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While previous reports have established the anti-inflammatory effects of hangeshashinto, the intracellular signal transduction pathways involved have yet to be elucidated. We aim to employ an experimental system using oral cancer cells to assess the impact of hangeshashinto on intracellular signal transduction pathways in response to stimulation by Porphyromonas gingivalis pathogen-associated molecular patterns (PAMP). Hangeshashinto demonstrated the ability to inhibit the production of interleukin (IL)-6 and IL-8 induced by P. gingivalis PAMP. Furthermore, hangeshashinto suppressed the activation of the IL-6 promoter stimulated by PAMP. Hangeshashinto, like Toll-like receptor (TLR) signaling inhibitors (resatorvid and C29) and an immunosuppressant (dexamethasone), exhibited the ability to suppress TLR-mediated activation of the transcription factor nuclear factor-κB (NF-κB) in response to PAMP stimulation. This study suggests that the anti-inflammatory effects of hangeshashinto may be attributed to the inhibition of TLR signal transduction pathways including NF-κB activation, thereby suppressing NF-κB-dependent gene expression.
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Ozonated glycerol is glycerol containing ozone, has no unpleasant odor, and has a long half-life. To apply ozonated glycerol for clinical use, ozonated macrogol ointment has been developed by adding macrogol ointment to ozonated glycerol to increase the retention in the affected area. However, the effects of ozone on this macrogol ointment were unclear. The viscosity of the ozonated macrogol ointment was approximately two times higher than that of ozonated glycerol. The effect of the ozonated macrogol ointment on the human osteosarcoma cell line Saos-2 (Saos-2 cells) proliferation, type 1 collagen production, and alkaline phosphatase (ALP) activity were studied. The proliferation of Saos-2 cells was assessed using MTT and DNA synthesis assays. Type 1 collagen production and ALP activity were studied using ELISA and ALP assays. Cells were treated for 24 h with or without 0.05, 0.5, or 5 ppm ozonated macrogol ointment. The 0.5 ppm ozonated macrogol ointment significantly elevated Saos-2 cell proliferation, type 1 collagen production, and ALP activity. These results also showed almost the same trend as for ozonated glycerol.
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Systemic osteosclerotic lesions are frequently caused by multiple bone metastases or systemic metabolic disorders. However, bone metastasis from gastric cancer is rare. Herein, we describe such a case, with radiographic and clinical findings resembling Paget's disease. The patient was an 80-year-old Japanese woman with a history of early gastric cancer, treated by partial gastrectomy 2 years prior. The patient sought medical care for chronic low back pain. On imaging, systemic sclerotic lesions were observed throughout the spine and pelvis, with an increase in bone mineral density from 0.86 g/cm3 (2 years prior) to 1.38g/cm3 (current visit) in the lumbar spine. Elevated serum levels of osteoblastic and osteolytic markers were identified. A bone biopsy was used to confirm the diagnosis of metastatic gastric cancer. The patient was treated with TS-1 and denosumab, with normalization of abnormal metabolic markers and alleviation of the back pain. Bone metastasis is reported in only 10% of cases of gastric cancer and, thus, is relatively rare. Therefore, our case of gastric cancer recurrence presenting with mixed osteoblastic and osteolytic bone lesions similar to Paget's disease is relevant to the report. Bone biopsy is necessary for an accurate diagnosis.
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Orento is a traditional Japanese medicinal kampo preparation that is also prescribed in oral care. In oral squamous cell carcinoma cell line CAL27, orento significantly inhibited periodontopathogenic bacterium Porphyromonas gingivalis lipopolysaccharide (LPS) and lipoproteins (PAMP)-stimulated production of interleukin (IL)-6. This suggests that orento negatively regulates PAMP-mediated toll-like receptor (TLR) signaling. Orento significantly suppressed PAMP-stimulated activation of the IL-6 promoter, indicating that orento may suppress the production of IL-6 by PAMP at the transcriptional level. Orento also suppressed TLR-mediated activation of transcription factor nuclear factor-kappa B (NF-kB) that was stimulated by PAMP. This finding indicates that orento may suppress the function and activation of factors involved in TLR signaling, thereby suppressing NF-kB-dependent expression of various genes. Orento suppressed IL-1 receptor-associated kinase (IRAK4), IRAK1, and c-Jun N-terminal kinase (JNK) phosphorylation in PAMP-stimulated CAL27 cells. This result indicates that orento is involved in the initiation of TLR signaling by PAMP and suppresses the downstream signaling pathways of myeloid differentiation primary response gene 88 (MyD88) such as mitogen-activated protein kinase (MAPK) and NF-kB cascades. These findings suggest that orento has an inhibitory effect on the production of inflammatory cytokines.
