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1.
PLoS One ; 14(10): e0223052, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31613887

RESUMO

To further investigate the role of the phosphate (Pi) transporter PIT1 in Pi homeostasis and tissue mineralization, we developed a transgenic mouse expressing the C-terminal influenza hemagglutinin (HA) epitope-tagged human PIT1 transporter under control of the cytomegalovirus/chicken beta actin/rabbit beta-globin gene (CAG) promotor and a loxP-stop-loxP (LSL) cassette permitting conditional activation of transgene expression (LSL-HA-hPITtg/+). For an initial characterization of this conditional mouse model, germline excision of the LSL cassette was performed to induce expression of the transgene in all mouse tissues (HA-hPIT1tg/+). Recombination was confirmed using genomic DNA obtained from blood samples of these mice. Furthermore, expression of HA-hPIT1 was found to be at least 10-fold above endogenous mouse Pit1 in total RNA isolated from multiple tissues and from cultured primary calvaria osteoblasts (PCOB) estimated by semi-quantitative RT-PCR. Robust expression of the HA-hPIT1 protein was also observed upon immunoblot analysis in most tissues and permits HA-mediated immunoprecipitation of the transporter. Characterization of the phenotype of HA-hPIT1tg/+ mice at 80 days of age when fed a standard chow (0.7% Pi and 1% calcium) showed elevated plasma Pi, but normal plasma iPTH, iFGF23, serum calcium, BUN, 1,25-dihydroxy vitamin D levels and urine Pi, calcium and protein excretion when compared to WT littermates. Likewise, no change in bone mineral density was observed upon uCT analysis of the distal femur obtained from these mice. In conclusion, heterozygous overexpression of HA-hPIT1 is compatible with life and causes hyperphosphatemia while bone and mineral metabolism of these mice are otherwise normal.


Assuntos
Efeito Fundador , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Camundongos Transgênicos/genética , Fosfatos/metabolismo , Fator de Transcrição Pit-1/genética , Transgenes , Actinas/genética , Actinas/metabolismo , Animais , Transporte Biológico , Densidade Óssea , Calcitriol/sangue , Galinhas , Citomegalovirus/genética , Citomegalovirus/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Crânio/citologia , Crânio/metabolismo , Fator de Transcrição Pit-1/metabolismo , Globinas beta/genética , Globinas beta/metabolismo
2.
PLoS One ; 14(8): e0220818, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31393940

RESUMO

Cytokeratin 19 (KRT19) protein is highly expressed in the epithelium of the gastrointestinal (GI) tract, hepatobiliary tissues, and pancreas of humans and mice. In the present study, we used an improved Cre (iCre) gene to enhance the efficiency of Cre expression in mammalian cells. We established a new transgenic Krt19-iCre bacterial artificial chromosome (BAC) mouse model using the BAC recombineering strategy. Site-specific iCre expression pattern was examined in embryos, adults, and elderly Krt19-iCre mice crossed with Tomato or LacZ reporter mice. Both iCre and reporter protein expressions in adult Krt19-iCre;Tomatoflox/+ (Krt19-iCre Tomato reporter) mice were observed mainly in the epithelial cells of the GI tract, hepatobiliary tissues, and pancreas. However, the expression in the intrahepatic and small pancreatic duct were lower than those in the common bile and large pancreatic duct. In the Krt19-iCre; LacZ reporter embryos, ß-galactosidase for the LacZ reporter was expressed in the glandular epithelial cells of the GI tract in 9.5-day embryos, 12-day embryos, and newborn mice. The reporter protein expression in Krt19-iCre-Tomato reporter mice was consistent with the KRT19 expression in human GI tissues. In conclusion, Krt19-iCre BAC transgenic mice can be used to investigate developmental and pathological conditions using the iCre-loxP system.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Sistema Digestório/metabolismo , Integrases/metabolismo , Queratina-19/metabolismo , Camundongos Transgênicos/metabolismo , Animais , Genes Reporter , Óperon Lac , Camundongos , Modelos Animais
3.
FASEB J ; 33(3): 3575-3589, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30452882

