RESUMO
BACKGROUND: The basidiomycete Phanerochaete carnosa is a white-rot species that has been mainly isolated from coniferous softwood. Given the particular recalcitrance of softwoods to bioconversion, we conducted a comparative transcriptomic analysis of P. carnosa following growth on wood powder from one softwood (spruce; Picea glauca) and one hardwood (aspen; Populus tremuloides). P. carnosa was grown on each substrate for over one month, and mycelia were harvested at five time points for total RNA sequencing. Residual wood powder was also analyzed for total sugar and lignin composition. RESULTS: Following a slightly longer lag phase of growth on spruce, radial expansion of the P. carnosa colony was similar on spruce and aspen. Consistent with this observation, the pattern of gene expression by P. carnosa on each substrate converged following the initial adaptation. On both substrates, highest transcript abundances were attributed to genes predicted to encode manganese peroxidases (MnP), along with auxiliary activities from carbohydrate-active enzyme (CAZy) families AA3 and AA5. In addition, a lytic polysaccharide monooxygenase from family AA9 was steadily expressed throughout growth on both substrates. P450 sequences from clans CPY52 and CYP64 accounted for 50% or more of the most highly expressed P450s, which were also the P450 clans that were expanded in the P. carnosa genome relative to other white-rot fungi. CONCLUSIONS: The inclusion of five growth points and two wood substrates was important to revealing differences in the expression profiles of specific sequences within large glycoside hydrolase families (e.g., GH5 and GH16), and permitted co-expression analyses that identified new targets for study, including non-catalytic proteins and proteins with unknown function.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Phanerochaete/genética , Picea/microbiologia , Populus/microbiologia , Transcriptoma , Madeira/microbiologia , Perfilação da Expressão Gênica , Phanerochaete/fisiologiaRESUMO
BACKGROUND AND OBJECTIVE: Single nucleotide polymorphisms (SNPs) of paraoxonase 1 (PON1) are known to be associated with the pathogenesis of osteoporosis and periodontitis. However, the effects of PON1 on the osteoblastic differentiation of periodontal ligament (PDL) cells are unclear. In this study, we examined the effects of PON1 on the osteoblastic differentiation of PDL cells, and analysed the role of PON1 SNPs on the pathogenesis of aggressive periodontitis (AgP) in the Japanese population. MATERIAL AND METHODS: Human PDL (HPDL) cells were exposed to the PON1 plasmid and PON1 inhibitor, 2-hydroxyquinoline, and cultured in mineralization medium. Expression of calcification-related genes and calcified nodule formation were assessed by real-time PCR, an alkaline phosphatase (ALPase) activity assay and Alizarin red staining. Sanger sequencing was performed to evaluate whether PON1 SNPs are associated with the pathogenesis of AgP in Japanese people. RESULTS: During osteoblastic differentiation of HPDL cells, expression of PON1 mRNA increased in a time-dependent manner. PON1 stimulated an increase in expression of mRNA for calcification-related genes, as well as ALPase activity. In contrast, 2-hydroxyquinoline clearly inhibited the expression of calcification-related genes, ALPase activity and calcified nodule formation in HPDL cells. Moreover, there was a statistically significant difference in the minor allele frequency of PON1 SNP rs854560 between the Japanese control database and patients with AgP in the Japanese population (P = .0190). CONCLUSION: PON1 induced cytodifferentiation and mineralization of HPDL cells, and PON1 SNP rs854560 may be associated with the pathogenesis of AgP in the Japanese population.