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Neoplasias de la Boca , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Interleucina-6/genética , Lipopolisacáridos/farmacología , Neoplasias de la Boca/genética , FN-kappa B/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos , Porphyromonas gingivalis/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Receptores Toll-Like , Línea Celular TumoralRESUMEN
Background: Orento, a traditional Japanese medicine, is known as Kampo medicine in Japan. We investigated the possible efficacy of Kampo medicine for periodontal disease. In this study, we examined the in vitro effects of orento on the proliferation of the inflammatory cytokines interleukin (IL)-6 and IL-8, the production of type 1 collagen, and the secretion of alkaline phosphatase (ALP) in the human osteosarcoma cell line Saos-2 (Saos-2 cells). Methods: The proliferation of Saos-2 cells was assessed by MTT assay. IL-6 and IL-8 levels, type 1 collagen production and ALP secretion were evaluated using enzyme-linked immunosorbent assay and ALP assays. Saos-2 cells were treated with or without 0.1, 1, 10, 100 and 1000 µg/mL of orento for 24 h. Results: Orento (10 µg/mL) significantly induced the proliferation of Saos-2 cells. At this concentration, orento suppressed IL-6 and IL-8 and enhanced type 1 collagen production and ALP secretion. Conclusions: These results indicate that orento controls the IL-6 and IL-8 secretion and cellular metabolism of osteoblasts, resulting in the secretion of early bone-related biomarkers.
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Histatins are salivary proteins with antimicrobial activities. We previously reported that histatin 3 binds to heat shock cognate protein 70 (HSC70), which is constitutively expressed, and induces DNA synthesis stimulation and promotes human gingival fibroblast (HGF) survival. However, the underlying mechanisms of histatin 3 remain largely unknown. Here, we found that the KRHH sequence of histatin 3 at the amino acid positions 5-8 was essential for enhancing p27(Kip1) (a cyclin-dependent kinase inhibitor) binding to HSC70 that occurred in a dose-dependent manner; histatin 3 enhanced the binding between p27(Kip1) and HSC70 during the G1/S transition of HGFs as opposed to histatin 3-M(5-8) (substitution of KRHH for EEDD in histatin 3). Histatin 3, but not histatin 3-M(5-8), stimulated DNA synthesis and promoted HGF survival. Histatin 3 dose-dependently enhanced both p27(Kip1) and HSC70 ubiquitination, whereas histatin 3-M(5-8) did not. These findings provide further evidence that histatin 3 may be involved in the regulation of cell proliferation, particularly during G1/S transition, via the ubiquitin-proteasome system of p27(Kip1) and HSC70.
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Proliferación Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Histatinas/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Ubiquitina/metabolismo , Sitios de Unión , Supervivencia Celular/fisiología , Células HEK293 , Humanos , Unión Proteica , Ubiquitinación/fisiologíaRESUMEN
BACKGROUND: Salivary histatins are bioactive peptides related to the innate immune system associated with antimicrobial activities. However, very little is known about the physiological and biological functions of histatins against host cells or their role in oral cell inflammation. Histatin 3 binds to heat shock cognate protein 70 (HSC70, a constitutively expressed heat shock protein (HSP)). It is unclear whether HSC70 is involved in the inflammatory response in oral cells. Injured oral cells release some intracellular proteins including HSC70. It is possible that released HSC70 induces toll-like receptor (TLR) activation, just as extracellular HSP70 (a stress inducible HSP) does, and that histatin 3 affects this process. Therefore, we tested the hypothesis that HSC70 activates TLR signaling and histatin 3 inhibits this activation and inflammatory cytokine production. METHODS: A nuclear factor (NF)-κB-dependent luciferase reporter plasmid was transfected into HEK293 cells stably expressing TLR2 with coreceptor CD14 (293-TLR2/CD14 cells) or stably expressing TLR4 with CD14 and the accessory molecule MD2 (293-TLR4/MD2-CD14 cells). The cells were stimulated with HSC70 in the presence or absence of histatin 3, and examined using luciferase assays. We also stimulated human gingival fibroblasts (HGFs) with HSC70 with or without histatin 3. Then, we analyzed the levels of inflammatory cytokines (interleukin (IL)-6 and IL-8) in the culture media. Cell proteins were