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1.
J Eur Acad Dermatol Venereol ; 30(8): 1379-83, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26446433

RESUMO

BACKGROUND: Hypohidrosis/anhidrosis are congenital or acquired sweating impairments. Among them, acquired idiopathic generalized anhidrosis/hypohidrosis (AIGA) is the most common, and characterized by favourable response to systemic corticosteroid, however, no clinical markers for disease severity or activity have been developed. OBJECTIVE: Our aim was to verify the usefulness of serum carcinoembryonic antigen (CEA) level monitoring as a clinical marker for disease activity of AIGA. METHODS: Ten cases of AIGA diagnosed at Asahikawa Medical University, from 1980 to 2014 were included in the study. CEA and/or CEACAM1 expression level was analysed using immunohistochemistry and enzyme-linked immunosorbent assay. RESULT: CEA expression was restricted to the apical membrane of glandular cells in eccrine sweat glands in most of the three types of cases we examined [healthy control, patients with atopic dermatitis (AD) or urticaria]. However, CEA expression was detected diffusely and much more intensively in eight of the 10 AIGA cases included in this study. CEACAM1-expression was much more restricted on the apical membrane of glandular cells of both the AIGA cases and the other control subjects. While serum CEA levels increased in all five AIGA cases examined (5.8-43.2 ng/mL), it remained within normal limits in all control subjects: nine healthy individuals; 10 cases of AD; 10 cases of idiopathic urticaria; four cases of normohidrotic cholinergic urticaria (Mann-Whitney's U-test, P < 0.05). The increased serum CEA levels in AIGA decreased in conjunction with improved sweating during methyl prednisolone pulse therapy or repeated bathing. CONCLUSION: Serum CEA level may serve as a clinical marker for AIGA activity.


Assuntos
Biomarcadores/sangue , Antígeno Carcinoembrionário/sangue , Hipo-Hidrose/diagnóstico , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hipo-Hidrose/sangue , Hipo-Hidrose/imunologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
3.
J Dent Res ; 97(4): 467-475, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29131694

RESUMO

Although many reports have demonstrated that ectopic pain develops in the orofacial region following tooth pulp inflammation, which often causes misdiagnosis and inappropriate treatment for patients with pulpitis, the precise mechanism remains unknown. In the present study, we hypothesized that the functional interaction between satellite glial cells and neurons mediated by interleukin 1ß (IL-1ß) in the trigeminal ganglion (TG) is involved in ectopic orofacial pain associated with tooth pulp inflammation. The digastric muscle electromyogram (D-EMG) activity elicited by capsaicin administration into the maxillary second molar tooth pulp was analyzed to evaluate the noxious reflex and was significantly increased in rats with inflammation of the maxillary first molar (M1) versus rats injected with saline. A significant increase in the expression of connexin43 (Cx43), a gap junction containing protein, was observed in activated satellite glial cells surrounding second molar-innervating neurons in the TG after M1 pulpitis. Daily administration of Gap26, a Cx43 mimetic peptide and inhibitor, in the TG significantly suppressed the enhancement of capsaicin-induced D-EMG activity and the percentage of Fluoro-Gold (FG)-labeled cells encircled by glial fibrillary acid protein-immunoreactive (IR) + Cx43-IR cells after M1 pulp inflammation ( P < 0.01). The percentage of FG-labeled cells encircled by glial fibrillary acid protein-IR + IL-1ß-IR cells, IL-1 type I receptor-IR cells labeled with FG, and TRPV1-IR cells labeled with FG significantly increased after M1 pulp inflammation ( P < 0.01). Daily administration of IL-1ra, an IL-1 receptor antagonist, into the TG significantly reduced the enhancement of capsaicin-induced D-EMG activity and the percentage of TRPV1-IR neurons labeled with FG after M1 pulp inflammation ( P < 0.01). The present findings suggest that satellite glial cell is activated in the TG via activated gap junctions composed of Cx43 following tooth pulp inflammation, which leads to the hyperactivation of remote neurons via IL-1ß mechanisms and results in ectopic tooth pulp pain in the adjacent tooth.


