RESUMO
More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.
Assuntos
Sistemas CRISPR-Cas , Fertilidade/genética , Edição de Genes , Regulação da Expressão Gênica/fisiologia , Testículo/metabolismo , Animais , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Knockout , TranscriptomaRESUMO
Transcription factor TEA domain family transcription factor 4 (Tead4) is one of the key factors involved in the differentiation of the trophectoderm (TE) in murine embryos. However, knowledge on the roles of TEAD4 in preimplantation development during bovine embryos is currently limited. This study examined the transcript and protein expression patterns of TEAD4 and attempted to elucidate the functions of TEAD4 during bovine preimplantation development using RNA interference. TEAD4 mRNA was found to be upregulated between the 16-cell and morula stages, and nuclear localization of the TEAD4 protein was detected at the morula stage, as well as in subsequent developmental stages. TEAD4 downregulation did not affect embryonic development until the blastocyst stage, and TEAD4-downregulated embryos were capable of forming the TE under both 5% and 21% O2 conditions. Results of gene expression analysis showed that TEAD4 downregulation did not affect the expression levels of POU class 5 transcription factor 1 (OCT-4), NANOG, caudal-type homeobox 2 (CDX2), GATA binding protein 3 (GATA3), and interferon-tau (IFNT). In conclusion, TEAD4 might be dispensable for development until the blastocyst stage and TE differentiation in bovine embryos.
Assuntos
Blastocisto/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Desenvolvimento Embrionário/fisiologia , Proteínas Musculares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Bovinos , Proteínas de Ligação a DNA/genética , Feminino , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Expressão Gênica , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas Musculares/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Interferência de RNA , Fatores de Transcrição/genéticaRESUMO
Oct-4, a member of the POU family of transcription factors, is a key factor that regulates the segregation of the inner cell mass (ICM) and the trophectoderm (TE) during the transition from morula to blastocyst in mice. However, little is known about its role in porcine early embryogenesis. To determine the function of OCT-4 in the ICM and TE segregation of porcine embryos, we studied the developmental morphology of porcine embryos using RNA interference technology. Our experiments demonstrated that when 1-cell stage embryos were co-injected with the small interfering RNA (siRNA)for targeted knockdown of OCT-4 (OCT-4-siRNA) and tetramethylrhodamine isothiocyanate (TRITC)-dextran conjugate (Dx), they failed to form blastocysts. Therefore, in this study, we constructed chimeric embryos comprising blastomeres that either expressed OCT-4 normally or showed downregulated OCT-4 expression by co-injection of OCT-4-siRNA and Dx into one blastomere in 2- to 4-cell stage embryos. In control embryos, which were co-injected with control siRNA and Dx, Dx-positive cells contributed to the TE lineage in almost all the blastocysts examined. In contrast, Dx-positive cells derived from a blastomere co-injected with OCT-4-siRNA and Dx were degenerated in almost half the blastocysts. This was probably due to the inability of these cells to differentiate into the TE lineage. Real-time RT-PCR analysis revealed no difference in the levels of SOX2, TEAD4, FGF4 and FGFR1-IIIc, all of which are known to be regulated by OCT-4, between the OCT-4-siRNA-injected morulae and the control ones. However, the level of CDX2, a molecule specifically expressed in the TE lineage, was significantly higher in the former than in the latter. Our results indicate that continuous expression of OCT-4 in blastomeres is essential for TE formation of porcine embryos.
Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Mórula/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Trofoblastos/metabolismo , Animais , Blastocisto/citologia , Linhagem da Célula/genética , Regulação para Baixo , Feminino , Mórula/citologia , Fator 3 de Transcrição de Octâmero/genética , RNA Interferente Pequeno , Suínos , Trofoblastos/citologiaRESUMO
Krüppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos.
