RESUMO
Significant variation exists in the surgical suture materials and techniques used for dermatological surgery. Many wound-closure techniques are now practised, including use of sutures, staples and topical adhesives. The focus of our review article is to summarize the latest evidence relating to suture materials and wound-closure techniques, considering the following areas: scar/cosmesis, pain, patient satisfaction, cost, infection and wound complications. We searched the databases Medline, PubMed and Embase using the keywords 'skin surgery', 'dermatologic surgery', 'sutures', 'suture techniques', 'suturing techniques' and 'surgical techniques' to identify relevant English-language articles. Absorbable superficial sutures may be a preferred alternative to nonabsorbable sutures by both patients and surgeons. Subcuticular sutures may be preferable to simple interrupted sutures for superficial wound closure, and there may also be a role for skin staples in dermatological surgery, particularly on the scalp. However, there remains limited evidence specific to dermatological surgery supporting the use of particular suture materials and suturing techniques. Further high-quality research is required, including multicentre randomized trials with larger cohorts.
Assuntos
Procedimentos Cirúrgicos Dermatológicos/instrumentação , Procedimentos Cirúrgicos Dermatológicos/métodos , Técnicas de Sutura , Suturas , Cicatriz/prevenção & controle , Análise Custo-Benefício , Humanos , Dor/prevenção & controle , Preferência do Paciente , Satisfação do Paciente , Infecção da Ferida Cirúrgica , Técnicas de Sutura/efeitos adversos , Técnicas de Sutura/economia , Suturas/economia , CicatrizaçãoRESUMO
This is the second part of a two-part series summarizing the latest evidence related to suture materials and wound closure techniques in dermatological surgery. We critically appraised evidence focusing on the following consequences of suture choice: scar/cosmesis, pain, patient satisfaction, cost, infection and wound complications. We searched the databases MEDLINE, PubMed and Embase using the keywords 'skin surgery', 'dermatological surgery', 'sutures', 'braided sutures', 'monofilament sutures' and 'antibacterial sutures' to identify relevant English-language articles. This part of the review assesses the evidence for different types of buried sutures, including braided vs. monofilament sutures, longer-absorbing sutures and antibacterial sutures. The majority of trials were noted to be of poor quality, single-centre (thus lacking external validity) and underpowered, which presents challenges in comparing suture techniques in skin surgery. Future large-scale, multicentre, randomized trials are needed, with both surgeon and patient-assessed validated outcomes.
Assuntos
Procedimentos Cirúrgicos Dermatológicos/instrumentação , Procedimentos Cirúrgicos Dermatológicos/métodos , Técnicas de Sutura , Suturas , Antibacterianos/administração & dosagem , Cicatriz/prevenção & controle , Análise Custo-Benefício , Humanos , Dor/prevenção & controle , Preferência do Paciente , Satisfação do Paciente , Absorção Subcutânea , Infecção da Ferida Cirúrgica/prevenção & controle , Técnicas de Sutura/efeitos adversos , Técnicas de Sutura/economia , Suturas/economia , CicatrizaçãoRESUMO
PURPOSE: During healthy pregnancy, a distinct but limited invasion of trophoblast cells into the uterus occurs. In contrast, excessive trophoblast invasion is associated with placental choriocarcinoma (CC). Overexpression of the cytoskeletal protein LASP-1 was shown to contribute to cancer aggressiveness. Here, the yet unknown role of LASP-1 in CC cells is analysed. METHODS: Expression of LASP-1 in human primary carcinoma was assessed by immunohistochemistry and confirmed in CC-derived cell lines by immunocytochemistry, RT-PCR and Western blot. After down-regulation of LASP-1 expression with specific si-RNA in CC-derived cell lines, migratory and proliferative activities were analysed by matrigel migration assay and WST-8 test. RESULTS: LASP-1 expression was detected in human primary choriocarcinoma and in JEG-3, JAR and BeWo cells. Knock down of LASP-1 resulted in a decreased expression of LASP-1 protein in JEG-3 and JAR cells accompanied by a diminished migration and a decreased proliferative activity of these two cell lines. Knockdown of LASP-1 in BeWo cells failed. In consequence, migratory function and proliferation was unaffected. CONCLUSION: This is the first study describing LASP-1 expression in CC cells. Detecting an affection of migratory processes after LASP-1 silencing, we propose that LASP-1 could impact on metastasis of CC cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Coriocarcinoma/genética , Proteínas do Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas com Domínio LIM/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Coriocarcinoma/metabolismo , Proteínas do Citoesqueleto/genética , Regulação para Baixo/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Proteínas com Domínio LIM/genética , Gravidez , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Trofoblastos/metabolismoRESUMO
