RESUMEN
Genetic risk factors play important roles in the etiology of oral, dental, and craniofacial diseases. Identifying the relevant risk loci and understanding their molecular biology could highlight new prevention and management avenues. Our current understanding of oral health genomics suggests that dental caries and periodontitis are polygenic diseases, and very large sample sizes and informative phenotypic measures are required to discover signals and adequately map associations across the human genome. In this article, we introduce the second wave of the Gene-Lifestyle Interactions and Dental Endpoints consortium (GLIDE2) and discuss relevant data analytics challenges, opportunities, and applications. In this phase, the consortium comprises a diverse, multiethnic sample of over 700,000 participants from 21 studies contributing clinical data on dental caries experience and periodontitis. We outline the methodological challenges of combining data from heterogeneous populations, as well as the data reduction problem in resolving detailed clinical examination records into tractable phenotypes, and describe a strategy that addresses this. Specifically, we propose a 3-tiered phenotyping approach aimed at leveraging both the large sample size in the consortium and the detailed clinical information available in some studies, wherein binary, severity-encompassing, and "precision," data-driven clinical traits are employed. As an illustration of the use of data-driven traits across multiple cohorts, we present an application of dental caries experience data harmonization in 8 participating studies (N = 55,143) using previously developed permanent dentition tooth surface-level dental caries pattern traits. We demonstrate that these clinical patterns are transferable across multiple cohorts, have similar relative contributions within each study, and thus are prime targets for genetic interrogation in the expanded and diverse multiethnic sample of GLIDE2. We anticipate that results from GLIDE2 will decisively advance the knowledge base of mechanisms at play in oral, dental, and craniofacial health and disease and further catalyze international collaboration and data and resource sharing in genomics research.
Asunto(s)
Caries Dental , Periodontitis , Caries Dental/genética , Caries Dental/prevención & control , Genómica , Humanos , Salud Bucal , FenotipoRESUMEN
AIM: To evaluate the efficacy of a prototype root canal dressing containing surface pre-reacted glass-ionomer (S-PRG) fillers on repairing induced periapical lesions in a rat model. Calcium hydroxide [Ca(OH)2 ] was applied as a comparison in the healing process. METHODOLOGY: The pulp chambers of the maxillary first molars in 64 male Wistar rats aged 16 weeks were opened to induce periapical lesions. After 28 days, the mesial canal of each tooth was prepared, irrigated with 2.5% sodium hypochlorite only (control group: irrigation) or followed by the respective dressing [Ca(OH)2 group, irrigation + Ca(OH)2 ; S-PRG group, irrigation + S-PRG] and restored with composite resin for 3 or 7 days (10/group). Four rats with healthy molars were used as blank controls. Descriptive analysis of the periapical radiographs, haematoxylin and eosin staining and immunohistochemical observation was performed 3 and 7 days after treatment. The periapical grey value, CD68 macrophages and osteoclasts (cathepsin-K) were quantified and statistically analysed with Tukey's honest significant difference test. A significant difference was achieved when P values were <0.05. RESULTS: S-PRG and Ca(OH)2 dressings were associated with increased periapical grey values and inhibited osteoclast activity at 3 and 7 days; a significant difference in radiographic results and the number of osteoclasts was obtained at 3 and 7 days compared with the control group (P < 0.05). Reparative tissue was observed histologically in the space of the periapical resorbed necrotic area after S-PRG and Ca(OH)2 treatment for 3 and 7 days. The number of macrophages was significantly decreased at 3 and 7 days in the S-PRG and Ca(OH)2 specimens when compared with the controls (P < 0.05). CONCLUSIONS: In a rat experimental model, the S-PRG root canal dressing was comparable to Ca(OH)2 in promoting the healing of experimentally induced periapical lesions. S-PRG paste has the potential to be used as an alternative intracanal dressing in teeth with apical periodontitis.
