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1.
Mol Cell Neurosci ; 100: 103401, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31491533

RESUMO

Reelin plays important roles in regulating neuronal development, modulating synaptic function, and counteracting amyloid ß toxicity. A specific proteolytic cleavage (N-t cleavage) of Reelin abolishes its biological activity. We recently identified ADAMTS-3 (a disintegrin and metalloproteinase with thrombospondin motifs 3) as the major N-t cleavage enzyme in the embryonic and early postnatal brain. The contribution of other proteases, particularly in the postnatal brain, has not been demonstrated in vivo. ADAMTS-2, -3 and -14 share similar domain structures and substrate specificity, raising the possibility that ADAMTS-2 and -14 may cleave Reelin. We found that recombinant ADAMTS-2 protein expressed in cultured cell lines cleaves Reelin at the N-t site as efficiently as ADAMTS-3 while recombinant ADAMTS-14 hardly cleaves Reelin. The disintegrin domain is necessary for the Reelin-cleaving activity of ADAMTS-2 and -3. ADAMTS-2 is expressed in the adult brain at approximately the same level as ADAMTS-3. We generated ADAMTS-2 knockout (KO) mice and found that ADAMTS-2 significantly contributes to the N-t cleavage and inactivation of Reelin in the postnatal cerebral cortex and hippocampus, but much less in the cerebellum. Therefore, it was suggested that ADAMTS-2 can be a therapeutic target for adult brain disorders such as schizophrenia and Alzheimer's disease.

2.
Am J Pathol ; 189(3): 677-686, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30553837

RESUMO

Localization of the abnormal and normal isoforms of prion proteins to detergent-resistant membrane microdomains, lipid rafts, is important for the conformational conversion. Lipid rafts are enriched in sialic acid-containing glycosphingolipids (namely, gangliosides). Alteration in the ganglioside composition of lipid rafts can affect the localization of lipid raft-associated proteins. To investigate the role of gangliosides in the pathogenesis of prion diseases, we performed intracerebral transmission study of a scrapie prion strain Chandler and a Gerstmann-Sträussler-Scheinker syndrome prion strain Fukuoka-1 using various knockout mouse strains ablated with ganglioside synthase gene (ie, GD2/GM2 synthase, GD3 synthase, or GM3 synthase). After challenge with the Chandler strain, GD2/GM2 synthase knockout mice showed 20% reduction of incubation time, reduced prion protein deposition in the brain with attenuated glial reactions, and reduced localization of prion proteins to lipid rafts. These results raise the possibility that the gangliosides may have an important role in prion disease pathogenesis by affecting the localization of prion proteins to lipid rafts.

3.
Xenotransplantation ; : e12468, 2018 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-30375053

RESUMO

The ultimate goal of regenerative medicine is the transplantation of a target organ generated by the patient's own cells. Recently, a method of organ generation using pluripotent stem cells (PSCs) and blastocyst complementation was reported. This approach is based on chimeric animal generation using an early embryo and PSCs, and the contribution of PSCs to the target organ is key to the method's success. However, the contribution rate of PSCs in target organs generated by different chimeric animal generation methods remains unknown. In this study, we used 8-cell embryo aggregation, 8-cell embryo injection, and blastocyst injection to generate interspecies chimeric mice using rat embryonic stem (ES) cells and then investigated the differences in the contribution rate of the rat ES cells. The rate of chimeric mouse generation was the highest using blastocyst injection, followed in order by 8-cell embryo injection and 8-cell embryo aggregation. However, the contribution rate of rat ES cells was the highest in chimeric neonates generated by 8-cell embryo injection, and the difference was statistically significant in the liver. Live functionality was confirmed by analyzing the expression of rat hepatocyte-derived drug-metabolizing enzyme. Collectively, these findings indicate that the 8-cell embryo injection method is the most suitable for generation of PSC-derived organs via chimeric animal generation, particularly for the liver.