BACKGROUND: LIM and SH3 protein 1 (LASP-1) is a nucleo-cytoplasmatic signalling protein involved in cell proliferation and migration and is upregulated in breast cancer in vitro studies have shown that LASP-1 might be regulated by prostate-derived ETS factor (PDEF), p53 and/or LASP1 gene amplification. This current study analysed the prognostic significance of LASP-1 on overall survival (OS) in 177 breast cancer patients and addressed the suggested mechanisms of LASP-1-regulation. METHODS: Nucleo-cytoplasmatic LASP-1-positivity of breast carcinoma samples was correlated with long-term survival, clinicopathological parameters, Ki67-positivity and PDEF expression. Rate of LASP1 amplification was determined in micro-dissected primary breast cancer cells using quantitative RT-PCR. Cell-phase dependency of nuclear LASP-1-localisation was studied in synchronised cells. In addition, LASP-1, PDEF and p53 expression was compared in cell lines of different tumour entities to define principles for LASP-1-regulation. RESULTS: We showed that LASP-1 overexpression is not due to LASP1 gene amplification. Moreover, no correlation between p53-mutations or PDEF-expression and LASP-1-status was observed. However, nuclear LASP-1-localisation in breast carcinomas is increased during proliferation with peak in G2/M-phase and correlated significantly with Ki67-positivity and poor OS. CONCLUSION: Our results provide evidence that nuclear LASP-1-positivity may serve as a negative prognostic indicator for long-term survival of breast cancer patients.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/mortalidade , Carcinoma/mortalidade , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma/diagnóstico , Carcinoma/genética , Carcinoma/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Feminino , Amplificação de Genes/fisiologia , Humanos , Proteínas com Domínio LIM , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida , Sobreviventes/estatística & dados numéricos , Fatores de Tempo , Distribuição TecidualRESUMO
There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbß3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after in-vitro -activation and -inhibition with their respective untreated controls using unbiased fluorescence two-dimensional differential in-gel electrophoresis. The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen's d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.
Assuntos
Ativação Plaquetária , Proteômica , Adulto , Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Humanos , Concentração de Íons de Hidrogênio , Masculino , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Selectina-P/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/metabolismo , Controle de QualidadeRESUMO
Maternal cold exposure of pregnant sheep promotes fetal growth, whereas nutrient restriction (NR) can reverse this effect. The present study was designed to establish whether cold exposure induced by winter shearing of the mother at 70 days gestation (term=147 days), with or without NR (induced by a 50% reduction in maternal food intake from 110 days gestation), has specific effects on mRNA abundance of hepatic genes related to growth and liver energy metabolism that could regulate postnatal body and liver growth. Measurements of hepatic gene expression for the GH secretagog receptor-1a (GHSR-1A), peroxisome proliferator-activated receptor (PPAR)alpha, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase activity together with glycogen content were made in the livers of offspring at 1 and 30 days of age. Maternal NR reduced liver mass at day 1, whereas offspring of cold-exposed mothers had larger livers at day 30 irrespective of maternal diet. Cold exposure resulted in the up-regulation of GHSR-1A mRNA abundance and reduced glucose-6-phosphatase activity at 1, but not 30 days of age, whereas IGF-II mRNA was decreased at 1 and 30 days. PPARalpha mRNA abundance was enhanced, while PEPCK was reduced in 30-day old offspring of cold-exposed mothers. NR caused reductions in IGF-I mRNA and, at 1-day postnatal age, down-regulated GHR, while, at 30 days, reduced GHSR-1A gene expression and hepatic glycogen content. In conclusion, we have shown that maternal cold exposure and NR have different effects on the hepatic GH-IGF and metabolic axis that may contribute to changes in liver growth over the first month of life.