Assuntos
Interleucina-1beta/farmacologia , Neuroglia/metabolismo , Neurônios/metabolismo , Pulpite/patologia , Gânglio Trigeminal/metabolismo , Animais , Capsaicina , Conexina 43/metabolismo , Eletromiografia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Canais de Cátion TRPV/metabolismo
4.
J Clin Invest ; 93(2): 543-9, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7509342

RESUMO

By measurements of NO2-/NO3- (NOx) production and Northern blot analysis, we studied the effects of a membrane-permeable cAMP derivative, 8-bromo-cAMP, on the expression of inducible nitric oxide synthase (iNOS) gene and the synthesis of NOx in cultured rat vascular smooth muscle cells (VSMCs). 8-bromo-cAMP stimulated NOx production and increased steady-state levels of iNOS mRNA in rat VSMC in a time- and dose-dependent manner. NG-monomethyl-L-arginine, a NOS inhibitor, completely blocked the 8-bromo-cAMP-induced NOx production, whose effect was partially, but significantly reversed by an excess L-arginine, but not by D-arginine. Compounds that increase intracellular cAMP levels (cholera toxin, forskolin, and 3-isobutyl-1-methylxanthine), all stimulated NOx production. Dexamethasone inhibited the stimulated NOx production, as well as the induction of iNOS mRNA by cAMP. Both actinomycin D and cycloheximide completely blocked the stimulated NOx production by cAMP. Actinomycin D abolished the cAMP-induced iNOS mRNA, whereas cycloheximide remarkably increased iNOS mRNA levels in the presence and absence of 8-bromo-cAMP (superinduction). Actinomycin D, but not dexamethasone, completely abolished the cycloheximide-induced iNOS mRNA. The half-life of cAMP-induced iNOS mRNA was approximately 2 h, whereas no decay in the cycloheximide-induced iNOS mRNA was observed during 12 h. These results demonstrate that iNOS gene is upregulated by cAMP and the superinduction of iNOS mRNA is attributable to increased mRNA stability in rat VSMC.


Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Aminoácido Oxirredutases/biossíntese , Aorta Torácica/enzimologia , Arginina/análogos & derivados , AMP Cíclico/metabolismo , Músculo Liso Vascular/enzimologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Arginina/farmacologia , Células Cultivadas , Toxina da Cólera/farmacologia , Colforsina/farmacologia , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Dexametasona/farmacologia , Indução Enzimática , Cinética , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico Sintase , RNA Ribossômico/biossíntese , RNA Ribossômico/isolamento & purificação , Ratos , Ratos Wistar , ômega-N-Metilarginina
5.
J Clin Invest ; 96(5): 2357-63, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7593623

RESUMO

In this study, we have identified the source of nitric oxide (NO) produced in the human inflammatory joints by analyzing expression of inducible NO synthase. In ex vivo organ cultures, both inflammatory synovium and cartilage from patients with rheumatoid arthritis produced NO. The NO production was suppressed by NG-monomethyl-L-arginine, an inhibitor of NO synthase. The amount of NO produced by the synovium correlated with the proportion of CD14+ cells in the corresponding tissue (r = 0.8, P < 0.05). Immunohistochemical analysis as well as in situ hybridization showed that inducible NO synthase was predominantly expressed in synovial lining cells, endothelial cells, chondrocytes, and to a lesser extent, in infiltrating mononuclear cells and synovial fibroblasts. The synovial lining cells and the infiltrating cells expressing inducible NO synthase were identified where CD14+ cells were located. Together with morphological features, this suggests that they are type A synoviocytes. NO production from freshly isolated synoviocytes and chondrocytes was up-regulated by in vitro stimulation with a combination of IL-TNF-beta, TNF-alpha, and LPS. In summary, the present results suggest that NO is produced primarily by CD14+ synoviocytes, chondrocytes, and endothelial cells in inflammatory joints of arthritides. NO production can be upregulated by cytokines present in inflamed joints. The increased NO production may thus contribute to the pathological features in inflammatory arthritides.


