RESUMEN
The effect of bacterial toxins, modifying the activity of regulatory N proteins of adenylate cyclase and probably other systems, on the mitogen-induced changes of cytosolic free Ca2+ concentration ([Ca2+]i) has been studied using Ca2+ fluorescent probe quin-2. It is shown that treatment of thymocytes with cholera toxin, E. coli heat-labile (HL) toxin or pertussis toxin abolishes the concanavalin A (con A)-induced rise of [Ca2+]i. The inhibitory effect of cholera and HL toxins can be explained by the toxin-induced rise of intracellular cAMP. The effect of pertussis toxin indicates the involvement of N proteins in the action of con A receptor and in generation of Ca2+-signal during the mitogenic activation of thymocytes.
Asunto(s)
Toxinas Bacterianas/farmacología , Calcio/metabolismo , Citoplasma/efectos de los fármacos , Proteínas de Escherichia coli , Mitógenos/farmacología , Proteínas/metabolismo , Timo/efectos de los fármacos , Toxina de Adenilato Ciclasa , Animales , Células Cultivadas , Concanavalina A/farmacología , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Enterotoxinas/farmacología , Escherichia coli , Toxina del Pertussis , Ratas , Timo/citología , Timo/metabolismo , Vibrio cholerae , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The effect of pertussis toxin on GTP-binding protein of bovine rod cell outer segments (transducin) was studied. Pertussis toxin was shown to ADP ribosylate either alpha subunit of free transducin or transducin-GDP complex, whereas GTP and its analogue Gpp(NH)p strongly inhibit ADP ribosylation of transducin. Pertussis toxin inhibits rod outer segment membrane GTPase and GTPase of homogeneous transducin by 40% and 70-80%, respectively. Activation of rod cell cyclic nucleotide phosphodiesterase by transducin is reduced after its preincubation with pertussis toxin. In transducin modified by pertussis toxin, 83% of GDP becomes tightly bound and cannot be exchanged with Gpp(NH)p. The stabilization of complex transducin-GDP after ADP ribosylation can explain the inhibitory effect of pertussis toxin on GTP hydrolysis by transducin, and on phosphodiesterase activation by guanyl nucleotides.
Asunto(s)
Nucleótidos de Guanina/metabolismo , Toxina del Pertussis , Células Fotorreceptoras/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Transducina/fisiología , Factores de Virulencia de Bordetella/farmacología , Adenosina Difosfato Ribosa/metabolismo , Animales , Bovinos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Activación Enzimática/efectos de los fármacos , GTP Fosfohidrolasas/antagonistas & inhibidores , GTP Fosfohidrolasas/metabolismo , Luz , Hidrolasas Diéster Fosfóricas/metabolismo , Segmento Externo de la Célula en Bastón/efectos de los fármacos , Transducina/efectos de los fármacosRESUMEN
The activity of B. pertussis toxin has been tested in the continuous culture of CHO (Chinese hamster ovary) cells. The in vitro method of testing B. pertussis toxin is rapid, highly sensitive and specific. The unit of activity of B. pertussis toxin is higher than in mouse tests by several orders. The specificity of the action of B. pertussis toxin on CHO cells has been confirmed by the test of the neutralization of the toxicity effect with antiserum.
