RESUMEN
OBJECTIVE: This study aimed to examine the effects of quercetin glycoside-containing beverages on cognitive function and cerebral blood flow (CBF) in adult men and women aged between 60 and 75 years. PATIENTS AND METHODS: Eighty healthy men and women with no cognitive impairment and aware of ageing-related forgetfulness underwent a placebo-controlled, randomized, double-blind, and parallel-group trial. They regularly consumed 500 mL of beverage containing 110 mg of quercetin glycoside as isoquercitrin for 40 weeks. Cognitive function assessment by Cognitrax was the endpoint of the study. The participants were assessed for CBF, health-related quality of life, as well as physical, biological, and hematological parameters, and lateral index. RESULTS: Cognitrax demonstrated that the reaction time significantly improved in the quercetin glycoside intake group. The CBF measurement suggested that quercetin glycoside intake could likely suppress the decrease in cerebral blood volume, CBF, and cerebral activity owing to stress alleviation and inhibition of the accumulation of amyloid ß (Aß), a waste product in the brain, although there were no significant differences between the groups. CONCLUSIONS: Quercetin glycoside intake as a beverage could improve reaction time and may potentially inhibit the decrease in CBF and suppress Aß accumulation.
Asunto(s)
Cognición , Quercetina , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Péptidos beta-Amiloides/farmacología , Circulación Cerebrovascular/efectos de los fármacos , Cognición/efectos de los fármacos , Método Doble Ciego , Glicósidos/farmacología , Calidad de Vida , Quercetina/farmacologíaRESUMEN
OBJECTIVE: Partially hydrolyzed guar gum (PHGG), a water-soluble dietary fiber produced by the controlled partial enzymatic hydrolysis of guar gum beans, has various physiological roles. PHGG is expected to influence the immune function and prevent infections. The objective of this study was to examine the effect of continuous ingestion of PHGG for 12 weeks on the development of cold-like symptoms. PATIENTS AND METHODS: A placebo-controlled, double blind, randomized, parallel-group comparative study was conducted. 96 healthy Japanese adults received 5.2 g PHGG or placebo daily for 12 weeks. Cold-like symptoms were assessed based on patient diary, and the levels of short-chain fatty acids (SCFAs) in stool and blood immune markers at baseline and at weeks 6 and 12. RESULTS: The cumulative number of "no symptoms" days for all symptoms was significantly larger in the PHGG than in the placebo group. The result of the analysis by severity of cold-like symptoms also showed significant differences, with the PHGG group having a lower severity of cold-like symptoms. Propionic acid at weeks 6 and 12 and n-butyric acid and total SCFAs at week 12 were significantly higher in the PHGG than in the placebo group. The Interferon-γ level was significantly lower at week 6 in the PHGG than in the placebo group. CONCLUSIONS: PHGG intake may affect immune function and suppress cold-like symptoms through the production of SCFAs in healthy adults.
Asunto(s)
Galactanos , Gomas de Plantas , Adulto , Fibras de la Dieta , Heces , Humanos , Hidrólisis , Mananos/uso terapéutico , Gomas de Plantas/uso terapéuticoRESUMEN
Previous studies have shown that neutralization of macrophage migration inhibitory factor (MIF) by anti-MIF antibody reduces intestinal inflammation in mice. In this study we tested whether or not anti-MIF autoantibody induced by DNA vaccine targeting MIF protects mice against experimental colitis. Mice were administered a MIF-deoxyribonucleic acid (DNA) vaccine by introducing oligonucleotides encoding helper T epitope into the cDNA sequence of murine MIF by in vivo electroporation. Preventive effects of this method against dextran sulphate sodium-induced (DSS) colitis were evaluated. Mice administered with MIF-DNA vaccine raised values of autoantibody significantly. The clinical and histological findings of colitis induced by 3·0% DSS solution were ameliorated significantly in mice treated with MIF-DNA vaccine compared with saline or pCAGGS-treated mice given DSS. Myeloperoxidase activity, infiltration of F4/80-positive staining cells and the levels of proinflammatory cytokines were suppressed in the colon of MIF-DNA vaccine treated mice compared with saline or pCAGGS-treated mice exposed to DSS. Our results suggest that immunization with helper T epitope DNA-vaccine targeting MIF may be a useful approach for the treatment of colitis including inflammatory bowel diseases.