Assuntos
Blastômeros/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ectogênese , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , Fatores de Transcrição/metabolismo , Zigoto/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Blastômeros/citologia , Bovinos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Feminino , Fertilização in vitro/veterinária , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Microinjeções/veterinária , Mórula/citologia , Mórula/metabolismo , Oócitos/citologia , Piruvato Desidrogenase (Lipoamida)/genética , Piruvato Desidrogenase (Lipoamida)/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Zigoto/citologiaRESUMO
Ovarian pulmonary-type small cell carcinoma is a rare and extremely aggressive neoplasm. We report the occurrence of an ovarian small cell carcinoma of pulmonary type in a 54-year-old woman. She underwent a total abdominal hysterectomy with a bilateral salpingo-oophorectomy and infracolic omentectomy. A diagnosis of stage IIIA pulmonary-type small cell carcinoma was rendered. The tumor appeared to be composed of a solid growth of small cells arranged in sheets and closely packed nests with insular arrangements separated by a fibrous stroma. The tumor cells had hyperchromatic nuclei with inconspicuous nucleoli and scanty cytoplasm. Rosette and rosette-like structures were scattered. Immunohistochemical staining showed positivity for synaptophysin, neural cell adhesion molecule (NCAM), and focally for chromogranin. Cytokeratin and neuron-specific enolase (NSE) were also positive. Over 80% of the tumor cells showed strong reactivity for MIB-1. Electron microscopy showed neuroendocrine granules. She was effectively treated with paclitaxel plus carboplatin after the surgery.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Neuroendócrino/patologia , Carcinoma de Células Pequenas/patologia , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Carboplatina/uso terapêutico , Carcinoma Neuroendócrino/diagnóstico por imagem , Carcinoma Neuroendócrino/cirurgia , Carcinoma Epitelial do Ovário , Carcinoma de Células Pequenas/diagnóstico por imagem , Carcinoma de Células Pequenas/cirurgia , Proteínas de Transporte/metabolismo , Feminino , Humanos , Queratinas/metabolismo , Pulmão/patologia , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/diagnóstico por imagem , Neoplasias Epiteliais e Glandulares/cirurgia , Moléculas de Adesão de Célula Nervosa/metabolismo , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/cirurgia , Paclitaxel/uso terapêutico , Tomografia Computadorizada por Raios X , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The objective of this study was to investigate the role of the POU family transcription factor, Oct-4, in the early development of porcine embryos. We attempted Oct-4 downregulation of porcine early embryos by RNA interference, and evaluated Oct-4 suppression of developmental competencies and gene transcripts in porcine embryos. Injection of specific siRNA resulted in a distinct decrease in Oct-4 mRNA and protein expression in porcine embryos until at least the morula stage. Although the porcine embryos injected with Oct-4 siRNA were able to develop to the morula stage, these embryos failed to form blastocysts. Gene transcripts of caudal-like transcription factor (Cdx2) and fibroblast growth factor 4 (Fgf4), which were involved in segregation of the trophectderm and functionalization of the inner cell mass, were unchanged by Oct-4 siRNA injection. Our results indicated that Oct-4 is an important factor for porcine embryos and, in particular, for the regulation of porcine blastocyst formation.
Assuntos
Fator 3 de Transcrição de Octâmero/genética , Suínos/embriologia , Suínos/genética , Animais , Desenvolvimento Embrionário/genética , Feminino , Fator 4 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Imuno-Histoquímica/veterinária , Gravidez , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transcrição GênicaRESUMO
In mouse embryos, segregation of the inner cell mass (ICM) and trophectoderm (TE) lineages is regulated by genes, such as OCT-4, CDX2 and TEAD4. However, the molecular mechanisms that regulate the segregation of the ICM and TE lineages in porcine embryos remain unknown. To obtain insights regarding the segregation of the ICM and TE lineages in porcine embryos, we examined the mRNA expression patterns of candidate genes, OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc, in blastocyst and elongated stage embryos. In blastocyst embryos, the expression levels of OCT-4, FGF4 and FGFR1-IIIc were significantly higher in the ICM than in the TE, while the CDX2, TEAD4 and GATA3 levels did not differ between the ICM and TE. The expression ratio of CDX2 to OCT-4 (CDX2/OCT-4) also did not differ between the ICM and TE at the blastocyst stage. In elongated embryos, OCT-4, NANOG, FGF4 and FGFR1-IIIc were abundantly expressed in the embryo disc (ED; ICM lineage), but their expression levels were very low in the TE. In contrast, the CDX2, TEAD4 and GATA3 levels were significantly higher in the TE than in the ED. In addition, the CDX2/OCT-4 ratio was markedly higher in the TE than in the ED. We demonstrated that differences in the expression levels of OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc genes between ICM and TE lineages cells become more clear during development from porcine blastocyst to elongated embryos, which indicates the possibility that in porcine embryos, functions of ICM and TE lineage cells depend on these gene expressions proceed as transition from blastocyst to elongated stage.