RESUMO

Soluble receptor for advanced glycation end products (sRAGE), shed from cell surfaces, is found in human circulation and has been implicated in cardiovascular disease. Its pathophysiological regulation and underlying mechanisms are scarcely understood. In endothelium-specific human RAGE transgenic mice, human sRAGE was detected in circulation, whereas its level was markedly increased after LPS treatment. That increase was preceded by a rapid rise in TNF-α level. Treatment with TNF-α also significantly increased serum sRAGE. In human microvascular endothelial cells or human umbilical vein endothelial cells with RAGE overexpression, TNF-α markedly induced RAGE shedding, which was dependent on MMP9 and ADAM10. TNF-α-stimulated MMP9 expression was completely dependent on JNK activation, with its inhibition partially effective in suppressing TNF-α-induced RAGE shedding. In contrast, TNF-α transiently induced activation transcription factor (ATF)4, a major component in unfolded protein response (UPR), whereas knockdown of ATF4 abrogated TNF-α-stimulated RAGE shedding. Protein levels of the pro and activated forms of ADAM10 were also decreased by ATF4 knockdown, whereas inhibition of other components of UPR, including XBP1 and ATF6, failed to block TNF-α-stimulated RAGE shedding. Although the endoplasmic reticulum stressors thapsigargin and tunicamycin induced markedly and sustained expression of ATF4 and XBP-1, they did not induce RAGE shedding to the same level as TNF-α, suggesting that ATF4 is necessary but not sufficient alone for TNF-α-mediated RAGE shedding. ATF4 inhibition did not affect TNF-α-stimulated MMP9 expression, whereas inhibition of JNK activity did not influence ADAM10 activation. Thus, inflammatory cascades including TNF-α induced RAGE shedding in endothelial cells in vivo and in vitro. JNK and ATF4 may be 2 platforms for regulation of TNF-α-stimulated RAGE shedding.-Miyoshi, A., Koyama, S., Sasagawa-Monden, M., Kadoya, M., Konishi, K., Shoji, T., Inaba, M., Yamamoto, Y., Koyama, H. JNK and ATF4 as two important platforms for tumor necrosis factor-α-stimulated shedding of receptor for advanced glycation end products.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos/metabolismo
4.
Heart Fail Rev ; 24(2): 269-277, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30284070

RESUMO

Iron deficiency (ID) is a common and ominous comorbidity in heart failure (HF) and predicts worse outcomes, independently of the presence of anaemia. Accumulated data from animal models of systemic ID suggest that ID is associated with several functional and structural abnormalities of the heart. However, the exact role of myocardial iron deficiency irrespective of systemic ID and/or anaemia has been elusive. Recently, several transgenic models of cardiac-specific ID have been developed to investigate the influence of ID on cardiac tissue. In this review, we discuss structural and functional cardiac consequences of ID in these models and summarize data from clinical studies. Moreover, the beneficial effects of intravenous iron supplementation are specified.


Assuntos
Anemia Ferropriva/complicações , Insuficiência Cardíaca/fisiopatologia , Coração/fisiopatologia , Ferro/sangue , Ferro/deficiência , Administração Intravenosa , Animais , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Comorbidade , Feminino , Coração/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/mortalidade , Hepcidinas/metabolismo , Homeostase/fisiologia , Humanos , Ferro/administração & dosagem , Ferro/uso terapêutico , Distúrbios do Metabolismo do Ferro/complicações , Masculino , Camundongos , Camundongos Transgênicos/metabolismo , Modelos Animais , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Receptores da Transferrina/metabolismo
5.
FASEB J ; 33(3): 3549-3561, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30423260

RESUMO

Excessive iron increases the incidence of diabetes and worsens diabetic complications. Reciprocally, diabetes induces iron loading, partially attributable to elevated intestinal iron export according to a recent report. Herein, we show that iron uptake and the mRNA expression of iron importer divalent metal transporter 1 (DMT1) were significantly increased in the duodenum of streptozotocin-induced diabetic mice. Immunofluorescence staining of human intestinal biopsies revealed increased brush border membrane (BBM) and decreased cytoplasmic DMT1 expression in patients with diabetes, suggesting translocation of DMT1. This pattern of DMT1 regulation was corroborated by immunoblotting results in diabetic mice showing that BBM DMT1 expression was increased by 210%, in contrast to a 60% increase in total DMT1. PKC mediates many diabetic complications, and PKCα activity was increased in diabetic mouse intestine. Intriguingly, diabetic mice with PKCα deficiency did not show increases in iron uptake and BBM DMT1 expression. High-glucose treatment increased plasma membrane DMT1 expression via the activation of PKCα in cultured IECs. Inhibition of PKCα potentiated the ubiquitination and degradation of DMT1 protein. We further showed that high glucose suppressed membrane DMT1 internalization. These findings demonstrate that PKCα promotes microvillus membrane DMT1 expression and intestinal iron uptake, contributing to diabetic iron loading.-Zhao, L., Bartnikas, T., Chu, X., Klein, J., Yun, C., Srinivasan, S., He, P. Hyperglycemia promotes microvillus membrane expression of DMT1 in intestinal epithelial cells in a PKCα-dependent manner.


