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M Brouwer

Publications and source records attributed to M Brouwer.

At least 73 records · Page 4Linked to original sources
PubMed1991

Regulation of expression of CD27, a T cell-specific member of a novel family of membrane receptors.

CD27 belongs to a newly defined family of transmembrane R, including the nerve growth factor R, two distinct TNF R and CD40. The function of CD27 is unknown, but on the basis of structural and functional properties, we postulate that it plays a role in the events subsequent to T cell activation, possibly as a cytokine R. We have analyzed the mechanisms underlying the regulation of CD27 protein expression. Membrane expression of CD27 strongly increases after T cell activation via the TCR/CD3 complex or the CD2 molecule. In contrast, direct stimulation of protein kinase C by phorbol esters markedly down-regulates CD27 surface expression. This down-regulation most likely does not result from CD27 phosphorylation, because both anti-CD3 mAb and PMA induce hyperphosphorylation of CD27 on serine residues. Rather, membrane expression seems to be regulated primarily at the RNA level. Stimulation of T cells with anti-CD3 mAb strongly increases steady state CD27 mRNA levels, whereas PMA treatment greatly reduces these transcript levels. Dissection of the TCR/CD3-induced signaling pathways showed that cytoplasmic cAMP as well as Ca2+ concentrations contribute to the increase of CD27 expression. These data indicate that upon Ag-specific T cell stimulation, membrane expression of CD27 is regulated at the RNA level through the joint action of distinct TCR/CD3-associated signaling pathways.

Antibodies, Monoclonal↗
PubMed1991

Human CD8+ T lymphocytes can be divided into CD45RA+ and CD45RO+ cells with different requirements for activation and differentiation.

The functional distinction between CD45RA+ and CD45RO+ cells within the human CD4+ T cell subset is well established. This study was undertaken to investigate whether a similar division can be made within the CD8+ T cell population. A quantitative comparison was made of the requirements for activation and differentiation of CD8+CD45RA+ and CD8+CD45RO+ cells. Stimulation of T lymphocytes with anti-CD3 mAb immobilized at high-density induced strong proliferation and CTL activity in both CD45RA+ and CD45RO+ cells. Suboptimal TCR/CD3 triggering, in contrast, induced substantially higher levels of proliferation and CTL activity in CD8+CD45RO+ cells compared with their CD45RA+ counterparts. Lymphokine secretion (i.e., Il-2 and TNF-alpha) was under any condition more readily induced in CD8+CD45RO+ cells. Markedly, proliferation of both CD8+CD45RA+ and CD8+CD45RO+ T cells initiated by anti-CD3 mAb immobilized at high densities was not inhibited by addition of anti-CD25 mAb, in contrast to proliferation induced by suboptimal anti-CD3 mAb concentrations. These findings show that a functional division between CD45RA+ and CD45RO+ T cells with distinct requirements for activation and differentiation may also be made in the CD8+ subset.

Antigens, CD↗
PubMed1991

T cell receptor/CD3 and CD28 use distinct intracellular signaling pathways.

Ligation of the T cell membrane antigen CD28 strongly enhances cytokine secretion in human T lymphocytes that are activated via T cell receptor (TcR)/CD3 or CD2 molecules. This study was undertaken to investigate whether, as has been indicated for activation via TcR/CD3, stimulation via CD28 is dependent on the activation of protein kinase C (PKC). Two inhibitors of PKC, 1-alkyl 2-methyl-glycerol and staurosporine, caused a dose-dependent inhibition of T cell proliferation induced by anti-CD3 monoclonal antibodies (mAb). The induction of interleukin (IL) 2 secretion was found to be more sensitive to the effects of the PKC inhibitors than the up-regulation of IL 2 receptor expression. In marked contrast, the anti-CD28 mAb-mediated enhancement of T cell proliferation and IL 2 secretion were insensitive to the action of either compound. We conclude that two independent signaling pathways may be operational in human T cells. The first used by TcR/CD3 depends on the activation of PKC, whereas the second is employed by CD28 and functions independently of PKC.

Alkaloids↗
PubMed1991

Interaction of copper-metallothionein from the American lobster, Homarus americanus, with glutathione.

