RESUMEN
Frequency of Treponema pallidum invasion into cerebrospinal fluid (CSF) has not been clear at this present. Since it is impossible to culture T. pallidum in vitro at this present, we need molecular based-approach to detect it in CSF. Additionally, neurosyphilis is usually a late sequela, however it might result in asymptomatic neurosyphilis even at primary or secondary syphilis. This study was to reveal the frequency of T. pallidum invasion into CSF especially at primary or secondary syphilis with polymerase chain reaction (PCR) test. All patients were visited the Aichi Medical University Hospital or Izumi ladies' clinic between 2016 and 2017. Clinical CSF samples were collected from patients with early and late stages of syphilis. The PCR was done using primers targeting the tpN47gene. CSF samples were collected from 9 patients (4 patients with primary syphilis, 3 with secondary syphilis, and 1 early latent syphilis and 1 with late latent syphilis). PCR showed positive reaction in 2 of 7 (28.6%) primary and secondary syphilis patients, in 1 of 1 (100%) early latent syphilis patients, and in 1 of 1 (100%) late latent syphilis patients. Despite its lack of sensitivity for use alone as a diagnostic test, this PCR test should be preferred for the diagnosis of neurosyphilis. Because, T. pallidum was detected in the 28.6% CSF of patients at primary and secondary syphilis, which indicated that they invade the central nervous system from the early stages of infection. However, studies in a larger population are required to confirm these preliminary results.
Asunto(s)
Neurosífilis/líquido cefalorraquídeo , Neurosífilis/diagnóstico , Sífilis/líquido cefalorraquídeo , Treponema pallidum/genética , Proteínas Bacterianas/genética , ADN Bacteriano/líquido cefalorraquídeo , Humanos , Técnicas de Diagnóstico Molecular , Neurosífilis/etiología , Neurosífilis/microbiología , Reacción en Cadena de la Polimerasa , Pruebas Serológicas , Sífilis/complicaciones , Sífilis/microbiologíaRESUMEN
The 90-kDa isoform of the lipid kinase PIP kinase Type I γ (PIPKIγ) localizes to focal adhesions (FAs), where it provides a local source of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Although PtdIns(4,5)P(2) regulates the function of several FA-associated molecules, the role of the FA-specific pool of PtdIns(4,5)P(2) is not known. We report that the genetic ablation of PIPKIγ specifically from FAs results in defective integrin-mediated adhesion and force coupling. Adhesion defects in cells deficient in FAPtdIns(4,5)P(2) synthesis are corrected within minutes while integrin-actin force coupling remains defective over a longer period. Talin and vinculin, but not kindlin, are less efficiently recruited to new adhesions in these cells. These data demonstrate that the specific depletion of PtdIns(4,5)P(2) from FAs temporally separates integrin-ligand binding from integrin-actin force coupling by regulating talin and vinculin recruitment. Furthermore, it suggests that force coupling relies heavily on locally generated PtdIns(4,5)P(2) rather than bulk membrane PtdIns(4,5)P(2).
Asunto(s)
Adhesiones Focales/metabolismo , Integrinas/metabolismo , Fosfatidilinositol 4,5-Difosfato/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Proteínas Portadoras/metabolismo , Adhesión Celular/genética , Células Cultivadas , Adhesiones Focales/química , Proteínas de la Membrana/metabolismo , Ratones , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Unión Proteica , Talina/metabolismo , Vinculina/metabolismoRESUMEN
Localized production of polyphosphoinositides is critical for their signaling function. To examine the biological relevance of specific pools of phosphatidylinositol 4,5-bisphosphate we compared the consequences of genetically ablating all isoforms of phosphatidylinositol phosphate (PIP) kinase type Iγ (PIPKIγ), encoded by the gene Pip5k1c, versus ablation of a specific splice isoform, PIPKIγ_i2, with respect to three reported PIPKIγ functions. Ablation of PIPKIγ_i2 caused a neuron-specific endocytosis defect similar to that found in PIPKIγ(-/-) mice, while agonist-induced calcium signaling was reduced in PIPKIγ(-/-) cells, but was not affected in the absence of PIPKIγ_i2. A reported contribution of PIPKIγ to epithelial integrity was not evident in PIPKIγ(-/-) mice. Given that mice lacking PIPKIγ_i2 live a normal lifespan whereas PIPKIγ(-/-) mice die shortly after birth, we propose that PIPKIγ-mediated metabotropic calcium signaling may represent an essential function of PIPKIγ, whereas functions specific to the PIPKIγ_i2 splice isoform are not essential for survival.