Assuntos
Duodeno/metabolismo , Células Epiteliais/metabolismo , Hiperglicemia/metabolismo , Microvilosidades/metabolismo , Proteína Quinase C-alfa/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Idoso , Animais , Transporte Biológico/fisiologia , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Ferro/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos/metabolismo , Pessoa de Meia-Idade , Ubiquitinação/fisiologia
6.
Stem Cells Transl Med ; 8(3): 236-246, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30474937

RESUMO

Circulating angiogenic cells (CACs) have been implicated in the repair of ischemic tissues, and their mobilization from bone marrow is known to be regulated by the activations of chemokine receptors, including CXCR2 and CXCR4. This study was conducted to investigate the role of N-acetylated proline-glycine-proline (Ac-PGP; a collagen-derived chemotactic tripeptide) on CAC mobilization and its therapeutic potential for the treatment of peripheral artery diseases. Ac-PGP was administered daily to a murine hind limb ischemia model, and the effects of Ac-PGP on blood perfusion and CAC mobilization (Sca1+ Flk1+ cells) into peripheral blood were assessed. Intramuscular administration of Ac-PGP significantly improved ischemic limb perfusion and increased limb salvage rate by increasing blood vessel formation, whereas Ac-PGP-induced blood perfusion and angiogenesis in ischemic limbs were not observed in CXCR2-knockout mice. In addition, Ac-PGP-induced CAC mobilization was found to occur in wild-type mice but not in CXCR2-knockout mice. Transplantation of bone marrow from green fluorescent protein (GFP) transgenic mice to wild-type mice showed bone marrow-derived cells homed to ischemic limbs after Ac-PGP administration and that GFP-positive cells contributed to the formation of ILB4-positive capillaries and α smooth muscle actin (α-SMA)-positive arteries. These results suggest CXCR2 activation in bone marrow after Ac-PGP administration improves blood perfusion and reduces tissue necrosis by inducing CAC mobilization. These findings suggest a new pharmaceutical basis for the treatment of critical limb ischemia. Stem Cells Translational Medicine 2019;8:236&246.


Assuntos
Neovascularização Fisiológica/fisiologia , Peptídeos/metabolismo , Receptores de Interleucina-8B/metabolismo , Animais , Capilares/metabolismo , Membro Posterior/metabolismo , Isquemia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos/metabolismo
7.
JCI Insight ; 3(21)2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385721

RESUMO

TGF-ß signals through a receptor complex composed of 2 type I and 2 type II (TGF-ßRII) subunits. We investigated the role of macrophage TGF-ß signaling in fibrosis after AKI in mice with selective monocyte/macrophage TGF-ßRII deletion (macrophage TGF-ßRII-/- mice). Four weeks after injury, renal TGF-ß1 expression and fibrosis were higher in WT mice than macrophage TGF-ßRII-/- mice, which had decreased renal macrophages. The in vitro chemotactic response to f-Met-Leu-Phe was comparable between bone marrow-derived monocytes (BMMs) from WT and macrophage TGF-ßRII-/- mice, but TGF-ßRII-/- BMMs did not respond to TGF-ß. We then implanted Matrigel plugs suffused with either f-Met-Leu-Phe or TGF-ß1 into WT or macrophage TGF-ßRII-/- mice. After 6 days, f-Met-Leu-Phe induced similar macrophage infiltration into the Matrigel plugs of WT and macrophage TGF-ßRII-/- mice, but TGF-ß induced infiltration only in WT mice. We further determined the number of labeled WT or TGF-ßRII-/- BMMs infiltrating into WT kidneys 20 days after ischemic injury. There were more labeled WT BMMs than TGF-ßRII-/- BMMs. Therefore, macrophage TGF-ßRII deletion protects against the development of tubulointerstitial fibrosis following severe ischemic renal injury. Chemoattraction of macrophages to the injured kidney through a TGF-ß/TGF-ßRII axis is a heretofore undescribed mechanism by which TGF-ß can mediate renal fibrosis during progressive renal injury.


