RESUMEN
Cyclin A is a cell cycle regulatory protein that functions in mitotic and S-phase control in mammalian cells. Using a genomic construction corresponding to the human cyclin A gene under the control of its own promoter, we have established stable transfectants overexpressing cyclin A protein. Experiments assisted by laser scanning image cytometry showed that this overexpression begins from late G1 phase onwards and is therefore cell cycle-regulated in this model. We demonstrated that this overexpression advances entry into S phase, leading to a contraction of the overall cell generation time. These results provide evidence that cyclin A can be a rate-limiting factor with respect to the control of the transition to S phase in mammalian cells.
Asunto(s)
Ciclinas/biosíntesis , Fase S , Ciclinas/genética , ADN/biosíntesis , Fase G1 , Células HeLa , Humanos , TransfecciónRESUMEN
Alteration of guinea pig keratinocyte membrane microviscosities (eta) by liposomes of varying composition was determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Measurements performed either with whole cell suspensions or Percoll-separated cell subpopulations, indicate a similar membrane microviscosity (eta = 3.37 poise +/- 10%) compared to those microviscosities reported for other cell types. Our findings show that treatment of guinea pig keratinocytes with liposomes composed of phospholipids results in a decreased membrane microviscosity (1.95 poise), whereas treatment of the cells with an emulsion of cholesterol hemisuccinate, or liposomes composed of cerebrosides, causes an increase in membrane microviscosity (3.85 poise and 5.55 poise +/- 10%, respectively). Changes in membrane fluidity had no significant effect on cell viability. A reduced membrane microviscosity resulted in a decrease in the binding of Concanavalin A to keratinocytes, whereas an increased microviscosity resulted in an increased binding of Concanavalin A. Furthermore, endocytosis of Concanavalin A bound to keratinocytes plasma membranes was not significantly affected by a reduced membrane microviscosity, whereas an increased membrane microviscosity completely blocked the endocytosis of Concanavalin A. Another novel observation was that membranes "fluidified" by phospholipid liposomes could be "rigidified" by treatment with cholesterol hemisuccinate and vice versa. Moreover, these alternate changes in membrane microviscosity resulted in simultaneous alternate changes in the binding of Concanavalin A to the keratinocyte surface.
Asunto(s)
Colesterol/farmacología , Concanavalina A/metabolismo , Endocitosis , Queratinocitos/metabolismo , Liposomas/farmacología , Fosfolípidos/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Separación Celular , Ésteres del Colesterol/farmacología , Concanavalina A/antagonistas & inhibidores , Polarización de Fluorescencia , Cobayas , Cinética , Liposomas/metabolismo , ViscosidadRESUMEN
A method is described to study quantitatively and rapidly the kinetics of endocytosis of a receptor-bound ligand by cells of various subclasses within a heterogeneous cell population. The time course of the internalization of the bound ligand is followed by labeling, with a fluorescent antibody, the ligand molecules exposed on the cell surface at various times of the endocytosis process, and by measuring with a flow cytofluorometer the fluorescence of a large number of individual cell within the total cell population. A convenient mathematical treatment of the data is proposed for analyzing the experimental results. This method is applied to the kinetic study of the endocytosis of rabbit antibodies (anti-mouse immunoglobulin) by B-lymphocytes within a mouse spleen cell suspension.
