Marcel Joniau

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.

Lipopolysaccharide-enhanced expression of interleukin-6 in dibutyryl cyclic AMP-differentiated rat C6 glioma

Abstract: Rat C6 glioma synthesizes a low basal level of interleukin‐6 (IL‐6). Stimulation with 10 µg/ml of lipopolysaccharide (LPS) and induction of differentiation with 1 mM N6,O2′‐dibutyryl cyclic AMP (dbcAMP) for 48 h increased the secreted activity to 400 and 800 U/ml, respectively. An LPS stimulation of dbcAMP‐differentiated cells strongly enhanced the secreted activity. Depending on the dbcAMP concentration, the cell number, and the stimulation time, the secreted IL‐6 level increased up to 120,000 U/ml. After 48 h of costimulation with 10 µg/ml of LPS and 1 mM dbcAMP, northern blotting and immunoassay demonstrated an eightfold increase in IL‐6 mRNA concentration and IL‐6 immunoreactivity, whereas titration of the biological activity indicated a 100‐fold increase in the secreted IL‐6 activity. The enhanced secretion of IL‐6 is correlated with the induction of differentiation. Chromatography on heparin‐Sepharose and on DEAE‐5PW separated the secreted activity into several fractions, indicating that they differ in heparin affinity and charge either by posttranslational modifications or by binding to a carrier protein. Each of the partially purified IL‐6‐like activities could be neutralized by an anti‐murine IL‐6 antibody. Our observations demonstrate that in vivo inflammatory signals can trigger astrocytes and their precursors to secrete substantially different levels of immunoregulatory cytokines depending on their degree of differentiation.

Ontogenetical studies on extracellular hemoglobins of Artemia salina.

Abstract Ontogeny of the three extracellular hemoglobins of zygogenetic races of Artemia salina was studied. Hb-II was the first hemoglobin to appear in the swimming nauplius at about 2 hr posthatching, followed by Hb-III at about 8 hr posthatching. Hb-I was detectable by benzidine-H2O2 staining of cellulose acetate electrophoregrams only after the seventh to ninth day posthatching, when the functional gills were formed. Hb-III was always lower in quantity than Hb-II and disappeared completely from the hemolymph between the thirtieth and fourtieth day posthatching. This disappearance of Hb-III was earlier in the female than in the male. Both Hb-I and Hb-II were found to remain for most of the life period in the hemolymph of both the male and female, but Hb-II decreased gradually or sometimes disappeared in the old female, whereas this hemoglobin was continuously the major species in the old male. The general ontogetical pattern described in the present study appears to agree with the observations using different races of Artemia salina including several parthenogenetic ones. We conclude that ontogenesis is the factor most responsible for the observed hemoglobin polymorphism in Artemia salina.

Structural basis for difference in heat capacity increments for Ca2+ binding to two α-lactalbumins

Abstract Thermodynamic parameters for the unfolding of as well as for the binding of Ca 2+ to goat α -lactalbumin (GLA) and bovine α -lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10mM Tris-HCl, pH 7.5 near 25°C. Among the different parameters available, the heat capacity increments (Δ C p ) offer the most direct information for the associated conformational changes of the protein variants. The Δ C p values for the transition from the native to the molten globule state are rather similar for both proteins, indicating that the extent of the corresponding conformational change is nearly identical. However, the respective Δ C p values for the binding of Ca 2+ are clearly different. The data suggest that a distinct protein region is more sensitive to a Ca 2+ -dependent conformational change in BLA than is the case in GLA. By analysis of the tertiary structure we observed an extensive accumulation of negatively charged amino acids near the Ca 2+ -binding site of BLA. In GLA, the cluster of negative charges is reduced by the substitution of Glu-11 by Lys. The observed difference in Δ C p values for the binding of Ca 2+ is presumably in part related to this difference in charge distribution.