Assuntos
Lesão Renal Aguda/patologia , Fibrose/metabolismo , Rim/metabolismo , Macrófagos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Lesão Renal Aguda/complicações , Animais , Células da Medula Óssea/citologia , Fatores Quimiotáticos/metabolismo , Fatores Quimiotáticos/fisiologia , Fibrose/etiologia , Rim/patologia , Masculino , Camundongos , Camundongos Transgênicos/metabolismo , Monócitos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
8.
Sci Rep ; 8(1): 14453, 2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30262904

RESUMO

Site-specific recombinases (SSR) are utilized as important genome engineering tools to precisely modify the genome of mice and other model organisms. Reporter mice that mark cells that at any given time had expressed the enzyme are frequently used for lineage tracing and to characterize newly generated mice expressing a recombinase from a chosen promoter. With increasing sophistication of genome alteration strategies, the demand for novel SSR systems that efficiently and specifically recombine their targets is rising and several SSR-systems are now used in combination to address complex biological questions in vivo. Generation of reporter mice for each one of these recombinases is cumbersome and increases the number of mouse lines that need to be maintained in animal facilities. Here we present a multi-reporter mouse line for loci-of-recombination (X) (MuX) that streamlines the characterization of mice expressing prominent recombinases. MuX mice constitutively express nuclear green fluorescent protein after recombination by either Cre, Flp, Dre or Vika recombinase, rationalizing the number of animal lines that need to be maintained. We also pioneer the use of the Vika/vox system in mice, illustrating its high efficacy and specificity, thereby facilitating future designs of sophisticated recombinase-based in vivo genome engineering strategies.


Assuntos
DNA Nucleotidiltransferases , Proteínas de Escherichia coli , Genes Reporter , Proteínas de Fluorescência Verde , Integrases , Camundongos Transgênicos , Recombinases , Animais , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Recombinases/genética , Recombinases/metabolismo
9.
Cytoskeleton (Hoboken) ; 75(9): 395-409, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29979496

RESUMO

The actin-based motor myosin Va transports numerous cargos, including the smooth endoplasmic reticulum (SER) in cerebellar Purkinje neurons (PNs) and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Toward that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the immunoglobulin G-binding domain of protein A, a tobacco etch virus cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of SER transport in PNs and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it together with associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va-binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including guanine nucleotide-binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates myosin Va, and Gnao1's localization to PN dendritic spines depends on myosin Va. The mouse model created here should facilitate the identification of novel myosin Va-binding partners, which in turn should advance our understanding of the roles played by this important myosin in vivo.


Assuntos
Cerebelo/fisiologia , Camundongos Transgênicos/metabolismo , Miosina Tipo V/metabolismo , Animais , Camundongos
10.
Int J Mol Sci ; 19(5)2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29762513

RESUMO

Melanocytes are pigment producing cells in the skin that give rise to cutaneous malignant melanoma, which is a highly aggressive and the deadliest form of skin cancer. Studying melanocytes in vivo is often difficult due to their small proportion in the skin and the lack of specific cell surface markers. Several genetically-engineered mouse models (GEMMs) have been created to specifically label the melanocyte compartment. These models give both spatial and temporal control over the expression of a cellular 'beacon' that has an added benefit of inducible expression that can be activated on demand. Two powerful models that are discussed in this review include the melanocyte-specific, tetracycline-inducible green fluorescent protein expression system (iDct-GFP), and the fluorescent ubiquitination-based cell cycle indicator (FUCCI) model that allows for the monitoring of the cell-cycle. These two systems are powerful tools in studying melanocyte and melanoma biology. We discuss their current uses and how they could be employed to help answer unresolved questions in the fields of melanocyte and melanoma biology.


