Association of a functional TNF variant with Plasmodium falciparum parasitaemia in a congolese population.

Several studies have provided evidence of both helpful and harmful effects of TNF on the outcome of Plasmodium falciparum malaria infection. Several TNF polymorphisms that are located within non-coding regions have been associated with parasitaemia, mild malaria or severe malaria. We investigated the association of TNF1304 (rs3093664), TNF-308 (rs1800629), TNF-238 (rs361525) and TNF-244 (rs673) with mild malaria and symptomatic maximum parasitaemia in a population-based design (n=310). We obtained nominal evidence for an association between symptomatic maximum parasitaemia and TNF-308, TNF-238, and TNF-244 on the one hand, and between the number of mild malaria attacks and TNF-244 on the other hand. After accounting for multiple tests, we confirmed the association of symptomatic maximum parasitaemia with TNF-244. We further provide bioinformatics and experimental evidence that TNF-244 has a cis-regulatory effect. This is the first report that emphasizes the potential role of TNF-244 in malaria.

The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C.

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.

Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes.

The subcellular distribution of the 43,000-D protein (43 kD or v1) and of some major cytoskeletal proteins was investigated in Torpedo marmorata electrocytes by immunocytochemical methods (immunofluorescence and immunogold at the electron microscope level) on frozen-fixed sections and homogenates of electric tissue. A monoclonal antibody directed against the 43-kD protein (Nghiêm, H. O., J. Cartaud, C. Dubreuil, C. Kordeli, G. Buttin, and J. P. Changeux, 1983, Proc. Natl. Acad. Sci. USA, 80:6403-6407), selectively labeled the postsynaptic membrane on its cytoplasmic face. Staining by anti-actin and anti-desmin antibodies appeared evenly distributed within the cytoplasm: anti-desmin antibodies being associated with the network of intermediate-sized filaments that spans the electrocyte, and anti-actin antibodies making scattered clusters throughout the cytoplasm without preferential labeling of the postsynaptic membrane. On the other hand, a dense coating by anti-actin antibodies became apparent on the postsynaptic membrane in homogenates of electric tissue pointing to the possible artifactual redistribution of a soluble cytoplasmic actin pool. Anti-fodrin and anti-ankyrin antibodies selectively labeled the non-innervated membrane of the cell. F actin was also detected in this membrane. Filamin and vinculin, two actin-binding proteins recently localized at the rat neuromuscular junction (Bloch, R. J., and Z. W. Hall, 1983, J. Cell Biol., 97:217-223), were detected in the electrocyte by the immunoblot technique but not by immunocytochemistry. The data are interpreted in terms of the functional polarity of the electrocyte and of the selective interaction of the cytoskeleton with the innervated and non-innervated domains of the plasma membrane.

Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells.

Annexin II is a Ca(2+)-dependent membrane-binding protein present in a wide variety of cells and tissues. Within cells, annexin II is found either as a 36-kD monomer (p36) or as a heterotetrameric complex (p90) coupled with the S-100-related protein, p11. Annexin II has been suggested to be involved in exocytosis as it can restore the secretory responsiveness of permeabilized chromaffin cells. By quantitative confocal immunofluorescence, immunoreplica analysis and immunoprecipitation, we show here the translocation of p36 from the cytosol to a subplasmalemmal Triton X-100 insoluble fraction in chromaffin cells following nicotinic stimulation. A synthetic peptide corresponding to the NH2-terminal domain of p36 which contains the phosphorylation sites was microinjected into individual chromaffin cells and catecholamine secretion was monitored by amperometry. This peptide blocked completely the nicotine-induced recruitment of p36 to the cell periphery and strongly inhibited exocytosis evoked by either nicotine or high K+. The light chain of annexin II, p11, was selectively expressed by adrenergic chromaffin cells, and was only present in the subplasmalemmal Triton X-100 insoluble protein fraction of both resting and stimulated cells. p11 can modify the Ca(2+)- and/or the phospholipid-binding properties of p36. We found that loss Ca2+ was required to stimulate the translocation of p36 and to trigger exocytosis in adrenergic chromaffin cells. Our findings suggest that the translocation of p36 to the subplasmalemmal region is an essential event in regulated exocytosis and support the idea that the presence of p11 in adrenergic cells may confer a higher Ca2+ affinity to the exocytotic pathway in these cells.

The tyrosyl residues in creatine kinase. Modification by iodine.

The effect of the iodination of tyrosyl residues in creatine kinase from rabbit muscle has been investigated at alkaline pH after reversible masking of the reactive thiol groups. The conversion of 4-5 tyrosyl residues to monoiodotyrosines as measured by spectrotitration and by radioactive iodine labelling resulted in almost total loss of enzymic activity. The modified enzyme was unable to bind its nucleotide substrates but no significant conformational change was revealed by optical rotatory dispersion or Stokes radius measurements. However, change in the reactivity of some non-essential thiol groups, presumably those located near the active thiol groups, was observed.

Comparative structural studies of the active site of ATP: guanidine phosphotransferases. The essential cysteine tryptic peptide of taurocyamine kinase from Arenicola marina.

