RESUMO
BACKGROUND: Control of periodontal tissue inflammation during orthodontic treatment is very important in achieving a favourable therapeutic goal. We previously demonstrated that orally applied bovine lactoferrin (bLF) inhibited LPS-induced bone resorption but not orthodontic force-induced tooth movement in vivo. This study is designed to examine the underlying mechanism of it. METHODS: We examined the inhibitory effects of bLF on the expression of RANKL, OPG, TNF-α and COX-2 in osteoblasts loaded with compressive stress (CS) in comparison with LPS stimulated osteoblasts. Formation of osteoclasts was evaluated by co-culture system. RESULTS: Both CS- and LPS-applications upregulated COX-2 and RANKL but downregulated OPG. TNF-α was upregulated in LPS-stimulated osteoblasts but downregulated in CS-loaded osteoblasts. NS398 (a specific inhibitor of COX-2) significantly inhibited CS-induced RANKL-upregulation but not LPS-induced RANKL upregulation, indicating a critical role of COX-2/PGE2 pathway in CS-induced osteoclastogenesis. bLF significantly downregulated LPS-induced upregulation of RANKL and eliminated OPG suppression but not affected in CS-induced changes. Moreover, bLF significantly decreased LPS-induced osteoclast formation, whereas bLF had no effect on PGE2-induced osteoclast formation. CONCLUSIONS: bLF can effectively suppress harmful bone destruction associated with periodontitis without inhibiting bone remodelling by CS-loading. Therefore, oral administration of bLF may be highly beneficial for control of periodontitis in orthodontic patients.
Assuntos
Lactoferrina/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Reabsorção Óssea , Linhagem Celular , Força Compressiva , Ciclo-Oxigenase 2/metabolismo , Humanos , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Epithelial-mesenchymal transition (EMT) is a crucial process in cancer progression that provides cancer cells with the ability to escape from the primary focus, invade stromal tissues and migrate to distant regions. Cell lines that lack E-cadherin show increased tumorigenesis and metastasis, and the expression levels of E-cadherin and Snail correlate inversely with the prognosis of patients suffering from breast cancer or oral squamous cell carcinoma (OSCC). Moreover, recent studies have shown that most EMT cases are regulated by soluble growth factors or cytokines. Among these factors, fibroblast growth factors (FGFs) execute diverse functions by binding to and activating members of the FGF receptor (FGFR) family, including FGFR1-4. Fibroblast growth factor receptor 1 is an oncoprotein that is involved in tumorigenesis, and PD173074 is known to be a selective inhibitor of FGFR1. However, the roles of FGFR1 and FGFR1 inhibitors have not yet been examined in detail. METHODS: Here, we investigated the expression of FGFR1 in head and neck squamous cell carcinoma (HNSCC) and the role of the FGFR1 inhibitor PD173074 in carcinogenesis and the EMT process. RESULTS: Fibroblast growth factor receptor 1 was highly expressed in 54% of HNSCC cases and was significantly correlated with malignant behaviours. Nuclear FGFR1 expression was also observed and correlated well with histological differentiation, the pattern of invasion and abundant nuclear polymorphism. Fibroblast growth factor receptor 1 was also overexpressed in EMT cell lines compared with non-EMT cell lines. Furthermore, treatment of HOC313 cells with PD173074 suppressed cellular proliferation and invasion and reduced ERK1/2 and p38 activation. These cells also demonstrated morphological changes, transforming from spindle- to cobble stone-like in shape. In addition, the expression levels of certain matrix metalloproteinases (MMPs), whose genes contain activator protein-1 (AP-1) promoter sites, as well as Snail1 and Snail2 were reduced following PD173074 treatment. CONCLUSION: Taken together, these data suggest that PD173074 inhibits the MAPK pathway, which regulates the activity of AP-1 and induces MET. Furthermore, this induction of MET likely suppresses cancer cell growth and invasion.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Pirimidinas/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/biossíntese , Fator de Transcrição AP-1/metabolismo , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imuno-Histoquímica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-met/biossíntese , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fator de Transcrição AP-1/biossínteseRESUMO
BACKGROUND: The fraction of exhaled nitric oxide (FENO) is reduced by anti-inflammatory treatment in asthma. However, the FENO level is also regulated by individual demographics and there is considerable variation among clinically stable patients. OBJECTIVE: We hypothesized that some demographics may be responsible for persistent FENO elevation despite inhaled corticosteroids (ICS) therapy in asthma. METHODS: This was a prospective observational study. We initially screened 250 stable asthmatics and determined the FENO cut-off point for identifying poorly controlled asthma defined by one of the following criteria: Asthma control test <20, or forced expiratory volume in one-second % of predicted <80%, or peak expiratory flow variability <80% (Study 1). After 12-weeks, 229 patients who maintained high or low FENO were selected and the independent factors which might contribute to a high FENO were examined (Study 2). RESULTS: A FENO level >39.5 p.p.b. yielded 67% sensitivity and 76% specificity for identifying the patients with poorly controlled asthma. The persistent high FENO group (≥ 40 p.p.b.) was more likely to be ex-smokers, to show evidence of atopy (positive specific IgE, higher serum IgE and blood eosinophils), and to have allergic comorbidities. Especially, past smoking history, blood eosinophils, and chronic rhinosinusitis were identified to be independent predictors of high FENO. Neither the dose of ICS nor other medication use showed any difference between the groups. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggested that past smoking history, blood eosinophilia, and chronic rhinosinusitis are involved in the persistent airway inflammation detected by FENO. Although their relative contributions on FENO values should be further quantified, clarification of the features of the subjects with high FENO might provide clues for adjustment of the treatment approach in asthma.
