RESUMO
Mammary tumors that spontaneously occurred in domestic Djungarian hamsters (Phodopus sungorus) were histologically examined. Forty-five mammary tumors included 14 adenomas, 18 adenocarcinomas, 1 lipid-rich carcinoma, 2 adenoacanthomas, 2 malignant adenomyoepitheliomas, 1 benign mixed tumor, and 7 "balloon cell" carcinosarcomas. The latter 4 types were newly recognized neoplasms in Djungarian hamsters. The relatively high incidence of spontaneous mammary carcinosarcomas in domestic Djungarian hamsters is intriguing. Carcinosarcomas exhibited anomalous histological features made up of a mixture of glandular cells, polygonal cells (including "balloon cells"), and sarcomatous spindle cells in varying proportions. Transitional features from glandular cells to polygonal cells and subsequently to sarcomatous spindle cells were observed. Using immunohistochemistry, we observed that glandular cells exhibited an epithelial phenotype (cytokeratin(+)/vimentin(-)), spindle cells exhibited a mesenchymal phenotype (cytokeratin(-)/vimentin(+)), and polygonal cells exhibited an intermediate phenotype (cytokeratin(+)/vimentin(+)). Reduction or loss of ß-catenin expression and gain of S100A4 expression were observed in polygonal and spindle cells. The polygonal cell population included a varying number of characteristic cells that were expanded by large intracytoplasmic vacuoles. Electron microscopy revealed that these "balloon cells" had large cytoplasmic lumens lined by microvilli. These observations suggest that epithelial-mesenchymal transition may account for the pathogenesis of mammary carcinosarcomas in Djungarian hamsters.
Assuntos
Adenocarcinoma/veterinária , Carcinoma/veterinária , Neoplasias Mamárias Animais/patologia , Phodopus , Doenças dos Roedores/patologia , Adenocarcinoma/patologia , Animais , Carcinoma/patologia , Cricetinae , Transição Epitelial-Mesenquimal , Feminino , Imuno-Histoquímica/veterinária , Queratinas/análise , Vimentina/análise , beta Catenina/análiseRESUMO
Tenascin-C (Tn-C) is an extracellular matrix glycoprotein implicated in the progression of several human cancers. In canine mammary carcinomas, accumulation of Tn-C has been recognized in 3 different areas: regions of proliferating myoepithelial cells in complex carcinoma, basement membrane zone in low-grade simple carcinoma, and reactive stroma in high-grade simple carcinoma. To identify the Tn-C synthesizing cells in these areas, we utilized double-labeling immunohistochemistry, branched DNA in situ hybridization, and in situ hybridization-immunohistochemistry double-labeling techniques. In complex carcinomas, Tn-C was generated by proliferating myoepithelial cells. Tn-C in low-grade simple carcinomas was also derived from myoepithelial cells existing as a basal monolayer. However, stromal Tn-C in high-grade carcinomas was mainly synthesized by fibroblasts/myofibroblasts, similar to human breast cancer. Thus, the origin of Tn-C in canine mammary carcinomas differs between low- and high-grade malignancies. The role of myoepithelial cell-generated Tn-C is not yet understood.
