Biomedical subjects

G Möller

Publications and source records attributed to G Möller.

At least 19 recordsLinked to original sources

PubMed1992

Control of glycoprotein synthesis. Characterization of (1-->4)-N-acetyl-beta-D-glucosaminyltransferases acting on the alpha-D-(1-->3)- and alpha-D-(1-->6)-linked arms of N-linked oligosaccharides.

Hen oviduct membranes contain at least three N-acetyl-beta-D-glucosaminyltransferases (GlcNAc-T) that attach a beta GlcNAc residue in (1-4)-linkage to a D-Man p residue of the N-linked oligosaccharide core, i.e., (1-->4)-beta-D-GlcNAc-T III which adds a "bisecting" GlcNAc group to form the beta-D-GlcpNAc-(1-->4)-beta-D-Man p-(1-->4)-D-GlcNAc moiety; (1-->2)-beta-D-GlcNAc-T IV which adds a GlcNAc group to the (1-->3)-alpha-D-Man arm to form the beta-D-GlcpNAc-(1-->4)-[beta-D- GlcpNAc-(1-->2)]-alpha-D-Man p-(1-->3)-beta-D-Man p-(1-->4)-D-GlcpNAc component; and (1-->4)-beta-D-GlcNAc-T VI which adds a GlcNAc group to the alpha-D-Man p residue of beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpNAc- (1-->2)]-alpha-D-Man p-R to form beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpNAc-(1-->4)]-[beta-D-GlcpNAc- (1-->2)]-alpha-D-Man p-R. We now report a novel (1-->4)-beta-D-GlcNAc-T activity (GlcNAc-T VI') in hen oviduct membranes that transfers GlcNAc to beta-D-GlcpNAc-(1-->2)-alpha-D-Man p-(1-->6)-beta-D-Man p-R to form beta-D-GlcpNAc-(1-->4)-[beta-D-GlcpNAc-(1-->2)]-alpha-D-Man p-(1-->6)- beta-D-Man p-R. The structure of the enzyme product was confirmed by 1H NMR spectroscopy, FAB-mass spectrometry and methylation analysis. Previous work with GlcNAc-T IV was carried out with biantennary substrates; we now show that hen oviduct membrane GlcNAc-T IV can also transfer GlcNAc to monoantennary beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->3)-beta-D-Man p-R to form beta-D-GlcpNAc-(1-->4)-[beta-D-GlcpNAc-(1-->2)]-alpha-D-Man p- (1-->3)-beta-D-Man p-R. The findings that GlcNAc-T VI' and IV have similar kinetic characteristics and that hen oviduct membranes can convert methyl beta-D-GlcpNAc-(1-->2)-alpha-D-Man p to methyl beta-D-GlcpNAc-(1-->4)-[beta-D-GlcpNAc-(1-->2)]-alpha-D-Man p suggest that these two activities may be due to the same enzyme. The R-group of the beta-D-GlcpNAc-(1-->2)-alpha-D-Man p-(1-->6)-beta-D-Man p (or Glcp)-R substrate has an important influence on GlcNAc-T VI' enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Man alpha 3R beta 2-N-acetylglucosaminyltransferase I using synthetic substrate analogues.

UDP-GlcNAc: Man alpha 3R beta 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M(r) 42,000). The Vmax for the pure enzyme with [Man alpha 6(Man alpha 3)Man alpha 6](Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc beta-Asn as substrate was 4.6 mumol min-1 mg-1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds an N-acetylglucosamine (GlcNAc) residue in beta 1-2 linkage to the Man alpha 3Man beta-terminus of the substrate. Several derivatives of Man alpha 6(Man alpha 3)Man beta-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the beta-linked mannose of Man alpha 6(Man alpha 3)Man beta-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the alpha 3-linked mannose (Man) of the substrate increases the KM 20-fold. Modifications on the alpha 6-linked mannose or on the core structure affect mainly the KM and to a lesser degree the Vmax, e.g., substitutions of the Man alpha 6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower the KM, whereas various other substitutions at the 3-position increase the KM slightly. Man alpha 6(Man alpha 3)4-O-methyl-Man beta 4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.

Animals↗

PubMed1992

Age-dependent responsiveness to interleukin-6 in B lymphocytes from a systemic lupus erythematosus-prone (NZB x NZW)F1 hybrid.

