RESUMO
The aim of this study was to conduct a POP biomonitoring programme for children in high-risk areas. We evaluated 247 serum samples from children between the ages of 6 and 12years old from two zones in Mexico: (1) indigenous zones, which included Cuatlamayan (CUA), Tocoy (TOC), and Santa Maria Picula (SAM); and (2) industrial zones, which included Tercera Chica (TC), Industrial San Luis (IND) and Rincon de San Jose (SJR); Mundo Nuevo (MN); and Alpuyeca (ALP). Our results showed that α-endosulfan was similar to CUA, TOC, SAM, TC and MN (178.6-306.9ng/g lipid). ß-Endosulfan levels were higher in ALP (901.5ng/g lipid), followed by CUA (139.9ng/g lipid) and TOC, SAM, TC and MN, which had similar levels (55.4-64.5ng/g lipid). For endosulfan sulfate, the ALP community had the highest concentration levels (1096.4ng/g lipid), whereas CUA and TOC (212.3 and 289ng/g lipid, respectively) had concentrations similar to those found in SAM and TC (99.5 and 119.1ng/g lipid, respectively). DDE levels were found in malaria-endemic areas of SAM, CUA and TOC (1782.2, 1358.3 and 57.0ng/g lipid), followed by MN (35.1ng/g lipid). HCB concentration levels were found to be higher in MN and SJR (691.8 and 575.4ng/g lipid, respectively), followed by CUA and TC (363.9 and 269.1ng/g lipid, respectively), with levels similar to those found in TOC and SAM (191.8 and 181.9ng/g lipid, respectively). Finally, PCB 101 concentration levels were found to be the highest in ALP (1032.7ng/g lipid), followed by similar levels of SJR and IND (567.5 and 327.3ng/g lipid, respectively) and TC and MN, with 109.1 and 144.5ng/g lipid, respectively. The evidence provided by this exploratory study indicates that the evaluation of the health risks posed to children living in contaminated areas is a high priority health issue.
Assuntos
Exposição Ambiental/estatística & dados numéricos , Poluentes Ambientais/metabolismo , Compostos Orgânicos/metabolismo , Criança , Pré-Escolar , Diclorodifenil Dicloroetileno/metabolismo , Endossulfano/metabolismo , Exposição Ambiental/análise , Monitoramento Ambiental , Feminino , Locais de Resíduos Perigosos , Humanos , Masculino , México , Bifenilos Policlorados/metabolismoRESUMO
Acute poisoning by organophosphate or carbamate is important to recognize since it can cause severe complications such as cardiorespiratory failure, coma, and even death in the absence of early management. Pharmacologically, the mode of action of these substances is based on an inhibition of cholinesterases; the clinical presentation therefore consists of a cholinergic syndrome. The typical clinical picture can be confirmed by the dosage of plasma cholinesterases. On a therapeutic viewpoint, atropine remains the antidote of choice. In high doses, it is the only molecule with a demonstrated effect for the specific treatment of such poisonings. Pralidoxime is clearly recommended in case of poisoning by an organophosphate, but is more discussed when carbamates are involved. The observation of a case of voluntary poisoning by a powerful carbamate, aldicarbe, offers s the opportunity to review the key elements of this type of poisoning. The rather loose inaugural presentation and the initial absence of diagnosis, underline the value of an adequate premature symptomatic care.
