RESUMO
Experimental glomerulonephritis results in hypertension that is sensitive to salt. Nevertheless, salt retention alone cannot explain the increase in blood pressure. Angiotensin antagonistic therapy reduces hypertension caused by puromycin amino nucleosides (PAN). We investigated the hypothesis that PAN modifies renal vascular reactivity through processes dependent on angiotensin. Long-Evans rats were given an intraperitoneal injection of either puromycin (150 mg/kg) or saline (controls). Group 1 was fed a normal sodium diet (NSD, n = 9). Group 2 was given 30 mg/L of quinapril (Q) in addition to NSD (NSD + Q; n = 6). Group 3 received a high sodium diet (HSD, n = 7), and Group 4 received HSD + Q (n = 7). Systolic blood pressure (SBP), plasma creatinine, proteinuria, and sodium balance were monitored for 12 days. On day 15, renal vascular reactivity was assessed by administering increasing doses of angiotensin II, acetylcholine (ACh), and sodium nitroprusside (SNP) directly into the renal artery. SBP progressively increased in all PAN groups. This increase in SBP was greater in the HSD groups and was not significantly altered by Q treatment. SBP increased by 22 ± 4% (NSD), 51 ± 5% (NSD + Q), 81 ± 10% (HSD), and 65 ± 8% (HSD + Q). The renal blood flow of PAN rats did not return to baseline despite their normal renal vasoconstrictor responses to angiotensin II. Additionally, they showed reduced renal vasodilator responses to SNP and Ach. The vasodilator responses to both vasodilators were surprisingly unaffected by the inhibition of the angiotensin-converting enzyme (ACE). Renal vasodilator responses to both endothelium-dependent and independent variables were reduced in early PAN-induced hypertension. We found that the angiotensin-mediated mechanism is not responsible for this altered renal vasoreactivity.
Assuntos
Angiotensina II , Rim , Animais , Angiotensina II/farmacologia , Ratos , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Masculino , Ratos Long-Evans , Pressão Sanguínea/efeitos dos fármacos , Puromicina/farmacologia , Nitroprussiato/farmacologia , Puromicina Aminonucleosídeo , Acetilcolina/farmacologia , Nefropatias/induzido quimicamenteRESUMO
OBJECTIVES: To compare, in vitro, resin cement excess removal techniques at the veneer-tooth interface. MATERIALS AND METHODS: Anterior human teeth were restored with ceramic veneers and randomly divided according to the following techniques (n = 10): removal of excess resin cement with brush and dental floss, followed by light-curing with Valo (Group 1) or Elipar (Group 2) for 1 min and 40 s; tack-curing with Valo (Group 3) or Elipar (Group 4) for 1 s; and tack-curing with Valo (Group 5) or Elipar (Group 6) for 5 s. The tack-curing was followed by removal of excess with probe and dental floss and light-curing for 1 min and 40 s. The area of excess resin cement (mm2) was measured in micro-CT images using AutoCAD program. The failures at the cervical margin in the X, Y, and Z axes (µm) of greater value were measured using the DataViewer program. The specimens were submitted to microleakage with 2% basic fuchsin. RESULTS: According to the Kruskal-Wallis and multiple comparison test, the highest area of excess resin cement was found in Group 1 (5.06 mm2), which did not differ statistically from Groups 2 (3.70 mm2) and 5 (2.19 mm2). Groups 2, 3 (1.73 mm2), 4 (1.14 mm2), and 5 (2.18 mm2) did not differ statistically. Group 6 (0.77 mm2) obtained the lowest value, which did not differ statistically from Groups 3 and 4. According to the Kruskal-Wallis and Dunn test, there was no significant difference in failures in X (p = 0.981), Y (p = 0.860), and Z (p = 0.638) axes and no significant difference in microleakage (p = 0.203) among the groups. CONCLUSIONS: Tack-curing for 1 s or 5 s, followed by removal of excess resin cement using a probe and a dental floss, tended to result in a lower amount of excess material around the margin. CLINICAL RELEVANCE: The technique used for resin cement excess removal influences the amount of excess leaved at the veneer-tooth interface. Tack-curing for 1 s or 5 s is recommended to mitigate the excess resin cement.