4.
Mediators Inflamm ; 2017: 6909415, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29138532

RESUMO

We investigated the regulatory roles of USP2 in mRNA accumulation of proinflammatory cytokines in macrophage-like cells after stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Human macrophage-like HL-60 cells, mouse macrophage-like J774.1 cells, and mouse peritoneal macrophages demonstrated negative feedback to USP2 mRNA levels after LPS stimulation, suggesting that USP2 plays a significant role in LPS-stimulated macrophages. USP2 knockdown (KD) by short hairpin RNA in HL-60 cells promoted the accumulation of transcripts for 25 of 104 cytokines after LPS stimulation. In contrast, limited induction of cytokines was observed in cells forcibly expressing the longer splice variant of USP2 (USP2A), or in peritoneal macrophages isolated from Usp2a transgenic mice. An ubiquitin isopeptidase-deficient USP2A mutant failed to suppress LPS-induced cytokine expression, suggesting that protein ubiquitination contributes to USP2-mediated cytokine repression. Although USP2 deficiency did not accelerate TNF receptor-associated factor (TRAF) 6-nuclear factor-κB (NF-κB) signaling, it increased the DNA binding ratio of the octamer binding transcription factor (Oct)-1 to Oct-2 in TNF, CXCL8, CCL4, and IL6 promoters. USP2 decreased nuclear Oct-2 protein levels in addition to decreasing the polyubiquitination of Oct-1. In summary, USP2 modulates proinflammatory cytokine induction, possibly through modification of Oct proteins, in macrophages following TLR4 activation.


Assuntos
Proteínas de Drosophila/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Células HL-60 , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
5.
Biochem Biophys Rep ; 9: 322-329, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28956020

RESUMO

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.

6.
J Biol Chem ; 292(37): 15378-15394, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28754693

RESUMO

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-ß expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist.


Assuntos
Células Dendríticas/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Antígeno 96 de Linfócito/agonistas , Macrófagos/efeitos dos fármacos , Modelos Imunológicos , Receptor 4 Toll-Like/agonistas , Animais , Sítios de Ligação , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Células Cultivadas , Biologia Computacional , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Desenho de Drogas , Humanos , Ligantes , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Simulação de Acoplamento Molecular , Fosforilação , Piridonas/química , Piridonas/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Organismos Livres de Patógenos Específicos , Relação Estrutura-Atividade , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
7.
Nat Commun ; 7: 10574, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26868148

RESUMO

Recent evidence has revealed that senescence induction requires fine-tuned activation of p53, however, mechanisms underlying the regulation of p53 activity during senescence have not as yet been clearly established. We demonstrate here that SCF(Fbxo22)-KDM4A is a senescence-associated E3 ligase targeting methylated p53 for degradation. We find that Fbxo22 is highly expressed in senescent cells in a p53-dependent manner, and that SCF(Fbxo22) ubiquitylated p53 and formed a complex with a lysine demethylase, KDM4A. Ectopic expression of a catalytic mutant of KDM4A stabilizes p53 and enhances p53 interaction with PHF20 in the presence of Fbxo22. SCF(Fbxo22)-KDM4A is required for the induction of p16 and senescence-associated secretory phenotypes during the late phase of senescence. Fbxo22(-/-) mice are almost half the size of Fbxo22(+/-) mice owing to the accumulation of p53. These results indicate that SCF(Fbxo22)-KDM4A is an E3 ubiquitin ligase that targets methylated p53 and regulates key senescent processes.


Assuntos
Senescência Celular , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Citometria de Fluxo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Células MCF-7 , Metilação , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Citoplasmáticos e Nucleares/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
8.
Adipocyte ; 2(4): 227-36, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24052898