Assuntos
Aldicarb/intoxicação , Carbamatos/intoxicação , Inseticidas/intoxicação , Comportamento Autodestrutivo , Humanos , Masculino , Pessoa de Meia-Idade , Comportamento Autodestrutivo/diagnósticoAssuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/sangue , Biomarcadores/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Apolipoproteínas E/genética , Diagnóstico Diferencial , HumanosRESUMO
Pseudomonas aeruginosa binds to human respiratory mucins by mechanisms involving flagellar component-receptor interactions. The adhesion of P. aeruginosa strain PAK is mediated by the flagellar cap protein, FliD, without the involvement of flagellin. Two distinct types of FliD proteins have been identified in P. aeruginosa: A type, found in strain PAK, and B type, found in strain PAO1. In the present work, studies performed with the P. aeruginosa B-type strain PAO1 indicate that both the FliD protein and the flagellin of this strain are involved in the binding to respiratory mucins. Using polyacrylamide-based fluorescent glycoconjugates in a flow cytometry assay, it was previously demonstrated that P. aeruginosa recognizes Le(x) (or Lewis x) derivatives found at the periphery of human respiratory mucins. The aim of the present work was therefore to determine whether these carbohydrate epitopes (or glycotopes) are receptors for FliD proteins and flagellin. The results obtained by both flow cytometry and a microplate adhesion assay indicate that the FliD protein of strain PAO1 is involved in the binding of glycoconjugates bearing Le(x) or sialyl-Le(x) determinants, while the binding of flagellin is restricted to the glycoconjugate bearing Le(x) glycotope. In contrast, the type A cap protein of P. aeruginosa strain PAK is not involved in the binding to glycoconjugates bearing Le(x), sialyl-Le(x), or sulfosialyl-Le(x) glycotopes. This study demonstrates a clear association between a specific Pseudomonas adhesin and a specific mucin glycotope and demonstrates that fine specificities exist in mucin recognition by P. aeruginosa.
Assuntos
Proteínas de Bactérias/metabolismo , Glicoconjugados/metabolismo , Antígenos CD15/metabolismo , Mucinas/química , Mucinas/metabolismo , Pseudomonas aeruginosa/fisiologia , Aderência Bacteriana , Proteínas de Bactérias/genética , Flagelina/genética , Flagelina/metabolismo , Citometria de Fluxo , Glicoconjugados/química , Humanos , Antígenos CD15/química , Mutação , Oligossacarídeos/síntese química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Mucosa Respiratória/metabolismo , Antígeno Sialil Lewis XRESUMO
In response to stimuli, endothelial cells release arachidonic acid, a lipid precursor of various vasoactive substances. We have investigated the relationships between cytosolic Ca2+ movements and arachidonic acid release in human umbilical vein endothelial cells. Histamine, a receptor-dependent agonist, and thapsigargin, a specific inhibitor of sarco-/endoplasmic Ca2+ pumps, time- and dose-dependently increased the release of [1-14C]-arachidonic acid. This release was inhibited by AACOCF3, a selective inhibitor of cytosolic phospholipase A2 (PLA2). In the absence of Ca2+ influx, arachidonic acid release was suppressed in both histamine- and thapsigargin-stimulated cells, despite marked elevations of cytosolic Ca2+ concentration ([Ca2+]i). In the presence of Ca2+ influx, arachidonic acid release was reduced in cells treated with BAPTA, an intracellular Ca2+ buffer, or with SK&F 96365, a receptor-operated Ca2+ channel blocker. Arachidonic acid release was analyzed as a function of the two successive phases of Ca2+ response to stimulation: Ca2+ peak and plateau phase, reflecting Ca2+ mobilization from internal stores and Ca2+ influx, respectively. The amount of arachidonic acid released was directly related to [Ca2+]i values measured at the influx phase with a 80 nM [Ca2+]i threshold, similar to that reported for PLA2 translocation. This suggests that Ca2+ entry from the extracellular space is essential for activating cytosolic PLA2 in human endothelial cells.