Assuntos
Temperatura Baixa , Privação de Alimentos , Fígado/metabolismo , Exposição Materna , Carneiro Doméstico/metabolismo , Somatomedinas/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Feminino , Glucose-6-Fosfatase/metabolismo , Glicogênio/metabolismo , Hormônio do Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , PPAR alfa/metabolismo , Gravidez , RNA Mensageiro/análiseRESUMO
Vegetation and peatland fires cause poor air quality and thousands of premature deaths across densely populated regions in Equatorial Asia. Strong El-Niño and positive Indian Ocean Dipole conditions are associated with an increase in the frequency and intensity of wildfires in Indonesia and Borneo, enhancing population exposure to hazardous concentrations of smoke and air pollutants. Here we investigate the impact on air quality and population exposure of wildfires in Equatorial Asia during Fall 2015, which were the largest over the past two decades. We performed high-resolution simulations using the Weather Research and Forecasting model with Chemistry based on a new fire emission product. The model captures the spatio-temporal variability of extreme pollution episodes relative to space- and ground-based observations and allows for identification of pollution sources and transport over Equatorial Asia. We calculate that high particulate matter concentrations from fires during Fall 2015 were responsible for persistent exposure of 69 million people to unhealthy air quality conditions. Short-term exposure to this pollution may have caused 11,880 (6,153-17,270) excess mortalities. Results from this research provide decision-relevant information to policy makers regarding the impact of land use changes and human driven deforestation on fire frequency and population exposure to degraded air quality.
Assuntos
Poluentes Atmosféricos , Poluição do Ar , Exposição Ambiental , Modelos Teóricos , Fumaça , Incêndios Florestais , Ásia , Feminino , Humanos , MasculinoRESUMO
The activation of the cGMP-dependent protein kinase and cAMP-dependent protein kinase by the diastereomers of guanosine 3',5'-monophosphorothioate, (Sp)-cGMPS and (Rp)-cGMPS, and 8-chloroguanosine 3',5'-monophosphorothioate, (Sp)-8-Cl-cGMPS and (Rp)-8-Cl-cGMPS, was investigated using the peptide Kemptide as substrate. The (Sp)-diastereomers, which have an axial exocyclic sulfur atom, bound to the cGMP-dependent protein kinase and stimulated its phosphotransferase activity. In contrast, the (Rp)-isomers, which have an equatorial exocyclic sulfur atom, bound to the enzyme without stimulation of its activity. (Rp)-cGMPS and (Rp)-8-Cl-cGMPS antagonized the activation of the cGMP-dependent protein kinase with a Ki of 20 microM and 1.5 microM, respectively. (Rp)-cGMPS also antagonized the activation of cAMP-dependent protein kinase with a Ki of 20 microM. In contrast, (Rp)-8-cGMPS ws a weak inhibitor of the cAMP-dependent protein kinase with a Ki of 100 microM. (Rp)-8-Cl-cGMPS appears to be a rather selective inhibitor of the cGMP-dependent protein kinase and may be a useful tool for studying the role of cGMP in broken and intact cell systems.
Assuntos
GMP Cíclico/análogos & derivados , Inibidores de Proteínas Quinases , Tionucleotídeos/farmacologia , Animais , Bovinos , GMP Cíclico/farmacologia , Ativação Enzimática , Cinética , Pulmão/enzimologia , Estrutura Molecular , Miocárdio/enzimologia , Proteínas Quinases/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Detailed studies of differences in distinct cGMP kinase isoforms are highly dependent on expression of large amounts of these enzyme isoforms that are not easily purified by conventional methods. Here cGMP-dependent protein kinases, the type I beta soluble form from human placenta, and the type II membrane-associated form from rat intestine, were each expressed in a baculovirus/Sf9 cell system and purified in milligram amounts by affinity chromatography. The expressed recombinant proteins displayed characteristics like those of their native counterparts. cGK I beta was expressed as a 76 kDa protein predominantly found in the cytosol fraction, whereas cGK II was expressed as an 86 kDa protein predominantly associated with the membrane fraction. The apparent Ka and Vmax of cGMP for activation of cGK I beta were 0.5 microM and 3.4 mumol/min/mg, and for cGK II were 0.04 microM and 1.8 mumol/min/mg.
