RESUMEN
Staphylococcus aureus produces a variety of exoproteins that interfere with host immune systems. We attempted to purify cytotoxins against human leukocytic cells from the culture supernatant of S. aureus by a combination of ammonium sulfate precipitation, ion-exchange chromatography on a CM-cellulose column and HPLC on a Mono S 5/50 column. A major protein possessing cytotoxicity to HL60 human promyelocytic leukemia cells was purified, and the protein was identified as α-hemolysin (Hla, α-toxin) based on its molecular weight (34 kDa) and N-terminal amino acid sequence. Flow cytometric analysis suggested differential cytotoxicity of Hla against different human peripheral blood leukocyte populations. After cell fractionation with density-gradient centrifugation, we found that peripheral blood mononuclear cells (PBMCs) were more susceptible to Hla than polymorphonuclear leukocytes. Moreover, cell surface marker analysis suggested that Hla exhibited slightly higher cytotoxicity against CD14-positive PBMCs (mainly monocytes) than CD3- or CD19-positive cells (T or B lymphocytes). From these results, we conclude that human leukocytes have different susceptibility to Hla depending on their cell lineages, and thereby the toxin may modulate the host immune response.
Asunto(s)
Toxinas Bacterianas/farmacología , Proteínas Hemolisinas/farmacología , Leucocitos/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Leucocitos/inmunologíaRESUMEN
The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/coronin-1 antibodies and HL60 cell lysates revealed that ß-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/coronin-1 regulates its interaction with actin.
Asunto(s)
Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfotreonina/metabolismo , Secuencia de Aminoácidos , Benzofenantridinas/farmacología , Electroforesis en Gel Bidimensional , Células HEK293 , Células HL-60 , Humanos , Proteínas de Microfilamentos/química , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fagocitosis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Serina/metabolismoRESUMEN
Elastic fibers play an important role in the characteristic resilience of many tissues. The assembly of tropoelastin into a fibrillar matrix is a complex stepwise process and the deposition and cross-linking of tropoelastin are believed to be key steps of elastic fiber formation. However, the detailed mechanisms of elastic fiber assembly have not been defined yet. Here, we demonstrate the relationship between deposition and the cross-linking/maturation of tropoelastin. Our data show that a C-terminal half-fragment of tropoelastin encoded by exons 16-36 (BH) is deposited onto microfibrils, yet we detect very limited amounts of the cross-linking amino acid, desmosine, an indicator of maturation, whereas the N-terminal half-fragment encoded by exons 2-15 (FH) was deficient for both deposition and cross-linking, suggesting that elastic fiber formation requires full-length tropoelastin molecules. A series of experiments using mutant BH fragments, lacking either exon 16 or 30, or a deletion of both exons showed that self-association of tropoelastin polypeptides was an early step in elastic fiber assembly. Immunofluorescence and Western blot assay showed that the treatment of cell culture medium or conditioned medium with beta-aminopropionitrile to inhibit cross-linking, prevented both the deposition and polymerization of tropoelastin. In conclusion, our present results support the view that self-association and oxidation by lysyl oxidase precedes tropoelastin deposition onto microfibrils and the entire molecule of tropoelastin is required for this following maturation process.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Tropoelastina/química , Animales , Western Blotting , Bovinos , Supervivencia Celular , Desmosina/química , Ensayo de Inmunoadsorción Enzimática , Exones , Fibrilinas , Proteínas de Microfilamentos/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
Fibulin-5 is believed to play an important role in the elastic fiber formation. The present experiments were carried out to characterize the molecular interaction between fibulin-5 and tropoelastin. Our data showed that the divalent cations of Ca(2+), Ba(2+) and Mg(2+) significantly enhanced the binding of fibulin-5 to tropoelastin. In addition, N-linked glycosylation of fibulin-5 does not require for the binding to tropoelastin. To address the fibulin-5 binding site on tropoelastin constructs containing, exons 2-15 and exons 16-36, of tropoelastin were used. Fibulin-5 binding was significantly reduced to either fragment and also to a mixture of the two fragments. These results suggested that the whole molecule of tropoelastin was required for the interaction with fibulin-5. In co-immunoprecipitation experiments, tropoelastin binding to fibulin-5 was enhanced by an increase of temperature and sodium chloride concentration, conditions that enhance the coacervation of tropoelastin. The binding of tropoelastin fragments to fibulin-5 was directly proportional to their propensity to coacervate. Furthermore, the addition of fibulin-5 to tropoelastin facilitated coacervation. Taken together, the present study shows that fibulin-5 enhances elastic fiber formation in part by improving the self-association properties of tropoelastin.
