Biomedical subjects

K E Magnusson

Publications and source records attributed to K E Magnusson.

At least 37 records · Page 2Linked to original sources

PubMed1999

Localization of the insulin receptor in caveolae of adipocyte plasma membrane.

The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.

3T3 Cells↗

PubMed1998

Organization and mobility of CD11b/CD18 and targeting of superoxide on the surface of degranulated human neutrophils.

A monoclonal IgM, specifically recognizing both CD11b and CD18 of human neutrophils, was used to examine the organization and mobility of CD11b/CD18 in the plasma membrane of human neutrophils degranulated by dihydrocytochalasin B (dhCB) treatment and fMet-Leu-Phe (fMLF) stimulation. Subcellular fractionation analysis of untreated or dhCB-treated control neutrophils indicated that 20% of CD11b/CD18 cosedimented with plasma membrane and the remainder with specific granules. In contrast, fMLF stimulation of dhCB-treated cells caused a major reorganization of CD11b/CD18, in which 60-70% of CD11b/CD18 sedimented in dense plasma membrane fractions that were also enriched in superoxide-generating NADPH oxidase activity. Similarly pretreated neutrophils were fixed, immunogold labeled, and examined by scanning electron microscopy. Immunogold particles were distributed uniformly over the symmetrically ruffled surface of unstimulated neutrophils. On dhCB-treated cells, immunogold was mostly uniformly distributed on a smooth membrane with a small percentage of particles lining up into linear arrays. After fMLF + dhCB stimulation, CD11b/CD18 gold label was more abundant on the cell surface and formed large aggregates on polarized membrane protrusions. However, when cells were adhered to an albumin-coated quartz surface and stimulated with fMLF in the presence of dhCB, immunogold was excluded on the articulated and rounded cell body but concentrated on the periphery of adherent lamellae. Fluorescence photobleaching recovery indicated that in unstimulated cells 38 +/- 3% of CD11b/CD18 was mobile (R) with a diffusion constant D of 3.1 +/- 0.3 x 10(-10) cm2/s. Treatment with dhCB raised R and D 24 and 74%, respectively. Stimulation using 1 microM fMLF with dhCB lowered D and R to near control levels. Since NADPH oxidase and CD11b/CD18 cosediment in high-density plasma membrane domains after fMLF + dhCB stimulation, we speculate that a stimulus-induced reorganization of CD11b/CD18 and NADPH oxidase to common membrane domains may occur in fMLF + dhCB-degranulated neutrophils.

CD18 Antigens↗

PubMed1998

Endogenous nitric oxide in MDCK-I cells modulates the Vibrio cholerae haemagglutinin/protease (HA/P)-mediated cytotoxicity.

Previously, we have shown that the Vibrio cholerae haemagglutinin/protease (HA/P) accounts for significant remaining toxicity of CVD110, an attenuated V. cholerae 01 El Tor live oral vaccine-strain. The present report demonstrates that endogenous nitric oxide (NO) production modulates HA/P-mediated cytotoxicity in Madin-Darby canine kidney cell strain I (MDCK-I) epithelial cells. The basal levels of endogenous NO suppressed the cytotoxicity of HA/P, whereas inhibition of NO production with nitro-L-arginine methyl-ester (L-NAME) made the MDCK-I cells susceptible even to low concentrations of the cytotoxin. The inhibition of NO production caused a reinforcement of the HA/P- mediated distortion of a tight junction-associated protein ZO-1 and increment of filamentous actin at the apical and the lateral membrane domains. The mechanism by which NO exerts its modulatory action is not likely to be from its direct interaction with the zinc-containing catalytic domain of HA/P, since two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetyl-D, L-penicillamine (SNAP), did not affect the proteolytic activity of HA/P. In conclusion, the endogenous NO in the MDCK-I cells has a modulating effect on the cytotoxicity of HA/P and thus protects the cells against the cytotoxin.

Animals↗

PubMed1998

The role of glutamine, serum and energy factors in growth of enterocyte-like cell lines.

BACKGROUND: Glutamine is routinely added to most cell cultures. Glutamine has been found to be the preferential nutrient to the rapidly replicating intestinal mucosa, but whether this is a metabolic effect or due to other properties of this amino acid is not determined. To study the importance of glutamine on the growth of two enterocyte-like cell lines, the effects of depriving the media or supplementing it with glutamine were assessed in media with different serum and energy supplements. METHODS: CaCo-2 and HT-29 cells were grown in serum-free medium, with fetal bovine or synthetic serum, and with or without glucose or galactose. The glutamine content was varied between 0 and 4 mM. All growth assays were performed in triplicate by counting in a hemocytometer. RESULTS: Both cell lines were dependent of serum factors for growth, but displayed distinct requirements on glutamine supplementation. Glutamine was an obligate supplement with dose-dependent correlation to growth (r = 0.87, p < 0.01) for CaCo-2 cells cultured in synthetic, but not in fetal bovine serum. In HT-29 cells, the correlation between glutamine and growth was significant (r = 0.68, p < 0.05) only in fetal bovine serum in the absence of galactose. CONCLUSION: This study shows that glutamine has different growth stimulating effects on two enterocyte-like cell lines studied. This could reflect different modes of action of glutamine on proliferation and differentiation in an enterocyte cell population.