Assuntos
Artrite Reumatoide/metabolismo , Cartilagem Articular/metabolismo , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico/biossíntese , Membrana Sinovial/metabolismo , Cartilagem Articular/patologia , Citocinas/metabolismo , Feminino , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Membrana Sinovial/patologia , Regulação para Cima
6.
J Clin Invest ; 91(4): 1367-73, 1993 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7682570

RESUMO

Endothelins (ET) produce endothelium-dependent vasodilation through nitric oxide (NO) synthesis. The present study was designed to elucidate the cellular mechanism by which ET induces synthesis and release of endothelium-derived NO by cultured bovine endothelial cells (EC). Binding studies revealed that bovine EC membrane had the binding sites of a novel agonist (BQ3020) for non-isopeptide-selective receptor subtype (ETB). Affinity labeling studies showed a major labeled band with the apparent molecular mass of 50 kD. Northern blot analysis demonstrated the expression of mRNA for ETB receptor. BQ3020 rapidly and dose dependently induced formation of inositol-1,4,5-triphosphate and increased intracellular Ca2+ concentrations in fura-2-loaded cells. Concomitantly, BQ3020 dose dependently stimulated production of both nitrate/nitrite (NOx) and cyclic GMP; a highly significant correlation existed between NOx and cGMP production. The stimulatory effect on NOx and cGMP production by ETB agonist was inhibited by NO synthase inhibitor monomethyl-L-arginine; this effect was reversed by coaddition of L-arginine, but not D-arginine. NOx and cGMP production stimulated by BQ3020 was inhibited by pretreatment with pertussis toxin. ETB agonist-induced NOx production was blocked by a calmodulin inhibitor and an intracellular Ca2+ chelator, but not by an extracellular Ca2+ chelator or a Ca2+ channel blocker. These data suggest that endothelins stimulate ETB receptor-mediated phosphoinositide breakdown via pertussis toxin-sensitive G-protein(s), which triggers release of intracellular Ca2+, thereby activating Ca2+/calmodulin-dependent NO synthase in EC.


Assuntos
Aminoácido Oxirredutases/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/ultraestrutura , Receptores de Endotelina/fisiologia , Animais , Arginina/metabolismo , Northern Blotting , Cálcio/análise , Cálcio/fisiologia , Calmodulina/fisiologia , Bovinos , GMP Cíclico/metabolismo , Endotelinas/farmacocinética , Endotélio Vascular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Inosina Trifosfato/metabolismo , Radioisótopos do Iodo , Nitratos/metabolismo , Óxido Nítrico Sintase , Nitritos/metabolismo , Fragmentos de Peptídeos/farmacocinética , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia
7.
Ann ICRP ; 45(2_suppl): 58-64, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28054488

RESUMO

Iitate, a village in Fukushima Prefecture, suffered little damage from the Great East Japan Earthquake that struck on 11 March 2011. However, all village residents were belatedly ordered to evacuate 1 month after the accident at Fukushima Daiichi nuclear power plant. My family and I had to evacuate to the city of Fukushima, located 40 km from our home in Iitate. This came at a time when my husband and I were planning to start research on Natsuhaze (a type of blueberry grown in Japan) after his retirement. There were conflicting reports. On one hand, the media reported that it was not possible to live in Fukushima. The village of Iitate organised a lecture by an expert to assuage the fear of the residents. The evacuation order 1 month after the disaster contradicted what the expert was saying, and appeared to amplify distrust among the residents. I tried to arrive at my own judgement by measuring the ambient radiation dose in and around my house. Participating in the International Commission on Radiological Protection dialogue seminars provided accurate understanding of the situation. Measurement of radiation doses of wild plants that my husband's father had been cultivating for over 30 years has given me many insights, and I had no concerns about returning to Iitate.


Assuntos
Proteção Radiológica , Terremotos , Acidente Nuclear de Fukushima , Japão , Centrais Nucleares
8.
Endocrinology ; 134(1): 222-8, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8275937