Asunto(s)
Ovario/efectos de los fármacos , Toxina del Pertussis , Factores de Virulencia de Bordetella/toxicidad , Animales , Células Cultivadas , Cricetinae , Relación Dosis-Respuesta a Droga , Femenino , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/toxicidad , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos CBA , Estimulación Química , Factores de Virulencia de Bordetella/aislamiento & purificaciónRESUMEN
Following the immunization of BALB/c mice with B. pertussis toxin, monoclonal antibodies (McAb) to the antigens of human epithelium, both dermal (the basal, superbasal or all epidermis levels) and thymic (the epithelium of the medullary zone, the cortical and medullary epithelium around Hassall's corpuscles), have been obtained. McAb have been obtained as the result of the polyclonal activation of autoreactive B-cells with B. pertussis toxin. McAb thus obtained can be used for the determination of the corresponding antigens in the epithelial tissues of the thymus and other organs in man, as well as for the diagnosis of tumors, histogenetically related to integumentary tissues of the epidermal genesis.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Epítopos/inmunología , Activación de Linfocitos/efectos de los fármacos , Toxina del Pertussis , Piel/inmunología , Timo/inmunología , Factores de Virulencia de Bordetella/farmacología , Animales , Reacciones Antígeno-Anticuerpo , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Epitelio/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Hibridomas/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB CRESUMEN
Hybridomas synthetizing monoclonal antibodies (McAb) to B. pertussis toxin (BPT) and endotoxin, or lipopolysaccharide (LPS), were obtained. The specificity of McAb to BPT was confirmed in the leukocytosis-stimulating factor neutralization test. Two hybridomas synthetized McAb, seemingly active against the common determinant of BPT and LPS. The McAb of one hybridoma reacted with the crude extract of BPT.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Hibridomas/inmunología , Toxina del Pertussis , Factores de Virulencia de Bordetella/inmunología , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Antígenos Bacterianos/inmunología , Inmunización , Técnicas Inmunológicas , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBARESUMEN
Transducin from bovine retinal rod outer segments possesses two sites responsible for the binding of guanyl nucleotides, one of which is specific only for GTP (GTP-site), while the other one may bind both GTP and GDP (GTP/GDP-site). Pertussis toxin covalently modifies the alpha-subunit of transducin as a result of which 83% of GDP bound at the GTP/GDP site of the protein remain tightly bound and are not displaced by Gpp(NH)p excess. The GTP-site in modified transducin binds Gpp(NH)p at the same rate and reveals the same sensitivity to rhodopsin as does native transducin. Presumably, the GTP/GDP site is localized in the alpha-subunit of transducin. The inhibiting effect of pertussis toxin on GTP hydrolysis by transducin and on stimulation of retinal rod outer segment phosphodiesterase by guanyl nucleotides is due to the tight binding of GDP in the active center of the protein after transducin ADP-ribosylation, which makes impossible the formation of a complex between GTP and the alpha-subunit of transducin.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Nucleótidos de Guanina/metabolismo , Proteínas de la Membrana/metabolismo , Toxina del Pertussis , Células Fotorreceptoras/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Factores de Virulencia de Bordetella/farmacología , Animales , Sitios de Unión , Bovinos , Técnicas In Vitro , Cinética , TransducinaRESUMEN
The modified method for the isolation and purification of B. pertussis toxin has been proposed. Chromatography with the use of hydroxylapatite and lentil lectin--Sepharose 4B has permitted the isolation of the preparation purified 600 times. Its molecular weight is about 90,000. The preparation has been found to possess leukocytosis-stimulating, histamine-sensitising and hemagglutinating activity. Electrophoretic analysis has revealed that the isolated substance consists of four subunits with molecular weights 28,400, 24,300, 21,800 and 15,200. This substance has proved to be capable of hydrolyzing NAD+, as well as of suppressing the GTPase activity of transducin, which is indicative of the covalent modification (ADP-ribosylyzing) of GTP-binding protein under the action of B. pertussis toxin. Two methods for the isolation of B. pertussis toxin (from liquid and solid growth media), as well as the isolation of the toxin from different B. pertussis strains, are evaluated.
