Tyrosine phosphorylation of I kappa B-alpha activates NF-kappa B without proteolytic degradation of I kappa B-alpha.

The transcription factor NF-kappa B regulates genes participating in immune and inflammatory responses. In T lymphocytes, NF-kappa B is sequestered in the cytosol by the inhibitor I kappa B-alpha and released after serine phosphorylation of I kappa B-alpha that regulates its ubiquitin-dependent degradation. We report an alternative mechanism of NF-kappa B activation. Stimulation of Jurkat T cells with the protein tyrosine phosphatase inhibitor and T cell activator pervanadate led to NF-kappa B activation through tyrosine phosphorylation but not degradation of I kappa B-alpha. Pervanadate-induced I kappa B-alpha phosphorylation and NF-kappa B activation required expression of the T cell tyrosine kinase p56ick. Reoxygenation of hypoxic cells appeared as a physiological effector of I kappa B-alpha tyrosine phosphorylation. Tyrosine phosphorylation of I kappa B-alpha represents a proteolysis-independent mechanism of NF-kappa B activation that directly couples NF-kappa B to cellular tyrosine kinase.

Stimulation of the T-cell antigen receptor-CD3 complex signaling pathway by the tyrosine phosphatase inhibitor pervanadate is mediated by inhibition of CD45: evidence for two interconnected Lck/Fyn- or zap-70-dependent signaling pathways.

The tyrosine phosphatase specific inhibitor pervanadate is a potent activator of T lymphocytes through induction of tyrosine phosphorylation and downstream events of the activation cascade. Using CD45- or CD3-negative variants of the Jurkat leukemic T-cell line we show that the different biochemical events induced by pervanadate appeared to be dependent on the presence at the cell surface of either CD45 or CD3. CD45-dependent events such as tyrosine phosphorylation of Shc, activation of nuclear factor-kappa B (NF-kappa B), activator protein-1 (AP-1), transcription factors, and stimulation of interleukin-2 (IL-2) promoter and of CD69 and CD25 surface expression paralleled activation of the tyrosine kinases lck and fyn. By contrast, stimulation of calcium influx, a CD3-dependent event, paralleled zap-70 activation. The data demonstrate that the T-cell antigen receptor-CD3 (TcR-CD3) complex is functionally linked to two different protein tyrosine kinase (PTK) modules with separate specific functions and that CD45 may be an important regulator of this coupling.

Jurkat T cells express a functional neutral endopeptidase activity (CALLA) involved in T cell activation.

We have characterized a T lymphocyte endopeptidase activity that hydrolyses succinyl-alanine-alanine-phenylalanine-paranitroanilide (Suc-Ala-Ala-Phe-pNa). Hydrolysis of this substrate by intact Jurkat T cells was markedly enhanced when exogenous aminopeptidase N was added to the incubation medium. It thus appears that the release of paranitroaniline from Suc-Ala-Ala-Phe-pNA results from the combination of two distinct enzymatic activities: (i) an endopeptidase activity that cleaves the substrate at the alanyl bond and (ii) an aminopeptidase activity that ultimately cleaves the phenylalanyl bond. This cleavage was further confirmed by HPLC analysis. Specific endopeptidase 24.11 inhibitors were shown to inhibit the endopeptidase activity. These features are reminiscent of the characteristics of neutral endopeptidase (NEP, also known as endopeptidase 24.11, CALLA or CD10). Anti-CD10 monoclonal antibodies (mAbs) recognized the CD10+ B cell line Raji, but not Jurkat cells as assessed by FACS analysis. This is probably due to a lack of sensitivity of this method, the level of NEP activity in Jurkat T cells being 3-5% of that measured in B cell lines. Anti-CD10 mAbs immunoprecipitated endopeptidase 24.11 activities in both Jurkat T cells and Raji B cells, demonstrating that T lymphocytes express a CALLA-related endopeptidase. We also demonstrate that T and B cell endopeptidases have the same molecular weight, that T cells express less functional CALLA mRNA than B cells and that there are at least two shorter transcripts (1.8 and 0.8 kb) in both T and B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of a T lymphocyte mutant defective in the protein kinase C signal transduction pathway.

