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1.
Nihon Yakurigaku Zasshi ; 156(4): 235-238, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-34193703

RESUMO

Sandhoff disease (SD) is a genetic disorder caused by a mutation in the ß-hexosaminidase B (HexB) gene in humans. This results in the massive accumulation of GM2 gangliosides in the nervous system, causing progressive neurodegeneration. The symptoms of SD include muscle weakness, seizures, and mental illness;along with loss of muscle coordination, vision, and hearing. In the most severe form, the onset begins during early infancy, and death usually occurs within 3-5 years of age. The established animal model, Hexb-deficient (Hexb-/-) mouse, shows abnormalities that resemble the severe phenotype found in human infants. We have previously reported that activated microglia causes astrogliosis in Hexb-/- mouse at the early stage of development that can be ameliorated via immunosuppression. Moreover, within the cerebral cortices of Hexb-/- mouse, reactive astrocytes were found to express adenosine A2A receptors in later inflammatory phases. Inhibiting this receptor with istradefylline decreases the number of activated microglial cells and inflammatory cytokines/chemokines. Thus, we underline the importance of the astrocytic A2A receptor as a sensor, in regulating microglial activation in the late phase of inflammation.


Assuntos
Doença de Sandhoff , Animais , Modelos Animais de Doenças , Gliose , Hexosaminidase B , Camundongos , Camundongos Knockout , Neuroglia , Doença de Sandhoff/tratamento farmacológico , Doença de Sandhoff/genética
2.
Zhonghua Gan Zang Bing Za Zhi ; 29(6): 585-590, 2021 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-34225436

RESUMO

Mdr2 knockout mice is a liver disease model, which causes cholestasis due to the lack of phospholipids in the bile. At present, it is not only used for the study of human homologous MDR3 gene, but also widely used as an animal model of liver diseases such as primary sclerosing cholangitis, liver fibrosis, progressive familial intrahepatic cholestasis, liver cancer. Herein, we review the Mdr2 knockout mice physiological characteristics and its application in liver disease research.


Assuntos
Colangite Esclerosante , Colestase Intra-Hepática , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Colangite Esclerosante/genética , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Fígado , Camundongos , Camundongos Knockout
3.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198511

RESUMO

The insulin receptor (IR) is critically involved in maintaining glucose homeostasis. It undergoes proteolytic cleavage by proprotein convertases, which is an essential step for its activation. The importance of the insulin receptor in liver is well established, but its role in pancreatic ß cells is still controversial. In this study, we investigated the cleavage of the IR by the proprotein convertase FURIN in ß cells and hepatocytes, and the contribution of the IR in pancreatic ß cells and liver to glucose homeostasis. ß-cell-specific Furin knockout (ßFurKO) mice were glucose intolerant, but liver-specific Furin knockout (LFurKO) mice were normoglycemic. Processing of the IR was blocked in ßFurKO cells, but unaffected in LFurKO mice. Most strikingly, glucose homeostasis in ß-cell-specific IR knockout (ßIRKO) mice was normal in younger mice (up to 20 weeks), and only mildly affected in older mice (24 weeks). In conclusion, FURIN cleaves the IR non-redundantly in ß cells, but redundantly in liver. Furthermore, we demonstrated that the IR in ß cells plays a limited role in glucose homeostasis.


Assuntos
Furina/deficiência , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Fígado/metabolismo , Receptor de Insulina/metabolismo , Animais , Furina/metabolismo , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Homeostase , Camundongos Knockout , Proteólise , Receptor de Insulina/deficiência , Transdução de Sinais
4.
Nat Commun ; 12(1): 4075, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210972

RESUMO

Long noncoding RNAs (lncRNAs) are known to regulate DNA damage response (DDR) and genome stability in proliferative cells. However, it remains unknown whether lncRNAs are involved in these vital biological processes in post-mitotic neurons. Here, we report and characterize a lncRNA, termed Brain Specific DNA-damage Related lncRNA1 (BS-DRL1), in the central nervous system. BS-DRL1 is a brain-specific lncRNA and depletion of BS-DRL1 in neurons leads to impaired DDR upon etoposide treatment in vitro. Mechanistically, BS-DRL1 interacts with HMGB1, a chromatin protein that is important for genome stability, and is essential for the assembly of HMGB1 on chromatin. BS-DRL1 mediated DDR exhibits cell-type specificity in the cortex and cerebellum in gamma-irradiated mice and BS-DRL1 knockout mice show impaired motor function and concomitant purkinje cell degeneration. Our study extends the understanding of lncRNAs in DDR and genome stability and implies a protective role of lncRNA against neurodegeneration.