Assuntos
Baculoviridae/genética , Proteínas Quinases Dependentes de GMP Cíclico/genética , Expressão Gênica , Isoenzimas/genética , Animais , Células Cultivadas , Cromatografia de Afinidade , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/isolamento & purificação , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Feminino , Humanos , Intestinos/enzimologia , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Placenta/enzimologia , Ratos , Proteínas Recombinantes , Spodoptera/metabolismo , Frações Subcelulares/enzimologiaRESUMO
Activation of cyclic GMP-dependent protein kinase (cGK) is an important event in the regulation of blood pressure and platelet function. Upstream signals are the generation of nitric oxide (NO) by NO synthases and the subsequent rise in cyclic GMP levels mediated by NO-dependent guanylyl cyclases (GCs). The identification of new cGK activators by high throughput screening (HTS) may lead to the development of a novel class of therapeutics for the treatment of cardiovascular diseases. Therefore, a homogeneous, nonradioactive assay for cGK activity was developed using a biotinylated peptide derived from vasodilator-stimulated phosphoprotein (VASP), a well-characterized natural cGK substrate. The phosphorylated peptide could be detected by a VASP-specific monoclonal phosphoserine antibody and a fluorescent detection system consisting of a europium-labeled secondary antibody and allophycocyanin (APC)-labeled streptavidin. Fluorescence resonance energy transfer (FRET) from europium to APC was detected in a time-resolved fashion (TR-FRET). Activation and inhibition constants for known substances determined by this new fluorescence-based assay correlated well with published results obtained by conventional radioactive cGK activity assays. The assay proved to be sensitive, robust, highly specific for cGK, and suitable for HTS in 96- and 384-well formats. This assay is applicable to purified enzymes as well as to complex samples such as human platelet extracts.
Assuntos
Carbazóis , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Indóis , Espectrometria de Fluorescência/métodos , Alcaloides/farmacologia , Sequência de Aminoácidos , Automação , Plaquetas/enzimologia , Plaquetas/metabolismo , Western Blotting , Moléculas de Adesão Celular/metabolismo , GMP Cíclico/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Células Jurkat , Proteínas dos Microfilamentos , Modelos Biológicos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Fatores de TempoRESUMO
1. The modulation of the guanosine 3':5'-cyclic monophosphate (cyclic GMP)- and adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase activities by the diastereomers of 8-bromo-beta phenyl-1, N2-ethenoguanosine 3':5'-cyclic monophosphorothioate, ((Rp)- and (Sp)-8-bromo-PET-cyclic GMPS) was investigated by use of purified protein kinases. In addition, the effects of (Rp)-8-bromo-PET-cyclic GMPS on protein phosphorylation in intact human platelets and on [3H]-noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries were also studied. 2. Kinetic analysis with purified cyclic GMP-dependent protein kinase (PKG) type I alpha and I beta, which are expressed in the rat tail artery, revealed that (Rp)-8-bromo-PET-cyclic GMPS is a competitive inhibitor with an apparent Ki of 0.03 microM. The activation of purified cyclic AMP-dependent protein kinase (PKA) type II was antagonized with an apparent Ki of 10 microM. 3. In human platelets, (Rp)-8-bromo-PET-cyclic GMPS (0.1 mM) antagonized the activation of the PKG by the selective activator 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate (8-pCPT-cyclic GMP; 0.2 mM) without affecting the activation of PKA by (Sp)-5, 6-dichloro-1-beta-D-ribofurano-sylbenzimidazole- 3':5'-cyclic monophosphorothioate ((Sp)-5,6-DCl-cyclic BiMPS; 0.1 mM). 4. (Rp)-8-bromo-PET-cyclic GMPS was not hydrolysed by the cyclic GMP specific phosphodiesterase (PDE) type V from bovine aorta but potently inhibited this PDE. 5. The corresponding sulphur free cyclic nucleotide of the two studied phosphorothioate derivatives, 8-bromo-beta-phenyl-1, N2-ethenoguanosine-3':5'-cyclic monophosphate (8-bromo-PET-cyclic GMP), had no effect on electrically-induced [3H]-noradrenaline release but concentration-dependently decreased the stimulation-induced vasoconstriction. (Rp)-8-bromo-PET-cyclic GMPS (3 microM) shifted the vasoconstriction response to the right without affecting stimulation evoked tritium overflow. 