Assuntos
Asma/fisiopatologia , Demografia , Óxido Nítrico/análise , Corticosteroides/uso terapêutico , Adulto , Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Expiração , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoAssuntos
Cálculos/complicações , Colangite/etiologia , Divertículo/complicações , Duodenopatias/complicações , Doença Aguda , Idoso , Cálculos/diagnóstico , Cálculos/cirurgia , Colangite/diagnóstico , Colestase/etiologia , Divertículo/cirurgia , Duodenopatias/cirurgia , Endoscopia Gastrointestinal , Humanos , MasculinoRESUMO
OBJECTIVE: Passive smoking is the involuntary inhalation of cigarette smoke (CS) and has an adverse impact on oral health. We examined the effect of CS exposure on saliva and salivary glands (SGs). METHODS: Cigarette smoke-exposed rats were intermittently housed in an animal chamber with whole-body exposure to CS until killed. Whole saliva was collected before CS exposure (0 day), and 15 and 30 days after the start of CS exposure. Saliva secretion was stimulated by administration of isoproterenol and pilocarpine after anesthesia. SGs were collected on 31 days. RESULTS: The increase in body weight of the CS-exposed rats was less than that of the control rats. Salivary flow rates did not differ at 0, 15 or 30 days after the start of CS exposure. However, the amylase and peroxidase activities and total protein content in the saliva were significantly lower in 15-day CS-exposed rats than in 15-day control rats. Histological examination of the SGs of CS-exposed rats showed vacuolar degeneration, vasodilation and hyperemia. CONCLUSION: These results suggest that CS exposure has adverse impacts on salivary composition and SGs, which could aggravate the oral environment.
Assuntos
Saliva/metabolismo , Glândulas Salivares/patologia , Proteínas e Peptídeos Salivares/análise , Poluição por Fumaça de Tabaco/efeitos adversos , Amilases/análise , Animais , Cotinina/análise , Dilatação Patológica/induzido quimicamente , Exposição por Inalação/efeitos adversos , Masculino , Peroxidase/análise , Ratos , Ratos Wistar , Saliva/química , Glândulas Salivares/irrigação sanguínea , Glândulas Salivares/efeitos dos fármacos , Taxa Secretória , Estimulação QuímicaRESUMO
BACKGROUND: Exhaled breath condensate (EBC) is thought to contain substances of the lower airway epithelial lining fluid (ELF) aerosolized by turbulent flow. However, contamination by saliva may affect the EBC when collected orally. OBJECTIVE: The purpose of this study was to compare the cytokine expression levels in EBC with those in saliva, and to clarify the influence of saliva on cytokine measurements of EBC. METHODS: EBC and saliva samples were obtained from 10 adult subjects with stable asthma. To estimate differences in the contents of substances between EBC and saliva, the total protein concentration of each sample was measured. Further, we also measured the total protein concentration of ELF obtained from another patient group with suspected lung cancer using a micro sampling probe during bronchoscopic examination and roughly estimated the dilution of EBC by comparing the total protein concentration of EBC and ELF from those two patient groups. The cytokine expression levels of EBC and saliva from asthmatic group were assessed by a cytokine protein array. RESULTS: The mean total protein concentrations in EBC, saliva and ELF were 4.6 microg/ml, 2,398 microg/ml and 14,111 microg/ml, respectively. The dilution of EBC could be estimated as 1:3000. Forty cytokines were analyzed by a cytokine protein array and each cytokine expression level of EBC was found to be different from that of saliva. Corrected by the total protein concentration, all cytokine expression levels of EBC were significantly higher than those of saliva. CONCLUSION: These results suggest that the salivary influence on the cytokine assessment in EBC may be negligible.
