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Biomedical subjects

W Völker

Publications and source records attributed to W Völker.

At least 19 recordsLinked to original sources

Staphylococcus aureus induces caspase-independent cell death in human peritoneal mesothelial cells.

Bacterial peritonitis remains a serious complication of peritoneal dialysis. Although Staphylococcus epidermidis is the most common pathogen involved, infections with Staphylococcus aureus lead to severe peritoneal damage and are often associated with a dramatic loss of mesothelial cells. Induction of cell death appears to be involved in peritoneal damage and mesothelial cell loss during bacterial infections. Using cultured human peritoneal mesothelial cells (HMCs), we investigated the ability of different S. epidermidis and S. aureus strains to damage the HMC monolayer and to trigger cell death. We show that only a subgroup of live S. aureus isolates, characterized by an invasive and alpha-hemolysin-producing phenotype, induces cell death. None of the tested S. epidermidis strains, which were not invasive or hemolytic, had a cytotoxic effect. After host cell invasion, S. aureus resided within phagocytic vacuoles, and HMCs were apparently able to degrade staphylococci. However, even after prolonged infection, a high percentage of S. aureus remained alive within HMCs and might be released after host cell death. Cell death induced by S. aureus was accompanied by apoptotic alterations, such as DNA fragmentation, but was independent of endogenous FasL and tumor necrosis factor-alpha death ligand expression. Moreover, caspases were not involved in S. aureus-induced mesothelial cell death. In conclusion, our data indicate that mesothelial cell death might represent a major mechanism of S. aureus-induced damage of the peritoneum during bacterial peritonitis.

Apoptosis↗

Generalized arteriopathy in patients with cervical artery dissection.

OBJECTIVE: To make an ultrastructural comparison of superficial temporal artery (STA) biopsy specimens from patients with spontaneous cervical artery dissection (sCAD) and controls. METHODS: The authors used light microscopic examination of semithin sections and electron microscopic examination of ultrathin sections of STA biopsy specimens from patients with sCAD and controls. RESULTS: STA biopsy specimens from patients with sCAD taken around the time of the dissection showed a zone of connective tissue weakening with fissuring at the junction between the tunica media (TM) and the tunica adventitia (TA) in seven of nine specimens and erythrocyte infiltration in eight of nine specimens but in none of the control specimens. Light microscopy demonstrated transparent circular spots that, on electron microscopy, turned out to represent erythrocytes and other cellular components at different stages of degradation. Occasionally, scattered immune cells were found in specimens from patients with sCAD. In addition, smooth muscle cells of the synthetic phenotype, some of them showing extensive vacuolation were more common in the TM of STA biopsy specimens from patients with sCAD than in control specimens. CONCLUSIONS: Signs of tissue weakening along the TM/TA junction in STA biopsy specimens of patients with sCAD but not in controls suggest the presence of a generalized arteriopathy leading to impairment of the stability of the arterial wall in patients with sCAD. Limiting factors of the study are that some control biopsies were obtained from autopsies and that the anticoagulation status of patients and controls were not completely comparable.

Adult↗

Characterization of glycosaminoglycans during tooth development and mineralization in the axolotl, Ambystoma mexicanum.

Glycosaminoglycans (GAGs) involved in the formation of the teeth of Ambystoma mexicanum were located and characterized with the cuprolinic blue (CB) staining method and transmission electron microscopy (TEM). Glycosaminoglycan-cuprolinic blue precipitates (GAGCB) were found in different compartments of the mineralizing tissue. Various populations of elongated GAGCB could be discriminated both according to their size and their preferential distribution in the extracellular matrix (ECM). GAGCB populations that differ in their composition could be attributed not only to the compartments of the ECM but also to different zones and to different tooth types (early-larval and transformed). Larger precipitates were only observed within the dentine matrix of the shaft of the early-larval tooth. The composition of the populations differed significantly between the regions of the transformed tooth: pedicel, shaft and dividing zone. In later stages of tooth formation, small-sized GAGCBs were seen as intracellular deposits in the ameloblasts. It is concluded that the composition of GAGCB populations seems to play a role in the mineralization processes during tooth development in A. mexicanum and influence qualitative characteristics of the mineral in different tooth types and zones, and it is suggested that GAGs might be resorbed by the enamel epithelium during the late phase of enamel formation.

