RESUMEN
Lipopolysaccharide (LPS) is the principal component of the outer membrane of gram-negative bacteria. The prior oral administration of LPS attenuates inflammatory responses, such as intestinal injury and atopic dermatitis, in mouse models; however, the underlying mechanism remains unclear. Here, we examined the effect of topical LPS application on allergic contact dermatitis and its mechanism of action using a murine contact hypersensitivity (CHS) model. Prolonged LPS application to the skin significantly suppressed 2,4-dinitrofluorobenzene (DNFB)-induced CHS. LPS application to the skin also reduced the phagocytosis of fluorescein isothiocyanate (FITC)-dextran by Langerhans and dendritic cells. Cutaneous cell migration into the skin-draining lymph nodes (LNs) induced by FITC painting was reduced by LPS application. During the CHS response, DNFB application induced T-cell proliferation and inflammatory cytokine production in skin-draining LNs, whereas prolonged LPS application inhibited DNFB-induced T-cell growth and interferon gamma production, indicating suppression of DNFB-induced sensitization. These results suggest that prolonged LPS application suppressed DNFB-induced sensitization and subsequently CHS response. Our findings imply that topical application of LPS may prevent allergic dermatitis such as CHS.
Asunto(s)
Dermatitis por Contacto/tratamiento farmacológico , Factores Inmunológicos/farmacología , Lipopolisacáridos/farmacología , Linfocitos/efectos de los fármacos , Piel/efectos de los fármacos , Administración Cutánea , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Dermatitis por Contacto/etiología , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/patología , Dextranos/metabolismo , Dinitrofluorobenceno/administración & dosificación , Oído , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Células de Langerhans/citología , Células de Langerhans/efectos de los fármacos , Células de Langerhans/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Activación de Linfocitos/efectos de los fármacos , Linfocitos/citología , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Fagocitosis/efectos de los fármacos , Cultivo Primario de Células , Piel/inmunología , Piel/patologíaRESUMEN
We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins.
Asunto(s)
Penicillium chrysogenum/enzimología , Penicillium chrysogenum/genética , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Clonación Molecular , ADN de Hongos/química , ADN de Hongos/genética , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of Penicillium chrysogenum 31B. PcRGL4A optimal activity occurred between pH 7-8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved catalytic and four conserved substrate-binding residues as determined by X-ray crystallography of the Aspergillus aculeatus RG lyase. Recombinant PcRGL4A (rPcRGL4A) expressed in Escherichia coli demonstrated specific activity against rhamnogalacturonan (RG) but not homogalacturonan. Analysis of the RG reaction products by high-performance anion-exchange chromatography revealed that rPcRGL4A cleaved the substrate in an endo-manner and that the major final product was an RG tetrasaccharide with 4-deoxy-4,5-unsaturated galacturonic acid at the nonreducing end. Based on these results, PcRGL4A was classified as an endo-acting RG lyase (EC 4.2.2.23). Divalent cations were not essential for the enzymatic activity of rPcRGL4A, but addition of calcium ions to the reaction mixture increased enzymatic activity. rPcRGL4A demonstrated a preference for RG lacking galactose decoration.
Asunto(s)
Penicillium chrysogenum/enzimología , Polisacárido Liasas/metabolismo , Dominio Catalítico , Cromatografía por Intercambio Iónico , Clonación Molecular , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Polisacárido Liasas/química , Polisacárido Liasas/genética , Polisacárido Liasas/aislamiento & purificación , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
We reported previously that a single ingestion of an alcohol extract of grains of paradise (GP, Aframomum melegueta), a species of the ginger family, increases energy expenditure (EE) through the activation of brown adipose tissue, a site of sympathetically mediated metabolic theromogenesis. The present study aimed to examine a daily ingestion of GP extract on whole-body EE and body fat in humans. Whole-body EE and body fat content were measured before and after daily oral ingestion of GP extract (30 mg/d) for 4 wk in 19 non-obese female volunteers aged 20-22 y in a single-blind, randomized, placebo-controlled, crossover design. Four-week daily ingestion of GP and a placebo decreased and increased slightly the visceral fat area at the umbilicus level, respectively. The GP-induced change was significantly different from that induced by the placebo (p<0.05), and negatively correlated with the initial visceral fat area (r=-0.64, p<0.01). Neither GP nor placebo ingestion affected subcutaneous or total fat. The daily ingestion of GP, but not the placebo, increased whole-body EE (p<0.05). These results suggest that GP extract may be an effective and safe tool for reducing body fat, mainly by preventing visceral fat accumulation.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Grasa Intraabdominal/efectos de los fármacos , Extractos Vegetales/administración & dosificación , Zingiberaceae/química , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Adulto , Índice de Masa Corporal , Peso Corporal/efectos de los fármacos , Estudios Cruzados , Femenino , Humanos , Grasa Intraabdominal/metabolismo , Obesidad/tratamiento farmacológico , Método Simple Ciego , Adulto JovenRESUMEN