RESUMO

The anti-diabetic effects of Brazilian propolis were examined using ob/ob mice. Although repeated injection of an ethanol extract of Brazilian propolis (100 mg/kg, ip, twice a week for 12 weeks) did not affect body weight gain and food intake of ob/ob mice, blood glucose and plasma cholesterol levels were significantly attenuated. Moreover, the propolis extract partially restored glucose tolerance and insulin resistance, indicating anti-diabetic properties of the extract. The propolis-treated mice exhibited lower weight gain in mesenteric adipose tissue, while weight gains in inguinal and epididymal adipose tissues were not modulated. Flow cytometric and microscopic analyses suggested that the extract promoted accumulation of eosinophils into mesenteric and epididymal adipose tissues. Alternatively, the ratio of M1-like macrophages to M2-like macrophages in mesenteric adipose tissue was reduced by the propolis injection, coincident with the decrement of the number of interleukin-12A(+) cells. Levels of M1 macrophage markers, such as Itgax and Il12b transcripts, were decreased in the vascular stromal fraction of mesenteric adipose tissue, whereas those of pan-macrophage markers Emr1 and Cd68 were not influenced. Microarray and subsequent gene ontology term analyses suggested that propolis attenuated immune activation in mesenteric adipose tissues. Taken together, this indicates that Brazilian propolis improves diabetes in ob/ob mice, presumably through modification of immune cells in mesenteric adipose tissues.

9.
Biomed Res ; 34(4): 189-95, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23995055

RESUMO

We have reported that in ganglioside GM3-deficient (GM3(-/-)) mice, spontaneous alternation behavior assessed by a Y-maze task was significantly lower, and total arm entries were significantly higher than in wild-type mice. The objective of the present study was to examine the role of nicotinic acetylcholine receptor (nAChR) signaling in impairment of spontaneous alternation behavior of GM3(-/-) mice. Nicotine treatment (0.3, 1.0 mg/kg, s.c.) dose dependently improved the spontaneous alternation deficit without affecting total arm entries in GM3(-/-) mice. The nicotine-induced (1.0 mg/kg, s.c.) improvement was significantly abolished by the nAChR antagonist mecamylamine (1.0 mg/kg, i.p.). The α4ß2 nAChR antagonist dihydro-ß-erythroidine (2.5, 10.0 mg/kg, i.p.) dose dependently counteracted the nicotine-induced improvement of spontaneous alternation in GM3(-/-) mice, whereas the α7 nAChR antagonist methyllycaconitine (2.5, 10.0 mg/kg, i.p.) did not. In addition, the α4ß2 nAChR agonist RJR-2403 (5.0, 10.0 mg/kg, s.c.) dose dependently and significantly improved the spontaneous alternation deficit, whereas the α7 nAChR agonist PNU120596 (0.3, 1.0, 3.0 mg/kg, i.p.) did not. These findings revealed that nicotine improved spontaneous alternation behavior of GM3(-/-) mice via the activation of α4ß2, but not α7, nAChR. Thus, the ganglioside GM3 might be responsible for α4ß2 nAChR signaling in the spontaneous alternation behavior.


Assuntos
Comportamento Animal , Gangliosídeo G(M3)/deficiência , Receptores Nicotínicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia
10.
FASEB J ; 27(12): 4940-53, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24005904

RESUMO

Macrophages play a critical role in chronic inflammation and metabolic diseases. We identified a longer splice variant of ubiquitin specific protease (USP) 2-69 as a novel molecule that modulates pathways implicated in metabolic disorders. Expression levels of aP2/FABP4 and PAI-1/SERPINE1 genes were increased by 4- and 1.8-fold, respectively, after short hairpin RNA-mediated knockdown (KD) of the USP2 gene, and such expression was alleviated by overexpression of USP2-69 in human myeloid cell lines. Supernatants derived from USP2-KD cells induced IL6 (∼6-fold) and SAA3 (∼15-fold) in 3T3-L1 adipocytes to suggest the anti-inflammatory properties of USP2. In addition, we observed a 30% decrease in the number of macrophages in mesenteric adipose tissue derived from USP2-69 transgenic mice fed a high-fat diet for 14 wk compared with that in their C57BL/6 littermates (P<0.01), which was consistent with a ∼40% decrease in transcription of aP2 and PAI-1. The aP2 locus exhibited elevated chromatin accessibility (>2.1-fold), methylation of histone H3 lysine 4 (>4.5-fold), and acetylation of histone H4 (>2.5-fold) in USP2-KD cells. Transfection of isopeptidase-mutated USP2-69 did not alter chromatin conformation on the aP2 locus in USP2-KD cells. Our results suggest that USP2-69 suppresses meta-inflammatory molecules involved in the development of type-2 diabetes.


