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K Hedman

Publications and source records attributed to K Hedman.

At least 73 records · Page 4Linked to original sources
PubMed1988

Inactivation of human immunodeficiency virus in serum specimens as a safety measure for diagnostic immunoassays.

Since the human immunodeficiency virus (HIV) may be transmitted accidentally to laboratory personnel analyzing patient sera, the efficiency of a non-ionic detergent, Triton X-100, in inactivation of HIV in human serum as a safety measure was studied. Semliki Forest virus, an enveloped toga virus, was used as a model virus to create optimal treatment conditions. In the presence of 50% serum, complete inactivation (i.e. no residual virus detected, greater than 7 log reduction of virus titre) was achieved by incubation with 0.2% Triton X-100 for 1 h at 37 degrees C. Under these conditions HIV was also completely inactivated (i.e. no residual infectious virus detected, greater than or equal to 5 log reduction of virus titre). Both treated and untreated serum specimens were also tested with several enzyme immunoassays used in virological laboratories to determine whether the inactivation treatment interfered with the assays. The treated specimens, further diluted as recommended for each assay, were subjected to 15 enzyme immunoassays for microbial antibodies and antigens (HIV IgG, hepatitis A IgG and IgM, hepatitis B s, c, and e antigens and antibodies, cytomegalovirus IgG, mumps virus IgG, poliovirus IgG, rubellavirus IgM, toxoplasma IgG, and chlamydia IgG). Clearly decreased sensitivity was found only with two hepatitis B tests (e antigen and antibody to the surface antigen). It is concluded that safe inactivation of HIV in serum is achieved by 0.2% Triton X-100, but the treatment may decrease the sensitivity of some tests in which low specimen dilution is used.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Ultrastructural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts.

We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PA-containing focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5% FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion.

Cell Adhesion↗
PubMed1988

Pre-lysosomal divergence of alpha 2-macroglobulin and transferrin: a kinetic study using a monoclonal antibody against a lysosomal membrane glycoprotein (LAMP-1).

We have used electron microscopic immunocytochemistry to compare the distribution of LAMP-1, a marker for lysosomal membranes, with the intracellular localization of alpha 2-macroglobulin (alpha 2-M) and transferrin at various time points after their endocytosis into cultured NIH 3T3 cells. The purposes of this study were (a) to determine how soon endocytic ligands reach lysosomal organelles, (b) to examine whether the intermediate endocytic vesicles gained lysosomal markers gradually or in a precipitous, discrete event, and (c) to examine the relationship, if any, between the pathway of recycling ligands and lysosomes. At early time points (0-5 min) after initiation of endocytosis, most structures containing alpha 2-M labeled with colloidal gold (receptosomes) were not labeled by anti-LAMP-1 detected using ferritin bridge or peroxidase immunocytochemistry. At late time points (greater than or equal to 15 min), the structures containing alpha 2-M (lysosomes) were strongly labeled by anti-LAMP-1. In contrast, transferrin that was directly labeled with ferritin was mostly located in LAMP-1-negative structures at all time points studied. The proportion of alpha 2-M-gold containing vesicles strongly labeled for LAMP-1 roughly paralleled the proportion of alpha 2-M-gold-containing structures positive for cytochemically detectable acid phosphatase. Our data indicate that ligands such as transferrin that are internalized through coated pits and receptosomes, but not delivered to lysosomes, do not traverse a lysosomal organelle compartment as marked by LAMP-1 content. Ligands such as alpha 2-M that are destined for lysosomal delivery reach a LAMP-1-positive organelle compartment only after they traverse LAMP-1-negative, non-lysosomal vesicles previously described as receptosomes.

Acid Phosphatase↗
PubMed1987

Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry.