RESUMO
Primary chondrosarcoma was found in the quadrate lobe of the liver of a 6-year-old, intact, male Golden Retriever. At 6 months after partial hepatectomy, recurrence in the liver occurred. The dog died of its systemic metastases 10 months thereafter. Histologically, the hepatic mass revealed neoplastic chondrocytes with abundant chondroid matrix, and there were few myxoid areas where the cellularity and pleomorphism of the neoplastic cells were more prominent. The neoplastic cells were positive for periodic acid-Schiff and were immunohistochemically positive for vimentin and S-100 protein; the matrix was deeply stained for alcian blue and was metachromatic for toluidine blue stain. This tumor might be derived from pluripotent mesenchymal cells in the connective tissue of the liver. To the best of our knowledge, in all mammalians, including humans, this is the first report of extraskeletal chondrosarcoma primarily arising in the liver.
Assuntos
Condrossarcoma/veterinária , Doenças do Cão/patologia , Neoplasias Hepáticas/veterinária , Animais , Condrossarcoma/patologia , Cães , Neoplasias Hepáticas/patologia , MasculinoRESUMO
The aim of this study was to assess the importance of immunohistochemical thymidylate synthase (TS) expression level as a prognostic marker in tongue cancer patients. In 140 patients with primary squamous cell carcinoma (SCC) of the tongue, intratumoural TS expression was evaluated by immunohistochemistry. The level of TS expression was determined by a semiquantitative scoring system, ranging from 1+ to 3+ according to the ratio of TS-positive cells. Of 140 patients, 64 (45.7%), 49 (35.0%) and 27 (19.3%) were assessed as 1+, 2+ and 3+, respectively. Univariate analyses demonstrated that both disease-free survival (DFS) and overall survival (OS) were significantly lower in patients with a TS 3+ tumour than in those with a TS 1+/2+ tumour (DFS: P = 0.0082, OS: P = 0.0100). In a multivariate analysis using the Cox regression model, cervical lymph-node status and TS expression level were selected as independent factors for DFS and OS. Maintenance adjuvant chemotherapy by oral 5-fluorouracil (5-FU) significantly improved DFS and OS in patients with a TS 1+/2+ tumour (DFS: P = 0.0027, OS: P = 0.0398). These data suggest that the level of immunohistochemical TS expression is an independent prognosticator in patients with tongue SCC, and may be useful in the selection of patients who would benefit from oral 5-FU adjuvant chemotherapy.
Assuntos
Carcinoma de Células Escamosas/enzimologia , Timidilato Sintase/análise , Neoplasias da Língua/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/secundário , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , Fluoruracila/uso terapêutico , Seguimentos , Previsões , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Timidilato Sintase/antagonistas & inibidores , Resultado do TratamentoRESUMO
Thymidylate synthase (TS), an enzyme involved in the DNA synthesis, is a critical target for fluoropyrimidines. We investigated the relationship between the level of tumoural TS expression and response to tegafur/uracil (UFT) in 26 patients with oral squamous cell carcinoma. The patients received peroral administration of UFT alone preoperatively. In biopsy specimens, TS expression level was assessed by the immunohistochemical staining and graded as 1+, 2+ or 3+ according to the frequency of strongly-stained tumour cells. Out of 26 tumours, 10 (38.5%), 10 (38.5%) and 6 (23.1%) cases were categorized as 1+, 2+ and 3+, respectively. The response to UFT was histologically evaluated by a grading system according to the extent of degenerative or necrotic cancer cells in surgical specimens. Results showed patients with the lower TS expression had the higher response and there was a statistically significant association between TS expression and response to UFT (P=0.031). This finding suggests that TS expression is a predictor of chemosensitivity to UFT in oral squamous cell carcinomas.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/enzimologia , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/enzimologia , Timidilato Sintase/biossíntese , Idoso , Análise de Variância , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma de Células Escamosas/patologia , Diferenciação Celular/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Estatísticas não Paramétricas , Tegafur/administração & dosagem , Uracila/administração & dosagemRESUMO
The status of cyclin D1 and glycogen synthase kinase 3beta (GSK-3beta) was investigated in 41 patients with T1 and T2 squamous cell carcinomas (SCCs) of the tongue. Out of the 41 SCCs, 27 (65.9%) showed overexpression of cyclin D1 in comparison with normal lingual epithelia by an immunohistochemical method. Cyclin D1 gene amplification was detected in only two (9.1%) of 22 informative cases of the SCCs by differential PCR. Expression of GSK-3beta, which was found to regulate proteosomal degradation of cyclin D1 protein, was reduced in 16 cases (39.0%) of the SCCs relative to normal epithelia, and the intensity of GSK-3beta staining showed an inverse association with cyclin D1. These findings suggest that overexpression of cyclin D1 primarily results from stabilization due to reduction of GSK-3beta, but not cyclin D1 gene amplification, in lingual SCCs. Kaplan-Meier analysis demonstrated that the patients with high cyclin D1 and reduced GSK-3beta expression had a significantly lower 5-year survival than the patients with low cyclin D1 and non-reduced GSK-3beta expression (P=0.014). The cyclin D1 and GSK-3beta coupled assessment was more valuable for the prediction of prognosis than assessment based on cyclin D1.