PubMed Health⌕ Search

Biomedical subjects

A Olofsson

Publications and source records attributed to A Olofsson.

17 recordsLinked to original sources
PubMed1992

Transforming growth factor-beta 1, -beta 2, and -beta 3 secreted by a human glioblastoma cell line. Identification of small and different forms of large latent complexes.

Transforming growth factor-beta 1 (TGF-beta 1) has been found to occur as latent high molecular weight complexes, with or without an associated component denoted latent TGF-beta 1-binding protein (LTBP). We show here that a human glioblastoma cell line (U-1240 MG) secretes all isoforms of TGF-beta s found in mammalian cells (TGF-beta 1, -beta 2, and -beta 3). Approximately 26% of the secreted TGF-beta is in an active form. Latent TGF-beta s were partially purified from medium conditioned by the U-1240 MG cell line using anion exchange chromatography. Analysis of the different fractions by immunoblotting using antisera against precursor parts of the different TGF-beta isoforms, and against LTBP, revealed that not only TGF-beta 1 but also other isoforms of TGF-beta may occur in high molecular weight forms containing LTBP. In addition, each one of the TGF-beta isoforms occurred in smaller forms not containing LTBP. Interestingly, each of the TGF-beta isoforms was also seen in complexes of about 210 kDa containing associated component(s) distinct from LTBP. These results indicate that each of the different isoforms of TGF-beta is synthesized and secreted by this glioblastoma cell line in several different high molecular weight latent forms; the biological importance of the various latent TGF-beta complexes is discussed.

Carrier Proteins↗
PubMed1992

Oligomer formation of staphylococcal alpha-toxin analyzed by electron microscopy and image processing.

The 12S oligomeric form of Staphylococcus aureus alpha-toxin has been studied with electron microscopy after incubation of the toxin with membrane preparations or liposomes. The target material originated from human platelets. Different electron microscopic preparation techniques were used including negative staining, freeze-fracture and vitrification in liquid ethane. Analysis of micrographs with image processing methods revealed two groups of ring-like structures corresponding to alpha-toxin oligomers. One form measured 75 A in diameter and had a high stain density in the central protein deficient part while the other was larger with a diameter of 100 A and less stain accumulation in the center. The conditions under which the latter were formed suggest that this corresponds to an inactive loosely-bound form of the toxin. The high stain density in the smaller particle is consistent with the presence of a penetrating pore in this structure.

Bacterial Toxins↗
PubMed1991

Oligomerisation of cell-bound staphylococcal alpha-toxin in relation to membrane permeabilisation.

We have studied the kinetics of staphylococcal alpha-toxin oligomerisation in relation to membrane permeabilisation, using as targets cultured adrenocortical Y1 cells, rabbit red blood cells (RRBC), human platelets, and liposomes prepared of lipids extracted from platelets. After isolation of membranes from toxin-treated cells, oligomeric toxin was detected (i) by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography or Western blotting, and (ii) by electron microscopy of negatively stained specimens. alpha-Toxin was found to oligomerise on all membranes independently of the temperature. On RRBC and Y1 cells most of the membrane associated toxin appeared converted to the oligomeric form. Hexamers were always present along with membrane permeabilisation. However, hexamers were also detected at conditions when membrane permeabilisation did not occur; at low temperature, in the presence of high concentrations of Ca2+, and after pretreatment of cells with concanavalin A (Con A). Addition of a neutralising monoclonal antibody (MAb) to cell-bound toxin collected it into aggregates much larger than the hexamers. By contrast hexameric toxin remained after addition of a non-neutralising MAb. Our data suggest that the active toxin species is not monomeric, and support the hypothesis that alpha-toxin permeabilises membranes by forming hexameric protein-lined transmembrane channels.

