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1.
Cytokine ; 54(3): 282-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21414799

RESUMO

Interleukin 6 (IL-6) and nitric oxide (NO) are important mediators of the inflammatory response. We report that in human peripheral blood mononuclear cells (PBMCs), NO exerts a biphasic effect on the expression of IL-6. Using sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) as NO-donating compounds, we observed that both mRNA and protein levels of IL-6 increased at lower (≤10µM) and decreased at higher (>100µM) concentrations of NO donors. Changes in the expression of IL-6 correlated with changes in the activity of NF-κB, which increased at lower and decreased at higher concentrations of both NO donors as shown by the electrophoretic mobility shift assay (EMSA). The effects of NO on NF-κB activity were cGMP-dependent because they were reversed in the presence of ODQ, the inhibitor of soluble guanylyl cyclase (sGC), and KT5823, the inhibitor of cGMP-dependent protein kinase (PKG). Moreover, the membrane permeable analog of cGMP (8-Br-cGMP) mimicked the effect of the NO donors. These observations show that NO, depending on its concentration, may act in human PBMCs as a stimulator of IL-6 expression involving the sGC/cGMP/PKG pathway.


Assuntos
GMP Cíclico/metabolismo , Interleucina-6/biossíntese , Leucócitos Mononucleares/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Carbazóis/farmacologia , Núcleo Celular/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Citocinas/metabolismo , Vetores Genéticos , Humanos , Inflamação , Interleucina-6/metabolismo , Leucócitos Mononucleares/citologia , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/metabolismo , S-Nitrosoglutationa/metabolismo
2.
Immunobiology ; 216(3): 423-30, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20656372

RESUMO

Inflammation involves the cooperation of various cells and biologically active molecules. An important intracellular messenger molecule participating in the regulation of the process is cyclic GMP (cGMP), which is synthesized by guanylyl cyclases (GCs). The GC family comprises cytosolic (soluble) and membrane-bound (particulate) enzymes. The aim of this study was to determine whether and how the synthesis of cGMP by various forms of GC affects the expression of inflammatory cytokines depending on the activity of the transcription factors NF-κB (nuclear factor-κB) and AP-1 (activator protein-1). We established that in rat peripheral blood mononuclear cells (PBMCs), synthesis of cGMP was elevated by sodium nitroprusside (SNP), the activator of soluble GC, and by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), the activators of particulate GC-A and GC-B, respectively. Stimulation of various GCs differently affected the expressions of the cytokines IL-1ß, IL-6, and TNF-α in control cells and in cells activated by bacterial endotoxin (LPS). In control PBMCs their expression was elevated by stimulation of soluble, but not particulate, GC. SNP caused an increase in NF-κB activity, but had no influence on the activity of AP-1. The cells treated with LPS decreased the expressions of IL-1ß, IL-6, and TNF-α in response to stimulation of particulate GC-A, but not other guanylyl cyclases. This inhibitory effect was a result of suppression of the activities of NF-κB and AP-1. Both effects that of SNP and of ANP, were cGMP dependent, as shown using its membrane-permeable analog 8-Br-cGMP. The implementation of specific inhibitors showed that the stimulatory effect of SNP was mediated by soluble GC and cGMP-dependent protein kinase (PKG-I). However, PKG-I was not involved in the inhibition of NF-κB and AP-1 activities by ANP in LPS-activated cells. Taken together, these results for the first time indicate that various GCs and various cGMP-dependent signaling pathways can modulate the activity of AP-1 and/or NF-κB and thus affect the expressions of IL-1ß, IL-6, and TNF-α, which play important roles in the development of inflammation.


Assuntos
Citocinas/genética , Guanilato Ciclase/metabolismo , Leucócitos Mononucleares/imunologia , Fator de Transcrição AP-1/metabolismo , Animais , Fator Natriurético Atrial/farmacologia , Western Blotting , GMP Cíclico/análogos & derivados , GMP Cíclico/biossíntese , GMP Cíclico/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Citocinas/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Guanilato Ciclase/antagonistas & inibidores , Inflamação/genética , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/imunologia , Peptídeo Natriurético Tipo C/farmacologia , Nitroprussiato/farmacologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Solubilidade
3.
Biomed Pharmacother ; 64(1): 16-20, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19758783