Assuntos
Massa Celular Interna do Blastocisto/metabolismo , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Trofoblastos/metabolismo , Animais , Massa Celular Interna do Blastocisto/citologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trofoblastos/citologiaRESUMO
BACKGROUND: Spermatozoa become mature and competent for fertilization during transit from the caput epididymis to the cauda epididymis. However, detailed molecular mechanisms of epididymal sperm maturation are still unclear. Here, we focused on multiple epididymis-enriched genes: lipocalin family genes (Lcn5, Lcn6, Lcn8, Lcn9, and Lcn10) and Ly6 family genes (Ly6g5b and Ly6g5c). These genes are evolutionarily conserved in mammals and form clusters on chromosomes 2 and 17 in the mouse, respectively. OBJECTIVE: To clarify whether these genes are required for epididymal sperm maturation and acquisition of fertilizing ability, we generated knockout (KO) mice using the CRISPR/Cas9 system and analyzed their phenotype. MATERIALS AND METHODS: We generated four lines of KO mice: Lcn9 single KO, the lipocalin family quadruple KO (Lcn5, Lcn6, Lcn8, and Lcn10), quintuple KO (Lcn5, Lcn6, Lcn8, Lcn10, and Lcn9), and double KO of Ly6 family genes (Ly6g5b and Ly6g5c). RESULTS: Although the Lcn9 single KO did not affect male fertility, the quadruple KO and quintuple KO male mice were subfertile and mostly infertile, respectively, with a reduced amount of ADAM3, an essential protein for sperm binding to the zona pellucida. Further analysis revealed that the quintuple KO spermatozoa lack the CMTM2A/B that are required for ADAM3 maturation. Intriguingly, Ly6g5b and Ly6g5c double KO male mice also showed subfertility with reduced sperm ADAM3. CONCLUSION: These results suggest epididymal secretory proteins are involved in ADAM3 maturation and acquisition of sperm fertilizing ability.
RESUMO
Adenomatoid tumors are rare benign mesothelial tumors of the genital tract, and only a few cases of uterine adenomatoid tumors treated at laparoscopic surgery have been reported. Herein is reported the case of a laparoscopically resected uterine adenomatoid tumor with coexisting endometriosis. A 34-year-old nulliparous woman with suspected uterine fibroma and endometrial cysts underwent laparoscopic surgery in which both the uterine tumor and the endometrial cysts were enucleated. Enucleation of the uterine tumor was difficult, and, therefore, the border between the tumor and normal myometrium was divided using a harmonic scalpel for tumor resection. Microscopic examination of the tumor showed irregularly proliferating smooth muscle cells and many round hiatuses lined by epithelial-like cells. These epithelial-like cells were immunohistochemically positive for mesothelin and podoplanin, and negative for CD34, which suggests that the tumor was an adenomatoid tumor. This may be the fourth reported case of an adenomatoid tumor resected using the laparoscopic approach.