Transferrin is taken up by receptor-mediated endocytosis into intracellular vesicles and tubules, and then recycles rapidly to the plasma membrane (diacytosis). We applied double-label cytochemistry to study whether the recycling structures containing transferrin fuse with the intracellular membranous structures that deliver newly synthesized membrane glycoproteins from the ER to the plasma membrane (exocytosis) or whether they remain independent. KB and Vero cells were infected with the temperature-sensitive transport mutant 0-45 of vesicular stomatitis virus (VSV). Temperature-regulated exocytosis of membrane glycoprotein "G" occurred simultaneously with diacytosis of transferrin. The exocytic "G" protein, as detected by immunoperoxidase electron microscopy, passed through the cisternal Golgi stacks and vacuolar, tubular, vesicular, and pit-like structures of the Golgi system. A transferrin-ferritin conjugate used in ultrastructural double-label experiments was detected in diacytic vesicles and tubules that accumulated in the proximal (trans-reticular) Golgi area of the cell. The ferritin-labeled vesicles/tubules were often close to and intermixed with the VSV-"G" containing membranous structures, but in most cases at early times (15-20 min) the transferrin and VSV-"G" containing vesicular structures remained distinct. At later times (30-45 min), the two labels were occasionally found in the same structures. These results indicate that rapid recycling of endocytosed materials and exocytosis of membrane glycoproteins to the cell surface usually occur in distinct vesicles, possibly along the same general morphologic exit pathway.

Exocytosis↗
PubMed1987

Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma cells.

We have previously purified from cultured JEG-3 choriocarcinoma cells an Mr 75,000 protein, originally detected using antibodies to a retrovirus-related synthetic peptide. Using polyclonal antibodies, we have now localized this protein immunocytochemically in JEG-3 cells at both light and electron microscopic levels. In immunofluorescence microscopy of saponin-permeabilized cells, the antigen appeared as dots and short strands at the apical cell surface. In pre-embedding immunoperoxidase electron microscopy, the Mr 75,000 protein was specifically localized to microvilli on the apical cell surface. Immunoferritin electron microscopy was used to assess more quantitatively the antigen distribution in the plane of the plasma membrane, and to define the position of the antigenic site(s) with respect to the membrane. The immunoferritin results confirmed the microvillus specificity of the Mr 75,000 protein and showed that the antigenic portion of the protein is within a few nanometers from, and on the cytoplasmic side of, the lipid bilayer. In detergent extraction experiments, the Mr 75,000 antigen was highly enriched in the soluble fractions. These results demonstrate that the Mr 75,000 protein is a membrane protein highly specific for microvilli.

Cell Compartmentation↗
PubMed1986

Basement membrane matrix in vitro: focal binding of exogenous fibronectin to the matrix of teratocarcinoma-derived endodermal cells.

Interaction of exogenous fibronectin with the basement membrane-like PYS-2 cell matrix, lacking fibronectin and hyaluronic acid but containing heparan sulfate proteoglycan, was studied in vitro. Both human plasma fibronectin and fibronectin in fetal calf serum bound to PYS-2 matrix; also, fragments of fibronectin containing heparin-binding domains but lacking the collagen-binding domain bound to the matrix. In immunoelectron microscopy the bound fibronectin was found as 20-40 nm globules or patches. Distribution of fibronectin differed from that of laminin and correlated best with that of heparan sulfate proteoglycan. The results suggest that the binding of fibronectin to basement membrane matrices is not due to random adherence but involves specific interactions with other components.

Animals↗
PubMed1986

Single-serum radial hemolysis to detect recent rubella virus infection.

In the radial hemolysis test for rubella virus-specific antibodies, sera obtained shortly after primary rubella produce a characteristically altered hemolysis zone, termed soft hemolysis. The occurrence of soft hemolysis was analyzed statistically in 6,723 sera. The specificity of soft hemolysis for infection with rubella virus was 99.0%. Its sensitivity in primary infections was 97.3%, and the predictive value for rubella serodiagnosis (increase in IgG level or detectable IgM) was 91.2%. The temporal association of soft hemolysis with rubella was determined with sera obtained from 129 persons during late convalescence (70-310 days after onset of symptoms). Soft hemolysis disappeared by 190 days after the onset of symptoms and the presence of ordinary hemolysis ruled out primary rubella in the preceding 70-100 days. Soft hemolysis was caused by IgG antibody to rubella virus, and thus represents a new serodiagnostic principle. This technique, together with an Igm assay, allows rapid diagnosis of recent rubella from a single serum sample.

Antibodies, Viral↗
PubMed1986

The organelles of the trans domain of the cell. Ultrastructural localization of sialoglycoconjugates using Limax flavus agglutinin.