Assuntos
Cerâmica , Cimentos de Resina , Humanos , Pescoço , Puromicina , Microtomografia por Raio-XRESUMO
ABSTRACT: Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.
Assuntos
Agregação Plaquetária , Trombose , Camundongos , Humanos , Animais , Trombina/metabolismo , Tromboxanos , Puromicina/efeitos adversosRESUMO
Staphylococcus aureus is highly prevalent among patients with atopic dermatitis (AD), and this pathogen may trigger and aggravate AD lesions. The aim of this study was to determine the prevalence of S. aureus in the nares of pediatric subjects and verify the phenotypic and molecular characteristics of the isolates in pediatric patients with AD. Isolates were tested for antimicrobial susceptibility, SCCmec typing, and Panton-Valentine Leukocidin (PVL) genes. Lineages were determined by pulsed-field gel electrophoresis and multilocus sequence typing (MLST). AD severity was assessed with the Scoring Atopic Dermatitis (SCORAD) index. Among 106 patients, 90 (85%) presented S. aureus isolates in their nares, and 8 also presented the pathogen in their skin infections. Two patients had two positive lesions, making a total of 10 S. aureus isolates from skin infections. Methicillin-resistant S. aureus (MRSA) was detected in 24 (26.6%) patients, and PVL genes were identified in 21 (23.3%), including 6 (75%) of the 8 patients with skin lesions but mainly in patients with severe and moderate SCORAD values (P=0.0095). All 24 MRSA isolates were susceptible to trimethoprim/sulfamethoxazole, while 8 isolates had a minimum inhibitory concentration (MIC) to mupirocin >1024 μg/mL. High lineage diversity was found among the isolates including USA1100/ST30, USA400/ST1, USA800/ST5, ST83, ST188, ST718, ST1635, and ST2791. There was a high prevalence of MRSA and PVL genes among the isolates recovered in this study. PVL genes were found mostly among patients with severe and moderate SCORAD values. These findings can help clinicians improve the therapies and strategies for the management of pediatric patients with AD.
Assuntos
Animais , Masculino , Camundongos , Ratos , Nefropatias/metabolismo , Rim/metabolismo , Podócitos/metabolismo , Transdução de Sinais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica , Redes Reguladoras de Genes , Immunoblotting , Nefropatias/induzido quimicamente , Nefropatias/genética , Rim/patologia , Rim/fisiopatologia , Microscopia Eletrônica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Puromicina , Podócitos/patologia , Podócitos/ultraestrutura , Proteômica/métodos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Proteinuria is critical in the tubulointerstitial changes that ultimately lead to renal insufficiency. Increased protein filtration has direct toxic effects on tubular epithelial cells, leading to epithelial mesenchymal transition (EMT) to a myofibroblast phenotype. Angiotensin II and transforming growth factor (TGF)-ß1 are the main mediators of EMT. Calcitriol may exert a potential renoprotective effect by reducing the activity of the renin angiotensin system by suppressing renin gene expression and also by inhibiting the proinflammatory nuclear factor-κB pathway. The present study investigated the benefits of calcitriol treatment in a puromycin-induced proteinuric nephropathy model. Uninephrectomized adult male Wistar rats received intraperitoneal administration of a single dose of puromycin (100 mg/kg) or vehicle. After eight weeks, the animals were divided into two groups and received vehicle or calcitriol (0.5 µg/kg) for four weeks. The vehicle-treated, proteinuric rats developed progressive proteinuria and tubulointerstitial fibrosis after 12 weeks. Increased collagen deposition and fibrosis were significantly ameliorated by calcitriol treatment. Calcitriol was effective in preventing an increase in the EMT markers, α-smooth muscle actin and fibroblast-specific protein 1, reducing macrophage infiltration as evidenced by levels of ED-1. In addition, calcitriol increased the anti-inflammatory cytokine interleukin-10 and reduced the pro-oxidant p47 phox enzyme. These effects were paralleled by a reduction in TGF-ß/Smad3 expression. Calcitriol may have therapeutic potential in the proteinuric nephropathy model used in the present study by inhibiting the TGF-ß1 axis.