Assuntos
Doenças do Cão/patologia , Neoplasias Mamárias Animais/patologia , Animais , Progressão da Doença , Doenças do Cão/metabolismo , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Hibridização In Situ/veterinária , Neoplasias Mamárias Animais/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Tenascina/metabolismoRESUMO
Routinely diagnosed simple solid carcinoma (SSC) of the canine mammary gland comprises a heterogeneous group of tumors. Seventy-two cases that had been diagnosed as SSC based on hematoxylin and eosin-stained tissue sections were reclassified immunohistochemically on the basis of myoepithelial markers p63 and α-smooth muscle actin, as well as a luminal epithelial marker cytokeratin 8. Only 23 cases (32%) were true SSC, composed only of luminal epithelial cells, whereas 11 cases (15%) were malignant myoepithelioma (MM), composed predominantly of myoepithelial cells, and 38 cases (53%) were biphasic carcinoma (BC), characterized by biphasic proliferation of luminal epithelial and basal/myoepithelial components. As the pathological parameters were compared between the reclassified tumor types, infiltrative potential, vascular/lymphatic invasion, lymph node metastasis, and Ki-67 labeling index were higher in true SSC compared with MM and BC, suggesting that the former may exhibit a poorer prognosis compared with the latter two.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma/veterinária , Doenças do Cão/patologia , Neoplasias Mamárias Animais/patologia , Mioepitelioma/veterinária , Animais , Carcinoma/metabolismo , Carcinoma/patologia , Doenças do Cão/metabolismo , Cães , Células Epiteliais/patologia , Feminino , Imuno-Histoquímica/veterinária , Metástase Linfática , Neoplasias Mamárias Animais/metabolismo , Mioepitelioma/metabolismo , Mioepitelioma/patologiaRESUMO
BACKGROUND: While adalimumab is a mainstay of treatment for moderate to severe chronic plaque psoriasis, the data regarding optimal treatment intervals for therapeutic maintenance are limited. OBJECTIVE: We compared the clinical efficacy of biweekly maintenance administration of adalimumab with that of monthly treatment. METHODS: 17 psoriasis patients treated with adalimumab 40 mg every other week with initial loading dose of 80 mg until week 24 were assigned to the maintenance therapy with adalimumab 40 mg either every other week (n = 7), or every month (n = 10). The treatment efficacy was evaluated by the proportion of patients who achieved PASI 75 from the baseline at weeks 36, 48 and 60. There was no selection bias between the two groups. RESULTS: At week 24, all the patients except for one in each group achieved PASI 75. In both groups, all the patients who achieved PASI 75 at week 24 maintained PASI 75 responses at week 60. Regarding two patients who did not achieve PASI 75 at week 24, one biweekly treated patient experienced a gradual increase in therapeutic response while one monthly treated patient showed exacerbation after week 24. CONCLUSION: Monthly adalimumab treatment seems to be a reasonable treatment option for patients who responded well to initial standard adalimumab treatment for 24 weeks. Since there are several limitations in this study, including the number of patients, observation period, and patients' characteristics, large randomized controlled trials are needed to confirm these results.
Assuntos
Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Psoríase/tratamento farmacológico , Adalimumab , Adulto , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/efeitos adversos , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Esquema de Medicação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do TratamentoRESUMO
PURPOSE: Few studies have evaluated the influence of antioxidant agents on the optical and mechanical properties of ceramic laminate veneers after dental bleaching. Thus, this in vitro study aimed to evaluate the influence of antioxidant agents on the color stability and mechanical properties, such as nanohardness (HIT), elastic modulus (Eit*), and degree of conversion (DC) of the bonding interface components after dental bleaching in ceramic laminate veneer luting. METHODS AND MATERIALS: A total of 143 bovine teeth were distributed into experimental groups, according to the procedure method (unbleached or bleached with Whiteness HP Maxx 35%), antioxidant type (control, 10% ascorbic acid, or 10% α-tocopherol), and luting period (24 hours or 14 days) (n=13). The Tetric N-Bond Universal adhesive system and Variolink Esthetic LC resin cement were used as luting agents to lute IPS e.max ceramic restorations (0.6 mm in thickness) to enamel. A UV-visible spectrophotometer was used to assess color stability before and after UV-B artificial accelerated aging for 252, 504, and 756 hours (n=8). The HIT and Eit* of the adhesive and resin cement were measured using a nanohardness tester under a load of 1000 µN, and the DC was measured using a micro-Raman spectrometer (n=5). The color stability and mechanical properties were measured and evaluated by twoway and one-way ANOVA, respectively, and Tukey test (α=0.05). RESULTS: Distinct aging periods exerted significant changes on the color stability of the restorations luted in enamel associated with ascorbic acid, bleached and unbleached, and the bleached enamel under no antioxidant agent action, for the experimental groups evaluated after 14 days (p<0.05). The use of the α-tocopherol antioxidant solution after the bleaching process for 24 hours did not alter the optical and mechanical properties of the adhesive interface of the laminate restorations compared to those of the control group (p>0.05). CONCLUSION: The use of a 10% α-tocopherol antioxidant solution produced promising results, suggesting that it could be mediately used after tooth bleaching to lute ceramic laminate veneers.