Assuntos
Tumor Adenomatoide/cirurgia , Endometriose/cirurgia , Doenças Ovarianas/cirurgia , Neoplasias Uterinas/cirurgia , Tumor Adenomatoide/complicações , Tumor Adenomatoide/patologia , Adulto , Endometriose/complicações , Endometriose/patologia , Feminino , Procedimentos Cirúrgicos em Ginecologia , Humanos , Laparoscopia , Doenças Ovarianas/complicações , Doenças Ovarianas/patologia , Resultado do Tratamento , Neoplasias Uterinas/complicações , Neoplasias Uterinas/patologiaRESUMO
Atypical polypoid adenomyoma is a rare uterine tumor composed of atypical endometrial glands, which often exhibit squamous metaplasia, and a cellular smooth muscle stroma. Although atypical polypoid adenomyoma is categorized as a benign lesion, it is reportedly associated with endometrial cancer, and it shows persistence and recurrence even after conservative medical treatment. We present a rare case of atypical polypoid adenomyoma that possibly underwent a serial pathological change from endometrial hyperplasia to carcinoma in a 40-year-old woman with no history of pregnancy. She was diagnosed with atypical polypoid adenomyoma during polypectomy surgery. After resecting the atypical polypoid adenomyoma, endometrial hyperplasia complex was detected. This condition eventually progressed from atypical hyperplasia complex to endometrial adenocarcinoma, and total abdominal hysterectomy was performed. A patient with atypical polypoid adenomyoma who wishes to preserve her fertility should be carefully monitored for endometrial carcinoma. If endometrial hyperplasia is detected in such a patient, a meticulous follow-up examination by performing endometrial biopsy is mandatory.
Assuntos
Adenomioma/diagnóstico , Neoplasias Uterinas/diagnóstico , Adenomioma/patologia , Adenomioma/cirurgia , Adulto , Diagnóstico Diferencial , Progressão da Doença , Hiperplasia Endometrial/diagnóstico , Hiperplasia Endometrial/patologia , Feminino , Humanos , Histerectomia , Neoplasias Uterinas/patologia , Neoplasias Uterinas/cirurgiaRESUMO
Supraclavicular lymph node metastasis is a rare presentation of primary fallopian tube carcinoma. A 76-year-old woman presented with an enlarged supraclavicular lymph node. A biopsy was performed, and its findings confirmed metastatic adenocarcinoma. Subsequent exploratory laparotomy revealed right fallopian tube carcinoma as the primary lesion; consequently, right salpingo-oophorectomy was performed. After adjuvant chemotherapy, she underwent a laparotomy with total abdominal hysterectomy, left salpingo-oophorectomy, pelvic and para-aortic lymph node sampling, and omentectomy. Supraclavicular lymph node metastasis was thought to be, although rarely, the first manifestation of primary fallopian tube carcinoma (PFTC). When supraclavicular lymph node metastasis of an unknown origin is encountered, the possibility of PFTC should be considered.
Assuntos
Adenocarcinoma/secundário , Neoplasias das Tubas Uterinas/patologia , Linfonodos/patologia , Adenocarcinoma/terapia , Idoso , Biópsia , Quimioterapia Adjuvante , Neoplasias das Tubas Uterinas/terapia , Feminino , Humanos , Histerectomia , Excisão de Linfonodo , Metástase Linfática , Terapia Neoadjuvante , Ovariectomia , Tomografia por Emissão de Pósitrons , Resultado do TratamentoRESUMO
Primary ovarian lymphoma is a rare disease, and its definition is still controversial. Many cases of primary ovarian lymphoma are diagnosed as diffuse large B-cell type, whereas the precursor lymphoblastic type is extremely rare. Herein, we describe a case of precursor B-cell lymphoblastic lymphoma of the ovary that was successfully treated with surgery and chemotherapy. A 19-year-old woman presented ovarian tumors and paraaortic lymphoadenopathy, which were suggestive of advanced ovarian cancer. Exploratory laparotomy and right salpingo-oophorectomy were performed, and the diagnosis of precursor B-cell lymphoblastic lymphoma was established. The patient was treated with a combination of chemotherapy and is currently disease-free 1 year after surgery. To our best knowledge, this is the fourth reported case of precursor lymphoblastic lymphoma of the ovary.
