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Biomedical subjects

G Fröman

Publications and source records attributed to G Fröman.

31 records · Page 2Linked to original sources
PubMed1987

Isolation and characterization of a fibronectin receptor from Staphylococcus aureus.

Attachment of bacteria to the host tissue is considered a first step in the development of many infections. Previous studies have shown that fibronectin, a protein shown to mediate substrate adhesion of eukaryotic cells, also binds to some pathogenic bacteria and mediates the tissue adherence of these prokaryotes. In the present communication, we report on the isolation and characterization of a fibronectin receptor from Staphylococcus aureus strain Newman. A 210-kDa fibronectin binding protein was isolated from a bacterial lysate by affinity chromatography followed by gel chromatography. Additional smaller peptides with fibronectin binding properties were also obtained. These peptides seem to represent degradation products of the large receptor protein since the former dominated when the purification was carried out in the absence of protease inhibitors. Furthermore, degradation of the purified receptor protein by staphylococcal V8 protease generated a large number of peptides that retained fibronectin binding activity. This observation also suggests that the large receptor protein contains several binding sites for fibronectin, and analysis of the binding of the 29-kDa amino-terminal fibronectin fragment to the 210-kDa receptor adsorbed in microtiter wells suggests that one receptor molecule can bind six to nine fibronectin molecules.

Amino Acids↗
PubMed1987

Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus.

The gene encoding the fibronectin-binding protein (FNBP) from Staphylococcus aureus strain 8325-4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5-kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin-Sepharose followed by ion-exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I-labelled 165-kd polypeptide, and unlabeled 165- and 87-kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp-gene encoding the fibronectin-binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG-binding domains of protein A followed by a fibronectin-binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.

Cloning, Molecular↗
PubMed1987

Binding of fibronectin to Escherichia coli isolated from bovine mastitis from different geographical regions.

Seventy strains of Escherichia coli, isolated from bovine mastitis in Australia, Denmark, Norway and the U.S.A., were tested for their ability to bind fibronectin. Fifty-three strains (76%) interacted with iodinated fibronectin at a level exceeding 5% of the total radioactivity added. Binding of the amino-terminal (29 kD) fragment of fibronectin was tested for 15 strains, and 6 strains (40%) bound greater than 5%. Bacteria binding the 29 kD fragment at greater than or equal to 19% of the added protein, consistently showed "high" attachment to bovine skin fibroblasts. These cells were shown by immunofluorescence to produce extracellular matrix containing fibronectin. Strains binding lower amounts of fibronectin or 29 kD fragment adhered poorly to these fibroblasts.

Animals↗
PubMed1987

Binding of fibronectin, fibrinogen and type II collagen to streptococci isolated from bovine mastitis.

Binding of 125I-labelled fibronectin, fibrinogen and type II collagen to group B (S. agalactiae), group C (S. dysgalactiae and S zooepidemicus), group E (S. uberis) and nontypable streptococci isolated from bovine mastitis was studied. S. agalactiae and S. uberis were found to bind low levels of all three proteins, while S. zooepidemicus bound high levels. Binding of the proteins to S. dysgalactiae varied, i.e. fibronectin was high, fibrinogen moderate and collagen low. Nontypable strains showed moderate or low binding of all proteins. Both hydrophobic and hydrophilic strains were found to bind fibronectin. For S. dysgalactiae the specific fibronectin binding ranged from 70% to 10% and for S. zooepidemicus it was more than 80% and this binding was sensitive to papain treatment. The binding of 29K-fibronectin fragment to one S. dysgalactiae strain showed an affinity of KD = 2.6 x 10(-8) M and the number of binding sites per colony forming unit (CFU) was calculated at 11,000.

Animals↗
PubMed1984

Binding of Escherichia coli to fibronectin. A mechanism of tissue adherence.

Four out of 17 enterotoxigenic strains of Escherichia coli isolated from infantile diarrhea bound 125I-fibronectin. This binding, which was inhibited by unlabeled fibronectin but not by other proteins, appears to involve two classes of receptors, one of which binds the ligand reversibly. Consistent with the presence of two classes of receptors the bacteria bound to at least two distinct sites of the fibronectin molecule, one being the amino-terminal domain which also contains the binding sites for Gram-positive bacteria and the other located outside this domain. The E. coli strain expressing fibronectin receptors adhered to fibroblasts and to fibronectin but not to ovalbumin-coated coverslips. Bacteria grown at 40 degrees C did not express fibronectin receptors and did not adhere to either substrate. Saturation of receptors with fibronectin blocked adhesion to both fibronectin-coated coverslips and to cultured fibroblasts. These data suggest that binding to fibronectin represents a mechanism of tissue adherence of E. coli.

Animals↗
PubMed1982

Effect of cholesterol on the reconstituted D-glucose transport system of human erythrocyte membranes.

Human erythrocyte membranes were solubilized by cholate. Reconstitution into phospholipid liposomes containing different amounts of cholesterol was performed by chromatographic removal of cholate. Glucose transport was determined as the differential uptake of radioactive D- and L-glucose. The reconstituted glucose transport activity decreased with increasing concentration of cholesterol up to about 20 mole percent. Further increase in cholesterol concentration resulted in an increase in the glucose transport activity.