Asunto(s)
Periodontitis Periapical , Materiales de Obturación del Conducto Radicular , Animales , Vendajes , Hidróxido de Calcio , Cavidad Pulpar , Masculino , Periodontitis Periapical/terapia , Ratas , Ratas Wistar , Irrigantes del Conducto RadicularRESUMEN
Fms-like tyrosine kinase 3 (FLT3) is highly expressed in mixed-lineage leukemia (MLL) gene-rearranged acute lymphoblastic leukemia (MLL+ALL) with a dismal prognosis. We previously reported that FLT3 ligand (FL) stimulation induced cell cycle arrest in MLL+ALL cells leading to resistance against anti-leukemic agents. Given that FL stimulation enhanced transforming growth factor (TGF)ß1 mRNA levels in MLL+ALL cells, we extensively examined the effect of TGFß1 on the cell cycle progression and chemosensitivity in MLL+ALL cells, and found that TGFß1 stimulation induced MLL+ALL cells into cell cycle arrest resistant to arabinosyl cytosine; its effect was markedly enhanced in synergy with FL. Thus, it is likely that TGFß1 and FL, both abundantly produced by bone marrow stromal cells, function in a coordinated manner to render MLL+ALL cells chemoresistant, which should lead to the development of minimal residual disease (MRD) resulting in relapse. The use of inhibitors against FLT3 and TGFß1 may become a useful strategy for eradicating MRD in MLL+ALL.
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Resistencia a Antineoplásicos/fisiología , Proteínas de la Membrana/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Western Blotting , Línea Celular Tumoral , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/fisiología , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Proteína de la Leucemia Mieloide-Linfoide/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Periodontitis is a multifactorial disease in which bacterial, lifestyle, and genetic factors are involved. Although previous genetic association studies identified several susceptibility genes for periodontitis in European populations, there is little information for Asian populations. Here, we conducted a genome-wide association study and a replication study consisting of 2,760 Japanese periodontitis patients and 15,158 Japanese controls. Although single-nucleotide polymorphisms that surpassed a stringent genome-wide significance threshold (P < 5 × 10(-8)) were not identified, we found 2 suggestive loci for periodontitis: KCNQ5 on chromosome 6q13 (rs9446777, P = 4.83 × 10(-6), odds ratio = 0.82) and GPR141-NME8 at chromosome 7p14.1 (rs2392510, P = 4.17 × 10(-6), odds ratio = 0.87). A stratified analysis indicated that the GPR141-NME8 locus had a strong genetic effect on the susceptibility to generalized periodontitis in Japanese individuals with a history of smoking. In conclusion, this study identified 2 suggestive loci for periodontitis in a Japanese population. This study should contribute to a further understanding of genetic factors for enhanced susceptibility to periodontitis.
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Periodontitis/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Mapeo Cromosómico , Cromosomas Humanos Par 16/genética , Cromosomas Humanos Par 7/genética , Femenino , Interacción Gen-Ambiente , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Intrones/genética , Japón , Canales de Potasio KCNQ/genética , Desequilibrio de Ligamiento/genética , Masculino , Persona de Mediana Edad , Periodontitis/clasificación , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Receptores Acoplados a Proteínas G/genética , Fumar , Tiorredoxinas/genética , Adulto JovenRESUMEN
BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.
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Pérdida de Hueso Alveolar/prevención & control , Infecciones por Bacteroidaceae/microbiología , Canavalia , Fitoterapia/métodos , Extractos Vegetales/uso terapéutico , Porphyromonas gingivalis/efectos de los fármacos , Adhesinas Bacterianas/efectos de los fármacos , Pérdida de Hueso Alveolar/microbiología , Animales , Canavalia/química , Canavanina/análisis , Canavanina/farmacología , Canavanina/toxicidad , Clorhexidina/análogos & derivados , Clorhexidina/farmacología , Clorhexidina/toxicidad , Cromatografía Líquida de Alta Presión , Cistatinas/farmacología , Cistatinas/toxicidad , Cisteína Endopeptidasas/efectos de los fármacos , Progresión de la Enfermedad , Células Epiteliales/efectos de los fármacos , Cisteína-Endopeptidasas Gingipaínas , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Células KB , Leupeptinas/farmacología , Leupeptinas/toxicidad , Masculino , Pruebas de Sensibilidad Microbiana , Mucosa Bucal/citología , Mucosa Bucal/efectos de los fármacos , Extractos Vegetales/análisis , Ratas , Ratas Wistar , Organismos Libres de Patógenos EspecíficosRESUMEN
As ~75% of the Werner syndrome (WS) patients recognized between 1904 and 2008 all over the world are of Japanese origin, the most case reports and clinical studies on WS has been published in Japanese journals. Thus, the detailed English-written clinical review on the recent WS case reports has been warranted. Although WS has been characterized by a variety of clinical manifestations mimicking premature aging, the recent longevity and delayed age-associated manifestations observed both from Japanese WS and general population may suggest a common environmental effect on some gene(s) other than WRN and may give us a newer pathophysiological look at WS and also natural aging through the molecular dysfunction of WRN.