RESUMO

A proline-rich polypeptide complex (PRP) with immunoregulatory and procognitive activities shows beneficial effects in the Alzheimer's disease (AD). The mechanism of action of PRP is not yet fully clarified, we have shown that the PRP complex inhibits overproduction of reactive oxygen species, nitric oxide and proinflammatory cytokines induced by lipopolysaccharide (LPS). LPS stimulation exerts its inflammatory effects through the activation of the classical nuclear factor-kappaB (NF-kappaB) pathway. The results presented in this study showed the ability of PRP to inhibit the NF-kappaB activity induced by LPS while it increased activity of NF-kappaB in untreated cells. Examining the effect of PRP on IkappaB it was shown that relative level of IkappaB was lowered in the presence of PRP. It seems that in cells untreated with LPS, PRP can activate proteasome system and stimulate IkappaB degradation. Our results suggest that the regulatory effect of PRP on inflammatory processes may be associated with the influence of PRP on NF-kappaB translocation. Inhibitory effect of PRP on NF-kappaB activity might, at least in part, contribute to the beneficial therapeutic effects in the case of Alzheimer's disease.


Assuntos
Mediadores da Inflamação/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Domínios Proteicos Ricos em Prolina , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/fisiopatologia , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Humanos , Quinase I-kappa B/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
4.
Breast Cancer Res Treat ; 110(2): 269-71, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17851760

RESUMO

Cancer-associated retinopathy (CAR) is a rare paraneoplastic syndrome usually associated with small-cell lung carcinoma and serum autoantibodies against recovering. We report the breast cancer woman with visual impairments and electrophysiological abnormalities characteristic of CAR. Her serum contained high-titer antibodies against alpha-enolase but not against other retinal proteins. This suggests that anti-enolase antibodies could be responsible for the development of CAR symptoms.


Assuntos
Neoplasias da Mama/complicações , Neoplasias da Mama/patologia , Carcinoma/complicações , Carcinoma/patologia , Retina/metabolismo , Doenças Retinianas/diagnóstico , Doenças Retinianas/patologia , Neoplasias da Mama/imunologia , Carcinoma/imunologia , Linhagem Celular Tumoral , Eletrofisiologia/métodos , Evolução Fatal , Feminino , Humanos , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/patologia , Pessoa de Meia-Idade , Metástase Neoplásica , Fosfopiruvato Hidratase/metabolismo , Doenças Retinianas/imunologia , Carcinoma de Pequenas Células do Pulmão/complicações , Carcinoma de Pequenas Células do Pulmão/patologia , Visão Ocular
5.
Arch Immunol Ther Exp (Warsz) ; 55(4): 261-5, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17659379

RESUMO

INTRODUCTION: Cancer-associated retinopathy (CAR) is a paraneoplastic neurological syndrome resulting in progressive loss of vision and clinical signs of retinal degeneration. It is associated with various types of cancer and is also considered to be an autoimmune disorder that involves cross-reaction between autoantibodies and retinal proteins. The aim of this study was to establish whether immunoreactivity to retinal antigens (RAs) observed in patients with breast cancer is accompanied by any visual impairments. MATERIALS AND METHODS: Sera of 295 patients with diagnosed breast cancer were screened for the presence of anti-RAs antibodies using immunoblotting. Cellular immunoreactivity to RAs present in retinal extracts and to purified recoverin and arrestin was determined by means of a lymphocyte proliferation assay. Six patients with high-titer antibodies to RAs then underwent ophthalmic and neurological examinations. RESULTS: Four serum samples contained high-titer antibodies to a 46-kDa protein, most probably retinal alpha-enolase, three had antibodies to a 48-kDa protein identified as retinal arrestin, while 56-, 43-, 41-, and 34-kDa antigens were recognized only by one serum sample each. Moreover, weak cellular response to all the RAs tested was observed in one patient and another patient responded only to retinal extract. Two of the examined patients displayed symptoms of CAR. CONCLUSIONS: Immunoreactivity to RAs in patients with breast cancer may also be present in cases without clinical signs of CAR.


Assuntos
Arrestina/imunologia , Autoanticorpos/sangue , Doenças Autoimunes/imunologia , Neoplasias da Mama/complicações , Proteínas do Olho/imunologia , Síndromes Paraneoplásicas/imunologia , Fosfopiruvato Hidratase/imunologia , Doenças Retinianas/imunologia , Transtornos da Visão/imunologia , Idoso , Arrestina/química , Doenças Autoimunes/fisiopatologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/fisiopatologia , Eletrorretinografia , Potenciais Evocados Visuais , Proteínas do Olho/química , Feminino , Humanos , Pessoa de Meia-Idade , Peso Molecular , Oftalmoscopia , Síndromes Paraneoplásicas/fisiopatologia , Fosfopiruvato Hidratase/química , Doenças Retinianas/fisiopatologia , Transtornos da Visão/fisiopatologia , Acuidade Visual , Campos Visuais
6.
Int J Cardiol ; 122(2): 111-6, 2007 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-17270295