Organisms have harnessed the unique chemistry of copper for a variety of purposes. However, that same chemistry makes this essential metal toxic at elevated concentrations. Metallothioneins (MTs), a family of small metal-binding proteins, are thought to play a crucial role in the regulation of this reactive ion. Here we report that copper-metallothioneins from the American lobster, Homarus americanus, interact with the tripeptide glutathione (gamma-Glu-Cys-Gly). Glutathione in the cytosolic fraction prepared from the digestive gland of the American lobster coelutes with copper-metallothionein during size-exclusion chromatography. The latter protein can be separated into three isoforms by anion-exchange chromatography. All three isoforms belong to the class I MTs. CuMT-I and -II are very similar, whereas CuMT-III is distinct from isoforms I and II. The interaction between glutathione and MT isoforms was examined by ultrafiltration experiments and size-exclusion HPLC. CuMT-III forms a stable 1:1 complex with glutathione, with a dissociation constant of 1 microM. CuMT-I/II makes a transient complex with glutathione, which releases copper as a copper-glutathione complex. This complex can function as the source of Cu(I) in the restoration of the oxygen-binding capacity of copper-free hemocyanin. These studies suggest that metallothionein and glutathione are intricately linked in the biochemistry of copper regulation.

Amino Acid Sequence↗
PubMed1991

Regulation of T-cell differentiation by CD2 and CD28 accessory molecules.

It was analysed to what extent the functional T-cell responses that result from T-cell receptor (TcR)/CD3 triggering differ from responses that are induced after simultaneous ligation of CD2 and CD28 accessory molecules. To allow a quantitative comparison of these activation pathways, purified lymphocytes were stimulated with either graded densities of immobilized anti-CD3 monoclonal antibodies (mAb) or with increasing amounts of anti-CD28 mAb in the presence of a constant concentration of anti-CD2 mAb. Both activation systems were sensitive to the regulatory properties of CD11a/CD18 molecules. T-cell stimulation via CD2/CD28 molecules induced a more potent release of interleukin-2 (IL-2) and more pronounced T helper (Th) cell responses than T-cell stimulation via the TcR/CD3 complex, whereas CD25 expression was more readily initiated after T-cell activation via the TcR/CD3 complex. Optimal Th cell differentiation was detected under conditions of suboptimal receptor occupancy whereas, in contrast, optimal cytolytic T lymphocyte (CTL) differentiation required optimal TcR/CD3 or CD2/CD28 engagement. The findings indicate that T-cell differentiation can be influenced in a qualitative manner by the strength of the activation signal provided, and suggest that antigen-specific T-cell responses might be regulated in a quantitative manner through CD2 and CD28 accessory molecules.

Antigens, CD↗
PubMed1990

Generation of alloreactive cytolytic T lymphocytes by immobilized anti-CD3 monoclonal antibodies. Analysis of requirements for human cytolytic T-lymphocyte differentiation.

Requirements for the induction of human cytolytic T-lymphocyte (CTL) activity were studied in a monocyte-free T-cell activation system that uses immobilized anti-CD3 monoclonal antibodies (mAb) as a stimulus. Alloreactive CTL with specificity for HLA-A and -B locus antigens could be demonstrated within 2 days after the initiation of activation. CTL induction in purified T cells initiated by an optimal concentration of immobilized anti-CD3 mAb was not enhanced by the addition of monocytes or exogeneous cytokines, whereas addition of anti-CD25 mAb largely blocked the response. Upon suboptimal anti-CD3 mAb stimulation, addition of recombinant interleukin (rIL)-2, rIL-1 and rIL-4, but not recombinant interferon-gamma (IFN-gamma) or rIL-6, potentiated the development of CTL activity. Finally it was shown that immobilized anti-CD3 mAb induced significant levels of CTL activity in both purified CD4+ and CD8+ cells. This study indicates that the requirement for cytokines in the differentiation of CTL precursors depends on the strength of the activation signal delivered through the T-cell receptor.

Antibodies, Monoclonal↗
PubMed1989

Allosteric control in Limulus polyphemus hemocyanin: functional relevance of interactions between hexamers.