Assuntos
Neoplasias Ovarianas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Adulto , Feminino , Humanos , Neoplasias Ovarianas/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapiaRESUMO
BACKGROUND: Increased triglyceride (TG)-rich lipoproteins and decreased HDL that are implicated in the progression of atherosclerotic vascular diseases, are present in serum samples of patients undergoing haemodialysis (HD) therapy. Therefore, it is important to measure serum TG-rich lipoprotein concentrations to prevent the diseases. METHODS: The cholesterol concentrations of lipoprotein classes in serum samples from the HD patients (n = 18) and healthy subjects (n = 18) were analysed by our recently developed method of high-performance liquid chromatography (HPLC), in which the lipoprotein classes were separated using an anion-exchange column, and the cholesterol concentrations of each of those were measured enzymatically using a post-column reaction. The ability of fractionated lipoprotein cholesterol determination by this HPLC method is mostly equivalent to the determination ability of an ultracentrifugation (UC). RESULTS: HDL, LDL, and TG-rich lipoproteins, i.e. IDL, VLDL and chylomicrons, were well separated in the chromatograms. HDL cholesterol concentrations in the HD patients were significantly lower than in the healthy subjects (P < 0.0001), and IDL cholesterol concentrations and VLDL cholesterol concentrations in the HD patients were significantly higher than in the healthy subjects (P < 0.05). Profiles of these measured lipoprotein values were consistent with the previously reported lipoprotein values, measured ultracentrifugally characteristic of HD patients. CONCLUSION: These results suggest that the HPLC method may be sufficiently applied to the assessment of serum lipoprotein profile in HD patients in place of the other method including an UC.
Assuntos
Colesterol/sangue , Lipoproteínas/sangue , Diálise Renal , Idoso , Análise Química do Sangue/métodos , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/etiologia , Estudos de Casos e Controles , HDL-Colesterol/sangue , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Cromatografia Líquida de Alta Pressão/métodos , Quilomícrons/sangue , Feminino , Humanos , Lipoproteínas/classificação , Masculino , Pessoa de Meia-Idade , Diálise Renal/efeitos adversos , Fatores de Risco , Triglicerídeos/sangueRESUMO
The 17beta-hydroxysteroid dehydrogenases (HSDs) are enzymes that catalyze the reduction of 17-ketosteroids or the oxidation of 17beta-hydroxysteroids. 17beta-HSD type 12, the most recently cloned member of this gene family, was classified into the 17beta-HSD family based on sequence homology, rather than steroid catalyzing activity. Meanwhile, it has been reported that 17beta-HSD type 12 may be involved in fatty acid synthesis. To better understand the role of 17beta-HSD type 12 in lipid metabolism, we determined the detailed systemic distribution and tissue localizations of 17beta-HSD type 12, which, due partly to the lack of antibodies, had not yet been studied. We carried out these investigations by quantitative reverse transcription (RT)-PCR, Northern blot analysis, and immunohistochemistry, using an antibody against 17beta-HSD type 12 that we have generated. 17beta-HSD type 12 is highly expressed in organs related to lipid metabolism such as liver, kidney, heart and skeletal muscle. 17beta-HSD type 12 is also detected in endocrine-related organs such as pancreas, pituitary gland, adrenal gland, testis and placenta, and in the gastrointestinal tract, which point to the possible involvement of 17beta-HSD type 12 in the regulation of lipid biosynthesis and steroid metabolism. These results support previous reports and solidify the possibility that 17beta-HSD type 12 may play critical roles in the physiological processes, such as fatty acid synthesis, in addition to the steroid metabolism.