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Envejecimiento Prematuro/fisiopatología , Exodesoxirribonucleasas/genética , RecQ Helicasas/genética , Síndrome de Werner/epidemiología , Adulto , Envejecimiento Prematuro/genética , Femenino , Humanos , Japón/epidemiología , Masculino , Mutación , Síndrome de Werner/genética , Síndrome de Werner/patología , Helicasa del Síndrome de WernerRESUMEN
The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p < 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.
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Factor 2 de Crecimiento de Fibroblastos/uso terapéutico , Regeneración Tisular Guiada Periodontal/métodos , Periodontitis/cirugía , Adulto , Pérdida de Hueso Alveolar/diagnóstico por imagen , Pérdida de Hueso Alveolar/cirugía , Proceso Alveolar/efectos de los fármacos , Índice de Placa Dental , Método Doble Ciego , Femenino , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Estudios de Seguimiento , Encía/patología , Hemorragia Gingival/clasificación , Recesión Gingival/clasificación , Humanos , Masculino , Persona de Mediana Edad , Pérdida de la Inserción Periodontal/clasificación , Índice Periodontal , Ligamento Periodontal/efectos de los fármacos , Bolsa Periodontal/clasificación , Placebos , Radiografía , Proteínas Recombinantes , Colgajos Quirúrgicos , Movilidad Dentaria/clasificación , Resultado del TratamientoRESUMEN
AIM: Carnitine plays an essential role in fat oxidation in skeletal muscles; therefore carnitine influx could be crucial for muscle metabolism. OCTN2, a sodium-dependent solute carrier, is assumed to transport carnitine into various organs. However, OCTN2 protein expression and the functional importance of carnitine transport for muscle metabolism have not been studied. We tested the hypothesis that OCTN2 is expressed at higher levels in oxidative muscles than in other muscles, and that the carnitine uptake capacity of skeletal muscles depends on the amount of OCTN2. METHODS: Rat hindlimb muscles (soleus, plantaris, and the surface and deep portions of gastrocnemius) were used for Western blotting to detect OCTN2. Tissue carnitine uptake was examined by an integration plot analysis using l-[(3)H]carnitine as a tracer. Tissue carnitine content was determined by enzymatic cycling methods. The percentage of type I fibres was determined by histochemical analysis. RESULTS: OCTN2 was detected in all skeletal muscles although the amount was lower than that in the kidney. OCTN2 expression was significantly higher in soleus than in the other skeletal muscles. The amount of OCTN2 was positively correlated with the percentage of type I fibres in hindlimb muscles. The integration plot analysis revealed a positive correlation between the uptake clearance of l-[(3)H]carnitine and the amount of OCTN2 in skeletal muscles. However, the carnitine content in soleus was lower than that in other skeletal muscles. CONCLUSION: OCTN2 is functionally expressed in skeletal muscles and is involved in the import of carnitine for fatty acid oxidation, especially in highly oxidative muscles.
Asunto(s)
Carnitina/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Animales , Transporte Biológico , Western Blotting , Ácidos Grasos/metabolismo , Miembro Posterior , Masculino , Fibras Musculares de Contracción Lenta/metabolismo , Oxidación-Reducción , Ratas , Ratas Wistar , Miembro 5 de la Familia 22 de Transportadores de Solutos , TritioRESUMEN
The present study investigated the effects of intensive interval training during 20-day of unloading on local muscle oxygenation kinetics evaluated by near infrared spectroscopy technique (NIRS). Eleven adult men completed 20-day unloading and were divided into two groups; the control (CON) group and training (TR) group. The TR group engaged in exercise training sessions that consisted of one-legged submaximal cycle exercise using the unloaded leg at 60 approximately 80% of VO(2peak) with intermittent rest periods, 25 min/day every other day. All subjects performed isometric knee extension exercise at 50% of their maximum voluntary contraction force before and after unloading. NIRS Delta[deoxy-Hb/Mb] signal was recorded from m. vastus lateralis and was fitted to an exponential equation in order to determine the kinetics parameters. The time constant (tau) of the % Delta[deoxy-Hb/Mb] was unchanged in the TR group, while it significantly increased in the CON group after unloading (pre, 5.0+/-1.0; post, 7.4+/-1.0 s). It is concluded that 20-day unloading increased the tau, suggesting deterioration of capacity for oxidative phosphorylation and oxygen utilization in a skeletal muscle. Additionally, the preservation of tau in the TR group suggested that intensive interval training could have an impact on the maintenance of muscle oxidative metabolism during unloading.