Asunto(s)
Colitis/prevención & control , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Vacunas de ADN/uso terapéutico , Animales , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Colitis/inducido químicamente , Citocinas/análisis , Sulfato de Dextran/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Peroxidasa/análisisRESUMEN
Sepsis is a common and frequently fatal condition and there is an urgent need for new therapies that will further reduce sepsis-induced mortality. Macrophage migration inhibitory (MIF) factor is important in the regulation of innate and adaptive immunity and is believed to play a key regulatory role in sepsis and autoimmune disease. As MIF deficiency or immunoneutralization protects mice or rats from fatal endotoxic shock or other inflammatory diseases, we examined whether DNA vaccination against this molecule would also be protective. DNA vaccines can stimulate both humoral and cellular immunity simultaneously and have been shown to be effective against a variety of pathogens or cytokine-driven pathologies. Mice were immunized with a MIF/tetanus toxin (TTX) DNA vaccine and sepsis was then induced by lipopolysaccharide or cecal ligation and puncture. The MIF/TTX DNA-vaccinated mice were protected from the lethal effect of sepsis compared with control-vaccinated mice in both models. Compared with the control-vaccinated mice, the MIF/TTX DNA-vaccinated mice also showed significantly lower serum tumor necrosis factor (TNF)-alpha protein levels and reduced mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, macrophage inflammatory protein-2 and Toll-like receptor-4 in the lungs. Thus, the MIF/TTX DNA vaccine may be useful for the prophylaxis of septic shock.
Asunto(s)
Terapia Genética/métodos , Factores Inhibidores de la Migración de Macrófagos/genética , Choque Séptico/terapia , Vacunas de ADN/administración & dosificación , Animales , Anticuerpos/análisis , Biomarcadores/sangre , Ciego/lesiones , Quimiocina CXCL2/sangre , Interleucina-6/sangre , Ligadura , Lipopolisacáridos/farmacología , Pulmón/inmunología , Factores Inhibidores de la Migración de Macrófagos/inmunología , Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Choque Séptico/inmunología , Piel/lesiones , Receptor Toll-Like 4/sangre , Factor de Necrosis Tumoral alfa/sangre , Cicatrización de HeridasRESUMEN
In order to clarify the role of cytokines in the remodelling of the grafted tendon for ligament reconstruction we compared the responses to interleukin (IL)-1beta, platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-beta1 of extrinsic fibroblasts infiltrating the frozen-thawed patellar tendon in rats with that of the normal tendon fibroblasts, in regard to the gene expression of matrix metalloproteinase (MMP)-13, using Northern blot analysis. We also examined, immunohistologically, the local expression of IL-1beta, PDGF-BB, and TGF-beta1 in fibroblasts infiltrating the frozen-thawed patellar tendon. Northern blot analysis showed that fibroblasts derived from the patellar tendon six weeks after the freeze-thaw procedure in situ showed less response to IL-1beta than normal tendon fibroblasts with respect to MMP-13 mRNA gene expression. The immunohistological findings revealed that IL-1beta was over-expressed in extrinsic fibroblasts which infiltrated the patellar tendon two and six weeks after the freeze-thaw procedure in situ, but neither PDGF-BB nor TGF-beta1 was over-expressed in these extrinsic fibroblasts. Our findings indicated that IL-1beta had a close relationship to matrix remodelling of the grafted tendon for ligament reconstruction, in addition to the commencement of inflammation during the tissue-healing process.