Assuntos
17-Hidroxiesteroide Desidrogenases/análise , 17-Hidroxiesteroide Desidrogenases/metabolismo , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Primers do DNA , Feminino , Humanos , Masculino , Especificidade de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, has a pro-apoptotic effect on colon adenocarcinoma cells via COX-independent mechanisms. MATERIALS AND METHODS: The pro-apoptotic effect of N-(2-Aminoethyl)-4-[5- (4-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide (TT101), a new derivative of celecoxib, was investigated on the HT-29 and SW480 colon adenocarcinoma cells. Cell proliferation and viability were assessed by incorporation of 5-bromo-2'-deoxyuridine and by the 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, respectively. Apoptosis was detected by identifying DNA fragmentation. Production of prostaglandin E2 by the HT-29 cells was analyzed. RESULTS: TT101 inhibited the proliferation of HT-29 and SW480 cells by inducing apoptosis more potently than celecoxib in a concentration-dependent manner. The COX-2 inhibitory effect of TT101 was weaker than that of celecoxib. CONCLUSION: A slight modification of celecoxib enhanced the pro-apoptotic effect on colon adenocarcinoma cells.
Assuntos
Adenocarcinoma/tratamento farmacológico , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Adenocarcinoma/patologia , Caspases/metabolismo , Neoplasias do Colo/patologia , Dinoprostona/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HT29/efeitos dos fármacos , HumanosRESUMO
The functions of POU class 5 transcription factor 1 (Oct-4) and caudal-type homeobox 2 (Cdx2) in the differentiation of the murine inner cell mass (ICM) and trophectoderm (TE) have been described in detail. However, little is known about the roles of OCT-4 and CDX2 in preimplantation bovine embryos. To elucidate their functions during early development in bovine embryos, we performed OCT-4 and CDX2 downregulation using RNA interference. We injected OCT-4- or CDX2-specific short interfering RNAs (siRNAs) into bovine zygotes. The rate of blastocyst development of OCT-4-downregulated embryos was lower compared with uninjected or control siRNA-injected embryos. Gene expression analysis revealed decreased CDX2 and fibroblast growth factor 4 expression in OCT-4-downregulated embryos. CDX2-downregulated embryos developed to the blastocyst stage; however, in most cases, blastocoel formation was delayed. Gene expression analysis revealed decreased GATA3 expression and elevated NANOG expression in CDX2-downregulated embryos. In conclusion, OCT-4 and CDX2 are essential for early development and gene expression involved in differentiation of ICM and TE lineages in bovine embryos.
Assuntos
Massa Celular Interna do Blastocisto/citologia , Fator de Transcrição CDX2/metabolismo , Diferenciação Celular , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Trofoblastos/citologia , Animais , Massa Celular Interna do Blastocisto/metabolismo , Fator de Transcrição CDX2/genética , Bovinos , Células Cultivadas , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fator 3 de Transcrição de Octâmero/genética , Trofoblastos/metabolismoRESUMO
The terminal oxygenase component of the biphenyl dioxygenase (BphA1A2 complex) was over-expressed with a novel over expression system in recombinant Rhodococcus strain and purified. The purified enzyme has been crystallized by the hanging drop vapor diffusion method and subjected to X-ray diffraction analysis. The crystals belong to the tetragonal system in the space group P4(1)2(1)2 or P4(3)2(1)2 and diffract to better than 2.2A resolution.
Assuntos
Proteínas Ferro-Enxofre/química , Oxigenases/química , Rhodococcus/enzimologia , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Interpretação Estatística de Dados , Regulação Enzimológica da Expressão Gênica , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/isolamento & purificação , Oxigenases/genética , Oxigenases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Rhodococcus/genéticaRESUMO
Cell profiles determined by the thin-layer advanced cytology assay system (TACAS™), a liquid-based cytology technique newly developed in Japan, were analyzed in this study. Hybrid capture 2 (HC-2) was also performed using the liquid-based samples prepared by TACAS to ascertain its ability to detect human papillomavirus (HPV). Cell collection samples from uterine cervix were obtained from 359 patients and examined cytologically. A HC-2 assay for HPV was carried out in the cell specimens. All specimens were found to show background factors such as leukocytes. After excluding the 5 unsatisfactory cases from the total 354 cases, 82 cases (23.2%) were positive and 272 cases (76.8%) were negative for HPV. Cell specimens from 30 HPV-positive cases and 166 HPV-negative cases were subjected to 4 weeks of preservation at room temperature. Then, when subsequently re-assayed, 28 cases (93.3%) in the former group were found to be HPV positive and 164 cases (98.8%) in the latter group were found to be HPV negative. These results supported the excellent reproducibility of TACAS for HPV testing. A reasonable inference from the foregoing analysis is that TACAS may be distinguished from other liquid-based cytological approaches, such as ThinPrep and SurePath, in that it can retain the cell backgrounds. Furthermore, this study raises the possibility that cell specimens prepared using TACAS could be preserved for at least 4 weeks prior to carrying out a HC-2 assay for HPV.