The phorbol ester TPA is a potent protein kinase C (PKC) activator and a cofactor in the activation of the human Jurkat leukemic T cell line. We have studied the implication of the PKC signaling pathway in the process of T cell activation by generating TPA resistant mutants of Jurkat. These mutants were obtained by recovery of cells that survived a growth arrest induced by TPA. Several cellular phenomena dependent on TPA were dramatically altered in the mutated cells. The mutants were unable to form homoaggregates upon TPA stimulation. Moreover, they did not produce interleukin-2 after activation through engagement of the T cell receptor, in the presence of TPA. These results suggest that the PKC signaling pathway activated by TPA is defective in these cells. In an attempt to define and locate the defect present in the mutants, we have analysed the biochemical properties of PKC, the cellular receptor of TPA. The increase in kinase activity and the translocation of the enzyme to the plasma membrane after stimulation by TPA appeared to be normal in the mutants. We hypothesize that a metabolic step, critical for the completion of T cell activation, distinct from protein kinase C, is impaired in the mutant cells.

A phosphatidic acid-sensitive intracellular pool of calcium is released by anti-CD3 in Jurkat T cells.

Jurkat T cells, loaded with the fluorescent calcium probe Indo 1, responded to exogenous phosphatidic acid (PA) by transiently increasing their cytosolic Ca2+ concentration. This effect was dose-dependent, remained unmodified when external Ca2+ was chelated with EGTA, and was totally inhibited when cells were first exposed to CD3 monoclonal antibodies, indicating that it was solely due to the release of an intracellular pool, which is also mobilized during a stimulation via the CD3 T-cell receptor (TcR) molecular complex. CD3- and phytohaemagglutinin (PHA)-stimulated Jurkat cells also produced PA, the dose-responses and kinetics of which were consistent with those of calcium release. Moreover, diacylglycerol (DAG) kinase inhibitors abrogated PA production and lowered calcium release by CD3-stimulated cells. PA did not induce any apparent increase in inositol triphosphates (IP3), nor did it modify the increase entailed by activation of the CD3 pathway, pointing out that IP3 can be supplemented in mobilizing calcium from intracellular stores. Conversely, a first exposure to PA only partially inhibited the CD3- or ionomycin-induced internal release of calcium, suggesting either a rapid restoration of the PA-sensitive stores, or a contribution of other mediators, such as IP3, in the CD3 activation pathway.

Dynamics of human sperm acrosome reaction: relation with in vitro fertilization.

OBJECTIVE: Acrosomal status has been studied on human sperm prepared for in vitro fertilization (IVF) and related to the rate of fertilization. DESIGN AND PATIENTS: A group of 41 men with normal classical semen parameters, included in the IVF program of University of Nice for feminine tubal obstruction (n = 37) or unexplained infertility (n = 4), were evaluated in a prospective study and compared with a control group of 10 fertile donors. MAIN OUTCOME MEASURES: Evaluation of acrosome status (spontaneous and A23187-induced acrosome loss) after 6 hours incubation in Ménézo's B2 medium was made by flow cytometry on suspended cells with a new immunofluorescence test recently reported by the authors based on a monoclonal antibody GB24. RESULTS: Spontaneous acrosome loss remained low even after 6 hours capacitation (mean + 1 SD, 6.5% + 4.9%). Response to A23187 increased with the duration of preincubation with a marked response after 6 hours (29.5% + 8.9%). Low spontaneous acrosome loss (less than mean + 1 SD) and high response to A23187 (greater than mean - 1 SD) were observed in 25 out of 26 cases of group A with a high fertilization rate (greater than 50% fertilized oocytes). A high level of spontaneous acrosome loss and/or a lack of response to A23187 was observed in 2 of 7 cases of group B (fertilization rate less than 50%) and 6 of 8 cases of group C (unexplained unsuccessful fertilization). CONCLUSION: Impaired acrosomal status can be associated with unexplained unsuccessful fertilization.

Inhibitors of chymotrypsin-like activities selectively block the mitotic pathway in rat hepatoma cells.

We provide evidence that both covalent and non-covalent inhibitors of chymotrypsin-like activities inhibit the insulin-induced DNA replication, while the hormonal metabolic effects such as induction of tyrosine aminotransferase activity or increase of amino-acid transport remain unchanged. Besides, the protease inhibitors that we tested were without any effect on both the autocatalytic phosphorylation of insulin receptors and the tyrosine kinase activity towards poly(glutamate/tyrosine). The inhibitory effect of protease inhibitors on DNA synthesis was also visible when fibroblast growth factor (FGF) was used to commit cells in the proliferative cycle. This observation proves that the involvement of a putative protease is not restricted to the insulin mitogenic pathway. Finally, we observed that Fao cells totally escaped the inhibitory action of a covalent inhibitor of chymotrypsin after having been exposed to insulin for 10 h. We propose that a chymotrypsin-like activity is involved in the intracellular signalling leading to the proliferation of rat hepatoma cells up to a non-return point situated in the middle of G1 (6-8 h).

Evaluation of the human sperm acrosome reaction using a monoclonal antibody, GB24, and fluorescence-activated cell sorter.