The subcellular distribution of sialic acid was determined at the ultrastructural level using Limax flavus agglutinin (LFA). This lectin, which is specific for N-acetylneuraminic acid and N-glycolylneuraminic acid, was covalently conjugated to horseradish peroxidase (HRP). The conjugates (LFA-HRP) were applied to aldehyde-fixed, saponin-permeabilized 3T3 cells in pre-embedding labeling electron microscopy. Peroxidase label was detected in a patchy distribution at the cell surface, and in plasma-membrane-coated pits, endocytic vesicles (receptosomes), multivesicular bodies, and lysosomes. Smooth-surfaced tubular and vesicular structures, similar to those that participate in membrane recycling, were labeled. In the Golgi complex, more than half of the cisternae contained label--typically only one cisterna on the cis side was unlabeled. Heavily labeled structures of the trans Golgi included a reticular membranous system with coated regions--50-80 nm diameter vesicular or pit-like profiles and larger coated vacuoles. Smooth 200-300 nm vacuoles were labeled on the trans side of the Golgi stack. Similar structures have been previously shown to participate in the exocytosis of plasma membrane and secretory glycoproteins from the Golgi stacks. These findings identify those intracellular organelles that are functionally at the level of, or distal to, the sialyltransferase-containing membranes of the Golgi, and distinguish them from the pre-Golgi membranous structures. The LFA-HRP conjugate is an indicator for this functional trans domain of the cell, and should be applicable for ultrastructural double-label experiments as a cis versus trans marker of the exocytic pathway.

Cell Membrane↗
PubMed1985

Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts.

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called 'link proteins' were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.

Cartilage, Articular↗
PubMed1985

Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins.

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.

Animals↗
PubMed1985

The Mr 95,000 gelatin-binding protein in differentiated human macrophages and granulocytes.

Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well-characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross-reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin-binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.

Adult↗
PubMed1984

Binding of fibronectin by the acute phase reactant C-reactive protein.

Following tissue injury, the concentration of C-reactive protein (CRP) is known to increase in plasma rapidly, while that of fibronectin often decreases. We now report that CRP immobilized onto polystyrene surfaces binds soluble plasma fibronectin (Kd = 1.5 X 10(-8) M). The binding of fibronectin by CRP was relatively sensitive to ionic conditions, being maximal at physiological NaCl concentrations. A decrease of pH from neutral to 5-6 greatly enhanced the binding of fibronectin by CRP. Ca2+ ions at greater than 1 mM inhibited binding. No binding was observed between fibronectin and CRP in soluble phase. CRP was found also to bind fibrinogen, which competed with fibronectin for CRP-binding sites. This was shown to explain why fibronectin was effectively bound from serum but not from plasma by immobilized CRP. The amount of CRP immobilized was critical in binding fibronectin; a too dense molecular layer of CRP inhibited the binding, as did the postsaturation of free surfaces with albumin, which itself was not bound by CRP. Soluble fibronectin agglutinated CRP-coated latex particles. Most or all of the CRP-binding activity in the fibronectin molecule was localized to the 120-140-kilodalton fragment, which also contains cell-binding and heparin-binding domains of fibronectin. The results provide a link between acute phase response and tissue repair.

C-Reactive Protein↗
PubMed1984

Integrity of the pericellular fibronectin matrix of fibroblasts is independent of sulfated glycosaminoglycans.

The pericellular matrix fibers of cultured human fibroblasts contain fibronectin, other glycoproteins, and heparan and chondroitin sulfate proteoglycans. In the present study, cell-free pericellular matrices were isolated from metabolically labeled fibroblast cultures. The isolated matrices were digested with heparinase from Flavobacterium heparinum, and then analyzed for sulfated glycosaminoglycans (GAGs). Nitrous acid degradation was used to distinguish the N-sulfated GAGs (heparan sulfate) from chondroitin sulfate. Fibronectin and the other major matrix polypeptides were studied using gel electrophoresis, enzyme immunoassay and immunofluorescence. Upon heparinase digestion, greater than 95% of sulfated GAGs were degraded in the matrix without detectable release of fibronectin or other matrix polypeptides or alteration of the fibrillar matrix structure. We conclude that in fibroblast cultures the integrity of the fibrillar matrix is independent of sulfated GAGs. Together with earlier observations, this suggests that filamentous polymerization of fibronectin forms the backbone of early connective tissue matrix.