Ambystoma mexicanum↗

Atherosclerosis in apolipoprotein E-deficient mice is decreased by the suppression of endogenous sex hormones.

To elucidate the influence of gonadotropins, endogenous sex hormones and testosterone on atherosclerosis, 4-week-old male and female apoE-deficient mice received either 100 microg subcutaneous injections of the gonadotropin-releasing hormone (GnRH) antagonist Cetrorelix every 48 hours or a subcutaneous implantation of a permeable silastic tube with 35 mg of testosterone. Control mice received either subcutaneous injections of saline, a silastic implant with saline, or no treatment. The animals were sacrificed after eight weeks of treatment; blood was obtained by cardiac puncture and the aorta was taken out and prepared. The suppression of testosterone led to an increase in atherosclerosis in both the sinus aortae and the ascending aorta despite increases of cholesterol in male and decreases of HDL cholesterol in female mice. Treatment with testosterone led to small but significant increases of cholesterol levels and atherosclerotic lesions in male mice. Female mice showed no change in lipids and fewer atherosclerotic lesions. In conclusion, the suppression of gonadotropins appears to have a moderate anti-atherogenic effect. The effect of testosterone appears to be either neutral or opposed by gonadotropins.

Animals↗

In vitro modelling of tissue using isolated vascular cells on a synthetic collagen matrix as a substitute for heart valves.

BACKGROUND: Tissue engineering is a promising approach for obtaining lifetime durability in biological heart valves. Basic questions with respect to the selection of suitable cell populations as well as scaffolds remain unsolved. The purpose of this study was to develop a tissue-like substitute in vitro for replacement of diseased valves in vivo. METHODS: Smooth-muscle cells (SMCs) were isolated from human and porcine aortic tissue using the 'explant technique' and endothelial cells from collagenase digestion. Seeding and cultivation of isolated cells was performed on a type-I collagen matrix. The scaffold-cell specimen was investigated using light and electron microscopy. Cupromeronic blue and immunoprecipitation were used for ultracytochemical staining. RESULTS: SMCs were allowed to grow to multilayers and migrate into the collagen network. We found a tissue-like morphology in these samples characterised by several layers of cells, spaces between the cell layers filled with newly formed extracellular matrix components, compartmentalisation of proteoglycans and their association with fibrilar matrix and the cell surface. Endothelium cells covered the SMCs of the scaffold with a histological topography similar to heart valves. CONCLUSIONS: This is an approach for in vitro modelling of tissue-like substitutes and preparing plane multicellular tissues as substitutes for heart valves. This model may also be used for cell biological investigations of cell-matrix interactions.

Animals↗

Effects of tibolone on the endometrium.

OBJECTIVE: To investigate the effects of 24 months of tibolone treatment on the endometrium in postmenopausal women. DESIGN: An open, prospective, multicenter study with a treatment duration of 24 months. POPULATION: One hundred and fifty healthy postmenopausal women. METHODS: Women were treated with one tablet of tibolone (2.5 mg Org OD14) daily. Endometrial biopsies were taken at baseline, 12 months (n = 112) and 24 months (n = 62) to investigate endometrial histology by means of light microscopy. RESULTS: No stimulation of atrophic endometrium was observed in 98.2% and 91.9% of the analyzed women after 12 and 24 months, respectively. A change from an atrophic endometrial pattern to a weakly proliferative pattern was seen in 1.8% (95% confidence interval (CI) 0.22-6.45) and 6.5% (95% CI 1.20-7.51) of the women after 12 and 24 months, respectively. In one woman, the endometrial pattern after 24 months was classified as simple hyperplasia (1.6%; 95% CI 0.01-3.20). The low incidence of proliferation and/or hyperplasia corresponds to that seen in other clinical trials in which women have been treated with placebo. Vaginal bleeding and/or spotting was reported by 18 women (12%), all showing an atrophic endometrium at all assessments, except for one woman with a proliferative endometrium at 24 months. CONCLUSION: In the majority of women (92%), the endometrium remained atrophic during 24 months of tibolone treatment.