The cellular mechanisms for the production of IgG anti-DNA antibodies were studied. Culture of T and B cells from old (NZB x NZW)F1 mice led to the production of IgG anti-DNA antibodies. We found that direct cell contact was partly necessary for the production of IgG anti-DNA antibodies. Fixation of the T cells showed that lymphokines were largely responsible for the antibody synthesis. Antibodies to mouse interleukin-6 (IL-6) inhibited the production of these antibodies in the T-B cell coculture. Human IL-6 could induce small "resting" B cells from the old (NZB x NZW)F1 mice to produce IgG anti-DNA antibodies in a dose-dependent fashion. The response was inhibited by an anti-human IL-6 monoclonal antibody. Large or small B cells from young (B/W)F1 mice or Balb/c mice were not induced by IL-6 to antibody production. Therefore, the capacity of the B/W mice to produce the IgG anti-DNA antibodies correlated with the ability of the B cells to respond to IL-6 and with the age at which the mice begin to have signs of the disease.

Animals↗

PubMed1992

Hospital care versus home care for rehabilitation after hip replacement.

A pilot study was done to assess the feasibility of reducing the hospital stays of patients with total hip replacement (THR). The length of hospital stay for these patients depends largely on how rehabilitation, mostly physical therapy, is organized. This study shows that not more than a half hour per postoperative day was devoted to care services and rehabilitation activities. It is feasible and less expensive to reduce substantially hospital stay by planned physical therapy in the patient's home. These results have prompted a randomized controlled clinical trial to assess hospital versus home rehabilitation.

Aged↗

PubMed1992

Qualitative shift of lymphokine production in response to stimulation, as a consequence of preactivation in vivo or in vitro.

Lymphokine production, analysed at the single cell level, was compared in resting and primed T-cell populations. Cells were preactivated in vitro by repeated mitogen stimulations, or isolated as large, low density cells naturally activated in vivo, from normal spleens of unimmunized animals. A similar qualitative shift in the pattern of lymphokines synthesized after restimulation was found as a result of in vivo and in vitro preactivation of cells. Repeated stimulations in vitro resulted in a qualitative shift in the lymphokines produced in response to activation, from a dominance of IL-2 during the first and second culture, to a dominance of IL-4 and IL-5 in the later stimulations. In vivo activation lead to a similar separation of lymphokine production as primarily IL-2 was made by small resting cells, while large cells preferentially produced IL-4 and IL-5. IFN-gamma was produced by both small and large cells. Preactivation in vitro lead to a more rapid appearance of lymphokines during restimulation. In contrast, the in vivo naturally activated cells responded with a slow onset of lymphokine production when stimulated in vitro.

Animals↗

PubMed1992

Primary stimulation of CD4+ cells in the presence of IL-4 or IFN-gamma alters the frequencies of cytokine-producing cells at restimulation.

The induction of specific effector functions in naive T cells may be directed by accessory signals during activation. These could be elicited through binding to cell surface molecules or through factors secreted by antigen-presenting cells or other simultaneously activated cells. We have investigated the influence of CD8+ cells and of exogenously added cytokines (interleukin (IL)-2, IL-4 and interferon (IFN)-gamma) on the cytokine production in splenic CD4+ T cells. IL-2, IL-4, IL-5 and IFN-gamma production in CD4+ cells was measured at the single cell level during primary mitogen stimulation in vitro in the presence or absence of factors or CD8+ cells. On day 5 the cells were restimulated with mitogen alone and analysed to evaluate the short-term development of cytokine-producing cells in such cultures. Preactivation in the presence of either exogenous IL-4 or IFN-gamma led to an increased production of IL-4 and IFN-gamma respectively at restimulation, and the effects of both IL-4 and IFN-gamma were augmented by IL-2. After preactivation in the presence of IL-2 and IL-4, every third CD4+ cell could be induced to produce IL-4. Exogenous IL-4 or IFN-gamma further decreased each other's production. Depletion of CD8+ cells before activation resulted in a slight increase of IL-4-producing cells, indicating that simultaneous activation of CD8+ cells will influence lymphokine production in CD4+ cells. The results suggest that the pattern of lymphokines induced in naive cells may be influenced by factors secreted by preactivated CD4+ and CD8+ cells, and that naive cells are preferentially 'recruited' to produce similar cytokines.

Animals↗

PubMed1992

Control of O-glycan synthesis: specificity and inhibition of O-glycan core 1 UDP-galactose:N-acetylgalactosamine-alpha-R beta 3-galactosyltransferase from rat liver.