RESUMO

BACKGROUND: Inflammatory immune mechanisms are involved in the pathogenesis and progression of chronic heart failure (CHF), and also promote the development of generalised body wasting seen in this syndrome. We examined the activity of nuclear factor kappa-B (NF-kappaB), the major mediator of immune response, in peripheral blood mononuclear cells (PBMC) isolated from cachectic and non-cachectic patients with CHF. METHODS: Using electromobility shift assay, NF-kappaB activity was assessed in nuclear fractions of PBMC isolated from 43 patients with systolic CHF (88% men, age: 64 years [median], left ventricular ejection fraction [LVEF]: 30%, ischaemic CHF aetiology: 79%, NYHA class [I/II/III/IV]: 2/21/19/1, 10 patients with cardiac cachexia) and 12 healthy adult subjects. RESULTS: As compared to healthy controls, NF-kappaB activity in PBMC was increased in patients with CHF (P<0.05), in particular in those with severe CHF (NYHA class III-IV) (P<0.05). NF-kappaB activity in PBMC in CHF patients was not related either to age, sex, CHF aetiology, LVEF, or any clinical parameters reflecting disease severity (haemoglobin, LDL cholesterol, sodium and creatinine levels) (all P>0.1). Regardless of the severity of CHF expressed as NYHA class, patients with cardiac cachexia demonstrated significantly reduced NF-kappaB activity in PBMC as compared to both non-cachectic CHF patients (P<0.001) and healthy controls (P<0.05). CONCLUSION: The activity of NF-kappaB system in peripheral immune cells is augmented in patients with advanced CHF, whereas it is diminished in those with cardiac cachexia. The significance of derangements within NF-kappaB system in PBMC for immune phenomena seen in cachectic and non-cachectic CHF patients remains further studies.


Assuntos
Caquexia/sangue , Caquexia/epidemiologia , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/epidemiologia , Leucócitos Mononucleares/metabolismo , NF-kappa B/sangue , Idoso , Comorbidade , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Insuficiência Cardíaca/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade
7.
Biochim Biophys Acta ; 1773(2): 209-18, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17141339

RESUMO

It has been shown that cyclic GMP (cGMP) modulates the inflammatory responses of macrophages, but the underlying molecular mechanisms are still poorly understood. Looking for proteins potentially regulated by cGMP in rat peritoneal macrophages (PMs), in this study we analyzed expression and activity of cGMP-hydrolyzing and cGMP-regulated phosphodiesterases (PDEs). It was found that freshly isolated peritoneal exudate macrophages (PEMs) express enzymes belonging to families PDE1-3, PDE5, PDE10, and PDE11. Analysis of substrate specificity, sensitivity to inhibitors, and subcellular localization showed that PDE2 and PDE3 are the main cGMP-regulated PDE isoforms in PEMs. The profile of PDE expression was altered by maintaining PEMs in culture and treatment with bacterial endotoxin (LPS). After 24 h culture, PDE5 was not present and the levels of PDE2, PDE3, and PDE11 were markedly decreased. However, their expression and activity was recovered after treatment of cultured cells with LPS. A similar pattern of changes was observed for the expression of TNFalpha, but not for guanylyl cyclase A (GC-A). LPS up-regulated PDE expression also in resident peritoneal macrophages (RPMs), although not all PDEs present in PEMs were detected in RPMs. Taken together, our results show that in rat PMs expression of cGMP-dependent PDEs positively correlates with the activation state of cells. Moreover, the fact that most of these PDEs hydrolyze also cAMP indicates that cGMP can play a role of potent regulator of cAMP signaling in macrophages.


Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/enzimologia , Regulação para Cima/efeitos dos fármacos , 3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Inibidores de Fosfodiesterase/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Especificidade por Substrato/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Regulação para Cima/genética
8.
Biochim Biophys Acta ; 1760(11): 1618-23, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17045402

RESUMO

The nitric oxide/soluble guanylyl cyclase/cGMP-dependent protein kinase (NO/sGC/PKG) cascade has been shown to affect important functions of circulating neutrophils. We demonstrate that neutrophils isolated from rats treated intraperitoneally with peptone protease cannot use this signaling pathway. Although PKG was detected at both the mRNA and protein levels in peripheral blood neutrophils (PBNs) of control rats, it was expressed neither in PBNs nor in peritoneal exudate neutrophils (PENs) of provoked rats. Also, mRNA of the alpha and beta chains of heterodimeric sGC was present in PBNs, but absent in PENs. Consistently, PBNs responded to activators of sGC with cGMP synthesis, while PENs did not. These results showed that neutrophils recruited by a provoking agent lost PKG and, in the case of PENs, also sGC and thus the capacity to respond to NO with cGMP signaling. We speculate that such downregulation of the sGC/PKG pathway is likely a result of the high activity of inducible NO synthase observed in inflammatory neutrophils.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Neutrófilos/enzimologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de GMP Cíclico/genética , Guanilato Ciclase/genética , Masculino , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Peptonas/farmacologia , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais , Guanilil Ciclase Solúvel
9.
J Immunoassay Immunochem ; 27(4): 331-40, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16981646

RESUMO

Serum autoantibodies to visual arrestin, also termed S-antigen, have been shown to accompany several autoimmune-related diseases. However, they were also detected in sera of healthy individuals; there is lack of a sensitive and fast method for evaluation of putative differences between those two groups of antibodies. We show that, using biosensor technology based on surface plasmon resonance (SPR), it was possible to characterize real-time interactions of immune sera with immobilized arrestin. Binding characteristics revealed different interaction kinetics of antiarrestin antibodies present in two distinct rabbit sera and, thus, broadened results of immunoblotting analysis. Therefore, we suggest that SPR-based biosensor technology might be a valuable method for monitoring and evaluation of antiarrestin antibodies in patients' sera.


Assuntos
Arrestina/imunologia , Autoanticorpos/sangue , Ressonância de Plasmônio de Superfície/métodos , Animais , Complexo Antígeno-Anticorpo/análise , Reações Antígeno-Anticorpo , Técnicas Biossensoriais , Bovinos , Humanos , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Coelhos , Sensibilidade e Especificidade , Fatores de Tempo
10.
Postepy Hig Med Dosw (Online) ; 59: 324-33, 2005 Jul 08.
Artigo em Polonês | MEDLINE | ID: mdl-16012393

RESUMO

Arrestins are cytosolic proteins involved in the termination of signaling by activated G protein-coupled receptors (GPCR). Four different arrestins are identified in mammals to date. Two of these are specific to retinal photoreceptor cells, while the other two (beta-arrestin 1 and 2) are ubiquitously expressed. Recently, several new regulatory functions of arrestins in such processes as receptor ubiquitination, signaling through kinases, and others have been discovered.


Assuntos
Arrestinas/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Animais , Arrestinas/metabolismo , Humanos , Células Fotorreceptoras/metabolismo , beta-Arrestina 1 , beta-Arrestinas
11.
Ophthalmic Res ; 36(2): 120-3, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15017109

RESUMO

Multiple sclerosis (MS) is frequently accompanied by visual symptoms including those related to retinal disorders. Since they may be a consequence of an autoimmune reaction, we examined whether sera of patients with diagnosed MS and changes in visual-evoked potentials contain antibodies against retinal antigens (retAgs). Immunoblot analysis revealed that MS sera recognized mainly a 46-kD antigen, a 41-kD antigen, retinal arrestin, to a smaller extent also 70-, 56-, 43-, and 36-kD proteins. Patients whose sera showed the highest reactivity with 41- and 46-kD antigens had deficiencies in visual acuity, visual fields, ophthalmoscopy, and electroretinograms. Our observation suggests that antibodies to these retAgs may play a role in the origin of ophthalmologic impairment in MS.


Assuntos
Autoanticorpos/sangue , Autoantígenos/imunologia , Esclerose Múltipla/imunologia , Retina/lesões , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peso Molecular , Esclerose Múltipla/complicações , Transtornos da Visão/etiologia , Acuidade Visual
12.
Acta Biochim Pol ; 50(3): 837-48, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14515164