Exchangeability of phospholipids between anionic, zwitterionic and cationic membranes

Abstract The spontaneous transport of phospholipid molecules between artificial vesicles has been monitored by free-flow electrophoresis. First, the transfer of traces of the negatively charged [ 3 H] dimyristoylphosphatidylglycerol (DMPG) between anionic and zwitterionic or cationic and zwitterionic vesicles is followed. Anionic vesicles were generated from dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylglycerol (molar ratio 9/1), neutral ones from DMPC and cationic ones from DMPC and 4-(17-tritriacontyl)- N -methylpyridiniumchloride mixed in a molar ratio of 9/1. A halftime of 50 min was calculated for the transfer of DMPG from the anionic donors to the neutral acceptors. In the opposite direction transfer proceeded much slower ( t 1/2 = 900 min). On the other hand, DMPG moved with a t 1/2 of 520 min from the neutral towards the cationic vesicle population, whereas no transfer at all occurred in the reverse direction. In contrast, with the zwitterionic [ 3 H] DMPC, similar t 1/2 values (≈100 min) were found in the above-mentioned donor—acceptor systems. These results demonstrate that membrane charges can strongly modulate the transfer capacity of anionic phospholipids.

Spontaneous phospholipid transfer between artificial vesicles followed by free-flow electrophoresis

Abstract The application of continuous free-flow electrophoresis in the liposome field is described. The technique is able to distinguish between artificial phospholipid vesicles as a function of their surface charge. This made possible to follow intervesicular interactions between differently charged bilayer structures. As an example, we present evidence for a relatively fast, spontaneous phospholipid transfer between dimyristoylphosphatidylcholine and mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol vescles.

Reaction of α-tubulin with lodotyrosines 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.

Spontaneous intervesicular transfer of anionic phospholipids differing in the nature of their polar headgroup

Abstract A set of synthetic negatively charged phospholipids with different polar headgroups was inverstigated with respect to their spontaneous transferability between ‘fluid’ small unilamellar vesicles. As donor vesicles dimyristoylphosphatidylcholine mixed with an anionic lipid in a 9:1 molar ratio was used whereas the recipient particles solely consisted of dimyristoylphosphatidylcholine. The progress of the anionic lipid transfer was followed by continuous free-flow electrophoresis. In a first approach, the different anionic phospholipids used were esters of phosphatidic acid and simple alcohols (from ethanol to hexanol), polyalcohols (ethyleneglycol, glycerol, erythritol) and serine. In spite of their different nature, the effect of these anionic lipids on the melting behavior of the various types of mixed donors is limited. At 33°C in 5 mM Tes (pH 7.0), 10 mM potassium chloride, dimyristoylphosphatidic acid transfers with a halftime of 156 min. This transfer rate gradually decreases upon progressive addition of methyl(ene) residues in the polar headgroup. In contrast, all polyalcohol and serine derivatives move at a faster rate. These different transfer rates are correlated with the hydrophobic/hydrophilic character of the polar domains. We also proved that an additional methylene group present in the fatty acyl chains has a stronger reducing effect on the speed of the transfer process than if it is present in the polar moiety. The identical activation energies found for dimyristoylphosphatidic acid and -phosphatidylbutanol further indicate that the butyl chain in the bilayer is not shielded from the aqueous environment. Secondly, also pH affects the transfer of dimyristoylphosphatidic acid: above pH 7.0 transfer occurs at a relatively fast speed whereas below pH 4.5 it is considerably retarded. These observations are discussed in terms of hydrogen bonding, electrostatic interactions, as well as the intrinsic hydration properties of the different dissociation states of the phosphate group.

The reaction of 4-(p-sulfophenylazo)-2-mercuriphenol with the thiol groups of proteins

1. 1.|A new water-soluble azomercurial, 4-(p-sulphenylazo)-2-mercuriphenol, has been prepared and tested as a thiol probe. 2. 2.|Upon reaction with the thiol groups of native proteins, the pK of the phenolic group of the reagent underwent a shift depending on the environment of the thiol group. An ovalbumin derivative, containing about 3 moles of azomercurial per mole of protein, showed two pK values for the phenolic group. 3. 3.|Using the large differences in visible absorption between bound and free dye, resulting from the pK shifts, a spectrophotometric thiol titration of β-lactoglobulin could be carried out. The thiol titration of bovine serum albumin was disturbed by adsorption of excess dye.

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 andp‐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.