Assuntos
Calcitriol/farmacologia , Rim/efeitos dos fármacos , Nefrite Intersticial/tratamento farmacológico , Proteinúria/tratamento farmacológico , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Vitaminas/farmacologia , Actinas/genética , Actinas/metabolismo , Animais , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Ectodisplasinas/genética , Ectodisplasinas/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibrose , Expressão Gênica , Interleucina-10/agonistas , Interleucina-10/genética , Interleucina-10/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Nefrectomia , Nefrite Intersticial/induzido quimicamente , Nefrite Intersticial/metabolismo , Nefrite Intersticial/patologia , Proteinúria/induzido quimicamente , Proteinúria/metabolismo , Proteinúria/patologia , Puromicina , Ratos , Ratos Wistar , Sistema Renina-Angiotensina/efeitos dos fármacos , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Although homologs of the ATP-dependent Lon protease exist in all domains of life, the relevance of this protease in archaeal physiology remains a mystery. In this study, we have constructed and phenotypically characterized deletion and conditional lon mutants in the model haloarchaeon Haloferax volcanii to elucidate the role of the unusual membrane-bound LonB protease in archaea. Hvlon could be deleted from the chromosome only when a copy of the wild type gene was provided in trans suggesting that Lon is essential for survival in this archaeon. Successful complementation of the lethal phenotype of ΔHvlon was attained by expression of the heterologous protease gene Nmlon from the haloalkaliphilic archaeon Natrialba magadii, meaning that the biological function of Lon is conserved in these organisms. Suboptimal cellular levels of Lon protein affected growth rate, cell shape, cell pigmentation, lipid composition and sensitivity to various antibiotics. The contents of bacterioruberins and some polar lipids were increased in the lon mutants suggesting that Lon is linked to maintenance of membrane lipid balance which likely affects cell viability in this archaeon. The phenotypes associated to a membrane-bound LonB protease mutant were examined for the first time providing insight on the relevance of this protease in archaeal physiology.
Assuntos
Proteínas Arqueais/genética , Haloferax volcanii/enzimologia , Lipídeos de Membrana/metabolismo , Peptídeo Hidrolases/genética , Antibacterianos/farmacologia , Proteínas Arqueais/metabolismo , Bacitracina/farmacologia , Sequência de Bases , Expressão Gênica , Regulação da Expressão Gênica em Archaea , Haloferax volcanii/efeitos dos fármacos , Lovastatina/farmacologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Novobiocina/farmacologia , Peptídeo Hidrolases/metabolismo , Pigmentação , Ligação Proteica , Puromicina/farmacologiaRESUMO
Biometric parameters, glycemia and activity levels of plasma neutral aminopeptidase (APN) and dipeptidyl peptidase IV (DPPIV) were measured in monosodium glutamate obese and food-deprived rats (MSG-FD), to analyze the involvement of these enzymes in such situations. Plasma APN was distinguished as sensitive (PSA) (K(m) = 7.8 x 10(-5) mol/l) and predominantly insensitive (APM) (K(m) = 21.6 x 10(-5) mol/l) to puromycin, whereas DPPIV was sensitive (DPPIV-DS) (K(m) = 0.24 x 10(-5) mol/l) and predominantly insensitive (DPPIV-DI) (K(m) = 7.04 x 10(-5) mol/l) to diprotin A. Although unchanged in the MSG and food-deprived animals, APM activity levels were closely correlated with body mass, Lee index, and mass of retroperitoneal fat pad in the food deprived, but not in the MSG animals. DPPIV-DI activity levels decreased by 33% and were correlated with body mass, Lee index, and mass of periepididymal fat pad in the food-deprived MSG rats. These data suggest that APM and DPPIV-DI are respectively related to the downregulation of somatostatin in food-deprived rats, and to the recovery of energy balance in MSG obese rats during food deprivation.