Blood Glucose↗
PubMed1981

The stereospecific D-glucose transport activity of cholate extracts from human erythrocyte membranes.

The glucose transport protein of human erythrocyte membranes was solubilized with cholate to facilitate rapid reconstitution and direct glucose transport measurements. This may simplify the isolation of the native glucose transporter. In most experiments the membranes were prepared from fresh blood within 8 h, frozen in liquid nitrogen and stored at -70 degrees C to minimize proteolytic degradation. Solubilization with 25 mM cholate in the presence of 200 mM NaCl at pH 8.4 for 12 min at room temperature gave a high D-glucose transport activity. The solubilized mixture contained 20% of the total membrane protein, only 6% of the polypeptides of molecular weight around 90000, 23% of the polypeptides of molecular weight around 55000, 30% of the phospholipids and at least 6% of the stereospecific D-glucose transport activity. At cholate concentrations up to 22 mM the ratio of solubilized phospholipids to cholate increased steeply, concomitant with an increase in solubilized activity. Above 30 mM cholate the activity diminished. At 4 degrees C the activity of the extract decreased rapidly within the first day and slowly during the next few days. The initial changes seem to have produced a fairly stable, but not native form or fragment of the transporter. When 20 mM EDTA and 5 mM dithioerythritol were included in the solubilization mixture a high activity was preserved for about one day.

Blood Glucose↗
PubMed1980

The glucose transport activity of human erythrocyte membranes. Reconstitution in phospholipid liposomes and fractionation by molecular sieve and ion exchange chromatography.

Human erythrocyte membranes, at a protein concentration of 1-2 g/l, were solubilized with 0.12 M cholate in the presence of 0.06 M phospholipid (egg yolk phospholipids or phosphatidylcholine). More than 40% of the protein was solubilized. Cholate was removed by molecular sieve chromatography, whereby liposomes formed. These liposomes exchanged D-glucose faster than L-glucose. The recovery of glucose transport activity in the reconstituted system was estimated to be higher than 16%. The liposomes were heterogenous in size, as shown by molecular sieve chromatography on Sepharose 4B, and small liposomes predominated. In liposomes formed with phosphatidylcholine, the distribution of glucose transport activity did not parallel the distribution of protein or phospholipid, and the activity was found mainly in the smallest liposomes. The proteins were incorporated mainly in the liposomes that eluted at the lowest ionic strength upon ion exchange chromatography. The glucose transport activity separated into three main peaks upon ion exchange chromatography of egg yolk phospholipid liposomes. The activity eluted at low ionic strength. The liposomes contained proteins mainly from the 3- and 4.5-regions (nomenclature according to Steck, T.L. (1974) J. Cell Biol. 62, 1-19). The activity peaks were highest in the first part of the chromatogram. The protein distribution did not coincide with the variation in activity over each peak. Therefore, it cannot be excluded that a minor component not seen in the electrophoretic analyses might be responsible for the glucose transport activity.

Biological Transport, Active↗
PubMed1980

A molecular sieving method for preparing erythrocyte membranes.

The most widely used methods for preparing erythrocyte membranes are time-consuming, since they involve repeated steps of centrifugation and washing. We have found that membranes can be prepared in a rapid and simple way by molecular-sieve chromatography on a Sepharose 4B column. Two sizes of columns have been used, one 9-ml column to prepare about 1 mg of membrane proteins in 10 min and one 2-litre column for larger amounts (up to 200 mg in 15 min). The optimal pH and phosphate concentration for the separation of hemoglobin from the membranes were found to be 7.5 and 5 mM, respectively. Sodium dodecyl sulphate (SDS) electrophoretic analysis of the membrane proteins prepared by centrifugation and by this new method shows similar protein compositions.

Blood Proteins↗
PubMedDate not supplied

Vaginal microbiological flora and sexually transmitted diseases in women with recurrent or current vulvovaginal candidiasis.

A history of recurrent vulvovaginal candidiasis (RVVC) was reported by 102 women, while current vulvovaginal candidiasis (VVC) was diagnosed in 83 of the same 996 women. They had all attended two family planning and one youth clinic, respectively. Two women, without RVVC or VVC, matched for age for each case of RVVC, were selected as a comparison group (COMP). Recurrent, but not current VVC, was associated with a history of sexually transmitted disease. Those with current, but not with recurrent, VVC had significantly more often genital warts and bacteriuria (> 10(5) bacteria/ml), but significantly less often bacterial vaginosis than the COMP women. Both VVC and RVVC were inversely correlated to a vaginal flora change with a mixed anaerobic vaginal flora. Those with VVC had a greater number of lactobacilli on vaginal cultures, than those with RVVC and the women in the COMP group. VVC and a history of RVVC both occurred more frequently in women with a lactobacilli-predominated vaginal flora, as compared with those with a flora change with a mixture of anaerobic and facultative anaerobic bacteria.

Bacteria↗