Assuntos
Ácido Araquidônico/metabolismo , Cálcio/fisiologia , Endotélio Vascular/metabolismo , Ácidos Araquidônicos/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Citosol/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Histamina/farmacologia , Humanos , Imidazóis/farmacologia , Metabolismo dos Lipídeos , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Tapsigargina/farmacologiaRESUMO
Recent studies have demonstrated that, unlike cholesterol, cholesterol oxidized at position 7 can reduce the maximal endothelium-dependent relaxation of isolated rabbit aortas (Circulation. 1997;95:723-731). The aim of the current study was to determine whether cholesterol oxides reduce the release of nitric oxide (NO) from human umbilical vein endothelial cells (HUVECs). The amount of NO released by histamine-stimulated HUVECs was determined by differential pulse amperometry using a nickel porphyrin- and Nafion-coated carbon microfiber electrode. The effects of cholesterol (preserved from oxidation by butylated hydroxytoluene), 7-ketocholesterol, 7beta-hydroxycholesterol, 5alpha,6alpha-epoxycholesterol, 19-hydroxycholesterol (60 microg/mL), and alpha-lysophosphatidylcholine (10 microg/mL) were compared. Pretreatment of HUVECs with cholesterol, 5alpha,6alpha-epoxycholesterol, or 19-hydroxycholesterol did not alter histamine-activated NO production. In contrast, pretreatment with 7-ketocholesterol or 7beta-hydroxycholesterol significantly decreased NO release. The inhibitory effect of 7-ketocholesterol was time and dose dependent and was maintained in the presence of L-arginine. In the absence of serum, lysophosphatidylcholine also reduced NO production. In ionomycin-stimulated cells, pretreatment with 7-ketocholesterol did not inhibit NO release. These results demonstrate that cholesterol derivatives oxidized at the 7 position, the main products of low density lipoprotein oxidation, reduce histamine-activated NO release in HUVECs. Such an inhibitory effect of cholesterol oxides may account, at least in part, for the ability of oxidized low density lipoprotein to reduce the endothelium-dependent relaxation of arteries.
Assuntos
Colesterol/farmacologia , Endotélio Vascular/metabolismo , Óxido Nítrico/metabolismo , Arginina/farmacologia , Células Cultivadas , Colesterol/análogos & derivados , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Histamina/farmacologia , Humanos , Hidroxicolesteróis/farmacologia , Ionomicina/farmacologia , Cetocolesteróis/farmacologia , Lisofosfatidilcolinas/farmacologia , Veias UmbilicaisRESUMO
The causal relationships between cytosolic free-Ca2+ concentration ([Ca2+]i) increases and production of nitric oxide (NO) have been investigated mostly with indirect methods and remain unclear. Here we demonstrate, by direct real-time measurements of [NO] with a porphyrinic microsensor, that Ca2+ entry, but not an increase in [Ca2+]i, is required for triggering of NO production in human endothelial cells. Histamine, ranging from 0.1 to 100 microM, increased both NO production and [Ca2+]i when given in a single dose. However, histamine caused increased NO release but induced progressively smaller [Ca2+]i changes when cumulatively added. In the absence of a transmembrane Ca2+ gradient, no significant NO release was detectable, despite the marked Ca2+ peak induced by histamine. Inhibition of Ca2+ entry by SK&F 96365 abolished histamine-elicited NO production but only reduced the transient [Ca2+]i rise. The suppression of the sustained [Ca2+]i response under these two conditions suggests that NO release was closely associated with Ca2+ entry from the extracellular space. In addition, membrane depolarization, achieved by increasing the extracellular K+ concentration from 5 to 130 mM, reduced both the amplitude of histamine-induced sustained [Ca2+]i elevation and NO production. These results lead us to propose that the availability of numerous Ca2+ ions around the internal side of the plasma membrane would promote the association between nitric oxide synthase and calmodulin, thereby activating the enzyme.
Assuntos
Cálcio/metabolismo , Endotélio Vascular/metabolismo , Histamina/farmacologia , Óxido Nítrico/biossíntese , Técnicas Biossensoriais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Citosol/metabolismo , Endotélio Vascular/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Potássio/metabolismoRESUMO
Vascular endothelial cells have been found to produce a relaxant mediator, identified as nitric oxide (NO) and implicated in numerous physiological functions. Subsequently, there has been an intensive search for accurate and specific detection methods to measure biological NO production. In the present study, we compared three approaches to evaluate NO production, based respectively on the Griess reaction (that quantifies nitrites and nitrates after their reduction), on the hemoglobin reaction (that quantifies oxyhemoglobin to methemoglobin transformation by NO), and on the electrochemical NO detection with a porphyrinic micro-probe. Comparison was made both under standard conditions and biological conditions, through calibration curves and measurements of histamine-induced NO production by cultured human endothelial cells and its modulation by L-arginine and N(omega)-monomethyl-L-arginine. We demonstrated that these three methods differ in terms of sensitivity and selectivity. The hemoglobin reaction and nitrate measurements suffer from a lack of specificity. Nitrite determination by the Griess reaction was hardly suitable for kinetic studies but it remains useful for the evaluation of basal NO production. The electrochemical technique, although it does not allow measurement of basal NO production, is the only one to exhibit great sensitivity and specificity and to allow instantaneous and non destructive measurements. This study brings up the potential hazards and pitfalls that may be associated with the various methods.