Assuntos
Tecido Adiposo/metabolismo , Antígenos CD13/sangue , Dipeptidil Peptidase 4/sangue , Privação de Alimentos/fisiologia , Obesidade/metabolismo , Glutamato de Sódio/farmacologia , 2-Naftilamina/análogos & derivados , 2-Naftilamina/metabolismo , 2-Naftilamina/farmacologia , Animais , Glicemia/metabolismo , Peso Corporal/fisiologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Feminino , Hiperglicemia/metabolismo , Masculino , Oligopeptídeos/farmacologia , Puromicina/farmacologia , Ratos , Ratos WistarRESUMO
Septic shock was formerly recognized as a consequence of Gram-negative bacteraemia, but at present the incidence of Gram-positive sepsis seems to be more relevant, contributing for more than 50% of cases. Staphylococcal aureus can induce toxic shock in humans through the production of potent toxins termed Staphylococcal enterotoxins, from which Staphylococcal enterotoxin type B (SEB) is one of most studied. Platelets are reported to participate in pathogenesis of severe sepsis, but the exact role of platelets in this event is poorly investigated, particularly that caused by Gram-positive bacteria. Therefore, we have used the model of platelet adhesion to fibrinogen-coated plates to investigate the actions of SEB on human platelets. Ninety-six-well microtiter plates were coated with human fibrinogen (50 microg/mL), and human washed platelet suspension (6 x 10(6) platelets) was added to each well. Adherent platelets were quantified through measurement of acid phosphatase activity. Staphylococcal enterotoxin B (0.0001-30 microg/mL, incubated for 5 to 60 min) time- and dose-dependently inhibited platelet adhesion. This response was modified neither by the protein synthesis inhibitor puromycin (0.01 and 0.1 mM) nor by the superoxide scavengers superoxide dismutase (SOD, 100 units/mL) and polyethylene glycol-SOD (30 U/mL). The peroxide hydrogen (H(2)O(2)) scavenger catalase polyethylene glycol (1000 U/mL) significantly attenuated the platelet adhesion inhibition by SEB. The cAMP and cGMP levels were not changed by SEB (0.0001-30 microg/mL, 60 min). Our findings suggest that H(2)O(2) at least partly contributes to the inhibitory responses of human platelet adhesion by SEB.
Assuntos
Plaquetas/efeitos dos fármacos , Enterotoxinas/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Animais , Plaquetas/citologia , Plaquetas/enzimologia , Catalase/farmacologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Enterotoxinas/sangue , Humanos , Nucleotídeos Cíclicos/sangue , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Polietilenoglicóis/farmacologia , Puromicina/farmacologia , Superóxido Dismutase/farmacologiaRESUMO
Acid (aspartyl), basic (arginyl) and neutral (alanyl) aminopeptidases degrade angiotensins, vasopressin, oxytocin, bradykinin and enkephalins. These peptides regulate memory, energy homeostasis, water-salt balance and blood pressure, functions that are mainly exerted in the hippocampus and hypothalamus, and that can be affected by diabetes mellitus. To evaluate the relationship between the diabetes mellitus and processing and inactivation roles of these representative aminopeptidases, we measured their activities in both brain structures of control and streptozotocin-diabetic rats. Hypothalamic soluble aspartyl and arginyl aminopeptidases presented significant decreased activity levels in diabetic rats, which were mitigated by insulin therapy. In addition to membrane-bound puromycin sensitive and insensitive alanyl aminopeptidases, its soluble puromycin sensitive form did not differ between diabetic and control rats in both brain structures. Glucose and/or insulin did not seem to alter in vitro the hypothalamic activities of soluble aspartyl and arginyl aminopeptidases. The implied hypothalamic control of regulatory peptide activity by aspartyl and arginyl aminopeptidases supports the hypothesis that the hydrolytic ability of these enzyme types could be a common link for the disruptions of water-salt balance, blood pressure and energy homeostasis in diabetes mellitus.
