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1.
Biochemistry (Mosc) ; 84(10): 1166-1176, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31694512

RESUMO

The aim of this study was to evaluate changes in the content of sphingoid bases - sphingosine (SPH), sphinganine, and sphingosine-1-phosphate (SPH-1-P) - and in expression of genes encoding enzymes involved in their metabolism in the brain structures (hippocampus, cortex, and cerebellum) and spinal cord of transgenic FUS(1-359) mice. FUS(1-359) mice are characterized by motor impairments and can be used as a model of amyotrophic lateral sclerosis (ALS). Lipids from the mouse brain structures and spinal cord after 2, 3, and 4 months of disease development were analyzed by chromatography/mass spectrometry, while changes in the expression of the SPHK1, SPHK2, SGPP2, SGPL1, ASAH1, and ASAH2 genes were assayed using RNA sequencing. The levels of SPH and sphinganine (i.e., sphingoid bases with pronounced pro-apoptotic properties) were dramatically increased in the spinal cord at the terminal stage of the disease. The ratio of the anti-apoptotic SPH-1-P to SPH and sphinganine sharply reduced, indicating massive apoptosis of spinal cord cells. Significant changes in the content of SPH and SPH-1-P and in the expression of genes related to their metabolism were found at the terminal ALS stage in the spinal cord. Expression of the SGPL gene (SPH-1-P lyase) was strongly activated, while expression of the SGPP2 (SPH-1-P phosphatase) gene was reduced. Elucidation of mechanisms for the regulation of sphingolipid metabolism in ALS will help to identify molecular targets for the new-generation drugs.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Proteína FUS de Ligação a RNA/metabolismo , Esfingolipídeos/metabolismo , Medula Espinal/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Esfingolipídeos/química
2.
Brain Nerve ; 71(11): 1289-1301, 2019 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-31722315

RESUMO

Protein misfolding crucially underlies the pathogenesis of amyotrophic lateral sclerosis (ALS). Mutant superoxide dismutase 1 (SOD1) and TAR DNA-binding protein 43kDa (TDP-43) are major causal proteins for familial and sporadic ALS, respectively, provoking diverse pathogenic pathways in both intracellular and extracellular environments. Of note, cell-to-cell spreading behavior is implicated in the progression of neurodegeneration, suggesting application in immunotherapies including vaccination, and antibody application as a molecular targeting therapy, due to strict antigen-specificity. Although immunotherapy of intravenous application of full-length immunoglobulin is aimed at targeting the extracellular proteins because of low access to the cytosol, it is therefore necessary to generate expression vectors for variable single chain fragments (scFv) that target intracellular proteins. Despite the advantages of scFv, such as low molecular size and the ability to apply molecular modifications adding proteolytic signals, safety and efficacy should be cautiously estimated in preclinical studies, using appropriate animal models. In ALS, we firstly succeeded in the vaccination of mutant SOD1 transgenic mice, which was followed by accumulating evidence showing the efficacy of immunization against misfolded SOD1. In TDP-43 proteinopathy, we are developing immunotherapy using intrabodies with proteolytic properties against mislocalized or aggregated forms of TDP-43 inside cells.


Assuntos
Esclerose Amiotrófica Lateral/terapia , Imunoterapia , Animais , Proteínas de Ligação a DNA/genética , Camundongos , Camundongos Transgênicos , Mutação , Dobramento de Proteína , Superóxido Dismutase-1/genética
3.
Medicine (Baltimore) ; 98(42): e17557, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31626119