Adult↗

[(23)sodium MRI for infarct imaging of the human heart].

PURPOSE: Sodium is elevated in acute/subacute myocardial infarction due to three distinct mechanisms: Breakdown of ion homeostasis with accumulation of intracellular sodium, extracellular edema formation and, during scar formation, increase of extracellular vs. intracellular space as cardiomyocytes are replaced by connective tissue. 23Na MRI has previously been shown to have the potential to demonstrate myocardial infarction in an animal model. Aim of this study was, therefore, to demonstrate myocardial infarction with the use of 23Na-MRI in patients. MATERIAL AND METHODS: 10 patients were examined 14 +/- 7 days after first myocardial infarction using a 23Na surface coil at 1.5 T. Double angulated short axis images of the entire heart were imaged using an ECG-triggered 3d-FLASH-sequence (FOV, 450 mm; matrix, 64 x 128; spatial resolution, 3.5 x 7 mm2; slice thickness, 16 mm; 32 acquisitions). Areas of elevated sodium signal intensity were correlated with infarct-related wall motion abnormalities imaged by Cine MRI in breathhold-technique. RESULTS: All patients showed an area of elevated sodium signal intensity that correlated well with the clinically determined localization of myocardial infarction as well as with regional wall motion abnormalities detected by Cine MRI. CONCLUSIONS: Elevated 23Na MR image signal intensity demonstrates subacute myocardial infarction in patients.

Aged↗

Differentiation of coronary smooth muscle cells to a cell cycle-arrested hypertrophic growth status by a synthetic non-toxic heparin-mimicking compound.

Studies on the mode of action of basic fibroblast growth factor (bFGF) identified an essential role of heparan sulfate and heparin-like molecules in the formation of distinct bFGF-heparan sulfate-bFGF-receptor complexes that are required for bFGF-induced signal transduction. In coronary smooth muscle cells that express 6-8 ng bFGF mg(-1) cell protein, the heparan sulfate chains of membrane-associated proteoheparan sulfate are implicated in bFGF signaling and thus are involved in the regulation of proliferation and differentiation of vascular smooth muscle cells. We studied the mode of action of a synthetic non-sulfated heparin-mimicking compound termed RG-13,577 (poly-4-hydroxyphenoxy acetic acid, Mr approximately 5 kD) and found a dose-dependent antiproliferative effect that was characterized by a block of G(1)/S-phase transition indicated by a marked (80%) reduction of [3H]thymidine incorporation at a concentration of 5 microg ml(-1) RG-13,577. Cell cycle analysis showed a block of cell division in the G(1)-phase. In response to RG-13,577 the cells were converted into a hypertrophic growth status within 72 h as judged from a doubling of the cellular protein content and measurement of cell and nucleus size. The increased cell protein content resulted from a de novo synthesis and was also associated with an increase in the incorporation of [35S]sulfate into cell-associated proteoglycans, including the proteoheparan sulfate coreceptor of bFGF. In contrast, the compound-induced G(1)-phase arrest was associated with an extensive downregulation of the cellular and pericellular bFGF level. The reduced bFGF content was accompanied by downregulation of the bFGF signaling-involved protein kinase C-alpha and MAP kinase, abrogation of MAP kinase phosphorylation and overexpression of protein kinase C-gamma. RG-13,577 failed to elicit apoptotic reactions at a concentration range of 0.5-10 microg ml(-1) and its effect was reversible upon removal of the compound. It appears that RG-13,577 induces a phenotype transformation of coronary SMC into a metabolically active hypertrophic status that could promote repair processes after balloon angioplasty (PTCA) without stimulating cell proliferation. Development of non-toxic polyanionic compounds may provide an effective strategy to inhibit cell proliferation associated with restenosis following balloon angioplasty and coronary artery bypass surgery.