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Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Señalización del Calcio , Células Cultivadas , Ratones , Ratones Noqueados , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Isoformas de Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5ß1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.
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Astrocitos/metabolismo , Fibronectinas/metabolismo , Integrinas/metabolismo , Neovascularización Fisiológica , Vasos Retinianos/crecimiento & desarrollo , Vasos Retinianos/metabolismo , Animales , Movimiento Celular , Matriz Extracelular/metabolismo , Fibronectinas/deficiencia , Fibronectinas/genética , Heparitina Sulfato/metabolismo , Integrina alfa5beta1/química , Integrina alfa5beta1/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oligopéptidos/química , Fosfatidilinositol 3-Quinasas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vasos Retinianos/inervación , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Background: The German word "kurort" means cure (kur) and area (ort), whereby a patient's health improves through walking in areas full of nature. A single session of kurort health walking (kurort) decreased high blood pressure and improved mental health. However, its continuing effect with repeat sessions remains unclear. Methods and Results: The subjects participated twice in kurort health walking in specially designated courses in Gifu City (n=242). Systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse rate (PR) were measured before and after kurort health walking. Mental health was assessed using a 10-item checklist after kurort health walking. Both basal SBP and DBP before walking were significantly decreased more in the second session than in the first. In both the first and second sessions SBP and DBP decreased, but the decrease in SBP (∆SBP) by kurort was significantly greater in the SBP ≥140- than in the SBP <140-mmHg group, SBP inversely correlated with ∆SBP, the decrease in DBP (∆DBP) was significantly greater in the DBP ≥90- than in the DBP <90-mmHg group, and DBP inversely correlated with ∆DBP. Mental health was similarly improved after both the first and second kurort. Conclusions: Basal SBP and DBP decreased more in the second than in the first kurort. The decrease in SBP and DBP, and improvement of mental health was noted after both sessions.
RESUMEN
Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of alpha5beta1 or alphav integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud-derived somites, and severe vascular defects resembling the phenotype of alpha5 integrin-deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that alphavbeta3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.
Asunto(s)
Ácido Aspártico/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Oligopéptidos/química , Reticulina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Línea Celular Transformada , Dimerización , Disulfuros/química , Embrión de Mamíferos , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fibronectinas/genética , Heterocigoto , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
OBJECTIVE: The aim of this study was to examine the efficacy and safety of a novel immune-enhancing enteral formula, Prem-8, which contains lactoferrin as an immunonutrient. DESIGN, SETTING, PATIENTS: A multicenter, randomized controlled trial was conducted in 5 hospitals in Japan, and 71 tube-fed bedridden patients with serum albumin concentrations between 2.5 and 3.5 g/dL were allocated to Prem-8 (n = 38) or control formula (n = 33) groups for an observation period of 12 weeks. MEASURES OF OUTCOME: Efficacy was evaluated by comparing immunological (natural killer cell activity, neutrophil-phagocytic activity, neutrophil-sterilizing activity, and C-reactive protein), and nutritional (anthropometric measurements and serum levels of nutritional assessment proteins and total cholesterol) variables. Safety was assessed by comparing the incidence of adverse events. In a secondary analysis, patients were subgrouped according to the amount of protein supplemented (1 g/kg/d) so that immunological and nutritional variables and safety could be further compared. RESULTS: Natural killer activity and neutrophil functions were normal for both groups throughout the study period, without significant between-group differences at any point. Nutritional status was stably maintained in both groups, although the body mass index at 12 weeks was marginally lower in the Prem-8 group than in the control group (p < 0.01). The incidence of adverse events were comparable between both groups, but the incidence of fever in the Prem-8 group (7/14) was significantly lower than in the control group (10/11) in a subgroup of patients whose supplemented protein was less than 1 g/kg/d (p < 0.05). CONCLUSION: Prem-8 did not demonstrate superiority to the control formula with respect to immunological and nutritional variables, whereas the body mass index of patients in the Prem-8 group marginally decreased. However, Prem-8 had a favorable effect on the incidence of fever in a subgroup of patients with low protein intake.