Animals↗
PubMed1991

A role of the latent TGF-beta 1-binding protein in the assembly and secretion of TGF-beta 1.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized as latent complexes with high molecular weights. The large latent complex of TGF-beta 1 in platelets is composed of three components, i.e. the mature TGF-beta 1, which is non-covalently associated with a disulphide-bonded complex of the N-terminal remnant of the TGF-beta 1 precursor (TGF-beta 1-latency associated peptide) and the latent TGF-beta 1 binding protein (LTBP). The TGF-beta 1-latency associated peptide is sufficient for the latency of TGF-beta 1, whereas the functions of LTBP remain to be elucidated. In a human erythroleukemia cell line, HEL, the production of the latent form of TGF-beta 1 was induced more than 100-fold by phorbol 12-myristate 13-acetate. Analysis by Northern blotting revealed that both the TGF-beta 1 precursor and LTBP were induced in a coordinated fashion. Analysis by immunoprecipitation using antibodies against LTBP and the TGF-beta 1 precursor dimer revealed that LTBP has a molecular size of 205 kd under reducing conditions in this cell type, i.e. similar to that from cells transfected with cDNA for LTBP, but larger than the platelet form (125-160 kd). Limited tryptic digestion of LTBP in HEL cells and analysis by SDS-PAGE showed protein bands of similar sizes to those of platelet LTBP, suggesting that the difference in molecular sizes of LTBP involves cell-specific processing. The biosynthesis of the latent TGF-beta 1 was studied by pulse-chase analysis. LTBP became covalently associated with the TGF-beta 1 precursor within 15 min after synthesis in this cell line. Secretion of the large latent TGF-beta 1 complex was observed as early as 30 min after the synthesis of LTBP; at the same time, a free form of LTBP not bound to the TGF-beta 1 precursor was seen. In contrast, the TGF-beta 1 precursor remained inside the cells in an unprocessed form for a longer time period and the TGF-beta 1 precursor dimer without LTBP was secreted only very slowly. Furthermore, the results of partial tryptic digestion of this molecule suggested that it contained improper disulphide bonding. These results suggest that LTBP plays a critical role in the assembly and secretion of the latent TGF-beta 1.

Carrier Proteins↗
PubMed1991

The structure of Staphylococcus aureus alpha-toxin: effects of trypsin treatment.

Staphylococcus aureus alpha-toxin was treated with trypsin, which inactivates the toxin. Two-dimensional crystals of the modified protein were produced on preformed lipid layers. The projection structure obtained by electron crystallographic analysis of a large number of crystals showed tetragonal p4 symmetry and a resolution of approximately 12 A. The fragments of the toxin, 17 and 18 kDa large, were arranged in a way resembling those observed earlier for the native protein (Olofsson et al., J. Mol. Biol. 214, 299-306, 1990). However, after trypsin treatment the stain-deficient region corresponding to one alpha-toxin monomer shows two separated subdomains of similar size. This separation is probably related to the inability of the modified toxin to undergo the conformational change thought to be essential for the membrane-damaging effect.

Bacterial Toxins↗
PubMed1990

Crystalline layers and three-dimensional structure of Staphylococcus aureus alpha-toxin.

Interaction of the pore-forming protein alpha-toxin from Staphylococcus aureus with lipid components from platelet membranes induces crystal formation of the toxin oligomers. Structure analysis of crystalline areas in either sodium phosphotungstic acid or a sodium phosphotungstic acid/glucose mixture has been performed with electron microscopy and image processing. Ordered domains extending up to a few micrometers were observed, particularly after application of alpha-toxin to pre-formed lipid layers. The crystals, showing tetragonal symmetry, formed either separate two-dimensional sheets or three-dimensional piles of layers. The corresponding unit cell parameter of the single layer was a = b = 109.4 A (standard deviation 2.1 A, n = 21). Incubation of the toxin with intact membranes or extracted lipids as well as application of the lipid layer technique resulted in congruous crystalline properties. The projected averaged alpha-toxin oligomer shows cyclic symmetry with a stain-filled space in the centre. The bulk of the three-dimensional model consists of four asymmetric protein units forming a ring. In addition, a small domain covers the central cavity at the face of the protein opposite to the underlying lipid. The conditions under which the tetragonal arrays are formed on the lipid layers suggest that the alpha-toxin molecule is in a conformation binding to a hydrophobic surface rather than fully inserted into a lipid bilayer.