Hemocyanin of the horseshoe crab Limulus polyphemus is composed of 48 oxygen-binding subunits, which are arranged in eight hexameric building blocks. Allosteric interactions in this oligomeric protein have been examined by measurement of high-precision oxygen-equilibrium curves, using an automated Imai cell. Several models were compared in numerical analysis of the data. A number of conclusions can be drawn with confidence. (1) Oxygen binding by Limulus hemocyanin cannot satisfactorily be described by the two-state MWC model [Monod, J., Wyman, J., & Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118] for allosteric transitions with either the hexamer or dodecamer as the allosteric unit. (2) Of the models tested, the data sets can be best described by an extended MWC model that allows for an equilibrium, within the 48-subunit ensemble, between cooperative hexamers and cooperative dodecamers. The model invokes T and R states for both hexamers (T6 and R6) and dodecamers (T12 and R12). Allosteric effectors modulate oxygen affinity and cooperativity by affecting the R to T equilibria within hexamers and dodecamers and by shifting the equilibria between hexamers and dodecamers. (3) The fitted model parameters show that under most conditions the intersubunit contacts within T-state hexamers are more constrained than those within T-state dodecamers. (4) The oxygen affinities of the hexameric and dodecameric R states are the same, but under all conditions examined the conformation of the fully oxygenated molecule is that of the dodecameric R state. (5) Between pH 7.4 and pH 8.5 the dodecameric T state has a higher affinity for oxygen than the hexameric T state, allowing for "T-state cooperativity".(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1989

Structural and functional diversity of copper-metallothioneins from the American lobster Homarus americanus.

The role of copper metallothionein (CuMT) in copper metabolism and metalloenzyme activation is poorly understood. We have chosen marine crustaceans, in which a direct correlation exists between levels of Cu(I)MT and Cu(I)-hemocyanin during the molt cycle (Engel and Brouwer, Biol. Bull. 173, 239-251, 1987) as unique model systems to study the involvement of MTs in metalloprotein activation and degradation. We have isolated three low-molecular weight, cysteine-rich copper proteins from the American lobster Homarus americanus, which we designate as CuMT-1, CuMT-2, and CuMT-3, respectively. As a first attempt to fully characterize these proteins, we have determined the sequence of the first 56 amino acids of CuMT-1. The results show this protein to belong to the class I MTs, i.e., related in primary structure to equine renal MT. CuMT-1 cannot transfer its copper to copper-depleted apohemocyanin. CuMT-2 belongs to the same class of MTs as CuMT-1, but CuMT-3 does not. The latter can reactivate lobster hemocyanin containing reduced amounts of Cu(I). Spectroscopic studies show that Cu(I) transfer from CuMT-3 to apohemocyanin initially results in the formation of distorted binuclear-copper sites, which subsequently slowly return to their native stereochemical configuration. Finally, we present evidence that shows that the class I MTs in marine crustacea are involved in the sequestration of elevated levels of heavy-metal ions. These observations strongly suggest that the different forms of MT have different biological functions.

Amino Acid Sequence↗
PubMed1989

Studies on the monocyte dependence of T-cell proliferation induced by monoclonal antibodies directed against regions I and II of the CD2 antigen.

We produced three murine monoclonal antibodies (mAb) directed against the human T-cell differentiation antigen CD2 (formerly T11) and analysed their epitope specificity by E-rosette inhibition, cross-blocking and proliferation-induction experiments. When added together, two mAb (both IgG1 kappa) were able to induce proliferation in peripheral blood T cells. This proliferation was found to be strictly dependent on the presence of monocytes. The polymorphism of Fc receptors (FcR) expressed on human monocytes for murine IgG1 antibodies, which is readily demonstrable in anti-CD3-induced T-cell activation, was not found in the anti-CD2-driven system. Moreover, mAb directed against the 40,000 MW FcRII were unable to inhibit proliferation induced by the mitogenic anti-CD2 combination. Still, F(ab')2 fragments of the mitogenic anti-CD2 antibody combination could not initiate T-cell mitogenesis, despite their ability to induce a rise in the free intracellular [Ca2+]. The contributions of monocytes and of antibody Fc moieties to T-cell proliferation, induced by combinations of anti-CD2 mAb, will be discussed.