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Ciclismo/fisiología , Músculo Esquelético/metabolismo , Consumo de Oxígeno/fisiología , Resistencia Física/fisiología , Adulto , Prueba de Esfuerzo , Femenino , Frecuencia Cardíaca , Humanos , Contracción Isométrica , Cinética , Masculino , Músculo Esquelético/fisiología , Fosforilación Oxidativa , Espectroscopía Infrarroja Corta , Muslo/fisiologíaRESUMEN
BACKGROUND AND OBJECTIVE: A blood supply is indispensable for the regeneration of damaged or lost periodontal ligament (PDL) tissue. Mesenchymal stem cell-like activity of cells derived from the PDL has been identified by their capacity to form fibrous and osseous tissue and cementum. However, it remains to be clarified whether the cells have an ability to build the capillary network of blood vessels. This study evaluated the potential of cells derived from the PDL to construct a blood vessel-like structure and examined how growth factors controlled the multipotency of the cells. MATERIAL AND METHODS: The ability of a swine PDL fibroblast cell line, TesPDL3, to construct a blood vessel-like structure was evaluated on and in the self-assembling peptide scaffold, PuraMatrix(TM). In addition, the ability of the cells to form mineralized nodules was evaluated on type I collagen-coated plastic plates. In some cases, fibroblast growth factor (FGF)-2 and bone morphogenetic protein (BMP)-2 were added to these cultures. The status of the expression of vascular and osteoblastic cell-specific markers in the cells was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence analyses. RESULTS: The TesPDL3 cells not only formed mineralized nodules in response to BMP-2 stimulation but also constructed tube-like structures in response to FGF-2 stimulation. Intriguingly, FGF-2 inhibited the BMP-2-induced formation of mineralized nodules. Conversely, BMP-2 inhibited the FGF-2-induced formation of tube-like structures. CONCLUSION: Periodontal ligament fibroblasts have the potential to differentiate not only into osteoblastic but also into vascular cell lineages. The destiny of the cells was reciprocally regulated by BMP-2 and FGF-2.
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Proteína Morfogenética Ósea 2/farmacología , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Multipotentes/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Ligamento Periodontal/citología , Animales , Proteína Morfogenética Ósea 2/antagonistas & inhibidores , Calcificación Fisiológica/fisiología , Técnicas de Cultivo de Célula , Línea Celular , Células Clonales/citología , Células Clonales/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Proteína smad7/fisiología , Sus scrofa , Andamios del TejidoRESUMEN
An efficient system for Agrobacterium-mediated transformation of Lilium x formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing beta-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 microM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.
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Lilium/genética , Plantas Modificadas Genéticamente/fisiología , Rhizobium/metabolismo , Medios de Cultivo , Vectores Genéticos , Concentración de Iones de Hidrógeno , Lilium/crecimiento & desarrollo , Lilium/microbiología , Meristema/crecimiento & desarrollo , Meristema/microbiología , Meristema/fisiología , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/microbiologíaRESUMEN
Tacrolimus (FK506) has a neuroprotective action on cerebral infarction produced by cerebral ischemia, however, detailed mechanisms underlying this action have not been fully elucidated. We examined temporal profiles of survival-and death-related signals, Bad phosphorylation, release of cytochrome c (cyt.c), activation of caspase 3 and DNA fragmentation in the brain during and after middle cerebral artery occlusion (MCAo) in mice, and then examined the effect of tacrolimus on these signals. C57BL/6J mice were subjected to transient MCAo by intraluminal suture insertion for 60 min. Tacrolimus (1 mg/kg, i.p.) was administered immediately after MCAo. There were biphasic increases in the release of cyt.c in the ischemic core and penumbra; with the first increase toward the end of the occlusion period and the second increase 3-12 h after reperfusion. Tacrolimus significantly inhibited the increase of cytosolic cyt.c during ischemia and reperfusion. Phosphorylated Bad, Ser-136 (P-Bad(136)) and Ser-155 (P-Bad(155)) were detected 30 min after MCAo and after reperfusion in the ischemic cortex, respectively. Tacrolimus increased P-Bad(136) during ischemia and prolonged P-Bad(155) expression after reperfusion. Tacrolimus also decreased caspase-3 and terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling-positive cells, and reduced the size of infarct 24 h after reperfusion. Our study provided the first evidence that the neuroprotective action of tacrolimus involved inhibition of biphasic cyt.c release from mitochondria, possibly via up-regulation of Bad phosphorylation at different sites after focal cerebral ischemia and reperfusion.