Asunto(s)
Fibroblastos/efectos de los fármacos , Interleucina-1/farmacología , Metaloproteinasa 13 de la Matriz/metabolismo , Ligamento Rotuliano/efectos de los fármacos , Animales , Becaplermina , Northern Blotting , Fibroblastos/fisiología , Interleucina-1/metabolismo , Ligamento Rotuliano/metabolismo , Ligamento Rotuliano/fisiología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
We have previously reported on suppression of the PGE2 production in PMA- and calcium ionophore A23187-stimulated macrophages isolated from vitamin E-treated rats. To further study the mechanism, we examined the effect of vitamin E on phospholipase A2 activity in both intact macrophages and cell-free homogenates measuring the release of [14C]arachidonic acid. In macrophages from vitamin E-treated rats, arachidonic acid release in intact cells as stimulated with PMA and calcium ionophore A23187 was hardly detected. In the cell-free homogenates, increase in phospholipase A2 activity of cytosol and particulate fractions by PMA and A23187 was partially suppressed. In unstimulated macrophages, most of phospholipase A2 was recovered in the cytosol fraction. The partially purified cytosolic phospholipase A2 showed a molecular mass 95 kDa on TSK gel G3000SW gel-filtration and on Western blot analysis using anti-rabbit platelet phospholipase A2 monoclonal antibody RHY-5. The activity of cytosolic 95 kDa phospholipase A2 was not inhibited in vitro by vitamin E. From these results, it was suggested that vitamin E needs intact macrophages to suppress arachidonic acid release.
Asunto(s)
Ácido Araquidónico/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Fosfolipasas A/metabolismo , Vitamina E/farmacología , Animales , Radioisótopos de Carbono , Sistema Libre de Células/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Dinoprostona/metabolismo , Activación Enzimática/efectos de los fármacos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/metabolismo , Masculino , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A2 , Ratas , Ratas WistarRESUMEN
After the cDNA of human macrophage migration inhibitory factor (MIF) was cloned in 1989, this protein has been re-evaluated as a pro-inflammatory cytokine, pituitary hormone and glucocorticoid-induced immunoregulatory protein. We previously reported the expression of MIF in the basal cell layers of the epidermis, but its pathophysiological function in the skin has not been well understood. In this study, we examined the expression of MIF during the wound healing of rat skin injured by excision. Reverse transcription-polymerase chain reaction in combination with Southern blot analysis revealed that the increase of MIF mRNA expression was biphasic. The maximum peaks were observed at 3 and 24 h after the injury. Similarly, maximal increases of the serum MIF level were observed at 3 and 24 h after the injury. Immunohistochemical analysis at 12 h after injury demonstrated enhanced expression of MIF protein in the whole epidermal lesion of the wound tissue. By the Boyden chamber assay, we demonstrated that MIF had a chemotactic effect on freshly prepared keratinocytes from rat skin. Additionally, cultured fibroblasts from the skin wound lesion secreted a higher amount of MIF in response to lipopolysaccharide compared to those of the normal skin. Furthermore, administration of anti-MIF antibodies induced a delay of wound healing in vivo. Taken together, these results suggest that MIF contributes to the wound healing process of skin tissue.
Asunto(s)
Epidermis/metabolismo , Fibroblastos/metabolismo , Factores Inhibidores de la Migración de Macrófagos/biosíntesis , Piel/metabolismo , Cicatrización de Heridas/fisiología , Animales , Anticuerpos/farmacología , Southern Blotting , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Epidermis/lesiones , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Inmunohistoquímica , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Lipopolisacáridos/farmacología , Factores Inhibidores de la Migración de Macrófagos/sangre , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/inmunología , Factores Inhibidores de la Migración de Macrófagos/farmacología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/lesiones , Factores de TiempoRESUMEN
D-Dopachrome tautomerase converts 2-carboxy-2,3-dihydroindole-5, 6-quinone (D-dopachrome) into 5,6-dihydroxyindole. The amino acid sequence of this protein is 27% identical with that of macrophage migration inhibitory factor, which is known as a cytokine, pituitary hormone, and glucocorticoid-induced immunomodulator. In this study, we isolated and sequenced a 3490 bp-long genomic DNA of mouse D-dopachrome tautomerase that consists of three exons and two introns. By two procedures, 5' rapid amplification of cDNA ends and cap site labeling, we determined the transcription initiation site, which is located 46 bp upstream of the translation initiation site. The possible polyadenylation sequence (AATAAA) is located 180 bp downstream of the termination codon. Computer-assisted analysis of the nucleotide sequence revealed a number of regulatory motifs, including multiple sites for Sp1, C/EBP, NF-Y, and USF. Although the precise pathophysiological functions of D-dopachrome tautomerase remain to be elucidated, the present results will contribute not only to elucidation of the mechanism of gene expression, but also to understanding of the molecular function of this protein.