Bacterial Toxins↗
PubMed1990

TGF-beta 1 binding protein: a component of the large latent complex of TGF-beta 1 with multiple repeat sequences.

TGF-beta occurs in a latent complex of high Mr. We report the cDNA cloning and an initial structural and functional characterization of a component of the large latent TGF-beta 1 complex, denoted TGF-beta 1 binding protein (TGF-beta 1-BP). Most of the sequence of fibroblast TGF-beta 1-BP is made up of cysteine-rich repeats of two different kinds; there are 16 EGF-like repeats and three repeats with a distant resemblance to EGF, but of a distinct type hitherto not found in any other protein. beta-hydroxylated asparagine residues were identified in two of the EGF-like repeats. TGF-beta 1-BP purified from human platelets is considerably smaller than the fibroblast form (125-160 kd vs. 170-190 kd), suggesting that there is alternative splicing of the TGF-beta 1-BP gene or that TGF-beta 1-BP undergoes cell-specific proteolysis. TGF-beta 1-BP was found not to bind and inactive TGF-beta 1; its role in the latent complex is discussed.

Amino Acid Sequence↗
PubMed1988

Digitization of electron micrographs: a comparison of three different types of scanners.

Some aspects of digitization of electron micrographs have been investigated. The performances of a flat-bed, a rotating drum, and a diode array scanner have been evaluated. Estimates have been achieved for resolution, mechanical and optical stability, and optical density response. It is concluded that for routine transmission electron microscopy of, for example, negatively stained biologic specimens, a diode array scanner produces data good enough to obtain resolutions at a level normally expected. High speed is the major advantage with this type of equipment. However, for high-resolution work it is necessary to use a conventional scanner with a relatively slow scan speed.

Analog-Digital Conversion↗
PubMed1988

The projection structure of alpha-toxin from Staphylococcus aureus in human platelet membranes as analyzed by electron microscopy and image processing.

Most strains of Staphylococcus aureus produce alpha-toxin, a 33-kDa membrane active protein which is considered to be an important virulence factor of this bacterium. When alpha-toxin interacts with membranes an oligomeric from of the toxin can be seen by electron microscopy as characteristic ring structures in the membrane. A two-dimensional study of these annular structures, incorporated in membranes of human platelets, was performed, introducing a partly new method for rotational alignment of individual particles. It is shown that the averaged oligomer consists of six subunits. At neutral pH the outer diameter of the ring is about 75 A. The stain-filled pore or cavity in the center has a diameter of about 25 A. The size of the hexamer is increased if the pH is lowered.

Blood Platelets↗
PubMed1987

Relations between speech intelligibility and psychoacoustical modulation transfer function (PMTF).

The modulation transfer function, MTF, has proved to be a powerful measure for predicting speech intelligibility in speech transmission channels. We extended it to include the ear, by measuring the psycho-acoustical MTF, i.e. the PMTF. Tone thresholds of 11 normal-hearing and 20 hearing-impaired subjects were measured in presence of unmodulated and intensity modulated noise. Octave frequencies from 500 to 4,000 Hz were used. The noise was octave filtered around the frequency of the probe tone. Six modulation frequencies from 1 to 50 Hz were used. From these results the PMTFs were calculated, as well as the corresponding psycho-acoustical speech transmission indices, i.e. the PSTIs. The subjects' speech discrimination scores in quiet and speech reception thresholds in noise were also measured. A correlation coefficient of 0.85 between the speech discrimination score in quiet and the PSTI was obtained. For the speech reception threshold in noise and the PSTI, the correlation was 0.71. The first of these two figures is promising, but our method needs some improvement, as it gave some problems due to fatigue effects.