Asunto(s)
Antiinfecciosos/farmacología , Nutrición Enteral/métodos , Fiebre/epidemiología , Alimentos Formulados , Lactoferrina/farmacología , Estado Nutricional , Anciano , Anciano de 80 o más Años , Antiinfecciosos/efectos adversos , Reposo en Cama , Índice de Masa Corporal , Proteína C-Reactiva/inmunología , Proteínas en la Dieta/administración & dosificación , Nutrición Enteral/efectos adversos , Femenino , Humanos , Japón , Células Asesinas Naturales/inmunología , Lactoferrina/efectos adversos , Lactoferrina/inmunología , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Albúmina Sérica/metabolismoRESUMEN
BACKGROUND/AIMS: In both SHARP and Asia-Pacific Study, sorafenib was proved to improve the overall survival of the patients with hepatocellular carcinoma. However, factors contributing to the improvement of overall survival of the patients treated by sorafenib have not been fully evaluated. In this study, patient-derived, background liver disease-derived and tumor-derived factors before treatment were evaluated whether they have contributed to the improvement of the overall survival. METHODOLOGY: Forty-seven cases with HCC treated by sorafenib between Sept 2009 and Feb 2011 were included in this analysis. The survival of these cases was analyzed by Kaplan-Meier Method. Factors used for univariate analysis were two patient-derived parameters, two background liver disease-derived, five tumor-derived. Factors related to the over-all survival were analyzed by multivariate analysis using Cox regression model. RESULTS: In the multivariate analysis, only background liver disease-derived parameter Child-Pugh class A vs. B, (p=0.007, HR=0.21 (0.07-0.65)) was significant. No other parameters including tumor-derived factors were statistically significant by multivariate analysis. CONCLUSIONS: We undertook the statistical analysis on the three categories. Surprisingly, no tumor derived parameter contributed to the overall survival. Background liver disease-derived parameter rather than tumor-derived parameter was found to define the prognosis of patients with advanced HCC treated by sorafenib.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Femenino , Humanos , Japón , Estimación de Kaplan-Meier , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Niacinamida/uso terapéutico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Sorafenib , Factores de Tiempo , Resultado del TratamientoRESUMEN
Fibronectin, a 250-kDa eukaryotic extracellular matrix protein containing an RGD motif plays crucial roles in cell-cell communication, development, tissue homeostasis, and disease development. The highly complex fibrillar fibronectin meshwork orchestrates the functions of other extracellular matrix proteins, promoting cell adhesion, migration, and intracellular signaling. Here, we demonstrate that CagL, a 26-kDa protein of the gastric pathogen and type I carcinogen Helicobacter pylori, mimics fibronectin in various cellular functions. Like fibronectin, CagL contains a RGD motif and is located on the surface of the bacterial type IV secretion pili as previously shown. CagL binds to the integrin receptor alpha(5)beta(1) and mediates the injection of virulence factors into host target cells. We show that purified CagL alone can directly trigger intracellular signaling pathways upon contact with mammalian cells and can complement the spreading defect of fibronectin(-/-) knock-out cells in vitro. During interaction with various human and mouse cell lines, CagL mimics fibronectin in triggering cell spreading, focal adhesion formation, and activation of several tyrosine kinases in an RGD-dependent manner. Among the activated factors are the nonreceptor tyrosine kinases focal adhesion kinase and Src but also the epidermal growth factor receptor and epidermal growth factor receptor family member Her3/ErbB3. Interestingly, fibronectin activates a similar range of tyrosine kinases but not Her3/ErbB3. These findings suggest that the bacterial protein CagL not only exhibits functional mimicry with fibronectin but is also capable of activating fibronectin-independent signaling events. We thus postulate that CagL may contribute directly to H. pylori pathogenesis by promoting aberrant signaling cross-talk within host cells.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Forma de la Célula/efectos de los fármacos , Fibronectinas/metabolismo , Helicobacter pylori , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/química , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Proteínas Inmovilizadas/farmacología , Integrinas/metabolismo , Ratones , Oligopéptidos/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK)-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. RESULTS: Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry. CONCLUSION: Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectinâintegrin-beta1âFAK/SrcâEGFR/PDGFRâPI3-kinaseâVav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.