Cells, Cultured↗
PubMed1984

Altered hemolysis in single radial hemolysis from a single serum sample as an indicator of recent primary rubella virus infection.

The quality of hemolysis in the single radial hemolysis (SRH) test was observed to be altered in a small proportion (5.7%) of sera (N = 9628) studied for rubella rubella antibodies. Three different types of altered hemolysis were identified. Two of these types, the "soft margin" (SM) or the "soft zone" (SZ), occurred singly or in combination in 97% of paired sera (N = 321) taken 3-30 days after primary rubella infection (diagnostic seroconversion). For comparison, diagnostic increases of rubella antibodies (N = 77), including both primary and reinfections, contained these types of altered hemolysis in 80% of the cases. Of the remaining 20% of the samples (N = 15), rubella antibodies of IgM class were looked for in seven cases but not found. After primary rubella, SZ was always present in the first serum sample and disappeared rapidly within 20 days while SM persisted longer. The predictive value of the different types of hemolysis was estimated from 96 primary sera, which had altered hemolysis. In this material, SZ was followed by a diagnostic increase of antibodies in 87% of the cases; the prognostic value of the SM was significant but lower. These data show that the appearance of SM or SZ in a single serum sample is a useful marker of recent primary rubella. On the other hand, a normal hemolysis in SRH can be used to exclude recent primary rubella, but not reinfection, with a high degree of probability.

Antibodies, Viral↗
PubMed1983

Intracellular vesicles involved in the transport of Semliki Forest virus membrane proteins to the cell surface.

The route of transport of Semliki Forest virus (SFV) membrane glycoproteins to the plasma membrane was studied using immunoperoxidase electron microscopy. SFV glycoproteins were localized in cultured BHK-21 fibroblasts infected with a temperature-sensitive mutant ts-1 of SFV, which shows a temperature-dependent, reversible defect in the transport of membrane glycoproteins to the cell surface. At 39 degrees C (restrictive temperature) the viral proteins were retained in the endoplasmic reticulum and the nuclear membrane. After shift of the infected cultures to 28 degrees C (permissive temperature) the proteins were synchronously transported to the Golgi complex. In the Golgi complex the labeled proteins were first (at 2.5 min) detected in large Golgi-associated vacuoles (GAV). Subsequently, i.e., at 5-30 min, the viral glycoproteins appeared in the cisternal stack: at 5 min the label was found in one or two of the proximal cisternae whereas at 15 or 30 min also the more distal cisternae were partially or uniformly labeled. At all time points examined after the temperature-shift, peroxidase label was found in 50 nm vesicles which were frequently coated. At 30 min, in addition to the 50 nm vesicles, larger 80 nm vesicles, which often had a cytoplasmic coat were labeled in the Golgi region. These results identify two major size classes of both coated and smooth vesicles which appear to function in the transport of the viral membrane proteins from the endoplasmic reticulum via distinct GAV and the stacked Golgi cisternae to the plasma membrane.

Animals↗
PubMed1983

Chondroitin sulfate at the plasma membranes of cultured fibroblasts.

We have previously shown that in confluent human fibroblast cultures chondroitin sulfate proteoglycan is a component of the fibronectin-containing pericellular matrix fibers. In the present work the distribution of chondroitin sulfate was studied in subconfluent cell cultures using antibodies that bind to a chemically defined carbohydrate fragment of chondroitinase ABC-modified chondroitin sulfate proteoglycan. Using immunofluorescence microscopy, we observed, in addition to the fibrillar matrix staining, chondroitin sulfate diffusely distributed at the cell surface. In indirect immunoferritin electron microscopy this staining corresponded to patchy binding of ferritin close (24 nm) to the outer aspect of the plasma membrane. The patchy organization appeared uniform in all cell surfaces. The cell surface chondroitin sulfate could not be removed from the plasma membrane by agents that dissociate electrostatic interactions. These data show that in fibroblasts chondroitin sulfate is a component of the outer aspect of the plasma membrane, and raise the possibility of an integral plasma membrane chondroitin sulfate proteoglycan.

Animals↗