The specificity of glycosyltransferases is a major control factor in the biosynthesis of O-glycans. The enzyme that synthesizes O-glycan core 1, i.e., UDP-galactose:N-acetylgalactosamine-alpha-R beta 3-galactosyltransferase (beta 3-Gal-T; EC 2.4.1.122), was partially purified from rat liver. The enzyme preparation, free of pyrophosphatases, beta 4-galactosyltransferase, beta-galactosidase, and N-acetylglucosaminyltransferase I, was used to study the specificity and inhibition of the beta 3-Gal-T. beta 3-Gal-T activity is sensitive to changes in the R-group of the GalNAc alpha-R acceptor substrate and is stimulated when the R-group is a peptide or an aromatic group. Derivatives of GalNAc alpha-benzyl were synthesized and tested as potential substrates and inhibitors. Removal or substitution of the 3-hydroxyl or removal of the 4-hydroxyl of GalNAc abolished beta 3-Gal-T activity. Compounds with modifications of the 3- or 4-hydroxyl of GalNAc alpha-benzyl did not show significant inhibition. Removal or substitution of the 6-hydroxyl of GalNAc reduced activity slightly and these derivatives acted as competitive substrates. derivatives with epoxide groups attached to the 6-position of GalNAc acted as substrates and not as inhibitors, with the exception of the photosensitive 6-O-(4,4-azo)pentyl-GalNAc alpha-benzyl, which inhibited Gal incorporation into GalNAc alpha-benzyl. The results indicate that the enzyme does not require the 6-hydroxyl of GalNAc, but needs the 3- and the axial 4-hydroxyl as essential requirements for binding and activity. In the usual biochemical O-glycan pathway, core 2 (GlcNAc beta 6[Gal beta 3] GalNAc alpha-) is formed from core 1 (Gal beta 3GalNAc-R). We have now demonstrated an alternate pathway that may be of importance in human tissues.

Animals↗

PubMed1992

Increasing the efficiency of lipid-conjugated antibodies incorporation into the membrane of antigen presenting B cells.

We have introduced some modifications in the technique called "cell decoration" in order to increase the amount of lipid-conjugated antibodies which can be incorporated into the membrane of B cells. As shown by FACS analysis, we have obtained an approximately 4-fold increment in the amount of specific antibodies incorporated into the cell membrane. The procedure, which consists of successive changes of the medium that contains the lipid-conjugated antibodies, avoided changes on parameters that interfere with cell viability. The proposed modification resulted in an approximately 2-fold enhancement of the ability of decorated B cells to act as antigen presenting cells for specific T hybridomas.

Animals↗

PubMed1991

T and B cell collaboration: induction of motility in small, resting B cells by interleukin 4.

In this report we investigate if IL 4 can work as a chemoattractant factor by inducing locomotion in B cells. We found that murine recombinant IL 4 (rIL 4) induced motile morphology and migration through polycarbonate micropore filters of murine, splenic B cells at an optimal concentration of 3 ng/ml. Kinetic studies revealed optimal migration at 8-16 h, although a significant response could be detected already after 1 h. Flow cytometric studies confirmed that the migrated cells were indeed B cells. We also compared the activity of small, dense B cells and large, low-density B cells, based on Percoll gradient separation. We found no difference in IL 4-induced motility among the two groups. Furthermore, we looked at B cells activated in vitro by preculture in lipopolysaccharide (LPS) or IL 4. Our data indicate that both LPS and IL 4 can increase the general capacity for motility in B cells after preculture for 24 h. T and B cell collaboration requires close cell-cell contacts in order for T cell help to be administered to the B cell. One way of enhancing such cell contacts could be through directional cell migration induced by helper factors (chemotaxis). We suggest that IL 4 can play a role as a chemoattractant factor that enhances cell contacts between T helper cells and B cells.

Animals↗

PubMed1991

Differential regulation of lymphokine production in mitogen-stimulated murine spleen cells.

Activation of murine spleen cells in vitro with soluble anti-CD3 monoclonal antibody and phorbol 12-myristate 13-acetate (PMA) induced an initial production of interleukin 2 (IL2), interferon-gamma (IFN-gamma), IL4 and IL5, followed by a refractory state during which the T cells did not produce lymphokines when stimulated with some common mitogens. The refractory state was long-lasting, depended on the presence of anti-CD3 and PMA but could be reverted by incubation in fresh medium. Pokeweed mitogen (PWM) differed from other mitogens tested, since stimulation by PWM and PMA induced lymphokine production and proliferation also in the refractory cells. Furthermore, PWM stimulation selectively induced IL4 and IFN-gamma production but not IL2 and IL5, as detected by intracellular cytokine-specific immunofluorescence and in situ hybridization for mRNA. The results indicate differential regulation of lymphokine production in primary lymphocytes.