Assuntos
Oxirredutases do Álcool/metabolismo , Dano ao DNA , Instabilidade Genômica , Proteína HMGB1/metabolismo , Neurônios/metabolismo , RNA Longo não Codificante/metabolismo , Oxirredutases do Álcool/genética , Animais , Fenômenos Biológicos , Cerebelo , Cromatina , Feminino , Regulação da Expressão Gênica , Proteína HMGB1/genética , Masculino , Camundongos , Camundongos Knockout , Mutação , RNA Longo não Codificante/genética
5.
Nat Commun ; 12(1): 4071, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210974

RESUMO

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3' untranslated region (3'-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3'-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3'-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.


Assuntos
Embrião de Mamíferos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores CCR4/metabolismo , Regiões 3' não Traduzidas , Animais , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/genética , Receptores CCR4/genética , Canais de Cátion TRPP/metabolismo , Fatores de Transcrição
6.
Nat Commun ; 12(1): 4090, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215745

RESUMO

The transition from pluripotent to somatic states marks a critical event in mammalian development, but remains largely unresolved. Here we report the identification of SS18 as a regulator for pluripotent to somatic transition or PST by CRISPR-based whole genome screens. Mechanistically, SS18 forms microscopic condensates in nuclei through a C-terminal intrinsically disordered region (IDR) rich in tyrosine, which, once mutated, no longer form condensates nor rescue SS18-/- defect in PST. Yet, the IDR alone is not sufficient to rescue the defect even though it can form condensates indistinguishable from the wild type protein. We further show that its N-terminal 70aa is required for PST by interacting with the Brg/Brahma-associated factor (BAF) complex, and remains functional even swapped onto unrelated IDRs or even an artificial 24 tyrosine polypeptide. Finally, we show that SS18 mediates BAF assembly through phase separation to regulate PST. These studies suggest that SS18 plays a role in the pluripotent to somatic interface and undergoes liquid-liquid phase separation through a unique tyrosine-based mechanism.


Assuntos
Transição de Fase , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Núcleo Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Células HEK293 , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Tirosina
7.
Nat Commun ; 12(1): 4100, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215750

RESUMO

Tet3 is the main α-ketoglutarate (αKG)-dependent dioxygenase in neurons that converts 5-methyl-dC into 5-hydroxymethyl-dC and further on to 5-formyl- and 5-carboxy-dC. Neurons possess high levels of 5-hydroxymethyl-dC that further increase during neural activity to establish transcriptional plasticity required for learning and memory functions. How αKG, which is mainly generated in mitochondria as an intermediate of the tricarboxylic acid cycle, is made available in the nucleus has remained an unresolved question in the connection between metabolism and epigenetics. We show that in neurons the mitochondrial enzyme glutamate dehydrogenase, which converts glutamate into αKG in an NAD+-dependent manner, is redirected to the nucleus by the αKG-consumer protein Tet3, suggesting on-site production of αKG. Further, glutamate dehydrogenase has a stimulatory effect on Tet3 demethylation activity in neurons, and neuronal activation increases the levels of αKG. Overall, the glutamate dehydrogenase-Tet3 interaction might have a role in epigenetic changes during neural plasticity.


Assuntos
Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Dioxigenases/metabolismo , Glutamato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Ciclo do Ácido Cítrico , Dioxigenases/genética , Epigenômica , Expressão Gênica , Glutamato Desidrogenase/genética , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Complexo Cetoglutarato Desidrogenase/metabolismo , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Plasticidade Neuronal
8.
Nat Commun ; 12(1): 4105, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215755