Assuntos
Montagem e Desmontagem da Cromatina , Endopeptidases/genética , Macrófagos/metabolismo , Transcrição Genética , Proteases Específicas de Ubiquitina/genética , Adipócitos/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Endopeptidases/metabolismo , Epigênese Genética , Histonas/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Fator de Transcrição AP-2/genética , Fator de Transcrição AP-2/metabolismo , Proteases Específicas de Ubiquitina/metabolismo
11.
Biochem Biophys Res Commun ; 438(2): 301-5, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23886952

RESUMO

In previous studies, we identified a novel gene, factor for adipocyte differentiation 24 (fad24), which plays an important role during the early stages of adipogenesis in mouse 3T3-L1 cells. Moreover, overexpression of fad24 increased the number of smaller adipocytes in white adipose tissue and improved glucose metabolic activity in mice, thus indicating that fad24 functions as a regulator of adipogenesis in vivo. However, the physiological roles of fad24 in vivo are largely unknown. In this study, we attempted to generate fad24-deficient mice by gene targeting. No fad24-null mutants were recovered after embryonic day 9.5 (E9.5). Although fad24-null embryos were detected in an expected Mendelian ratio of genotypes at E3.5, none of the homozygous mutants developed into blastocysts. In vitro culture experiments revealed that fad24-null embryos develop normally to the morula stage but acquire growth defects during subsequent stages. The number of nuclei decreased in fad24-deficient morulae compared with that in wild-type ones. These results strongly suggested that fad24 is essential for pre-implantation in embryonic development, particularly for the progression to the blastocyst stage.


Assuntos
Adipogenia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Blastocisto/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Células 3T3-L1 , Animais , Cruzamentos Genéticos , Células-Tronco Embrionárias/citologia , Feminino , Genótipo , Glucose/metabolismo , Heterozigoto , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mórula/metabolismo , Mutação , Fatores de Tempo
12.
Biochem Biophys Res Commun ; 406(4): 524-8, 2011 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-21333627

RESUMO

The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. Although its expression is tightly controlled during early embryo development and postnatal development and maturation in the brain, the physiological role of ganglioside GM3 in the regulation of neuronal functions has not been elucidated. In the present study, we examined motor activity, cognitive and emotional behaviors, and drug administration in juvenile GM3-knockout (GM3-KO) mice. GM3-KO male and female mice showed hyperactivity in the motor activity test, Y-maze test, and elevated plus maze test. In the Y-maze test, there was significantly less spontaneous alternation behavior in GM3-KO male mice than in wild-type mice. In the elevated plus maze test, the amount of time spent on the open arms by GM3-KO male mice was significantly higher than that of sex-matched wild-type mice. In contrast, there was no significant difference between GM3-KO and wild-type female mice in these tests. Thus, juvenile GM3-KO mice show gender-specific phenotypes resembling attention-deficit hyperactivity disorder (ADHD), namely hyperactivity, reduced attention, and increased impulsive behaviors. However, administration of methylphenidate hydrochloride (MPH) did not ameliorate hyperactivity in either male or female GM3-KO mice. Although these data demonstrate the involvement of ganglioside GM3 in ADHD and the ineffectiveness of MPH, the first-choice psychostimulant for ADHD medication, our studies indicate that juvenile GM3-KO mice are a useful tool for neuropsychological studies.