Animals↗

PubMed1991

Helper interleukins are produced by both CD4 and CD8 splenic T cells after mitogen stimulation.

We have earlier described (Cardell, S. and Sander, B., Eur. J. Immunol. 1990. 20:389) mitogen-induced production of interleukin (IL)2, IL4 and IL5 mRNA by murine spleen cells, analyzed by in situ hybridization. In the present study we have investigated the potential of CD8 T cells to produce these interleukins, normally associated with the helper function of CD4 T cells. When concanavalin A (Con A)-activated spleen cells were restimulated with Con A and phorbol 12-myristate 13-acetate (PMA), higher levels of IL2, IL4 and IL5 mRNA were induced, as detected both by increased frequencies of positive cells, and by more mRNA per cell. Four-to-six-day Con A blasts were enriched for CD4+ or CD8+ T cells, and restimulated with Con A and PMA. Both CD4 and CD8 cells were found to produce all three kinds of mRNA when restimulated. The frequencies of IL2 mRNA-containing CD8 cells were half of those found for CD4 cells (3.5% as compared to 7%). On the average 1% of the CD8 cells were induced to produce IL4 and IL5 mRNA, while 9% and 3% of the activated CD4 cells contained IL4 and IL5 mRNA, respectively. CD4 and CD8 cells displayed different sensitivities to the reagents when tested alone. Con A induced the synthesis of IL4 and IL5 in CD4 cells, but not CD8 cells, independently of PMA. PMA alone induced extensive thymidine incorporation in CD8 cells, but not in CD4 cells, in the absence of detectable lymphokine mRNA. The results suggest that some CD8 cells have the capacity to give help in immune responses, by secretion of IL2, IL4 and IL5.

Animals↗

PubMed1991

Interferon-gamma and growth factor production by murine T cells derived from three different lymphoid tissues.

Various antigen-presenting cells and the environment in different lymphoid tissues have been suggested to influence the type of lymphokine produced by T cells. We have investigated the mitogen-induced proliferation, interferon-gamma (IFN-gamma) and growth factor production by cells isolated from spleen, mesenteric and peripheral (axillary, brachial and inguinal) lymph nodes (LN). We found that stimulation with concanavalin A or staphylococcus enterotoxin B induced IFN-gamma synthesis in spleen cells but not in LN cells. Proliferation and growth factor production were comparable in the three cell populations. The addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA), which is commonly used as a substitute for accessory cells, did not influence the IFN-gamma synthesis by LN cells. The growth factor production was, on the other hand, elevated by the addition of PMA. A high number of IFN-gamma-producing peripheral LN cells were obtained if they were stimulated in the presence of splenic adherent cells. The growth factor synthesis was marginally affected by the presence of these cells. Thus, splenic adherent cells provide a co-stimulatory signal to the T cell necessary for IFN-gamma synthesis.

Animals↗

PubMed1991

Antigen processing and presentation by small and large B cells.

We have investigated the ability of different cells from non-immunized mice of the BALB/c strain to present antigen to two ovalbumin-specific I-Ad-restricted T hybridomas. Lipopolysaccharide-activated B-cell blasts were found to be the most efficient antigen-presenting cells. Purified small and dense splenic B cells also stimulated the hybridomas, although not to the same extent as the activated blasts, but comparable to non-fractionated spleen cells. Glutaraldehyde-treated B cells failed to present antigen, whereas F(ab')2 anti-mouse IgM-treated B cells exhibited markedly increased ability to present antigen. Using flow cytometry, we further purified the resting B cells by sorting the small lymphocytes to ensure that the ability of these cells to activate the hybridomas was not due to contamination with large non-resting B cells. The sorted small B cells retained the ability of antigen presentation. Their resting state was confirmed by the fact that they did not incorporate [3H]-thymidine as shown by autoradiographic analysis.

Animals↗

PubMed1991

Membrane-incorporated immunoglobulin receptors increase the antigen-presenting ability of B cells.

Monoclonal antibodies specific for ovalbumin were conjugated to palmitate and inserted into the membrane of normal spleen B cells. Their presence in the membrane, as well as their ability to bind ovalbumin, was established by immunofluorescence. The so called anti-ovalbumin-'decorated' B cells were tested for their ability to act as antigen-presenting cells for ovalbumin-specific I-Ad-restricted T-cell hybridomas. It was found that the antibody-decorated B cells presented antigen more efficiently than non-decorated B cells.