Asunto(s)
Endocitosis , Linfocitos/fisiología , Receptores de Droga/fisiología , Animales , Citometría de Flujo , Fluoresceínas , Cinética , Ligandos , Linfocitos/citología , Masculino , RatonesRESUMEN
An immunodetection technique has been developed to map with high resolution the methylated sites of human chromosomes. We have used this method to define the methylated areas of chromosomes from normal donors and from leukemia cell lines. The chromosomes were exposed for a short time to UV light to induce mild denaturation. The methylated sites were detected in situ by using monoclonal antibodies against 5-methylcytosine (prepared in mouse), and fluorescein-conjugated antimouse immunoglobulins. The chromosomes from normal cells exhibited a fluorescent pattern with RCT banding, although some differences from previously reported patterns could be detected. With this method we have been able to show the presence of two types of R-bands: High fluorescence R-band (HFR) and low fluorescence R-band (LFR). Chromosomes from leukemia cell lines exhibited low global staining with disrupted RCT banding of the chromosomes. The decreased level of the methylation status of the chromosomes from leukemia cells was confirmed by detection of 5-methylcytosines on total immobilized DNA. Thus, we have shown that this method can be used to determine the methylated status of chromosomes and, in turn, to map not only the structural (banding) but also the functional (methylation status) properties of the different chromosome domains in normal and pathologic human cells.
Asunto(s)
Cromosomas Humanos/genética , Citosina/análogos & derivados , Metilación de ADN , Técnica del Anticuerpo Fluorescente Indirecta , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , 5-Metilcitosina , Adulto , Animales , Anticuerpos Monoclonales/análisis , Cromosomas Humanos/inmunología , Cromosomas Humanos/efectos de la radiación , Citosina/análisis , Citosina/inmunología , Metilación de ADN/efectos de la radiación , Enzimas de Restricción del ADN , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Telómero/inmunología , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
The effect of botulinum Toxin (BoTx), which blocks the mechanism of release of acetylcholine at neuromuscular junctions and induces paralysis of muscles stimulated by nerves, is known to be Ca2+-dependent. Amplitude of muscular contractions evoked by nerve impulse was studied in BoTx poisoned preparations. The present report notes that an increase in Ca2+ concentration in vitro delays paralysis of muscular contractions of the frog evoked by nerve impulse. The restorative effect of different drugs on this paralysis has been tested: 4-aminopyridine, ATXII (toxin isolated and purified from the sea anemone Anemonia sulcata tentacles) and a crude venom isolated from the scorpion Androctonus australis antagonize the BotX induced paralysis at physiological concentrations of Ca2+ (Cao2+ = 2 mM), whereas the restorative effect observed with tetra-ethylammonium or guanidine occurs at higher concentrations of Ca2+ (Cao2+ = 4 mM), as in mammals. ATXII restores in vivo the activity of a BoTx paralysed muscle of guinea pig and this effect is more efficient if the interval between the injection of BoTx and ATXII is shortened. These results on the frog and guinea pig are in agreement with those obtained on other biological preparations by several investigators. Moreover it is suggested that the antagonism of BoTx induced paralysis is a consequence of the increase in Ca2+ at the nerve ending. The efficiency of 4-aminopyridine and animal toxins is explained by an action on the nerve ending, by increasing Ca2+ from an interval compartment of the cell, whereas antagonism produced by guanidine and tetraethylammonium involves uptake of Ca2+ from the external medium. The bathing medium must be at a higher concentration of Ca2+ than usual. This explains the differences in antagonism obtained by these drugs and toxins in vitro and in vivo.
Asunto(s)
Toxinas Botulínicas/toxicidad , Calcio/fisiología , Unión Neuromuscular/efectos de los fármacos , Parálisis/inducido químicamente , Toxinas Biológicas/farmacología , Animales , Antitoxina Botulínica , Venenos de Cnidarios/farmacología , Cobayas , Técnicas In Vitro , Cinética , Neurotoxinas/farmacología , Rana esculenta , Venenos de Escorpión/farmacología , Factores de TiempoRESUMEN
A biotinyl-derivative of 17 beta-estradiol has been used to demonstrate a site of recognition and binding of estradiol located on the plasma membrane of human breast cancer MCF-7 cells by using the biotin/avidin-FITC system. The specificity of this binding has been shown by a displacement of the fluorescent label by 17 beta-estradiol. No displacement was observed when testosterone was added. Quantification of this phenomenon has been shown by laser scanning cytometry while preserving the cells adhesiveness to their growth support as well as their membrane integrity. An analysis by confocal laser scanning microscopy suggested that the fluorescence distribution on MCF-7 cells treated with estradiol-biotin was on the cell periphery. The results obtained are in favour of the recognition and binding site of 17 beta-estradiol located on the plasma membrane of MCF-7 cells and they would indicate that the biological activity of estradiol, among others, could be initiated by an interaction with the membrane.