RESUMO

BACKGROUND: Mild cognitive impairment (MCI) and Alzheimer's disease (AD) are neurodegenerative diseases associated with aging. The major clinical features of both are progressive memory loss and progressive cognitive loss. The objective of this systematic review protocol is to provide the methods for evaluating the effectiveness of acupuncture in the treatment on cognitive deficits in transgenic mouse. METHODS AND ANALYSIS: We will search the electronic databases of PubMed, Web of Science, Embase, PsycINFO, as well as the Chinese databases such as Chinese Biomedicine Literature (CBM), Chinese Medical Current Content (CMCC), Chinese Scientific Journal Database (VIP), WanFang Database and China National Knowledge Infrastructure (CNKI) from their inceptions to July 2019. RevMan 5.3 software will be used for the data synthesis and the quality of each study was assessed independently by use of the CAMARADES checklist. RESULTS: This review will provide a high-quality synthesis based on present evidence of acupuncture treatment for AD and MCI in transgenic mouse models. CONCLUSIONS: This systematic review will provide evidence for weather acupuncture is an effective intervention for AD and MCI in transgenic mouse models. ETHICS AND DISSEMINATION: Ethical approval is not necessary since this protocol is only for systematic review and does not involve privacy data or conduct an animal experiment. This protocol will be disseminated by a peer-review journal or conference presentation. TRIAL REGISTRATION NUMBER: PROSPERO CRD42019142985. STRENGTHS AND LIMITATIONS OF THIS STUDY: This systematic review will be the first to provide new knowledge underlying the effectiveness to improve cognitive function of acupuncture treatment for AD and MCI in transgenic mouse models. The result of this systematic review may provide experimental and theoretical basis for the future clinical application of acupuncture in the treatment of AD.The limitation of this systematic review may come from language barriers, because only English and Chinese can be included. Also, this study includes various kinds of acupuncture treatments which may result in essential heterogeneity.


Assuntos
Terapia por Acupuntura/métodos , Doença de Alzheimer/terapia , Cognição/fisiologia , Disfunção Cognitiva/terapia , Doença de Alzheimer/complicações , Doença de Alzheimer/fisiopatologia , Animais , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/fisiopatologia , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos
4.
Br J Anaesth ; 123(6): 818-826, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31570162

RESUMO

BACKGROUND: Studies in developing animals show that a clinically relevant anaesthesia exposure increases neuronal death and alters brain structure. In the hippocampal dentate gyrus, the anaesthetic isoflurane induces selective apoptosis among roughly 10% of 2-week-old hippocampal granule cells in 21-day-old mice. In this work, we queried whether the 90% of granule cells surviving the exposure might be 'injured' and integrate abnormally into the brain. METHODS: The long-term impact of isoflurane exposure on granule cell structure was studied using a transgenic mouse model fate-mapping approach to identify and label immature granule cells. Male and female mice were exposed to isoflurane for 6 h when the fate-mapped granule cells were 2 weeks old. The morphology of the fate-mapped granule cells was quantified 2 months later. RESULTS: The gross structure of the dentate gyrus was not affected by isoflurane treatment, with granule cells present in the correct subregions. Individual isoflurane-exposed granule cells were structurally normal, exhibiting no changes in spine density, spine type, dendrite length, or presynaptic axon terminal structure (P>0.05). Granule cell axon terminals were 13% larger in female mice relative to males; however, this difference was evident regardless of treatment (difference of means=0.955; 95% confidence interval, 0.37-1.5; P=0.010). CONCLUSIONS: A single, prolonged isoflurane exposure did not impair integration of this age-specific cohort of granule cells, regardless of the animal's sex. Nonetheless, although 2-week-old cells were not affected, the results should not be extrapolated to other age cohorts, which may respond differently.


Assuntos
Anestésicos Inalatórios/efeitos adversos , Hipocampo/efeitos dos fármacos , Isoflurano/efeitos adversos , Neurônios/efeitos dos fármacos , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos
5.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(9): 1017-1022, 2019 Sep 30.
Artigo em Chinês | MEDLINE | ID: mdl-31640958

