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1.
Bioorg Med Chem ; 28(7): 115376, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32088125

RESUMO

Sphingomyelin synthase 2 (SMS2) has attracted attention as a drug target for the treatment of various cardiovascular and metabolic diseases. The modification of a high throughput screening hit, 2-quinolone 10, enhanced SMS2 inhibition at nanomolar concentrations with good selectivity against SMS1. To improve the pharmaceutical properties such as passive membrane permeability and aqueous solubility, adjustment of lipophilicity was attempted and 1,8-naphthyridin-2-one 37 was identified as a potent and selective SMS2 inhibitor. A significant reduction in hepatic sphingomyelin levels following repeated treatment in mice suggested that compound 37 could be an effective in vivo tool for clarifying the role of SMS2 enzyme and developing the treatment for SMS2-related diseases.


Assuntos
Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Animais , Linhagem Celular , Descoberta de Drogas , Inibidores Enzimáticos , Humanos , Masculino , Camundongos
2.
J Med Chem ; 62(20): 9175-9187, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31550153

RESUMO

Dysferlinopathies, which are muscular diseases caused by mutations in the dysferlin gene, remain serious medical problems due to the lack of therapeutic agents. Herein, we report the design, synthesis, and structure-activity relationships of a 2,6-disubstituted 3H-imidazo[4,5-b]pyridine series, which was identified from the phenotypic screening of chemicals that increase the level of dysferlin in myocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs). Optimization studies with cell-based phenotypic assay led to the identification of a highly potent compound, 19, with dysferlin elevation effects at double-digit nanomolar concentrations. In addition, the molecular target of our chemical series was identified as tubulin, through a tubulin polymerization assay and a competitive binding assay using a photoaffinity labeling probe.


Assuntos
Imidazóis/química , Distrofia Muscular do Cíngulo dos Membros/tratamento farmacológico , Piridinas/química , Moduladores de Tubulina/uso terapêutico , Sítios de Ligação , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Disferlina/metabolismo , Células Hep G2 , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Simulação de Acoplamento Molecular , Distrofia Muscular do Cíngulo dos Membros/patologia , Proteína MyoD/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Estrutura Terciária de Proteína , Piridinas/farmacologia , Piridinas/uso terapêutico , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia
3.
Stem Cells Transl Med ; 8(10): 1017-1029, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31250983

RESUMO

Dysferlinopathy is a progressive muscle disorder that includes limb-girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG-132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient-derived induced pluripotent stem cells. According to the screening, nocodazole was found to effectively increase the level of dysferlin in cells, which, in turn, enhanced membrane resealing following injury by laser irradiation. Moreover, the increase was due to microtubule disorganization and involved autophagy rather than the proteasome degradation pathway. These findings suggest that increasing the amount of misfolded dysferlin using small molecules could represent an effective future clinical treatment for dysferlinopathy. Stem Cells Translational Medicine 2019;8:1017-1029.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Pluripotentes Induzidas/transplante , Células Musculares/metabolismo , Distrofia Muscular do Cíngulo dos Membros/tratamento farmacológico , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Fenótipo
4.
Sci Rep ; 7(1): 6050, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28729619

RESUMO

Purinergic P2Y 2 receptors, G-protein coupled receptors that primarily couple with Gαq/11-proteins, are activated equipotently by adenosine-5'-triphosphate (ATP) and uridine-5'-triphosphate. Evidence suggests that P2Y 2 agonists make potential drug candidates for the treatment of cardiovascular diseases. However, selective non-nucleotide, small-molecule P2Y 2 agonists have yet to be developed. In this report, we discuss Compound 89, a novel non-nucleotide allosteric P2Y 2 agonist that was active in signal transduction and gene induction, and in our in vitro cardiac hypertrophy model. Compound 89 exhibited selective P2Y 2 agonistic activity and potentiated responses to the endogenous agonist ATP, while exhibiting no agonistic activities for four other Gαq/11-coupled human P2Y (hP2Y) receptors and one representative Gαi/o-coupled hP2Y12 receptor. Its P2Y 2 agonistic effect on mouse P2Y 2 receptors suggested non-species-specific activity. Compound 89 acted as a pure positive allosteric modulator in a Ca2+ mobilization assay of neonatal rat cardiomyocytes; it potentiated ATP-induced expression of genes in the nuclear receptor 4A family (negative regulators of hypertrophic stimuli in cardiomyocytes). Additionally, Compound 89 attenuated isoproterenol-induced cardiac hypertrophy, presumably through dose-dependent interaction with pericellular ATP. These results indicate that Compound 89 is potentially efficacious against cardiomyocytes and therefore a good proof-of-concept tool for elucidating the therapeutic potential of P2Y2 activation in various cardiovascular diseases.