Antibodies, Monoclonal↗
PubMed1989

Immobilized anti-CD3 monoclonal antibodies induce accessory cell-independent lymphokine production, proliferation and helper activity in human T lymphocytes.

Monoclonal antibodies (mAb) directed against the human CD3 molecular complex are able, when immobilized on the plastic of microtitre wells, to induce accessory cell-independent T-cell proliferation. In this study, we show that the anti-CD3 mAb CLB-T3/3 induces strong T-cell stimulation that is proportional to the density of the immobilized antibody. T cells, optimally stimulated with plastic-immobilized CLB-T3/3, showed a five-fold higher proliferation compared to cells that were stimulated with soluble anti-CD3 in the presence of accessory cells. The difference in magnitude of proliferation was found to be correlated with the expression of the CD25 (TAC) antigen and the production of interleukin (IL)-2, but not with the number of high-avidity IL-2 receptors expressed on the surface of these differentially activated cells. In addition, immobilized CLB-T3/3 initiated the production of interferon-gamma (IFN-gamma), but not of IL-4, in purified T lymphocytes. Coated anti-CD3 mAb induced helper activity in T cells for IgM and IgG production by B lymphocytes. Whereas addition of IL-1 or IL-2 had only a moderate effect on T-cell proliferation induced by immobilized anti-CD3 mAb, helper activity was strongly enhanced in the presence of these factors. This T-cell activation system may prove useful for a standardized analysis of both activation requirements and immunoregulatory capacities of human T cells.

Antibodies, Monoclonal↗
PubMed1988

Regulatory properties of LFA-1 alpha and beta chains in human T-lymphocyte activation.

Lymphocyte function-associated antigen-1 (LFA-1) is a heterodimer composed of an alpha and beta chain that is expressed on the surface of most leukocytes and is an essential molecule for adhesion reactions between cells participating in the immune response. A putative ligand for LFA-1 is the intercellular adhesion molecule ICAM-1 (refs 3-5). Leukocyte adhesion abnormality is found in patients with LFA-1 deficiency. It is not clear whether binding of ligand to the LFA-1 molecule merely spatially orientates cells towards each other or can also induce signals that regulate cell activation and differentiation. We have recently developed a T-cell proliferation assay which uses immobilized anti-CD3 monoclonal antibodies as stimulant and is independent of LFA-1-mediated cellular adhesion. As there is no interference by anti-LFA-1 monoclonal antibodies with the adhesion-dependent activation steps, this T-cell activation system allows us to investigate whether transmembrane signals are induced by binding of ligand to LFA-1 on T cells. Our data indicate that binding of ligand to LFA-1 results in the transduction of regulatory signal across the plasma membrane, rather like other molecules (CD2, CD4, CD8) (refs 8-11) with signal-modifying properties involved in the adhesion of T cells to target/stimulator cells. Indeed, adhesion molecules might generally be important in signal transduction, even in cells not belonging to the immune system.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Signals involved in T cell activation. T cell proliferation induced through the synergistic action of anti-CD28 and anti-CD2 monoclonal antibodies.

Monoclonal antibodies (mAb) directed against the T cell differentiation antigen CD28 (Tp44) induce proliferation of resting T lymphocytes in the presence of phorbol esters. Moreover, it has been reported that such antibodies augment and sustain T cell proliferation induced by soluble antigens, phytohemagglutinin and anti-CD3 mAb. Recently, we have shown that in monocyte-depleted T cell suspensions, anti-CD28 mAb 9.3 and Kolt-2 were able to circumvent the requirement for interleukin 2(IL2) in T cell proliferation induced by soluble anti-CD3 antibodies. Apart from the synergy of anti-CD28 antibodies with phorbol myristate acetate and anti-CD3 antibodies, we found that anti-CD28 mAb were able to induce T cell mitogenesis in combination with an E rosette-blocking anti-CD2 antibody. In this report, we show that antibodies directed against different epitopes on the CD2 antigen can synergize with anti-CD28 mAb. Furthermore, we demonstrate that proliferation induced through the synergistic action of anti-CD28 mAb with anti-CD2 antibodies can be induced in the absence of accessory cells and is accompanied by the production of IL2 and the expression of IL2 receptors. We were unable to induce detectable Ca2+ mobilization through the simultaneous binding of anti-CD28 and anti-CD2 mAb. Taken together, these data show that IL2-dependent proliferation can be induced through the simultaneous binding of anti-CD28 and anti-CD2 antibodies, possibly through phosphatidyl inositol-independent pathways. The observations may provide further insight into the activation mechanisms of human T cells.