Assuntos
Aminopeptidases/metabolismo , Encefalopatias Metabólicas/enzimologia , Encefalopatias Metabólicas/etiologia , Diabetes Mellitus Experimental/complicações , Hipocampo/enzimologia , Hipotálamo/enzimologia , Aminopeptidases/análise , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Encefalopatias Metabólicas/fisiopatologia , Antígenos CD13/análise , Antígenos CD13/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Doenças do Sistema Endócrino/enzimologia , Doenças do Sistema Endócrino/etiologia , Doenças do Sistema Endócrino/fisiopatologia , Glutamil Aminopeptidase/análise , Glutamil Aminopeptidase/metabolismo , Hipocampo/fisiopatologia , Homeostase/fisiologia , Hipotálamo/fisiopatologia , Insulina/metabolismo , Insulina/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neuropeptídeos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Ratos , Ratos Wistar , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
Trypanosomes are outstanding examples of the importance of mRNA metabolism in the regulation of gene expression, as these unicellular eukaryotes mostly control protein synthesis by post-transcriptional mechanisms. Here, we show that mRNA metabolism in these organisms involves recruitment of mRNAs and proteins to microscopically visible ribonucleoprotein granules in the cytoplasm. These structures engage transcripts that are being translated and protect mRNAs from degradation. Analysis of the protein composition of trypanosomal mRNA granules indicated that they contain orthologous proteins to those present in P bodies and stress granules from metazoan organisms. Formation of mRNA granules was observed after carbon-source deprivation of parasites in axenic culture. More important, mRNA granules are formed naturally in trypanosomes present in the intestinal tract of the insect vector. We suggest that trypanosomes make use of mRNA granules for transient transcript protection as a strategy to cope with periods of starvation that they have to face during their complex life cycles.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Transporte de RNA , Ribonucleoproteínas/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/metabolismo , Motivos de Aminoácidos , Animais , Carbono/farmacologia , Cicloeximida/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Privação de Alimentos , Trato Gastrointestinal/efeitos dos fármacos , Insetos Vetores/efeitos dos fármacos , Insetos Vetores/parasitologia , Modelos Biológicos , Parasitos/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Puromicina/farmacologia , Transporte de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/efeitos dos fármacos , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/citologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.
Assuntos
Cinamatos , Vetores Genéticos/genética , Higromicina B/análogos & derivados , Puromicina , Transfecção/métodos , Trypanosoma cruzi/genética , Animais , Dados de Sequência MolecularRESUMO
This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.
Assuntos
Animais , Vetores Genéticos , Transfecção , Trypanosoma cruzi , Dados de Sequência Molecular , PuromicinaRESUMO
The main objective of this study was to determine if the components of the kallikrein-kinin system are released into the venous effluent from isolated perfused rat hearts. To assess the contribution of kinins and the vascular and cardioprotective effects of the ACE inhibitor ramipril, we determined the status of cardiac kallikrein (CKK), potent kinin-generating enzyme, in rats with right ventricular hypertrophy induced by chronic volume overload and left ventricular hypertrophy by aortic banding. CKK was measured as previously described (Nolly, H.L., Carbini, L., Carretero, O.A., Scicli, A.G., 1994). Kininogen by a modification of the technique of Dinitz and Carvalho (1963) and kinins were extracted with a Sep-Pak C18 cartridge and measured by RIA. CKK (169 +/- 9 pg Bk/30 min), kininogen (670 +/- 45 pg Bk/30 min) and immunoreactive kinins (62 +/- 10 pg Bk/30 min) were released into the perfusate. The release was almost constant over a 120 min period. Pretreatment with the protein synthesis inhibitor puromycin (10 mg i.p.) lowered the release of kallikrein (42 +/- 12 pg Bk/30 min, p < 0.001) and kininogen (128 +/- 56 pg Bk/30 min, p < 0.001). Addition of