RESUMEN
Microfluidic microphysiological systems (MPSs) or "organs-on-a-chip" are a promising alternative to animal models for drug screening and toxicology tests. However, most microfluidic devices employ polydimethylsiloxane (PDMS) as the structural material; and this has several drawbacks. Cyclo-olefin polymers (COPs) are more advantageous than PDMS and other thermoplastic materials because of their low drug absorption and autofluorescence. However, most COP-based microfluidic devices are fabricated by solvent bonding of the constituent parts. Notably, the remnant solvent can affect the cultured cells. This study employed a photobonding process with vacuum ultraviolet (VUV) light to fabricate microfluidic devices without using any solvent and compared their performance with that of solvent-bonded systems (using cyclohexane, dichloromethane, or toluene as the solvent) to investigate the effects of residual solvent on cell cultures. Quantitative immunofluorescence assays indicated that the coating efficiencies of extracellular matrix proteins (e.g., Matrigel and collagen I) were lower in solvent-bonded COP devices than those in VUV-bonded devices. Furthermore, the cytotoxicity of the systems was evaluated using SH-SY5Y neuroblastoma cells, and increased apoptosis was observed in the solvent-processed devices. These results provide insights into the effects of solvents used during the fabrication of microfluidic devices and can help prevent undesirable reactions and establish good manufacturing practices.
RESUMEN
Phospholipase A(2) (PLA(2)) not only plays a role in the membrane vesiculation system but also mediates membrane-raft budding and fission in artificial giant liposomes. This study aimed to demonstrate the same effects in living cells. Differentiated Caco-2 cells were cultured on filter membranes. MDCK cells were challenged with Influenza virus. The MDCK cultures were harvested for virus titration with a plaque assay. Alkaline phosphatase (ALP), a membrane-raft associated glycosylphosphatidylinositol (GPI)-anchored protein, was 70% released by adding 0.2 mmol/l lysophosphatidylcholine, which was abolished by treatment with a membrane-raft disrupter, methyl-beta-cyclodextrin. Activation of calcium-independent PLA(2) (iPLA(2)) by brefeldin A increased the apical release of ALP by approximately 1.5-fold (p<0.01), which was blocked by PLA(2) inhibitor bromoenol lactone (BEL). BEL also reduced Influenza virus production into the media (<10%) in the MDCK culture. These results suggest that cells utilize inverted corn-shaped lysophospholipids generated by PLA(2) to modulate plasma membrane structure and assist the budding of raft-associated plasma membrane particles, which virus utilizes for its budding. Brush borders are enriched with membrane-rafts and undergo rapid turnover; thus, PLA(2) may be involved in the regulatory mechanism in membrane dynamism. Further, iPLA(2) may provide a therapeutic target for viral infections.