RESUMO

CCCH zinc finger proteins resolve immune responses by degrading the mRNAs of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-6. Here we report that one such family member, monocyte chemotactic protein-induced protein 3 (MCPIP3, also named ZC3H12C or Regnase-3), promotes skin inflammation by simultaneously enhancing TNF in macrophages and repressing IL-6 in plasmacytoid dendritic cells (pDCs). MCPIP3 is positively associated with psoriasis pathogenesis, and highly expressed by macrophages and pDCs. MCPIP3-deficient macrophages produce less TNF and IL-12p40. However, MCPIP3-deficient pDCs secrete significantly more IL-6. This enhanced intradermal IL-6 may alleviate imiquimod-induced skin inflammation. As a result, MCPIP3-deficient mice are protected from imiquimod-induced psoriasiform lesions. Furthermore, early exposure to pDC-derived IL-6 suppresses macrophage-derived TNF and IL-12p40. Mechanistically, MCPIP3 could directly degrade mRNAs of IL-6, Regnase-1, and IκBζ. In turn, Regnase-1 could degrade MCPIP3 mRNAs. Our study identifies a critical post-transcriptional mechanism that synchronizes myeloid cytokine secretion to initiate autoimmune skin inflammation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinas/metabolismo , Dermatite/metabolismo , Endorribonucleases/metabolismo , Inflamação/metabolismo , Células Mieloides/metabolismo , Ribonucleases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Quimiocina CCL2 , Células Dendríticas , Endorribonucleases/deficiência , Endorribonucleases/genética , Epigenômica , Humanos , Imiquimode , Inflamação/patologia , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Psoríase , Ribonucleases/deficiência , Ribonucleases/genética , Pele/patologia , Fator de Necrose Tumoral alfa/metabolismo
9.
Nat Commun ; 12(1): 4288, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257283

RESUMO

The commonly mutated human KRAS oncogene encodes two distinct KRAS4A and KRAS4B proteins generated by differential splicing. We demonstrate here that coordinated regulation of both isoforms through control of splicing is essential for development of Kras mutant tumors. The minor KRAS4A isoform is enriched in cancer stem-like cells, where it responds to hypoxia, while the major KRAS4B is induced by ER stress. KRAS4A splicing is controlled by the DCAF15/RBM39 pathway, and deletion of KRAS4A or pharmacological inhibition of RBM39 using Indisulam leads to inhibition of cancer stem cells. Our data identify existing clinical drugs that target KRAS4A splicing, and suggest that levels of the minor KRAS4A isoform in human tumors can be a biomarker of sensitivity to some existing cancer therapeutics.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células A549 , Animais , Western Blotting , Proliferação de Células , Citometria de Fluxo , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas de Ligação a RNA/genética
10.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199520

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss of function of PKD1 (polycystin 1) or PKD2 (polycystin 2). The Ca2+-activated Cl- channel TMEM16A has a central role in ADPKD. Expression and function of TMEM16A is upregulated in ADPKD which causes enhanced intracellular Ca2+ signaling, cell proliferation, and ion secretion. We analyzed kidneys from Pkd1 knockout mice and found a more pronounced phenotype in males compared to females, despite similar levels of expression for renal tubular TMEM16A. Cell proliferation, which is known to be enhanced with loss of Pkd1-/-, was larger in male when compared to female Pkd1-/- cells. This was paralleled by higher basal intracellular Ca2+ concentrations in primary renal epithelial cells isolated from Pkd1-/- males. The results suggest enhanced intracellular Ca2+ levels contributing to augmented cell proliferation and cyst development in male kidneys. Enhanced resting Ca2+ also caused larger basal chloride currents in male primary cells, as detected in patch clamp recordings. Incubation of mouse primary cells, mCCDcl1 collecting duct cells or M1 collecting duct cells with dihydrotestosterone (DHT) enhanced basal Ca2+ levels and increased basal and ATP-stimulated TMEM16A chloride currents. Taken together, the more severe cystic phenotype in males is likely to be caused by enhanced cell proliferation, possibly due to enhanced basal and ATP-induced intracellular Ca2+ levels, leading to enhanced TMEM16A currents. Augmented Ca2+ signaling is possibly due to enhanced expression of Ca2+ transporting/regulating proteins.