Assuntos
Endotélio Vascular/metabolismo , Óxido Nítrico/biossíntese , Células Cultivadas , Colorimetria/métodos , Eletroquímica/métodos , Hemoglobinas , Humanos , Nitratos/análise , Óxido Nítrico/análise , Nitritos/análise , Sensibilidade e Especificidade , Veias UmbilicaisRESUMO
The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cystic fibrosis. Local factors in the respiratory tract, such as osmolarity or iron concentration, might influence this colonization. In the present work, we have observed that overall levels of adhesion of two nonmucoid, nonpiliated strains of P. aeruginosa, 1244-NP and PAK-NP, to human airway mucins were higher when these strains were grown in a minimal medium of low osmolarity (M9) than when they were grown in a rich medium of higher osmolarity (tryptic soy broth [TSB]). However, increasing the NaCl concentration of M9 to increase the osmolarity did not modify the level of binding. In order to find out whether these differences were due to variations in nutrients, the influence of iron concentration was investigated: the levels of binding of the two strains increased after TSB was depleted of iron and decreased after iron was added to M9. Since the outer membranes from the two strains have been shown to contain proteins reacting with human bronchial mucins, we compared the mucin-binding proteins expressed by the two strains grown in different media. When the nonpiliated strains 1244-NP and PAK-NP were grown in the different media, previously observed mucin-binding bands were detected in the 42- to 48-kDa range but additional mucin-binding bands in the 77- to 85-kDa range were detected when these strains were grown in M9 or iron-deprived TSB. These results demonstrate that the adhesion of P. aeruginosa and the expression of mucin-binding proteins in the outer membranes of nonpiliated P. aeruginosa are affected by the iron content of the medium in which the bacteria are grown and not by the osmolarity.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Mucinas/metabolismo , Pseudomonas aeruginosa/citologia , Sistema Respiratório/microbiologia , Humanos , Pseudomonas aeruginosa/metabolismo , Sistema Respiratório/metabolismoRESUMO
The effects of the imidazole compound SK&F 96365 on Ca2+ movements and production of nitric oxide (NO) and von Willebrand factor (vWF) have been investigated in human endothelial cells. Changes in cytosolic Ca2+ concentration ([Ca2+]i) were measured with Fura-2. Real-time production of NO was monitored with a porphyrinic microsensor and the release of vWF with an enzyme-linked immunosorbent assay. Irrespective of the transmembrane Ca2+ gradient, 30 microM SK&F 96365 doubled [Ca2+]i suggesting a Ca2+ release from intracellular stores. The SK&F 96365-induced [Ca2+]i rise was not accompanied by detectable NO and vWF production, while 1 microM thapsigargin enhanced [Ca2+]i 2.5 times, doubled the secretion of vWF and increased the NO production to 10 +/- 4 nM (n = 5). Pretreatment with SK&F 96365 prevented thapsigargin from increasing [Ca2+]i, NO production and vWF secretion. To investigate the mechanism by which SK&F 96365 released Ca2+ from internal pools, its effect and that of thapsigargin on the ATP-dependent 45Ca2+ uptake into platelet membrane vesicles were compared. SK&F 96365 as thapsigargin, dose-dependently reduced the initial rate of 45Ca2+ uptake. In conclusion, we demonstrate that, in the absence of Ca2+ entry from the extracellular space, the [Ca2+]i increase elicited by SK&F 96365 or thapsigargin is not sufficient to initiate NO synthesis and vWF secretion. This confirms the important role of Ca2+ influx in endothelial secretion processes.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Imidazóis/farmacologia , Óxido Nítrico/biossíntese , Fator de von Willebrand/biossíntese , Trifosfato de Adenosina/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Plaquetas/enzimologia , Plaquetas/ultraestrutura , Cálcio/farmacocinética , Radioisótopos de Cálcio/farmacocinética , ATPases Transportadoras de Cálcio/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Células Cultivadas/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/enzimologia , Humanos , Membranas Intracelulares/metabolismo , Tapsigargina/farmacologia , Veias Umbilicais/citologia , Fator de von Willebrand/metabolismoRESUMO