Animals↗

Cholesterol-induced changes of type VIII collagen expression and distribution in carotid arteries of rabbit.

Lipoproteins play a major role in cardiovascular disease and atherosclerosis. In the vascular wall, they strongly influence the organization of extracellular matrix. The present study set out to investigate the changes in the extracellular matrix of the vessel wall induced by atherogenic diet, focusing on type VIII collagen, a vascular collagen that has not previously been investigated in detail. The influence of cholesterol diet on the expression, distribution, and deposition of type VIII collagen was examined in carotid arteries of New Zealand White rabbits. Carotid arteries of rabbits receiving diet supplemented with 1% cholesterol for 6 weeks and those on the same regimen followed by normal chow for 1 day, 10 days, 5 weeks, and 12 weeks were studied and compared with controls not exposed to the cholesterol diet. Carotid arteries of normocholesterolemic rabbits contained type VIII collagen-expressing cells in all layers, with focal accumulations of expressing cells in the subendothelial areas, the outer medial zone, and the adventitia. In response to cholesterol diet, type VIII collagen synthesis was reduced in media and adventitia and the distribution patterns changed. Expressing cells were found predominantly in the endothelium, and type VIII collagen accumulated in the intimal space. Immunogold labeling for electron microscopy revealed that type VIII collagen in the intima is associated with microfibrils extending from the internal elastic lamina. Withdrawal of cholesterol resulted in reestablishment of the normal distribution pattern. Northern and Western blot analyses supported the immunoconfocal and in situ hybridization data, demonstrating decreased type VIII collagen expression in response to cholesterol diet and progressive recovery to normal levels with time after withdrawal of cholesterol. Our study demonstrates that type VIII collagen is modulated in the presence of cholesterol. The data indicate that type VIII collagen is specifically remodeled during early experimental atherosclerosis, implying a role for this extracellular matrix component in neointimal growth.

Animals↗

Copper-induced inflammatory reactions of rat carotid arteries mimic restenosis/arteriosclerosis-like neointima formation.

The pathogenesis of arteriosclerosis and of restenosis after angioplasty is linked with an inflammatory and fibroproliferative response of the arterial tissue. We have induced a non-infectious inflammation by implanting a silicon-copper cuff around rat carotid arteries. The copper ions released from the oxidized copper initiate and mimic all morphological features of post-angioplasty restenotic and arteriosclerotic lesions. The copper-induced lesions were analyzed by electron and light microscopy, immunohistochemical methods and quantified by morphometry. During the first phase of copper-induced tissue reaction (3 days), macrophages and polymorphonuclear leucocytes invaded through the endothelium, accumulated in the subendothelial space and triggered the proliferation of smooth muscle cells which then migrated from the tunica media through the lamina elastica interna into the intima. Within 3 weeks, the accumulated smooth muscle cells, macrophages, leucocytes and newly synthesized extracellular matrix formed a circular mostly eccentric fibrotic thickening that narrows the vessel lumen by 30-40%. The accompanying structural disorganization of the medial layer led to focal rupture and aneurysm-like dilatation of the vessel wall in 3 of 11 animals between day 20 and 43. The neointima progressively increased in thickness over time leading to corresponding reduction of the vessel lumen. The carotid arteries of control animals and animals treated with copper-free silicon cuffs showed no abnormal pathological appearance. Our results show that inflammation-inducing agents can contribute to and simulate restenosis- and arteriosclerosis-like lesions and that the copper-cuff model may be useful in the exploration of new approaches to intervention.

Angioplasty, Balloon↗

Perlecan is responsible for thrombospondin 1 binding on the cell surface of cultured porcine endothelial cells.