Animals↗

PubMed1998

Children with celiac disease express inducible nitric oxide synthase in the small intestine during gluten challenge.

BACKGROUND: Childhood celiac disease in Sweden is presently seen at an incidence of around 1/250 and is thus one of the commonest chronic diseases in children. It has recently been shown that children with untreated celiac disease have increased levels of nitrate/nitrite in the urine, most likely reflecting an increased production of nitric oxide in the inflamed mucosa. Nitric oxide is produced from L-arginine by an inducible or a constitutive nitric oxide synthase. The inducible nitric oxide synthase (iNOS) can be stimulated in various cells by, for instance, inflammatory mediators. The present study has been done to find a possible source of nitric oxide in the small intestine that could result in the increased levels of nitrate/nitrite in the urine in children with active celiac disease. METHODS: Small-intestinal biopsy specimens from children with active celiac disease were labeled with rabbit-anti-human antibodies to iNOS and visualized with fluorescent pig anti-rabbit antibodies. The specimens were then analyzed with confocal microscopy to assess the labeling pattern. RESULTS: In all of seven specimens from children with increased levels of nitrate/nitrite in the urine, we detected antibodies to iNOS, whereas in five of six control specimens--that is, from children with normal nitrate/nitrite levels--we could not detect any iNOS. CONCLUSIONS: Children with active celiac disease have a gluten-induced nitric oxide production in the small intestine reflected by increased urine levels of nitrate/nitrite and iNOS expression in the intestine. We conclude that the increased production of nitric oxide could presumably, directly or indirectly, result in injury of the small-intestinal tissue.

Adolescent↗

PubMed1998

Significantly increased levels of nitric oxide products in urine of children with celiac disease.

BACKGROUND: Celiac disease is characterized by morphologic and functional aberrations of the small intestinal mucosa, i.e., crypt hyperplasia, villous atrophy, infiltration of intraepithelial lymphocytes, and alteration of permeability. Nitric oxide has been shown to affect mucosal permeability after ischemia-reperfusion, but little is known about the regulatory role of nitric oxide in celiac disease. The purpose of this study was to assess nitric oxide production in children with celiac disease and in control subjects. METHODS: The sum of nitrite and nitrate in the urine was measured with a colorimetric method in 137 children with a median age of 3 years, 84 patients and 53 reference children, all of whom underwent a small intestinal biopsy to confirm or overrule suspicion of celiac disease. RESULTS: Median urinary nitrite-nitrate concentration in celiac children was 3323 microM (4147 +/- 1102; mean +/- SEM) at first clinical examination and 2501 microM (2939 +/- 386) after gluten challenge, which was significantly higher than concentrations in reference children (1029 microM; 1174 +/- 116) and in children with celiac disease on a gluten-free diet (882 microM; 1369 +/- 360) (p < 0.0001). CONCLUSIONS: A gluten-containing diet is associated with an increased nitrite-nitrate secretion in the urine in children with celiac disease, presumably as a result of nitric oxide synthase activation and nitric oxide production in the diseased small intestinal mucosa.

Adolescent↗

PubMed1997

Aggregation of PDGF-beta receptors in human skin fibroblasts: characterization by image correlation spectroscopy (ICS).

Receptor aggregation is believed to be an important, early step when growth factors such as PDGF stimulate proliferation and differentiation of cell populations. To investigate receptor aggregation, we utilized a novel biophysical technique, image correlation spectroscopy, to study the distribution and aggregation state of PDGF-beta receptors on the surface of human dermal fibroblasts under various experimental conditions. It was found that the cell surface receptors were pre-clustered at 4 degrees C and receptor aggregation increased for samples measured at 37 degrees C. Treatment with PDGF-BB had no measurable effect on the receptor aggregation state. The results also indicate that additions of 10% serum or an inhibitor of tyrosine kinase activity, may disperse the receptors. The results of this study are consistent with organization of PDGF-beta receptors in pre-existing membrane domains.

Becaplermin↗

PubMed1997

Role of annexins in endocytosis of antigens in immature human dendritic cells.