Pseudomonas aeruginosa binds to different glycoconjugates in vitro. As six other bacteria, it binds to several glycolipids, mainly asialo GM1 and asialo GM2. Asialo GM1 has been reported to exist at the surface of cystic fibrosis cells. The binding of P. aeruginosa to asialo GM1 involves the pili, especially the C-terminal part of pilin that recognizes the GaINAc(beta 1,4) Gal sequence of asialo GM1.P. aeruginosa may also bind to sialylated membrane-bound glycoproteins. Human salivary and respiratory mucins are also recognized by P. aeruginosa. Mucins represent the main components of mucus. The peptide part (apomucin) of this broad family of secreted glycoproteins is encoded by several mucin genes. The apomucins are covered by a large number of carbohydrate chains that can be remarkably different and represent a mosaic of sites for attachment of microorganisms. The binding of P. aeruginosa to mucins involves outer membrane proteins and mucin carbohydrate chains that are structurally different from the carbohydrate recognized by pillin. Airway and salivary mucins secreted by patients suffering from cystic fibrosis (CF) show alterations in their carbohydrate moiety. The increased sulfation of airway mucins seems to correspond to a primary defect. Other abnormalities such as increased sialylation or fucosylation have also been detected. The binding of P. aeruginosa to airway or salivary mucins is increased in CF. However, the precise link between the carbohydrate alterations and the increased binding of P. aeruginosa to CF mucins remains to be elucidated.
Assuntos
Glicoconjugados/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistema Respiratório/microbiologia , Animais , Aderência Bacteriana , Fibrose Cística/microbiologia , Glicolipídeos/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Sistema Respiratório/metabolismoRESUMO
1. Changes in membrane lipid composition and metabolism could participate in myocardial membrane dysfunction in essential or experimental hypertension. Phospholipid-bound fatty acid profile and metabolism are altered in cultured heart myocytes of newborn genetically hypertensive rats. The present study was designed to investigate the participation of phospholipase A2 in these modifications. 2. Phospholipase A2 activity of cultured cardiomyocytes of neonate spontaneously hypertensive rats and normotensive control Wistar-Kyoto rats was compared. The enzyme activity was measured using 2-[1-14C]arachidonyl-phosphatidylethanolamine as substrate. In both strains, Ca(2+)-dependent and independent phospholipase A2 activities were present. Only the Ca(2+)-dependent enzyme activity was altered in spontaneously hypertensive rat cardiomyocytes. With 0.2 mmol/l substrate and 5 mmol/l Ca2+, the phospholipase A2 activities were 79.0 +/- 13.4 and 26.0 +/- 3.6 nmol h-1 mg-1 of protein in spontaneously hypertensive and Wistar-Kyoto rat cardiomyocytes respectively (n = 10 in both cases, P = 0.001). The maximum velocity of the enzyme was three times higher in spontaneously hypertensive rat than in Wistar-Kyoto rat, without changes in the apparent affinity of the enzyme for its substrate. 3. The present results demonstrate an enhanced phospholipase A2 activity in cultured heart muscle cells of spontaneously hypertensive rats, which could be genetically determined.