Thrombospondin 1 (TSP1), a high molecular weight glycoprotein of the extracellular matrix, interacts with glycosaminoglycan at the cell surface of porcine endothelial cells (Schön et al., Eur.J. Cell Biol. 59, 329-339 (1992)). In this study we identified and characterized the heparan sulfate proteoglycan (HSPG) responsible for TSP1 binding and uptake in endothelial cells and investigated some properties of the TSP1-proteoglycan interaction. Porcine endothelial cells synthesize proteoglycans containing heparan sulfate (HS) or chondroitin/dermatan sulfate (CS/DS). CS/DS-containing compounds are present predominantly in the culture medium. On Sepharose CL-4B the cellular proteoglycan fraction yielded two HS-containing compounds with a Kav = 0.18 and Kav = 0.55. Only the larger HS-containing component was sensitive to alkaline treatment and was also found in the medium fraction. Trypsin treatment of endothelial cells revealed that the large HS-containing component represents a cell surface-associated proteoglycan, whereas the smaller fraction represents a pool of intracellular HS-chains. The cellular HSPG is partially localized at the apical cell surface but also incorporated and tightly bound to the subendothelial matrix. Deglycosylation of the high molecular weight HSPG resulted in the identification of a core protein of about 400 kDa. Using specific antibodies, in ELISA assays and in immunoblot analysis we observed that the large HSPG is identical to the extracellular matrix proteoglycan, perlecan. Immunohistochemical studies confirmed the location of perlecan on the apical cell surface and additionally as a dense fibrillar network surrounding the cells. Purified perlecan bound to TSP1 in a dose-dependent manner and the binding was mediated by its glycosaminoglycan side chains. In competition assays using various sulfated polysaccharides, heparin potently inhibited binding of perlecan to TSP1 immobilized on nitrocellulose. Dermatan sulfate was a less effective inhibitor. Calcium bound to TSP1 was found to influence its capacity for binding perlecan. The present data provide evidence that perlecan is required for binding and concentrating TSP1 at the apical surface of vascular endothelial cells during receptor-mediated endocytosis.

Animals↗

Pyrogallol red-vanadium complex-a new stain for electron microscopy.

We report on the application of a pyrogallol red-vanadium complex (PR-V) for ultracytochemical staining of proteinaceous structures in animal tissues and cell cultures. This dye may be used as a general purpose stain in electron microscopy. In contrast to osmium tetroxide, the price of the material is low and no toxic vapors are produced. The PR-V complex was prepared by addition of vanadium (IV) oxide sulfate to pyrogallol red dissolved in acetate buffer (pH 5.6). The formation of the complex was indicated by a color change from purple-red (lambda max = 520 nm) to violet (lambda max = 539 nm) which occurred at equimolar concentrations of the dye and the metal salt. Under these conditions PR-V was stable for several days. The mechanism of PR-V binding was checked in dot blots using different proteins as well as heparin for control. While heparin remained unstained, proteins were stained in a dose-dependent manner. Deamination of proteins with nitric oxide strongly reduced PR-V staining in dot blots as well as in cell cultures. Optimal staining results of animal cells and tissues were obtained in specimens that had been mildly fixed for at least 1 h or longer with a mixture of 0.1% glutaraldehyde and 1.0% paraformaldehyde dissolved in phosphate-buffered saline, pH 7.2, washed with acetate buffer, pH 5.6, and subsequently treated with PR-V in the presence of 50% ethanol at room temperature. Control specimens without PR-V but treated en bloc with uranyl acetate or sodium molybdate showed similar contrast but less details in the ultrastructure of the tissue. All specimens were embedded in epoxy resin and ultratain sections were stained conventionally with uranyl and lead salt solutions. In electron micrographs, membrane-associated particles, stress fibers and filaments of the cell cortex, collagen fibrils, tight junctions and desmosomes, and other proteinaceous components were clearly visualized only in the PR-V-treated specimens. In conclusion, the ability to bind selectively and specifically to protein-aceous structures makes PR-V a versatile stain to study the localization and distribution of these structures in cells and tissues at the ultrastructural level.