RESUMO

An endothelium-derived vasoactive peptide, endothelin (ET)-1, is a potent constrictor and mitogen for vascular smooth muscle cells (VSMC). To determine whether vascular ET receptor subtypes phenotypically change during in vitro culture conditions, we studied the expression of ET receptor subtypes, ET-induced phosphoinositide breakdown, and DNA synthesis in cultured rat VSMCs during serial passages. Binding studies using [125I]ET-1 as a radioligand revealed that the early passage (10th-15th) VSMCs possess predominantly ETA receptors, whereas the late passage (30th-35th) VSMCs possess predominantly ETB receptors in addition to ETA receptors. Northern blot analysis using cDNAs for rat ETA and ETB receptors as probes also demonstrated the predominant expression of ETA receptor mRNA in the early passage and ETB receptor mRNA in the late passage, whereas only ETA receptor mRNA was expressed in intact medium of rat aorta. ET-1 had a greater effect than ET-3 in stimulating inositol 1,4,5-trisphosphate formation, whereas ET-1 and ET-3 almost equipotently stimulated insitol 1,4,5-trisphosphate formation in the late passage VSMC even in the presence of an ETA receptor antagonist. ET-1-induced DNA synthesis was almost completely inhibited by an ETA receptor antagonist in the early passage VSMC. In contrast, ET-1, ET-3, and an ETB receptor agonist remarkably stimulated DNA synthesis in the late passage VSMC, which was completely inhibited by a nonselective ET receptor antagonist, but not by an ETA receptor antagonist. Our data provide the first evidence that a phenotypic change in VSMC in culture is concomitantly associated with a change in the ET receptor subtype that potentiates mitogenic activity and suggest that switching the ET receptor subtype from ETA to ETB during phenotypic change may in part contribute to the development of vascular lesions, such as in atherosclerosis.


Assuntos
Músculo Liso Vascular/metabolismo , Receptores de Endotelina/metabolismo , Animais , Ligação Competitiva , Células Cultivadas , DNA/biossíntese , Inositol 1,4,5-Trifosfato/biossíntese , Masculino , Músculo Liso Vascular/citologia , Fenótipo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Endotelina/genética
9.
Endocrinology ; 133(6): 2474-80, 1993 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8243267

RESUMO

We studied the cellular mechanism by which natriuretic peptides inhibit the synthesis and release of endothelin-1 (ET-1) in cultured rat aortic endothelial cells (EC). Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) showed dose-dependent and equipotent effects on displacement of [125I]ANP binding and generation of cGMP production in rat EC, whereas C-type natriuretic peptide and biologically inactive ANP analog had lesser effects. ANP and BNP as well as 8-bromo-cGMP had potent inhibitory effects on immunoreactive ET-1 release, the transient increase in the intracellular Ca2+ concentration, and the formation of inositol 1,4,5-trisphosphate stimulated by thrombin in rat EC. A cGMP-dependent protein kinase inhibitor (KT5823), but not a cAMP-dependent protein kinase inhibitor (KT5720), completely abolished the inhibitory effect of ANP on thrombin-induced immunoreactive Et-1 release. Northern blot analysis using cDNA for rat prepro-ET-1 as a probe showed that ANP and 8-bromo-cGMP, but not C-type natriuretic peptide, inhibited thrombin-induced prepro-ET-1 mRNA expression, whose effect was abolished by KT5823. These data suggest that ANP and BNP inhibit the thrombin-induced synthesis and release of ET-1 in cultured rat aortic EC by blocking phosphoinositide breakdown, possibly via natriuretic peptides type A receptor-mediated cGMP-dependent mechanism.


Assuntos
Fator Natriurético Atrial/farmacologia , Endotelinas/antagonistas & inibidores , Endotélio Vascular/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Endotelina-1 , Endotelinas/metabolismo , Endotélio Vascular/citologia , Fosfatos de Inositol/biossíntese , Membranas Intracelulares/metabolismo , Peptídeo Natriurético Encefálico , Peptídeo Natriurético Tipo C , Inibidores de Proteínas Quinases , Precursores de Proteínas/metabolismo , Ratos , Trombina/farmacologia
10.
Hypertension ; 22(1): 34-9, 1993 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7686532