Assuntos
Anoctamina-1/genética , Doenças Renais Policísticas/genética , Canais de Cátion TRPP/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Proliferação de Células/genética , Cloretos/metabolismo , Di-Hidrotestosterona/farmacologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Caracteres Sexuais
11.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206478

RESUMO

Prevalences of Campylobacter (C.) jejuni infections are progressively rising globally. Given that probiotic feed additives, such as the commercial product Aviguard®, have been shown to be effective in reducing enteropathogens, such as Salmonella, in vertebrates, including livestock, we assessed potential anti-pathogenic and immune-modulatory properties of Aviguard® during acute C. jejuni-induced murine enterocolitis. Therefore, microbiota-depleted IL-10-/- mice were infected with C. jejuni strain 81-176 by gavage and orally treated with Aviguard® or placebo from day 2 to 4 post-infection. The applied probiotic bacteria could be rescued from the intestinal tract of treated mice, but with lower obligate anaerobic bacterial counts in C. jejuni-infected as compared to non-infected mice. Whereas comparable gastrointestinal pathogen loads could be detected in both groups until day 6 post-infection, Aviguard® treatment resulted in improved clinical outcome and attenuated apoptotic cell responses in infected large intestines during acute campylobacteriosis. Furthermore, less distinct pro-inflammatory immune responses could be observed not only in the intestinal tract, but also in extra-intestinal compartments on day 6 post-infection. In conclusion, we show here for the first time that Aviguard® exerts potent disease-alleviating effects in acute C. jejuni-induced murine enterocolitis and might be a promising probiotic treatment option for severe campylobacteriosis in humans.


Assuntos
Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/terapia , Campylobacter jejuni/fisiologia , Enterocolite/microbiologia , Enterocolite/terapia , Probióticos/uso terapêutico , Animais , Biomarcadores , Infecções por Campylobacter/diagnóstico , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Enterocolite/diagnóstico , Microbioma Gastrointestinal , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade , Mediadores da Inflamação/metabolismo , Interleucina-10/deficiência , Jejuno/microbiologia , Jejuno/patologia , Camundongos , Camundongos Knockout
12.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199897

RESUMO

Transthyretin (TTR), a 55 kDa evolutionarily conserved protein, presents altered levels in several conditions, including malnutrition, inflammation, diabetes, and Alzheimer's Disease. It has been shown that TTR is involved in several functions, such as insulin release from pancreatic ß-cells, recovery of blood glucose and glucagon levels of the islets of Langerhans, food intake, and body weight. Here, the role of TTR in hepatic glucose metabolism was explored by studying the levels of glucose in mice with different TTR genetic backgrounds, namely with two copies of the TTR gene, TTR+/+; with only one copy, TTR+/-; and without TTR, TTR-/-. Results showed that TTR haploinsufficiency (TTR+/-) leads to higher glucose in both plasma and in primary hepatocyte culture media and lower expression of the influx glucose transporters, GLUT1, GLUT3, and GLUT4. Further, we showed that TTR haploinsufficiency decreases pyruvate kinase M type (PKM) levels in mice livers, by qRT-PCR, but it does not affect the hepatic production of the studied metabolites, as determined by 1H NMR. Finally, we demonstrated that TTR increases mitochondrial density in HepG2 cells and that TTR insufficiency triggers a higher degree of oxidative phosphorylation in the liver. Altogether, these results indicate that TTR contributes to the homeostasis of glucose by regulating the levels of glucose transporters and PKM enzyme and by protecting against mitochondrial oxidative stress.


Assuntos
Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Dinâmica Mitocondrial , Pré-Albumina/fisiologia , Piruvato Quinase/metabolismo , Animais , Feminino , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 4/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piruvato Quinase/genética
13.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200187

RESUMO

The parasympathetic nervous system is critically involved in the regulation of tear secretion by activating muscarinic acetylcholine receptors. Hence, various animal models targeting parasympathetic signaling have been developed to induce dry eye disease (DED). However, the muscarinic receptor subtype (M1-M5) mediating tear secretion remains to be determined. This study was conducted to test the hypothesis that the M3 receptor subtype regulates tear secretion and to evaluate the ocular surface phenotype of mice with targeted disruption of the M3 receptor (M3R-/-). The experimental techniques included quantification of tear production, fluorescein staining of the ocular surface, environmental scanning electron microscopy, assessment of proliferating cells in the corneal epithelium and of goblet cells in the conjunctiva, quantification of mRNA for inflammatory cytokines and prooxidant redox enzymes and quantification of reactive oxygen species. Tear volume was reduced in M3R-/- mice compared to age-matched controls at the age of 3 months and 15 months, respectively. This was associated with mild corneal epitheliopathy in the 15-month-old but not in the 3-month-old M3R-/- mice. M3R-/- mice at the age of 15 months also displayed changes in corneal epithelial cell texture, reduced conjunctival goblet cell density, oxidative stress and elevated mRNA expression levels for inflammatory cytokines and prooxidant redox enzymes. The findings suggest that the M3 receptor plays a pivotal role in tear production and its absence leads to ocular surface changes typical for DED at advanced age.