The active cysteinyl residues of dimeric taurocyamine kinase from Arenicola marina were labelled with N-ethyl-[1-14C]maleimide. The resulting inactivated N-ethyl-[1-14C]succinimido enzyme was then subjected to tryptic hydrolysis. The peptide containing the labelled essential cysteinyl residue was isolated. The amino acid sequence of this peptide is Leu-Gly-Tyr-Leu-Gly-Thr-[14C]-Cys-Pro-Thr-Asn-Ile-Gly-Leu-Arg. This sequence is very similar to that of homologous ATP:guanidine phosphotransferases previously studied, arginine kinase from Homarus vulgaris muscle, creatine kinase from ox brain and ox muscle, and from rabbit muscle, and lombricine kinase from Lubricus terrestris.

An essential arginyl residue in yeast hexokinase.

The inactivation of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) by phenylglyoxal obeys pseudo first-order kinetics. Formation of a reversible enzyme-reagent complex prior to modification is suggested by the observed saturation kinetics. Loss of activity correlates with the incorporation of 1 mol of [14C]phenylglyoxal per mol 50 000 dalton subunit. No significant conformational change occurs concomitantly. Inactivation is attributable to modification of an arginyl residue. The pattern of protection by substrates and analogs favors an interaction of this essential residue with the terminal phosphoryl group of ATP or glucose 6-phosphate.

Yeast 3-phosphoglycerate kinase. Essential arginyl residues at the 3-phosphoglycerate binding site.

Yeast 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phospho-transferase, EC 2.7.2.3) is inactivated by phenylglyoxal. Loss of activity correlates with the modification of two arginyl residues, both of which are protected by all of the substrates. The modification is not accompanied by any significant conformational change as determined by optical rotatory dispersion. Ultraviolet difference spectrophotometry indicates that the inactivated enzyme retains its capacity for binding the nucleotide substrates whereas the spectral perturbation characteristic of 3-phosphoglycerate binding is abolished in the modified enzyme. The data suggest that at least one of the two essential arginyl residues is located at or near the 3-phosphoglycerate binding site. A likely role of this residue could be its interaction with the negatively charged phosphate or carboxylate groups of 3-phosphoglycerate.

Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca(2+) concentration.

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca(2+)-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-beta-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-beta-cyclodextrin complexes restored the Ca(2+)-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca(2+), annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca(2+) concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca(2+) concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.

Absence of fodrin (spectrin-like protein) under the pseudopod membrane in stimulated thyroid cells.

A fodrin-like protein purified from porcine thyroid cells and characterized by its properties identical to those of pig brain spectrin (F. Regnouf et al., Eur. J. Biochem. 153, 313-319 (1985)) has been localized by immunofluorescence and electron immunocytochemistry in porcine and rat thyroid. Fodrin-like polypeptides were detected in subplasmalemmal meshworks of microfilaments attached to isolated or in situ plasma membranes. In resting cells, fodrin was found under apical and basolateral membrane domains, whereas it was always absent under the pseudopod membrane domain induced by acute TSH stimulation in vitro, using monolayers of porcine cultured cells attached to collagen permeable substrates, as well as in vivo, using rats intravenously treated with TSH. Thyroid fodrin could be involved in exocytosis and membrane stabilization which occurs during the formation of pseudopods induced by TSH stimulation.

Annexin 1 is present in different molecular forms in rat cerebral cortex.

Previously we have purified annexin 1 [J. Neurochem. 56 (1991) 1985-1986] from pig cerebral cortex as a monomeric protein of 37 kDa. Here, the localization of annexin 1 was investigated in subcellular fractionations of rat cerebral cortex using immunodetection by a specific antibody. In contrast to synaptophysin, a specific synaptic vesicle integral membrane protein, annexin 1 is located in the synaptic plasma membrane fraction where it appears on SDS-PAGE as a polypeptide of 74 kDa. Annexin 1 is extracted also as a 74 kDa polypeptide from the purified synaptic plasma membranes. These results suggest for the 74 kDa molecular form an enzymatic dimerization of annexin 1 when associated to the membrane.

Translocation of cytosolic annexin 2 to a Triton-insoluble membrane subdomain upon nicotine stimulation of chromaffin cultured cells.

To gain a better understanding of the function of annexin 2, we have investigated the subcellular distribution of the monomeric and heterotetrameric forms of annexin 2 and their relationship to the cytoskeleton upon stimulation of chromaffin cells. Quantitative immunoblotting has revealed that in resting cells a large amount of annexin 2 is monomeric and cytosolic. Upon nicotine stimulation 80% of total annexin 2 becomes associated with a Triton-X100-insoluble fraction where the monomeric and the heterotetrameric forms are found. The translocation of monomeric annexin 2 is Ca2+-dependent and complete at 1 microM free Ca2+. We have shown that about 66% of the annexin 2 associated with the Triton-X100-insoluble fraction is soluble in octylglucoside while the remnants are insoluble in the detergent and remain likely associated with actin filaments and associated cytoskeleton proteins. The octylglucoside-soluble fraction contains integral proteins from the plasma membrane and from granule membrane, but does not contain caveolin. Moreover, upon nicotine stimulation, a redistribution of proteins was detected in this fraction. These dynamic processes appear concomitantly with the phosphorylation of annexin 2 in this compartment and with catecholamine release. It is suggested that the soluble octylglucoside fraction may represent a special lipidic membrane compartment where the NSF attachment proteins and the cytosolic proteins like annexin 2 and rab3a may become concentrated upon stimulation of the cell. The presence of annexin 2 is consistent with its proposed function on granule and target membrane proteins required for the close apposition of two distinct membranes and supports its functional role in the regulated exocytosis/endocytosis process.