Animals↗

Antibodies against proteins of streptococcal hyaluronate synthase bind to human fibroblasts and are present in patients with rheumatic fever.

Antibodies directed against the streptococcal 42 kDa hyaluronate synthase and a 56 kDa auxiliary protein bound to the surface of intact human fibroblasts in vitro. Staining was most prominent during the detachment phase of mitosis. In eukaryotic plasma membranes a 52 kDa protein was recognized by the antiserum against the 56 kDa streptococcal protein. Since the cross-reacting proteins could be involved in immunological mimicry between streptococcal and human antigens leading to heart cell necrosis, the reactivity of sera from patients with rheumatic fever was compared with that of sera from healthy or streptococcal infected persons. The sera from patients with rheumatic fever showed a higher reactivity against the 56 kDa protein than those from healthy persons or from patients with an antibiotic treated streptococcal infection. This difference was not observed for the 42 kDa protein. These sera were able to lead to cell lysis in the presence of complement.

Anti-Bacterial Agents↗

Inhibition of smooth muscle cell proliferation and neointimal growth by low-anticoagulant heparin.

Low-anticoagulant heparin (LA-heparin) obtained by affinity chromatography on antithrombin III Sepharose inhibits the proliferation of cultured arterial smooth muscle cells in an in vitro bioassay system as effectively as standard heparin. A growth inhibition of smooth muscle cells of about 60% is achieved when LA-heparin or heparin is added to the culture medium to a concentration of 50 micrograms/ml. In normolipemic rats LA-heparin suppresses the formation of neointimal thickenings and stenosis after balloon catheter-induced deendothelialization of the carotid artery. In terms of mass a dose of 5 mg/kg body weight/d given subcutaneously twice daily one week before and 2 weeks after balloon injury the cross sectional area of the neointima is reduced to 36% as compared with the nontreated control group (100%). This 64% reduction is statistically highly significant (p < 0.001). After treatment with 0.5 mg LA-heparin/kg/d the reduction of the neointima was 11% (p < 0.05). At a dose of 5 mg/kg body weight single or repeated administrations of LA-heparin caused only a small and transient increase in activated partial thromboplastin time values. The results show that subcutaneous administration of LA-heparin very effectively prevents smooth muscle cell proliferation and balloon catheter-induced neointimal growth. The well tolerated systemic application of this chemically non-modified LA-heparin might justify clinical trials for prevention of restenosis after percutaneous transluminal coronary angioplasty or other invasive cardiovascular interventions without complications of bleeding.

Angioplasty, Balloon↗

Lectin binding and uptake and glycoprotein characterization of isolated porcine aortic endothelial and smooth muscle cells.

Endothelial and smooth muscle cells were isolated from porcine aorta and kept in short-term culture. To determine the terminal carbohydrate composition of the plasma membranes from both cell populations, the cells were incubated with a panel of fluorescein-labelled lectins. Both cell populations shared a number of terminal carbohydrates, but the N-galactosamine specific lectin Wistaria floribunda agglutinin labelled only endothelial cells. A lectin which selectively labelled smooth muscle cells was not found. Western blot analysis of isolated endothelial cell membrane glycoproteins indicated that most membrane glycoproteins are labelled by Wistaria floribunda agglutinin.

Acetylgalactosamine↗

Binding and processing of multimeric vitronectin by vascular endothelial cells.