analyzed using enzyme-linked immunosorbent assay and Western blotting with antibodies of mitogen-activated protein kinases and NF-κB inhibitor IκB-α, respectively. Histatin 3-bound form of HSC70 was analyzed using limited V8 protease proteolysis. RESULTS: HSC70 induced NF-κB activation in a dose-dependent manner in 293-TLR2/CD14 and 293-TLR4/MD2-CD14 cells, and histatin 3 inhibited this process and when histatin 3 binding to HSC70 was precluded by 15-deoxyspergualin, which augmented NF-κB-triggered activation. In HGFs, histatin 3 also inhibited HSC70-induced inflammatory cytokine production, extracellular signal-regulated protein kinase phosphorylation, and degradation of IκB-α. Moreover, HSC70 in the presence of histatin 3 was relatively resistant to digestion by V8 protease compared with HSC70 in the presence of control peptide. CONCLUSIONS: Histatin 3 may be an inhibitor of HSC70-triggered activation of TLR signaling and inflammatory cytokine production and may be involved in inflammation processes noted in oral cells.
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In the present study, we investigated the effects of a Kampo medicine Orento (TJ-120) on the production of prostaglandin E(2) (PGE(2)), interleukin (IL)-6 and IL-8 by human gingival fibroblasts (HGFs) treated with lipopolysaccharide from Porphyromonas gingivalis (PgLPS). HGFs proliferation was dose-dependently decreased with Orento at days 3 and 7. However, treatment with PgLPS (10 ng/ml), Orento (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Orento suppressed PgLPS-induced PGE(2) production in a dose-dependent manner but did not alter basal PGE(2) level. In contrast, Orento did not alter PgLPS-induced IL-6 and IL-8 productions. These alterations by Orento were similar to those by a mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor, PD98059. A Orento showed no effect on cyclooxygenase (COX)-1 and COX-2 activities, and increased cytoplasmic phospholipase A(2) (cPLA(2)) expression and increased PgLPS-induced COX-2 expression. Orento suppressed PgLPS-induced mobility retardation of cPLA(2) band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, that is cPLA(2) phosphorylation and its activation, while Orento alone did not alter cPLA(2) phosphorylation. Orento suppressed PgLPS-induced extracellular signal-regulated kinase (ERK) phosphorylation, which is known to lead to ERK activation and cPLA(2) phosphorylation. These results suggest that Orento decreased PGE(2) production by inhibition of cPLA(2) phosphorylation and its activation via inhibition of ERK phosphorylation, and also that Orento may be useful to improve gingival inflammation in periodontal disease.
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Antiinflamatorios/farmacología , Dinoprostona/biosíntesis , Medicamentos Herbarios Chinos/farmacología , Encía/efectos de los fármacos , Interleucinas/biosíntesis , Magnoliopsida , Fitoterapia , Acetiltransferasas/metabolismo , Antiinflamatorios/uso terapéutico , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/uso terapéutico , Electroforesis en Gel de Poliacrilamida , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Flavonoides , Encía/metabolismo , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipopolisacáridos , Medicina Kampo , Enfermedades Periodontales/tratamiento farmacológico , Fosforilación , Factores de Transcripción/metabolismoRESUMEN
Chronic periodontitis is a widespread and major dental disease. Recent studies have analyzed a possible relationship between polymorphism of several genes and periodontitis. Histatins are salivary polypeptides with fungicidal activities against Candida albicans and yeast and bactericidal activities against Porphyromonas gingivalis and Streptococcus mutans. Histatins are part of the innate defense of the oral cavity. We examined the frequency of the polymorphism codon 23 of the histatin 3 gene (HIS2 allele) in relation to periodontitis in the Japanese population. The subjects were 143 Japanese individuals, of which 63 were healthy control subjects and 80 were periodontal patients. We isolated genomic DNA from lingual mucosal cells and tested them for single nucleotide polymorphism (SNP) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The incidence of polymorphism was analyzed statistically by Fisher's exact test. The results indicated that the gene polymorphism at codon 23 of the histatin 3 gene was not associated with periodontitis in the Japanese population (p = 0.166). Rather, if at all, it appeared to be associated with resistance to periodontitis.