Assuntos
Periodontite Agressiva/patologia , Arildialquilfosfatase/metabolismo , Arildialquilfosfatase/farmacologia , Diferenciação Celular/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/patologia , Adulto , Periodontite Agressiva/enzimologia , Fosfatase Alcalina/análise , Arildialquilfosfatase/genética , Reabsorção Óssea , Calcificação Fisiológica , Células Cultivadas , Feminino , Expressão Gênica , Frequência do Gene , Humanos , Hidroxiquinolinas/antagonistas & inibidores , Japão , Masculino , Osteoblastos/efeitos dos fármacos , Bolsa Periodontal , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Previous reports suggest that several serum biomarkers play roles in the pathogenesis, inflammatory response, and oxidative stress in periodontitis caused by bacterial infections, linking chronic periodontitis to atherosclerotic vascular disease (ASVD). The aim of this preliminary study was to investigate, in a Japanese cross-sectional community survey, potential serum biomarkers of periodontitis that are associated with ASVD and chronic periodontitis. MATERIAL AND METHODS: The study cohort included a total of 108 male subjects who underwent annual health examinations. Serum biomarkers (high-sensitivity C-reactive protein [hs-CRP], proprotein convertase subtilisin/kexin type 9 [PCSK9], interleukin-6, tumor necrosis factor-α, soluble CD14, myeloperoxidase, matrix metalloproteinase-3, adiponectin, total bilirubin [TBIL], and serum lipids) were analyzed to determine their association (if any) with periodontal parameters. Aortic stiffness was evaluated using the brachial-ankle aortic pulse wave velocity (PWV) index and the cardio-ankle vascular index (CAVI). RESULTS: The concentrations of PCSK9 and hs-CRP were increased (P = .001 and .042, respectively), and the concentration of TBIL was decreased (P = .046), in subjects with periodontal disease (determined as a probing depth of ≥4 mm in at least one site) compared with periodontally healthy subjects. The ratio of low-density lipoprotein cholesterol (LDL-C) to high-density lipoprotein cholesterol and the concentrations of triglycerides, remnant-like particles-cholesterol, and oxidized LDL were elevated in subjects with periodontal disease compared with periodontally healthy subjects (P = .038, .007, .002, and .049, respectively). Multivariate regression analyses indicated that the number of sites with a pocket depth of ≥4 mm was associated with the concentration of PCSK9 and inversely associated with the concentration of TBIL independently (standardized ß = .243, P = .040; standardized ß = -.443, P = .0002; respectively). Analysis of receiver operating characteristic curves of PCSK9 indicated moderate accuracy for predicting the presence of disease sites (probing depth ≥ 4 mm) (area under the curve = 0.740). No significance in the values of PWV and CAVI was observed between subjects with periodontal disease and periodontally healthy subjects. CONCLUSION: In Japanese male subjects, the concentrations of serum PCSK9 and TBIL were correlated with periodontal parameters. Moreover, PCSK9 could be a candidate biomarker for diagnosing chronic periodontitis, and may also have potential to evaluate the risk for periodontitis to cause ASVD. Longitudinal studies of larger populations are necessary to confirm the exact association of periodontitis with increased serum PCSK9 and decreased TBIL.
Assuntos
Bilirrubina/sangue , Periodontite Crônica/sangue , Pró-Proteína Convertase 9/sangue , Adiponectina/sangue , Adulto , Povo Asiático , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Periodontite Crônica/diagnóstico , Periodontite Crônica/enzimologia , Estudos de Coortes , Estudos Transversais , Humanos , Interleucina-6/sangue , Japão , Receptores de Lipopolissacarídeos/sangue , Lipoproteínas/sangue , Estudos Longitudinais , Masculino , Metaloproteinase 3 da Matriz/sangue , Pessoa de Meia-Idade , Triglicerídeos/sangue , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND AND OBJECTIVE: Full-mouth scaling and root planing (FM-SRP) acts as a potent inflammatory stimulus immediately after treatment; however, systemic inflammation typically improves in the long term. The contribution of FM-SRP to systemic biological and acute-phase responses is largely unknown. The purpose of this prospective intervention study was to assess the systemic and local biological responses after FM-SRP. MATERIAL AND METHODS: Thirty-one patients with generalized moderate-to-severe chronic periodontitis received 1-stage FM-SRP. Measurement of clinical parameters and body temperature as well as collection of subgingival plaque, peripheral blood and gingival crevicular fluid was performed before and after treatment 2 or 3 times. Quantification of periodontopathic bacteria in the sulcus and measurement of corresponding serum IgG titers were performed. Systemic and local inflammatory markers such as endotoxin, high-sensitive C-reactive protein (hs-CRP) and 6 inflammatory cytokines were assessed using high-sensitivity assays. RESULTS: Compared to baseline values, FM-SRP resulted in a substantial improvement in clinical parameters (P < .05), lower bacterial counts (P < .01) and a significant decrease of IgG titers against Porphyromonas gingivalis (P < .001) 6 weeks after treatment. Comparing baseline parameters to those at 1 day post-treatment, there was a statistically significant elevation in body temperature (P = .007). In addition, a 5-fold increase in hs-CRP (P < .001), a remarkable increase in interferon-γ (P < .001) and a slight increase in interleukin (IL)-12p70 (P = .001) were detected in serum samples. In the gingival crevicular fluid, marked increases in hs-CRP (P < .001), IL-5 (P = .001), IL-6, IL-12p70 and tumor necrosis factor-α (P < .001 for the latter 3 markers) were noted 1 day after treatment. Endotoxin levels were below measurable limits for most time points. CONCLUSION: FM-SRP resulted in clinical and microbiological improvement 6 weeks post-treatment, but produced a moderate systemic acute-phase response including elevated inflammatory mediators 1 day post-treatment.
