two subtypes. Each is involved in Erk activation signaling downstream of the cell growth factor receptor in cultured cells. Regarding their role in individual animals, we have previously reported that mice deficient in GAREM2, which is highly expressed in the brain, exhibit emotional changes. In this paper, we report an amino acid substitution mutation (K291R) in GAREM1, in a patient with idiopathic short stature, which indicates that the mutant exhibits dominant-negative properties. The GAREM K291R mutant did not promote Erk activation in EGF-stimulated cultured cells. Similar features were also observed in cells in which GAREM1 expression was suppressed by genome editing; along with Erk, phosphorylation of S6 kinase and 4EBP1, whose activation is necessary for cell proliferation and biological growth, were inhibited Furthermore, we generated mice deficient in GAREM1 and showed that the mutant mice are lighter in weight. Overall, the results of this paper suggest that GAREM1 is required for normal growth and for maintaing average body size in humans and mice.


Assuntos
Peso Corporal , Nanismo , Proteína Adaptadora GRB2 , Proteínas Adaptadoras de Transdução de Sinal , Animais , Peso Corporal/genética , Proteínas de Ciclo Celular , Linhagem Celular , Nanismo/genética , Fator de Crescimento Epidérmico/metabolismo , Proteína Adaptadora GRB2/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Fosforilação , Proteínas Quinases S6 Ribossômicas/metabolismo
15.
Biochem Biophys Res Commun ; 614: 198-206, 2022 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-35605301

RESUMO

Podocyte damage is a major pathological lesion leading to focal segmental glomerulosclerosis (FSGS). Podocytes damaged by cellular stress undergo hypertrophy to compensate for podocytopenia. It is known that cyclin-dependent kinase inhibitors induced by p53 ensure podocytes hypertrophy; however, its precise mechanism remains to be further investigated. In this study, we found that ubiquitin specific protease 40 (USP40) is a novel regulator of p53. Although USP40 knockout mice established in the present study revealed no abnormal kidney phenotype, intermediate filament Nestin was upregulated in the glomeruli, and was bound to and colocalized with USP40. We also found that USP40 deubiquitinated histidine triad nucleotide-binding protein 1 (HINT1), an inducer of p53. Gene knockdown experiments of USP40 in cultured podocytes revealed the reduction of HINT1 and p53 protein expression. Finally, in glomerular podocytes of mouse FSGS, upregulation of HINT1 occurred in advance of the proteinuria, which was followed by upregulation of USP40, p53 and Nestin. In conclusion, USP40 bound to Nestin deubiquitinates HINT1, and in consequence upregulates p53. These results provide additional insight into the pathological mechanism of podocyte hypertrophy in FSGS.


Assuntos
Glomerulosclerose Segmentar e Focal , Proteínas do Tecido Nervoso , Nestina , Podócitos , Proteína Supressora de Tumor p53 , Proteases Específicas de Ubiquitina , Animais , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Hipertrofia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina/genética , Nestina/metabolismo , Podócitos/metabolismo , Podócitos/patologia , Podócitos/fisiologia , Proteína Quinase C/antagonistas & inibidores , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Regulação para Cima
16.
Dev Growth Differ ; 64(7): 379-394, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36057539

RESUMO

When the regulation of axonal and dendritic growth is altered, the neuronal network becomes disordered, which may contribute to the development of psychiatric disorders. Some genome analyses have suggested relationships between mutations in strawberry notch homologue 1 (SBNO1) and neurodevelopmental disorders. However, the function of SBNO1 has not yet been reported. Here, SBNO1 expression pattern during the development of the cerebral cortex in mice was examined. SBNO1 was strongly expressed in the cortical plate and its expression was maintained at a low level during the postnatal stage. CRISPR/Cas9-based knockout of Sbno1 in Neuro2A cultured cells showed delayed growth of neurites. A cortical neuron-specific conditional knockout mouse was constructed, which resulted in hypotrophy of axon bundles and dendrites in cortical neurons. Thus, when mutated, SBNO1 is a candidate gene for psychiatric diseases, such as schizophrenia, as suggested by human genome studies.