Animals↗

PubMed1991

The influence of T cells on the immunoglobulin repertoire and the affinity maturation of the immune response against dextran B512 in C57BL/6 mice.

A collection of hybridomas from C57BL/6 mice producing antibodies to dextran B512 was analysed and found to reflect the immune response in vivo with regard to immunoglobulin class expression, T cell dependency and antibody affinity. IgM-, IgG-3, and IgG-2b, and IgA-producing hybridomas were found. IgG3-producing hybridomas were obtained from nude mice, indicating T cell independent IgG3 synthesis. All monoclonal antibodies were of kappa light chain. A major anti-dextran idiotype was expressed in many monoclonals. Secondary immune responses to dextran were also suppressed at the hybridoma cell level. However, hybridomas from secondary responses produced antibodies expressing the major idiotype, suggesting that anti-idiotype mediated suppression was not responsible for the reduced secondary response. Most monoclonals belonged to the VHJ558 family, but the IgG3-producing hybridomas showed a preferential use of genes from the VHX24 family. All monoclonals were directed against internal structures of the dextran molecule. The affinity for dextran of the IgG antibodies produced in secondary immune responses was drastically increased, even when the mice were immunized with thymus-independent forms of dextran, indicating that T helper cells need not be involved in affinity maturation of the immune response.

Animals↗

PubMed1991

The immunoglobulin receptors on B cells bind antigen, focus activation signals to them and initiate antigen-presentation.

We do not agree with the analysis of Langman and Cohn on the function of Ig receptors. We have reviewed the available literature regarding anti-Ig activation of B cells and found it contradictory and unconvincing. We have presented experimental evidence on the inability of Ig receptors on B cells to mediate activation or tolerogenic signals. We suggest that the Ig receptors serve to focus antigen to specific B cells so the B cells can be activated by TI antigens or helper T cells. The Ig molecules also bind foreign antigen and thereby initiate internalization and antigen processing. The processed peptides are exported to the membrane, where they associate with MHC class II antigens, thus transforming B cells into efficient antigen-presenting cells.

Animals↗

PubMed1991

The effects of interleukins 4 and 5 on the differentiation of B cells from (NZB x NZW)F1 mice.

We describe here that IL-4 and IL-5 together can induce the production of high amounts of IgG1 polyclonal antibodies by B cells from old (NZB x NZW)F1 mice even in the absence of LPS. The effect was less marked in young mice from the same strain or in normal Balb/c mice. The cells sensitive to the treatment with the interleukins were the large B cells. Old mice had higher proportions of such cells than young mice or normal Balb/c mice. Synthesis of anti-DNA antibodies of the IgM class was induced. Although IL-4 plus IL-5 induced a strong polyclonal IgG1 production, there were no IgG1 anti-DNA antibodies.

Animals↗

PubMed1990

Control of mucin synthesis: the peptide portion of synthetic O-glycopeptide substrates influences the activity of O-glycan core 1 UDPgalactose:N-acetyl-alpha-galactosaminyl-R beta 3-galactosyltransferase.

Synthetic O-glycopeptides containing one or two GalNAc residues attached to Ser or Thr were used as substrates to investigate the effect of peptide structure on the activity of crude preparations of UDP-Gal:GalNAc alpha-R beta 3-Gal-transferase from pig stomach and pig and rat colonic mucosa and of a partially purified enzyme preparation from rat liver. High-performance liquid chromatography used to separate enzyme products revealed that uncharged glycopeptides with an acetyl group at the amino-terminal end and a tertiary butyl or an amide group at the carboxy-terminal end were resistant to proteolysis in crude preparations. The activity of beta 3-Gal-transferase varied with the sequence and length of the peptide portion of the substrate, the presence of protecting groups, the attachment site of GalNAc, and the number of GalNAc residues in the substrate. The presence and position of Pro had little effect on enzyme activity; ionizing groups near the GalNAc unit interfered with enzyme activity. Since the GalNAc-Thr moieties in many of these O-glycopeptides have been shown to assume similar rigid conformations, the variation in enzyme activity indicates that the beta 3-Gal-transferase recognizes both the peptide and carbohydrate moieties of the substrate. Rat and pig colonic mucosal homogenates contain beta 3- and beta 6-GlcNAc-transferases that synthesize respectively O-glycan core 3 (GlcNAc beta 3GalNAc alpha-R) and core 4 [GlcNAc beta 6(GlcNAc beta 3)GalNAc alpha-R]. These enzymes also showed variations in activity with different peptide structures; these effects did not parallel those observed with beta 3-Gal-transferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