Asunto(s)
Oxidorreductasas Intramoleculares/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , Factores Inhibidores de la Migración de Macrófagos/química , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
In order to examine the possible role of vitamin E on the modulation of macrophages, we investigated the effect of vitamin E on O2- and PGE2 production in macrophages. The production of both PGE2 and O2- in rat peritoneal macrophages was dose-dependently stimulated by the addition of PMA and calcium ionophore A23187. However, the macrophages obtained after intraperitoneal injection of vitamin E for six successive days showed less PGE2 and O2- production when stimulated with PMA or A23187 as compared to those of control macrophages. O2- production in control macrophages stimulated with 139 nM PMA and 1 microM A23187 as 4.2 +/- 0.3 and 3.0 +/- 0.2 nmol/min per 10(6) cells, respectively. On the other hand, O2- production by the macrophages from vitamin E-treated rats was 1.5 +/- 0.4 nmol/min per 10(6) cells when stimulated with the PMA, and was not detectable when stimulated with A23187. As for the production of PGE2, control macrophages produced 2.59 +/- 0.70 ng/30 min per 10(6) cells when stimulated with PMA and 8.96 +/- 3.26 ng/30 min per 10(6) cells with the A23187, whereas PGE2 production by the macrophages from vitamin E-treated rats was reduced to 12-20% of the control. By analyzing alpha-tocopherol content and intracellular concentration of calcium ion [( Ca2+]i) in the macrophages isolated from control and vitamin E-treated rats, vitamin E treatment augmented alpha-tocopherol content (384.7 +/- 76.1 vs. 1.2 +/- 0.4 ng/10(6) cells) and decreased free [Ca2+]i when stimulated with A23187 (652 +/- 14 vs. 1201 +/- 223 nM).
Asunto(s)
Dinoprostona/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Superóxidos/metabolismo , Vitamina E/farmacología , Animales , Calcimicina/farmacología , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Fura-2 , Macrófagos/metabolismo , Masculino , Peritoneo , Ratas , Ratas Endogámicas , Acetato de Tetradecanoilforbol/farmacología , Vitamina E/administración & dosificaciónRESUMEN
Combined stimulation, by superoxide ions generated by the xanthine-xanthine oxidase reaction, and platelet-activating factor (PAF), induced cell differentiation of rat monocytic leukemia cells (c-WRT-LR) to macrophage-like mature cells. Monitoring of cytochrome c reduction revealed that PAF stimulation induced the release of superoxide ions from c-WRT-LR. To further investigate the effect of superoxide ions in the autocrine or paracrine mechanism in cell differentiation, molecular species of the oxygen radicals under PAF stimulation were examined using the EPR spin trap, 5,5'-dimethyl-1-pyrroline N-oxide (DMPO). PAF and/or phorbol myristate acetate caused the formation of EPR spectra, a combination of DMPO/.OOH and DMPO/.OH. Since both spectra were diminished in the presence of superoxide dismutase, it was concluded that DMPO/.OH was derived from superoxide ions. Mannitol and catalase suppressed cell differentiation induced by combined stimulation with PAF and oxygen radicals generated by the xanthine-xanthine oxidase reaction. Taken together, these results suggest that hydroxyl radicals generated by Fenton reaction from H2O2 may be involved in the mechanism of cell differentiation in rat monocytic leukemia cells.