Asunto(s)
Proteínas de la Matriz Extracelular/química , Tropoelastina/química , Animales , Sitios de Unión , Bovinos , Proteínas de la Matriz Extracelular/metabolismo , Glicosilación , Humanos , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Tropoelastina/metabolismoRESUMEN
Naturally occurring mutations are useful in identifying domains that are important for protein function. We studied a mutation in the elastin gene, 800-3G>C, a common disease allele for SVAS (supravalvular aortic stenosis). We showed in primary skin fibroblasts from two different SVAS families that this mutation causes skipping of exons 16-17 and results in a stable mRNA. Tropoelastin lacking domains 16-17 (Delta16-17) was synthesized efficiently and secreted by transfected retinal pigment epithelium cells, but showed the deficient deposition into the extracellular matrix compared with normal as demonstrated by immunofluorescent staining and desmosine assays. Solid-phase binding assays indicated normal molecular interaction of Delta16-17 with fibrillin-1 and fibulin-5. However, self-association of Delta16-17 was diminished as shown by an elevated coacervation temperature. Moreover, negative staining electron microscopy confirmed that Delta16-17 was deficient in forming fibrillar polymers. Domain 16 has high homology with domain 30, which can form a beta-sheet structure facilitating fibre formation. Taken together, we conclude that domains 16-17 are important for self-association of tropoelastin and elastic fibre formation. This study is the first to discover that domains of elastin play an essential role in elastic fibre formation by facilitating homotypic interactions.
Asunto(s)
Tejido Elástico/fisiología , Tropoelastina/química , Secuencia de Aminoácidos , Estenosis Aórtica Supravalvular/genética , Estenosis Aórtica Supravalvular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Microfibrillas/fisiología , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , Transfección , Tropoelastina/genética , Tropoelastina/metabolismoRESUMEN
Mönckeberg-type arteriosclerosis occurs as a complication in diabetic, uremic patients and in postmenoposal women. It has been shown that arterial calcification generates loss of elasticity in tunica media. We have already reported that the expression of tropoelastin (TE), the precursor protein of elastin, is suppressed by arterial calcification, although no changes of mRNA expression of the other elastic fiber components, such as fibrillins, was observed. We examined the effects of bisphosphonates, known as anti-osteoporotic drugs, in inorganic phosphate (Pi)-induced calcified bovine aortic smooth muscle cells (BASMCs) (in vitro arterial calcification model). Treatment with the bisphosphonate risedronate, significantly inhibited calcium deposition in the arterial calcification model. Risedronate also inhibited suppression of TE mRNA expression and the progression of osteopontin (OPN) and core binding factor-alpha1 (Cbfa1), an osteogenic transcription factor, by BASMCs calcification. Basically, bisphosphonates could inhibit phenotypic transition such as SMC to osteoblast-like cell. Inhibitory effects of bisphosphonates were also shown in female Sprague-Dawley rats with calcinosis induced by administration of an over-dose of vitamin D2 (in vivo arterial calcification model). It is known that arterial calcification is accelerated by oxidative low-density lipoprotein (oxLDL). Therefore we examined the effects of 7-ketocholesterol (7kc), a component of oxLDL, on in vitro arterial calcification. Thereupon, it was revealed that 7kc drastically accelerated Pi-induced calcification, and risedronate completely restored the calcification and mRNA expression accelerated by 7kc.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Ácido Etidrónico/análogos & derivados , Matriz Extracelular/metabolismo , Esclerosis Calcificante de la Media de Monckeberg/prevención & control , Tropoelastina/metabolismo , Animales , Bovinos , Ácido Etidrónico/uso terapéutico , Femenino , Humanos , Cetocolesteroles , Lipoproteínas LDL , Esclerosis Calcificante de la Media de Monckeberg/etiología , Esclerosis Calcificante de la Media de Monckeberg/metabolismo , Ratas , Ratas Sprague-Dawley , Ácido RisedrónicoRESUMEN