6. The NO donor, 3-morpholinosydnonimine (SIN-1) relaxed rat tail arteries precontracted with phenylephrine (1 microM). The SIN-1 concentration-relaxation curve was shifted in a parallel manner to the right by (Rp)-8-bromo-PET-cyclic GMPS, suggesting that the relaxation was mediated by a cyclic GMP/PKG-dependent mechanism. 7. The [3H]-noradrenaline release-enhancing effect and stimulation-induced decrease in vasoconstriction of forskolin were unaffected by (Rp)-8-bromo-PET-cyclic GMPS. Moreover, the forskolin concentration-relaxation curve was not changed in the presence of the PKG inhibitor, suggesting a high selectivity in intact cells for PKG- over PKA-mediated effects. 8. The results obtained indicate that (Rp)-8-bromo-PET-cyclic GMPS presently is the most potent and selective inhibitor of PKG and is helpful in distinguishing between cyclic GMP and cyclic AMP messenger pathways activation. Therefore, this phosphorothioate stereomer may be a useful tool for studying the role of cyclic GMP in vitro.
Assuntos
Plaquetas/efeitos dos fármacos , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , GMP Cíclico/análogos & derivados , Músculo Liso Vascular/efeitos dos fármacos , Norepinefrina/metabolismo , Fosfoproteínas/antagonistas & inibidores , Tionucleotídeos/farmacologia , Vasoconstrição/efeitos dos fármacos , Animais , Sequência de Bases , Plaquetas/enzimologia , Bovinos , Moléculas de Adesão Celular , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/síntese química , GMP Cíclico/farmacologia , Humanos , Técnicas In Vitro , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Estereoisomerismo , Tionucleotídeos/síntese química , Vasodilatadores/farmacologiaRESUMO
8-(p-Chlorophenylthio)-cGMP (8-pCPT-cGMP) and 8-bromo-cGMP were compared with respect to their chemical and biological properties in order to evaluate their potential as selective activators of cGMP-dependent protein kinase (cGMP-PK; EC 2.7.1.37) in intact human platelets. 8-pCPT-cGMP, 8-Br-cGMP and cGMP were shown to be potent and selective activators of purified bovine lung cGMP-PK and of cGMP-PK present in human platelet membranes when compared with the activation of cAMP-dependent protein kinase (cAMP-PK; EC 2.7.1.37). 8-pCPT-cGMP was not hydrolysed by the purified cGMP-stimulated phosphodiesterase (cGS-PDE), cGMP-inhibited phosphodiesterase (cGI-PDE) and Ca(2+)-calmodulin-dependent phosphodiesterase (CaM-PDE), whereas cGMP and, to a lesser extent, 8-Br-cGMP were hydrolysed by all three types of 3',5' cyclic nucleotide phosphodiesterases (EC 3.1.4.17) examined. Also, 8-pCPT-cGMP was not hydrolysed by a human platelet homogenate which contains a high level of the cGMP-specific cGMP-binding phosphodiesterase (cGB-PDE). Additionally, 8-pCPT-cGMP did not activate the cGS-PDE or inhibit the cGI-PDE, whereas half-maximal inhibition of cGI-PDE occurred at 8 microM 8-Br-cGMP. The apparent lipophilicity of 8-pCPT-cGMP was higher than that of 8-Br-cGMP. Extracellular application of 8-pCPT-cGMP to intact human platelets reproduced the pattern of protein phosphorylation induced by sodium nitroprusside (SNP), a cGMP-elevating inhibitor of platelet activation. Quantitatively, 8-pCPT-cGMP was more effective than 8-Br-cGMP in inducing phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein, a major substrate of cGMP-PK in intact platelets. As observed with SNP, pretreatment of human platelets with 8-pCPT-cGMP prevented the aggregation induced by thrombin. The results suggest that 8-pCPT-cGMP is a very potent and selective activator of cGMP-PK in cell extracts and in intact human platelets and, in this respect, is superior to 8-Br-cGMP and other cGMP analogs used for intact cell studies. The data also suggest that inhibition of platelet activation in intact human platelets by nitrovasodilators is mediated by cGMP-PK.