Assuntos
Crescimento Neuronal , Neurônios , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Humanos , Camundongos , Camundongos Knockout , Neuritos/metabolismo , Crescimento Neuronal/genética
17.
Proc Natl Acad Sci U S A ; 116(47): 23653-23661, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31694883

RESUMO

The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.


Assuntos
Glicoproteínas de Membrana/agonistas , RNA Nuclear Pequeno/imunologia , Receptor 7 Toll-Like/agonistas , Adulto , Alarminas/química , Animais , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Sequência de Bases , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Imunossupressores/síntese química , Imunossupressores/farmacologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , RNA/imunologia , RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/imunologia , Análise de Sequência de RNA , Receptor 7 Toll-Like/deficiência , Adulto Jovem
18.
Int J Mol Sci ; 23(6)2022 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-35328412

RESUMO

Brain natriuretic peptide (BNP) levels are increased in both patients with heart failure with preserved (HFpEF) and reduced ejection fraction (HFrEF), but the reasons for this remain unclear. Our purpose was to examine whether serum-induced BNP (iBNP) expression partly contributes to increased BNP in patients with HFpEF. BNP reporter cardiomyocytes from pBNP-luc-KI mice were stimulated with serum from patients with HFpEF or HFrEF (n = 114 and n = 82, respectively). Luciferase activity was examined as iBNP and the iBNP-to-BNP ratio was evaluated. Patient characteristics and clinical parameters were compared, and multivariate regression analysis was performed to determine independent predictors of the iBNP-to-BNP ratio. Female sex and frequencies of atrial fibrillation, hypertension and the use of a calcium channel blocker (CCB) were higher in HFpEF. The iBNP-to-BNP ratio was significantly higher in HFpEF (26.9) than in HFrEF (16.1, p < 0.001). Multivariate regression analysis identified the existence of HFpEF as an independent predictor of the iBNP-to-BNP ratio after adjusting for all other measurements (ß = 0.154, p = 0.032). Age, hemoglobin, CCB usage and deceleration time were also independent predictors (ß = 0.167, p = 0.025; ß = 0.203, p = 0.006; ß = 0.138, p = 0.049; and ß = 0.143, p = 0.049, respectively). These results indicate that the elevated BNP in patients with HFpEF is partly due to iBNP from the heart.


Assuntos
Fibrilação Atrial , Insuficiência Cardíaca , Disfunção Ventricular Esquerda , Animais , Biomarcadores , Feminino , Humanos , Camundongos , Peptídeo Natriurético Encefálico , Volume Sistólico
19.
Hinyokika Kiyo ; 68(2): 47-51, 2022 Feb.
Artigo em Japonês | MEDLINE | ID: mdl-35259863

RESUMO

A 75-year-old male visited a clinic with the chief complaint of pollakiuria. A computed tomography scan revealed, a left adrenal mass, and the patient was then referred to our hospital. Since a malignant tumor could not be ruled out. We performed laparoscopic left adrenal resection. Postoperative histopathological findings revealed the mass to be a bronchogenic cyst, which had no continuity with the normal adrenal gland. The postoperative course was uneventful, and recurrence has not been observed. Retroperitoneal bronchogenic cysts are rare and often difficult to diagnose preoperatively using imaging studies.


Assuntos
Neoplasias das Glândulas Suprarrenais , Cisto Broncogênico , Neoplasias das Glândulas Suprarrenais/patologia , Glândulas Suprarrenais , Idoso , Cisto Broncogênico/diagnóstico por imagem , Cisto Broncogênico/cirurgia , Humanos , Masculino , Espaço Retroperitoneal/diagnóstico por imagem , Espaço Retroperitoneal/patologia , Tomografia Computadorizada por Raios X
20.
J Biol Chem ; 295(51): 17632-17645, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33454003

RESUMO

Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre-mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Endotélio/metabolismo , Receptores Notch/metabolismo , Animais , Artérias/crescimento & desenvolvimento , Artérias/metabolismo , Região Branquial/irrigação sanguínea , Região Branquial/crescimento & desenvolvimento , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Embrião de Mamíferos/metabolismo , Endotélio/citologia , Feminino , Humanos , Camundongos , Camundongos Knockout , Morfogênese , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transdução de Sinais , Ativação Transcricional
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