GB24 is a mouse monoclonal antibody raised against human trophoblast microvilli, which recognizes an antigenic determinant on the acrosomal region of the human sperm head. By indirect immunofluorescence, reactivity of GB24 could not be detected on freshly ejaculated spermatozoa but was strongly positive after sperm permeabilization with acetone. On viable, motile spermatozoa, reactivity appeared after induction of the acrosomal reaction with the calcium ionophore A23187. These results suggest that the antigen recognized by GB24 is present on the inner acrosomal membrane. A quantitative evaluation assay of the acrosome reaction on viable spermatozoa by flow cytometry using GB24 and indirect immunofluorescence is proposed.

The Na+/K+/Cl- cotransport in C6 glioma cells. Properties and role in volume regulation.

The role of the Na+/K+/Cl- cotransporter in the regulation of the volume of C6 astrocytoma cells was analyzed using isotopic fluxes and cell cytometry measurements of the cell volume. The system was inhibited by 'loop diuretics' with the following order of potency: benzmetanide greater than bumetanide greater than piretanide greater than furosemide. Under physiological conditions of osmolarity of the incubation media, equal rates of bumetanide-sensitive inward and outward K+ fluxes were observed. Blockade of the Na+/K+/Cl- cotransporter with bumetanide did not lead to a modification in the mean cell volume. When C6 cells were incubated in an hyperosmotic solution, a cell shrinkage was observed. It was accompanied by a twofold increase in the activity of the Na+/K+/Cl- cotransport, which then catalyzed the net influx of K+. In spite of this increased activity, no cell swelling could be measured. Incubation of the cells in an iso-osmotic medium deprived of either Na+, K+ or Cl- also produced cell shrinkage. Large activations (up to tenfold) of the Na+/K+/Cl- cotransport together with a cell swelling back to the normal volume were observed upon returning ion-deprived C6 cells to a physiological solution. This cell swelling was completely prevented in the presence of bumetanide. It is concluded that the Na+/K+/Cl- cotransport system is one of the transport systems involved in volume regulation of glial cells. The system can either be physiologically quiescent or active depending on the conditions used. A distinct volume regulating mechanism is the Na+/H+ exchange system.

Velocity field of a Björk-Shiley valve prosthesis: influence of the disc orientation.

An experimental investigation was carried out on the development of physiological flows downstream of a Björk-Shiley valve prosthesis. The post-valvular velocity field was determined by an ultrasonic method in an elastic model of the aortic arch. The flow development in the ascending aorta was strongly dependent on the orientation of the tilting disc. The rotating direction of the vortices and the site of the maximum velocity were influenced by the orientation.

[In vitro determination of the pressure-diameter relationship and velocity profiles by ultrasonic technics. In vivo application]. / Détermination in vitro de la relation pression-diamètre et des profils de vitesse par des techniques ultrasonores. Application in vivo.

A good knowledge of arterial flow mechanics and of the phenomena associated with fluid-boundary interactions is necessary for the determination of some fundamental parameters such as velocity, pressure and pressure-diameter relationship during a cardiac cycle. Ultrasonic techniques were developed on a test bench and directly applied to animals without major modification. On such a test bench allowing a good simulation of physiological type flows, velocity field and pressure-diameter relationship were determined. In vivo application of these techniques allowed a systematic analysis of velocity profiles in the rabbit abdominal aorta and a precise approach of rheological properties of the vascular wall.

Velocity profiles in the wake of two prosthetic heart valves using a new cardiovascular simulator.

In this paper we present a study of the post valvular flow field on a new cardiovascular simulator including an elastic model of the aortic arch. Transverse and vertical two-dimensional velocity measurements are performed with an ultrasonic velocimeter. Two prosthetic heart valves are tested in the aortic position. The behaviour of the velocity vectors patterns during one pulsatile cycle is one of the most striking features of the flow.

The effect of unsteadiness on the flow through stenoses and bifurcations.

This paper is concerned with the influence of a stenosis or a bifurcation on the flow through a tube. In particular the effect of unsteadiness is investigated using simple pulsatile and physiological type flows (Fig. 1). The experimental investigations reported herein are concerned with velocity measurements and flow visualizations. (see formula in text) These measurements, performed in a 60 degrees bifurcation, have permitted the reconstruction of the three-dimensional velocity profiles. The importance of the secondary flow in the branching is analyzed for various values of the flow parameters. Results of tests show a strong influence of unsteadiness on flow characteristics and then on hemodynamic factors. One conclusion is the following: if hemodynamic factors play an important role in the problems of atherosclerosis, then, for macrocirculation studies, it is necessary to take into account unsteadiness and, in particular, the actual shape of the flow-time forcing function.