Assuntos
Colo do Útero/virologia , Técnicas Citológicas/métodos , Detecção Precoce de Câncer/métodos , Papillomaviridae/isolamento & purificação , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Feminino , Humanos , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
We compared the detection rate of cervical neoplasias between a liquid-based cytology (LBC) method using SurePath and the conventional method. We also studied the feasibility of human papillomavirus (HPV) typing by linear array assay. Cytological specimens from 1551 Japanese women were prepared using the conventional and SurePath methods; the cytological and histological results from biopsy samples were compared. HPV typing using an HPV linear array assay was carried out on residual specimens using the SurePath method. The cytodiagnostic results showed a concordance rate of 85.3% (Κ= 0.46) between the two methods. The sensitivity of lesions histopathologically diagnosed as CIN1 or above was not significantly different between the two methods (P = 0.575-1.000). The receiver operating characteristic curve analysis of the detectability in CIN2 or above revealed no significant difference between the two methods (P = 0.096). Among the 44 patients who underwent HPV typing using a linear array assay, 33 samples were eligible for HPV testing and were stored at ambient temperature. In conclusion, the SurePath and conventional methods have equivalent abilities for detecting cervical lesions. After preparation for cytological diagnosis, use of the remaining cells from the SurePath specimens to perform HPV typing using the linear array method could be feasible.
Assuntos
Alphapapillomavirus/genética , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/virologia , Citodiagnóstico/métodos , Genótipo , Infecções por Papillomavirus/virologia , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Adulto , Idoso , Feminino , Humanos , Japão , Pessoa de Meia-IdadeRESUMO
We have already demonstrated that celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, has a proapoptotic effect on synovial fibroblasts obtained from patients with rheumatoid arthritis (RA). Here we report on the development of two novel derivatives of celecoxib, N-(2-aminoethyl)-4-[5-(4-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (TT101) and 4-[5-(4-aminophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (TT201), including whether these compounds have a proapoptotic effect on synovial fibroblasts. Synovial fibroblasts were harvested from the synovial tissues of patients with RA or osteoarthritis (OA). Cell proliferation and cell viability were assessed by the incorporation of 5-bromo-2'-deoxyuridine and by the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, respectively. Apoptosis was detected by the identification of DNA fragmentation, and activation of caspase-3 was detected by the addition of a caspase-3 substrate to cell lysates. Production of prostaglandin E(2) by RA synovial fibroblasts was analyzed by enzyme-linked immunosorbent assay. TT101 inhibited the proliferation of RA and OA synovial fibroblasts in a concentration-dependent manner. It caused a marked decrease of cell viability and induced DNA fragmentation more potently than either celecoxib or SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide). TT101 also increased caspase-3 activity. The order of potency of the COX-2 inhibitory activity of these drugs in RA synovial fibroblasts was celecoxib = SC-236 > rofecoxib > TT201 > TT101. In conclusion, we developed TT101 with about a 5- to 10-fold stronger proapoptotic effect on RA and OA synovial fibroblasts compared with that of celecoxib. Although the mechanism of action of TT101 remains unclear, it may have potential as a novel antirheumatic agent.