Antibodies, Monoclonal↗
PubMed1987

Functional studies with anti-CD3 heavy chain isotype switch-variant monoclonal antibodies. Accessory cell-independent induction of interleukin 2 responsiveness in T cells by epsilon-anti-CD3.

A series of heavy chain isotype switch-variant anti-CD3 monoclonal antibodies (mAb) was used to study the proliferation requirements of purified T cells. None of the variant antibodies was able by itself to induce proliferation. In the presence of exogenous interleukin 2 (IL-2) strong mitogenesis was observed upon stimulation with epsilon-anti-CD3, whereas gamma 1, gamma 2b, gamma 2a, and alpha-anti-CD3 failed to induce T cell proliferation. All variant antibodies induced vigorous proliferation in combination with phorbol myristate acetate. Purified T cells, cultured in the presence of epsilon-anti-CD3, in the absence of IL-2, did not express detectable amounts of TAC-antigen (CD25). The binding of the variant antibodies to the CD3 antigen was evaluated in cross-blocking experiments. It was demonstrated that the epsilon-anti-CD3 antibody, in comparison with the other variant mAb, has a relatively low avidity for the CD3 antigen. In modulation experiments, the IgE variant antibody was unable to induce a substantial loss of CD3 antigen. T cell triggering was investigated at the level of Ca2+ mobilization by means of the dye Indo-1. In contrast to the gamma 1, gamma 2b, gamma 2a, and alpha mAb, which induced a rapid and high rise in the free intracellular calcium level, epsilon-anti-CD3 caused a slow and low rise. These studies indicate that the epsilon-anti-CD3 antibody has a low avidity for the CD3 antigen, compared with the other variant mAb, possibly as a result of monovalent binding. Apparently, the avidity and/or valency of CD3 antigen binding not only has a major influence on CD3 modulation and anti-CD3-induced Ca2+ mobilization, but also sets T cell requirements for IL-2 responsiveness.

Antibodies, Monoclonal↗
PubMed1986

Purification and characterization studies of cadmium-binding proteins from the American oyster, Crassostrea virginica.

The previously reported low molecular weight cadmium-binding protein (CdBP) from the American oyster, Crassostrea virginica, has been further purified and characterized by improved technical methods. The internal organ distribution of the protein within the oyster and effects of life cycle/season on CdBP production also have been evaluated. CdBP isolated by extended ion-exchange gradients or double ion-exchange chromatography followed by HPLC analysis possesses an electrophoretic Rf of about 0.7 and contains relatively little Zn, as previously reported. Cysteine, lysine, and glycine are the dominant amino acids. When ion-exchange columns are developed with NaCl gradients, the aromatic residues tryptophan, tyrosine, and phenylalanine are found to be present, but these may be largely removed depending upon whether the protein is denatured and carboxymethylated prior to analysis. The ultraviolet absorption spectrum of CdBP also was variable, with 250/280 nm ratios ranging from 17:1 immediately after ion-exchange chromatography to 2:1 following concentration procedures. Internal organ distribution studies showed that the visceral mass contained most of the Cd present with lesser amounts in the gills and mantle. In contrast with mammals, CdBP accounts for only about 30% of the total cell Cd burden in these tissues. Cu displacement of Cd from the protein is a particular problem during the summer spawning season and appears to stem from altered Cu metabolism during this period. Relative oyster dormancy during the winter also reduces CdBP production in response to Cd, and the protein is obtained most readily during the fall and spring.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1986

Cadmium and copper metallothioneins in the American lobster, Homarus americanus.