RESUMO

To elucidate whether cytokines induce nitric oxide synthase in vascular smooth muscle cells, we studied the effects of human recombinant interleukin-1 beta on the synthesis and release of nitric oxide in cultured rat vascular smooth muscle cells by measurement of NO2-/NO3- levels. Furthermore, we performed Northern blot analysis using subcloned polymerase chain reaction products as probes for constitutive and inducible nitric oxide synthase. Interleukin-1 beta dose dependently (1 to 20 ng/mL) stimulated NO2-/NO3- production as a function of time. Northern blotting demonstrated the interleukin-1 beta-induced expression of messenger RNA for an inducible but not for the constitutive nitric oxide synthase after 3 hours. NG-Monomethyl L-arginine completely blocked the interleukin-1 beta-induced NO2-/NO3- production, the effect of which was reversed by L-arginine but not by D-arginine. Dexamethasone inhibited the interleukin-1 beta-induced NO2-/NO3- production in a dose-dependent manner (10(-9) to 10(-7) M) and the interleukin-1 beta-inducible nitric oxide synthase messenger RNA levels. Neither a calmodulin inhibitor (W-7) nor a protein kinase C inhibitor (staurosporine) showed any effects on the induction of nitric oxide synthase transcripts or production of NO2-/NO3- stimulated by interleukin-1 beta, whereas cycloheximide and actinomycin D completely inhibited the basal and stimulated NO2-/NO3- production. These data demonstrate for the first time that interleukin-1 beta induces gene expression of inducible nitric oxide synthase and its de novo protein synthesis in rat vascular smooth muscle cells, thereby leading to generation of nitric oxide via Ca2+/calmodulin-independent and protein kinase C-independent mechanisms.


Assuntos
Aminoácido Oxirredutases/biossíntese , Interleucina-1/farmacologia , Músculo Liso Vascular/metabolismo , Alcaloides/farmacologia , Aminoácido Oxirredutases/genética , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Sequência de Bases , Northern Blotting , Calmodulina/antagonistas & inibidores , Calmodulina/fisiologia , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Indução Enzimática , Dados de Sequência Molecular , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase , Reação em Cadeia da Polimerase , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/farmacologia , Estaurosporina , Sulfonamidas/farmacologia , ômega-N-Metilarginina
11.
Hypertension ; 18(2): 165-70, 1991 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1909304

RESUMO

Release of endothelin-1, a novel potent vasoconstrictor peptide originally isolated from endothelial cells, from cultured bovine endothelial cells has been shown to be stimulated by arginine vasopressin and angiotensin II. To elucidate the cellular mechanism by which endothelin-1 is released by these vasoconstrictors, we tested the effects of several compounds on the agonist-induced endothelin-1 release and studied the changes of cytosolic free Ca2+ concentrations and phosphoinositide breakdown by these agonists in cultured bovine endothelial cells. Protein kinase C inhibitors (H-7, staurosporine), an intracellular Ca2+ chelator, and an inhibitor of phospholipase C (neomycin), all abolished the agonist-induced endothelin-1 release, whereas the Ca2+ channel blocker nicardipine was ineffective. Although synthetic 1,2-diglyceride (diolein) dose dependently stimulated endothelin-1 release, downregulation of protein kinase C after pretreatment with phorbol ester resulted in decreased effects to increase endothelin-1 release by the agonists. Both arginine vasopressin and angiotensin II induced immediate and transient increases in intracellular Ca2+ levels of fura-2-loaded endothelial cells as well as formation of inositol trisphosphate; the agonist-induced intracellular Ca2+ increases were not affected either by nicardipine or by chelating extracellular Ca2+. The arginine vasopressin- and angiotensin II-induced intracellular Ca2+ increases, inositol trisphosphate formation, and endothelin-1 release were completely abolished by V1-receptor antagonist and saralasin, respectively. It is concluded that arginine vasopressin and angiotensin II stimulate the release of endothelin-1 by a common mechanism, involving receptor-mediated mobilization of intracellular Ca2+ and activation of protein kinase C in endothelial cells.


Assuntos
Angiotensina II/farmacologia , Arginina Vasopressina/farmacologia , Endotelinas/biossíntese , Endotélio Vascular/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Alcaloides/farmacologia , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Diglicerídeos/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Ácido Egtázico/farmacologia , Endotélio Vascular/efeitos dos fármacos , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/farmacologia , Isoquinolinas/farmacologia , Neomicina/farmacologia , Nicardipino/farmacologia , Piperazinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases , Estaurosporina
12.
FEBS Lett ; 184(2): 202-6, 1985 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-3996585

RESUMO

Using a procedure involving DNase I affinity chromatography and Sephadex G-200 gel filtration, we partially purified a Ca2+-sensitive actin regulatory 90-kDa protein from bovine aorta. The 90-kDa protein existed in the form of a complex with actin on a DNase I column even in the presence of 5 mM EGTA, indicating that the 90-kDa protein binds tightly to actin in a Ca2+-insensitive manner. The isolation procedure described above indicates that the 90-kDa protein is present in smooth muscles including aorta, uterus and bladder, but not in skeletal and heart muscles. When added to G-actin before polymerization, the 90-kDa protein increases the initial rate of actin polymerization and lowers the final viscosity at Ca2+ concentrations greater than 10(-7) M. This decrease in viscosity is due to the generation of filaments which cannot be readily sedimented.