RESUMO

The aim of our studies was to establish which enzymes constitute the "cGMP pathway" in rat and guinea pig peritoneal macrophages (PM). We found that in guinea pig PM synthesis of the nucleotide was significantly enhanced in response to activators of soluble guanylyl cyclase (sGC) and it was only slightly stimulated by specific activators of particulate guanylyl cyclases (pGC). In contrast, rat PM responded strongly to atrial natriuretic peptide (ANP), the activator of pGC type A. The rat cells synthesized about three-fold more cGMP than an equal number of the guinea pig cells. The activity of phosphodiesterases (PDE) hydrolyzing cGMP was apparently regulated by cGMP itself in PM of both species and again it was higher in the rat cells than in those isolated from guinea pig. However, guinea pig PM revealed an activity of Ca(2+)/calmodulin-dependent PDE1, which was absent in the rat cells. Using Western blotting analysis we were unable to detect the presence of cGMP-dependent protein kinase 1 (PKG1) in PM isolated from either species. In summary, our findings indicate that particulate GC-A is the main active form of GC in the rat PM, while in guinea pig macrophages the sGC activity dominates. Since the profiles of the PDE activities in rat and guinea pig PM are also different, we conclude that the mechanisms regulating cGMP metabolism in PM are species-specific. Moreover, our results suggest that targets for cGMP other than PKG1 should be present in PM of both species.


Assuntos
Fator Natriurético Atrial/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Macrófagos Peritoneais/metabolismo , Animais , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Cobaias , Masculino , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Wistar , Frações Subcelulares
13.
Acta Biochim Pol ; 50(2): 367-76, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12833163

RESUMO

Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca(2+)-binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca(2+). Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca(2+)-dependent binding of recoverin to Phenyl-Sepharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca(2+)](free) (10 microM) than at low [Ca(2+)](free) (10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca(2+)](free) in recoverin and at low [Ca(2+)](free) in GCAP2. Such different changes of hydrophobicity evoked by Ca(2+) appear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.


Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/química , Proteínas do Olho , Lipoproteínas , Proteínas do Tecido Nervoso , Sefarose/análogos & derivados , Naftalenossulfonato de Anilina/química , Naftalenossulfonato de Anilina/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Bovinos , Transferência Ressonante de Energia de Fluorescência , Proteínas Ativadoras de Guanilato Ciclase , Hipocalcina , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Recoverina , Retina/química , Sefarose/metabolismo , Espectrometria de Fluorescência/métodos , Triptofano/química , Triptofano/metabolismo
14.
Postepy Hig Med Dosw ; 57(1): 19-32, 2003.
Artigo em Polonês | MEDLINE | ID: mdl-12765121

RESUMO

Nuclear factor kappa B (NF-kappa B) is common proinflammatory transcription factor involved in expression of c.a. 300 different genes. It is usually present in the cytosol as an inactive complex and upon activation translocates into the nucleus. The mechanisms of activation of NF-kappa B are complex and they involve several different signaling pathways and plethora of proteins. In this minireview we describe the main deciphered as well as suggested mechanisms regulating of NF-kappa B activity.


Assuntos
NF-kappa B/metabolismo , Animais , Núcleo Celular/metabolismo , Citosol/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Interleucina-1/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo
15.
Postepy Hig Med Dosw ; 57(6): 631-48, 2003.
Artigo em Polonês | MEDLINE | ID: mdl-15002161

RESUMO

Cyclic GMP is common second messenger in a plethora of processes. Its major intracellular receptors are the cGMP-dependent protein kinases (PKGs). In this minireview we summarise the main results of studies on structure and physiological role of PKGs.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , GMP Cíclico/fisiologia , Animais , Sistema Cardiovascular/enzimologia , Sistema Nervoso Central/enzimologia , Regulação da Expressão Gênica , Humanos , Intestinos/enzimologia , Rim/enzimologia , Nervos Periféricos/enzimologia , Sistemas do Segundo Mensageiro
16.
Arch Immunol Ther Exp (Warsz) ; 50(4): 289-94, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12371625