Brown adipose tissue (BAT) is responsible for cold- and diet-induced thermogenesis, and thereby contributes to the control of whole-body energy expenditure (EE) and body fat content. BAT activity can be assessed by fluoro-2-deoxyglucose (FDG)-positron emission tomography (PET) in human subjects. Grains of paradise (GP, Aframomum melegueta), a species of the ginger family, contain pungent, aromatic ketones such as 6-paradol, 6-gingerol and 6-shogaol. An alcohol extract of GP seeds and 6-paradol are known to activate BAT thermogenesis in small rodents. The present study aimed to examine the effects of the GP extract on whole-body EE and to analyse its relation to BAT activity in men. A total of nineteen healthy male volunteers aged 20-32 years underwent FDG-PET after 2 h of exposure to cold at 19°C with light clothing. A total of twelve subjects showed marked FDG uptake into the adipose tissue of the supraclavicular and paraspinal regions (BAT positive). The remaining seven showed no detectable uptake (BAT negative). Within 4 weeks after the FDG-PET examination, whole-body EE was measured at 27°C before and after oral ingestion of GP extract (40 mg) in a single-blind, randomised, placebo-controlled, crossover design. The resting EE of the BAT-positive group did not differ from that of the BAT-negative group. After GP extract ingestion, the EE of the BAT-positive group increased within 2 h to a significantly greater (P<0·01) level than that of the BAT-negative group. Placebo ingestion produced no significant change in EE. These results suggest that oral ingestion of GP extract increases whole-body EE through the activation of BAT in human subjects.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Suplementos Dietéticos , Metabolismo Energético/efectos de los fármacos , Extractos Vegetales/farmacología , Zingiberaceae/química , Tejido Adiposo , Tejido Adiposo Pardo/efectos de los fármacos , Adulto , Antropometría , Calorimetría Indirecta , Estudios Cruzados , Dieta , Fluorodesoxiglucosa F18 , Guayacol/análogos & derivados , Guayacol/metabolismo , Humanos , Cetonas/química , Cetonas/metabolismo , Masculino , Tomografía de Emisión de Positrones , Semillas/metabolismo , Método Simple Ciego , Temperatura , Factores de Tiempo , Adulto JovenRESUMEN
Although the role of Na(+) in several aspects of Ca(2+) regulation has already been shown, the exact mechanism of intracellular Ca(2+) concentration ([Ca(2+)](i)) increase resulting from an enhancement in the persistent, non-inactivating Na(+) current (I(Na,P)), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na(+) current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na(+) concentration ([Na(+)](i)) and biphasic [Ca(2+)](i) increase in CA1 pyramidal cells in acute hippocampal slices. The Ca(2+) response was tetrodotoxin- and extracellular Ca(2+)-dependent and ionotropic glutamate receptor-independent. The first phase of [Ca(2+)](i) rise was the net result of Ca(2+) influx through voltage-gated Ca(2+) channels and mitochondrial Ca(2+) sequestration. The robust second phase in addition involved reverse operation of the Na(+)-Ca(2+) exchanger and mitochondrial Ca(2+) release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non-inactivating Na(+) current and [Ca(2+)](i) regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with I(Na,P). Describing the magnitude, temporal pattern and sources of Ca(2+) increase induced by I(Na,P) may provide novel targets for antiepileptic drug therapy.
Asunto(s)
Calcio/metabolismo , Hipocampo/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Sodio/metabolismo , Veratridina/farmacología , Animales , Animales Recién Nacidos , Anticonvulsivantes/farmacología , Biofisica , Cloruro de Cadmio/farmacología , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Clonazepam/análogos & derivados , Clonazepam/farmacología , Estimulación Eléctrica/métodos , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/citología , Técnicas In Vitro , Ionóforos/farmacología , Masculino , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Ratas , Ratas Wistar , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Tapsigargina/farmacología , Tiazepinas/farmacologíaRESUMEN
Daily topical applications of the concentrate of sake (CS) have been shown to reduce epidermal barrier disruption in murine skin caused by ultraviolet B (UVB) radiation, while one of the components of sake, ethyl alpha-D-glucoside (alpha-EG), also reduces barrier disruption. We confirmed the effect of oral ingestion of various doses of CS on epidermal barrier disruption caused by UVB irradiation in hairless mice. Then, to identify the effective components, we quantitatively analyzed alpha-EG, organic acids, and glycerol, the main components of CS, and examined the effect of various concentration of each on barrier disruption. alpha-EG and organic acids showed comparable results to CS itself, and transepidermal water loss levels in murine skin were significantly decreased as compared with the control. Furthermore, an investigation of the dose dependency of these agents was performed and the results showed the significant effectiveness of alpha-EG. In addition, red wine concentrate (WC) and beer concentrate (BC) were examined in order to confirm the unique effects of CS. Similar effects were not found with WC and BC.
Asunto(s)
Bebidas Alcohólicas , Epidermis/efectos de la radiación , Oryza , Rayos Ultravioleta , Bebidas Alcohólicas/análisis , Animales , Cerveza/análisis , Epidermis/fisiología , Fermentación , Ratones , Ratones Pelados , Permeabilidad , Pérdida Insensible de Agua , Vino/análisisRESUMEN
Cisternal stacks are induced during hypoxia, which may be associated with intracellular Ca2+ regulation. Although neurons are divided internally in different compartments, little is known about regional differences in cisternal stack formation. We investigated the effects of hypoxic hypoxia and later reoxygenation on cisternal stack formation and other ultrastructual changes in the proximal dendrite, dendritic spine, and cell body of cerebellar Purkinje cells in rats. After brief hypoxic events, cisternal stacks appeared predominantly in the proximal dendrites and after longer hypoxic events in dendritic spines and cell body. Following reoxygenation, cisternal stacks disappeared first in the cell body, followed by the dendritic spines, then the proximal dendrites. These results showed that stack formation occurred at different degrees and time courses among the three regions, and the effect was reversible, which suggests that these compartments are differentially sensitive to hypoxia.