Assuntos
Túnica Conjuntiva/patologia , Síndromes do Olho Seco/patologia , Epitélio Corneano/patologia , Células Caliciformes/patologia , Receptor Muscarínico M3/fisiologia , Animais , Túnica Conjuntiva/metabolismo , Modelos Animais de Doenças , Síndromes do Olho Seco/etiologia , Síndromes do Olho Seco/metabolismo , Epitélio Corneano/metabolismo , Células Caliciformes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Lágrimas/metabolismo
14.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200296

RESUMO

Recent studies found that expression of NEDD4-2 is reduced in lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and that the conditional deletion of Nedd4-2 in lung epithelial cells causes IPF-like disease in adult mice via multiple defects, including dysregulation of the epithelial Na+ channel (ENaC), TGFß signaling and the biosynthesis of surfactant protein-C proprotein (proSP-C). However, knowledge of the impact of congenital deletion of Nedd4-2 on the lung phenotype remains limited. In this study, we therefore determined the effects of congenital deletion of Nedd4-2 in the lung epithelial cells of neonatal doxycycline-induced triple transgenic Nedd4-2fl/fl/CCSP-rtTA2S-M2/LC1 mice, with a focus on clinical phenotype, survival, lung morphology, inflammation markers in BAL, mucin expression, ENaC function and proSP-C trafficking. We found that the congenital deletion of Nedd4-2 caused a rapidly progressive lung disease in neonatal mice that shares key features with interstitial lung diseases in children (chILD), including hypoxemia, growth failure, sterile pneumonitis, fibrotic lung remodeling and high mortality. The congenital deletion of Nedd4-2 in lung epithelial cells caused increased expression of Muc5b and mucus plugging of distal airways, increased ENaC activity and proSP-C mistrafficking. This model of congenital deletion of Nedd4-2 may support studies of the pathogenesis and preclinical development of therapies for chILD.


Assuntos
Células Epiteliais/patologia , Pulmão/patologia , Ubiquitina-Proteína Ligases Nedd4/fisiologia , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/patologia , Animais , Animais Recém-Nascidos , Células Epiteliais/metabolismo , Feminino , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Fibrose Pulmonar/etiologia
15.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200797

RESUMO

Although sex differences in the brain are prevalent, the knowledge about mechanisms underlying sex-related effects on normal and pathological brain functioning is rather poor. It is known that female and male brains differ in size and connectivity. Moreover, those differences are related to neuronal morphology, synaptic plasticity, and molecular signaling pathways. Among different processes assuring proper synapse functions are posttranslational modifications, and among them, S-palmitoylation (S-PALM) emerges as a crucial mechanism regulating synaptic integrity. Protein S-PALM is governed by a family of palmitoyl acyltransferases, also known as DHHC proteins. Here we focused on the sex-related functional importance of DHHC7 acyltransferase because of its S-PALM action over different synaptic proteins as well as sex steroid receptors. Using the mass spectrometry-based PANIMoni method, we identified sex-dependent differences in the S-PALM of synaptic proteins potentially involved in the regulation of membrane excitability and synaptic transmission as well as in the signaling of proteins involved in the structural plasticity of dendritic spines. To determine a mechanistic source for obtained sex-dependent changes in protein S-PALM, we analyzed synaptoneurosomes isolated from DHHC7-/- (DHHC7KO) female and male mice. Our data showed sex-dependent action of DHHC7 acyltransferase. Furthermore, we revealed that different S-PALM proteins control the same biological processes in male and female synapses.