Assuntos
Colagem Dentária , Cimentos de Resina , Bovinos , Animais , Cimentos de Resina/química , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , alfa-Tocoferol , Facetas Dentárias , Cerâmica/uso terapêutico , Cerâmica/química , Ácido Ascórbico , Teste de Materiais , Porcelana Dentária/química , CorAssuntos
Neoplasias Pulmonares/tratamento farmacológico , Rituximab/uso terapêutico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/líquido cefalorraquidiano , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Encefalite/líquido cefalorraquidiano , Encefalite/induzido quimicamente , Humanos , Ipilimumab/administração & dosagem , Ipilimumab/efeitos adversos , Neoplasias Pulmonares/líquido cefalorraquidiano , Neoplasias Pulmonares/patologia , Masculino , Nivolumabe , Carcinoma de Pequenas Células do Pulmão/líquido cefalorraquidiano , Carcinoma de Pequenas Células do Pulmão/patologiaRESUMO
The endopiriform nucleus (EPN) is a large group of multipolar cells located in the depth of the piriform cortex (PC). Although many studies have suggested that the EPN plays a role in temporal lobe epilepsy, the normal function of the EPN remains to be elucidated. By using optical imaging of coronal brain slice preparations with voltage-sensitive dye, we found signal propagation from the PC or gustatory cortex (GC) to the EPN in normal medium. In our previous research, we failed to elicit a reliable signal reproducibly in the EPN by single stimulation either to the PC or GC. In our current research, we found that a double-pulse stimulation to either the PC or GC (interpulse interval: 20-100 ms) induced robust signal propagation to the EPN through excitation in the agranular division of the insular cortex (AI), with further extension to the claustrum. Finally, double site paired-pulse stimulation to the PC and GC also evoked excitation in the AI, claustrum, and EPN. These results suggest that the EPN has dual roles: 1) further processing of modality-specific olfactory and gustatory information from the PC and GC, respectively and 2) synergistic integration of PC-derived olfactory information and GC-derived gustatory information.
Assuntos
Condutos Olfatórios/fisiologia , Paladar/fisiologia , Animais , Estimulação Elétrica , Eletrofisiologia , Neurônios/fisiologia , Condutos Olfatórios/citologia , Ratos , Ratos WistarRESUMO
The aims of this study were to determine whether the appearance of stromal myofibroblasts and the expression of tenascin-C (Tn-C) correlate with the grade of malignancy in canine mammary tumors and to determine the main cellular source of Tn-C in these tumors. Single or double immunostaining using antibodies against α-smooth muscle actin (α-SMA) and Tn-C was performed on serial sections of normal canine mammary glands as well as those with lobular hyperplasia, simple adenoma, and simple carcinoma. Thirty-nine of 42 simple carcinomas (93%) exhibited stromal α-SMA-positive myofibroblasts and Tn-C expression. Only 6 of 11 cases of simple adenoma (55%) showed these changes, whereas no changes were observed in normal mammary gland tissue or cases of lobular hyperplasia. The distribution of stromal Tn-C correlated with the presence of myofibroblasts. However, Tn-C immunoreactivity was also occasionally observed in the basement membrane zone surrounding the myoepithelial layer in normal tissue, benign lesions, and tubulopapillary carcinomas. This pattern of staining was not related to the presence of myofibroblasts. The appearance of stromal myofibroblasts and expression of Tn-C were significantly correlated with higher histological grades of malignancy and vascular/lymphatic invasion in simple carcinomas. Stromal myofibroblasts appear to be a major cellular source of Tn-C and play an important role in the development of canine mammary tumors. The Tn-C expressed in the basement membrane zone of normal, hyperplastic, and neoplastic mammary tissue, which is likely produced by neighboring myoepithelial cells, may differ functionally from the Tn-C produced by myofibroblasts.