We examined the responses of basilar arteries taken from Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a type 2 diabetes model. Both the nitric oxide (NO)-mediated relaxation and the cyclic 3',5'-guanosine monophosphate (cGMP) production elicited by acetylcholine (ACh) were much weaker in OLETF rats than in age-matched control Long Evans Tokushima Otsuka (LETO) rats. The contraction induced by an NO synthase (NOS) inhibitor [N(G)-nitro-L-arginine (L-NNA)] was weaker in the OLETF group. In that group, application of apocynin, an NAD(P)H oxidase inhibitor, normalized (i) ACh-induced relaxation, (ii) L-NNA-induced contraction, and (iii) ACh-induced cGMP production to the LETO levels. Superoxide anion production was greater in basilar arteries from OLETF rats than in those from LETO rats. The protein expression of gp91(phox), an NAD(P)H oxidase subunit, was upregulated in the OLETF arteries (versus LETO ones). These results suggest that the existence of endothelial dysfunction in basilar arteries in type 2 diabetes is related to increased oxidative stress mediated via NAD(P)H oxidase. Possibly, an impairment of NO-dependent relaxation responses and a basal impairment of NO signaling may be responsible for the increased risk of adverse cerebrovascular events in type 2 diabetes.
Asunto(s)
Antioxidantes/fisiología , Arteria Basilar/fisiopatología , Endotelio Vascular/fisiopatología , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Óxido Nítrico/metabolismo , Acetofenonas/farmacología , Acetilcolina/fisiología , Animales , Diabetes Mellitus Tipo 2/fisiopatología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Masculino , Glicoproteínas de Membrana/genética , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Óxido Nítrico Sintasa/antagonistas & inhibidores , Estrés Oxidativo , Ratas , Ratas Endogámicas OLETF , Vasoconstricción/fisiología , Vasoconstrictores , Vasodilatación/fisiologíaRESUMEN
AIM: Vascular calcification is a common feature in patients with advanced atherosclerosis, postmenopausal women and patients with renal failure, which results in reduced elasticity of arteries. Pamidronate, a bisphosphonate, is used as a therapeutic agent for anti-osteoporosity, although there are adverse side effects, such as renal damage and aortic inflamed plaque rupture. In the present study, we demonstrated the effects of vitamin K(2) alone or in combination with pamidronate in an arterial calcification model induced using inorganic phosphate in cultured bovine aortic smooth muscle cells (BASMCs). METHODS: Calcification was induced by the addition of Pi (3 mM) in BASMCs. Calcium deposition was determined by Calcium C-test Wako and von Kossa staining. mRNA expression was assessed by semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: Calcium deposition assay and von Kossa staining showed that calcification could be inhibited in a dose-dependent manner by treatment with vitamin K(2) alone, and that its inhibitory effect was enhanced when combined with pamidronate. It was found that the expression of tropoelastin mRNA was synergistically enhanced by combined treatment with vitamin K(2) and pamidronate, and the expression matrix Gla protein mRNA and osteopontin mRNA expression were also enhanced and suppressed, respectively, by treatment with vitamin K(2) or pamidronate. Moreover, our data showed that the suppression of TE expression by siRNA significantly increased Pi-induced vascular calcification. CONCLUSION: Taken together, our study suggests that vitamin K(2) in combination with pamidronate synergistically inhibits arterial calcification via the increased expression of tropoelastin, which would be a useful marker for developing effective therapeutic or prophylactic agents for arterial calcification.
