Search PubMed⌕ Search

Biomedical subjects

M Joniau

Publications and source records attributed to M Joniau.

At least 37 records · Page 2Linked to original sources

Efficient expression of bovine alpha-lactalbumin in Saccharomyces cerevisiae.

A synthetic gene encoding the mature bovine alpha-lactalbumin fused to the preproregion of the yeast alpha-mating factor has been expressed and secreted at high level in Saccharomyces cerevisiae under the control of the alpha-mating promoter. Growth conditions were found to be critical for the expression: recombinant alpha-lactalbumin could only be detected in the medium provided the culture was grown at neutral pH. The secreted bovine alpha-lactalbumin is enzymatically active and identical to the whey protein, as confirmed by SDS/PAGE, IEF, ultraviolet and CD spectral analysis, and amino-terminal sequence determination.

Amino Acid Sequence↗

Stimulation of CRP secretion in HepG2 cells: cooperative effect of dexamethasone and interleukin 6.

This report described the capability of the human. human acute phase reactant, C-reactive protein (CRP). Its secretion is stimulated by interleukin 6 (IL-6) in a dose-dependent fashion and can further be positively modulated by dexamethasone. The way in which this glucocorticoid influences the CRP response depends on its time of application. Incubation of HepG2 cells simultaneously with IL-6 and dexamethasone increases the magnitude of CRP release significantly above that seen with IL-6 alone. After preincubation with dexamethasone, the kinetics of CRP release, induced by IL-6, are increased and approach that observed in the case of alpha 1-acid glycoprotein (alpha 1-AGP) without dexamethasone pretreatment. Conditions for optimal secretion of CRP were determined.

C-Reactive Protein↗

Effects of chlorpromazine on PMN-mediated activities in vivo and in vitro.

Polymorphonuclear neutrophils (PMN) play a central role in the acute inflammatory response and functions associated with phagocytosis and bacterial killing, including lysosomal enzyme release and superoxide anion (O2-) generation, are also implicated in tissue injury. We have studied the modulation by chlorpromazine (CPZ) on the effects of lipopolisaccharide (LPS) in vivo in mice. Pretreatment with CPZ (4 mg/kg) and, to lesser extent, promethazine, inhibited LPS-induced hypoferraemia and lethality in mice. We have also observed that CPZ (1-15 microns) inhibited lactoferrin release by PMN in vitro, suggesting that this effect could be responsible for the inhibition of hypoferraemia. We have also evaluated the effect of CPZ on other PMN functions implicated in tissue damage and inflammation, chemotaxis and O2- production. CPZ inhibited both activities, although it had chemokinetic activity per se. These data indicate that CPZ is a modular of PMN functions in vivo and in vitro and this effect could be directly implicated in the protective action of CPZ against endotoxic shock.

Animals↗

Potentialities of magnetoliposomes in studying symmetric and asymmetric phospholipid transfer processes.

Using high-gradient magnetophoresis, the non-protein-mediated transfer and exchange of phosphatidylglycerol (PG) molecules between sonicated phospholipid dispersions and magnetoliposomes is studied. The latter structures consist of nanometer-sized magnetite (Fe3O4) cores which are enwrapped by a phospholipid bilayer. Their dimensions are similar to those of small unilamellar vesicles (De Cuyper and Joniau (1988) Eur. J. Biophys. 15, 311-319). Using these particles, spontaneous lipid movements were studied in three different cases. In a first setup, symmetric exchange between dimyristoylphosphatidylglycerol (DMPG) magnetoliposomes, labelled with [3H]DMPG, and DMPG vesicles was followed. Within the time scale of the experiment (1 day) both the lipid molecules residing in the inner and outer leaflet of the magnetoliposomes participate in the exchange process, although 'flip-flop' movements have a retarding effect. In the second approach a unidirectional flux of DMPG from DMPG magnetoliposomes to distearoylphosphatidylglycerol (DSPG) acceptors is noted. In this case, the outer phospholipid leaflet of the magnetoliposomes (in contrast to the inner one) can be largely stripped off; the extent of depletion is determined by the relative amount of the DSPG receiving structures. Furthermore, it is found that with a 15-fold molar excess of receptors, the whole depletion course can be described by a single first-order rate expression. The reluctancy of the inner shell phospholipids to migrate is further illustrated by the virtual lack of transfer, observed with monolayer-coated Fe3O4 colloids. In the third case, asymmetric bidirectional PG transfer is followed between equimolar amounts of DMPG magnetoliposomes and dipentadecanoylphosphatidylglycerol vesicles. In the initial stage of the incubation period, the mmol PG/g Fe3O4 ratio decreases, but progressively restores later on. By quantitatively measuring the transfer rate of each of the individual components, this complex behavior could be unravelled.