Assuntos
Doenças do Cão/patologia , Neoplasias Mamárias Animais/patologia , Miofibroblastos/citologia , Tenascina/metabolismo , Animais , Proliferação de Células , Cães , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Mamárias Animais/classificação , Neoplasias Mamárias Animais/metabolismoRESUMO
Abdominal ultrasonographical and computed tomography examinations of a 12-year-old neutered female toy poodle revealed a protruding mass, approximately 2 cm in diameter, at the apex of the bladder. The mass was firm and haemorrhagic with a homogeneously brownish-yellow cut surface. Microscopically, it was unencapsulated and located in the muscle layer with invasion of the extra-muscular layer. It was composed of spindloid to oval neoplastic cells that formed irregular clefts and diffuse sheets that dissected bundles of collagen. Immunohistochemically, the neoplastic cells were positive for vimentin and lymphatic vessel endothelial hyaluronan receptor 1 antigens, but negative for cytokeratin AE1/AE3, factor VIII-related antigen, CD31, CD34, Prox-1, S100, desmin, α-smooth muscle actin and MyoD1. Negative immunolabelling for laminin antigen supported the absence of evidence of a basal lamina on ultrastructural examination. Based on these findings, this tumour was identified as a lymphangiosarcoma. To the best of our knowledge, this case is the first report of lymphangiosarcoma arising from the bladder in a dog.
Assuntos
Doenças do Cão/patologia , Linfangiossarcoma/veterinária , Neoplasias da Bexiga Urinária/veterinária , Animais , Cães , FemininoRESUMO
The L-P ring complex is thought to be a molecular bushing that supports flagellar motor rotation at about 10,000 revolutions per minute with presumably very little friction. Structural studies of this complex have been limited because only very small amount of samples are available. Therefore devising an efficient method of crystallization was essential. The addition of a phospholipid and its subsequent slow depletion by phospholipase A2 have been used to successfully grow well-ordered monolayer crystals that extend up to about 10 micrometers. The interaction of the L-P ring complex with lipid membranes was also visualized during this process.
Assuntos
Flagelos/ultraestrutura , Salmonella typhimurium/ultraestrutura , Fracionamento Celular , Cristalização , Microscopia EletrônicaRESUMO
A 2-year-old neutered female Shiba dog exhibited laboured breathing for 1 month. Computed tomography of the thoracic cavity revealed multiple nodules (2-5 mm diameter) in the lungs. Grossly, the lungs were firm and normal in shape. The nodules were grey-white in colour. Microscopically, the nodules were non-encapsulated and exhibited an irregular shape. They were composed of polygonal or spindle cells with indistinct cell borders arranged in sheets. The cells had large, round, hyperchromatic nuclei and abundant pale eosinophilic cytoplasm with no atypia. Intrapulmonary arterial emboli and infiltration into the bronchioles were observed. Immunohistochemically, the cells were positive for vimentin and negative for cytokeratin, glial fibrillary acidic protein and α-smooth muscle actin. Ultrastructurally, the cells displayed cytoplasmic processes, desmosomes and intermediate filaments. These findings led to a diagnosis of diffuse pulmonary meningotheliomatosis with sarcomatous transformation. To the best of our knowledge, this is the first report of diffuse pulmonary meningotheliomatosis in a dog.