Asunto(s)
Calcinosis/tratamiento farmacológico , Difosfonatos/farmacología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Vitamina K 2/farmacología , Calcinosis/prevención & control , Células Cultivadas , Difosfonatos/uso terapéutico , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Miocitos del Músculo Liso/citología , Pamidronato , Tropoelastina/genética , Regulación hacia Arriba/genética , Vitamina K 2/uso terapéuticoRESUMEN
OBJECTIVE: It is known that elastin mRNA is transcribed from a single gene. The variety of tropoelastin isoforms results from multiple alternative splicing of the primary transcript. The purpose of this study was to investigate the characteristics of elastic fiber assembled with tropoelastin isoform, which is full-length human tropoelastin (HTE), exon 26A missing tropoelastin (Delta26A), and exon 32 missing tropoelastin (Delta32). DESIGN AND METHODS: We demonstrated the process of elastic fiber assembly and the existence of elastic fiber resistant to pancreatic elastase with HTE, Delta26A, or Delta32 fiber using an in vitro model of elastic fiber assembly. These elastic fibers were evaluated by immunofluorescent staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. RESULTS: There were no big differences getting into the matrix among these tropoelastins in immunofluorescence microscopy and semi-quantitative analysis. In the comparison with the HTE, the Delta26A and the Delta32 significantly increased and decreased, respectively, the formation of cross-linking amino acids and the binding to scaffold proteins. Furthermore, it was found that it is difficult to degrade the Delta26A assembly with pancreatic elastase as compared with HTE or Delta32 assembly. CONCLUSION: The elastic fiber assembled with the tropoelastin isoforms was characterized using an in vitro model. The present study provides important information regarding the pathology of human diseases including emphysema and atherosclerosis.
Asunto(s)
Tropoelastina/metabolismo , Empalme Alternativo , Aminoácidos/química , Reactivos de Enlaces Cruzados/química , Exones , Fibrilinas , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Elastasa Pancreática/metabolismo , Isoformas de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tropoelastina/química , Tropoelastina/genéticaRESUMEN
OBJECTIVES: We developed an in vitro model of elastic fiber assembly that provides a comparison of the efficiency of different tropoelastin molecules to organize into fibers. DESIGN AND METHODS: Recombinant tropoelastin was added to ARPE-19 cell culture medium. The elastic fiber assembly was evaluated by immunofluorescence staining, the quantitative analysis of cross-linking amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. RESULTS: We confirmed that ARPE-19 cells express fibrillin-containing microfibrils and lysyl oxidase, but they do not express tropoelastin. Immunofluorescence staining showed a dose- and time-dependent increase in the extracellular matrix. The quantity of cross-linking amino acids and matrix-associated tropoelastin also increased together with the matrix-associated elastin. Moreover, the analysis of a radioimmunoprecipitation assay (RIPA) buffer-soluble fraction indicated that tropoelastin interacted with microfibrils and cross-linked elastin was detected as a super molecular complex. CONCLUSION: These observations indicate that this in vitro model is especially useful for the analysis of mechanisms of elastic fiber formation.