Liposomes↗

3-azido-L-tyrosine as a photoinhibitor of tubulin:tyrosine ligase. Role of thiol groups.

We have synthesized the photoactivatable probes 3-azido-L-tyrosine and p-azido-L-phenylalanine and studied their capacity to inhibit the incorporation of [3H]tyrosine into tubulin catalyzed by tubulin:tyrosine ligase. Without illumination, only 3-azido-L-tyrosine reversibly inhibits the enzyme. Upon illumination, both reagents irreversibly photoinactivate the enzyme in a similar way. The ligase can be protected against photoinactivation by reversibly blocking essential thiol groups with pCMB during illumination.

Affinity Labels↗

Reaction of alpha-tubulin with iodotyrosines catalyzed by tubulin:tyrosine ligase: carboxy-terminal labeling of tubulin with [125I]monoiodotyrosine.

We have studied the capacity of different iodinated derivatives of phenylalanine and tyrosine to inhibit the incorporation of [3H]tyrosine into tubulin catalyzed by tubulin:tyrosine ligase. In contrast to thyronine and its iodinated derivatives, iodotyrosines were efficient inhibitors. That they also functioned as substrates of the enzyme was shown by the effective incorporation of [125I]mono- and diiodotyrosine into tubulin. The label was shown to be located at the carboxy terminus. Labeling by this method conserves the polymerization capacity of tubulin in contrast with classical radioiodination methods involving oxidation.

Iodine Radioisotopes↗

Human granulocyte chemotactic peptide (IL-8) as a specific neutrophil degranulator: comparison with other monokines.

The influence of human monocyte-derived chemotactic peptide (GCP/IL-8) on degranulation of neutrophils was investigated in relation to that of other monokines. GCP/IL-8 promoted a dose-dependent release of lactoferrin from specific granules but had no effect on enzyme release from primary granules. From the other monokines that were tested, tumour necrosis factor alpha (TNF alpha) also induced degranulation, while IL-1 beta and IL-6 scored negatively. TNF alpha-induced lactoferrin release was enhanced by cytochalasin B pretreatment of the granulocytes, while GCP/IL-8-promoted degranulation was not. In contrast to GCP, TNF alpha also caused the release of LDH, suggesting a non-specific cell destruction. These observations further support the view that, unlike the other monokines, GCP/IL-8 is a true and specific granulocyte activator.

Biological Factors↗

Magnetoliposomes. Formation and structural characterization.

The adsorption of different types of phosphatidylglycerols onto magnetizable solid particles is studied. The super-paramagnetic magnetite spheres used have an average diameter of only 14 nm and are stabilized by lauric acid to keep them in solution. During incubation and dialysis of this water-based magnetic fluid in the presence of preformed sonicated phospholipid vesicles, magnetoliposomes are formed which are captured from solution with high efficiency by high-gradient magnetophoresis. Support for the bilayer character of the phospholipid coat is derived from both theoretical calculations and experimental data. Phospholipids which form the inner monolayer are adsorbed very quickly with their charged head-group orientated towards the iron oxide surface. The high-affinity character of the binding is reflected in the adsorption isotherms and is further illustrated by their non-extractability with high concentrations of Tween 20. The outer layer assembles through interaction with the exposed hydrocarbon chains. As compared to the inner layer, the phospholipids adsorb at a much slower rate and are displaced by Tween 20 concentrations which usually disrupt conventional membranes. The adsorption isotherms for this layer obey the Langmuir expression. The affinity constants, derived from them, progressively increase as the hydrophobic nature of the phosphatidylglycerols is more pronounced.