Assuntos
Plaquetas/efeitos dos fármacos , GMP Cíclico/análogos & derivados , Ativação Plaquetária/efeitos dos fármacos , Proteínas Quinases/análise , Tionucleotídeos/farmacologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/análise , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/antagonistas & inibidores , Plaquetas/enzimologia , GMP Cíclico/farmacologia , Ativação Enzimática , Humanos , Fosforilação , Agregação Plaquetária/efeitos dos fármacos , Inibidores de Proteínas QuinasesRESUMO
In the present study, the inhibitory effect of the cGMP analog (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate ((Rp)-8-pCPT-cGMPS) on the cGMP-dependent protein kinase-mediated protein phosphorylation in intact human platelets was investigated. In vitro phosphorylation experiments with the substrate kemptide demonstrated an inhibition of the cGMP-dependent protein kinase by (Rp)-8-pCPT-cGMPS with a Ki of 0.5 microM. In intact human platelets, (Rp)-8-pCPT-cGMPS antagonized the activation of the cGMP-dependent protein kinase by 8-pCPT-cGMP without affecting cAMP-dependent protein kinase or cGMP-regulated phosphodiesterases. The data obtained suggest that (Rp)-8-pCPT-cGMPS may be a useful tool for studying the role of cGMP in vitro and in intact cells.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Western Blotting , Bovinos , Fenômenos Químicos , Físico-Química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/antagonistas & inibidores , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , Oligopeptídeos/metabolismo , Fosforilação , Inibidores da Agregação Plaquetária/farmacologia , Tionucleotídeos/antagonistas & inibidores , Tionucleotídeos/farmacologiaRESUMO
NO and cGMP have emerged as important signal transduction mediators of the effects of certain hormones, inter-/intracellular signals, toxins and drugs. However, a major challenge is to define relevant criteria for determining which of the many NO and/or cGMP effects are dependent on cGMP-dependent protein kinases (cGKs). Important criteria include that: (1) the cell types/tissues investigated contain at least one form of cGK which is activated by the cGMP-elevating agent in the intact cell system; (2) specific activators/inhibitors of cGKs mimic/block the effects of cGMP-elevating agents in the intact cell system; and (3) the cGMP effect is absent or blunted in cGK-deficient systems, or can be reconstituted by the introduction of active cGKs. Previously, analysis of cGK activity in intact cells has been very difficult. However, the analysis of vasodilator-stimulated phosphoprotein (VASP) phosphorylation by polyclonal antibodies and newly developed monoclonal antibodies, each of which specifically recognize different phosphorylation sites, allows the quantitative measurement of cGK activity in intact cells. With the use of these methods, the properties of certain cGK mutants, cGK activators (cGMP, 8-Br-cGMP, 8-pCPT-cGMP) as well as various "specific cGK inhibitors" (KT 5823, Rp-8Br-PET-cGMPS, Rp-8-pCPT-cGMPS, H8 and H89) were investigated. Although these "specific cGK inhibitors" have been widely used to establish or rule out functional roles of cGKs, very few studies have actually addressed the efficiency/specificity of such compounds in intact cells. Our results demonstrate that these inhibitors are useful tools only when used in combination with other experimental approaches and biochemical evidence.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , GMP Cíclico/fisiologia , Isoenzimas/fisiologia , Óxido Nítrico/fisiologia , Animais , Proteínas Quinases Dependentes de GMP Cíclico/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , TransfecçãoRESUMO
A series of new analogues of 1-beta-D-ribofuranosylbenzimidazole 3',5'-phosphate (cBIMP) has been designed according to the properties predicted by the MNDO method, and synthesised from substituted benzimidazoles. Dipole vectors and HOMO and LUMO energies for each benzimidazole base were calculated by the MNDO method and the lipophilicities of the cBIMP derivatives were determined. In general, the cBIMP derivatives activate cAMP-dependent protein kinases I and II and preferentially bind to site B, especially for the type II kinase, with 2-trifluoromethyl-cBIMP and 5,6-difluoro-cBIMP exhibiting the highest site selectivity. Each cBIMP derivative can stimulate cGMP-stimulated cyclic phosphodiesterase (cGS-PDE), with 5,6-dimethyl-cBIMP being as potent as cGMP, and also inhibit cGMP-inhibited phosphodiesterase (cGI-PDE). Only the 2-trifluoromethyl-cBIMP and the Rp-phosphorothioates (cBIMPS) (equatorial P = S) were resistant to hydrolysis by cPDE. The Sp-phosphorothioates were hydrolysed slowly, if at all. In addition to exhibiting a high lipophilicity, the most active compounds for the induction of apoptosis and inhibition of proliferation were also resistant to cPDE (Sp-5,6-dichloro-cBIMPS) and/or were potent activators of cAMP-dependent protein kinase (5,6-dichloro-cBIMP).
Assuntos
AMP Cíclico/farmacologia , GMP Cíclico/farmacologia , Nucleotídeos Cíclicos/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Hidrólise , Estrutura Molecular , Proteínas Quinases/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
LASP-1 is a multidomain protein predominantly localized at focal contacts, where it regulates cytoskeleton dynamics and cell migration. However, in different tumor entities, a nuclear LASP-1 accumulation is observed, thought to have an important role in cancer progression. Until now, the molecular mechanisms that control LASP-1 nuclear import were not elucidated. Here, we identified a novel LASP-1-binding partner, zona occludens protein 2 (ZO-2), and established its role in the signal transduction pathway of LASP-1 nucleo-cytoplasmatic shuttling. Phosphorylation of LASP-1 by PKA at serine 146 induces translocation of the LASP-1/ZO-2 complex from the cytoplasm to the nucleus. Interaction occurs within the carboxyterminal proline-rich motif of ZO-2 and the SH3 domain in LASP-1. In situ proximity ligation assay confirmed the direct binding between LASP-1 and ZO-2 and visualized the shuttling. Nuclear export is mediated by Crm-1 and a newly identified nuclear export signal in LASP-1. Finally, dephosphorylation of LASP-1 by phosphatase PP2B is suggested to relocalize the protein back to focal contacts. In summary, we define a new pathway for LASP-1 in tumor progression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteína da Zônula de Oclusão-2/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/biossíntese , Humanos , Proteínas com Domínio LIM/biossíntese , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
LIM and SH3 protein 1 (LASP-1), initially identified from human breast cancer, is a specific focal adhesion protein involved in cell proliferation and migration. In the present work, we analysed the effect of LASP-1 on biology and function of human ovarian cancer cell line SKOV-3 using small interfering RNA technique (siRNA). Transfection with LASP-1-specific siRNA resulted in a reduced protein level of LASP-1 in SKOV-3 cells. The siRNA-treated cells were arrested in G(2)/M phase of the cell cycle and proliferation of the tumour cells was suppressed by 60-90% corresponding to around 70% of the cells being transfected successfully as seen by immunofluorescence. Moreover, transfected tumour cells showed a 40% reduced migration. LASP-1 silencing is accompanied by a reduced binding of the LASP-1-binding partner zyxin to focal contacts without changes in actin stress fibre and microtubule organisation or focal adhesion morphology as observed by immunofluorescence. In contrast, silencing of zyxin is not influencing cell migration and had neither influence on LASP-1 expression nor actin cytoskeleton and focal contact morphology suggesting that LASP-1 is necessary and sufficient for recruiting zyxin to focal contacts. The data provide evidence for an essential role of LASP-1 in tumour cell growth and migration, possibly through influencing zyxin localization.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas do Citoesqueleto/genética , Primers do DNA , Eletroforese em Gel Bidimensional , Feminino , Fase G2 , Inativação Gênica , Glicólise , Humanos , Imuno-Histoquímica , Proteínas com Domínio LIM , Neoplasias Ovarianas/metabolismo , RNA Interferente Pequeno , Espectrometria de Massas por Ionização por Electrospray , ZixinaRESUMO