ramiprilat (10 micrograms/ml) increased kinin release from 54 +/- 18 to 204 +/- 76 pg Bk/30 min (p < 0.001). Aortic banding of rats increased their blood pressure (BP) (p < 0.001), relative heart weight (RHW) (p < 0.001) and CKK (p < 0.001). Ramipril treatment induced a reduction in BP (p < 0.05) and RHW (p < 0.005) while CKK remained elevated. Aortocaval shunts increased their ANF plasma levels (p < 0.05), RHW (p < 0.001) and CKK (p < 0.01). Ramipril treatment induced a reduction in RHW (p < 0.05), while CKK and ANF increased significantly (p < 0.05). The present data show that the components of the kallikrein-kinin system are continuously formed in the isolated rat heart and that ramipril reduces bradykinin breakdown with subsequent increase in bradykinin outflow. The experiments with aorta caval shunt and aortic banding show that cardiac tissues increase their kinin-generating activity and this was even higher in ramipril-treated animals. This may suggest that the actual level of kinins is finely tuned to the local metabolic demands. In this experimental model of cardiac hypertrophy. ACE inhibitors potentiate the actions of kinins and probably try to normalise endothelial cell function.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Bradicinina/metabolismo , Coração/efeitos dos fármacos , Sistema Calicreína-Cinina/efeitos dos fármacos , Ramipril/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Derivação Arteriovenosa Cirúrgica , Fator Natriurético Atrial/sangue , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Direita/tratamento farmacológico , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Sistema Calicreína-Cinina/fisiologia , Calicreínas/metabolismo , Cininogênios/isolamento & purificação , Cininogênios/metabolismo , Masculino , Miocárdio/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Radioimunoensaio , Ramipril/uso terapêutico , Ratos , Ratos WistarRESUMO
Tumor necrosis factor alpha mediated cell death in L929 cells correlates with a late increase in reduction of the superoxide scavenger 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), suggesting an increase in MTT reduction per viable cell. This effect was studied in two TNF-sensitive and in five different TNF-resistant clones. Within 36 hrs TNF promoted a 7-fold increase in the reduction of MTT in TNF-sensitive cells. Exogenous ceramide induced a similar effect prior to cell death. Four of the five TNF-resistant clones were also resistant to ceramide and displayed no increase in MTT reduction with either TNF or ceramide. The remaining TNF-resistant clone was sensitive to ceramide, displaying an increase in MTT reduction. Our results suggest a late increase in superoxide production prior to cellular destruction during TNF and ceramide mediated cell death and support the notion that ceramide can serve as a second messenger for TNF in cell death.
Assuntos
Ceramidas/farmacologia , Estresse Oxidativo , Fator de Necrose Tumoral alfa/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Corantes , Resistência a Medicamentos , Sequestradores de Radicais Livres , Cinética , Células L , Camundongos , Mutagênese , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Puromicina/farmacologia , Proteínas Recombinantes/biossíntese , Sais de Tetrazólio , Tiazóis , TransfecçãoRESUMO
A glucantime sensitive Leishmania (V.) guyanensis strain was used to obtain in vitro resistant cell lines, by increments in glucantime concentrations employing both one step and stepwise protocols. Whereas the effective concentration of drug that inhibited the growth of wild type cells by 50% (EC50 value) was 0.20 mg Sb(v)/mL, the resistant cells were able to grow in glucantime concentrations greater than 8.0 mg/mL. The resistant cell lines were partially characterized by their in vitro response to glucantime, the stability of resistance phenotype, cross resistance to a range of drugs, and also by the analysis of total DNA fragments generated by restriction endonucleases and blot hybridization. Amplified DNA sequence similar to a P-glycoprotein analog from Leishmania tarentolae (ltpgpA gene) was observed in all the resistant cell lines obtained through the one-step protocol. These cell lines showed cross resistance to heavy metals but were sensitive to puromycin, vinblastine, and pentostam.