Assuntos
Periodontite Crônica/terapia , Raspagem Dentária , Mediadores da Inflamação/metabolismo , Aplainamento Radicular , Periodontite Crônica/microbiologia , Endotoxinas/sangue , Feminino , Seguimentos , Líquido do Sulco Gengival/química , Humanos , Imunoglobulina G/metabolismo , Japão , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do TratamentoRESUMO
BACKGROUND AND OBJECTIVE: The proteasome inhibitor, bortezomib, is known to induce osteoblastic differentiation in a number of cell lines, such as mesenchymal stem cells and osteoblastic precursor cells. As periodontal ligament (PDL) cells are multipotent, we examined whether bortezomib may induce the differentiation of PDL cells into hard-tissue-forming cells. MATERIAL AND METHODS: A mouse PDL clone cell line, MPDL22 cells, was cultured in mineralization medium in the presence or absence of bortezomib. Expression of calcification-related genes and calcified-nodule formation were evaluated by real-time PCR and Alizarin Red staining, respectively. RESULTS: Bortezomib increased the expression of calcification-related mRNAs, such as tissue nonspecific alkaline phosphatase isoenzyme (ALPase), bone sialoprotein (Bsp), runt-related transcription factor 2 (Runx2) and osteopontin, and calcified-nodule formation in MPDL22 cells. These effects were induced, in part, by increasing the cytosolic accumulation and nuclear translocation of ß-catenin, leading to an increase in expression of bone morphogenetic protein (Bmp)-2, -4 and -6 mRNAs. In addition, bortezomib enhanced BMP-2-induced expression of Bsp and osteopontin mRNAs and increased calcified-nodule formation in MPDL22 cells. CONCLUSION: Bortezomib induced cytodifferentiation and mineralization of PDL cells by enhancing the accumulation of ß-catenin within the cytosol and the nucleus and increasing the expression of Bmp-2, -4 and -6 mRNAs. Moreover, bortezomib enhanced the BMP-2-induced cytodifferentiation and mineralization of PDL cells, suggesting that bortezomib may be efficacious for use in periodontal regeneration therapy.
Assuntos
Bortezomib/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Fosfatase Alcalina/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 4/efeitos dos fármacos , Proteína Morfogenética Óssea 6/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Citosol/efeitos dos fármacos , Isoenzimas/efeitos dos fármacos , Camundongos , Osteopontina/efeitos dos fármacos , Ligamento Periodontal/citologia , beta Catenina/efeitos dos fármacosRESUMO
AIM: By describing the practice of a Japanese nurse practitioner, this descriptive case study discusses role development and outcomes before and after the intervention. BACKGROUND: One of the first Japanese nurse practitioners intervened at a nursing home during the government-designated trial period for nurse practitioner practice. CONCLUSION: Because of the nurse practitioner's meticulous observation and timely care provision to the residents in collaboration with the physician and the other staff in the facility, comparative data showed improvement in daily health status management of every resident and decreased deterioration of residents' health conditions requiring ambulance transfer and hospitalization.
Assuntos
Enfermagem Geriátrica , Profissionais de Enfermagem , Papel do Profissional de Enfermagem , Casas de Saúde , Avaliação de Resultados em Cuidados de Saúde , Idoso de 80 Anos ou mais , Feminino , Humanos , Japão , MasculinoRESUMO
BACKGROUND AND OBJECTIVE: T and B cells are known to be involved in the disease process of periodontitis. However, the role of natural killer T cells in the pathogenesis of periodontitis has not been clarified. MATERIALS AND METHODS: To examine the role of these cells, C57BL/6J (wild-type), CD1d(-/-) and α-galactosylceramide (αGC)-stimulated wild-type mice were orally infected with Porphyromonas gingivalis strain W83. RESULTS: Apart from CD1d(-/-) mice, the level of alveolar bone resorption was elevated by the infection and was further accelerated in αGC-stimulated mice. The infection induced elevated levels of serum amyloid A and P. gingivalis-specific IgG in the sera, although the degree of elevation was much smaller in the CD1d(-/-) mice. Infection-induced RANKL elevation was only observed in αGC-stimulated mice. Although the cytokines produced by splenocytes were mainly T-helper 1 type in wild-type mice, those in αGC-stimulated mice were predominantly T-helper 2 type. In the liver, the infection demonstrated no effect on the gene expression for interferon-γ, interleukin-4 and RANKL except αGC-stimulated mice in which the infection upregulated the gene expressions. CONCLUSION: This study is the first to show that natural killer T cells upregulated systemic and local inflammatory responses induced by oral infection with P. gingivalis, thereby contributing to the progression of alveolar bone resorption.