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Ciclina D1/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Neoplasias da Língua/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Ciclina D1/genética , Feminino , Seguimentos , Amplificação de Genes , Glicogênio Sintase Quinase 3 beta , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase/métodos , Prognóstico , Taxa de Sobrevida , Neoplasias da Língua/genética , Neoplasias da Língua/patologiaRESUMO
BACKGROUND: Recently, the usefulness of intratumoral microvessel density (IMD) and expression of several angiogenic factors as prognostic indicators have been demonstrated in several human solid tumors. METHODS: One hundred four patients with pancreatic ductal adenocarcinoma were examined retrospectively. The investigated clinicopathologic and immunohistologic data included staining for vascular endothelial growth factor (VEGF), thymidine phosphorylase (TP), basic fibroblast growth factor (bFGF), CD34 (for calculating IMD), p53, and Ki-67. RESULTS: Multivariate analysis for both overall and relapse-free survival revealed two independent variables, IMD and TP staining in stromal cells (TPs, P < 0.02). Whereas the frequency of hepatic metastasis was correlated significantly with cytoplasmic expression of TP or bFGF in tumor cells (TPc, bFGFc), IMD, and p53 status, local recurrence was significantly more common in patients with positive staining for TPs, bFGF in stromal cells (bFGFs), and for the pM category (P < 0.05). TPc, bFGFc, VEGF, and p53 expression correlated with IMD (P < 0.01), although TPs and bFGFs expression did not. VEGF and IMD status correlated with p53 expression (P < 0.001), although TP, bFGF, and Ki-67 status did not. CONCLUSIONS: TPs expression and IMD were revealed to be valuable tools for predicting overall and relapse-free survival in patients with pancreatic adenocarcinoma. Whereas TPc and bFGFc are likely to participate in hepatic metastasis by means of their angiogenic properties, TPs and bFGFs may be related to local tumor progression. Angiogenesis in human pancreatic carcinoma may be dependent on VEGF, TP, and bFGF. p53 abnormality is likely to take part in VEGF-related angiogenesis.
Assuntos
Adenocarcinoma/irrigação sanguínea , Neovascularização Patológica , Neoplasias Pancreáticas/irrigação sanguínea , Timidina Fosforilase/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Carcinoma Ductal Pancreático/irrigação sanguínea , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Fatores de Crescimento Endotelial/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Linfocinas/metabolismo , Masculino , Microcirculação , Pessoa de Meia-Idade , Análise Multivariada , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Análise de Sobrevida , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularAssuntos
Malformações Arteriovenosas/terapia , Regeneração Óssea/fisiologia , Embolização Terapêutica/instrumentação , Mandíbula/irrigação sanguínea , Adolescente , Angiografia Digital , Reabsorção Óssea/fisiopatologia , Cateterismo Periférico , Embolização Terapêutica/métodos , Veia Femoral , Seguimentos , Humanos , Estudos Longitudinais , Masculino , Mandíbula/fisiopatologia , Doenças Mandibulares/fisiopatologia , Radiografia Panorâmica , Tomografia Computadorizada por Raios XRESUMO
Since the ultrasonically activated scalpel (UAS) incorporates multiple functions, we have used it for hepatectomies. The present study discusses the noteworthy points and problems of use, and shows initial results. Intraoperative ultrasonography is an important tool for comprehending the positional relationship between the plane of division and the main blood vessels. It allows initial adjustment of coagulation and cutting effects according to the rigidity of the liver parenchyma by means of variable ultrasound levels and exchangeable blade tips, and offers good visibility of the cut surface in deep sites as long as adequate tension on the tissue and an upper position for the blade are maintained. 30 patients underwent hepatectomies using the UAS. The amount of blood loss for lobectomy was significantly less than that for partial lobectomy in normal livers in addition to a significant difference between normal and damaged livers in each group according to the extent of resection. There were no serious complications seen in all cases during the operation. The incidence of positive bile leakage was high. It is recommended that bile leakage testing be carried out as thoroughly as possible. There were 3 postoperative bile fistulas and 1 postoperative hemorrhage. In conclusion, although a dramatic improvement in blood loss and shortened operating time could not be obtained in all procedures, the safety and usefulness were demonstrated in lobectomy. The UAS can be considered as a surgical device that can contribute to the efficiency of hepatectomy, depending on the indications selected.