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Citocromos c/metabolismo , Inmunosupresores/farmacología , Ataque Isquémico Transitorio/metabolismo , Tacrolimus/farmacología , Proteína Letal Asociada a bcl/metabolismo , Análisis de Varianza , Animales , Western Blotting/métodos , Infarto Encefálico/tratamiento farmacológico , Infarto Encefálico/etiología , Infarto Encefálico/metabolismo , Infarto Encefálico/patología , Modelos Animales de Enfermedad , Inmunohistoquímica/métodos , Inmunosupresores/uso terapéutico , Etiquetado Corte-Fin in Situ , Ataque Isquémico Transitorio/complicaciones , Ataque Isquémico Transitorio/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Tacrolimus/uso terapéutico , Factores de TiempoRESUMEN
OBJECTIVES: The possible contribution of alpha1-protease inhibitor (alpha1-PI) and secretory leukocyte protease inhibitor (SLPI) in gingival crevicular fluid (GCF) to predict the periodontal disease activity was evaluated. DESIGN: GCF samples were collected at each site before scaling and root planning (SRP), 2 and 4 weeks after SRP. SUBJECTS AND METHODS: Forty-one sites that initially bled on probing in 21 patients with moderate to severe periodontitis were studied. Sites were classified according to the presence or absence of bleeding on probing (BOP) at 4 weeks. In GCF alpha1-PI and SLPI were determined by enzyme-linked immunosorbent assays. RESULTS: A significant decrease was observed in alpha1-PI at 2 and 4 weeks in BOP(-) sites and at 4 weeks in BOP(+) sites. SLPI significantly increased at 2 weeks in BOP(+) site, while SLPI did not significantly differ at both time points in BOP(-) sites and at 4 weeks in BOP(+) sites. GCF alpha1-PI was significantly less at 2 weeks in BOP(-) than in BOP(+) sites. CONCLUSION: At 2 weeks GCF alpha1-PI may reflect the healing response of the periodontal tissues following nonsurgical periodontal treatment. GCF SLPI levels may be influenced by healing.
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Líquido del Surco Gingival/enzimología , Periodontitis/enzimología , Proteínas/análisis , Proteínas y Péptidos Salivales/análisis , Inhibidores de Serina Proteinasa/análisis , alfa 1-Antitripsina/análisis , Adulto , Anciano , Raspado Dental , Femenino , Estudios de Seguimiento , Hemorragia Gingival/enzimología , Humanos , Masculino , Análisis por Apareamiento , Persona de Mediana Edad , Índice Periodontal , Periodontitis/terapia , Proteínas Inhibidoras de Proteinasas Secretoras , Aplanamiento de la Raíz , Inhibidor Secretorio de Peptidasas Leucocitarias , Cicatrización de HeridasRESUMEN
Werner syndrome (WS) is caused by mutations in the gene encoding RecQ type DNA helicase (WRN). We report a 53-year-old Japanese male with WS who initially presented with skin ulcers on the feet and the left elbow. The patient had a high-pitched voice, hoarseness, a characteristic bird-like facial appearance with a beak-shaped nose, canities and juvenile cataracts. Immunoblot analysis using a monoclonal antibody directed against the WS gene product DNA helicase revealed that the patient's leucocytes lacked this particular molecule, confirming the diagnosis of WS. This new immunoblot system therefore enables the diagnosis of WS to be made without the need to undertake more complex mutational analysis.