Assuntos
Perda do Osso Alveolar/imunologia , Infecções por Bacteroidaceae/imunologia , Células Matadoras Naturais/imunologia , Periodontite/microbiologia , Porphyromonas gingivalis/imunologia , Perda do Osso Alveolar/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos CD1d/imunologia , Galactosilceramidas/farmacologia , Imunoglobulina G/sangue , Inflamação/imunologia , Interferon gama/análise , Interleucina-4/análise , Células Matadoras Naturais/microbiologia , Fígado/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Periodontite/imunologia , Ligante RANK/análise , Ligante RANK/efeitos dos fármacos , Proteína Amiloide A Sérica/análise , Baço/imunologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
AIM: This paper describes the establishment of the first Japanese nurse practitioner graduate programme and legislative activities to institutionalize nurse practitioners in Japan. BACKGROUND: To address the super-ageing population, Oita University of Nursing and Health Sciences initiated the first academic graduate level nurse practitioner programme in Japan, based upon the global standard defined by the International Council of Nurses. CONCLUSION: In 2010, Oita University of Nursing and Health Sciences graduated the first nurse practitioner. We believe that nurse practitioners will be highly valued in Japan for thoughtful nursing care to the fragile elders living in rural and urban Japan.
Assuntos
Educação de Pós-Graduação em Enfermagem/organização & administração , Profissionais de Enfermagem/educação , Credenciamento/organização & administração , Humanos , JapãoRESUMO
BACKGROUND AND OBJECTIVE: Periodontal infection affects atherosclerotic diseases, such as coronary heart diseases. Mouse models have revealed that oral infection with Porphyromonas gingivalis induces changes in inflammatory- and lipid metabolism-related gene expression, regardless of the development of atherosclerotic lesions. However, the serum protein expression profile in the oral infection model has not been investigated. The present study aimed to analyse the effect of oral infection with P. gingivalis on the expression levels of multiple cytokines in the serum in apolipoprotein E-deficient mice by using a cytokine antibody array. MATERIAL AND METHODS: C57BL/6.KOR-Apoe(shl) mice were orally infected with P. gingivalis five times at 3 day intervals and were then killed. Splenocytes were isolated and analysed for proliferative activity and immunoglobulin G (IgG) production in response to in vitro restimulation with P. gingivalis. The expression levels of various cytokines in the sera were analysed using a mouse antibody array glass chip. RESULTS: Splenocytes from P. gingivalis-infected mice demonstrated significantly greater proliferation and IgG production in response to P. gingivalis compared with those from sham-infected mice. Antibody array analysis revealed the selective upregulation of matrix metalloproteinase 3, intercellular adhesion molecule 1, insulin-like growth factor binding protein 2 and chemokine (C-X-C motif) ligand 7 and the downregulation of interleukin-17, tumor necrosis factor-α and L-selectin. CONCLUSION: These data demonstrate that oral infection with P. gingivalis induces alterations in systemic cytokine production. These cytokines could play roles in the development not only of periodontitis but also of atherosclerosis.
Assuntos
Infecções por Bacteroidaceae/imunologia , Citocinas/sangue , Doenças da Boca/microbiologia , Porphyromonas gingivalis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Apolipoproteínas E/genética , Proliferação de Células , Quimiocinas CXC/sangue , Modelos Animais de Doenças , Imunoglobulina G/sangue , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Molécula 1 de Adesão Intercelular/sangue , Interleucina-17/sangue , Interleucina-6/sangue , Selectina L/sangue , Masculino , Metaloproteinase 3 da Matriz/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Doenças da Boca/imunologia , Baço/citologia , Baço/imunologia , Fator de Necrose Tumoral alfa/sangueRESUMO
OBJECTIVES: Following lesions in somatosensory pathways, deafferentation pain often occurs. Patients report that the pain is qualitatively complex, and its treatment can be difficult. Mirror visual feedback (MVF) treatment can improve deafferentation pain. We sought to classify the qualities of the pain in order to examine whether the potential analgesic effect of MVF depends on these qualities. METHODS: Twenty-two patients with phantom limb pain, or pain related to spinal cord or nerve injury, performed a single MVF procedure. Before and after the MVF procedure, we evaluated phantom limb awareness, movement representation of the phantom or affected/paralysed limb, pain intensity on an 11-point numerical rating scale (0-10) and the qualities of the pain [skin surface-mediated (superficial pain) vs deep tissue-mediated (deep pain)] using lists of pain descriptors for each of the two categories. RESULTS: Fifteen of the patients perceived the willed visuomotor imagery of the phantom or affected/paralysed limb after the MVF procedure. In most of the patients, a reduction in pain intensity and a decrease in the reporting of deep-pain descriptors were linked to the emergence of willed visuomotor imagery. CONCLUSIONS: In this pilot study, we roughly classified the pain descriptor items into two types for evaluating the qualities of deafferentation pain. We found that visually induced motor imagery by MVF was more effective for reducing deep pain than superficial pain. This suggests that the analgesic effect of MVF treatment does depend on the qualities of the pain. Further research will be required to confirm that this effect is a specific consequence of MVF.