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Histatinas/genética , Periodontitis/genética , Polimorfismo Genético/genética , Adulto , Anciano , Alelos , Codón/genética , ADN/análisis , Femenino , Frecuencia de los Genes/genética , Genotipo , Humanos , Inmunidad Innata/genética , Japón , Masculino , Persona de Mediana Edad , Mutación/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción/genética , Polimorfismo de Nucleótido Simple/genética , Adulto JovenRESUMEN
BACKGROUND AND OBJECTIVE: A major factor in the pathogenesis of periodontal disease, which is one of the biofilm infectious diseases, is thought to be lipopolysaccharide (LPS), owing to its ability to cause inflammation and promote tissue destruction. Moreover, the elimination of pathogens and their component LPSs is essential for the successful treatment of periodontal disease. Lipopolysaccharide tolerance is a mechanism that prevents excessive and prolonged responses of monocytes and macrophages to LPS. Since persistence of inflammation is necessary for inflammatory cytokine production, cells other than monocytes and macrophages are thought to maintain the production of cytokines in the presence of LPS. In this study, we investigated whether human gingival fibroblasts (HGFs), the most abundant structural cell in periodontal tissue, might be able to maintain inflammatory cytokine production in the presence of LPS bynot displaying LPS tolerance. MATERIAL AND METHODS: Human gingival fibroblasts were pretreated with LPS (from Porphyromonas gingivalis and Escherichia coli) and then treated with LPS, and the amounts of interleukin (IL)-6 and IL-8 in the cell culture supernatants were measured. The expression of negative regulators of LPS signalling (suppressor of cytokine signalling-1, interleukin-1 receptor-associated-kinase M and SH2 domain-containing inositol-5-phosphatase-1) was also examined in LPS-treated HGFs. RESULTS: Human gingival fibroblasts did not display LPS tolerance but maintained production of IL-6 and IL-8 when pretreated with LPS, followed by secondary LPS treatment. Lipopolysaccharide-treated HGFs did not express negative regulators. CONCLUSION: These results demonstrate that HGFs do not show LPS tolerance and suggest that this characteristic of HGFs sustains the inflammatory response in the presence of virulence factors.
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Fibroblastos/patología , Encía/patología , Interleucina-6/análisis , Interleucina-8/análisis , Periodontitis/patología , Actinas/análisis , Línea Celular , Células Cultivadas , Escherichia coli/inmunología , Fibroblastos/inmunología , Encía/inmunología , Humanos , Tolerancia Inmunológica/inmunología , Inositol Polifosfato 5-Fosfatasas , Quinasas Asociadas a Receptores de Interleucina-1/análisis , Interleucina-10/farmacología , Lipopolisacáridos/inmunología , Periodontitis/inmunología , Monoéster Fosfórico Hidrolasas/análisis , Porphyromonas gingivalis/inmunología , Piel/inmunología , Piel/patología , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/análisis , Factor de Crecimiento Transformador beta1/farmacología , Dominios Homologos src/inmunologíaRESUMEN
Histatins, a family of salivary proteins, have antimicrobial activity. Candida albicans, which is killed by histatins, induces oral candidiasis in individuals with compromised immune systems. Although the functional significance of histatins has been documented, their biological and physiological functions against host cells have not been clarified. In this study, we found that histatin 3, a member of the histatin family, binds to heat shock cognate protein 70 (HSC70). These proteins were co-localized in the cytoplasm and nucleus in human gingival fibroblasts following non-heat and heat shock. Histatin 3 induced stimulation of DNA synthesis and cell survival in human gingival fibroblasts in a dose-dependent manner. This DNA synthesis was found to be dependent on HSC70 by knockdown experiments. The effect of heat shock on DNA synthesis induced by histatin 3 was approximately 2-fold higher than that of non-heat shock. When the histatin 3 uptake into cells was inhibited by monodansylcadaverine or when histatin 3 binding to HSC70 was precluded by 15-deoxyspergualin, DNA synthesis by histatin 3 was approximately 2-fold less than that without monodansylcadaverine or 15-deoxyspergualin. Although HSC70 directly bound to p27(Kip1) (a cyclin-dependent kinase inhibitor), histatin 3 increased the binding between those proteins but not with a peptide capable of binding to HSC70. Moreover histatin 3 prevented ATP-dependent dissociation of HSC70-p27(Kip1). ATP was unable to form a histatin 3-HSC70(D10N)-p27(Kip1) complex (HSC70(D10N) is a mutant attenuating ATPase activity). These findings suggest that histatin 3 may be involved in cell proliferation through the regulation of HSC70 and p27(Kip1) in oral cells.