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Depuradores de Radicales Libres , Factor de Activación Plaquetaria/farmacología , Superóxidos/metabolismo , Animales , Catalasa/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Leucemia Experimental , Leucemia Mieloide , Manitol/farmacología , Ratas , Ratas Endogámicas , Superóxido Dismutasa/farmacología , Superóxidos/farmacología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
To clarify the role of vitamin E (alpha-tocopherol) for the induction of cyclooxygenase-2 (COX-2) in rat macrophages stimulated by lipopolysaccharide (LPS), vitamin E-enriched macrophages were prepared by intraperitoneal injection of vitamin E for 6 days at a rate of 5 mg per day. The production of PGE2 was increased in dose- and time-dependent manners by addition of LPS in both control and vitamin E-enriched peritoneal macrophages. The maximum effect of LPS was observed in 12 h at concentration of 5 micrograms/ml. By analyzing COX-2 mRNA level by Northern blot and COX-2 enzyme mass and phosphotyrosine by Western blot, it was revealed that the increase of PGE2 production reflected the induction of COX-2 expression through activation of tyrosine kinase. Vitamin E failed to inhibit PGE2 production in LPS-stimulated macrophages; however, genistein, a tyrosine kinase inhibitor, completely inhibited the production at 100 microM. These results suggest that vitamin E does not inhibit COX-2 expression via LPS-mediated tyrosine kinase signal transduction pathway.
Asunto(s)
Isoenzimas/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Vitamina E/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Antioxidantes/farmacología , Ciclooxigenasa 2 , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Genisteína , Isoflavonas/farmacología , Macrófagos Peritoneales/enzimología , Masculino , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
We expressed rat macrophage migration inhibitory factor (rMIF) in E. coli using the cDNA isolated from a rat liver cDNA library. rMIF specifically bound glutathione (dissociation constant = 500 microM). We purified rMIF homogeneously on SDS-PAGE by S-hexylglutathione Sepharose affinity column chromatography and Sephadex G-100 column chromatography. The amino-acid sequence of rMIF was highly homologous to that of human MIF from a T-cell line; only a single amino-acid residue was substituted if conservative amino-acid substitutions were involved. The molecular weight of rMIF was calculated to be 12.4 kDa and 23.6 kDa by SDS-PAGE and analytical ultracentrifugation, respectively. Thus, it was concluded that the native rMIF formed a homodimeric structure. Proton nuclear magnetic resonance (1H-NMR) study revealed that rMIF was less thermostable (the denaturing temperature was from 50-60 degrees C) than human MIF (the denaturing temperature is about 80 degrees C (Nishihira et al. (1993) Biochem. Mol. Biol. Int. 31, 841-850). The secondary structure of rMIF evaluated by 1H-NMR experiments revealed that the contents of alpha-helix, beta-strand, and coil were 13.8%, 55.6%, and 30.6%, respectively.