Assuntos
Proteínas de Transporte/isolamento & purificação , Proteínas dos Microfilamentos/isolamento & purificação , Músculo Liso Vascular/análise , Proteínas/isolamento & purificação , Fatores de Despolimerização de Actina , Animais , Bovinos , Destrina , Gelsolina , Peso Molecular
13.
Atherosclerosis ; 28(2): 111-9, 1977 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-199204

RESUMO

Elastin preparations were isolated from human thoracic aorta, from atherosclerotic and from grossly normal regions. A relatively mild procedure was used to avoid hot alkaline extraction and autoclaving. The elastase digest of the aortic elastin was chromatographed on a Sephadex G-100 column and separated into two fractions: A (larger molecular weight) and B (smaller molecular weight). The ratio of fraction A to total aortic elastin increased with age and the development of the atherosclerosis. Amino acid and sugar analyses showed that fraction A consistently contained more polar amino acids, hexose, hexosamine and L-fucose, and less sialic acid, in comparison with fraction B. Part of the elastin preparation was incubated with human low-density lipoprotein; a considerable amount of lipid, especially cholesterol, was transferred from the lipoprotein to the elastin. Estimation of protein and cholesterol in fractions A and B of the elastase hydrolyzate of incubated elastin showed that most of the cholesterol taken up by elastin had been in fraction A. The increased proportion of fraction A in aortic elastin derived from plaque areas appeared responsible for the marked lipid-binding capacity of plaque elastin.


Assuntos
Elastina/metabolismo , Lipoproteínas LDL/sangue , Fatores Etários , Idoso , Aminoácidos/análise , Aorta Torácica/metabolismo , Arteriosclerose/sangue , Sítios de Ligação , Colesterol/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/sangue , Triglicerídeos/sangue
14.
J Immunol Methods ; 202(2): 193-204, 1997 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-9107308

RESUMO

A novel cell display system was developed for cloning the variable region (V) genes of antigen-specific human antibodies. The system is based on an antibody library displayed on the surface of COS cells, using a plasmid vector designed to direct expression of membrane-bound antibodies. COS cells expressing antigen-specific antibodies were separated using a flow cytometer for their binding to a fluorescent dye-labeled antigen. To test the performance of this system. We cloned V genes of 4 antibodies directed against hepatitis B surface antigen (HBsAg) from a library prepared from peripheral blood lymphocytes of a vaccinated donor. These membrane-bound anti-HBsAg antibodies were easily converted to soluble forms, all of which showed a size similar to human serum IgG in SDS-PAGE and the same specific binding to HBsAg as membrane-bound forms in ELISA. All VH and VK gene segments of the 4 clones isolated in this study belonged to VHIII and VKI subgroups, respectively. These findings demonstrate the potential and selection capabilities of our cell display system for cloning the V genes of antigen-specific human antibodies.


Assuntos
Genes de Imunoglobulinas , Anticorpos Anti-Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular/métodos , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Transfecção
15.
Biochem Pharmacol ; 31(18): 2977-81, 1982 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-6291552

RESUMO

The interactions of psychotropic drugs with phospholipids, including lysophosphatidylcholine, phosphatidylinositol, and lysophosphatidylserine, were studied using calmodulin-dependent cyclic nucleotide phosphodiesterase partially purified from the cortex of hog brain. All the compounds used inhibited both calmodulin- and phospholipid-stimulated phosphodiesterase activity but not the basal activity. Fluphenazine was confirmed by kinetic analysis to be a competitive inhibitor, with both calmodulin and phospholipid. Using fluphenazine-Sepharose affinity chromatography, it was demonstrated that fluphenazine did not interact with the enzyme. The potencies of antipsychotics such as fluphenazine in inhibiting the various phospholipid-dependent activations decreased in the following order: lysophosphatidylcholine-, phosphatidylinositol-, and lysophosphatidylserine-dependent activation. On the other hand, antidepressant drugs exhibited similar inhibitory potencies towards lysophosphatidylcholine- and phosphatidylinositol-dependent activation. Antipsychotic and antidepressant drugs appear to have different characteristics with regard to lipid-drug interaction.