Assuntos
Hipertensão/enzimologia , Miocárdio/enzimologia , Fosfolipases A/metabolismo , Animais , Ácido Araquidônico/metabolismo , Cálcio/metabolismo , Células Cultivadas , Hipertensão/metabolismo , Miocárdio/metabolismo , Fosfolipases A2 , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Mineralocorticoid hormones regulate many physiological functions in the cardiovascular system. Although high affinity binding sites for aldosterone have been found in myocardium, aldosterone effects on pHi regulatory systems in cardiac cells have not been described. We have addressed this issue by using microspectrofluorimetric monitoring of intracellular pH in developing neonatal rat cardiomyocytes cultured for 2 weeks. Developmental changes in cell morphology were controlled by anti-myosin light chain antibody staining of the sarcomeric units using confocal laser scanning microscopy. The data obtained demonstrate that from early stages of the development, pHi in neonatal cardiac cells is regulated by three ion transporting mechanisms, namely, Na/H antiport, Na- and HCO3-dependent transporter and Cl/HCO3 exchanger. A 24-h treatment of the cells with aldosterone increases the activity of the Cl/HCO3 exchanger at day 6 of cell culture while the Na/H antiport activity is enhanced in the cells treated with the hormone at days 9 and 13 of culture. Thus, by affecting the activity of ionic transporters, aldosterone modulates acid-base balance in cardiac cells.
Assuntos
Aldosterona/farmacologia , Canais de Cloreto/metabolismo , Miocárdio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Transporte/metabolismo , Células Cultivadas , Ventrículos do Coração/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Microscopia Confocal , Ratos , Ratos WistarRESUMO
Genetic hypertension has been proposed to be associated with impaired lipid metabolism. To investigate whether lipid metabolism is altered in young rats of the spontaneously hypertensive Okamoto strain (SHR), we have compared the phospholipid fatty acid content and metabolism in cultured heart myocytes and fibroblasts from SHR and normotensive Wistar-Kyoto (WKY) newborn rats. The phospholipid-bound fatty acid profile and metabolism were altered in SHR cardiomyocytes and unchanged in SHR fibroblasts. In SHR myocytes, the fatty acid composition of the phospholipid fraction was modified, with a lowered proportion of linoleic (P < .05) and eicosapentaenoic acid (P < .001), resulting in a decreased polyunsaturated to saturated fatty acid ratio (1.16 +/- 0.08 in SHR v 1.44 +/- 0.08 in WKY, P < .02). The metabolism of radioactive arachidonate (C20:4) and linoleate (C18:2) also differed between SHR and WKY myocytes. Their release was increased (P < .004 and .05 for C20:4 and C18:2, respectively). The labeled phospholipid species also differed between the two strains, suggesting an altered phospholipid turnover in SHR. This study demonstrates modifications of phospholipid fatty acid profile and metabolism in spontaneously contractile cardiac cells from newborn prehypertensive SHR, in the absence of neural, hormonal, and hemodynamic influences.
Assuntos
Ácidos Graxos/metabolismo , Hipertensão/metabolismo , Miocárdio/metabolismo , Fosfolipídeos/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Ácidos Graxos Insaturados/metabolismo , Fibroblastos/metabolismo , Hipertensão/genética , Ratos , Ratos Endogâmicos SHRRESUMO
The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cases of cystic fibrosis. For this purpose, microtiter plate adhesion assays have been developed and have suggested that nonpilus adhesins of P. aeruginosa are the most important ones for binding to human respiratory mucins. In order to characterize these mucin-binding adhesins, outer membrane proteins (OMP) from two adhesive strains, 1244-NP and PAK-NP, and their poorly adhesive rpoN mutants, 1244-N3 and PAK-N1, were prepared by a mild extraction with Zwittergent 3-14. Mucin-binding adhesins were detected after polyacrylamide gel electrophoresis and blotting of the OMP on nitrocellulose replicas, using human bronchial mucins labeled with 125I. The binding properties of these OMP with lactotransferrin, another glycoprotein abundant in respiratory mucus, were also studied. Radiolabeled mucins detected four bands at 48, 46, 28, and 25 kDa with strain PAK-NP. With the nonmucoid strain 1244-NP, five bands were observed at 48, 46, 42, 28, and 25 kDa. The bands at 48 and 25 kDa were also visualized by radiolabeled lactotransferrin. These bands were partially or completely displaced by nonradiolabeled respiratory mucin glycopeptides but not by tetramethylurea, suggesting that they recognized carbohydrate sites. In contrast, the poorly adhesive strains showed weakly binding bands. These results demonstrate that outer membranes from two different nonpiliated P. aeruginosa strains express multiple adhesins with an affinity for human respiratory mucins and/or lactotransferrin.