Lobsters were fed cadmium-rich oysters for 28 days, and the induction of cadmium metallothionein and its relation to concentrations of cadmium, copper, and zinc in the digestive gland and gills was determined. A portion of the tissues also was retained for determining the cytosolic distribution of these metals by gel filtration and ion-exchange chromatography. The digestive gland contained a majority of the cadmium, copper, and zinc, and both cadmium and zinc were actively accumulated from the oysters. Gel chromatography of the digestive gland cytosol showed that initially cadmium and zinc were bound to macromolecules with molecular weights of greater than 70,000, approximately 45,000 and less than 5000, and for copper greater than 70,000, 10,000-7,000, and less than 5000. Therefore, only copper was bound to a protein with a molecular weight in the range of metallothionein (i.e., 10,000-7,000). However, after feeding on cadmium-laden oysters for 28 days, both cadmium and copper were bound to the metallothionein-like protein. Further purification of the cadmium/copper protein by ion-exchange chromatography showed that a large portion of the copper and all of the cadmium did not bind to DEAE-Sephacel. The induction of cadmium metallothionein in the digestive gland is correlated with tissue cadmium concentration. There is, however, a tissue threshold concentration of cadmium of 80 to 100 micrograms Cd/g wet weight required for induction. Coincident with the induction of the cadmium metallothionein was a cytosolic redistribution of copper. The distribution of zinc was not affected.

Animals↗
PubMed1986

Copper-metallothioneins in the American lobster, Homarus americanus: potential role as Cu(I) donors to apohemocyanin.

The physiological function of copper(I)-metallothionein is not well understood. The respiratory function of hemocyanin, a copper(I)-containing respiratory protein found in the hemolymph of many invertebrates, has been known a long time. However, the mechanism by which Cu(I) is inserted into the oxygen-binding site of apohemocyanin is completely unknown. This investigation tests the hypothesis that copper(I)-metallothionein may act as a Cu(I) donor to apohemocyanin. To this end, copper-binding proteins and hemocyanin were purified from the digestive gland and hemolymph of the American lobster, Homarus americanus. In the presence of beta-mercaptoethanol, the copper-binding proteins can be resolved into three components on DEAE-cellulose. The first two have been characterized as metallothioneins, based on their high cysteine content and lack of aromatic amino acid residues. The cysteine content of the third component is half of that of components I and II. In the absence of beta-mercaptoethanol the three proteins elute as a single protein complex during ion-exchange chromatography. Components I and II show a strong tendency to polymerize, a process that is accompanied by the loss of protein-bound copper. The purified proteins are not capable of transferring Cu(I) to the active sites of completely copper-free apohemocyanin. They are capable, however, of transferring Cu(I) to active sites of hemocyanin containing reduced amounts of Cu(I), suggesting that the conformational state of hemocyanin is the determining factor in the Cu(I) transfer mechanism.

Amino Acids↗
PubMed1985

The interaction of cytostatic drugs with adsorbents in aqueous media. The potential implications for liposome preparation.

A new method that involves the use of the cation exchange resin Dowex 50W-X4 to remove non-encapsulated drugs from liposome dispersions was investigated. Cytostatic drugs widely varying in their molecular structure can be removed from aqueous solutions by Dowex 50W-X4. The applicability of the resin to separate free from liposome-bound drugs was illustrated for a number of cytostatic drugs (cisplatin, doxorubicin, vincristine). The technique presented allows for a rapid, efficient and convenient procedure for the free drug removal from liposome dispersions without dilution of the liposomal preparation. Studies with liposome-encapsulated drugs will be facilitated by the use of this method, since it avoids many of the problems introduced by conventional methods as dialysis, gel filtration and centrifugation/washing. To elucidate the interaction mechanism of doxorubicin with Dowex 50W-X4, alternative adsorbents were studied for their doxorubicin binding properties. In the adsorption process of doxorubicin onto Dowex 50W-X4 both electrostatic (ion exchange) and hydrophobic effects play a role. The results indicate that hydrophobic contributions to the interaction are responsible for the high resistance offered by the binding forces against desorption of adsorbed doxorubicin. For other adsorbents the interactions are either mainly of an electrostatic or a hydrophobic nature.

Adsorption↗