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Biomedical subjects

P Samuelson

Publications and source records attributed to P Samuelson.

15 recordsLinked to original sources
PubMed1997

Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus.

The heterologous surface expression of the cholera toxin B subunit (CTB) from Vibro cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene encoding native CTB (103 amino acids) was introduced into gene constructs encoding chimeric receptors designed to be translocated and anchored on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capacity of the pentameric CTB to bind the GM1 ganglioside, both the surface accessibility and the functionality of the surface-displayed CTB receptors were evaluated. It could be concluded that the chimeric receptors were targeted to the cell wall of the staphylococci, since they could be released by lysostaphin treatment and, after subsequent affinity purification, identified as full-length products by immunoblotting. Surface accessibility of the chimeric receptors was demonstrated by a colorimetric assay and by immunofluorescence staining with a CTB-reactive rabbit antiserum. Pentamerization was investigated by using a monoclonal antibody described to be specific for pentameric CTB, and the functionality of the receptors was tested in a binding assay with digoxigenin-labelled GM1. It was concluded that functional CTB was present on both types of staphylococci, and for S. carnosus, the reactivity to the pentamer-specific monoclonal antibody and in the GM1 binding assay was indeed significant. The implications of the results for the design of live bacterial vaccine delivery systems intended for administration by the mucosal route are discussed.

Antibodies, Bacterial↗
PubMed1997

Flow cytometric quantification of surface-displayed recombinant receptors on staphylococci.

Surface display of recombinant proteins on bacteria and phages has become an important tool in bioscience. To evaluate the various host systems, a great need exists for quantitative methods to determine the densities of displayed proteins and peptides on the bacteria and phage surfaces. Here we describe how a method previously applied for quantification of surface proteins on mammalian cells has been adapted for quantification of chimeric receptors surface-displayed on bacteria; in this study, the bacteria being recombinant staphylococci. The presented method takes advantage of fluorescence-activated cell sorting (FACS) technology and a new type of nonfluorescent plastic beads, similar in size (2 microns in diameter) to bacterial cells, and thus suitable for generation of calibration curves from which the number of chimeric receptors can be obtained. The method was used to estimate the number of antigenic sites on two types of recombinant staphylococci, both carrying heterologous chimeric receptors, and it was found that the recombinant Staphylococcus carnosus cells carried approximately 10(4) surface-displayed antigenic sites, while recombinant Staphylococcus xylosus exposed approximately 3 x 10(3) sites per cell. The use of the deviced method for different applications is discussed.

Animals↗
PubMed1996

Surface display on staphylococci: a comparative study.

Two different host-vector expression systems, designed for cell surface display of heterologous receptors on Staphylococcus xylosus and Staphylococcus carnosus, respectively, were compared for the surface display of four variants of a 101 amino acid region derived from the G glycoprotein of human respiratory syncytial virus (RSV). Surface localization of the different chimeric receptors was evaluated by a colorimetric assay and by fluorescence-activated cell sorting. It was concluded that the S. carnosus system was better both in the ability to translocate inefficiently secreted peptides and in the number of exposed hybrid receptors. The potential use of the described staphylococci as live bacterial vaccine vehicles or alternatives to filamentous phages for surface display of protein libraries is discussed.

Flow Cytometry↗
PubMed1996

Surface display of a functional single-chain Fv antibody on staphylococci.

Two different host-vector expression systems designed for cell surface display of chimeric receptors on Staphylococcus xylosus and Staphylococcus carnosus have been evaluated for surface display of a mouse immunoglobulin G1(kappa) [IgG1(kappa)] anti-human IgE single-chain Fv (scFv) antibody fragment. To achieve surface anchoring of the chimeric receptors containing the scFv, the cell surface attachment regions from Staphylococcus aureus protein A were used in both expression systems. The different chimeric receptors could be recovered from cell wall extracts of both S. xylosus and S. carnosus, and surface localization was demonstrated by taking advantage of a serum albumin-binding reporter region present within the two types of receptors. In addition, the two different recombinant staphylococci carrying hybrid receptors containing the scFv were demonstrated to react with the antigen, which was human IgE, in whole-cell enzyme-linked immunosorbent assays. This is the first report of an antibody fragment expressed in a functional form anchored to the surface of gram-positive bacteria. The potential use of recombinant gram-positive bacteria as whole-cell diagnostic devices or alternatives to filamentous phages for surface display of scFv libraries is discussed.

Animals↗
PubMed1996

Cryotherapy after cruciate knee surgery. Skin, subcutaneous and articular temperatures in 8 patients.

We recorded temperature changes on the skin surface, subcutaneously and intra-articularly during cryotherapy after knee surgery by using Cryo-cuff compression dressings. Subcutaneous recordings on the contralateral knee were used as reference. 8 patients were examined. There was a reproducible decrease in skin temperature and subcutaneous temperature. Skin temperature had to be lowered to about 20 degrees C to obtain demonstrable intraarticular temperature changes.

Adolescent↗
PubMed1995

Hydrophobicity engineering to facilitate surface display of heterologous gene products on Staphylococcus xylosus.