Assuntos
Biorretroalimentação Psicológica/métodos , Causalgia/terapia , Adolescente , Adulto , Idoso , Causalgia/etiologia , Feminino , Humanos , Imagens, Psicoterapia/métodos , Masculino , Pessoa de Meia-Idade , Medição da Dor/métodos , Membro Fantasma/terapia , Projetos Piloto , Desempenho Psicomotor , Resultado do TratamentoRESUMO
In a cross-sectional study, visit-to-visit blood pressure (BP) variability was shown to be associated with artery remodelling. Here, we investigated the impact of visit-to-visit BP variability and average BP on the carotid artery remodelling progression in high-risk elderly according to different classes of antihypertension medication use/non-use. BP measurements and carotid ultrasound were performed in the common carotid artery in 164 subjects (mean age 79.7 years at baseline, 74.7% females) with one or more cardiovascular risk factors. Based on 12 visits (1 × /month for 1 year), we calculated visit-to-visit BP variability expressed as the standard deviation (s.d.), coefficient of variation (CV), maximum BP, minimum BP and delta (maximum-minimum) BP. We measured mean intima-media thickness (IMT) as well as stiffness parameter ß were measured at baseline and at the mean 4.2-year follow-up. In a multiple regression analysis, the maximum, minimum, s.d. and average of systolic BP (SBP) were significantly associated with a change in ß-values between the baseline and follow-up after adjustment for age, smoking, lower high-density lipoprotein level, baseline ß-value and follow-up period. There were no significant associations between the visit-to-visit BP variability measures and the change in mean IMT. Significant associations of maximum, minimum, s.d. and average SBP were found with increased ß-values in the subjects without calcium channel blocker (CCB) use and in the subjects using renin-angiotensin system inhibitors (RASIs). Thus, exaggerated visit-to-visit SBP variability and a high average SBP level were significant predictors of progression in carotid arterial stiffness in high-risk elderly without CCBs use and in those using a RASI.
Assuntos
Antagonistas de Receptores de Angiotensina/farmacologia , Pressão Sanguínea , Bloqueadores dos Canais de Cálcio/farmacologia , Artéria Carótida Primitiva/efeitos dos fármacos , Rigidez Vascular , Idoso , Idoso de 80 Anos ou mais , Antagonistas de Receptores de Angiotensina/uso terapêutico , Bloqueadores dos Canais de Cálcio/uso terapêutico , Artéria Carótida Primitiva/diagnóstico por imagem , Espessura Intima-Media Carotídea , Feminino , Humanos , Hipertensão/tratamento farmacológico , Masculino , Estudos ProspectivosRESUMO
Sphingomyelin phosphodiesterase 3 ( Smpd3), which encodes neutral sphingomyelinase 2 (nSMase2), is a key molecule for skeletal development as well as for the cytodifferentiation of odontoblasts and alveolar bone. However, the effects of nSMase2 on the cytodifferentiation of periodontal ligament (PDL) cells are still unclear. In this study, the authors analyzed the effects of Smpd3 on the cytodifferentiation of human PDL (HPDL) cells. The authors found that Smpd3 increases the mRNA expression of calcification-related genes, such as alkaline phosphatase (ALPase), type I collagen, osteopontin, Osterix (Osx), and runt-related transcription factor (Runx)-2 in HPDL cells. In contrast, GW4869, an inhibitor of nSMase2, clearly decreased the mRNA expression of ALPase, type I collagen, and osteocalcin in HPDL cells, suggesting that Smpd3 enhances HPDL cytodifferentiation. Next, the authors used exome sequencing to evaluate the genetic variants of Smpd3 in a Japanese population with aggressive periodontitis (AgP). Among 44 unrelated subjects, the authors identified a single nucleotide polymorphism (SNP), rs145616324, in Smpd3 as a putative genetic variant for AgP among Japanese people. Moreover, Smpd3 harboring this SNP did not increase the sphingomyelinase activity or mRNA expression of ALPase, type I collagen, osteopontin, Osx, or Runx2, suggesting that this SNP inhibits Smpd3 such that it has no effect on the cytodifferentiation of HPDL cells. These data suggest that Smpd3 plays a crucial role in maintaining the homeostasis of PDL tissue.