Assuntos
Ciclo Celular , Proteínas de Fluorescência Verde/genética , Melanócitos/citologia , Camundongos Transgênicos/genética , Animais , Linhagem da Célula , Proteínas de Fluorescência Verde/metabolismo , Melanócitos/metabolismo , Melanoma/patologia , Camundongos , Camundongos Transgênicos/metabolismo , Microscopia de Fluorescência/métodos
11.
Oncogene ; 37(29): 4046-4054, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29695833

RESUMO

The receptor tyrosine kinase Ret, a key gain-of-function mutated oncoprotein in thyroid carcinomas, has recently been implicated in other cancer types. While Ret copy number gains and mutations have been reported at low frequencies in breast tumors, we and others have reported that Ret is overexpressed in about 40% of human tumors and this correlates with poor patient prognosis. Ret activation regulates numerous intracellular pathways related to proliferation and inflammation, but it is not known whether abnormal Ret expression is sufficient to induce mammary carcinomas. Using a novel doxycycline-inducible transgenic mouse model with the MMTV promoter controlling Ret expression, we show that overexpression of wild-type Ret in the mammary epithelium produces mammary tumors, displaying a morphology that recapitulates characteristics of human luminal breast tumors. Ret-evoked tumors are estrogen receptor positive and negative for progesterone receptor. Moreover, tumors rapidly regress after doxycycline withdrawal, indicating that Ret is the driving oncoprotein. Using next-generation sequencing, we examined the levels of transcripts in these tumors, confirming a luminal signature. Ret-evoked tumors have been passaged in mice and used to test novel therapeutic approaches. Importantly, we have determined that tumors are resistant to endocrine therapy, but respond successfully to treatment with a Ret kinase inhibitor. Our data provide the first compelling evidence for an oncogenic role of non-mutated Ret in the mammary gland and are an incentive for clinical development of Ret as a cancer biomarker and therapeutic target.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias Mamárias Animais/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos Transgênicos/metabolismo , Receptores de Progesterona/metabolismo
12.
FASEB J ; 32(8): 4247-4257, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29509512

RESUMO

M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.


Assuntos
Cognição/fisiologia , Subunidades Proteicas/metabolismo , Receptor Muscarínico M1/metabolismo , Receptores de AMPA/metabolismo , Animais , Pareamento Cromossômico/fisiologia , Hipocampo/metabolismo , Aprendizagem/fisiologia , Masculino , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/metabolismo , Fosforilação/fisiologia , Receptores Muscarínicos/metabolismo
13.
Sci Rep ; 8(1): 957, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29343749

RESUMO

Low testosterone (T) in men, especially its free fraction, has been associated with loss of energy. In accordance, orchidectomy (ORX) in rodents results in decreased physical activity. Still, the mechanisms through which T stimulates activity remain mostly obscure. Here, we studied voluntary wheel running behavior in three different mouse models of androgen deficiency: ORX, androgen receptor (AR) knock-out (ARKO) and sex hormone binding globulin (SHBG)-transgenic mice, a novel mouse model of "low free T". Our results clearly show a fast and dramatic action of T stimulating wheel running, which is not explained by its action on muscle, as evidenced by neuromuscular studies and in a muscle-specific conditional ARKO mouse model. The action of T occurs via its free fraction, as shown by the results in SHBG-transgenic mice, and it implies both androgenic and estrogenic pathways. Both gene expression and functional studies indicate that T modulates the in vivo sensitivity to dopamine (DA) agonists. Furthermore, the restoration of wheel running by T is inhibited by treatment with DA antagonists. These findings reveal that the free fraction of T, both via AR and indirectly through aromatization into estrogens, stimulates physical activity behavior in male mice by acting on central DA pathways.


Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Condicionamento Físico Animal/fisiologia , Testosterona/metabolismo , Androgênios/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/metabolismo , Atividade Motora/fisiologia , Orquiectomia/métodos , Receptores Androgênicos/metabolismo , Corrida/fisiologia
14.
Postepy Biochem ; 64(1): 21-28, 2018 Jun 30.
Artigo em Polonês | MEDLINE | ID: mdl-30652834

RESUMO

The mouse is a popular animal model employed for studying metabolic alterations. The generation of fat-1 transgenic mice by Professor Jing X. Kang and collaborators has revolutionised the omega-3 research. Fat-1 mice are able to convert omega-6 fatty acids to omega-3 fatty acids due to gene fat-1 from Caenorhabditis elegans that encodes an omega-3 fatty acids desaturase. This mice model can endogenously synthesize omega-3 PUFA without ALA intake and the balancing quantity and quality of various confounding factors of different diets. Next, novel transgenic mice - "Omega mice" with the expression of the fat-1 and fat-2 transgenes were created to produce both omega-6 and omega-3 PUFA from a diet containing saturated fat or carbohydrates with essential fatty acids deficiency. Both transgenic mice are utilities for studying molecular mechanisms of omega-3 fatty acids and their metabolites in tumorigenesis and inflammatory disorders.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Camundongos Transgênicos/metabolismo , Modelos Animais , Animais , Carcinogênese , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Inflamação , Camundongos , Camundongos Transgênicos/genética
16.
Antioxid Redox Signal ; 28(12): 1105-1119, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28931313