RESUMO

OBJECTIVE: To generate a new strain of HBeAg transgenic mice using CRISPR/Cas9 technique. METHODS: Hepatitis B virus (HBV) HBeAg gene was cloned and inserted in the pliver-HBeAg expression frame at the site of Rosa26 gene using CRISPR/Cas9 and homologous recombination techniques to construct the pliver-HBeAg expression vector containing HBeAg gene. The linear DNA fragment containing HBeAg gene was obtained by enzyme digestion. Cas9 mRNA, gRNA and the donor vector were microinjected into fertilized eggs of C57BL/6J mice, which were then transplanted into the uterus of C57BL/6J female surrogate mice to obtain F0 generation mice. The F0 generation mice were identified by long fragment PCR to obtain F0 transgenic mice with HBeAg gene. The positive F0 generation mice were bred with wild-type C57BL/6J mice to produce the F1 mice, which were identified by PCR and sequencing. The positive F1 transgenic mice carrying HBeAg gene were backcrossed until the homozygous offspring transgenic mice were obtained. The genotypes of the offspring mice were identified. The expressions of HBeAg and HBeAb in the heterozygous and homozygous HBeAg transgenic mice were detected by automatic chemiluminescence immunoassay, immune colloidal gold technique and immunohistochemistry method. RESULTS: A total of 56 F0 mice were obtained, and 2 of them carried homologous recombined HBeAg gene. Six positive F1 mice were obtained, from which 22 homozygous and 29 heterozygous F2 generation HBeAg transgenic mice were obtained. High concentration of HBeAg protein was detected in the peripheral blood of all the positive HBeAg transgenic mice without HBeAb expression. HBeAg expression was detected in the hepatocytes of HBeAg transgenic mice. CONCLUSIONS: We obtained a new strain of HBeAg transgenic mice with stable expression of HBeAg in the hepatocytes and immune tolerance to HBeAg using CRISPR/Cas9 technique, which provide a new animal model for studying HBV.


Assuntos
Sistemas CRISPR-Cas , Antígenos E da Hepatite B/genética , Camundongos Transgênicos , Animais , Feminino , Vetores Genéticos , Vírus da Hepatite B , Camundongos , Camundongos Endogâmicos C57BL
6.
Expert Opin Investig Drugs ; 28(11): 967-975, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31661331

RESUMO

Introduction: The amyloid hypothesis of Alzheimer's disease (AD) states that brain accumulation of amyloid-ß (Aß) oligomers and soluble aggregates represents the major causal event of the disease. Several small organic molecules have been synthesized and developed to inhibit the enzyme (ß-site amyloid precursor protein cleaving enzyme-1 or BACE1) whose action represents the rate-limiting step in Aß production.Areas covered: We reviewed the pharmacology and clinical trials of major BACE1 inhibitors.Expert opinion: In transgenic mouse models of AD, BACE1 inhibitors dose-dependently lower Aß levels in brain and cerebrospinal fluid (CSF) but the evidence for attenuation or reversal cognitive or behavioral deficits is very scanty. In AD patients, BACE1 inhibitors robustly lower plasma and CSF Aß levels and reduce brain plaques but without cognitive, clinical, or functional benefit. To date, seventeen BACE1 inhibitors have failed in double-blind, placebo-controlled clinical trials in patients with mild-to-moderate or prodromal AD, or in cognitively normal subjects at risk of developing AD. Several of these studies were prematurely interrupted due to toxicity or cognitive and behavioral worsening compared to placebo-treated patients. Elenbecestat, the last BACE1 inhibitor remaining in late clinical testing for AD, was recently discontinued due to safety concerns.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Drogas em Investigação/administração & dosagem , Doença de Alzheimer/fisiopatologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Desenvolvimento de Medicamentos , Drogas em Investigação/farmacologia , Humanos , Camundongos , Camundongos Transgênicos
7.
Mol Biol (Mosk) ; 53(5): 755-773, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31661476

RESUMO

Dysregulated proinflammatory cytokine expression may result in the development of severe pathologies, such as rheumatoid arthritis, psoriasis, and neurodegenerative diseases. Transgenic mice and, in particular, those with controllable systemic overexpression of proinflammatory cytokines have recently become an essential instrument to study the molecular mechanisms underlying disease development. Importantly, many of the models are humanized by introducing a human cytokine gene, while leaving or removing the respective endogenous mouse gene. Humanized mice are especially valuable for biomedical research as they provide a relevant model to develop therapies based on blocking the pathogenic activity of a cytokine or to establish the functional significance of genome polymorphisms. The review discusses the available humanized mouse models with overexpression of key proinflammatory cytokines (TNF, IL-ip, and IL-6) and inflammatory cytokines with more specific functions (IL-8, IL-17, and IL-32) and their significance for basic and clinical research.