Asunto(s)
Hígado/metabolismo , Factores Inhibidores de la Migración de Macrófagos/aislamiento & purificación , Macrófagos/metabolismo , Animales , Escherichia coli/metabolismo , Factores Inhibidores de la Migración de Macrófagos/biosíntesis , Espectroscopía de Resonancia Magnética , Peso Molecular , Estructura Secundaria de Proteína , Ratas , UltracentrifugaciónRESUMEN
Macrophage migration inhibitory factor (MIF) was identified in rat peritoneal macrophages by Western blot analysis and its secretion into culture medium by enzyme-linked immunosorbent assay. We investigated the effect of vitamin E on MIF production in macrophages in response to phorbol 12-myristate-13-acetate (PMA), calcium ionophore A23187, and lipopolysaccharide (LPS). Intraperitoneal injections of vitamin E (5 mg per rat) for 6 successive days resulted in a significant increase of alpha-tocopherol content in peritoneal macrophages (478.3+/-90.7 ng/106 cells) compared with the control (1.5+/-0.5 ng/10(6) cells). For the control macrophages, MIF content of the medium (2.5x10(6) cells/18 ml) without stimulation was 2.27+/-0.20 ng/ml after 14 h culture, whereas stimulation with calcium ionophore A23187 (400 nM) and LPS (5.0 microg/ml) induced the elevation of MIF content to 3. 66+/-0.41 and 4.12+/-0.58 ng/ml, respectively. On the other hand, vitamin E-enriched macrophages without stimulation showed less MIF content (0.77+/-0.23 ng/ml) than the control. Similarly, the increase of MIF of vitamin E-treated macrophages was significantly suppressed after stimulation with calcium ionophore A23187 or LPS, compared with the control macrophages. From analysis of intracellular MIF content by Western blot, we found no alteration of intracellular MIF content of vitamin E-macrophages, in contrast to the decreased content of control stimulated-macrophages. Taken together, these results indicate that vitamin E may contribute to the regulation of immune responses through regulation of MIF secretion.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Vitamina E/farmacología , Animales , Calcimicina , Medios de Cultivo/análisis , Interleucina-6/análisis , Lipopolisacáridos , Macrófagos Peritoneales/metabolismo , Masculino , Ratas , Ratas Wistar , Acetato de Tetradecanoilforbol , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Macrophage migration inhibitory factor (MIF) is known as a pluripotent immunoregulatory cytokine involved in T-cell activation and inflammatory responses; however, no study on this protein in the peripheral nervous systems has been carried out. We here demonstrated for the first time expression of MIF mRNA and MIF protein in rat sciatic nerves by reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Immunohistochemical analysis revealed positive staining of MIF, which was largely observed in Schwann cells. Furthermore, we examined MIF mRNA expression in the sciatic nerves by Northern blot analysis in the case of nerve transection. In both proximal and distal segments, the level of MIF mRNA started to increase 12 h after the nerve transection. The level remained high from 24 h up to day 7 after the injury. During the period from days 14 to 21, MIF mRNA sharply decreased to the pre-transection level. In immunohistochemistry, positive staining of MIF was largely observed in axons as well as non-neuronal cells in proximal segments at day 4 after transection. In the distal segments, contrastingly, endoneurial fibroblasts or Schwann cells migrating into neuronal fibers showed positive staining with Wallerian degeneration. Although the precise functions of MIF in the peripheral nerves remain to be elucidated, the present results could represent a major departure from the current state of knowledge, revealing a novel function in the degenerative-regenerative process.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/análisis , Regeneración Nerviosa , Nervios Periféricos/fisiología , Animales , Axotomía , Northern Blotting , Inmunohistoquímica , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/inmunología , Masculino , Nervios Periféricos/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Wistar , Nervio Ciático/metabolismoRESUMEN
Macrophage migration inhibitory factor (MIF) is known as a proinflammatory cytokine, glucocorticoid-induced immunomodulator, and pituitary hormone, and contributes to broad-spectrum immune and inflammatory response. To investigate the expression of MIF in the central nervous system in an event of viral infection, we evaluated MIF mRNA expression in the mouse brain infected with Japanese encephalitis virus (JEV). In situ hybridization revealed that MIF mRNA expression was significantly up-regulated in the whole brain by intracranial JEV inoculation at 2 days post-inoculation (d.p.i.). Neurons as well as glial cells expressed MIF transcripts in which some of these cells were co-labeled by double staining for JEV antigens and MIF mRNA. At 4 d.p.i., when typical symptoms of encephalitis were observed, JEV antigen-positive cells were much increased in parallel with enhanced MIF mRNA, consistent with the results of Northern blot analysis. Reverse transcription-polymerase chain reaction showed that MIF mRNA was minimally changed at 1 d.p.i. in comparison with that at 0 d.p.i., but markedly up-regulated after 2 d.p.i. and sustained up to 4 d.p.i. On the other hand, a significant increase of tumor necrosis factor (TNF)-alpha mRNA was observed after only 3 d.p.i. These data suggest the possibility that MIF is involved in virus-induced encephalitis with regard to not only immune responses in the early stage, but also the exacerbation of inflammation in concert with TNF-alpha in the late stages. This is the first evidence demonstrating that MIF is up-regulated in the case of virus-induced encephalitis, which should contribute to the further understanding of the pathological mechanism of JEV-induced encephalitis.