RESUMO

AIM: Neuromuscular junction (NMJ) represents the morphofunctional interface between muscle and nerve. Several chronic pathologies such as aging and neurodegenerative diseases, including muscular dystrophy and amyotrophic lateral sclerosis, display altered NMJ and functional denervation. However, the triggers and the molecular mechanisms underlying the dismantlement of NMJ remain unclear. RESULTS: Here we provide evidence that perturbation in redox signaling cascades, induced by muscle-specific accumulation of mutant SOD1G93A in transgenic MLC/SOD1G93A mice, is causally linked to morphological alterations of the neuromuscular presynaptic terminals, high turnover rate of acetylcholine receptor, and NMJ dismantlement. The analysis of potential molecular mechanisms that mediate the toxic activity of SOD1G93A revealed a causal link between protein kinase Cθ (PKCθ) activation and NMJ disintegration. INNOVATION: The study discloses the molecular mechanism that triggers functional denervation associated with the toxic activity of muscle SOD1G93A expression and suggests the possibility of developing a new strategy to counteract age- and pathology-associated denervation based on pharmacological inhibition of PKCθ activity. CONCLUSIONS: Collectively, these data indicate that muscle-specific accumulation of oxidative damage can affect neuromuscular communication and induce NMJ dismantlement through a PKCθ-dependent mechanism. Antioxid. Redox Signal. 28, 1105-1119.


Assuntos
Junção Neuromuscular/metabolismo , Proteína Quinase C-theta/metabolismo , Superóxido Dismutase-1/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos/metabolismo , Neurônios Motores/metabolismo , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Oxirredução
17.
Biochem Biophys Res Commun ; 495(2): 1986-1991, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29223399

RESUMO

Genetically modified mice have been widely used in the field of ß-cell research. However, analysis of results gathered using genetically modified organisms should be interpreted carefully as the results may be confounded by several factors. Here, we showed the ectopic serotonin (5-HT) production in ß-cells of RIP-CreMgn, MIP-GFP, and MIP-Cre/ERT mice. These mice contained a human growth hormone (hGH) cassette to enhance transgene expression and hGH expression and Stat5 phosphorylation were detected in pancreatic islets of these mice. The expression level of tryptophan hydroxylase 1 (Tph1) was upregulated in pancreatic islets of transgenic mice with an hGH cassette but not in transgenic mice without an hGH cassette. Ectopic 5-HT production was not observed in ß-cell-specific prolactin receptor (Prlr) knockout mice or Stat5 knockout mice crossed with RIP-CreMgn. We further confirmed that 5-HT production in ß-cells of several transgenic mice was induced by hGH expression followed by the activation of the Prlr-Stat5-Tph1 pathway. These findings indicate that results obtained using transgenic mice containing the hGH cassette should be interpreted with care.


Assuntos
Linfócitos B/metabolismo , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/metabolismo , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Serotonina/genética , Serotonina/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL
18.
Neuroreport ; 29(2): 118-122, 2018 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-29251688

RESUMO

Retinal ganglion cells (RGCs) that express the photopigment melanopsin (mRGCs) are photosensitive and initiate the non-image-forming pathway, where the majority of their axons terminate in the suprachiasmatic nucleus (SCN). RGCs only make up approximately half of the cells in the ganglion cell layer of the retina; therefore, it is important to be able to distinguish them from other cell types. The transgenic Thy-1 YFP mouse line 16 (Thy-1 YFP-16) expresses yellow-fluorescent protein (YFP) in projection neurons, including RGCs. Our objective was to determine whether mRGCs are labeled with YFP in Thy-1 YFP-16 transgenic mice. Paraformaldehyde-fixed retinal wholemounts and frozen vertical sections were prepared from Thy-1 YFP-16 mice and fluorescently labeled with rabbit anti-melanopsin and guinea-pig anti-RNA binding protein with multiple splicing to identify mRGCs and total RGCs, respectively. Thy-1 YFP-16 mouse brains were sectioned coronally and imaged to view RGC axonal projections to the SCN. Confocal images of retinal preparations show that the majority (∼89%) of mRGCs are not YFP-positive in Thy-1 YFP-16 mice, where ∼11% expressed a weak fluorescent signal. In addition, there are almost no YFP-positive axons present in the SCN of coronal brain sections. We conclude that the majority of mRGC somas and axons are not labeled with YFP in the transgenic Thy-1 YFP-16 mouse line; therefore, this mouse model may not suitable for research involving mRGC visual pathways.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos Transgênicos/anatomia & histologia , Camundongos Transgênicos/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Opsinas de Bastonetes/metabolismo , Animais , Proteínas de Bactérias/genética , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Luminescentes/genética , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Vias Visuais/citologia , Vias Visuais/metabolismo
19.
Biomed Pharmacother ; 96: 1380-1388, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29169728