Asunto(s)
Tejido Elástico/fisiología , Epitelio Pigmentado Ocular/metabolismo , Tropoelastina/fisiología , Animales , Bovinos , Línea Celular , Desmosina/metabolismo , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Microfibrillas/metabolismo , Epitelio Pigmentado Ocular/citología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Primary localized cutaneous nodular amyloidosis is a rare form of cutaneous amyloidosis. Amyloid fibrils in primary localized cutaneous nodular amyloidosis have been reported to be originated from immunoglobulin light chains. Immunohistochemical studies on the lesional skins of four patients with primary localized cutaneous nodular amyloidosis demonstrated that amyloid deposits of all cases showed a positive reaction with the antibodies for beta2-microglobulin and advanced glycation end products as well as immunoglobulin light chain (kappa or lambda). No beta2-microglobulin and advanced glycation end product immunoreactivity was found in the amyloid deposits of other primary localized cutaneous amyloidosis (lichen amyloidosis and macular amyloidosis). Double immunofluorescence study of the lesional skin of primary localized cutaneous nodular amyloidosis showed that anti-kappa light chain, anti-beta2-microglobulin and anti-advanced glycation end product antibodies mostly reacted with the same area of amyloid deposit. Amyloid proteins were sequentially extracted with distilled water from one case of primary localized cutaneous nodular amyloidosis and recovered in the five water-soluble fractions (fractions I-V). Immunoblot assay of amyloid fibril proteins demonstrated that immunoreactive polypeptides with anti-kappa light chain antibody (29 kDa) and with anti-beta2-microglobulin antibody (12 kDa) were detected in fractions I-V, whereas immunoreactive polypeptide with anti-advanced glycation end product antibody (12 kDa) was detected exclusively in fractions III-V but not in fractions I and II. Two-dimensional polyacrylamide gel electrophoresis revealed that 12 kDa polypeptide in fractions I and II was electrophoretically identical with authentic beta2-microglobulin and that beta2-microglobulin in fractions III-V was advanced glycation end product-modified beta2-microglobulin with more acidic pI value. These results indicate that beta2-microglobulin is another major component of amyloid fibrils in primary localized cutaneous nodular amyloidosis and that beta2-microglobulin in primary localized cutaneous nodular amyloidosis is partly subjected to the modification of advanced glycation end product.
Asunto(s)
Amiloide/fisiología , Amiloidosis/metabolismo , Productos Finales de Glicación Avanzada/fisiología , Enfermedades de la Piel/metabolismo , Microglobulina beta-2/metabolismo , Anciano , Amiloidosis/patología , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Microscopía Electrónica , Enfermedades de la Piel/patologíaRESUMEN
Atherosclerosis is characterized by inflammatory metabolic change with lipid accumulation in the artery. Atherosclerotic plaque occurs at discrete locations in the arterial system and involves the proliferation of smooth muscle cells (SMCs) together with imbalance of the extracellular matrix elements, elastic fiber in particular. The role of elastin in arterial development and disease was confirmed by generating mice that lack elastin. Thus, elastin is a critical regulatory molecule that regulates the phenotypic modulation, proliferation and migration of SMCs. We estimated that elastin expression and SMC proliferation are coupled inversely: potent stimulators of cell proliferation may potentially inhibit elastin expression and potent inhibitors of cell proliferation can stimulate elastin expression. Moreover, elastin was found to be expressed maximally at the G(0) and minimally at the G(2)/M phase during the cell cycle, suggesting that its expression is regulated by the cell growth state. The elastin peptide VPGVG enhanced SMC proliferation, resulting in the reduction of elastin expression. The inhibition of elastin expression by elastin fragments may be reflected in the negative feedback regulatory mechanism. The relationship between cell proliferation and elastin expression may be changed in atherosclerosis. Areas of atherosclerotic plaque show abnormality of elasticity and permeability from the viewpoint of the physiological function of the arterial wall. The etiology was estimated to be that cholesterol and calcium are deposited on the elastic fiber, resulting in decreased elastin synthesis and cross-linking formation. In addition, these dysfunctions of elastin fiber are also associated, in that the down-regulation of elastin and its related components (fibrillin-1 and lysyl oxidase) are directly related to calcification in SMCs. The denatured arterial elastin by cholesterol and calcium accumulation was also susceptible to proteolytic enzymes such as elastase and matrix metalloproteinase (MMP). Therefore, metabolic change in elastic fiber induces decreased elasticity and is associated with essential hypertension. Vitamin K(2) is used in drug therapy against atherosclerosis, or calcification in diabetes mellitus or dialysis, due to its promotion of the carboxylation of the matrix Gla protein.