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Fibroblastos/metabolismo , Fase G1 , Encía/citología , Proteínas del Choque Térmico HSC70/metabolismo , Histatinas/metabolismo , Fase S , Proteínas y Péptidos Salivales/metabolismo , Adenosina Trifosfato/farmacología , Supervivencia Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , ADN/biosíntesis , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fase G1/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Guanidinas/farmacología , Proteínas del Choque Térmico HSC70/química , Respuesta al Choque Térmico/efectos de los fármacos , Humanos , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Fase S/efectos de los fármacosRESUMEN
Cross-linked B cell receptor (BCR) aggregates on the cell surface, then assembles into the "cap" where Ras is co-localized, and transduces various intracellular signals including Ras-ERK activation. BCR signals induce proliferation, differentiation, or apoptosis of B cells depending on their maturational stage. The adaptor protein BLNK binds various signaling proteins and Igalpha, a signaling subunit of the BCR complex, and plays an important role in the BCR signal transduction. BLNK was shown to be required for activation of ERK, but not of Ras, after BCR cross-linking, raising a question how BLNK facilitates ERK activation. Here we demonstrate that BLNK binds the active form of H-Ras, and their binding is facilitated by BCR cross-linking. We have identified a 10-amino acid Ras-binding domain within BLNK that is necessary for restoration of BCR-mediated ERK activation in BLNK-deficient B cells and for anti-apoptotic signaling. The Ras-binding domain fused with a CD8alpha-Igalpha chimeric receptor could induce prolonged ERK phosphorylation, transcriptional activation of Elk1, as well as the capping of the receptor in BLNK-deficient B cells. These results indicate that BLNK recruits active H-Ras to the BCR complex, which is essential for sustained surface expression of BCR in the form of the cap and for the signal leading to functional ERK activation.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Proteínas ras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis , Antígenos CD8/biosíntesis , Células COS , Diferenciación Celular , Proliferación Celular , Pollos , Chlorocebus aethiops , Humanos , Ratones , FosforilaciónRESUMEN
Histatins are salivary proteins found and expressed in human salivary glands. They play a role in the non-immune system of antimicrobial defense, for instance, against Candida albicans. The transcriptional regulatory sequences of the histatin gene, HIS1, have remained obscure for a long time. Here, we cloned the putative promoter from human genomic DNA and tested it in a luciferase reporter system. This promoter is much more active in salivary gland cells than in other cell types. Analysis of deletion mutants revealed that the region encompassing -2254 to -1748 is a strong positive transcriptional element, and its functional core sequence (termed HTN27 box) works in correct and reverse orientations in synergy with downstream sequences, the region spanning -680 to +28 and a proximal promoter. The plus single-stranded HTN27 box is specifically bound by a 100 kDa protein that is present in HSG cells, but not in HeLa cells. These findings indicate that the regulation of the histatin gene expression may be intricate, and it seems to have a cell-type preference in the salivary gland cells.
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Regulación de la Expresión Génica , Histatinas/genética , Regiones Promotoras Genéticas/genética , Saliva/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Clonación Molecular , Perfilación de la Expresión Génica , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Nucleótidos/genética , Especificidad de Órganos , Unión Proteica , Eliminación de SecuenciaRESUMEN
Periodontitis, which is a widespread and major dental disease, is a multifactorial, lifestyle-related disease and has been analyzed for gene polymorphism. We examined the frequency of the polymorphisms of the pro-inflammatory cytokine genes IL-1 A (-889) and IL-1 B (+3953) in relation to periodontitis in the Japanese population. We also examined whether polymorphism of TLR2 (Arg677Trp) and TLR4 (Asp299Gly), which are receptors recognized by periodontopathic bacteria, may also be associated with periodontitis. The subjects were 92 Japanese individuals, among whom 43 had periodontitis and 49 were healthy controls. We isolated genomic DNA from lingual mucosal cells and tested them for single nucleotide polymorphisms by polymerase chain reaction-restriction fragment length polymorphism. The incidence of polymorphisms was analyzed statistically by Fisher's exact test, and the sensitivity and specificity of the gene polymorphisms were calculated. The purpose was to determine whether such polymorphisms might be effectively used in the diagnosis of periodontitis. However, we found no evidence that the gene polymorphism of IL-1A (p = 0.082), IL-1 B (p = 0.180), TLR2 (p = 1.000) or TLR4 (p = 1.000) and overall gene polymorphism in any of the genes (p = 0.752) correlate with periodontitis. The sensitivity (14.0%) and specificity (83.7%) of the mutations found in all of the genes were low. Therefore, we advise against using the analyses of polymorphism of these genes to detect periodontitis in the Japanese population.