Assuntos
Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Agonistas do Receptor Purinérgico P2/farmacologia , Quinolonas/farmacologia , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2/química , Quinolonas/química , Ratos
5.
J Control Release ; 237: 1-13, 2016 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-27369865

RESUMO

Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab' fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after intravenous administration in normal mice. In particular, 1µg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected intramuscularly. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by intramuscular injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.


Assuntos
Imunoconjugados/uso terapêutico , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Miostatina/genética , Doença Arterial Periférica/terapia , RNA Interferente Pequeno/uso terapêutico , Animais , Antígenos CD/imunologia , Células Cultivadas , Feminino , Imunoconjugados/genética , Imunoconjugados/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Doença Arterial Periférica/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Terapêutica com RNAi , Ratos , Receptores da Transferrina/imunologia
6.
Eur J Nucl Med Mol Imaging ; 41(4): 624-33, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24258009

RESUMO

PURPOSE: Intravascular ultrasonography (IVUS) and (18)F-FDG PET have been used to evaluate the efficacy of antiatherosclerosis drugs. These two modalities image different characteristics of atherosclerotic plaques, and a comparison of IVUS and PET images with histology has not been performed. The aim of this study was to align IVUS and PET images using anatomic landmarks in Watanabe heritable hyperlipidaemic (WHHL) rabbits, enabling comparison of their depiction of aortic atherosclerosis. Cellular (18)F-FDG localization was evaluated by (3)H-FDG microautoradiography (micro-ARG). METHODS: A total of 19 WHHL rabbits (7 months of age) were divided into three groups: baseline (n = 6), 3 months (n = 4), and 6 months (n = 9). PET, IVUS and histological images of the same aortic segments were analysed. Infiltration by foamy macrophages was scored from 0 to IV using haematoxylin and eosin (H&E) and antimacrophage immunohistochemical staining, and compared with (3)H-FDG micro-ARG findings in two additional WHHL rabbits. RESULTS: IVUS images did not identify foamy macrophage deposition but revealed the area of intimal lesions (r = 0.87). (18)F-FDG PET revealed foamy macrophage distribution in the plaques. The intensity of (18)F-FDG uptake was correlated positively with the degree of foamy macrophage infiltration. Micro-ARG showed identical (3)H-FDG accumulation in the foamy macrophages surrounding the lipid core of the plaques. CONCLUSION: F-FDG PET localized and quantified the degree of infiltration of foamy macrophages in atherosclerotic lesions. IVUS defined the size of lesions. (18)F-FDG PET is a promising imaging technique for evaluating atherosclerosis and for monitoring changes in the composition of atherosclerotic plaques affecting their stability.


Assuntos
Fluordesoxiglucose F18/farmacocinética , Células Espumosas/diagnóstico por imagem , Placa Aterosclerótica/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/farmacocinética , Ultrassonografia de Intervenção , Animais , Aorta/diagnóstico por imagem , Aorta/patologia , Placa Aterosclerótica/patologia , Coelhos
7.
Stem Cell Rev Rep ; 7(1): 43-52, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20376577