The multifunctional adhesive glycoprotein vitronectin (VN) undergoes a unique conformational transition from the plasma form into a multimeric form that represents the reactive heparin-binding form. In this study we investigated the interaction of multimeric vitronectin (VNmult) or VN-gold conjugates (which are equivalent in biochemical properties) with confluent and subconfluent monolayers of porcine endothelial cells. Time-dependent direct binding of radiolabeled VNmult to the luminal face of endothelial cells at 37 degrees C was observed which was competed by heparin, whereas plasma VN showed hardly any binding. At 4 degrees C binding of VNmult remained cell-associated, whereas after 6 hr at 37 degrees C a major portion of the ligand was translocated through cells and was associated with the subcellular matrix. Cytochemical studies with VN-gold conjugates were performed to demonstrate uptake of VNmult. At 4 degrees C only surface decoration of cells with gold label was seen, which was totally reversible in the presence of heparin. Subsequent incubation for various time intervals at 37 degrees C revealed disappearance of gold label from the surface and accumulation of conjugates in a perinuclear distribution inside the cells as judged both by electron microscopy and after silver enhancement by light microscopy. Cross-sections of endothelial cells demonstrated the inclusion of VN-gold conjugates in coated pits, endosomes, and in lysosomal compartments close to the nucleus. Within 2-6 hr a portion of VN-gold conjugates had accumulated with proteoglycans at the matrix face. These data provide strong evidence for specific routing of a portion of VNmult from the circulation into extravascular spaces, where the protein is believed to fulfill major adhesive and regulatory functions particularly as co-factor in plasminogen activation and immune defense.

Animals↗

Stimulation of the biosynthesis of lactosamine repeats in glycoproteins in differentiating U937 cells and its suppression in the presence of NH4Cl.

We prepared a mouse monoclonal antibody, 2D5, which recognized a highly glycosylated human lysosomal membrane antigen. The apparent molecular mass of this antigen was cell type dependent and ranged between 100 kDa and 130 kDa. The difference was due to a variation in the carbohydrate moiety, since upon removal of the N-linked oligosaccharides the size of the glycoprotein was reduced to approximately 50 kDa in all cases. The high carbohydrate contents, subcellular localization and N-terminal sequence indicated a high similarity or identity of this antigen with the lamp-2 protein. In U937 cells several agents known to elicit differentiation induced synthesis of a larger form of the lamp antigen. Thus, treatment of cells with calcitriol resulted in a shift in its average molecular mass from 115 kDa to 130 kDa. The difference was due to an increase in the contents of lactosamine repeats. In subcellular membranes from calcitriol-treated cells the specific activity of the UDP-N-acetylglucosamine: N-acetyllactosamine N-acetylglucosaminyltransferase was enhanced 3-fold. The enhancement was accompanied with an elongation of lactosamine repeats in N-linked oligosaccharides in the 46 kDa mannose 6-phosphate receptor and the homing receptor, the leucocyte antigen CD44. In contrast, the apparent size of the leucocyte antigen CD43 which bears numerous O-linked oligosaccharides was not changed indicating a selectivity in the modulation of the formation of lactosamine repeats in N- and O-linked carbohydrates. It is shown further that the synthesis of lactosamine repeats in U937 cells is impeded in the presence of NH4Cl.

Amino Acid Sequence↗

Colocalization of a large heterodimeric proteoglycan with basement membrane proteins in cultured cells.

A novel large heterodimeric dermatan sulfate proteoglycan with core proteins of 460 and 300 kDa, respectively, had been described as a secretory product of human fetal skin fibroblasts (Breuer et al., J. Biol. Chem. 266, 13224-13232 (1991)). Pulse-chase experiments showed a preferential association of the proteoglycan with the cell membrane. Immunogold labeling indicated its localization in fibrils on the cell surface as well as in fibrillar extensions from the cell body. Immunofluorescence studies yielded a fibrillar and punctate staining pattern which was also seen in cultured human and porcine endothelial cells. Dot-like structures were observed in transformed human keratinocytes. Various immunocytochemical double-labeling experiments indicated a remarkable colocalization of the proteoglycan with fibronectin, laminin, perlecan, and type IV collagen whereas only occasionally a colocalization with chondroitin-6-sulfate was found. No evidence for an enrichment of the proteoglycan in vinculin-containing structures was obtained. These results suggest that the proteoglycan is a widely distributed macromolecule which can associate with basement membrane components. Preliminary findings in rat cornea supported this conclusion.

Basement Membrane↗