Assuntos
Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Regulação para Cima , Animais , Artrite Reumatoide/genética , Citocinas/biossíntese , Humanos , Camundongos , Camundongos Transgênicos , Doenças Neurodegenerativas/genética , Psoríase/genética
8.
Nat Neurosci ; 22(10): 1731-1742, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501572

RESUMO

Mitochondria vary in morphology and function in different tissues; however, little is known about their molecular diversity among cell types. Here we engineered MitoTag mice, which express a Cre recombinase-dependent green fluorescent protein targeted to the outer mitochondrial membrane, and developed an isolation approach to profile tagged mitochondria from defined cell types. We determined the mitochondrial proteome of the three major cerebellar cell types (Purkinje cells, granule cells and astrocytes) and identified hundreds of mitochondrial proteins that are differentially regulated. Thus, we provide markers of cell-type-specific mitochondria for the healthy and diseased mouse and human central nervous systems, including in amyotrophic lateral sclerosis and Alzheimer's disease. Based on proteomic predictions, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neuronal mitochondria. We also characterize cell-type differences in mitochondrial calcium buffering via the mitochondrial calcium uniporter (Mcu) and identify regulator of microtubule dynamics protein 3 (Rmdn3) as a determinant of endoplasmic reticulum-mitochondria proximity in Purkinje cells. Our approach enables exploring mitochondrial diversity in many in vivo contexts.


Assuntos
Encéfalo/citologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Astrócitos/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Células Cultivadas , Cerebelo/citologia , Ácidos Graxos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Membranas Mitocondriais/metabolismo , Proteômica , Células de Purkinje/metabolismo
9.
J Biochem ; 166(5): 383-392, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504625

RESUMO

The apelin receptor (APJ), a receptor for apelin and elabela/apela, induces vasodilation and vasoconstriction in blood vessels. However, the prolonged effects of increased APJ-mediated signalling, involving vasoconstriction, in smooth muscle cells have not been fully characterized. Here, we investigated the vasoactive effects of APJ gain of function under the control of the smooth muscle actin (SMA) gene promoter in mice. Transgenic overexpression of APJ (SMA-APJ) conferred sensitivity to blood pressure and vascular contraction induced by apelin administration in vivo. Interestingly, ex vivo experiments showed that apelin markedly increased the vasoconstriction of isolated aorta induced by noradrenaline (NA), an agonist for α- and ß-adrenergic receptors, or phenylephrine, a specific agonist for α1-adrenergic receptor (α1-AR). In addition, intracellular calcium influx was augmented by apelin with NA in HEK293T cells expressing APJ and α1A-AR. To examine the cooperative action of APJ and α1A-AR in the regulation of vasoconstriction, we developed α1A-AR deficient mice using a genome-editing technique, and then established SMA-APJ/α1A-AR-KO mice. In the latter mouse line, aortic vasoconstriction induced by a specific agonist for α1A-AR, A-61603, were significantly less than in SMA-APJ mice. These results suggest that the APJ-enhanced response requires α1A-AR to contract vessels coordinately.


Assuntos
Receptores de Apelina/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Vasoconstrição , Animais , Humanos , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Músculo Liso Vascular/química
10.
Cell Physiol Biochem ; 53(3): 496-507, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31486324