Assuntos
Aciltransferases/fisiologia , Lipoilação , Plasticidade Neuronal , Neurônios/fisiologia , Processamento de Proteína Pós-Traducional , Sinapses/fisiologia , Transmissão Sináptica , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Fatores Sexuais
16.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200896

RESUMO

The goals of this study are to develop a high purity patented silk fibroin (SF) film and test its suitability to be used as a slow-release delivery for insulin-like growth factor-1 (IGF-1). The release rate of the SF film delivering IGF-1 followed zero-order kinetics as determined via the Ritger and Peppas equation. The release rate constant was identified as 0.11, 0.23, and 0.09% h-1 at 37 °C for SF films loaded with 0.65, 6.5, and 65 pmol IGF-1, respectively. More importantly, the IGF-1 activity was preserved for more than 30 days when complexed with the SF film. We show that the IGF-1-loaded SF films significantly accelerated wound healing in vitro (BALB/3T3) and in vivo (diabetic mice), compared with wounds treated with free IGF-1 and an IGF-1-loaded hydrocolloid dressing. This was evidenced by a six-fold increase in the granulation tissue area in the IGF-1-loaded SF film treatment group compared to that of the PBS control group. Western blotting analysis also demonstrated that IGF-1 receptor (IGF1R) phosphorylation in diabetic wounds increased more significantly in the IGF-1-loaded SF films group than in other experimental groups. Our results suggest that IGF-1 sustained release from SF films promotes wound healing through continuously activating the IGF1R pathway, leading to the enhancement of both wound re-epithelialization and granulation tissue formation in diabetic mice. Collectively, these data indicate that SF films have considerable potential to be used as a wound dressing material for long-term IGF-1 delivery for diabetic wound therapy.


Assuntos
Bombyx/química , Diabetes Mellitus Experimental/fisiopatologia , Sistemas de Liberação de Medicamentos , Fibroblastos/efeitos dos fármacos , Fibroínas/química , Fator de Crescimento Insulin-Like I/administração & dosagem , Cicatrização/efeitos dos fármacos , Animais , Bandagens , Preparações de Ação Retardada , Feminino , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Reepitelização , Receptores para Leptina/fisiologia
17.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203675

RESUMO

Recently, we found that the deletion of TRPC5 leads to increased inflammation and pain-related behaviour in two animal models of arthritis. (-)-Englerin A (EA), an extract from the East African plant Phyllanthus engleri has been identified as a TRPC4/5 agonist. Here, we studied whether or not EA has any anti-inflammatory and analgesic properties via TRPC4/5 in the carrageenan model of inflammation. We found that EA treatment in CD1 mice inhibited thermal hyperalgesia and mechanical allodynia in a dose-dependent manner. Furthermore, EA significantly reduced the volume of carrageenan-induced paw oedema and the mass of the treated paws. Additionally, in dorsal root ganglion (DRG) neurons cultured from WT 129S1/SvIm mice, EA induced a dose-dependent cobalt uptake that was surprisingly preserved in cultured DRG neurons from 129S1/SvIm TRPC5 KO mice. Likewise, EA-induced anti-inflammatory and analgesic effects were preserved in the carrageenan model in animals lacking TRPC5 expression or in mice treated with TRPC4/5 antagonist ML204.This study demonstrates that while EA activates a sub-population of DRG neurons, it induces a novel TRPC4/5-independent analgesic and anti-inflammatory effect in vivo. Future studies are needed to elucidate the molecular and cellular mechanisms underlying EA's anti-inflammatory and analgesic effects.


Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Sesquiterpenos de Guaiano/farmacologia , Canais de Cátion TRPC/metabolismo , Analgésicos/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Carragenina , Células Cultivadas , Cobalto/metabolismo , Modelos Animais de Doenças , Edema/patologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Hiperalgesia/tratamento farmacológico , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/patologia , Masculino , Camundongos Knockout , Dor/complicações , Dor/tratamento farmacológico , Dor/patologia , Fenótipo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Sesquiterpenos de Guaiano/uso terapêutico
18.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205849