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Interleucina-1alfa/genética , Interleucina-1beta/genética , Periodontitis/inmunología , Polimorfismo Genético/genética , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Arginina/genética , Ácido Aspártico/genética , Glicina/genética , Humanos , Japón , Persona de Mediana Edad , Mutación/genética , Periodontitis/genética , Periodontitis/microbiología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple/genética , Sensibilidad y Especificidad , Triptófano/genéticaRESUMEN
In the present study, we investigated the anti-inflammatory effects of a Kampo medicine Shosaikoto (TJ-9) using in vitro periodontal disease model, in which human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) produce IL-6, IL-8 and prostaglandin E2 (PGE2). Treatment with PgLPS (10 ng/ml), TJ-9 (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Moreover, TJ-9 did not alter LPS-induced IL-6 and IL-8 productions. However, TJ-9 significantly suppressed LPS-induced PGE2 production in a dose-dependent manner but TJ-9 alone did not affect basal PGE2 level. Western blotting demonstrated that TJ-9 decreased cyclooxygenase-2 (COX-2) expression in a dose-dependent manner but not phospholipase A2. Moreover, TJ-9 selectively and dose-dependently inhibited COX-2 activity. These results suggest that TJ-9 decreased PGE2 production by inhibition of both COX-2 expression and activity and that TJ-9 may be useful to improve gingival inflammation in periodontal disease.
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Antiinflamatorios no Esteroideos , Medicamentos Herbarios Chinos/farmacología , Fibroblastos/efectos de los fármacos , Encía/citología , Encía/efectos de los fármacos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/toxicidad , Medicina Kampo , Western Blotting , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/biosíntesis , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Gingivitis/tratamiento farmacológico , Gingivitis/patología , Humanos , Indicadores y Reactivos , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Porphyromonas gingivalis/química , Sales de Tetrazolio , TiazolesRESUMEN
PURPOSE: To investigate whether genomic DNA can be purified in sufficient quantity and quality from the oral cavity. METHODS: One milliliter of peripheral blood and saliva were collected. The buccal and lingual mucosal cells were also obtained using 10 strokes with a swab or a toothbrush, respectively. All materials were centrifuged and the cells were lysed by adding sodium dodecyl-sulfate and proteinase K. The DNAs were extracted with phenol and precipitated with ethanol followed by electrophoresing on 0.8% agarose gel. The purified DNAs were digested with restriction enzyme Dpn I and Mbo I, respectively. Amplification of the IL-1A gene by PCR was carried out using the purified DNAs and electrophoresing on polyacrylamide gel. RESULTS: DNA was obtained from lingual mucosal cells collected with a toothbrush. Only about one-thirtieth of the recovered DNA was of non-human origin (bacterial contaminants from the oral cavity). Judging from the PCR amplifications of the IL-1A gene, the DNA extracted from lingual cells was of sufficient quality, in all respects indistinguishable from the DNAs extracted from the other specimen, such as peripheral blood, saliva and buccal mucosal cells collected with a swab, and in sufficient quantity. Our results indicate that it is possible to purify DNAs from lingual mucosal cells collected with a toothbrush in a simple and safe manner. Compared to DNA samples from patients by blood extraction, the described method also had the advantage of being painless and not inducing mental distress.