Asunto(s)
Neoplasias de la Mama/metabolismo , Membrana Celular/metabolismo , Estradiol/metabolismo , Sitios de Unión , Unión Competitiva , Biotina , Línea Celular , Estradiol/análogos & derivados , Femenino , Fluoresceína-5-Isotiocianato , Fluoresceínas , Humanos , Rayos Láser , Microscopía Fluorescente , Células Tumorales CultivadasRESUMEN
Cell viability and cell proliferation are endpoints that can be used to identify cytotoxic effects. In a study of the cytotoxicity of four biomaterials and drugs, these two criteria were determined by different techniques. There were notable similarities and differences among the different methods used. Cell viability, which was determined by the trypan blue exclusion test, spectrophotometric microtitration (neutral red) and flow cytometry (fluorescein diacetate) gave similar results. However, the neutral red assay was found to be the most sensitive method for determining the cytotoxicity of these biomaterials and drugs. Cell proliferation measurement, by cell counts and quantitative protein estimation (coomassie blue), revealed important variations between the two methods and indicated poor sensitivity for the protein assay. A slight variability in the determination of the inhibitory concentration 50 (IC(50)) for the two drugs was observed for all the techniques.
RESUMEN
Human chromosomes can be observed after coloration with ADN specific fluorochromes. Measurement of fluorescence intensity may be done by flow cytometry and it allows achievement of flow karyotypes. It is possible to define a standard karyotype and-by comparison-to bring to the fore chromosomes abnormalities (translocation, deletion, polysomy). Various genomic abnormalities are observed with leukemia. Flow cytometry allows a multiparameter analysis which could be used to detect rare events, unknown in classic karyotyping. With Flow Cytometry and cell sorting, the abnormal chromosomes could be separated and secondly observed after Q banding or analysed after molecular hybridization to confirm leukemia diagnosis or prognostic. Flow cytometry, an analytical technology already used in onco-hematology, allows, which chromosome analysis associated with other technics (molecular biology,...) a new approach of particular diagnosis.
Asunto(s)
ADN de Neoplasias/genética , Cariotipificación/métodos , Aberraciones Cromosómicas , Bandeo Cromosómico , ADN de Neoplasias/análisis , Citometría de Flujo , Humanos , Leucemia/diagnóstico , Hibridación de Ácido NucleicoRESUMEN
Flow cytometry (FMC) is an analytical and preparative technique whereas image analysis is only applied to cell analysis. Recently, image analysis has been adapted as a preparative method using a new technique: image cytometry for analysis and sorting (ICAS). FCM and ICAS are complementary. Flow cytometry allows rapid, quantitative and precise study of fluorescence and light scattering in a large number of cells in suspension, while ICAS analyses fewer cells (adherent cells or tissue) on the basis of fluorescence, morphology and size. ICAS can use these criteria to destroy unwanted cells and hence sort selected cells. ICAS can also be used for confocal microscopy and laser surgery.
Asunto(s)
Adhesión Celular , Citometría de Flujo/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Separación Celular/instrumentación , Separación Celular/métodos , Citometría de Flujo/instrumentación , Técnica del Anticuerpo Fluorescente , Procesamiento de Imagen Asistido por Computador/instrumentación , Procesamiento de Señales Asistido por Computador , Manejo de Especímenes , SuspensionesRESUMEN
After reviewing basic technical considerations, we discuss some applications of flow cytometry in French laboratories. This methodology is used in several areas: oncology, cellular pharmacotoxicology, molecular biology and genetics, immunology, as well as cellular biology and physiology. We also examine the evolution of this technique in two directions: on the one hand, the appearance of increasingly sophisticated instruments; on the other, the development of less expensive and less complicated apparatuses principally directed at clinical applications.