Assuntos
Hepatectomia/instrumentação , Perda Sanguínea Cirúrgica/prevenção & controle , Desenho de Equipamento , Hemostasia Cirúrgica , Humanos , Cirrose Hepática/cirurgia , UltrassomRESUMO
To investigate the incidence of cleft lip or palate or both (CLP) in Japan, 303738 babies born in 1532 institutions between 1994 and 1995 were examined and 437 (0.14%) were found to have abnormalities. Of these babies, 32.1% had cleft lip, 43.3% had cleft lip and palate, and 24.8% had cleft palate (Table 2). These results show that the incidence of cleft lip and palate has declined compared with the period from 1981 to 1982.
Assuntos
Fenda Labial/epidemiologia , Fissura Palatina/epidemiologia , Feminino , Humanos , Incidência , Lactente , Japão/epidemiologia , Masculino , Idade MaternaRESUMO
To clarify the pathologic value of endogenous biotin in the salivary gland, we examined in a series of neoplasms of the salivary gland by immunohistochemical staining the distribution of endogenous biotin and of biotin-binding enzymes, namely, acetyl CoA carboxylase (AC), which is a cytosolic enzyme, and pyruvate carboxylase (PC), which is a mitochondrial enzyme. In pleomorphic adenoma, we found biotin and PC in ductal epithelial elements, while AC was found mainly in myoepithelial elements. Carcinoma ex pleomorphic adenoma, adenocarcinoma and mucoepidermoid carcinoma were frequently immunopositive for biotin, PC and AC, while adenoid cystic carcinoma was rarely immunopositive for biotin, PC or AC. These results indicate that endogenous biotin might be associated with the mitochondrial enzyme, which is present at high levels in ductal cells of the salivary gland. However, the neoplastic cells in adenoid cystic carcinoma seemed to have an unusual expression of biotin and related enzymes.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Biotina/metabolismo , Piruvato Carboxilase/metabolismo , Ductos Salivares/enzimologia , Neoplasias das Glândulas Salivares/enzimologia , Acetil-CoA Carboxilase/análise , Adenocarcinoma/enzimologia , Adenolinfoma/enzimologia , Adenoma Pleomorfo/enzimologia , Biotina/análise , Carcinoma Adenoide Cístico/enzimologia , Carcinoma Mucoepidermoide/enzimologia , Carcinoma de Células Escamosas/enzimologia , Citosol/enzimologia , Humanos , Imuno-Histoquímica , Fígado/imunologia , Mitocôndrias/enzimologia , Piruvato Carboxilase/análise , Glândulas Salivares/enzimologia , Distribuição TecidualRESUMO
Sp1 is one of the well documented transcription factors, but the whole structure of human Sp1 has not been determined yet. In the present study, we isolated several cDNAs representing two forms of human Sp1 mRNA with different 5'-terminal structures in HepG2 cells. Isolation of a genomic clone established that one of the cDNAs represents the mRNA having consecutive alignment of exons, which allowed deducing the complete amino acid sequence for human Sp1. Another cDNA clone had a surprising structure that possessed an alignment of exons 3-2-3. Both reverse transcriptase-polymerase chain reaction and RNase protection assays confirmed accumulation of the two forms of Sp1 mRNA in HepG2 cells. Because Southern blot analysis suggested that exon 3 is of a single copy in the genome, the cDNA clone having the duplicated sequences for exon 3 appeared to reflect the trans-splicing between pre-mRNAs of human Sp1.