Assuntos
Adesinas Bacterianas , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Lactoferrina/metabolismo , Lectinas , Mucinas/metabolismo , Pseudomonas aeruginosa/patogenicidade , Humanos , Escarro/metabolismoRESUMO
We have isolated, from newborn rats, heart cultures enriched in contractile muscle cells (M) and cultures of fibroblast-like cells (F). M cultures respond to simulated ischemia by an arrest of beating activity, by a decrease in beta oxydation rate, ATP and phosphocreatine content and by a loss of membrane phospholipids associated with neutral lipids accumulation. F cells in contrast do not respond to oxygen deprivation. Firstly, we observed that cocultures of M and F cells respond to oxygen deprivation by an arrest of beating activity and a decrease in cellular ATP content, but failed to exhibit any significant loss of membrane phospholipids. Secondly, we demonstrated that culture medium conditioned by F cells is able to inhibit the reaction of M cells to simulated ischemia thus suggesting that fibroblasts produce a diffusible factor able to block phospholipase activation. Heat treatment and trypsinisation failed to abolish this activity, indicating that the phospholipase inhibitory factor is probably not a polypeptide.
Assuntos
Lipídeos de Membrana/metabolismo , Miocárdio/metabolismo , Fosfolipídeos/metabolismo , Animais , Ácido Araquidônico/metabolismo , Células Cultivadas , Doença das Coronárias/metabolismo , Ácidos Graxos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Miocárdio/citologia , Fosfolipases A/antagonistas & inibidoresRESUMO
Newborn spontaneously hypertensive rats (SHR) develop cardiac hypertrophy before a rise in blood pressure. Cytosolic pH (pHi) has been discovered to modulate cell growth and proliferation; therefore, we have investigated pHi in myocytes and fibroblasts from 3- to 4-day-old SHR and normotensive Wistar (W) and Wistar-Kyoto controls (WKY). The ratio of heart to body weight was higher in SHR than in W and WKY (7.56 +/- 0.10 v 6.21 +/- 0.10 and 5.98 +/- 0.14 mg/g in 10, 5, and 7 groups of 20 to 40 animals; P less than .001 for both). Cytosolic pH, determined with the fluorescent probe BCECF, was measured from the sixth to the eighth day in culture on confluent cells. The mean pHi was higher in myocytes from SHR than in those from W or WKY rats (7.19 +/- 0.03, N = 30, v 7.09 +/- 0.03 and 7.11 +/- 0.02, N = 25 and 30; P = .008 and .024, respectively). In contrast, pHi was similar in fibroblasts from the three strains (7.21 +/- 0.03, 7.18 +/- 0.03, and 7.19 +/- 0.02, N = 15, 15, and 14, in SHR, W, and WKY rats, respectively). External acidification induced similar decreases in pHi from SHR and WKY myocytes, maintaining higher pHi values in SHR myocytes along the entire external pH (pHo) range studied. The inhibition of Na+/H+ exchange by the amiloride derivative, ethylisopropylamiloride, decreased the steady-state pHi of myocytes independently of the initial pHi values. This study demonstrated a cytosolic alkalinization in contractile cardiac cells from SHR before a significant rise in blood pressure and in the absence of hemodynamic influences and specific plasma factors.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fibroblastos/fisiologia , Coração/fisiologia , Hipertensão/fisiopatologia , Miocárdio/citologia , Animais , Cardiomegalia/fisiopatologia , Células Cultivadas , Citoplasma/fisiologia , Concentração de Íons de Hidrogênio , Hipertensão/patologia , Contração Miocárdica , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Exposure to silica can induce fibrosis and/or emphysema. Various factors such as proteases, other hydrolases and oxidants may be involved in the destruction of lung parenchyma. On the other hand, antiproteases play an important role in the protection of lung parenchyma against the action of proteases. We have developed an animal model of silicosis in monkey Macacus cynomolgus and followed these factors by bronchoalveolar lavage (BAL). We have studied glycosidases activities, elastase-like activity, immunoreactive alpha 1-protease inhibitor (alpha 1PI), neutrophil elastase inhibitory capacity (NEIC) and myeloperoxidase. Bronchoalveolar cells in serial BAL were also