Asunto(s)
Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Alergia e Inmunología , Animales , ADN/análisis , ADN de Neoplasias/análisis , Francia , Humanos , Neoplasias/patologíaRESUMEN
Flow cytogenetic is widely used since 1975, and essentially contributes to karyotype analysis and chromosome sorting. The principles of experimentation and its possibilities and limitations are now well known. Recently several new technologies have appeared. What attitude should the cytometrist adopt regarding PCR, microdissection of chromosomes, in situ hybridization, slit-scan flow cytometry or image analysis?
Asunto(s)
Cromosomas , Citogenética/métodos , Citometría de Flujo/métodos , Animales , Fraccionamiento Celular/métodos , Línea Celular , Cromosomas Humanos , Citogenética/instrumentación , Citogenética/tendencias , ADN/análisis , Células Eucariotas/ultraestructura , Femenino , Citometría de Flujo/instrumentación , Citometría de Flujo/tendencias , Humanos , Cariotipificación/métodos , Masculino , Micromanipulación , Manejo de EspecímenesRESUMEN
Haemopoietic stem cells present in the spleen of adult mice were analysed by grafting X-irradiated animals with polystyrene-nonadherent (NABS) and polystyrene-adherent (ABS) B-enriched splenocytes from syngeneic donors. The progeny of the haemopoietic stem cells present in NABS and ABS subsets were studied with respect to size, surface markers, and response to mitogens and antigens. Ninety-six per cent of the precursors of the myeloid cell lineage (CFU-S) were present in the NABS fraction (50-fold enrichment). The presence in NABS of progenitors of functional T and B lymphocytes was also demonstrated. Twelve days after grafting with NABS, more than 80% of the recipient splenocytes were large and nonadherent granulocyte-like cells. These cells had surface similarities with NABS from normal mice, since both populations reacted with peanut agglutinin and with a rabbit anti-NABS (RAN) serum.
Asunto(s)
Linfocitos B/citología , Granulocitos/citología , Células Madre Hematopoyéticas/citología , Bazo/citología , Linfocitos T/citología , Animales , Formación de Anticuerpos , Linfocitos B/inmunología , Adhesión Celular , División Celular , Femenino , Hematopoyesis , Cinética , Activación de Linfocitos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos , Bazo/efectos de la radiación , Bazo/trasplante , Linfocitos T/inmunologíaRESUMEN
We have shown that Scorpion venom restores the neuro-muscular transmission inhibited by Botulinum toxin in the Frog. The effectiveness of Scorpion venom was antagonized by excess magnesium.
Asunto(s)
Antitoxina Botulínica , Unión Neuromuscular/efectos de los fármacos , Venenos de Escorpión/farmacología , Transmisión Sináptica/efectos de los fármacos , Animales , Anuros , Calcio/farmacología , Técnicas In Vitro , Magnesio/farmacología , Unión Neuromuscular/fisiologíaRESUMEN
We have shown that, if Scorpion venom is acting a skeletal muscle indirectly by releasing Acetycholine and directly by inducing an increase in intracellular free calcium, the main action of toxin II isolated from Anemonia Sulcata tentacles is presynaptic.
Asunto(s)
Venenos de Cnidarios/farmacología , Unión Neuromuscular/efectos de los fármacos , Venenos de Escorpión/farmacología , Acetilcolina/metabolismo , Animales , Anuros , Calcio/farmacología , Contracción Muscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Anémonas de Mar , Transmisión Sináptica/efectos de los fármacos , Tubocurarina/farmacologíaRESUMEN
The binding levels of a highly soluble rhodamine-labeled lipopolysaccharide (LPS-Rh) on different LPS-responsive and nonresponsive murine spleen cells were measured with a cytofluorometer (CFM). In all the experiments, a preferential binding of LPS-Rh to LPS-responsive cells was observed. Furthermore, only a proportion of B cells from the responder mouse strain was selectively stained after addition of LPS-Rh. These findings suggested that LPS-Rh binds to specific components of the cell membrane, involved in the triggering of B-cell mitogenicity.