Spatial proximity of a tyrosyl and a lysyl residue in the active site region of yeast 3-phosphoglycerate kinase.

The effect of 7-chloro-4-nitrobenzofurazan on yeast 3-phosphoglycerate kinase causes a modification of one tyrosyl residue concomitantly with a total loss of activity of the enzyme. The modification is not accompanied by any significant conformational change. A total protection against inactivation is observed with the substrates : furthermore, AMP, tripolyphosphate and pyrophosphate afford an effective protection. At pH 9, a shift in the absorbance spectrum of the tyrosine O-nitrobenzofurazan derivative of 3-phosphoglycerate kinase is observed. It can be related to the transfer of the reagent from tyrosine to lysine. The N-nitrobenzofurazan derivative is also completely inactive. It is concluded that a lysine residue is located close to the essential tyrosyl residue.

Interaction between microtubule-associated protein tau and spectrin.

Tau factor, one of the microtubule-associated proteins (MAPs), is shown here to bind to spectrin. Evidence for an interaction between these two proteins is provided by spectrin affinity chromatography of brain MAPs, gel overlay of electrophoresed MAPs with 125I-labelled spectrin, incorporation of tau factor in human erythrocyte ghosts, and demonstration that tau inhibits the F-actin cross-linking activity of tetrameric spectrin. The wide distribution of both tau and spectrin-like proteins in eukaryotic cells is in favor of the possible biological significance of this interaction. The results suggest that tau could be one of the proteins involved in the concerted regulation of microtubule and actin networks in the membrane vicinity.

Interaction of yeast 3-phosphoglycerate kinase with negatively charged carriers.

The aim of this study was to investigate the possibility of an interaction of yeast 3-phosphoglycerate kinase with negatively charged carriers such as polyanionic agents or a polarized electrode. Various polyanions were found to promote enzyme aggregation as judged by ultracentrifugation measurements and chemical modification. The data obtained suggest that these interactions are mediated through the N-terminal domain of the protein. However, the most striking property of 3-phosphoglycerate kinase described here is concerned with its significant dipolar moment as evidenced by electrocapillary measurements, which allows an orientation of the macromolecule in an electric field. Further, the enzyme could be absorbed by a negatively charged surface, first by hydrophobic links and then oriented perpendicularly to the surface. Therefore, the intrinsic properties of yeast 3-phosphoglycerate kinase agree with the formation of an enzyme-membrane complex and afford the ability for a specific orientation of the molecule at the lipid bilayer surface or in the cytoplasm.

Electrical stimulation of transforming growth factor-beta 1 secretion by human dermal fibroblasts and the U937 human monocytic cell line.

The in vitro effects on human dermal fibroblasts and the U937 human monocytic cell line of three phases of electrical microcurrents generated by the ACE Stimulator were investigated. The growth and viability of growing and confluent dermal fibroblasts were not directly influenced by the separate microcurrent phases. One form of microcurrent (designated phase 1) stimulated both dermal fibroblasts and U937 cells to secrete transforming growth factor-beta 1 (TGF-beta 1), which is an important regulator of cell-mediated inflammation and tissue regeneration, but none of the three phases stimulated secretion of the pro-inflammatory cytokine interleukin-6 by U937 cells. The stimulation of TGF-beta 1 secretion in these experiments was not dramatic (a median increase over control levels of 20-30%), although it could be biologically significant.

Molecular characterization of novel trispecific ErbB-cMet-IGF1R antibodies and their antigen-binding properties.

Therapeutic antibodies are well established drugs in diverse medical indications. Their success invigorates research on multi-specific antibodies in order to enhance drug efficacy by co-targeting of receptors and addressing key questions of emerging resistance mechanisms. Despite challenges in production, multi-specific antibodies are potentially more potent biologics for cancer therapy. However, so far only bispecific antibody formats have entered clinical phase testing. For future design of antibodies allowing even more targeting specificities, an understanding of the antigen-binding properties of such molecules is crucial. To this end, we have generated different IgG-like TriMAbs (trispecific, trivalent and tetravalent antibodies) directed against prominent cell surface antigens often deregulated in tumor biology. A combination of surface plasmon resonance and isothermal titration calorimetry techniques enables quantitative assessment of the antigen-binding properties of TriMAbs. We demonstrate that the kinetic profiles for the individual antigens are similar to the parental antibodies and all antigens can be bound simultaneously even in the presence of FcγRIIIa. Furthermore, cooperative binding of TriMAbs to their antigens was demonstrated. All antibodies are fully functional and inhibit receptor phosphorylation and cellular growth. TriMAbs are therefore ideal candidates for future applications in various therapeutic areas.