We have evaluated the uptake of a soluble protein antigen, denitrophenylated human serum albumin (DNP-HSA), and two different intracellular bacteria; Chlamydia trachomatis serovar L2 and Mycobacterium tuberculosis strain H37Ra, by immature human dendritic cells. These were generated by culturing progenitor cells from blood in the presence of cytokines (granulocyte-macrophage colony-stimulating factor and interleukin-4). Dendritic cells play a crucial part in antigen presentation for the induction of T-cell-dependent immune responses in various tissues. Recently, macropinocytic and phagocytic activity has been shown for immature dendritic cells of mouse, rat and human origin. In the present study, macropinocytosis characterized the uptake of the soluble protein-antigen DNP-HSA, whereas the C. trachomatis were ingested via receptor-mediated endocytosis in coated pits, and opsonized M. tuberculosis via phagocytosis. To follow the intracellular routes of the antigens, their positions were compared with the localization of annexins, a family of Ca(2+)-and phospholipid-binding proteins, involved in membrane fusion, aggregation and transport of different vesicles. To elucidate further the intracellular pathway of the antigens, two other proteins, lysosome-associated membrane protein-1 (LAMP-1) and cathepsin D, were labelled. They are known to colocalize with major histocompatibility complex class II compartments in the immature dendritic cells. We observed a distinct translocation of annexin V to DNP-HSA containing endosomes, and annexin III to vesicles with C. trachomatis. Furthermore, annexin III, IV and V redistributed to phagosomes with M. tuberculosis. Both LAMP-1 and cathepsin D colocalized with DNP-HSA endosomes, and with phagosomes with M. tuberculosis. Thus, immature human dendritic cells have the capacity to phagocytose. Moreover, the handling of these antigens by dendritic cells may represent three distinct intracellular pathways, albeit some properties and compartments are shared.

Annexins↗

PubMed1997

YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane.

Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia, the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK-mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK-mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.

Animals↗

PubMed1997

Oral immunoglobulin treatment in Crohn's disease.

Two patients with severe Crohn's disease who failed to respond to traditional medical treatment were given immunoglobulin orally. We used the 6-h urinary recovery of mixture of polyethyleneglycols PEG 400 and 1000 (molecular weight range 282-1250 Da) to test the intestinal permeability. Both patients showed a similar permeability pattern with an increased leakage of large-sized PEGs before treatment and a lower urinary recovery of probes after treatment with immunoglobulin. This might indicate that the inflammatory process in Crohn's disease can be affected from the luminal side of the mucosa.

Administration, Oral↗

PubMed1996

Modulation of virulence factor expression by pathogen target cell contact.

Upon contact with the eukaryotic cell, Yersinia pseudotuberculosis increased the rate of transcription of virulence genes (yop), as determined by in situ monitoring of light emission from individual bacteria expressing luciferase under the control of the yopE promoter. The microbe-host interaction triggered export of LcrQ, a negative regulator of Yop expression, via the Yop-type III secretion system. The intracellular concentration of LcrQ was thereby lowered, resulting in increased expression of Yops. These results suggest a key role for the type III secretion system of pathogenic bacteria to coordinate secretion with expression of virulence factors after physical contact with the target cell.

Bacterial Adhesion↗

PubMed1996

In vitro effects of ethanol on polymorphonuclear leukocyte membrane receptor expression and mobility.

The hampered inflammation and host defense seen in alcoholics may be due to impairment of functional responses of neutrophil polymorphonuclear leukocytes (PMN). We have shown that ethanol inhibits the oxidative metabolism of PMN induced by surface receptor dependent stimuli, such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and opsonized zymosan. Because the unresponsiveness might be due to reduced numbers of surface receptors, we assessed the expression of CR1, Fc-gamma, and fMLP receptors as well as membrane fluidity after treatment of PMN with ethanol in vitro. Ethanol impaired the induced expression of CR1 and fMLP receptors to 71% and 51% of control, respectively, but did not affect the resting level of CR1 nor Fc-gamma receptor expression. Furthermore, the mobility of cell membrane glycoconjugates was increased by ethanol. However, phagocytosis, a functional response dependent on membrane rheology, was unaffected. Because the results indicated an effect of ethanol on mobilization of receptors from intracellular stores, we assessed lactoferrin release, which was reduced to 59%. Thus, ethanol appeared to hamper the upregulation of PMN surface receptors or functional subsets of those stored in granules. Ethanol also increased the mobility of the cell membrane. These reactions were accompanied by reductions in the functional responses mediated by either class of receptors.

Cells, Cultured↗

PubMed1996

Liver cell uptake and degradation of soluble immunoglobulin G immune complexes in vivo and in vitro in rats.