Asunto(s)
Lisofosfolípidos/química , Microdominios de Membrana/química , Fosfolipasas A2/química , Ensamble de Virus , Fosfatasa Alcalina/metabolismo , Animales , Calcio/metabolismo , Membrana Celular , Células Cultivadas/virología , Perros , Humanos , Lisofosfolípidos/metabolismo , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Orthomyxoviridae/fisiología , Fosfolipasas A2/metabolismo , Replicación Viral/efectos de los fármacos , beta-Ciclodextrinas/farmacologíaRESUMEN
Intestinal alkaline phosphatase (IAP) is a brush-border membrane ectoenzyme (BBM-IAP) that is released into the lumen (L-IAP) after a high-fat diet. We examined the effects of oil feeding and the addition of mixed-lipid micelles on the formation of L-IAP in oil-fed rat intestine, Caco-2 cell monolayers, and mouse intestinal loops. We localized IAP in the duodenum of rats fed corn oil using fluorescence microscopy with enzyme-labeled fluorescence-97 as substrate. Four hours after oil feeding, L-IAP increased approximately 10-fold accompanied by the loss of BBM-IAP, consistent with BBM-IAP release. Rat IAP isozyme mRNAs progressively increased 4-6 h after oil feeding, followed by the increase of IAP activity in the subapical location at 6 h, consistent with the restoration of IAP protein. Postprandial lipid-micelle components, sodium taurocholate with or without oleic acid, mono-oleylglycerol, cholesterol, or lysophosphatidylcholine (lysoPC) were applied singly or as mixed-lipid micelles to the apical surface of polarized Caco-2 cell monolayers. LysoPC increased L-IAP >10-fold over basal release. LysoPC released IAP into the apical medium more than other intestinal brush-border enzymes, 5'-nucleotidase, sucrase, aminopeptidase N, and lactase, without comparable lactate dehydrogenase release or cell injury. LysoPC increased human IAP mRNA levels by 1.5-fold in Caco-2 cells. Luminally applied lysoPC also increased release of IAP preferentially in mouse intestinal loops. These data show that lysoPC accelerates the formation of L-IAP from BBM-IAP, followed by enhanced IAP synthesis, suggesting the role that lysoPC might play in the turnover of brush-border proteins.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Aceite de Maíz/administración & dosificación , Intestinos/enzimología , Lisofosfatidilcolinas/metabolismo , Fosfatasa Alcalina/genética , Animales , Antígenos de Neoplasias/metabolismo , Células CACO-2 , Duodeno/enzimología , Células Epiteliales/enzimología , Proteínas Ligadas a GPI , Humanos , Isoenzimas , Masculino , Ratones , Ratones Endogámicos C57BL , Micelas , Microvellosidades/enzimología , Periodo Posprandial , Transporte de Proteínas , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Clustering of fibroblasts into spheroids induces a massive proinflammatory, proteolytic and growth-factor response, named nemosis, which promotes tumor cell invasiveness and differentiation of leukemia cells. We have now sought to investigate mechanisms leading to the formation of multicellular spheroids and subsequent activation of fibroblasts (nemosis). Cell lines either lacking fibronectin expression (FN-/-) or expressing FN with a mutated integrin-binding site (FNRGE/RGE) were unable to form compact spheroids. Furthermore, inhibition of FN synthesis by siRNA or functional inhibition of FN or its integrins impaired spheroid formation (alpha5, beta1) and quenched fibroblast activation (alphaV). The integrin ligand GRGDSP hexapeptide interfered with spheroid formation and induced activation of fibroblasts. Surprisingly, a 70 kDa FN fragment, which prevents deposition of FN matrix but does not interfere with FN-integrin interaction, prevented spheroid formation only marginally and did not block the activation. Our results present a new mechanism of fibroblast activation, which is initiated by interaction of FN with its integrin receptors.
Asunto(s)
Fibroblastos/citología , Fibronectinas/metabolismo , Integrinas/metabolismo , Fibroblastos/metabolismo , Fibronectinas/análisis , Fibronectinas/genética , Humanos , Integrina alfa5/metabolismo , Integrina alfaV/metabolismo , Integrina beta1/metabolismo , Esferoides CelularesRESUMEN
Phosphatidylcholine (PC) and its hydrolysates are considered to stimulate intestinal lipid absorption, however, their exact effects on lipoproteins and apolipoprotein (apo) metabolism remain ambiguous. This study aimed to further differentiate the effects of them using fully differentiated enterocyte-like Caco-2 cells. Lipid micelles (oleic acid 0.6, cholesterol 0.05, monooleylglycerol 0.2, taurocholate 2 in mmol/l) with or without choline, PC, and lysoPC (0.2 mmol/l each) were applied apically to Caco-2 cells. (3)H-oleic acid and (14)C-cholesterol were added to the micelles when necessary. Secreted lipoproteins were analyzed by a HPLC method. LysoPC had the most potent promoting effect on lipid uptake, and lipoprotein and apolipoprotein B-48 secretion among the molecules tested. LysoPC doubled the output of cholesterol and triglyceride as the lipoprotein component, but PC did not. On the other hand, PC only increased the secretion of apoA-IV in the presence of lipid micelles. These findings confirm that the alteration of PC by PLA(2) hydrolysis is intrinsically involved in the intestinal lipid absorption process and suggest that PC and its hydrolysis are coordinately associated with not only lipid absorption efficiency but also lipoprotein output and metabolism.