Assuntos
Antiprotozoários/farmacologia , Leishmania guyanensis/efeitos dos fármacos , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Antimônio/farmacologia , Tartarato de Antimônio e Potássio/farmacologia , Gluconato de Antimônio e Sódio/farmacologia , Arseniatos/farmacologia , DNA de Protozoário/análise , DNA de Protozoário/genética , Resistência a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Dosagem de Genes , Genes de Protozoários , Leishmania/genética , Leishmania guyanensis/genética , Antimoniato de Meglumina , Óxidos/farmacologia , Puromicina/farmacologia , Homologia de Sequência do Ácido Nucleico , Vimblastina/farmacologiaRESUMO
We have studied the in vitro formation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) using a partially purified ppGpp synthetase I (PSI) from Escherichia coli BGA8, a polyamine auxotrophic strain. A comparison of the enzyme obtained from polyamine-supplemented or deprived bacteria showed similar requirements for the reaction, Mg+2 optimum levels and sparing effect of spermidine. No differences in the inhibitory effects of tetracycline, puromycin and fusidic acid were detected either. However, a modified subcellular distribution, as well as a larger specific activity and a larger stimulation by streptomycin was observed when PSI was prepared from polyamine-depleted bacteria. The role of ribosome assembly and subunit distribution on the altered properties of the enzyme are discussed.
Assuntos
Escherichia coli/metabolismo , Ligases/metabolismo , Poliaminas/farmacologia , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Ácido Fusídico/farmacologia , Guanosina Tetrafosfato/biossíntese , Ligases/antagonistas & inibidores , Ligases/isolamento & purificação , Magnésio/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Ribossomos/metabolismo , Espermidina/farmacologia , Estreptomicina/farmacologia , Frações Subcelulares/enzimologia , Tetraciclina/farmacologiaRESUMO
Proteolysis of endogenous proteins may play a key role in the adaptation of T. cruzi to the different host environments to which it is exposed during its complex life cycle. For this reason, we have attempted to study the intracellular pathways of protein degradation in the non infective epimastigotes form (EP strain) of T. cruzi. Following intracellular proteolysis by pulse chase experiments with 35 S methionine, we observed a significant inhibition (50%) of the degradation of endogenous proteins in log phase parasites in the presence of inhibitors of lysosomal functions, such as chloroquine and E 64. A significant increase in proteolysis was observed in stationary phase parasites which was reverted to log phase values by supplementing the chase medium with 0.5% glucose or 10% serum, or in the presence of chloroquine. Under this condition of nutritional stress, we could observe an increase in the activity of acid proteases. A significant increase in the degradation rates was observed when abnormal proteins were induced in the parasite by amino acid analogs and puromycin. This increase was not affected by E 64, suggesting the participation of non lysosomal mechanisms in the degradation of rapidly degradable abnormal proteins. Under these conditions, we could observe an increase in high molecular weight conjugates of ubiquitin with respect to endogenous proteins. These results suggest the importance of lysosomal mechanisms in the degradation of cellular proteins in nutritional optimal conditions and during nutritional deprivation, and the possible involvement of the ubiquitin system in the degradation of high turnover proteins.
Assuntos
Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Cloroquina/farmacologia , Cisteína Endopeptidases/metabolismo , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Peso Molecular , Inibidores de Proteases/farmacologia , Puromicina/farmacologia , Fatores de Tempo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Ubiquitinas/metabolismoRESUMO
Proteolysis of endogenous proteins may play a key role in the adaptation of T. cruzi to the different host environments to which it is exposed during its complex life cycle. For this reason, we have attempted to study the intracellular pathways of protein degradation in the non infective epimastigotes form (EP strain) of T. cruzi. Following intracellular proteolysis by pulse chase experiments with 35 S methionine, we observed a significant inhibition (50 percent) of the degradation of endogenous proteins in log phase parasites in the presence of inhibitors of lysosomal functions, such as chloroquine and E 64. A significant increase in proteolysis was observed in stationary phase parasites which was reverted to log phase values by supplementing the chase medium with 0.5 percent glucose