Assuntos
Doenças do Cão/patologia , Neoplasias Pulmonares/veterinária , Pulmão/patologia , Sarcoma/veterinária , Animais , Doenças do Cão/diagnóstico por imagem , Cães , Feminino , Pulmão/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Sarcoma/diagnóstico por imagem , Sarcoma/patologia , Tomografia Computadorizada por Raios XRESUMO
OBJECTIVE: Tyrosine kinases and its receptors play important roles in growth, migration, and invasion of malignant cells. Among those, there are only few reports examining the expression pattern of Eph/ephrin signalling system in oesophageal carcinoma. The prognostic importance of ephrin-B2 ligand (EFNB2) and its receptor EphB4, and its correlation with clinicopathologic characteristics are yet to be delineated in patients with oesophageal carcinoma. MATERIALS AND METHODS: EFNB2 gene and EphB4 receptor gene were examined of mRNA specimens in 61 patients with oesophageal squamous cell carcinoma using reverse-transcriptase polymerase chain reaction. EFNB2 protein was selectively examined using an immunohistochemical analysis. RESULTS: EFNB2 mRNA expression was detected in 38 (62.3%) and EphB4 expression was found in 44 (72.1%) out of 61 cancer tissues analysed. There was a statistically significant correlation between EFNB2 expression and number of lymph node metastasis (P<0.05), and a trend toward statistical significance for correlation between EFNB2 expression and American Joint Committee on Cancer Classification Stage (P<0.1), indicating that EFNB2 expression was up-regulated by advancement of the disease process. EFNB2 protein was strongly expressed in tumour with high mRNA EFNB2 expression and was weakly expressed in tumour with low mRNA expression in some representative tumours. The 5-year overall survival rate (23%) of patients with positive EFNB2 gene expression was significantly worse than 55% of negative expression (P<0.05). The results of multivariate analysis of prognosticators for survival showed that positive EFNB2 gene expression (P<0.01) and number of lymph node metastasis (P<0.05) were identified as significant factors indicative of a poorer survival. CONCLUSIONS: EFNB2 gene expression may be a biological marker and a useful prognostic indicator in patients with oesophageal squamous cell carcinoma.
Assuntos
Carcinoma de Células Escamosas/genética , Efrina-B2/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , RNA Neoplásico/genética , Receptor EphB4/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Efrina-B2/biossíntese , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Receptor EphB4/biossíntese , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida/tendênciasRESUMO
A 21-year-old neutered female domestic shorthaired cat was presented with a history of inappetence, vomiting and haematuria. The cat was humanely destroyed at the owner's request and a necropsy examination was performed. A 0.8 × 0.5 × 0.5 cm mass was located in the left lobe of the pancreas. The mass was gelatinous in nature and the external and cut surfaces were pale yellow in colour. Microscopically, the mass was non-capsulated and comprised an accumulation of extracellular stromal mucin containing suspended neoplastic columnar epithelial cells forming tubular structures. Immunohistochemically, these cells diffusely expressed cytokeratin (CK) AE1/AE3, CK7 and carcinoembryonic antigen and were partially positive for CK19 and trypsin, but negative for vimentin. The tumour was diagnosed as a colloid carcinoma. The clinical presentation in this case was caused by chronic renal failure complicated by secondary renal hyperparathyroidism and associated metastatic calcinosis. To the best of our knowledge, this is the first report of colloid carcinoma arising from the pancreas in a cat.
Assuntos
Adenocarcinoma Mucinoso/veterinária , Doenças do Gato/patologia , Neoplasias Pancreáticas/veterinária , Animais , Biomarcadores Tumorais/análise , Gatos , FemininoRESUMO
A 5-year-old male miniature dachshund was presented with a dermal nodule on the left forelimb that increased to 5 mm in diameter over a 2-month period. Grossly, the nodule was firm, and both the external and cut surfaces were homogeneously pale pink in colour. Microscopically, the nodule was comprised of mainly plump endothelial cells and inflammatory cells; among the latter, lymphocytes were predominant, with few scattered plasma cells, mast cells and macrophages. Lymphoid follicles with germinal centres were often observed. Mitotic figures were not observed amongst the endothelial cells. Immunohistochemically, the endothelial cells were positive for vimentin, factor VIII-related antigen and CD31, and the surrounding cells were positive for smooth muscle actin. Lymphocytes expressed CD3 or BLA36. These findings led to a diagnosis of cutaneous angiolymphoid hyperplasia. To the best of our knowledge, this is the first report of a cutaneous proliferative disorder comprising an admixture of proliferating vascular endothelial cells and lymphocytic infiltration with follicle formation in a dog.