Asunto(s)
Linfocitos B/metabolismo , Lipopolisacáridos/metabolismo , Activación de Linfocitos , Receptores Inmunológicos , Animales , Linfocitos B/inmunología , Linfocitos B/fisiología , Adhesión Celular , Membrana Celular/metabolismo , Citometría de Flujo , Receptores de Lipopolisacáridos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C3H , Mitógenos/farmacología , Receptores Inmunológicos/fisiología , Rodaminas/metabolismo , Solubilidad , Linfocitos T/metabolismoRESUMEN
We concluded from our results that contractures which occurred when NaCl is replaced by other substitutes could be due to increased Ca entry. This ionic disturbance does not produce any increase of the resting acetylcholine output such as that described for the innervated longitudinal muscle obtained from the ileum. In this environment, venom from the Scorpion (Androctonus australis) is shown to have no effect.
Asunto(s)
Contracción Muscular/efectos de los fármacos , Sodio/deficiencia , Ponzoñas/farmacología , Acetilcolina/metabolismo , Animales , Calcio/farmacología , Galopamilo/farmacología , Cobayas , Íleon/efectos de los fármacos , Íleon/metabolismo , Lantano/farmacología , Potasio/farmacología , EscorpionesRESUMEN
ATX II is a toxin extracted from tentacles of Anemonia sulcata. It was known that this protein displays neurotoxic effects on frog isolated neuromuscular preparation (Fig. 1, 2) and that muscular contractures observed with ATX II are blocked by d-tubocurarine (Fig. 3) or on a 40-days-denervated gastrocnemius (Fig. 4). Part of these experiments has already appeared. 1. These effects of ATX II depend on calcium concentration in the bathing medium, as is the case for transmitter release. The same results were observed when we substituted strontium to calcium. 2. On an intact sciatic sartorius preparation, ATX II does not act on the amplitude of the miniature endplate potentials (mepps, Fig. 6). The muscular action potential is not modified by this toxin. 3. ATX II increases the frequency of the mepps (Fig. 5). The evoked transmitter release (quantal content) after ATX II is also largely increased (Fig. 7). 4. In conclusion, it is suggested that ATX II acts indirectly on the muscle through an increase in acetylcholine release from the motor nerve terminals.
Asunto(s)
Cnidarios/fisiología , Venenos de Cnidarios , Unión Neuromuscular/efectos de los fármacos , Neurotoxinas/farmacología , Anémonas de Mar/fisiología , Animales , Estimulación Eléctrica , Electrofisiología , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , RanidaeRESUMEN
Laser scanning cytometry for analysis and preparation is viewed by some as a blend between flow cytometry and image analysis, since it allows to measure and localize fluorescence and to obtain morphologic or morphometric information on adherent or suspended cells or tissues. However, laser scanning cytometry has additional capabilities such as kinetic studies (slow or rapid) of live cells or measurement of fluorescence recovery after photoblinding. Most of these studies can be performed with great accuracy on localized zones by adding a confocal microscope system and performing three-dimensional image reconstruction. Studies on some of the novel possibilities of laser scanning cytometry are very scant.
Asunto(s)
Citometría de Flujo/métodos , Fluorescencia , Canales de Calcio , Separación Celular , Colorantes Fluorescentes , HumanosRESUMEN
We studied the effect of staurosporine, a potent inhibitor of protein kinase C (PKC) activity, on the phorbol ester- or monoclonal antibody (mAb)-induced modulation of CD3 and CD4 surface antigens. Staurosporine (10(-5) M) completely inhibited phorbol ester-induced modulation but had no effect on that induced by mAb. These results indicate that the down-regulation of CD3 and CD4 observed after activation of the cells by the corresponding mAb is independent from PKC-mediated phosphorylations, and thus that the activation of PKC is sufficient but not necessary to induce the modulation of CD3 and CD4 antigens.