RESUMEN
The photoinduced electron transfer of a series of meta- and para-linked triphenylamine-naphthalimide dyads, N-{3- and 4-[bis(4-R-substituted phenyl)amino]phenyl}-1,8-naphthalimide, 1m,p (R = H), 2m,p (R = Me), 3m,p (R = OMe), and 4m,p (R = NMe2) was investigated in toluene and DMF. The singlet charge-transfer (CT) states were observed in all cases. The decay rates were found to be faster in DMF (tau = 6.5 ps to 100 ps) than those in toluene (tau = 190 ps to 7 ns). The long-lived triplet CT states were observed in toluene for 3 (ca. 10% contribution, tau = 670 ns for 3m, 240 ns for 3p). No long-lived species were detected in DMF. The decay rates were somewhat faster in the para-isomers than in the meta-isomers in most cases. The photolysis of 5 (p-phenylene extended analogue of 3, R = OMe) gave a singlet CT state and a locally excited triplet state on the naphthalimide chromophore.
RESUMEN
BACKGROUND AND OBJECTIVES: Aging and malnutrition are known to influence immune functions. The aim of this study was to investigate the relationship of aging and malnutrition to innate immune functions in tube-fed bedridden patients. METHODS AND STUDY DESIGN: A cross-sectional survey was performed in 71 tube-fed bedridden patients aged 50-95 years (mean age±SD, 80.2±8.5 years) with serum albumin concentrations between 2.5 and 3.5 g/dL. We evaluated associations of age and nutritional variables with natural-killer cell activity, neutrophilphagocytic activity, and neutrophil-sterilizing activity. Nutritional variables included body mass index, weightadjusted energy intake, total lymphocyte count, and serum concentrations of albumin, transferrin, prealbumin, total cholesterol, C-reactive protein, and zinc. RESULTS: Natural-killer cell activity, neutrophil-phagocytic activity, and neutrophil-sterilizing activity were normal or increased in 67 (94%), 63 (89%), and 69 (97%) patients, respectively. Multiple linear regression analysis with a backward elimination method showed that natural-killer cell activity correlated negatively with aging and lymphocyte counts (p<0.01 for both) but positively with body mass index and transferrin (p<0.01 for both). Neutrophil-phagocytic and neutrophil-sterilizing activities were not associated with any variables. CONCLUSIONS: In tube-fed bedridden patients with hypo-albuminemia, natural-killer cell activity may be associated with aging, body mass index, transferrin, and lymphocyte counts.
Asunto(s)
Envejecimiento/inmunología , Nutrición Enteral , Inmunidad Innata , Estado Nutricional/inmunología , Anciano , Anciano de 80 o más Años , Índice de Masa Corporal , Estudios Transversales , Ingestión de Energía , Femenino , Humanos , Células Asesinas Naturales/inmunología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Fagocitos/inmunología , Albúmina Sérica/análisis , Transferrina/análisisRESUMEN
AIM: Rev-Erb ß gene plays crucial roles in circadian rhythm, lipid and glucose metabolism, and several diseases. The molecular mechanisms of the transcriptional regulation of Rev-Erb ß that generate and determine the phase of the circadian oscillation remain unclear. METHODS: We analyzed the Rev-Erb ß promoter by luciferase reporter assays, real-time bioluminescence monitoring assays and electrophoretic mobility shift assays. RESULTS: Luciferase reporter assays indicated that only the 5' region and exon 1 have obvious promoter activity. Real-time bioluminescence monitoring assays revealed that E1, E2, E3, D boxes are important for maintenance of the amplitude of Rev-Erb ß oscillation. Based on EMSA results, REV-ERBß binds ROREs in the Bmal1 promoter region and inhibits Bmal1 promoter activity. CONCLUSION: We provide direct evidence that three E-boxes and one D-box located in the first intron are crucial for the phase of circadian oscillation in Rev-Erb ß expression and that the sequences upstream from its transcription start site function as a promoter with no circadian regulation. We also found that the E1 box affects the Rev-Erb ß oscillation phase. Our results offer new insight into the role of Rev-Erb ß in the circadian rhythm system.