Assuntos
Hiperplasia Angiolinfoide com Eosinofilia/veterinária , Doenças do Cão/patologia , Animais , Cães , MasculinoRESUMO
A 5-year-old female domestic shorthair cat was presented with abdominal distension and serum biochemical evaluation indicated a high concentration of oestradiol (32.81 pg/ml). Exploratory laparotomy revealed a large cystic mass in the right ovary with cystic fluid containing a high level of oestradiol (18.80 pg/ml). The tumour was composed of immature neuroectodermal tissue, mature cartilage, smooth muscle, adipose tissue and aggregated, poorly differentiated mesenchymal cells. It contained cysts of various sizes that were lined by epithelium of different types. The basal layer of the lining epithelium was shown to express aromatase by immunohistochemistry. The findings suggest that this was a novel, malignant, oestrogen-secreting teratoma and that the aromatase-positive, neoplastic cells may have been the source of elevated levels of serum oestrogen.
Assuntos
Doenças do Gato/patologia , Estrogênios/sangue , Neoplasias Ovarianas/veterinária , Teratoma/veterinária , Animais , Biomarcadores Tumorais/análise , Gatos , Feminino , Imuno-HistoquímicaRESUMO
A total of 22 genes have been identified in the carcinoembryonic antigen (CEA) gene family. The protein products of this family are highly homologous and include CEA, biliary glycoprotein, nonspecific cross-reacting antigen 50/90 (NCA 50/90), NCA 95, and pregnancy-specific beta-glycoprotein. We used a monoclonal antibody with high affinity to develop a specific enzyme-linked immunosorbent assay (ELISA) method for NCA 50/90 in serum and plasma. Our calibrators were based on affinity-purified recombinant protein from a baculovirus expression system. No significant reactivity with purified CEA, recombinant NCA 95, or recombinant biliary glycoprotein was found by Western blot analysis or in the ELISA method. Only 1 of 15 sera from pregnant women (chorionic gonadotropin > 1000 ng/ml) was positive in the NCA 50/90 ELISA, suggesting that this method does not detect pregnancy-specific glycoprotein. A cutoff value of 18 ng/ml was established based on the 95% value of serum and plasma from 147 healthy volunteers. Only 3 of 31 serum and plasma samples from patients with clinically inactive breast cancer were elevated above the cutoff value, but 44% of 136 samples from patients with clinically active breast cancer were positive. NCA 50/90 measurements were elevated in 7 of 25 patients with active breast cancer whose CEA and CA 15-3 values were below cutoff, and NCA 50/90 values do not correlate with CEA in breast cancer. In addition, we found sensitivities of 70, 39, and 42% for lung cancer, colon cancer, and leukemia, respectively. The sensitivity for non-small cell lung cancer was 85%, however, compared to 50% for small cell lung cancer. Serum from leukemia patients showed an overall sensitivity of 43%, but 71% (10 of 14) sera from patients with chronic myelogenous leukemia were positive compared to, for example, chronic lymphocytic leukemia where 0 of 7 sera had NCA 50/90 values above the cutoff. These studies suggest that NCA 50/90 may have clinical utility in the management of patients with a variety of cancers.