Assuntos
Periodontite Agressiva/genética , Ligamento Periodontal/citologia , Esfingomielina Fosfodiesterase/fisiologia , Adulto , Periodontite Agressiva/enzimologia , Fosfatase Alcalina/metabolismo , Calcificação Fisiológica , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Feminino , Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Immunoblotting , Japão , Masculino , Osteocalcina/metabolismo , Osteopontina/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Esfingomielina Fosfodiesterase/genéticaRESUMO
NaS2 is a Na+-coupled transporter for sulfate that belongs to the SLC13 gene family. This transporter was originally cloned from high endothelial venule endothelial cells, but nothing is known about the functional characteristics of this transporter except that it transports sulfate in a Na+-coupled manner. Northern blot analysis indicates that NaS2 is expressed most robustly in placenta. In the present study, we cloned NaS2 from rat placenta and characterized its transport function in detail using the Xenopus laevis oocyte expression system. Rat NaS2 consists of 629 amino acids and is highly similar to human NaS2. In situ hybridization studies with mouse placental sections show that NaS2 transcripts are expressed primarily in trophoblasts of the labyrinth zone. The expression of the transporter is confirmed in primary cultures of trophoblasts isolated from human placenta. When expressed in X. laevis oocytes, rat NaS2 mediates Na+-coupled transport of sulfate. The transport of sulfate is inhibited by oxyanions of selenium, chromium, arsenic, molybdenum, and phosphorous, suggesting that the transporter may mediate the transport of these oxyanions in addition to sulfate. The Kt for sulfate is 153+/-30 microM and the Na+:sulfate stoichiometry is 3:1. The transport process is electrogenic as evidenced from the inhibition of the uptake process by K+-induced depolarization. We conclude that NaS2 is a placenta-specific Na+-coupled, electrogenic, transporter for sulfate expressed in trophoblasts and that it is also responsible for the transport of oxyanions of the micronutrients selenium and chromium.
Assuntos
Ânions/metabolismo , Cromo/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Selênio/metabolismo , Sulfatos/metabolismo , Simportadores/metabolismo , Trofoblastos/metabolismo , Sequência de Aminoácidos , Animais , Biblioteca Gênica , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Transportadores de Sulfato , Xenopus laevisRESUMO
BACKGROUND: Cancer treatments that appear promising in tissue culture are often less effective in solid tumors, in part because of the proliferative and microenvironmental heterogeneity that develops in these tumors as they grow. Heterogeneous tumor models are thus needed for drug screening. PURPOSE: Our goal was to develop and test for drug evaluation a solid tumor model based on cell growth inside biocompatible hollow fibers. METHODS: Building on the experience of Hollingshead and co-workers with a sparse-cell, hollow-fiber tumor model, we tested six human tumor cell lines for in vitro growth inside 450-microns internal-diameter polyvinylidine fluoride fibers and examined them histologically. Human SW620 colon carcinoma cells grown in hollow fibers were also examined using electron microscopy, and their doxorubicin sensitivity was assessed. A colorimetric assay based on sulforhodamine B was adopted to replace the more cumbersome clonogenic cell survival assay. RESULTS: Five of the human tumor cell lines tested grew to confluence, forming heterogeneous in vitro tumors with subpopulations of viable and necrotic cells. For SW620 hollow-fiber tumors, maximum viable cell populations in excess of 10(8) cells/mL were obtained after 8 days of growth. This viable cell density remained roughly constant for 3-4 days, permitting dose-response experiments over this time interval. Tumor cells in hollow fibers were much more resistant to a 4-hour doxorubicin exposure than were tumor cells in monolayers: LC50 values (i.e., the drug concentrations at which the plating efficiency equals one-half the plating efficiency of untreated cells) of 3.5 microM and 0.16 microM were obtained for hollow-fiber tumors and monolayers, respectively. LC50 values decreased when drug exposure time was increased. Results from the colorimetric assay were in agreement with those from the clonogenic assay. CONCLUSION: The successful growth of tumor cells to confluence in hollow fibers and the feasibility of performing in vitro drug dose-response experiments with a relatively easy colorimetric assay demonstrate the potential of the hollow-fiber solid tumor model as a tool for experimental therapeutic research. IMPLICATION: Hollow-fiber solid tumors may prove useful for experimental drug evaluation.