Immune complexes were formed between dinitrophenylated human serum albumin (DNP-HSA) and polyclonal rabbit immunoglobulin G (IgG) anti-DNP antibodies at antibody excess. The antigen was labelled with isotope (125I-tyramine-cellobiose) or fluorochrome, (6-[fluorescein-5-(and-6)-carboxamido] hexanoic-acid, succinimidyl ester). The radiolabelled antigen, native or antibody complexed, was given intravenously to rats. Radioactivity was measured in various organs at 1 hour following injection. The liver was the main site for removal of the antigen as well as of the immune complexes. Within the liver, immune complexes were mainly associated with nonparenchymal liver cells, the total recovery from Kupffer cells being about 10 times greater than from the liver endothelial cells. The uncomplexed radiolabelled antigen was readily degraded by both cells types. After IgG complexing, the degradation decreased, both in Kupffer cells and in liver endothelial cells. In vitro experiments with isolated liver cells, showed that IgG complexing increased antigen uptake to about the same extent in Kupffer cells and in liver endothelial cells. The degradation of both antigen and immune complexes was less efficient in vitro than in vivo. Immune complex uptake in vitro was shown also by confocal fluorescence microscopy in Kupffer cells and in liver endothelial cells. Also in vitro, only minor uptake was found in the hepatocytes. We conclude that both liver endothelial cells and Kupffer cells are involved in the hepatic handling of soluble IgG immune complexes, but we found no evidence for substantial uptake by hepatocytes.

Animals↗

PubMed1996

Vibrio cholerae hemagglutinin/protease (HA/protease) causes morphological changes in cultured epithelial cells and perturbs their paracellular barrier function.

In this report, we describe the cytotoxic activity of the cholera hemagglutinin/protease (HA/protease). A concentrated protein sample from the 37 degrees C overnight culture supernatant of CVD110, a delta ctxA, delta zot, delta Ace and hlyA::(ctxB mer) mutant of El Tor biotype Ogawa serotype strain E7946 caused morphological changes in cultured MDCK-I epithelial cells and altered their arrangement of filamentous actin (F-actin) and Zonula occludens-associated protein ZO-1. The drastic morphological changes can be inhibited by Zincov, a specific bacterial metalloprotease inhibitor. The cytotoxic fractions of the sample after FPLC gelfiltration fractionation showed two visible protein bands with molecular weights of approximately 34- and 32 kDa. Microsequencing of these two proteins revealed that they were the cholera HA/protease.

Actins↗

PubMed1996

The N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) increases the lateral diffusion of complement receptor 1 (CR1/CD35) in human neutrophils; a causative role for oxidative metabolites?

The effects of the N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) on the lateral mobility of the complement receptor type 1 (CR1/CD35) in glass-adherent human neutrophils were investigated, using fluorescence recovery after photobleaching (FRAP) and confocal microscopy (CSLM). It was found that addition of 0.1-1 microM fMLF increased the diffusion constant (D) of CR1/CD35 to 167-228% of controls. No effect was observed on the receptor distribution or the mobile fraction of receptors. The effect of fMLF on the lateral diffusion of CR1/CD35 could be totally inhibited by addition of pertussis toxon (PD, 250 ng/ml) or of the free radical scavenger enzymes superoxide dismutase (SOD, 2000 U/ml) and catalase (CAT, 200 U/ml), added together the results show that oxidative metabolites produced by neutrophils in response to fMLF can modulate CR1/CD35 diffusion, and indicate a regulatory role for oxygen radicals in phagocytosis.

Catalase↗

PubMed1996

Studies with wortmannin and cytochalasins suggest a pivotal role of phosphatidylinositols in the regulation of tight junction integrity.

Wortmannin, a selective inhibitor of phosphatidylinositol 3-kinase (P13K), was found to give a dose and time-dependent, bimodal effect-initial increase, followed by decrease on the tight junction integrity of MDCK-1 monolayers, as assessed by electrical resistance measurement of the epithelia. Moreover, dihydrocytochalasin B inhibited the wortmannin-induced alteration, whereas cytochalasin B had a negligible influence on the wortmannin effect. Wortmannin was also found to cause changes in the cytoskeleton structure. These alterations were also seen when wortmannin was combined with cytochalasin B. However, in accordance with the electrical resistance measurements, dihydrocytochalasin B was able to abolish wortmannin-induced filamentous (F-) actin changes. These findings suggest that the P13K, phosphatidylinositols, and filamentous actin rearrangements, in combination, play an important role in the modulation of the junctional integrity.

Androstadienes↗

PubMed1996

Modulation of the junctional integrity by low or high concentrations of cytochalasin B and dihydrocytochalasin B is associated with distinct changes in F-actin and ZO-1.

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2-1.2 microM) and higher concentrations (1.5 microM and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain 1 (MDCK-1) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 microgram/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.

Actins↗

PubMed1996

YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signalling associated with phagocytosis.

The PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild-type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild-type yopH restored the anti-phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopHC403A was without effect. The PTPase inhibitor orthovanadate impaired the anti-phagocytic effect of the wild-type strain, further demonstrating the importance of bacteria-derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild-type Y. pseudotuberculosis caused a YopH-dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface-located protein invasin. Co-infection with the phagocytosable strain and the wild-type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH-mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis-linked signalling in the phagocyte.

Animals↗