Asunto(s)
Toxina del Pertussis , Factores de Virulencia de Bordetella/aislamiento & purificación , Animales , Técnicas Bacteriológicas/instrumentación , Hemaglutinación/efectos de los fármacos , Histamina/inmunología , Hidrólisis , Leucocitosis/inducido químicamente , Ratones , Ratones Endogámicos CBA , Microscopía Electrónica , Peso Molecular , NAD/metabolismo , Temperatura , Factores de Virulencia de Bordetella/análisis , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Effect of B. pertussis lymphocytosis-promoting factor (LPF) on the lympho-hematopoietic system of mice was studied. The injection of LPF was shown to sharply enhance endogenous colony formation and to induce a severe depletion of thymus cells, reaching its maximum of day 4. Thymocytes obtained on day 2 or 3 after the injection of LPF produced a suppressive effect on endogenous colony formation. The proliferative activity of hematopoietic stem cells sharply increased under the influence of LPF, though it had no radioprotective action. On the following day after the injection of LPF a steep rise in the number of hematopoietic stem cells was observed in the blood of mice: their content increased 20-fold in comparison with the control level. These data may be important for the evaluation of the side effects of pertussis vaccine on the lympho-hematopoietic system.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Linfocitos/efectos de los fármacos , Toxina del Pertussis , Factores de Virulencia de Bordetella/farmacología , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/efectos de la radiación , Trasplante de Médula Ósea , Recuento de Células/efectos de los fármacos , Recuento de Células/efectos de la radiación , Ensayo de Unidades Formadoras de Colonias , Células Madre Hematopoyéticas/efectos de la radiación , Interfase/efectos de los fármacos , Interfase/efectos de la radiación , Linfocitos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Bazo/efectos de los fármacos , Bazo/efectos de la radiación , Bazo/trasplante , Timo/efectos de los fármacos , Timo/efectos de la radiación , Factores de Tiempo , Factores de Virulencia de Bordetella/aislamiento & purificaciónRESUMEN
The effects of choleragen- and pertussis toxin (PT)-induced ADP-ribosylation on the GTP-binding protein transducin (TD) from retinal rod outer segments (ROS) have been studied. It has been shown that both toxins cause inhibition of the TD GTPase activity. PT inhibited the GTPase by 30-40% in "native" ROS and by 70-80% in homogeneous TD. Choleragen, in contrast with PT, had no effect on the GTPase activity of homogeneous TD, but was as effective as PT in membrane preparations. The effects of both toxins on the GTPase activity of TD were found to be dependent on the chemical structure of the guanyl nucleotide present in the vehicle. The data obtained suggest that PT and choleragen differ in their specificity for the TD-guanyl nucleotide complex. The former can interact with free TD as well as with the TD-GDP complex, while the latter affects only the TD-GTP complex.
Asunto(s)
Toxinas Bacterianas/farmacología , GTP Fosfohidrolasas/antagonistas & inhibidores , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Células Fotorreceptoras/enzimología , Segmento Externo de la Célula en Bastón/enzimología , Animales , Bovinos , Toxina del Cólera/farmacología , Nucleótidos de Guanina/metabolismo , Técnicas In Vitro , NAD/metabolismo , Toxina del Pertussis , Segmento Externo de la Célula en Bastón/metabolismo , Especificidad por Sustrato , Transducina , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The content of trace elements necessary for the normal growth of bacteria was found to have no effect on the intracellular concentration of Ca2+ and Al3+. The content of Cu2+, Fe3+, Mn2+, Mg2+ was considerably reduced. The addition of Mg2+ at different concentrations to this culture medium stimulated the capacity of cells for accumulating not only Mg2+, but also some other ions. Their maximum intracellular concentration was observed when the concentration of Mg2+ in the culture medium was 41 mM. The growth of microbial cells in the standard culture medium containing Mg2+ at a concentration of 4 mM was accompanied by the increased consumption of elements actively participating in redox reactions (Cu2+, Fe3+, Mn2+). Shifts in the ionic composition of microbial cells were manifested by the morphological features of B. pertussis, linked with the increased synthesis of crystalloid structures. The influence of Mn2+, Al3+, Zn2+ at different concentrations on the ionic composition and morphology of B. pertussis was studied.
Asunto(s)
Bordetella pertussis/metabolismo , Bordetella pertussis/ultraestructura , Oligoelementos/metabolismo , Medios de Cultivo/metabolismo , Relación Dosis-Respuesta a Droga , Iones , Microscopía Electrónica , Oligoelementos/análisisRESUMEN
A colorimetric method for the rapid determination of the quantitative content of microbial mass in B. pertussis suspensions has been developed. The method is based on the indirect determination of carbon in microbial suspensions by its oxidation with the mixture consisting of potassium bichromate in concentrated sulfuric acid and the subsequent colorimetric analysis of the products of this reaction. The method ensures sufficient accuracy, the determination procedure is simple, takes not more than 2 hours and requires no complex reagents. The results thus obtained are well comparable with those obtained by the classical gravimetric method. The new method permits the determination of microbial mass in B. pertussis suspensions with a minimum concentration of 0.5 mg/ml. The method is recommended for the determination of dry microbial mass in B. pertussis suspensions.