RESUMO

The mechanism that regulates the terminal maturation of hematopoietic stem cells into erythroid cells is poorly understood. Therefore, identifying genes and surface markers that are restricted to specific stages of erythroid maturation will further our understanding of erythropoiesis. To identify genes expressed at discrete stages of erythroid development, we screened for genes that contributed to the proliferation and maturation of erythropoietin (EPO)-dependent UT-7/EPO cells. After transducing erythroid cells with a human fetal liver (FL)-derived lentiviral cDNA library and culturing the cells in the absence of EPO, we identified 17 candidate genes that supported erythroid colony formation. In addition, the mouse homologues of these candidate genes were identified and their expression was examined in E12.5 erythroid populations by qRT-PCR. The expression of candidate erythroid marker was also assessed at the protein level by immunohistochemistry and ELISA. Our study demonstrated that expression of the Apoa-1 gene, an apolipoprotein family member, significantly increased as hematopoietic stem cells differentiated into mature erythroid cells in the mouse FL. The Apoa-1 protein was more abundant in mature erythroid cells than hematopoietic stem and progenitor cells in the mouse FL by ELISA. Moreover, APOA-1 gene expression was detected in mature erythroid cells from human peripheral blood. We conclude that APOA-1 is a novel marker of the terminal erythroid maturation of hematopoietic stem cells in both mice and humans.


Assuntos
Apolipoproteína A-I/metabolismo , Diferenciação Celular , Células Eritroides/citologia , Células Eritroides/metabolismo , Células-Tronco Hematopoéticas/citologia , Animais , Apolipoproteína A-I/genética , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células , Separação Celular , Eritropoetina/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Estudos de Associação Genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lentivirus/genética , Fígado/citologia , Fígado/metabolismo , Camundongos , Receptores da Eritropoetina/metabolismo , Transdução de Sinais , Transdução Genética
8.
Mol Cell Biochem ; 319(1-2): 181-7, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18709335

RESUMO

The gene transduction method is a very powerful tool, not only in basic science but also in clinical medicine. Regenerative medicine is one field that has close connection with both basic and clinical. Recently, it has been reported that induced pluripotent stem (iPS) cells can be produced from somatic cells by a three or four gene transduction. We have also recently reported that lentiviral gene transfer of the tal1/scl gene can efficiently differentiate non-human primate common marmoset ES cells into hematopoietic cells without the support of stromal cells. In this study, we constructed a high-performance human fetal liver-derived lentiviral expression library, which contains a high number of individual clones, in order to develop a very helpful tool for understanding early hematopoiesis and/or hepatocytosis for future regenerative medicine. Our lentiviral cDNA library consisted of more than 8 x 10(7) individual clones, and their average insert size was >2 kb. DNA sequence analysis for each individual inserted cDNAs revealed that >60% contained the full-length protein-coding regions for many genes including cytokine receptors, cytoplasmic proteins, protein inhibitors, and nuclear factors. The transduction efficiency on 293T cells was 100% and the average size of an integrated cDNA was ~1.1 kb. These results suggest that our lentiviral human fetal liver cDNA expression library could be a very helpful tool for accelerating the discovery of novel genes that are involved in early hematopoiesis and hepatopoiesis and to make the use of iPS cells more efficient in the field of regenerative medicine.


Assuntos
Feto , Biblioteca Gênica , Hematopoese/genética , Lentivirus , Animais , Callithrix , Linhagem Celular , Humanos , Transdução Genética
9.
J Vet Med Sci ; 68(3): 297-301, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16598179

RESUMO

Growth arrest and DNA damage-inducible protein 45 (GADD45) plays an important role in suppressing multistep carcinogenesis. In this report, we describe the isolation of the complete wild-type feline GADD45 cDNA from feline tissues. Expression of feline GADD45 mRNA was detected in the liver, spleen, kidney, lung, and testis. The predicted amino acid sequences encoded by the full-length feline GADD45 cDNA display sequence homology with those from other vertebrates, and as in the case of human GADD45, cell growth suppression was observed by ectopic expression of feline GADD45. However, no mutations were detected by sequence analysis of feline GADD45 in several feline lymphoma cell lines, indicating that the GADD45 mutation might be uncommon in feline oncogenesis.