RESUMO

BACKGROUND/AIMS: Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). The relationship between blood bank storage and RBC necroptosis was explored in this study. METHODS: Human RBCs were stored in standard blood bank additive solutions (AS-1, AS-3, or AS-5) for 1 week and hemolysis was evaluated in the context of necroptosis inhibitors and reactive oxygen species (ROS) scavengers. Activation of key factors including RIP1, RIP3, and MLKL was determined using immunoprecipitations and western blot. RBC vesiculation and formation of echinocytes was determined using phase-contrast microscopy. The effect of necroptosis and storage on RBC clearance was determined using a murine transfusion model. RESULTS: Necroptosis is associated with increased RBC clearance post-transfusion. Moreover, storage in AS-1, AS-3, or AS-5 sensitizes RBCs for necroptosis. Importantly, storage-sensitized RBCs undergo necroptosis in response to multiple PFTs, regardless of specificity for CD59. Storage-sensitized RBCs undergo necroptosis via NADPH oxidase-generated ROS. RBC storage led to RIP1 phosphorylation and necrosome formation in an NADPH oxidase-dependent manner suggesting the basis for this sensitization. In addition, storage led to increased RBC clearance post-transfusion. Clearance of these RBCs was due to Syk-dependent echinocyte formation. CONCLUSION: Storage-induced sensitization to RBC necroptosis and clearance is important as it may be relevant to hemolytic transfusion reactions.


Assuntos
Antígenos CD59/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Necrose/metabolismo , Adjuvantes Imunológicos , Animais , Apoptose/fisiologia , Bancos de Sangue , Western Blotting , Morte Celular/genética , Morte Celular/fisiologia , Células Cultivadas , Hemólise/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/genética , Fosforilação/fisiologia , Espécies Reativas de Oxigênio/metabolismo
11.
Exp Parasitol ; 206: 107767, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31520603

RESUMO

Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.


Assuntos
Ovalbumina/análise , Receptores de Antígenos de Linfócitos T/imunologia , Schistosoma mansoni/metabolismo , Linfócitos T/imunologia , Animais , Western Blotting , Galinhas , Citocinas/análise , Citocinas/metabolismo , Eletroforese em Gel de Ágar , Feminino , Citometria de Fluxo , Interleucina-17/análise , Interleucina-17/metabolismo , Interleucina-2/análise , Interleucina-2/metabolismo , Interleucina-6/análise , Interleucina-6/metabolismo , Fígado/parasitologia , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/imunologia , Ovalbumina/metabolismo , Óvulo/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/genética , Transcrição Reversa , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Baço/citologia , Linfócitos T/citologia
12.
Chem Commun (Camb) ; 55(76): 11458-11461, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31535684

RESUMO

We report a polymer-based sensor that rapidly detects cancer based on changes in serum protein levels. Using three ratiometric fluorescence outputs, this simple system identifies early stage and metastatic lung cancer with a high level of accuracy exceeding many biomarker-based assays, making it an attractive strategy for point-of-care testing.


Assuntos
Biomarcadores Tumorais/sangue , Proteínas Sanguíneas/análise , Corantes Fluorescentes/química , Neoplasias Pulmonares/diagnóstico por imagem , Polímeros/química , Animais , Fluorescência , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Neoplasias Experimentais/sangue , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/secundário , Testes Imediatos
13.
Artigo em Chinês | MEDLINE | ID: mdl-31550758

RESUMO

Objective: To study the response characteristics of the posterior intralaminar nucleus (PIN) of auditory thalamus in VGluT2-Cre transgenic adult mice when exposed to white noise and 10K pure tone stimulation. Methods: All adult male Vglut2-Cre mice (8-12 weeks) were used in this study between Oct, 2017 and Oct, 2018. Using the calcium signal fiber photometry method, optic fiber was employed to locate on PIN by injecting AAV-hSyn-DIO-GCaMP6m virus, and thereafter, the activity of the target cluster neurons during different acoustic stimuli was recorded. Matlab was used for data processing and statistical analysis. Results: (1)In both white noise and 10 kHz pure tone as a continuous three-second stimulation, the peak amplitude of calcium signal activity generated in PIN by white noise was superior to that of pure tone, the statistic result showed significantly difference (n=6, t=2.404, P=0.037 1) . (2)In addition, when white noise and 10K pure tone played as consecutive 3 or 5 pips within three-second stimulation, the stimulus-following ability in a consecutive 3 pulses play within 3 seconds was far better than a consecutive 5 pips play within 3 seconds (in both white noise and 10 kHz pure tone), yet consecutive 3 pips play showed greater signal attenuation speed than that in consecutive 5 pips play, the statistic result showed significantly difference (n=6, t=2.748 P=0.033 4) .(3)Regardless of the intra-group comparisons between white noise and 10 kHz pure tone stimulation, PIN showed better signal response in a consecutive 3 pips play than consecutive 5 pips play or a continuous three-second stimulation. When came to the statistical analysis, the acoustic response degree of a continuous three-second stimulation was an intermediate between two others, both consecutive 3 or 5 pips play showed significantly difference. Conclusions: The results suggest that under the same acoustic intensity, VGluT2-Cre transgenic adult mice's PIN shows greater signal response in white noise than pure tone. PIN shows greater signal attenuation to repetition play of 10 kHz pure tone, which implies PIN shows stronger adaptation to 10 kHz pure tone than to white noise. Lastly, PIN is more responsive to a complex sound information (white noise) than to simple sound information (pure tone).