Asunto(s)
Arteriosclerosis/patología , Tejido Conectivo/patología , Matriz Extracelular/patología , Miocitos del Músculo Liso/patología , Animales , HumanosRESUMEN
Vascular calcification is a common feature of advanced atherosclerosis resulting in reduced elasticity of elastic arteries. However, the relationship between elastic fibers and vascular calcification at the molecular and cellular levels remains unknown. We investigated the expression of major elastic fiber components such as tropoelastin (TE) and fibrillin-1 (FBN1) and elastin-related enzyme, lysyl oxidase (LO), in a calcification model using beta-glycerophosphate (beta-GP) in cultured bovine aortic smooth muscle cells (BASMCs). Ten mM of beta-GP stimulated calcium deposition in a time-dependent manner. As determined by Western blot analysis, 10 mM of beta-GP time-dependently decreased TE and FBN1 protein levels. TE, FBN1, and LO mRNA levels, assessed by reverse transcription-polymerase chain reaction, were also decreased by exposure to 10 mM beta-GP. Furthermore, we investigated whether the processes of calcification in BASMCs directly control these regulations. In experiments using levamisole, an alkaline phosphatase inhibitor, and DMDP, a bisphosphonate, both inhibitors inhibited down-regulation during beta-GP-induced calcification, suggesting that the down-regulation of TE, FBN1, and LO directly relates to calcium deposition. In cases of vascular calcification, the decreased expression of TE, FBN1, and LO may be partially responsible for decreased vascular elasticity and also for the decreased formation of new elastic fibers.
Asunto(s)
Calcinosis/metabolismo , Proteínas de Microfilamentos/genética , Músculo Liso Vascular/metabolismo , Proteína-Lisina 6-Oxidasa/genética , Tropoelastina/genética , Fosfatasa Alcalina/antagonistas & inhibidores , Animales , Aorta/citología , Bovinos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Fibrilinas , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Glicerofosfatos/farmacología , Levamisol/farmacología , Proteínas de Microfilamentos/metabolismo , Músculo Liso Vascular/citología , Proteína-Lisina 6-Oxidasa/metabolismo , Tropoelastina/metabolismoRESUMEN
Little is known about the relationship at the molecular and cellular levels between vascular calcification and elastic fibers essential for elasticity. To gain a better understanding of the physiological function of elastin in vascular calcification, we developed a calcification model on cultured bovine retinal-pigmented-epithelial cells (RPEs) that do not express endogenous tropoelastin. The addition of inorganic phosphate (NaH2PO4; Pi) induced calcium deposition in RPEs. The Pi-induced calcification, as assessed by the o-cresolphthalein complexone method, Goldenbergs method, and von Kossa staining, was completely inhibited by treatment with clodronate (DMDP) and phosphonoformic acid (PFA) and was weakly suppressed by treatment with levamisole. Moreover, the osteopontin mRNA expression was upregulated in the Pi-induced calcification of RPEs. These reactions in RPEs were characteristically consistent with those already established in cultured bovine aortic smooth muscle cells (BASMCs). Furthermore, bacterially expressed tropoelastin inhibited calcium deposition in RPEs as well as in BASMCs. Finally, Pi-induced calcification was partially suppressed after the addition of tropoelastin due to elastic fiber formation. In conclusion, we suggest that this calcification model in RPEs is useful for analyzing the relation between elastic fibers and vascular calcification, and that tropoelastin and elastic fibers may contribute to the inhibition of vascular calcification.