Asunto(s)
Ritmo Circadiano , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Regiones Promotoras Genéticas , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Factores de Transcripción ARNTL/genética , Animales , Secuencia de Bases , Proteínas CLOCK/genética , Cartilla de ADN , Elementos de Facilitación Genéticos , Ratones , Células 3T3 NIHRESUMEN
Host cell entry by the food-borne pathogen Campylobacter jejuni has been reported as one of the primary reasons of tissue damage in infected humans, however, molecular invasion mechanisms and cellular factors involved in this process are widely unclear. Here we used knockout cell lines derived from fibronectin(-/-), integrin beta1(-/-), and focal adhesion kinase (FAK)(-/-) deficient mice and corresponding wild-type (WT) controls, to study C. jejuni-induced signaling cascades involved in the bacterial invasion process. Using high resolution scanning electron microscopy, GTPase pull-downs, G-LISA, and gentamicin protection assays we found that each of these host cell factors is indeed required for activation of the small Rho GTPase member Rac1 and maximal host cell invasion of this pathogen. Interestingly, membrane ruffling, tight engulfment of bacteria and invasion were only seen during infection of WT control cells, but not in fibronectin(-/-), integrin beta1(-/-), and FAK(-/-) knockout cell lines. We also demonstrate that C. jejuni activates FAK autophosphorylation activity at Y-397 and phosphorylation of Y-925, which is required for stimulating two downstream guanine exchange factors, DOCK180 and Tiam-1, which are upstream of Rac1. Small interfering (si) RNA studies further show that DOCK180 and Tiam-1 act cooperatively to trigger Rac1 activation and C. jejuni invasion. Moreover, mutagenesis data indicate that the bacterial fibronectin-binding protein CadF and the intact flagellum are involved in Rho GTPase activation and host cell invasion. Collectively, our results suggest that C. jejuni infection of host epithelial target cells hijacks a major fibronectin â integrin beta1 â FAK â DOCK180/Tiam-1 signaling cascade, which has a crucial role for Rac1 GTPase activity and bacterial entry into host target cells.
Asunto(s)
Campylobacter jejuni/patogenicidad , Interacciones Huésped-Patógeno/fisiología , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/fisiología , Secuencia de Bases , Infecciones por Campylobacter/etiología , Campylobacter jejuni/genética , Campylobacter jejuni/ultraestructura , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Línea Celular , Activación Enzimática , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Fibronectinas/deficiencia , Fibronectinas/genética , Fibronectinas/fisiología , Quinasa 1 de Adhesión Focal/deficiencia , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/fisiología , Genes Bacterianos , Factores de Intercambio de Guanina Nucleótido/fisiología , Interacciones Huésped-Patógeno/genética , Humanos , Integrina beta1/genética , Integrina beta1/fisiología , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Modelos Biológicos , Mutación , Neuropéptidos/fisiología , ARN Interferente Pequeño/genética , Transducción de Señal , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Virulencia/genética , Virulencia/fisiología , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/fisiología , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/fisiologíaRESUMEN
AIM: PPARgamma (peroxisome proliferator-activated receptor gamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes associated with lipid metabolism. Herein, we show that expression levels of the novel PPARgamma transcript exhibit circadian oscillation. To study the mechanisms controlling PPARgamma expression, a novel PPARgamma gene promoter was cloned and characterized. METHODS: We analyzed the novel PPARgamma promoter by luciferase reporter assays and gel shift analysis. RESULTS: Surprisingly, it was not an intron but rather the novel first exon of PPARgamma that was found to have functional minimal promoter activity. Luciferase reporter assays and gel shift assays revealed that the novel first exon is essential for novel PPARgamma promoter activation and that DBP (albumin gene D-site binding protein) and E4BP4 (E4 promoter A binding protein 4) bind directly to D-sites in the novel first exon. CONCLUSION: Our results demonstrate that the PAR-bZIP (bZIP, basic leucine zipper) family and E4BP4 are the main regulatory factors involved in oscillation of novel PPARgamma expression. This regulatory mechanism clearly differs from that of the circadian expression of PPARalpha.