Assuntos
Acústica , Neurônios , Tálamo , Estimulação Acústica , Animais , Limiar Auditivo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/fisiologia , Som , Tálamo/fisiologia
14.
DNA Cell Biol ; 38(11): 1303-1312, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31553232

RESUMO

Growth differentiation factor 5 (GDF5) was reported to regulate brown adipogenesis; however, its effects on insulin sensitivity, full metabolic syndrome spectrum, and the thermogenesis in subcutaneous white adipose tissue (sWAT) have not been elucidated yet. We thus generated fatty acid-binding protein 4 (Fabp4)-GDF5 transgenic (TG) mice and showed that GDF5 TG mice developed a relative lean phenotype on a high-fat diet (HFD) and showed increased insulin sensitivity. Over expression of GDF5 in adipose tissues greatly promoted the thermogenic process in sWAT after cold or ß3-agonist treatment. In TG mice, sWAT showed an important thermogenic effect as the thermogenic gene expression was markedly increased, which was consistent with the typical features of beige adipocytes. Moreover, knockdown of the protein GDF5 impaired browning program in sWAT after thermogenic stimuli. Enhanced mitogen-activated protein kinase (MAPK)/activating transcription factor 2 (ATF2) signaling was also identified in sWAT of HFD-fed GDF5 mice, and thermogenesis in mature adipocytes induced by GDF5 protein could be partly blocked by a p38 MAPK inhibitor. Taken together, our data suggest that GDF5 could improve insulin sensitivity and prevent metabolic syndrome, the adaptive thermogenesis in sWAT could mediate the obesity resistance effects of GDF5 in mice and partially resulted in the activation of the p38 MAPK signaling pathway.


Assuntos
Tecido Adiposo Branco/fisiologia , Fator 5 de Diferenciação de Crescimento/fisiologia , Termogênese/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adipogenia/fisiologia , Tecido Adiposo Branco/metabolismo , Animais , Células Cultivadas , Fator 5 de Diferenciação de Crescimento/genética , Resistência à Insulina/genética , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/genética , Obesidade/metabolismo , Transdução de Sinais/genética
15.
Nat Cell Biol ; 21(9): 1102-1112, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481792

RESUMO

The classical model of tissue renewal posits that small numbers of quiescent stem cells (SCs) give rise to proliferating transit-amplifying cells before terminal differentiation. However, many organs house pools of SCs with proliferative and differentiation potentials that diverge from this template. Resolving SC identity and organization is therefore central to understanding tissue renewal. Here, using a combination of single-cell RNA sequencing (scRNA-seq), mouse genetics and tissue injury approaches, we uncover cellular hierarchies and mechanisms that underlie the maintenance and repair of the continuously growing mouse incisor. Our results reveal that, during homeostasis, a group of actively cycling epithelial progenitors generates enamel-producing ameloblasts and adjacent layers of non-ameloblast cells. After injury, tissue repair was achieved through transient increases in progenitor-cell proliferation and through direct conversion of Notch1-expressing cells to ameloblasts. We elucidate epithelial SC identity, position and function, providing a mechanistic basis for the homeostasis and repair of a fast-turnover ectodermal appendage.