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ADN Helicasas/metabolismo , Immunoblotting/métodos , Úlcera Cutánea/etiología , Síndrome de Werner/diagnóstico , Exodesoxirribonucleasas , Femenino , Humanos , Persona de Mediana Edad , Mutación/genética , RecQ Helicasas , Helicasa del Síndrome de WernerRESUMEN
The protein B23 is a major nucleolar phosphoprotein comprising two isoforms, B23.1 and B23.2, which differ only in their carboxyl-terminal short sequences, the N-terminal 255 residues being identical in both forms. Both B23.1 and B23.2 stimulated immunoaffinity-purified calf thymus DNA polymerase alpha in a dose-dependent manner. The stimulatory effect of protein B23.1, the longer isoform, was found to be 2-fold greater than that of B23.2. Purified DNA polymerase alpha bound tightly to a protein B23.1-immobilized column, while it bound weakly to a protein B23.2-immobilized column. Surface plasmon resonance studies by BIAcore further showed that protein B23.1 bound to the DNA polymerase alpha-(dA).(dT) complex more tightly than did protein B23.2. The protein B23 isoforms appear to interact directly with the DNA polymerase alpha protein and not through the bound nucleic acid. These observations indicated that protein B23 physically bound to the DNA polymerase alpha and stimulated the enzyme activity. Product analyses showed that protein B23 greatly enhanced the reaction both in amount and length of product DNA, whereas it did not significantly alter the processivity of polymerization. In contrast, protein B23 effectively protected DNA polymerase alpha from heat inactivation. These results suggest that protein B23 stabilizes DNA polymerase alpha that is detached from product DNA, allowing the enzyme to be recruited for further elongation. Moreover, experiments using various C-terminal deletion mutants of protein B23 indicated that 12 amino acids at the C-terminal end of B23.1, which are absent in B23.2, may be essential for the full stimulation of the DNA polymerase alpha.
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ADN Polimerasa I/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Estabilidad de Enzimas , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Nucleofosmina , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Human B-lymphoblastoid cell lines transformed by Epstein-Barr (EBV-LCLs) are considered to be immortalized, although most of them show a normal diploid karyotype. Recently, we and others have shown that only part of EBV-LCLs is immortalized by developing strong telomerase activity that stabilizes the telomeres. In this study, we investigated the change in karyotypes during immortalization. All the eight immortalized cell lines developed clonal chromosomal aberrations accompanied by the development of strong telomerase activity. Interestingly, abnormal chromosomes were not shared among the immortalized cell lines. These results strongly suggest that chromosomal rearrangements and induction of strong telomerase activity are two events that take place in parallel in the process of immortalization of EBV-LCLs, and indicate that EBV-LCLs are clearly divided into two distinct groups, pre-immortal cell lines mostly with a normal diploid karyotype and post-immortal cell lines with a clonally abnormal karyotype.
Asunto(s)
Linfocitos B/ultraestructura , Transformación Celular Viral , Aberraciones Cromosómicas , Herpesvirus Humano 4/genética , Telomerasa/metabolismo , Linfocitos B/enzimología , Línea Celular , Humanos , Cariotipificación , TelómeroAsunto(s)
Antígenos Nucleares , ADN Helicasas/fisiología , Reparación del ADN , Síndrome de Werner/genética , Animales , ADN Helicasas/genética , ADN Polimerasa III/fisiología , Replicación del ADN , Proteínas de Unión al ADN/fisiología , Exodesoxirribonucleasas , Genoma , Humanos , Autoantígeno Ku , Proteínas Nucleares/fisiología , RecQ Helicasas , Recombinación Genética , Proteína de Replicación A , Telómero , Proteína p53 Supresora de Tumor/fisiología , Helicasa del Síndrome de WernerRESUMEN
BACKGROUND: Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S-phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family. Although biochemical studies showed that the gene product of WRN (WRNp) interacts with proteins that participate in DNA metabolism, its precise biological function remains unclear. RESULTS: Using immunocytochemistry, we found that WRNp forms distinct nuclear foci in response to DNA damaging agents, including camptothecin (CPT), etoposide, 4-nitroquinolin-N-oxide and bleomycin. The presence of aphidicolin inhibited CPT-induced WRNp foci strongly but not bleomycin-induced foci. These WRNp foci overlapped with the foci of replication protein A (RPA) almost entirely and with the foci of Rad51 partially, implicating cooperative functions of these proteins in response to DNA damage. We also found that WRNp foci partially co-localize with sites of 5-bromo-2'-deoxy-uridine incorporation. CONCLUSIONS: These findings suggest that WRNp form nuclear foci in response to aberrant DNA structures, including DNA double-strand breaks and stalled replication forks. We propose that WRNp takes part in the homologous recombinational repair and in the processing of stalled replication forks.