Assuntos
Comportamento Animal , Gangliosídeo G(M3)/fisiologia , Atividade Motora , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Emoções , Feminino , Gangliosídeo G(M3)/genética , Hipercinese/genética , Hipercinese/fisiopatologia , Masculino , Aprendizagem em Labirinto , Metilfenidato/administração & dosagem , Camundongos , Camundongos Knockout
13.
EMBO J ; 29(20): 3558-70, 2010 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-20834228

RESUMO

Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1+/- nor Chk2-/- mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1+/-Chk2-/- and Chk1+/-Chk2+/- mice have a progressive cancer-prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra-S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double-mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53- or Rb-dependent oncogene-induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.


Assuntos
Ciclo Celular/fisiologia , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose/fisiologia , Células Cultivadas , Senescência Celular , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Aberrações Cromossômicas , Dano ao DNA , Reparo do DNA , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Deleção de Genes , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/patologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética
14.
Biol Pharm Bull ; 31(11): 2143-5, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18981588

RESUMO

The present study was conducted to determine whether cutaneous itch involves mu-opioid receptors in either of the spinal cord or lower brainstem or in both regions in mice. An intraplantar injection of serotonin hydrochloride (100 nmol/site) induced biting, an itch-related behavior. The behavior was inhibited by subcutaneous (0.3-1 mg/kg) and intracisternal (1--10 nmol/site), but not intrathecal (1--10 nmol/site), injections of naloxone hydrochloride. An intradermal injection of serotonin (100 nmol/site) to the rostral back induced scratching, an itch-related behavior, which was inhibited by subcutaneous (1 mg/kg) and intracisternal (10 nmol/site) injections of naloxone. These results suggest that mu-opioid receptor in the lower brainstem, but not spinal cord, is a site of central pruritogenic action of opioids and is involved in the facilitatory regulation of itch signaling.


Assuntos
Comportamento Animal/efeitos dos fármacos , Cisterna Magna/metabolismo , Naloxona , Antagonistas de Entorpecentes , Prurido/tratamento farmacológico , Animais , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/metabolismo , Cisterna Magna/efeitos dos fármacos , Injeções Espinhais , Injeções Subcutâneas , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos ICR , Naloxona/administração & dosagem , Naloxona/farmacologia , Naloxona/uso terapêutico , Antagonistas de Entorpecentes/administração & dosagem , Antagonistas de Entorpecentes/farmacologia , Antagonistas de Entorpecentes/uso terapêutico , Prurido/metabolismo , Receptores Opioides mu/metabolismo , Serotonina/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo
15.
J Pharmacol Sci ; 106(4): 667-70, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18403901

RESUMO

The common adverse effect of centrally-injected mu-opioid receptor (mu-OR) agonists is pruritus. This study was conducted using mice to examine whether different subtypes of mu-OR would be responsible for pruritus and analgesia. Intracisternal injections of morphine and morphine-6beta-glucronide (M6G), but not M3G, produced an antinociceptive effect. Morphine, but neither M6G nor M3G, induced facial scratching, a pruritus-related response. Facial scratching following morphine was not affected by the mu(1)-OR antagonist naloxonazine at doses that inhibited the antinociceptive effects. The results suggest that different subtype and/or splice variants of mu-OR are separately involved in pruritus and antinociception of opioids.


Assuntos
Analgésicos Opioides/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Morfina/administração & dosagem , Limiar da Dor/efeitos dos fármacos , Prurido/induzido quimicamente , Receptores Opioides mu/agonistas , Analgésicos Opioides/toxicidade , Animais , Relação Dose-Resposta a Droga , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Endogâmicos ICR , Morfina/toxicidade , Derivados da Morfina/administração & dosagem , Naloxona/administração & dosagem , Naloxona/análogos & derivados , Antagonistas de Entorpecentes/administração & dosagem , Medição da Dor , Prurido/metabolismo , Prurido/prevenção & controle , Receptores Opioides mu/metabolismo , Fatores de Tempo
16.
Cancer Sci ; 99(3): 608-14, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18167132