Assuntos
Splicing de RNA , RNA Mensageiro/genética , Fator de Transcrição Sp1/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Éxons , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fator de Transcrição Sp1/genética , Células Tumorais CultivadasRESUMO
The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalian cells showed four-fold bands on non-reduced SDS-PAGE, though it was homogeneous under reduced conditions. It contains three cysteine residues, Cys-25, Cys-103, and Cys-184, which may be concerned with this heterogeneity. Therefore, we examined the formation of a disulfide bond in the recombinant Shh-N and identified three kinds of disulfides with a combination of peptide mapping and NH(2)-terminal amino acid sequencing analysis. Among them, one type of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be separated from the other Shh-Ns using reverse phase HPLC and had no activity of alkaline phosphatase induction in C3H10T1/2 cells. This molecule could also be made by denaturation of the purified Shh-N with guanidine-HCl under non-reduced conditions. On the other hand, the reduced Shh-N and the reduced S-methylated Shh-N at cysteine residues showed approximately 10-fold higher activity compared to the originally purified Shh-N. These results suggested that Shh-N was synthesized as an active form whose three cysteine residues did not form disulfide and inactivated finally by forming a disulfide bond between Cys-103 and Cys-184.
Assuntos
Proteínas/química , Transativadores , Animais , Dissulfetos , Proteínas Hedgehog , Camundongos , Conformação Proteica , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The FAD7 gene is expressed preferentially in the chlorophyllous tissues of unwounded plants. Wounding activates the expression of the FAD7 gene not only in chlorophyllous tissues, but also in nonchlorophyllous tissues of stems and roots. Our previous study suggested that wound-responsive transcriptional activation by the FAD7 promoter in leaves/stems and roots is brought about by a jasmonic acid (JA)-independent and JA-dependent signaling pathway, respectively. In this paper, we show that a specific region (from -259 to -198) in the FAD7 promoter is required for wound-activated expression of this gene in leaves and stems, while another region (from -521 to -363) is necessary not only for wound-activated but also for JA-responsive expression of this gene in roots. Thus, different regulatory regions of the FAD7 promoter mediate distinct wound-induced expression of this gene in leaves/stems and roots. Gel mobility shift assays revealed the wound-inducible DNA-binding activity to the -242/-223 region in both stem and leaf nuclear extracts. In fact, deletion of this region abolished wound response of the FAD7 promoter, suggesting the in vivo role of this site. Furthermore, we detected root nuclear factors interacting with the region from -433 to -363 of this promoter. Wounding and methyl jasmonate treatments induced differently these DNA-binding activities. These results suggest that different regulatory mechanisms mediate the wound-induced expression of the FAD7 gene in aerial and subterranean organs.
Assuntos
Ácidos Graxos Dessaturases/genética , Regulação da Expressão Gênica de Plantas , Nicotiana/fisiologia , Plantas Tóxicas , Regiões Promotoras Genéticas , Acetatos/farmacologia , Sequência de Bases , Ciclopentanos/farmacologia , Proteínas Nucleares/metabolismo , Oxilipinas , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , Nicotiana/genética , Transcrição GênicaRESUMO
A 58 year-old man was admitted to Kimitsu Chuo Hospital complaining of epigastralgia. Abdominal ultrasound and computed tomography revealed a polypoid lesion at the neck of the gallbladder. Given the pre-operative diagnosis of gallbladder carcinoma, we resected the gallbladder along with the extrahepatic bile duct. There was a papillary tumor (25 x 16 mm) at the neck of the gallbladder. Histopathological examinations showed a subserosal nodular proliferation of uniform small tumor cells. Grimelius staining was slightly positive and Fontana-Masson staining was negative. Most of the tumor cells stained positively for chromogranin A and neuron-specific enolase (NSE), and some of the tumor cells were positive for pancreatic polypeptide. The presence of neurosecretory intracytoplasmic granules was proven ultrastructurally. It was diagnosed as a classical carcinoid tumor of the gallbladder. We reviewed the Japanese reported cases and discussed the difference in clinicopathological findings between classical and atypical carcinoid tumors of the gallbladder. Classical carcinoids of the gallbladder have neither a metastatic nor invasive character, and an extremely favorable prognosis compared with atypical carcinoids. The difference in character between classical and atypical carcinoids of the gallbladder is thought to be derived from their histogenetic origin.