Asunto(s)
ADN Helicasas/efectos de los fármacos , ADN Helicasas/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Mutágenos/farmacología , Proteínas/ultraestructura , Transactivadores , Síndrome de Werner/metabolismo , 4-Nitroquinolina-1-Óxido/farmacología , Afidicolina/metabolismo , Bleomicina/farmacología , Camptotecina/farmacología , Línea Celular , Núcleo Celular/ultraestructura , Daño del ADN/fisiología , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Etopósido/farmacología , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación Fluorescente in Situ , Recombinasa Rad51 , Síndrome de Werner/genética , Síndrome de Werner/patologíaRESUMEN
Werner syndrome (WS) is a rare autosomal recessive genetic disorder causing premature aging and rare cancers. A gene responsible for WS (WRN) encodes a protein with 1432 amino acids (a.a.) homologous to the E. coli RecQ-type DNA helicase. Transcriptional activation facilitated nucleolar localization of human WRN protein (hWRNp) and serum starvation induced translocation of hWRNp from the nucleoli to the nucleoplasm in human cultured cells, suggesting a nucleolar-nucleoplasm trafficking of hWRNp depending on transcriptional state. Mutant hWRNp lacking the C-terminal 30 a.a. residues (Delta1403-1432) failed to localize in the nucleolus, whereas Delta1405-1432 can migrate into the nucleolus. Here we identify a region putative for nucleolar localization signal (NoLS) containing a sequence of two positively charged amino acids (Arg(1403)-Lys(1404)) in the C-terminal area of hWRNp. By contrast, the mouse homolog (mWRNp) exists only in the nucleoplasm. We show that the inability of mWRNp to migrate into the nucleolus is due to a difference of a sequence in the region corresponding to the NoLS of hWRNp. In addition, mouse cells cannot recognize the NoLS of hWRNp. Our study suggests that defect in nucleolar function of hWRNp may be linked to the premature aging which is not observed in mWRN(-/-) mice.
Asunto(s)
Nucléolo Celular/enzimología , ADN Helicasas/metabolismo , Señales de Localización Nuclear/metabolismo , Secuencia de Aminoácidos , Animales , Núcleo Celular/enzimología , Células Cultivadas , ADN Helicasas/genética , Exodesoxirribonucleasas , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Células HeLa , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/ultraestructura , Ratones , Datos de Secuencia Molecular , Señales de Localización Nuclear/genética , RecQ Helicasas , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Helicasa del Síndrome de WernerRESUMEN
BACKGROUND, AIMS: The present study was undertaken to assess the periodontal status of a rural Japanese population and to study the correlation between the periodontal status and the serum antibody titers for Porphyromonas gingivalis (Pg) fimbriae. METHOD: A total of 236 individuals were examined for their periodontal conditions by the use of the community periodontal index for treatment needs (CPITN), and serum antibody titers for Pg fimbriae in their peripheral blood samples were evaluated using the enzyme-linked immunosorbent assay. RESULTS: There was a substantially larger proportion of edentulous subjects in the age group older than 60 years. The remaining teeth were 24.1, 23.2, 11.1 and 10.1 per person in the 40-49, 50-59, 60-69 and > or = 70 age groups, respectively. The % of sextants with a CPITN code of missing sextant (MS) increased towards elderly and reached >60% in the age group of > or = 70 years, as the % of the CPITN 2, 1 or 0 sextant decreased. The % of CPITN 4 and 3 sextants did not differ between different age groups and were about 6-8% and 15-20%, respectively. The % of CPITN 1 or 0 sextants was higher in female subjects than in male subjects in the 60-69 and > or = 70 age groups, while the % of CPITN 4 or 3 sextants was higher in male subjects than in female subjects in all age groups. There was no significant difference between various age groups in the mean serum antibody titers for Pg fimbriae. The mean anti-Pg fimbriae antibody titers was significantly higher for the subjects with a maximum CPITN code 4 (max.-CPITN 4 subject) than for the subjects with lower maximum CPITN codes. The antibody titers varied extensively among the max.-CPITN 4 or 3 subjects, but not among the max.-CPITN 2/1/0 or MS subjects. CONCLUSIONS: The present study demonstrated that tooth loss is a remarkable event in elderly subjects and that oral prophylaxis and mechanical debridement should be mandatory in the population examined. It was also demonstrated that the serum antibody titers against Pg fimbriae could be useful for screening individuals with moderate to severe periodontitis.