Assuntos
Doenças do Gato/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfoma/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Gatos , Linhagem Celular Tumoral , Clonagem Molecular , DNA Complementar/genética , Genes Supressores , Linfoma/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Homologia de Sequência de Aminoácidos , Transfecção/veterinária , Proteínas GADD45
10.
Vet Parasitol ; 136(2): 147-54, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16309840

RESUMO

Three beagle dogs were inoculated with mice spleen/liver homogenate infected with Ehrlichia species detected from Ixodes ovatus (EIO) and one dog was used as a control. All three infected dogs did not show clinical signs of disease except for mild pyrexia throughout the 41-day study period. Splenomegaly was observed from Day 7 post-inoculation (p.i.) in two of the dogs. Hematological and biochemical abnormalities included mild thrombocytopenia, hypoproteinaemia, hypoalbuminaemia and increased C-reactive protein values. One of the dogs' splenic aspirate sample was PCR-positive for Ehrlichia Day 7 p.i. and another dogs' blood and bone marrow aspirate sample was PCR-positive Day 41 p.i. Sequence analysis of the PCR products showed 100% homology with the 16SrRNA partial gene sequence of Ehrlichia sp. HF565. Antibody titers to EIO were observed in all three experimentally infected dogs starting from the first week p.i. and cross-reactivity with Ehrlichia canis was detectable in one of the dogs starting Day 7 p.i. These data suggest that infection of dogs with EIO is possible, though is probably of low pathogenic importance. Cross-reactivity of EIO infected dog serum with E. canis raises the likelihood of false E. canis seropositive dogs.


Assuntos
Vetores Aracnídeos/microbiologia , Doenças do Cão/parasitologia , Ehrlichia/patogenicidade , Ehrlichiose/veterinária , Ixodes/microbiologia , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/sangue , Proteína C-Reativa/análise , Reações Cruzadas , DNA Bacteriano/química , Doenças do Cão/diagnóstico , Cães , Ehrlichia/genética , Ehrlichia/imunologia , Ehrlichia/isolamento & purificação , Ehrlichia canis/genética , Ehrlichia canis/imunologia , Ehrlichiose/diagnóstico , Ehrlichiose/parasitologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Contagem de Plaquetas/veterinária , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico 16S/genética
11.
Mol Cell Biol ; 25(10): 4046-61, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15870277

RESUMO

Elimination of the regulatory mechanism underlying numeral homeostasis of centrosomes, as seen in cells lacking p53, results in abnormal amplification of centrosomes, which increases the frequency of chromosome segregation errors, and thus contributes to the chromosome instability frequently observed in cancer cells. We have previously reported that p53(-/-) mouse cells in prolonged culture undergo genomic convergence similar to that observed during tumor progression; early-passage p53(-/-) cells are karyotypically heterogeneous due to extensive chromosome instability associated with centrosome amplification, while late-passage p53(-/-) cells are aneuploid yet karyotypically homogeneous and chromosomally stable. Moreover, they contain numerically normal centrosomes. Through the microarray analysis of early- and late-passage p53(-/-) cells, we identified the BubR1 spindle checkpoint protein, which plays a critical role in suppression of centrosome amplification and stabilization of chromosomes in late-passage p53(-/-) cells. Up-regulation of BubR1 augments the checkpoint function, which effectively senses the spindle/chromosome aberrations associated with centrosome amplification. We further found that BubR1 transcription is largely controlled by p53. In early-passage p53(-/-) cells, BubR1 expression is low and the checkpoint function in response to microtubule toxin is considerably compromised. In late-passage cells, however, regaining of BubR1 expression restores the checkpoint function to mitotic aberrations caused by microtubule toxin. Our studies demonstrate the molecular aspect of genomic convergence in cultured cells, providing critical information for understanding the stepwise progression of tumors.


Assuntos
Centrossomo/metabolismo , Amplificação de Genes , Regulação da Expressão Gênica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas de Ciclo Celular , Divisão Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Amplificação de Genes/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Proteínas Quinases/deficiência , Proteínas Serina-Treonina Quinases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fuso Acromático/fisiologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
12.
J Vet Med Sci ; 67(3): 249-54, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15805726

RESUMO

OmpL1 is a 31-kDa outer membrane protein characterized in 1993 and known to be expressed only in pathogenic Leptospira spp. Recombinant OmpL1 (GST-rOmpL1) was expressed for use as an ELISA antigen for the detection of anti-Leptospira antibodies. In immunoblot analysis, the protein reacted with sera of dogs infected with three different serotypes of Leptospira interrogans, while did not react with sera of dogs both uninfected negative controls and infected with Borrelia burgdorferi, which is closely related to Leptospira spp. Moreover, in ELISA using GST-rOmpL1, the optical density (O.D.) values from the positive controls were very high (1.125 +/- 0.549). In contrast, the O.D. values from clinically healthy dogs and dogs with diseases other than leptospirosis were very low (0.109 +/- 0.046 and 0.089 +/- 0.046, respectively). These data suggest that the detection of anti-Leptospira antibodies by ELISA using the GST-rOmpL1 protein can be applied for diagnosis of canine leptospirosis.


Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Doenças do Cão/diagnóstico , Leptospira/imunologia , Leptospirose/veterinária , Animais , Primers do DNA , Doenças do Cão/imunologia , Doenças do Cão/microbiologia , Cães , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Immunoblotting/veterinária , Leptospirose/diagnóstico , Leptospirose/imunologia , Plasmídeos/genética , Reação em Cadeia da Polimerase/veterinária , Proteínas Recombinantes/metabolismo
13.
J Vet Med Sci ; 66(7): 797-805, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15297751

RESUMO

To evaluate the presence of centrosome amplification and the resulting chromosomal instability in cat tumors, a newly established feline lymphoma cell line and four already established feline lymphoma cell lines were examined using immunohistochemical analysis of centrosomes. The number of chromosomes were subsequently counted by metaphase spread. Moreover, to explore whether mutational inactivation of the p53 gene or inactivation of the P53 protein caused by mdm2 gene overexpression, occurred in the feline lymphoma cell lines, mutational analysis of the feline p53 gene was carried out. The expression of feline mdm2 mRNA was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Centrosome amplification and chromosomal instability was observed in three out of the five feline lymphoma cell lines. Of these three feline lymphoma cell lines, one had aberrations in the P53 amino-acid sequence, whereas the others had none. There was no significant difference in the expression of mdm2 mRNA between peripheral blood mononuclear cells (PBMC) obtained from a normal cat and that of the five feline lymphoma cell lines. These findings indicate that centrosome amplification also occurs in cat tumors and is strongly correlated with chromosomal instability, suggesting that the immunostaining of centrosomes could be an alternative method for the examination of the chromosomal instability. Furthermore, this study suggests the presence of unknown mechanism that leads to the centrosome amplification in feline lymphomas.


Assuntos
Doenças do Gato/genética , Centrossomo/fisiologia , Instabilidade Cromossômica/genética , Amplificação de Genes/genética , Linfoma/veterinária , Animais , Gatos , Análise Mutacional de DNA , DNA de Neoplasias/análise , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Genes p53 , Imuno-Histoquímica/veterinária , Vírus da Leucemia Felina/genética , Vírus da Leucemia Felina/isolamento & purificação , Linfoma/genética , Metáfase/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de DNA/veterinária , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo
14.
J Vet Med Sci ; 64(11): 973-9, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12499680

RESUMO

To detect the anti-P53 antibodies of dogs with tumors, a GST-recombinant canine (rc) P53 fusion protein was expressed and purified. Immunoblot analysis was performed using this GST-rcP53 fusion protein as an antigen and serum samples from dogs suffering from tumors as primary antibodies. Out of 16 serum samples obtained from various tumor cases, four samples showed reaction with GST-rcP53. In contrast, serum from other 12 dogs with tumors, four dogs with non-neoplastic diseases and two control healthy dogs (as controls) did not show any reaction with GST-rcP53 in immunoblotting. The p53 gene mutation and the P53 protein expression were examined, using the tumor tissues to explore the relationship between the existence of the GST-rcP53 bands, gene mutations of p53 and the accumulation of P53 protein. One case, which showed a clear GST-rcP53 band, had a point mutation of the p53 cDNA and showed nuclear accumulation of P53 protein. These results suggest that the anti-P53 antibodies are also produced in tumor dogs with p53 gene mutations.


Assuntos
Anticorpos/imunologia , Doenças do Cão/imunologia , Mutação/genética , Neoplasias/imunologia , Neoplasias/veterinária , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Animais , Anticorpos/sangue , Análise Mutacional de DNA , DNA Complementar/genética , Doenças do Cão/genética , Cães , Imuno-Histoquímica , Dados de Sequência Molecular
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