Adsorption↗

Enhanced oxygen metabolism of peritoneal macrophages in the presence of murine neuroblastoma cells is partly caused by enkephalins.

A bioluminescent technique was used to show that murine neuroblastoma (NB) cells or cell-free extracts (H variant) were able to enhance the release of reactive oxygen intermediates (ROI) from peritoneal macrophages in vitro. L-variant NB cells were ineffective. Physiological concentrations of met-enkephalin produced the same effect in vitro but not leu-enkephalin. When both H- and L-variant cells, or their extracts, were incubated together with macrophages, ROI production was not increased. Similar findings were detectable when met- and leu-enkephalin were cultured together with macrophages. In vivo, preliminary studies gave the same results. The concentration rate of met- to leu-enkephalin was higher in H-variant than in L-variant NB cells. We conclude from our results that met-enkephalin can enhance the release of ROI from peritoneal macrophages. The difference in the effects produced by the H and L variants is due to differences in the concentrations of enkephalins released.

Animals↗

Cellular immunity changes caused by LDH virus: analogy with observations of neuroblastoma-bearing mice.

Evidence is presented that LDH virus infection of mice results in drastic changes in several immune activities. Serum IFN titer and splenic NK activity are increased during the acute phase of infection. NK stimulation is mediated by IFN-alpha,beta since injection of an antibody against murine IFN-alpha,beta is able to abolish the effect. IL-2 production is inhibited throughout the study period following injection of LDH virus (14 days), although a partial recovery is observed during the second week. Similarly, IL-2 receptor expression and MLC responsiveness are suppressed. This suppression lasts for 2 and 7 days respectively after injection. Addition of recombinant IL-2, but not of indomethacin, to the MLC cultures restores the proliferation rate. Not only proliferation but also cytotoxic cell generation in MLC is diminished during the first week after LDH virus injection. Again, this response is normalized at day 14. Additional observations indicate that LDH virus is present in murine neuroblastoma. This explains some of the previously described effects of this tumor on the cellular immune system of the host.

Animals↗

Immune response to murine neuroblastoma: effects of in vitro culture of the tumor.

The humoral and cellular immune response of neuroblastoma (NB)-bearing mice has been investigated and shown to be dependent on the treatment of the NB tumor cells before implantation. Established lines and clones derived from the original tumor, as well as C1300 cells passaged in vitro, provoke an immune reaction which differs markedly from the one induced by the original C1300 tumor. Indeed, only the latter induces the formation of high amounts of circulating immune complexes (CIC), is able to trigger peritoneal macrophages in the host and to induce suppression, as shown in mixed lymphocyte cultures. After subculture of the tumor cells in vitro, none of these effects were observed. The pattern of immune response produced by tumor cells grown in vitro can be converted into the original type of response by repeated passaging of the tumor cells in vivo.

Animals↗

NK activity in neuroblastoma bearing mice: in vivo versus in vitro propagated tumor variants.

Two days after inoculation of A/J mice with in vivo propagated C1300 neuroblastoma (NB) cells, a 4-5 fold increase of the splenic natural killer (NK) activity is noticed. A similar rise of NK activity is also displayed by the host lymph node, bone marrow and peritoneal cells, reflecting a general stimulation of NK cells. These effector cells only lyse classical NK target cells. They are of low specific gravity, nylon wool non-adherent and insensitive to anti-Thy treatment, suggesting that they are indeed NK cells. In spite of the ability of in vivo maintained NB cells to increase the NK activity, they are not lysed by NK cells, nor do they have NK-recognizable target structures as demonstrated by competition studies. In contrast with the previously mentioned experiments, NB cells that have been cultured in vitro for at least one week, do not increase the NK activity. During further growth of both tumor variants, the NK activity gradually decreases. In vivo experiments revealed that the presence of a large NB tumor load exerts a negative effect on the induction of active NK effectors. This effect is not due to the intervention of splenic suppressor cells.

Animals↗

Immunocytochemical investigation of native matrix granules of the rat heart mitochondrion.

We have examined the ultrastructure and the protein content of native matrix granules (NMG) in rat heart mitochondria, by postembedding immunocytochemistry. Cytochrome c oxidase was found to be present in these granules. It is believed that these granules contain incomplete inner mitochondrial membrane fractions, which can be incorporated in the membrane after stimulation of the metabolism.