studied. Six monkeys were exposed to quartz aerosols (100 mg.m-3) for 18 wks. They were followed until they developed X-ray changes, which occurred between 21-64 wks after the end of the dust exposure. Cellular "silicotic nodules" were observed in lung biopsies. A control animal underwent serial BAL. Changes were seen in the differential cell count. The release of superoxide anion by bronchoalveolar cells obtained during the experiment was increased. Separation on a gradient of Percoll showed the presence of young macrophages, which exhibited enhanced release of superoxide anion as compared to the totality of bronchoalveolar cells. The biochemical analysis of BAL fluids obtained during and after the period of dust exposure showed an increase in glycosidases, alpha 1PI and NEIC. Some free elastase-like activity was simultaneously detected in BAL fluids from exposed animals but not from the control. This elastase-like activity was very low compared to NEIC. The increase in enzymatic and antiprotease activities occurred at different points in time for each animal, suggesting large differences in individual responses to dust, but occurred before the chest X-ray abnormalities.
Assuntos
Líquido da Lavagem Broncoalveolar/enzimologia , Líquido da Lavagem Broncoalveolar/patologia , Glicosídeo Hidrolases/metabolismo , Peroxidases/metabolismo , Inibidores de Proteases/metabolismo , Silicose/patologia , Acetilglucosaminidase/metabolismo , Animais , Biópsia , Feminino , Elastase de Leucócito , Pulmão/patologia , Macaca fascicularis , Elastase Pancreática/metabolismo , Silicose/enzimologia , Superóxidos/metabolismo , alfa 1-Antitripsina/metabolismoRESUMO
Catecholamines are known to exert deleterious effects on heart cells and to provoke biochemical alterations similar to those observed during myocardial infarction. In order to investigate the mechanisms of these effects, we have studied in cultures of muscle (M) and fibroblast-like (F) cells derived from newborn rat hearts, the action of isoproterenol on membrane lipid metabolism and on prostaglandin production. We showed in F cells that beta-agonist stimulation produced a striking loss of membrane phospholipids and a moderate hydrolysis of cell triglycerides. In addition, isoproterenol treatment induced a significant stimulation of the secretion of prostacyclin but not of prostaglandin E2 by F cells. None of these effects were potentiated by oxygen deprivation. In contrast, M cells, which are sensitive to ischemia, failed to respond to isoproterenol treatment. These results suggest that catecholamines and hypoxia may exert combined deleterious effects on heart tissue by acting separately on the different target cells in vivo.
Assuntos
Dinoprostona/biossíntese , Isoproterenol/farmacologia , Metabolismo dos Lipídeos , Miocárdio/metabolismo , Oxigênio/metabolismo , Animais , Animais Recém-Nascidos , Ácidos Araquidônicos/metabolismo , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glucose/metabolismo , Coração/efeitos dos fármacos , Hidrólise , Masculino , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos , Triglicerídeos/metabolismoRESUMO
Fatty acid utilization by muscle and nonmuscle heart cells in culture has been investigated in the 7-day-old Zucker rat to determine if this tissue could contribute to the lower energy expenditure reported in obese rats at the onset of obesity. The partitioning of oleate to oxidation and esterification products and the effect of genotype on this partitioning according to cell types were studied. Results showed that the fatty acid beta-oxidation and its esterification in neutral lipid was decreased by 30% in beating muscle cells from obese animals when compared with those from lean animals. In contrast, nonmuscle cells exhibited a decreased beta-oxidation alone. A similar fatty acid composition of the phospholipids was found in non-muscle cells of obese animals and their lean litter mates. In muscle cultures, palmitic and oleic acids are lower in cells of obese rats than in those of lean rats. The present study indicates that a defect in energy metabolism could be found in heart cells at the onset of obesity, suggesting that this defect is determined by intrinisic factor(s).