Asunto(s)
Calcinosis/metabolismo , Calcio/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Animales , Bovinos , Células Cultivadas , Ácido Clodrónico/farmacología , Foscarnet/farmacología , Levamisol/farmacología , Músculo Liso Vascular/metabolismo , Osteopontina , Fosfatos/farmacología , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/efectos de los fármacos , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Sialoglicoproteínas/genética , Tropoelastina/metabolismo , Tropoelastina/farmacología , Regulación hacia ArribaRESUMEN
In cases of vascular calcification, the expression of tropoelastin is down-regulated, which most likely decreases elastic fiber formation. However, the function of tropoelastin in vascular calcification remains unknown. We investigated whether tropoelastin affects the induction of vascular calcification. Calcification was induced using inorganic phosphate in cultured bovine aortic smooth muscle cells. The increase in tropoelastin due to the addition of recombinant bovine tropoelastin (ReBTE; 1 or 10 microg/ml) or beta-aminopropionitrile (25 microg/ml) significantly inhibited calcification at day 6, as assessed by the o-cresolphthalein complexone method. The addition of an elastin-derived peptide, VGVAPG peptide (0.1-1,000 nM), inhibited calcification at day 6 in a dose-dependent manner. In addition, these responses of beta-aminopropionitrile, ReBTE, and VGVAPG peptide were confirmed using von Kossa staining. To examine whether ReBTE inhibited calcium deposition via the elastin binding protein, lactose and elastin-specific antibody were used. The combination of lactose (20 mM) or this antibody (50 microg/ml) with ReBTE (10 microg/ml) attenuated the inhibition of calcification. These results suggest that increased tropoelastin inhibits vascular calcification in this model via the interaction between tropoelastin and elastin binding protein.
Asunto(s)
Calcinosis/prevención & control , Elastina/administración & dosificación , Músculo Liso Vascular/fisiopatología , Receptores de Superficie Celular/efectos de los fármacos , Tropoelastina/administración & dosificación , Animales , Aorta , Calcinosis/fisiopatología , Calcio/metabolismo , Bovinos , Células Cultivadas , Regulación hacia Abajo , Modelos Animales , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Receptores de Superficie Celular/fisiologíaRESUMEN
The direct separation of several stereoisomers (enantiomers and geometrical isomers) of furan derivatives, important intermediates for the synthesis of physiologically active natural products, was achieved using capillary gas chromatography/mass spectrometry with a per-O-methyl-beta-cyclodextrin, supercritical fluid chromatography and high-performance liquid chromatography with a tris(3,5-dimethylphenylcarbamate) of cellulose or amylose for the chiral stational phases, respectively. The temperature dependence of the peak resolution (Rs) and the retention factor (k) over the range of 110-130 degrees was studied using crotyl furfuryl ether in gas chromatography. Successive increases in the Rs value and of the difference between the k value of the E-isomer and the k value of the Z-isomer were observed when the gradient temperature was decreased. The per-O-methyl-beta-cyclodextrin column was suitable for use with volatile furan ethers whose molecular masses are between 150 and 180. In conclusion, the separation of thermally unstable furan derivatives was accomplished using supercritical fluid chromatography and high-performance liquid chromatography.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cromatografía con Fluido Supercrítico/métodos , Furanos/aislamiento & purificación , Cromatografía de Gases y Espectrometría de Masas/métodos , Furanos/química , Espectroscopía de Resonancia Magnética , Estereoisomerismo , TemperaturaRESUMEN
It has recently been suggested that RhoA plays an important role in the enhancement of Ca2+ sensitization observed in smooth muscle contraction. In the present study, the expression of rhoA mRNA in the bronchial smooth muscle of antigen-induced airway hyperresponsive rats was compared with that of control animals. Reverse transcription-polymerase chain reaction experiments using total RNA from these tissue specimens and the specific primers revealed rhoA mRNA to be expressed in bronchial smooth muscle of the rat. The rhoA mRNA expression in bronchial smooth muscle of the hyperresponsive rats was significantly increased in comparison to that of control animals. It is thus possible that upregulation of RhoA protein might be involved in the mechanism underlying the increased contractility of the bronchial smooth muscle which occurs with airway hyperresponsiveness.