Protein engineering has been employed to investigate the effect of specific amino acid changes on the targeting of heterologous proteins to the outer cell surface of the Gram-positive bacterium Staphylococcus xylosus. Three different variants, corresponding to a 101 amino acid region of the major glycoprotein (G protein) of human respiratory syncytial virus (RSV), were generated in which multiple hydrophobic phenylalanine residues were either substituted or deleted. The different G protein fragments were expressed as one part of recombinant receptors designed for surface display on S. xylosus cells. The engineered variants of the RSV G protein hybrid receptors were, in contrast to a non-engineered fragment, efficiently targeted to the outer cell surface of recombinant S. xylosus cells as determined by different methods, including fluorescence-activated cell sorting. In addition, immunization of mice with live recombinant S. xylosus demonstrated that surface exposure was required to generate receptor-specific antibodies. The present strategy of hydrophobic engineering should be of general interest in surface-display applications and for secretion of proteins otherwise difficult to translocate through host cell membranes.

Amino Acid Sequence↗
PubMed1995

Cell surface display of recombinant proteins on Staphylococcus carnosus.

A novel expression system for surface display of heterologous proteins on Staphylococcus carnosus cells has been developed. Taking advantage of the promoter and secretion signals, including a propeptide region, from the lipase gene of Staphylococcus hyicus and the cell wall-spanning and membrane-binding region of protein A from Staphylococcus aureus, efficient surface display of an 80-amino-acid peptide from a malaria blood stage antigen could be achieved. A serum albumin binding protein from streptococcal protein G was used both as a general reporter molecule and to increase the accessibility of the surface-displayed proteins. Immunoblotting, immunogold staining, and immunofluorescence on intact recombinant S. carnosus cells verified the presence of the propeptide, the malaria antigen, and the albumin-binding reporter protein on the bacterial surface. For the first time, fluorescence-activated cell sorting was used to analyze the presence of surface-displayed hybrid receptors on gram-positive bacteria.

Antigens, Protozoan↗
PubMed1994

Long-range interactions between DNA-bound ligands.

We have studied the interaction of the A:T specific minor-groove binding ligand 4',6-diamidino-2-phenylindole (DAPI) with synthetic DNA oligomers containing specific binding sites in order to investigate possible long-range interactions between bound ligands. We find that DAPI binds cooperatively to the oligomers. The degree of cooperativity increases with increasing number of binding sites and decreases with the separation between them. This dependence is paralleled by changes in the induced circular dichroism spectrum of DAPI, which decreases in intensity at 335 nm and increases at 365 nm. These results are consistent with an allosteric interaction of DAPI with DNA, where bound ligands cooperatively alter the structure of the DNA molecule. This structural change seems possible to induce under various conditions, including physiological. One consequence of allosteric binding is that ligands bound at a distance from each other sense each other's presence and influence each others' properties. If some regulatory proteins induce the same conformational change as DAPI, novel mechanisms for controlling gene expression can be anticipated.

Adenine↗
PubMed1993

The effect of prophylactic nitroglycerin infusion on the incidence of regional wall-motion abnormalities and ST segment changes in patients undergoing coronary artery bypass surgery.

The effects of nitroglycerin (NTG) on regional wall-motion abnormalities, ST segment changes, and the incidence of myocardial infarction (MI), cardiac failure, and mortality were studied in 30 patients undergoing coronary artery bypass. Patients received continuous infusions of either normal saline or NTG (1 microgram/kg per min [low dose] or 2 micrograms/kg per min [high dose]) beginning at anesthesia and continuing for 4 hours postoperatively. The occurrence of wall-motion abnormalities as detected by transesophageal echocardiography was 38 events in the normal saline group (N = 10). Significantly fewer events (p < 0.05) were recorded in the low dose and high dose NTG groups (20 events and 15 events, respectively) compared to controls. There were no significant differences between the control and NTG groups with regard to the incidence of Holter ST segment events or the incidence of MI, cardiac failure, or cardiac death. NTG reduced the incidence of echocardiographic wall-motion abnormalities in a dose dependent manner while having no significant effect on other parameters examined.

Coronary Artery Bypass↗
PubMed1993

Naproxen and paracetamol compared with naproxen only in coxarthrosis. Increased effect of the combination in 18 patients.

In a double-blind study of 18 patients with coxarthrosis the effect of 3 naproxen doses (0.5, 1.0, and 1.5 g daily) and 2 naproxen doses combined with paracetamol (0.5 g + 4 g daily and 1.0 g + 4 g daily) was investigated. Plasma levels of naproxen and paracetamol were measured (HPLC), and clinical assessment of pain, joint movement, activity of daily life and side-effects were performed at the end of the 5 treatment periods. A relationship was found between the 3 naproxen doses, naproxen plasma levels, pain at rest, and pain during movement. The combined treatment was more effective than treatment with the same naproxen dose alone. The effect of naproxen (0.5 g daily) combined with paracetamol (4 g daily) did not differ from that obtained during treatment with higher naproxen doses only. Furthermore, the effect of the highest naproxen dose was not better than the effect of the lower naproxen dose (1.0 g daily) combined with paracetamol. The main finding was that treatment with naproxen and paracetamol is more effective than treatment with higher naproxen doses alone.

Acetaminophen↗