Asunto(s)
Encéfalo/metabolismo , Virus de la Encefalitis Japonesa (Especie) , Factores Inhibidores de la Migración de Macrófagos/genética , ARN Mensajero/biosíntesis , Animales , Antígenos Virales/análisis , Encéfalo/virología , Corteza Cerebral/metabolismo , Corteza Cerebral/virología , Modelos Animales de Enfermedad , Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Viral/virología , Regulación de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Ratones , ARN Mensajero/análisis , Regulación hacia ArribaRESUMEN
The C-terminal region of rat glutathione S-transferase P (GST-P) was deleted by either carboxypeptidase (CPase) A and B or site-specific truncation to evaluate the role of the region in the catalytic mechanism. The C-terminal sequence from the 201st to 209th amino-acid residues is Arg-Pro-Ile-Asn-Gly-Asn-Gly-Lys-Gln. When seven of the C-terminal amino-acid residues from the C-terminus were removed by the CPases, the catalytic activity decreased in parallel with the amino-acid removal, amounting to less than 5% of that of the wild-type GST-P. On the other hand, a decrease of the catalytic activity was observed in a different manner when the C-terminal sequence was site-specifically truncated. The VmaxGSH/KmGSH values of the mutants withthree (GSTd207-209), four (GSTd206-209) and seven (GSTd203-209) C-terminal amino-acid residues deleted, were comparable or similar to that of the wild-type GST-P, whereas those of five (GSTd205-209), six (GSTd204-209), and eight (GSTd202-209) amino-acid residue-truncated mutants decreased to 43%, 40%, and 19% of that of the wild-type GST-P, respectively. Similar results were obtained as for VmaxCDNB/KmCDNB. The nine amino-acid residue-truncated mutant showed no catalytic activity. Heat treatment at 50 degrees C for 5 min had little effect on the catalytic activities of the wild-type GST-P and GSTd204-209, whereas those of GSTd207-209, GSTd206-209, GSTd203-209 and GSTd202-209 decreased to 22%, 27%, 18% and 10%, respectively, compared to the catalytic activity of the non-treated enzymes. Considering these results, it is concluded that the C-terminal region, Arg201-Gln209, has an important role in stabilizing the active-site conformation.
Asunto(s)
Glutatión Transferasa/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Arginina , Sitios de Unión , Carboxipeptidasas , Catálisis , Estabilidad de Enzimas , Glutatión Transferasa/genética , Glicina , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutación , Conformación Proteica , RatasRESUMEN
Two crystal forms of the macrophage migration inhibitory factor (MIF) from human lymphocytes have been obtained by the hanging drop method of vapor diffusion from ammonium sulfate solution. A trigonal crystal form belongs to the space group P3(1)21 (P3(2)21), with unit cell dimensions of a = b = 96.4 A and c = 105.5 A. Assuming that the asymmetric unit contains two or three dimers, the Vm value is calculated as 2.9 or 1.9, respectively. A second crystal form is orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions a = 68.4 A, b = 68.8 A and c = 86.8 A. The Vm value is 2.1 for two dimers in the asymmetric unit. Both crystals diffract to at least 1.9 A and are suitable for X-ray crystallographic structure determination at a high resolution.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/química , Secuencia de Bases , Cristalografía por Rayos X , Cartilla de ADN/química , Humanos , Linfocitos/química , Datos de Secuencia Molecular , Proteínas RecombinantesRESUMEN
OBJECTIVE: Macrophage migration inhibitory factor (MIF), which plays a pivotal role in the control of inflammatory responses, was first characterized as a T-cell cytokine, but later was also found as a pituitary peptide released in response to infection and stress. However, MIF's role and expression in the myocardium has never been reported. The goal of this study is to examine MIF in the myocardium. METHODS AND RESULTS: MIF protein and mRNA levels were assayed using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Increased MIF concentrations were detected in the sera of patients with acute myocardial infarction (AMI). In cultured rat cardiac myocytes, significant amounts of MIF were produced in response to