Assuntos
Fosfolipídeos/metabolismo , Psicotrópicos/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Calmodulina/antagonistas & inibidores , Cromatografia de Afinidade , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Flufenazina/farmacologia , Técnicas In Vitro , Cinética , Lisofosfatidilcolinas/metabolismo , Fosfatidilinositóis/metabolismo , Suínos
16.
Biochem Pharmacol ; 44(12): 2409-11, 1992 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-1472106

RESUMO

We have studied the cellular mechanism responsible for induction of preproendothelin (preproET)-1 mRNA and release of ET-1 by thrombin in cultured bovine endothelial cells (ECs). Thrombin induced an immediate and dose-dependent formation of inositol-1,4,5-trisphosphate (IP3) with a concomitant increase in intracellular Ca2+ concentration ([Ca2+]i). The thrombin-induced ET-1 release was abolished either by a phospholipase C inhibitor, a protein kinase C (PKC) inhibitor, or an intracellular Ca(2+)-chelator, whereas a Ca(2+)-channel antagonist was ineffective. A selective thrombin inhibitor (argatroban) decreased IP3 formation and the increase in [Ca2+]i and ET-1 release stimulated by thrombin. Northern blot analysis revealed that thrombin-induced expression of preproET-1 mRNA was inhibited completely by a PKC inhibitor and partially by argatroban. These data suggest that thrombin is involved in the mechanism of preproET-1 mRNA expression and subsequent ET-1 release, possibly through activation PKC and mobilization of intracellular Ca2+ resulting from the receptor-mediated phosphoinositide breakdown in ECs.


Assuntos
Endotelinas/biossíntese , Endotelinas/metabolismo , Endotélio/metabolismo , Precursores de Proteínas/metabolismo , Trombina/farmacologia , Animais , Arginina/análogos & derivados , Cálcio/metabolismo , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Endotelina-1 , Regulação da Expressão Gênica , Inositol 1,4,5-Trifosfato/biossíntese , Ácidos Pipecólicos/farmacologia , RNA Mensageiro/metabolismo , Sulfonamidas , Trombina/antagonistas & inibidores
17.
Biochem Pharmacol ; 43(11): 2385-92, 1992 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-1351727

RESUMO

Compounds with medium relative molecular masses active against human immunodeficiency virus (HIV) were synthesized. Sulfated alkyl oligosaccharides such as sulfated octadecyl maltohexaoside, sulfated dodecyl laminaripentaoside and sulfated dodecyl laminari-oligomer caused 50% inhibition of virus infection in the EC50 range of 0.4-0.7 microgram/mL in vitro using the MT-4 cell line and HIV-1HTLV-IIIB virus isolate, though sulfated oligosaccharides without alkyl groups showed low anti-HIV activities. This anti-HIV activity was close to the EC50 of 0.43 microgram/mL for a highly active sulfated polysaccharide curdlan sulfate which was reported to inhibit completely the HIV infection at a concentration as low as 3.3 micrograms/mL. These compounds were also active against HIV-2 and a clinically isolated HIV-1 with reduced AZT sensitivity. For such sulfated alkyl oligosaccharides, the mechanism of inhibition of HIV infection was assumed to be the inhibition of HIV binding to the cell and to some extent the interaction of the alkyl portion with the lipid bilayer of the virus.