RESUMO

A key molecular event in the pathogenesis of Parkinson's disease is mitochondrial damage caused by α-synuclein (α-syn). Mitochondria mediates both necrosis and apoptosis, which are associated with morphological changes. However, the mechanism by which α-syn alters mitochondrial morphology remains unclear. To address this issue, we investigated mitochondrial permeability transition pore (mPTP) opening and changes in cardiolipin (CL) levels in mitochondria isolated from the brain of Thy1α-syn mice. Cytoplasmic cytochrome C and cleaved caspase-3 protein levels were upregulated in the brain of transgenic mice. Morphological analysis by atomic force microscopy (AFM) suggested a correlation between mitochondrial morphology and function in these animals. Incubation of isolated mitochondria with recombinant human α-synuclein N terminus (α-syn/N) decreased mitochondrial CL content. An AFM analysis showed that α-syn/N induced mitochondrial swelling and the formation of pore-like structures, which was associated with decreased mitochondrial transmembrane potential and complex I activity. The observed mitochondrial dysfunction was abrogated by treatment with the mPTP inhibitor cyclosporin A, although there was no recovery of CL content. These results provide insight into the mechanism by which α-syn/N directly undermines mitochondrial structure and function via modulation of mPTP opening and CL levels, and suggests that morphological analysis of isolated mitochondria by AFM is a useful approach for evaluating mitochondrial injury.


Assuntos
Camundongos Transgênicos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , alfa-Sinucleína/metabolismo , Animais , Apoptose/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Citocromos c/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica/métodos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Doença de Parkinson/metabolismo
20.
Int J Mol Sci ; 18(10)2017 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-28974016

RESUMO

Regulated autophagy is involved in the repair of renal ischemia-reperfusion injury (IRI). Fat-1 transgenic mice produce ω3-Polyunsaturated fatty acids (ω3-PUFAs) from ω6-Polyunsaturated fatty acids (ω6-PUFAs) without a dietary ω3-PUFAs supplement, leading to a high accumulation of omega-3 in various tissues. ω3-PUFAs show protective effects against various renal injuries and it has recently been reported that ω3-PUFAs regulate autophagy. We assessed whether ω3-PUFAs attenuated IR-induced acute kidney injury (AKI) and evaluated its associated mechanisms. C57Bl/6 background fat-1 mice and wild-type mice (wt) were divided into four groups: wt sham (n = 10), fat-1 sham (n = 10), wt IRI (reperfusion 35 min after clamping both the renal artery and vein; n = 15), and fat-1 IRI (n = 15). Kidneys and blood were harvested 24 h after IRI and renal histological and molecular data were collected. The kidneys of fat-1 mice showed better renal cell survival, renal function, and pathological damage than those of wt mice after IRI. In addition, fat-1 mice showed less oxidative stress and autophagy impairment; greater amounts of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, Beclin-1, and Atg7; lower amounts of p62; and, higher levels of renal cathepsin D and ATP6E than wt kidneys. They also showed more adenosine monophosphate-activated protein kinase (AMPK) activation, which resulted in the inhibition of phosphorylation of the mammalian target of rapamycin (mTOR). Collectively, ω3-PUFAs in fat-1 mice contributed to AMPK mediated autophagy activation, leading to a renoprotective response.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Lesão Renal Aguda/metabolismo , Autofagia , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Ômega-3/metabolismo , Camundongos Transgênicos/genética , Traumatismo por Reperfusão/metabolismo , Lesão Renal Aguda/complicações , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/metabolismo , Estresse Oxidativo , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia
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