Asunto(s)
Bordetella pertussis/análisis , Técnicas Bacteriológicas , Carbono/análisis , Colorimetría/métodos , Gravedad Específica , Espectrofotometría Ultravioleta , SuspensionesRESUMEN
The character of changes in the infrared spectra of E. coli in the process of their mechanical disintegration has been studied. The destruction of E. coli cell structures has been shown to produce no changes in the optical density of the main analytical absorption bands in infrared spectra. This fact suggests that the infrared absorption spectra of E. coli are the sum of the spectra of all chemical components of the cell, which is confirmed by the infrared spectral study of E. coli cell-wall preparations. Similar results have been obtained in the study of the preparations of B. pertussis cell walls, protoplasts and intact cells.
Asunto(s)
Bordetella pertussis/análisis , Escherichia coli/análisis , Espectrofotometría Infrarroja , Membrana Celular/análisis , Pared Celular/análisis , Protoplastos/análisisRESUMEN
The authors present the results of studying the protective and sensitizing properties of a new preparation made of a ultrasonic disintegrate of pertussis microbes treated by ethyl ether. As shown by electron microscopy, the preparation consisted of the cell wall elements (the membrane), remnants of the cytoplasm and protectosome, i.e. it represented a vaccine consisting of cell fragments. In crude and sorbed condition it possessed marked protective properties (a test on mice). The content of protective units in the adsorbed preparation increased 1.5-3 times. The vaccine produced no sensitizing action, and its histamine-sensitizing activity was 3-5 times lower by protein and 5-10 times--by IOU than that of the whole-cell vaccine prepared form the same microbial suspension.
Asunto(s)
Bordetella pertussis/citología , Vacuna contra la Tos Ferina , Animales , Bordetella pertussis/inmunología , Pared Celular/inmunología , Estudios de Evaluación como Asunto , Liberación de Histamina , Ratones , Sonicación , Tos Ferina/prevención & controlRESUMEN
A possibility of obtaining a fraction of cytoplasmic membranes maximally purified of the cell wall material was for the first time shown on a model of Gram negative bacteria (B. pertussis). The results of electron microscopy, chemical analysis for the presence of the cell wall material--hexosamines, and of the activity of the respiratory chain enzymes served as control.
Asunto(s)
Bordetella pertussis/ultraestructura , Fraccionamiento Celular , Membrana Celular/análisis , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Hexosaminas/análisis , Protoplastos/ultraestructuraRESUMEN
B. pertussis suspension was tested by De Voe et al. method (1970) and its modification with the solutions of a definite ionic composition and a lysozyme. The best results were obtained by the following modification elaborated by the authors. The microbes were grown on the casein-carbon agar for 36 hours and were washed with chilled 0.5 M NaCl. The suspension was washed 4 times with the same solution and then the precipitate was suspended in saccharose solution (0.5 M). In 2 hours the saccharose was replaced by a solution of salts with lysozyme. After a 2-hour incubation at 35 degrees C the substance was centrifugated for 20 minutes and the precipitate suspended in the tris-buffer at pH 7.8. The following changes were observed: after the washing and incubation with saccharose there was seen a strong stretching and separation of the cell wall (CW) from the cytoplasmic membrane (CPM); cells without the CW were rarely revealed; 2) after the lysozyme treatment there were many cells of spherical shape (phasic-contrast microscopy) without any CW, limited by the CPM only. Morphologically they were no different from the true protoplasts of the Gram-positive bacteria. The chemical analysis also confirmed a possibility of obtaining the true protoplasts of the Gram-negative bacteria.