Assuntos
Antivirais/farmacologia , Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Oligossacarídeos/farmacologia , Polissacarídeos/farmacologia , beta-Glucanas , Sequência de Aminoácidos , Sequência de Carboidratos , Linhagem Celular Transformada/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glucanos/farmacologia , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Dados de Sequência Molecular
18.
Autoimmunity ; 14(3): 205-14, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-7687154

RESUMO

We studied the effects of interleukins (IL) on in vitro IgM and IgG anti-DNA antibody production by splenic B cells from autoimmune disease-prone NZBxNZW (NZB/W) F1 mice. It was found that different interleukins regulate phenotypically distinct B cells producing separate isotype of anti-DNA antibodies. IL-2 slightly but significantly inhibited the production of IgM anti-DNA antibodies. IL-4 and IL-6 significantly enhanced the antibody production, but the effects were not so marked and inconsistent, particularly with respect to IL-6. By contrast, the effects of IL-5 were remarkable, particularly on splenic B cells from young mice. As for IgG anti-DNA antibodies, IL-6, but not other interleukins, markedly up-regulated the antibody production by splenic B cells from mice over 6 months of age, in a dose dependent fashion. Thus, the ability of B cells to produce IgG anti-DNA antibodies appears to be dependent on the surface expression of IL-6 receptor (IL-6R) at the ages when the mice begin to develop the disease. Studies of the surface phenotypes showed that while the IL-5-sensitive major IgM anti-DNA producers were CD5+Lp-3(CD43)-sIgM+, the IL-6-sensitive major IgG anti-DNA producers were CD5-Lp-3+sIgM-. However, significant amounts of IgG antibodies were also produced, in the presence of IL-6, by CD5+Lp-3+sIgM+, but not by CD5-Lp-3+sIgM+ B cells from 6-month-old mice. We suggest that surface phenotypes of anti-DNA antibody producers change from CD5+Lp-3-sIgM+IL-5R+, CD5+Lp-3+sIgM+IL-6R+ and subsequently to CD5-Lp-3+sIgM-(sIgG+)IL-6R+ in NZB/W F1 mice with aging.


Assuntos
Anticorpos Antinucleares/biossíntese , Antígenos CD/análise , Linfócitos B/imunologia , Interleucinas/farmacologia , Lúpus Eritematoso Sistêmico/imunologia , Animais , Antígenos CD5 , Células Cultivadas , DNA/imunologia , Antígenos H-2/genética , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Camundongos , Camundongos Endogâmicos NZB , Ratos
19.
Eur J Pharmacol ; 225(1): 79-82, 1992 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-1311692

RESUMO

Receptor binding and cyclic GMP generation by three distinct natriuretic peptides (ANP, BNP, CNP) were studied in a cultured rat glioma cell line (C6). Binding studies revealed the presence of high-affinity binding sites for three natriuretic peptides with almost comparable affinities. In contrast, CNP and BNP were almost equipotent in stimulating intracellular cyclic GMP generation over the low concentration range, but CNP caused further elevation in the high concentration range, whereas ANP was minimally effective. Our data suggest that the glioma cells possess receptors more responsive to CNP than ANP and BNP despite no apparent correlation between receptor binding affinities and cyclic GMP responses.


Assuntos
Fator Natriurético Atrial/farmacologia , GMP Cíclico/biossíntese , Proteínas do Tecido Nervoso/farmacologia , Receptores de Superfície Celular/análise , Animais , Fator Natriurético Atrial/metabolismo , Sítios de Ligação , Glioma/metabolismo , Peptídeo Natriurético Encefálico , Peptídeo Natriurético Tipo C , Proteínas do Tecido Nervoso/metabolismo , Ratos , Receptores do Fator Natriurético Atrial , Células Tumorais Cultivadas
20.
Eur J Pharmacol ; 176(2): 225-8, 1990 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-2155800

RESUMO

We studied the effects of synthetic porcine big endothelin (ET), a putative 39-residue intermediate deduced from cDNA sequence analysis, on receptor binding activity in cultured rat vascular smooth muscle cells as well as its vasoconstrictive activity in rat pulmonary artery. Big ET was about two orders of magnitude less active than ET in the receptor binding and vasoconstriction assays. Our data are consistent with the contention that big ET is an intermediate which can be converted into fully bioactive 21-residue mature ET.


Assuntos
Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Vasoconstrição/efeitos dos fármacos , Animais , Células Cultivadas , Cromatografia por Troca Iônica , Endotelina-1 , Radioisótopos do Iodo , Músculo Liso Vascular/citologia , Peptídeos/farmacologia , Precursores de Proteínas/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Ratos , Receptores de Endotelina , Suínos
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