Assuntos
Ameloblastos/citologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Ectoderma/citologia , Incisivo/citologia , Animais , Divisão Celular/fisiologia , Células Epiteliais/citologia , Camundongos Transgênicos , Transdução de Sinais/fisiologia , Células-Tronco/citologia
16.
Nat Cell Biol ; 21(9): 1164-1172, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481796

RESUMO

Single-cell measurement of chromatin states, including histone modifications and non-histone protein binding, remains challenging. Here, we present a low-cost, efficient, simultaneous indexing and tagmentation-based ChIP-seq (itChIP-seq) method, compatible with both low cellular input and single cells for profiling chromatin states. itChIP combines chromatin opening, simultaneous cellular indexing and chromatin tagmentation within a single tube, enabling the processing of samples from tens of single cells to, more commonly, thousands of single cells per assay. We demonstrate that single-cell itChIP-seq (sc-itChIP-seq) yields ~9,000 unique reads per cell. Using sc-itChIP-seq to profile H3K27ac, we sufficiently capture the earliest epigenetic priming event during the cell fate transition from naive to primed pluripotency, and reveal the basis for cell-type specific enhancer usage during the differentiation of bipotent cardiac progenitor cells into endothelial cells and cardiomyocytes. Our results demonstrate that itChIP is a widely applicable technology for single-cell chromatin profiling of epigenetically heterogeneous cell populations in many biological processes.


Assuntos
Cromatina/metabolismo , Células Endoteliais/metabolismo , Processamento de Proteína Pós-Traducional/genética , Análise de Sequência de DNA , Animais , Sítios de Ligação , Imunoprecipitação da Cromatina/métodos , Epigenômica/métodos , Histonas/metabolismo , Camundongos Transgênicos , Análise de Sequência de DNA/métodos
17.
Zhonghua Yi Xue Za Zhi ; 99(34): 2701-2705, 2019 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-31505723

RESUMO

Objective: To established Apc(loxp/loxp)+Kras(LSL-G12D/-)+Smad4(loxp/loxp) transgenic mouse model that mimick the occurrence and development of human sporadic colorectal cancer(CRC) and its liver metastasis. Methods: C57BL/6-Apc(tm1Tyj)/J(Apc(loxP)), B6.129S4-Kras(tm4Tyj)/J(Kras(LSL-G12D)), 129S6-Smad4(tm2.1Cxd)/J(Smad4(loxP)) and C57BL/6J mice were crossed, and genotype with Apc(loxP/loxP)+Kras(LSL-G12D/-)and Apc(loxP/loxP)+Kras(LSL-G12D/-)+Smad4(loxP/loxP)were generated. Genotypes of the mice were identified by PCR and real-time quantitative PCR. The mice were divided into Apc(loxP/loxP)+Kras(LSL-G12D/-)+Smad4(loxP/loxP) group (n=20) and Apc(loxP/loxP)+Kras(LSL-G12D/-)group(n=24). Lentivirus expressing Cre enzyme and IRES-luciferase were injected into the submucosa of colon or rectum of the transgenic mice under colonoscopy. Intraabdominal injection of D-luciferase into mice every 4 weeks, imaging with small animal in vivo imaging system(IVIS). The tumor size, tumorigenesis rate and metastasis ratio were analyzed. At the end of the 20th week, the colorectal lesions and metastatic tissues of mice were stained with hematoxylin-eosin(HE) and the pathological changes were observed under microscope. Results: Apc(loxp/loxp)+Kras(LSL-G12D/-)+Smad4(loxp/loxp) and Apc(loxp/loxp)+Kras(LSL-G12D/-)transgenic mice were successfully bred. The colorectal stem cells of the transgenic mouse mutated leading tumor lesion and liver metastatic under the induction of Lentivirus(Cre-IRES-luciferase). The primary and metastatic foci of colorectal carcinoma and liver metastasis in mice were proved to be adenocarcinoma and liver metastatic carcinoma by histopathological examination. The primary tumor size inApc(loxP/loxP)+Kras(LSL-G12D/-)+Smad4(loxP/loxP) group and Apc(loxP/loxP)+Kras(LSL-G12D/-)group was(3.52±0.26) and(3.45±0.20)mm, respectively,without significant difference(t=0.872, P=0.388).The tumorigenesis rate was 70.0% and 50.0% respectively, and there was no significant difference between the two groups(χ(2)=0.440, P=0.507). The metastasis rate of two groups were 58.3% and 8.3%respectively(Fisher's exact test, P=0.027). Conclusions: In this study, the colorectal carcinogenesis and its spontaneously metastasis to the liver of CRC were induced by Lentivirus(Cre-IRES-luciferase) in our established transgenic mice,which successfully simulated the occurrence and development of human sporadic CRC and its liver metastasis.


Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Neoplasias Pancreáticas , Animais , Progressão da Doença , Humanos , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)
18.
Hypertension ; 74(4): 817-825, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31422694

RESUMO

The BBSome-a complex consisting of 8 Bardet-Biedl syndrome proteins-is involved in the regulation of various cellular processes. Recently, the BBSome complex has emerged as an important regulator of cardiovascular function with implications for disease. In this study, we examined the role of the BBSome in vascular smooth muscle and its effects on the regulation of cardiovascular function. Smooth muscle-specific disruption of the BBSome through tamoxifen-inducible deletion of Bbs1 gene-a critical component of the BBSome complex-reduces relaxation and enhances contractility of vascular rings and increases aortic stiffness independent of changes in arterial blood pressure. Mechanistically, we demonstrate that smooth muscle Bbs1 gene deletion increases vascular angiotensinogen gene expression implicating the renin-angiotensin system in these altered cardiovascular responses. Additionally, we report that smooth muscle-specific Bbs1 knockout mice demonstrate enhanced ET-1 (endothelin-1)-induced contractility of mesenteric arteries-an effect reversed by blockade of the AT1 (angiotensin type 1 receptor) with losartan. These findings highlight the importance of the smooth muscle BBSome in the control of vascular function and arterial stiffness through modulation of renin-angiotensin system signaling.


Assuntos
Pressão Sanguínea/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Rigidez Vascular/fisiologia , Angiotensinogênio/genética , Angiotensinogênio/metabolismo , Animais , Aorta/fisiologia , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Vasodilatação/fisiologia
20.
J Agric Food Chem ; 67(35): 9782-9788, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31390859

RESUMO

Sulforaphane, a potent antioxidant compound, is unstable at ambient temperature, whereas its precursor glucoraphanin is stable and metabolized to sulforaphane. Thus, we hypothesized that glucoraphanin-rich diet could effectively induce antioxidant enzyme activities and investigated the protective effects of long-term intake of a glucoraphanin-enriched kale (GEK) diet on skin aging in senescence-accelerated mouse prone 1 (SAMP1) mice. The senescence grading score was significantly lower after treatment with GEK for 39 weeks than that of the control mice. GEK also suppressed the thinning of the dorsal skin layer. Moreover, the GEK treatment enhanced the collagen production and increased the nuclear translocation of Nrf2 and HO-1 expression level in the skin tissue. TßRII and Smad3 expressions were clearly higher in the GEK-treated group than in the control group. Thus, GEK suppressed senescence in SAMP1 mice by enhancing the antioxidant activity and collagen production via the TßRII/Smad3 pathway, suggesting its practical applications for protection against skin aging.


Assuntos
Brassica/metabolismo , Glucosinolatos/metabolismo , Imidoésteres/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Extratos Vegetais/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Envelhecimento da Pele/fisiologia , Proteína Smad3/metabolismo , Animais , Antioxidantes/metabolismo , Brassica/química , Colágeno/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Transdução de Sinais , Envelhecimento da Pele/genética , Proteína Smad3/genética , Fatores de Tempo
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