Assuntos
Antígenos de Diferenciação Mielomonocítica/sangue , Antígenos de Neoplasias/sangue , Neoplasias da Mama/sangue , Moléculas de Adesão Celular , Neoplasias do Colo/sangue , Neoplasias Pulmonares/sangue , Glicoproteínas de Membrana/sangue , Animais , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Antígenos de Neoplasias/análise , Antígenos de Superfície/sangue , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Cinética , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Valores de ReferênciaRESUMO
Fluorescence live-cell RNA imaging to monitor the intracellular localization and dynamics of the target RNA is a challenging subject. One of the difficulties to achieve this is to establish a precise method to enable a fluorescent labeling to the target RNA in living cells. Technologies to reduce the background fluorescence and to detect the RNA with high sensitivity are also necessary to visualize and analyze the intracellular localization and dynamic of the target RNA precisely. Especially in monitoring single-molecule motion, a special setup of a microscope system is required. Such technical problems make the live-cell RNA imaging to be a difficult subject. We recently developed a methodology to label and to visualize a target RNA in living cells with low background fluorescence by using a probe that is based on an RNA-binding protein domain PUM-HD (pumilio homology domain) and a fluorescent protein reconstitution method. A noteworthy property of PUM-HD to apply RNA probes is that this protein domain can be modified to recognize a particular 8-base RNA sequence by inducing tailor-made designed mutagenesis. The fluorescent protein reconstitution method allows us to detect the target RNA with high signal-to-noise ratio. Using the probe based on PUM-HD, a fluorescent protein reconstitution method, and a homebuilt fluorescent microscope system, we succeeded in single-molecule observation of a target RNA in living cells. In this chapter, the techniques to establish the probe and to observe the motion of single-molecule RNA are described.
Assuntos
Proteínas Luminescentes/química , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Sondas RNA/química , Proteínas de Ligação a RNA/química , RNA/análise , Actinas/genética , Animais , Sequência de Bases , Sítios de Ligação , Sobrevivência Celular , Corantes Fluorescentes/química , Humanos , Modelos Moleculares , Imagem Molecular/métodos , Domínios Proteicos , RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
Xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) supplemented with an electron donor could catalyze the cis-trans isomerization of 3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide, 3-(5-nitro-2-furyl)-2-phenylacrylamide and 3-(5-nitro-2-furyl)-2-(2-furyl)acrylonitrile. The direction of isomerization (cis leads to trans, cis in equilibrium trans or trans leads to cis) is dependent on the chemical structure of these nitrofuran derivatives. Lipoyl dehydrogenase (NADH:lipoamide oxidereductase, EC 1.6.4.3), DT-diaphorase (NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2) and liver microsomes could also catalyze the conversion of cis-3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide to its trans isomer in the presence of an appropriate electron donor. Such isomerizing activity of these enzymes is much higher than their nitro-reducing activity. In addition, the cis-trans isomerization of some nitrofuran derivatives was demonstrated with the liver slices and the small intestines of rats. A new cis-trans isomerization mechanism which is based on transfer of a single electron by an enzyme system to a nitrofuran derivative to give the radical-anion was proposed. This postulated mechanism was supported by the preliminary experiments using pulse radiolysis technique.
Assuntos
Di-Hidrolipoamida Desidrogenase/metabolismo , Microssomos Hepáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Nitrofuranos , Quinona Redutases/metabolismo , Xantina Oxidase/metabolismo , Animais , Transporte de Elétrons , Radicais Livres , Técnicas In Vitro , Intestino Delgado/metabolismo , Fígado/metabolismo , Masculino , Modelos Químicos , Ratos , EstereoisomerismoRESUMO
By means of differential cDNA expression cloning, we earlier isolated a novel rat cDNA and its protein, named hepassocin, which is upregulated during liver regeneration. Using the rat cDNA as a probe, we have now isolated human hepassocin cDNA encoding a protein of 312 amino acids, which has 81.4% and 83.8% identity, respectively, to rat hepassocin before and after elimination of its signal peptide. Dot blot analysis revealed that hepassocin mRNA was strongly expressed in adult liver, fairly strongly in fetal liver, and weakly in pancreas, but not in other tissues. Recombinant human hepassocin produced in Chinese hamster ovary (CHO) cells by the dihydrofolate reductase-methotrexate (DHFR--MTX) gene amplification method is a homodimer (68 kDa) and has mitogenic activities in hepatocytes of various animal species including rat, mouse, rabbit and dog, and the activity was lost with 2-mercaptoethanol treatment. These results suggest that hepassocin is a potent regulator in liver cell growth not only in rats but also in humans. Computer searches revealed that human hepassocin as well as rat hepassocin has a characteristic disulfide structure close to that of fibrinogen-gamma. We assume that this newly identified growth factor exerts functions in association with an extracellular matrix such as fibrinogen.