or 10 percent serum, or in the presence of chloroquine. Under this condition of nutritional stress, we could observe an increase in the activity of acid proteases. A significant increase in the degradation rates was observed when abnormal proteins were induced in the parasite by amino acid analogs and puromycin. This increase was not affected by E 64, suggesting the participation of non lysosomal mechanisms in the degradation of rapidly degradable abnormal proteins. Under these conditions, we could observe an increase in high molecular weight conjugates of ubiquitin with respect to endogenous proteins. These results suggest the importance of lysosomal mechanisms in the degradation of cellular proteins in nutritional optimal conditions and during nutritional deprivation, and the possible involvement of the ubiquitin system in the degradation of high turnover proteins
Assuntos
Animais , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Cloroquina/farmacologia , Cisteína Proteases/metabolismo , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Peso Molecular , Inibidores de Proteases/farmacologia , Puromicina/farmacologia , Fatores de Tempo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Ubiquitina/metabolismoRESUMO
The presence in myoblasts of an intracellular receptor specific for 1,25-dihydroxyvitamin D-3 [1,25(OH)2D3) and 1,25(OH)2D3-dependent changes in myoblast Ca2+ transport and phospholipid metabolism which are suppressed by RNA and protein synthesis inhibitors have been shown. In agreement with these observations, incubation of chick embryo myoblasts, precultured for 24 h in a medium containing low levels of vitamin D-3 metabolites, with 1,25(OH)2D3 at conditions which induce maximum cell responses (10(-10) M, 24 h) markedly stimulated the incorporation of [3H]leucine into total cell proteins and this effect was abolished when sterol treatment was performed in the presence of cycloheximide or puromycin. To investigate whether 1,25(OH)2D3 selectively stimulates the de novo synthesis of muscle cell proteins, mixtures of myoblast proteins from control and sterol-treated cultures labelled with [14C]leucine and [3H]leucine, respectively, were separated by SDS-polyacrylamide gel electrophoresis and isoelectric focussing. Examination of 3H/14C ratios in gel fractions revealed that 1,25-(OH)2D3 stimulates the production of proteins of molecular masses (isoelectric points) of 9 kDa (4.1 and 8.5), 17 kDa (7.5), 30 kDa (7.2), 40 kDa (5.5), 55 kDa (4.5) and 100 kDa (8.6). Cell fractionation studies showed the following subcellular distribution: 9 kDa (85% cytosol, 15% microsomes); 17 and 100 kDa (100%, 1200 X g pellet); 30 kDa (65% cytosol, 35% mitochondria); 40 kDa (100% microsomes); 55 kDa (65% microsomes, 35% mitochondria). Marker enzyme data indicated that this distribution is not due to cross-contamination between fractions. Affinity chromatography of double-labelled myoblast proteins on an immobilized lectin showed that the 55 kDa protein contains carbohydrate. Labelling of myoblast proteins with 45CaCl2 after their separation on SDS-polyacrylamide gels showed in addition that the 1,25(OH)2D3-dependent proteins of 9, 17, 40 and 100 kDa are major Ca2+-binding components of the cells. Synthesis of these proteins may mediate the effects of the sterol on myoblast calcium metabolism.
Assuntos
Calcitriol/farmacologia , Proteínas Musculares/biossíntese , Músculos/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Embrião de Galinha , Cicloeximida/farmacologia , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/biossíntese , Ponto Isoelétrico , Microssomos/metabolismo , Mitocôndrias/metabolismo , Peso Molecular , Músculos/efeitos dos fármacos , Músculos/embriologia , Fosfatidilcolinas/biossíntese , Puromicina/farmacologiaRESUMO
Intraperitoneal inoculation of BIO.A mice with P. brasiliensis induces an acute inflammatory infiltrate in which 40-50% of the cells are PMN leucocytes. Previous depletion of serotonin, prostaglandin, histamine and complement does not alter the course of inflammation. Complement-derived factors appear to have no active participation in the process since C5-deficient mice depleted or not by Cobra venom factor (CoF) show the same kind of cellular influx. On the other hand, peritoneal cells incubated (6 h) with the fungus release a soluble factor that induces in vivo an active chemotaxis of PMN cells when inoculated i.p. The factor has the following characteristics: a) it is produced by adherent cells; b) it is protein in nature; c) its production is inhibited by incubation of peritoneal cells with 10 micrograms/ml puromycin and d) it has a molecular weight less than 15 000 daltons, as determined by gel filtration through a Sephadex G-75 column.