hypoxia and hydrogen peroxide (H(2)O(2)), but not to angiotensin II, endothelin-1, interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNFalpha). H(2)O(2)-induced MIF production increased in a time- and dose-dependent manner and was completely abolished in the presence of catalase. H(2)O(2) also induced MIF mRNA expression. The H(2)O(2)-induced MIF production was completely inhibited by the protein kinase C (PKC) inhibitor GF109203X, partially inhibited by the tyrosine kinase inhibitor herbimycin A, and uninhibited by calcium chelation or phorbol ester-sensitive PKC down-regulation. This suggests that H(2)O(2)-induced MIF production is mediated by an atypical PKC isoform. DNA microarray analysis revealed that 52 genes were preferentially expressed in response to MIF. Of these, the MIF-induced expression of both glutathione S-transferase (GST) and lipopolysaccharide-induced CXC chemokine (LIX) mRNAs was confirmed using RT-PCR analysis. CONCLUSION: The present results suggest that MIF is expressed by the myocardium in response to redox stress and may play a role in the pathogenesis of myocardial ischemia.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/fisiología , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Análisis de Varianza , Animales , Benzoquinonas , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación de la Expresión Génica , Humanos , Peróxido de Hidrógeno/farmacología , Indoles/farmacología , Lactamas Macrocíclicas , Factores Inhibidores de la Migración de Macrófagos/análisis , Factores Inhibidores de la Migración de Macrófagos/genética , Masculino , Maleimidas/farmacología , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidación-Reducción , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Quinonas/farmacología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rifabutina/análogos & derivadosRESUMEN
Human epidermal cells are capable of secreting various cytokines with immunologic, inflammatory, and proliferative properties. In a previous study, by reverse transcription-polymerase chain reaction and immunohistochemical analysis, we have shown that human epidermal keratinocytes express macrophage migration inhibitory factor and identified its presence in the cytoplasm. In this study, we detected an increased serum macrophage migration inhibitory factor level by enzyme-linked immunosorbent assay after a single total-body ultraviolet B exposure in vivo, indicating that human keratinocytes respond and release this cytokine in response to ultraviolet B irradiation. Moreover, we evaluated the effect of ultraviolet B on migration inhibitory factor production in cultured human epidermal keratinocytes and epidermal sheets. The results of enzyme-linked immunosorbent assay and northern blot analyses showed that migration inhibitory factor production of cultured keratinocytes was increased by ultraviolet B exposure. During the past few years, migration inhibitory factor was found to have a variety of biologic functions, such as being essential for T cell activation and induction of inflammatory cytokines. In this context, these results should encourage further investigation on the pathophysiologic role of migration inhibitory factor in cutaneous inflammatory reactions and immune responses.
Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/biosíntesis , Rayos Ultravioleta , Supervivencia Celular/efectos de la radiación , Humanos , Inmunohistoquímica , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Piel/citología , Factores de Tiempo , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
We identified macrophage migration inhibitory factor (MIF) mRNA expression in human cornea, and demonstrated its immunohistological localization. Reverse transcription-polymerase chain reaction analysis revealed that MIF mRNA was expressed in both the corneal epithelial and endothelial cells. Immunohistochemical study using the polyclonal antibody prepared from immunizing a rabbit with human recombinant MIF showed that MIF was present in the basal cells of corneal epithelium and endothelial cells. The fact that MIF exists in those cells of the cornea indicates that MIF may play an important role in corneal cell immunity and cellular differentiation.