Most platelet agonists activate and elevate the cytosolic free calcium concentration in human platelets through receptor-dependent mechanisms that are antagonized by cAMP- and cGMP-elevating agents. Nitrovasodilators such as nitroprusside and endothelium-derived relaxing factor are potent cGMP-elevating platelet inhibitors. In the present study, the role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of ADP- and thrombin-evoked calcium elevation and activation of human platelets was investigated. Preincubation of platelets with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP; a membrane-permeant selective activator of the cGMP-dependent protein kinase that does not significantly affect cGMP-regulated phosphodiesterases) inhibited the thrombin-induced phosphorylation mediated by myosin light chain kinase and protein kinase C. Nitrovasodilator-induced protein phosphorylation in human platelets was distinct from that induced by cAMP-elevating prostaglandins and could be mimicked by 8-pCPT-cGMP. Preincubation of human platelets with nitrovasodilators or 8-pCPT-cGMP inhibited the ADP- and thrombin-evoked calcium elevation in the presence and absence of external calcium. Nitrovasodilators and 8-pCPT-cGMP also inhibited the agonist-induced Mn2+ influx, but stopped-flow experiments indicated that the ADP receptor-operated cation channel was not significantly inhibited. These results suggest that in human platelets nitrovasodilators inhibit the agonist-induced calcium mobilization from intracellular stores and the secondary store-related calcium influx but not the ADP receptor-operated cation channel. The results also suggest that these nitrovasodilator effects are mediated by cGMP and the cGMP-dependent protein kinase.
Assuntos
Alprostadil/farmacologia , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , GMP Cíclico/fisiologia , Molsidomina/análogos & derivados , Nitroprussiato/farmacologia , Proteínas Quinases/fisiologia , Vasodilatadores/farmacologia , Difosfato de Adenosina/farmacologia , Plaquetas/metabolismo , Compartimento Celular , Citoplasma/metabolismo , Humanos , Técnicas In Vitro , Ativação do Canal Iônico , Cinética , Molsidomina/farmacologia , Fosfoproteínas/metabolismo , Receptores de Superfície Celular/fisiologia , Trombina/farmacologiaRESUMO
Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mitogen-activated protein kinase-activated protein kinase (MAPKAP) 2 is associated with signaling events involved in platelet aggregation and regulation of microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for the inhibition of platelet aggregation. Stimulation of washed platelets with 8-para-chlorophenylthio-cGMP, a cGK specific activator, resulted in a time-dependent phosphorylation of Hsp27. This is supported by the ability of cGK to phosphorylate Hsp27 in vitro to an extent comparable with the cGK-mediated phosphorylation of its established substrate vasodilator-stimulated phosphoprotein. Studies with Hsp27 mutants identified threonine 143 as a yet uncharacterized phosphorylation site in Hsp27 specifically targeted by cGK. To test the hypothesis that cGK could inhibit platelet aggregation by phosphorylating Hsp27 and interfering with the MAPKAP kinase phosphorylation of Hsp27, the known MAPKAP kinase 2-phosphorylation sites (Ser15, Ser78, and Ser82) as well as Thr143 were replaced by negatively charged amino acids, which are considered to mimic phosphate groups, and tested in actin polymerization experiments. Mimicry at the MAPKAP kinase 2 phosphorylation sites led to mutants with a stimulating effect on actin polymerization. Mutation of the cGK-specific site Thr143 alone had no effect on actin polymerization, but in the MAPKAP kinase 2 phosphorylation-mimicking mutant, this mutation reduced the stimulation of actin polymerization significantly. These data suggest that phosphorylation of Hsp27 and Hsp27-dependent regulation of actin microfilaments contribute to the inhibitory effects of cGK on platelet function.