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ADN/genética , Genoma/genética , Mucosa Bucal/citología , Lengua , ADN/sangre , Humanos , Interleucina-1alfa/genética , SalivaRESUMEN
BASH/BLNK/SLP-65 is an adaptor protein necessary for the B cell receptor (BCR) signal transduction. Here we report the identification through the yeast two-hybrid system of a novel 26-kDa protein, BASH N-terminus-associated protein 1 (BNAS1), which interacts with the conserved and functionally important N-terminal domain of BASH/BLNK/SLP-65. BNAS1 presumably contains four transmembrane domains and the leucine zipper (LZ) motif, and is expressed ubiquitously. The association of BNAS1 with BASH/BLNK/SLP-65 through its LZ motif in vertebrate cells was demonstrated by immunoprecipitation assay. Confocal microscopy revealed that exogenously expressed BNAS1 is localized to the endoplasmic reticulum (ER) and the nuclear envelope. BASH/BLNK/SLP-65 alone was present diffusely in the cytoplasm, but localized to the same position as BNAS1 when co-expressed with BNAS1. Their co-localization was dependent on the domains containing the LZ motif of both molecules. BCR-signaled transcriptional activation of Elk-1 was suppressed by over-expression of BNAS1 in DT40 chicken B cells, and conversely augmented in the BNAS1-deficient DT40 cells, which was restored by BNAS1 reconstitution. This augmentation of Elk-1 activation in the BNAS1-deficient cells was abolished selectively by Jun N-terminal kinase (JNK) inhibitor, suggesting that BNAS1 regulates Elk-1 activation through JNK. Taken together, these results suggest that BNAS1 interacts with BASH/BLNK/SLP-65 at the ER and/or the outer nuclear membrane and is involved in the regulation of the signal transmission via mitogen-activated protein kinases leading to Elk-1 activation.
Asunto(s)
Linfocitos B/inmunología , Proteínas Portadoras/inmunología , Activación de Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Fosfoproteínas/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Western Blotting , Núcleo Celular/metabolismo , Pollos , Retículo Endoplásmico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Marcación de Gen , Humanos , Proteínas de la Membrana/genética , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Transporte de Proteínas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos , Proteína Elk-1 con Dominio ets/inmunologíaRESUMEN
BACKGROUND, MATERIALS AND METHODS: It has recently become clear that RUNX3 expression is frequently silenced by aberrant methylation in gastric cancers. In this study, we investigated the methylation status of the RUNX3 gene in 92 resected primary colorectal cancers using methylation-specific PCR (MSP) and correlated the results with the clinicopathological features of affected patients. RESULTS: Aberrant promoter methylation of the RUNX3 gene was detected in 31 out of 92 (34%) colorectal cancers. A significant difference in histology (p = 0.028) was also found on comparing the RUNX3 methylation of poorly-differentiated colorectal cancers to that of other differentiated ones. CONCLUSION: RUNX3 aberrant methylation might play an important role in colorectal cancers, especially in poorly-differentiated colorectal cancers.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Metilación de ADN , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Diferenciación Celular/fisiología , Subunidad alfa 3 del Factor de Unión al Sitio Principal , Femenino , Humanos , Masculino , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras GenéticasRESUMEN
A B cell-specific adaptor protein, BASH (also known as BLNK or SLP-65), is crucial for B cell receptor (BCR) signaling. BASH binds to various signaling intermediates, such as Btk, PLCgamma2, Vav, and Grb2, through its well defined motifs. Although functional significance of such interactions has been documented, BASH-mediated signal transduction mechanism is not fully understood. Using the yeast two-hybrid system, we have identified a novel protein that binds to a conserved N-terminal domain of BASH, which we named BNAS2 (BASH N terminus associated protein 2). From its deduced amino acid sequence, BNAS2 is presumed to contain four transmembrane domains, which are included in a central MARVEL domain, and to localize to endoplasmic reticulum. BNAS2 was co-precipitated with BASH as well as Btk and ERK2 from a lysate of mouse B cell line. In the transfected cells, the exogenous BNAS2 was localized in a mesh-like structure in the cytoplasm resembling that of endoplasmic reticulum (ER) and nuclear membrane. BASH was co-localized with BNAS2 in a manner dependent on its N-terminal domain. RT-PCR analysis indicated that BNAS2 mRNA is expressed ubiquitously except for plasma cells. In chicken B cell line DT40, overexpression of BNAS2 resulted in an enhancement of BCR ligation-mediated transcriptional activation of Elk1, but not of NF-kappaB, in a manner dependent on the dose of BNAS2. Thus BNAS2 may serve as a scaffold for signaling proteins such as BASH, Btk, and ERK at the ER and nuclear membrane and may facilitate ERK activation by signaling from cell-surface receptors.