RESUMO

Numerous reports indicate that cyclic 3',5' guanosine monophosphate (cGMP) is involved in the regulation of immune processes. However, the mechanisms responsible for the synthesis of this nucleotide and its signaling pathways in immune cells are still not well recognized. The aim of our studies was to establish: 1) which form of guanylyl cyclase (GC) synthesizes cGMP in murine lymphoid organs and 2) whether the same organs express the isoforms PKG1alpha and/or PKG1beta of protein kinase G, known as possible target for synthesized cGMP. Cells isolated from thymus, lymph nodes, and spleen were treated with activators (SNP, ANP, CNP, STa) of soluble or particulate cyclases. Sodium nitroprusside (SNP) elevated intracellular cGMP 2-fold in thymic and lymph node cells and about 10-fold in spleen cells. Atrial natriuretic peptide (ANP) caused modest but statistically significant increases of cGMP in cells of all three organs. Additionally, spleen cells elevated their cGMP content about 2-fold in response to C-type natriuretic protein (CNP). In cellular homogenates of the all analyzed organs, the antibody anti-PKG1beta stained the 78 kDa band corresponding to the molecular mass of PKG1. Only homogenates of spleen cells were stained by the antibody recognizing PKG1alpha. Our results indicate that in the investigated organs cGMP may be synthesized mainly by soluble GC in response to nitric oxide. The modest increase of cGMP upon stimulation by ANP suggests that in all these organs either exists only a small subpopulation of cells that express particulate cyclase GC-A or GC-A is expressed at very low level. In spleen cells, however, cyclase GC-B appears to be the more active enzyme. Elevated cGMP concentration may in turn activate PKG1beta in thymus, lymph node, and spleen cells and also PKG1alpha in spleen cells.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/biossíntese , Regulação Enzimológica da Expressão Gênica , Tecido Linfoide/metabolismo , Animais , Western Blotting , Células Cultivadas , GMP Cíclico/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Isoenzimas/metabolismo , Tecido Linfoide/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL
17.
Adv Exp Med Biol ; 514: 319-32, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12596930

RESUMO

Lowered concentration of Ca2+ ions, resulting from illumination of the photoreceptor cell, is the signal for resynthesis of cGMP by retina-specific guanylyl cyclase (retGC). This Ca2+-dependent activation of retGC is mediated by Ca2+-binding proteins named GCAPs (guanylyl cyclase-activating proteins) and contributes to the recovery of photoreceptor cell to the dark state. Three different GCAPs (GCAP1, GCAP2 and GCAP3) are identified in vertebrate retina to date. In this chapter we describe their discovery, methods of purification, properties, and possible modes of action.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Regulação da Expressão Gênica , Guanilato Ciclase/metabolismo , Receptores de Superfície Celular , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Proteínas Ativadoras de Guanilato Ciclase , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Isoformas de Proteínas , Estrutura Terciária de Proteína , Retina/enzimologia , Homologia de Sequência de Aminoácidos
18.
Acta Biochim Pol ; 49(4): 891-7, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12545195

RESUMO

Intact rat peritoneal macrophages (rPM) treated with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases (PDEs), accumulated more cGMP than untreated cells. A PDE activity toward [(3)H]cGMP was detected in the soluble and particulate fractions of rPM. The hydrolysis of cGMP was Ca(2+)/calmodulin-independent but increased in the presence of cGMP excess. Similar results were obtained when [(3)H]cAMP was used as a substrate. The hydrolytic activity towards both nucleotides was inhibited in the presence of IBMX. Therefore, the PDEs of families 2, 5, 10 and 11 are potential candidates for cGMP hydrolysis in the rPM. They may not only regulate the cGMP level in a feedback-controlled way but also link cGMP-dependent pathways with those regulated by cAMP.


Assuntos
GMP Cíclico/metabolismo , Macrófagos Peritoneais/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Fator Natriurético Atrial/farmacologia , Cálcio/metabolismo , Calmodulina/metabolismo , Células Cultivadas , GMP Cíclico/farmacologia , Hidrólise , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Wistar , Solubilidade
19.
Acta Biochim Pol ; 49(4): 899-905, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12545196

RESUMO

The Ca(2+)-dependent activation of retina-specific guanylyl cyclase (retGC) is mediated by guanylyl cyclase-activating proteins (GCAPs). Here we report for the first time detection of a 19 kDa protein (p19) with GCAP properties in extracts of rat retina and pineal gland. Both extracts stimulate synthesis of cGMP in rod outer segment (ROS) membranes at low (30 nM) but not at high (1 microM) concentrations of Ca(2+). At low Ca(2+), immunoaffinity purified p19 activates guanylyl cyclase(s) in bovine ROS and rat retinal membranes. Moreover, p19 is recognized by antibodies against bovine GCAP1 and, similarly to other GCAPs, exhibits a Ca(2+)-dependent electrophoretic mobility shift.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Glândula Pineal/metabolismo , Retina/metabolismo , Animais , Anticorpos/imunologia , Western Blotting , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/isolamento & purificação , Bovinos , Membrana Celular/enzimologia , Eletroforese em Gel de Poliacrilamida , Guanilato Ciclase/metabolismo , Proteínas Ativadoras de Guanilato Ciclase , Camundongos , Peso Molecular , Ratos , Proteínas Recombinantes , Retina/citologia , Retina/enzimologia , Segmento Externo da Célula Bastonete/citologia , Segmento Externo da Célula Bastonete/metabolismo
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