RESUMO

Tamoxifen is an antagonist of estrogen receptor, which is used widely as an estrogen receptor-positive breast cancer drug that blocks growth signals and provokes apoptosis. However, recent studies have revealed that tamoxifen induces apoptosis even in estrogen receptor-negative cells. In the present study, we synthesized several tamoxifen derivatives to augment the apoptosis-inducing effect of tamoxifen and evaluated the apoptosis-inducing pathway. The estrogen receptor-positive human leukemia cell line HL-60 and estrogen receptor-negative human leukemia cell line Jurkat were treated with tamoxifen and synthesized tamoxifen derivatives, and thereafter subjected to cell viability-detection assays. Tamoxifen derivatives, as well as the lead compound tamoxifen, decreased the cell viability despite the expression of estrogen receptor. Among all of the synthesized tamoxifen derivatives, ridaifen-B had more potent cancer cell-damaging activity than tamoxifen. Ridaifen-B fragmented Jurkat cell DNA and activated caspases, suggesting that the ridaifen-B-induced apoptosis pathway is estrogen receptor independent. Moreover, mitochondrial involvement during ridaifen-B-induced apoptosis was estimated. Ridaifen-B significantly reduced mitochondrial membrane potential, and overexpression of Bcl-2 inhibited ridaifen-B-induced apoptosis. These results suggest that the induction of apoptosis by ridaifen-B, a novel tamoxifen derivative, is dependent on mitochondrial perturbation without estrogen receptor involvement.


Assuntos
Antineoplásicos Hormonais/farmacologia , Apoptose , Mitocôndrias/efeitos dos fármacos , Pirrolidinas/farmacologia , Tamoxifeno/análogos & derivados , Antineoplásicos Hormonais/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Células Jurkat , Mitocôndrias/metabolismo , Pirrolidinas/síntese química , Espécies Reativas de Oxigênio/metabolismo , Receptores Estrogênicos/metabolismo , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Tamoxifeno/síntese química , Tamoxifeno/farmacologia
17.
Bioorg Med Chem ; 15(24): 7599-617, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17904372

RESUMO

Two new synthetic pathways to the anti-cancer agent tamoxifen and its derivatives were developed. The first route involved the aldol reaction of benzyl phenyl ketone with acetaldehyde followed by Friedel-Crafts substitution with anisole in the presence of Cl(2)Si(OTf)(2) to produce 1,1,2-triaryl-3-acetoxybutane, a precursor of the tamoxifen derivatives. The second one utilized the novel three-component coupling reaction among aromatic aldehydes, cinnamyltrimethylsilane, and aromatic nucleophiles using HfCl(4) as a Lewis acid catalyst to produce 3,4,4-triarylbutene, that is also a valuable intermediate of the tamoxifen derivatives. The former strategy requires a total of 10 steps from the aldol formation to the final conversion to tamoxifen, whereas the latter needs only three or four steps to produce tamoxifen and droloxifene including the installation of the side-chain moiety and the base-induced double-bond migration to form the tetra-substituted olefin structure. This synthetic strategy seems to serve as a new and practical pathway to prepare not only the tamoxifen derivatives but also the other SERMs (selective estrogen receptor modulators) including estrogen-dependent breast cancer and osteoporosis agents.


Assuntos
Aldeídos/química , Aminoácidos Aromáticos/química , Antineoplásicos/síntese química , Cinamatos/química , Éteres Fenílicos/química , Moduladores Seletivos de Receptor Estrogênico/síntese química , Tamoxifeno/síntese química , Compostos de Trimetilsilil/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Estrutura Molecular , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/química , Tamoxifeno/farmacologia , Fatores de Tempo
18.
J Invest Dermatol ; 127(8): 2042-7, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17429442