Amobarbital↗

Immune response to murine neuroblastoma: effects of in vitro culture of the tumor. Mitogen and mixed lymphocyte culture responses, interleukin-2 production, suppression and natural killer activity.

The cellular immune response in A/J mice inoculated with in vitro cultured neuroblastoma (NB) cells is completely different from the response obtained after inoculation of in vivo passaged NB cells. Indeed, inoculation of in vivo grown tumor cells leads to a strongly decreased MLC and PHA response of the host spleen cells. This depressed response is not reversed by in vitro addition of indomethacin, but correlates with a low IL-2 production by the spleen cells. In addition, non-specific suppressor cells are present in the spleen and in the peritoneal cavity. The splenic suppressor cells are of low specific gravity and they are nylon wool adherent. The suppressor capacity is not reduced by anti-Thy plus rabbit complement treatment. Another effect of in vivo maintained NB cells is that the natural killer (NK) activity of the spleen cells of the host is increased 4-5 fold 2 days after inoculation of the tumor cells. On the contrary, in vitro passaged NB cells do not induce a reduced MLC and PHA responsiveness, and the IL-2 production of the host spleen cells remains nearly normal. No non-specific suppression can be demonstrated and the NK activity is not augmented 2 days after inoculation. We can conclude that it is very important in which condition tumor cells are maintained when used to study anti-tumor immunity.

Animals↗

Thermodynamics of the Ca2+ binding to bovine alpha-lactalbumin.

Bovine alpha-lactalbumin contains one strong Ca2+-binding site. The free energy (delta G0), enthalpy (delta H0), and entropy (delta S0) of binding of Ca2+ to this site have been calculated from microcalorimetric experiments. The enthalpy of binding was dependent on the metal-free bovine alpha-lactalbumin concentration. At 0.8 mg ml-1, metal-free bovine alpha-lactalbumin delta H0 was -110 +/- 6 kJ mol-1. At this concentration the binding constant was estimated from a mathematical analysis of the titration curves to be greater than 10(7) M-1. This means that delta G0 is smaller than -40 kJ mol-1 and delta S0 is less negative than -235 J.K-1 mol-1. The binding of Ca2+ is therefore enthalpy-driven. From binding experiments as a function of temperature, a delta Cp value of -4.1 kJ.K-1 mol-1 was calculated. This value is dependent on the protein concentration. A tentative explanation for this large value is given.

Animals↗

Chemiluminescence assay of phagocyte activity in mice bearing neuroblastoma: effect of in vitro culture of the tumor.

Phagocyte activity in mice bearing neuroblastoma (NB) has been assayed using a chemiluminescence (CL) technique and proves to be dependent on the pretreatment of the NB cells used to induce the tumor. Indeed, original NB C 1300 tumor, which is maintained by serial in vivo passages induces highly triggered peritoneal phagocytes, producing large amounts of reactive oxygen intermediates (ROI). On the contrary, C 1300 cells cultured in vitro for a few passages and established lines as well as clones derived from the original tumor fail to induce this triggering. However, this decreased phagocyte stimulation can be regained at full extent after a few in vivo passages of the tumor cells.

Animals↗

Circulating immune complexes in murine neuroblastoma: quantification and shifts in immunoglobulin composition.

Circulating immune complexes (CIC) in sera obtained from neuroblastoma (NB)-bearing A/J mice were quantitated using a modified conglutinin (Kg) RIA technique which--in this case--gives very reproducible results. It shows that CIC levels in these sera increase almost linearly as a function of time after tumor challenge. Three weeks after inoculation values ranging from about 4 to up to 10 times those of normal sera are obtained, depending on the type of inoculum (NB tumor or established NB cell lines). However, when tumor is adapted to cell culture, these CIC levels, measured at a constant time interval after challenge, decrease on continued subcultivation in vitro. Furthermore, analyses of the immunoglobulin classes involved in the complexes show that this decrease is more pronounced for the IgG2a and IgG2b subclasses than for IgG1 and IgM, resulting in a shift of the ratio of immunoglobulin classes as a function of subcultivation of these tumor-derived cells.

Animals↗