RESUMO

The ability of spermatozoa to swim towards an oocyte and fertilize it depends on precise K+ permeability changes. Kir5.1 is an inwardly-rectifying potassium (Kir) channel with high sensitivity to intracellular H+ (pHi) and extracellular K+ concentration [K+]o, and hence provides a link between pHi and [K+]o changes and membrane potential. The intrinsic pHi sensitivity of Kir5.1 suggests a possible role for this channel in the pHi-dependent processes that take place during fertilization. However, despite the localization of Kir5.1 in murine spermatozoa, and its increased expression with age and sexual maturity, the role of the channel in sperm morphology, maturity, motility, and fertility is unknown. Here, we confirmed the presence of Kir5.1 in spermatozoa and showed strong expression of Kir4.1 channels in smooth muscle and epithelial cells lining the epididymal ducts. In contrast, Kir4.2 expression was not detected in testes. To examine the possible role of Kir5.1 in sperm physiology, we bred mice with a deletion of the Kcnj16 (Kir5.1) gene and observed that 20% of Kir5.1 knock-out male mice were infertile. Furthermore, 50% of knock-out mice older than 3 months were unable to breed. By contrast, 100% of wild-type (WT) mice were fertile. The genetic inactivation of Kcnj16 also resulted in smaller testes and a greater percentage of sperm with folded flagellum compared to WT littermates. Nevertheless, the abnormal sperm from mutant animals displayed increased progressive motility. Thus, ablation of the Kcnj16 gene identifies Kir5.1 channel as an important element contributing to testis development, sperm flagellar morphology, motility, and fertility. These findings are potentially relevant to the understanding of the complex pHi- and [K+]o-dependent interplay between different sperm ion channels, and provide insight into their role in fertilization and infertility.


Assuntos
Infertilidade Masculina/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Espermatozoides/metabolismo , Animais , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Infertilidade Masculina/patologia , Masculino , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Músculo Liso/metabolismo , Oócitos/crescimento & desenvolvimento , Potássio/metabolismo , Motilidade Espermática/genética , Espermatozoides/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
19.
Science ; 373(6550)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210853

RESUMO

The mechanisms by which macrophages regulate energy storage remain poorly understood. We identify in a genetic screen a platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF)-family ortholog, Pvf3, that is produced by macrophages and is required for lipid storage in fat-body cells of Drosophila larvae. Genetic and pharmacological experiments indicate that the mouse Pvf3 ortholog PDGFcc, produced by adipose tissue-resident macrophages, controls lipid storage in adipocytes in a leptin receptor- and C-C chemokine receptor type 2-independent manner. PDGFcc production is regulated by diet and acts in a paracrine manner to control lipid storage in adipose tissues of newborn and adult mice. At the organismal level upon PDGFcc blockade, excess lipids are redirected toward thermogenesis in brown fat. These data identify a macrophage-dependent mechanism, conducive to the design of pharmacological interventions, that controls energy storage in metazoans.


Assuntos
Adipócitos/imunologia , Dieta Hiperlipídica , Proteínas de Drosophila/metabolismo , Metabolismo Energético , Linfocinas/metabolismo , Macrófagos/imunologia , Obesidade/imunologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Termogênese , Tecido Adiposo Marrom/imunologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Hemócitos/imunologia , Fígado/imunologia , Linfocinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Crescimento Derivado de Plaquetas/genética , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Science ; 373(6550)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210852

RESUMO

Large-scale human exome sequencing can identify rare protein-coding variants with a large impact on complex traits such as body adiposity. We sequenced the exomes of 645,626 individuals from the United Kingdom, the United States, and Mexico and estimated associations of rare coding variants with body mass index (BMI). We identified 16 genes with an exome-wide significant association with BMI, including those encoding five brain-expressed G protein-coupled receptors (CALCR, MC4R, GIPR, GPR151, and GPR75). Protein-truncating variants in GPR75 were observed in ~4/10,000 sequenced individuals and were associated with 1.8 kilograms per square meter lower BMI and 54% lower odds of obesity in the heterozygous state. Knock out of Gpr75 in mice resulted in resistance to weight gain and improved glycemic control in a high-fat diet model. Inhibition of GPR75 may provide a therapeutic strategy for obesity.


Assuntos
Índice de Massa Corporal , Exoma/genética , Obesidade/genética , Receptores Acoplados a Proteínas G/genética , Animais , Variação Genética , Humanos , Camundongos , Camundongos Knockout , Análise de Sequência de DNA , Ganho de Peso/genética
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