RESUMO

Thromboxane A2 (TXA2), a metabolite of arachidonic acid produced by cyclooxygenase and thromboxane synthase, is thought to participate in chronic dermatitis. This study investigated the involvement of TXA2 in cutaneous itch. An intradermal injection of U-46619, a stable analogue of TXA2, elicited scratching, an itch-associated response, in mice. Dose-response curve was bell shaped with a maximum effect at 10 nmol per site. The action of U-46619 was inhibited by a coinjection of the TP antagonist ONO-3708 and was abolished by TP receptor deficiency. TP receptor was mainly expressed in nerve fiber in the skin and keratinocytes. Thromboxane synthase was also expressed in keratinocytes. U-46619 increased intracellular Ca2+ ion concentration in primary cultures of dorsal root ganglion neurons and keratinocytes. The results suggest that TXA2 synthesized by keratinocytes acts as an itch mediator. It may elicit itch through the activation of TP receptors on primary afferents and keratinocytes; keratinocytes may produce itch mediators including TXA2. Thus, thromboxane synthase inhibitor and TP receptor antagonists will be candidates for antipruritic medicines.


Assuntos
Prurido/etiologia , Receptores de Tromboxanos/fisiologia , Tromboxano A2/fisiologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Gânglios Espinais/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Masculino , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Receptores de Tromboxanos/análise , Tromboxano-A Sintase/análise
19.
Mol Endocrinol ; 21(7): 1713-21, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17456791

RESUMO

The growth-retarded (grt) mouse has an autosomal recessive, fetal-onset, severe thyroid hypoplasia related to TSH hyporesponsiveness. Through genetic mapping and complementation experiments, we show that grt is a missense mutation of a highly conserved region of the tyrosylprotein sulfotransferase 2 (Tpst2) gene, encoding one of the two Tpst genes implicated in posttranslational tyrosine O-sulfation. We present evidence that the grt mutation leads to a loss of TPST2 activity, and TPST2 isoform has a high degree of substrate preference for TSH receptor (TSHR). The expression of TPST2 can restore TSH-TSHR-mediated cAMP production in fibroblasts derived from grt mice. Therefore, we propose that the tyrosine sulfation of TSHR by TPST2 is crucial for TSH signaling and resultant thyroid gland function.


Assuntos
Hipotireoidismo Congênito/enzimologia , Hipotireoidismo Congênito/genética , Nanismo/enzimologia , Nanismo/genética , Mutação de Sentido Incorreto , Sulfotransferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Hipotireoidismo Congênito/complicações , Primers do DNA/genética , Nanismo/complicações , Feminino , Teste de Complementação Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Dados de Sequência Molecular , Fenótipo , Processamento de Proteína Pós-Traducional , Receptores da Tireotropina/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Especificidade por Substrato , Sulfotransferases/deficiência , Sulfotransferases/metabolismo , Tireotropina/metabolismo
20.
Mamm Genome ; 17(5): 407-16, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16688531

RESUMO

The ICGN mouse is a model for nephrotic syndrome (NS) which presents with proteinuria, hyperlipidemia, and edema. In this study we attempted to identify the gene(s) responsible for NS. By analyzing albuminuria in 160 (ICGN x MSM)F(1) x ICGN backcross progenies, we found that NS in the ICGN mouse is caused by more than one gene. We then performed a quantitative trait locus (QTL) analysis and detected a QTL with a very high LOD score peak in the telomeric region of Chr 15. By analyzing the nucleotide sequence of 22 genes located close to the QTL, we found that the tensin2 gene of the ICGN mouse possessed an 8-nucleotide deletion mutation in exon 18, leading to a frameshift and giving rise to a terminal codon at a premature position. Analyses of in situ hybridization and immunohistochemistry revealed that tensin2 was expressed in podocytes and tubular epithelial cells in normal mice but not in the ICGN mouse. These data raise the possibility that a mutation of the tensin2 gene is responsible for NS of the ICGN mouse and tensin2 is a prerequisite for the normal kidney function.


Assuntos
Síndrome Nefrótica/congênito , Síndrome Nefrótica/genética , Fosfoproteínas Fosfatases/deficiência , Albuminúria/genética , Animais , Cruzamentos Genéticos , Modelos Animais de Doenças , Feminino , Mutação da Fase de Leitura , Deleção de Genes , Imuno-Histoquímica , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fosfoproteínas Fosfatases/genética , Locos de Características Quantitativas , Tensinas
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