Assuntos
Células Tumorais Cultivadas/citologia , Ensaio Tumoral de Célula-Tronco/métodos , Antineoplásicos/farmacologia , Materiais Biocompatíveis , Divisão Celular/efeitos dos fármacos , Colorimetria , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Humanos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
In preceding studies (Hikosaka et al., 1996; Sakai et al., 1998) we have shown that the presupplementary motor area (pre-SMA), an anterior part of the medial premotor cortex, is active during visuo-motor sequence learning. However, the paradigm required the subjects first to acquire correct visuo-motor association and then to acquire correct sequence, and it was still unknown which of the two processes the pre-SMA is involved in. To further characterize the role of pre-SMA, we have conducted another series of functional magnetic resonance imaging experiments using three learning paradigms. The three were the same in that they involved a visuo-motor association component, but they differed in terms of the involvement of sequential components; one involved no sequence learning, whereas the other two involved learning of motor sequence or perceptual sequence. Comparison of the learning conditions with the any-order button press condition revealed pre-SMA activation in all three paradigms. The pre-SMA activation remained unchanged during learning of visuo-motor associations but decreased during learning of sequences, suggesting that the pre-SMA is related to visuo-motor association rather than sequence. The decrease of pre-SMA activation in the sequential paradigms may reflect the process by which individual visuo-motor associations were replaced by the formation of sequential procedural memory, which occurs outside the pre-SMA. Thus activation of the pre-SMA was related to the extent to which the task performance depended on conscious visuo-motor associations.
Assuntos
Mapeamento Encefálico , Aprendizagem/fisiologia , Córtex Motor/fisiologia , Desempenho Psicomotor/fisiologia , Adulto , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estimulação LuminosaRESUMO
Successful motor behavior requires making appropriate response (response selection) at the right time (timing adjustment). Earlier psychological studies have suggested that the response selection and timing adjustment processes are performed serially in separate stages. We tested this hypothesis using functional magnetic resonance imaging. The subjects performed a choice reaction time task in four conditions: two (on-line response selection required or not) by two (on-line timing adjustment required or not). We found that the neural correlates for the two processes were indeed separate: the anterior medial premotor cortex (presupplementary motor area) was selectively active in response selection, whereas the cerebellar posterior lobe was selectively active in timing adjustment. However, the functional separation was only partial in that the lateral premotor cortex and the intraparietal sulcus were active equally for response selection and timing adjustment. The lateral premotor cortex was most active when both processes were required, suggesting that it integrates the information on response selection and the information on timing adjustment; alternatively, it might contribute to the allocation of attentional resources during dual information processing. The intraparietal sulcus was equally active when either response selection or timing adjustment was required, suggesting that it modifies, rather than integrates, these processes. Furthermore, our results suggest that these activations related to response selection and timing adjustment were distinct from sensory or motor processes.
Assuntos
Percepção Auditiva/fisiologia , Mapeamento Encefálico , Percepção Visual/fisiologia , Adulto , Comportamento de Escolha/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tempo de Reação/fisiologia , Análise e Desempenho de TarefasRESUMO
Rhythm is determined solely by the relationship between the time intervals of a series of events. Psychological studies have proposed two types of rhythm representation depending on the interval ratio of the rhythm: metrical and nonmetrical representation for rhythms formed with small integer ratios and noninteger ratios, respectively. We used functional magnetic resonance imaging to test whether there are two neural representations of rhythm depending on the interval ratio. The subjects performed a short-term memory task for a seven-tone rhythm sequence, which was formed with 1:2:4, 1:2:3, or 1:2.5:3.5 ratios. The brain activities during the memory delay period were measured and compared with those during the retention of a control tone sequence, which had constant intertone intervals. The results showed two patterns of brain activations; the left premotor and parietal areas and right cerebellar anterior lobe were active for 1:2:4 and 1:2:3 rhythms, whereas the right prefrontal, premotor, and parietal areas together with the bilateral cerebellar posterior lobe were active for 1:2.5:3.5 rhythm. Analysis on individual subjects revealed that these activation patterns depended on the ratio of the rhythms that were produced by the subjects rather than the ratio of the presented rhythms, suggesting that the observed activations reflected the internal representation of rhythm. These results suggested that there are two neural representations for rhythm depending on the interval ratio, which correspond to metrical and nonmetrical representations.