Hearing Loss, Sensorineural↗
PubMed1986

Intraepithelial migration of mucosal mast cells in hay fever: ultrastructural observations.

Evidence has been presented suggesting that a migration of nasal mast cells from the mucosal connective tissue stroma into the epithelium is part of the mucosal response in birch pollen allergy. In a previous study, the identification of these intraepithelial cells as tissue mast cells rather than blood basophils was based on light microscopical morphology and histochemistry. We have now studied the ultrastructure of these cells in mucosal biopsies taken before and during the birch pollen season. Intraepithelial cells with basophil or metachromatic granules were only observed in biopsies taken during the season. Some of these cells had the ultrastructural appearance of tissue mast cells, including cytoplasmic lipid droplets and a granular substructure composed of multilamellar arrays and scrolls, serving to distinguish human mast cells from blood basophils. The ultrastructural traits of the remaining cells were heterogeneous, some reminiscent of human blood basophils, others of globule leucocytes of other species, but entirely typical blood basophils could not be identified. The results thus support our previous suggestion that a migration of mucosal mast cells from the connective tissue stroma into the epithelium is part of the human allergic mucosal response. It cannot be determined whether the ultrastructural heterogeneity of these cells is the result of an adaptation to the intraepithelial environment of one single mast cell type or to the existence of an ultrastructurally distinct mucosal mast cell.

Basophils↗
PubMedDate not supplied

The three-dimensional structure of trypsin-treated Staphylococcus aureus alpha-toxin.

Trypsin treatment of staphylococcal alpha-toxin cleaves the molecule into two roughly equally sized parts, which results in inactivation of the toxin. Tetragonal arrays of oligomers, closely resembling the native ones, can however be formed on lipid layers. From tilted views of negatively stained crystals a 3D structure to 23 A resolution has been determined by electron microscopy and image processing. On comparison with the 3D structure of the native alpha-toxin (Olofsson et al., J. Mol. Biol. 214, 299-306, 1990) the subdomains are more separated, confirming the differences found when comparing the projection maps (Olofsson et al., J. Struct. Biol. 106, 199-204, 1991). The tryptic cleavage takes place in a postulated hinge region. The results are consistent with the hypothesis that the conformational change required for inducing the membrane permeabilizing property takes place in this region. Furthermore, we present a refined projection map at approximately 10 A resolution based on the analysis of a large number of crystals using unbending methods.

Bacterial Toxins↗
PubMedDate not supplied

Strong suppression by mononuclear leukocytes from cord blood of human newborns on maternal leukocytes associated with differences in sensitivity to prostaglandin E2.

We have tested peripheral mononuclear leukocytes (PML) from the cord blood of newborns, from sera of their mothers, and from sera of nonrelated nonpregnant adult women for sensitivity to suppressive exogenous prostaglandin E2 (PGE2). Endogenous PG production was simultaneously inhibited by indomethacin 2.8 microM. The phytohemagglutinin-stimulated (PHA-simulated) uptake of tritiated thymidine (3H-TdR) by PML from the mothers and the nonpregnant women was suppressed by the exogenous PGE2 at a concentration of 1.4 x 10(-8) M, 100 times less than the one required to suppress the PML from newborns (1.4 x 10(-6) M). In addition, 1.4 x 10(-7) M or less of PGE2 reversed the suppression of neonatal PML to stimulation. The maternal PML were reversed into stimulation at 1.4 x 10(-9) of exogenous PGE2. The amount of endogenous PGE2 synthesized by 1 x 10(6) fresh, nonstimulated neonatal PML according to gas chromatography-mass spectrometry assay was 5 ng (1.4 x 10(-8) M). The synthesis increased to 27 ng/10(6) cells after 18 hours' incubation. These concentrations are similar to the ones of exogenous PGE2 at which neonatal PML were slightly stimulated but the maternal cells were still suppressed. Preincubation for 18 h at 37 degrees C decreased the PGE2 induced suppression of the adult PML but did not change the response of the neonatal PML.

Adult↗