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Antígenos/inmunología , Bronquios/metabolismo , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/metabolismo , Músculo Liso/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Proteína de Unión al GTP rhoA/genéticaRESUMEN
We evaluated the antioxidant activity of natural compounds in water-soluble and lipid-soluble phases and found that ferulic acid, quercetin and caffeic acid showed stronger activity in the water-soluble phase. Various fractions isolated from Bidens pilosa showed this activity mainly in the water-soluble phase. Antioxidant activity in the lipid-soluble phase of propolis depended on the lipophilic extraction.
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Antioxidantes/farmacología , Ácidos Cafeicos/farmacología , Ácidos Cumáricos/farmacología , Medicamentos Herbarios Chinos/química , Radicales Libres/antagonistas & inhibidores , Lípidos , Própolis/química , Quercetina/farmacología , Agua , Antioxidantes/aislamiento & purificación , Bidens , Ácidos Cafeicos/aislamiento & purificación , Ácidos Cumáricos/aislamiento & purificación , Quercetina/aislamiento & purificación , SolubilidadRESUMEN
Bidens pilosa L. var. radiata Scherff: (BP) is a plant used as a traditional folk medicine. BP, cultivated with only green manure on Miyako Island, Okinawa prefecture, was processed to powder and is referred to as MMBP. We have reported that MMBP has antioxidant, anti-inflammatory, and anti-allergy properties. In this study, we investigated the effects of MMBP on several experimental gastric lesions induced by HCl/EtOH, a non-steroidal anti-inflammatory drug, or cold-restraint stress, comparing these results with those of rutin or anti-ulcerogenic drugs (cimetidine or sucralfate) based on the lesion index and hemorrhage from the gastric lesions. Orally administered MMBP prevented the progression of the gastric lesions. Moreover, treatment with MMBP, rutin, or sucralfate, which had potent antioxidative activity, inhibited increases in the levels of thiobarbituric acid reactive substances (TBARS) in the gastric mucosal lesions. The inhibition of the gastric mucosal TBARS content by MMBP may have been due to the antioxidant effects of MMBP. These results indicate that MMBP prevents the progression of acute gastric mucosal lesions, possibly by suppressing oxidative stress in the gastric mucosa.
Asunto(s)
Antiulcerosos/uso terapéutico , Bidens , Extractos Vegetales/uso terapéutico , Úlcera Gástrica/tratamiento farmacológico , Animales , Antiulcerosos/aislamiento & purificación , Antiulcerosos/farmacología , Bidens/toxicidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/patología , Masculino , Ratones , Ratones Endogámicos ICR , Componentes Aéreos de las Plantas , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/toxicidad , Úlcera Gástrica/patologíaRESUMEN
The medical mechanism against type I allergies is to block the release or production of chemical mediators from mast cells or to block the H(1)-receptor signaling. We previously reported that the anti-allergic action of the dry powder from Bidens pilosa L. var. radiata SCHERFF treated with the enzyme cellulosine (eMMBP) was dependent on the inhibition of histamine release from mast cells. Here, we investigate that the effect of fractions in eMMBP on the histamine-induced contraction in guinea pig ileum and on the release of histamine in rat peritoneal mast cells. The histamine-induced contraction in guinea pig ileum is dose-dependently inhibited by ketotifen, an antagonist of H(1)-receptor. Fractions contained caffeic acid, caffeoylquinic acid and fractions contained flavonoids such as hyperin and isoquercitrin in eMMBP inhibit histamine release from mast cells, but only flavonoids such as hyperin, isoquercitrin and rutin suppress the histamine-induced contraction in guinea pig ileum. Moreover, the histamine-induced contraction was not affected by caffeic acid, however, such contraction was significantly inhibited by rutin. These results suggest that the primary antagonists of H(1)- receptor are different from the components in eMMBP that inhibit histamine release, and that these components participate in the anti-allergic activity of eMMBP.