Assuntos
Mapeamento Encefálico , Encéfalo/fisiologia , Memória de Curto Prazo/fisiologia , Neurônios/fisiologia , Periodicidade , Retenção Psicológica/fisiologia , Adulto , Cerebelo/fisiologia , Feminino , Lobo Frontal/fisiologia , Lateralidade Funcional , Humanos , Imageamento por Ressonância Magnética , Masculino , Lobo Parietal/fisiologia , Córtex Pré-Frontal/fisiologiaRESUMO
We selected for study an anthracycline-resistant mutant from the archaebacteria Haloferax volcanii. This resistance was reversed by a Ca(2+)-channel antagonist, nifedipine (NDP). This resistance and its reversal by NDP suggest P-glycoprotein (Pgp) to be responsible for maintaining an anticancer drug concentration below the cytotoxic level. Using rhodamine 123 (RH123) as a substrate for Pgp, we then examined whether the resistance to anthracyclines in this bacteria might involve a Pgp-like anthracycline efflux pump. RH123 accumulation by the bacteria was determined with flow cytometry. A steady-state RH123 accumulation by the resistant cells revealed approx. one-fifteenth of that by the wild-type cells, which could be remarkably enhanced by NDP. The other modulators of Pgp, diltiazem and verapamil, also enhanced RH123 accumulation in resistant cells. The uncoupler FCCP completely restored RH123 accumulation in resistant cells to the wild-type cell level. RH123 unidirectional efflux from resistant cells after its preloading revealed much greater than that from wild-type cells, which was remarkably inhibited by FCCP. These confirmed that RH123 low accumulation involves its active efflux mechanism. Taken together, the present study indicated that lower evolutionary archaebacteria might also express a Pgp-like protein very similar to mammalian Pgp.
Assuntos
Archaea/metabolismo , Doxorrubicina/metabolismo , Glicoproteínas de Membrana/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Animais , Archaea/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Divisão Celular/efeitos dos fármacos , Diltiazem/farmacologia , Doxorrubicina/farmacologia , Resistência Microbiana a Medicamentos , Sinergismo Farmacológico , Citometria de Fluxo , Corantes Fluorescentes , Mamíferos , Nifedipino/farmacologia , Rodamina 123 , Rodaminas/metabolismo , Células Tumorais Cultivadas , Verapamil/farmacologiaRESUMO
The lipophilic cation tetraphenylphosphonium (TPP+) has been extensively utilized as the probe for the membrane potential (Vm) in various cells. For application to mammalian cells, however, two serious problems require resolution: (1), correction of TPP+ binding to intracellular constituents and (2), estimation of the considerable TPP+ accumulation in mitochondria. We propose here a simple corrective method for the TPP+ binding and its accumulation. TPP+ distribution is assumed as: (1), two compartments (a cytosolic and a mitochondrial space); (2), a proportional relationship between TPP+ bound amount and its unbound concentration in each compartment. We theoretically derived the simple equation: Vm = - RT/F ln(C/Mphys ratio/C/Mabol ratio) where R, T and F have their usual thermodynamic significance. Here, the C/M ratio is defined as the ratio of TPP+ concentration of apparent intracellular to extracellular space. The suffixes phys and abol, respectively, mean the physiological and solely Vm-abolished conditions. This equation was checked with hepatocytes, because estimating hepatocytes Vm with TPP+ distribution is not considered possible because of the relatively high mitochondrial content. The selective Vm abolition was achieved by permeabilization with 20 microM of amphotericin B. The Vm value was, thus, estimated to be -38.6 +/- 0.3 mV, compatible with those obtained with microelectrodes in other laboratories. Vm in hepatocytes is composed of transmembrane K+ diffusion potential (-20.6 +/- 0.3 mV) and electrogenic Na+/K(+)-ATPase (-19.6 +/- 0.4 mV). Addition of rheogenic L-alanine caused a transient but significant depolarization (from control to -34 +/- 0.3 mV). These results taken together indicate that hepatocyte Vm can be accurately determined with the present simple method, so that it may possibly be applicable to the evaluation of Vm in other mammalian cells.
Assuntos
Membrana Celular/metabolismo , Fígado/metabolismo , Oniocompostos/química , Compostos Organofosforados/química , Anfotericina B/farmacologia , Animais , Digitonina/farmacologia , Masculino , Matemática , Potenciais da Membrana , Mitocôndrias Hepáticas/metabolismo , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Ouabaína/farmacologia , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
We have previously reported a sequence of events which occurs during the recovery phase of the murine epidermal Langerhans cells (ELCs) after ultraviolet-B irradiation. We found that an ATPase-positive round cell divides, dendrites are gradually formed, and paired dendritic cells are eventually separated as the post-irradiation time elapses. We wondered if a series of events similar to this might occur in the normal murine epidermis without irradiation. In this study, we could identify exactly the same phases of the ELC mitotic cycle in normal mouse ear skin.