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Biomedical subjects

B Akerström

Publications and source records attributed to B Akerström.

At least 73 records · Page 4Linked to original sources
PubMed1987

Use of protein G for preparation and characterization of rabbit antibodies against rat adipose tissue hormone-sensitive lipase.

The newly described immunoglobulin G-binding streptococcal surface protein, protein G, was used to prepare and characterize rabbit antibodies. The antibodies were directed against rat hormone-sensitive lipase, the rate-limiting enzyme in the hydrolysis of the triacylglycerols stored in adipose tissue. Antiserum was obtained after two injections with 20 micrograms enzyme protein, and the immunoglobulin fraction was obtained using a protein G-based solid-phase radioimmunoassay. The hydrolysis of acylglycerols by the enzyme was inhibited by the antibodies, and the enzyme could be efficiently removed from a solution using the antibodies and heat-killed streptococci expressing surface protein G. By Western blot and detection with 125I-protein G, the antibodies were found to selectively bind to hormone-sensitive lipase and to a smaller extent to two minor contaminants, possibly proteolytic fragments of the lipase. The amount of 125I-labelled protein G bound to the lipase on the blot was quantitatively related to the amount of enzyme protein down to the detection limit 10 ng.

Adipose Tissue↗
PubMed1987

Purification of streptococcal protein G expressed by Escherichia coli by high performance liquid affinity chromatography using immobilized immunoglobulin G and albumin.

A one-step HPLC method was developed for the purification of protein G, a cell wall molecule from group C and G streptococci with immunoglobulin G- and albumin-binding properties. Lysed Escherichia coli bacteria infected with lambda-phages containing the protein G gene from group G streptococci were used as a starting material for the preparations. The lysate was applied to a column with immobilized human immunoglobulin G or human serum albumin. Protein G was selectively bound and eluted at pH 2.0. A 750-fold purification was achieved. Sodium dodecylsulfate + polyacrylamide gel electrophoresis showed that the highly purified protein G consisted of three sets of doublets with the apparent molecular weight of 64 and 67, 56 and 58, and 45 and 47 kilodaltons, respectively. A specific method for quantitation of small amounts of protein G was developed and used for specific tracing of the protein after the affinity chromatography. Goat polyclonal antibodies were bound to an antigen coated to the plastic walls of microtiter plates, causing the Fc-region of the immunoglobulins to be directed outwards. Unknown samples of protein G were then allowed to compete with radio-iodinated protein G (solid phase radioassay) or protein G coupled to alkaline phosphatase (enzyme linked sorbent assay) for the Fc-regions.

Bacterial Proteins↗
PubMed1986

Developmental and tissue-specific expression of alpha 1-microglobulin mRNA in the rat.

A rat liver cDNA library was constructed in the lambda gt11 expression vector. Three clones expressing alpha 1-microglobulin, an immunosuppressive plasma protein, were detected by screening with rabbit antiserum against rat alpha 1-microglobulin. The alpha 1-microglobulin activity from one of the clones, 6b, was confirmed with monoclonal antibodies in a solid phase radioimmunoassay. The nucleotide sequence of the fragment (165 base pairs) was determined, and the translated amino acid sequence (55 amino acids) showed a 75% homology to human alpha 1-microglobulin (position 122-176). Southern blots of restriction endonuclease-digested rat DNA indicated two distinct genes with alpha 1-microglobulin homology when probed with radioactive cDNA fragment from clone 6b. Northern blots showed the presence of a single mRNA species in rat liver, and the level was low in 1-month-old animals, increased to reach a maximum during adulthood (3 months), and decreased with aging (12 months). The alpha 1-microglobulin concentration in rat serum showed the same age dependence between 1 and 12 months, with the highest values at 3 months. Embryonic development (8.5-day to 17.5-day) was studied using total fetal RNA, and expression of alpha 1-microglobulin mRNA was detected in low amounts only at day 15.5. alpha 1-Microglobulin mRNA levels, studied by an RNA dot blot assay, were high in liver and kidney, low in brain and testis, and none were found in hypothalamus and spleen cells.

Aging↗
PubMed1986

A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties.

Protein G, an IgG-binding molecule, was prepared from the cell walls of a group G streptococcal strain, G-148. The protein could be extracted from the cells by papain digestion and purified by the sequential use of ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Sepharose-coupled human IgG, and gel chromatography on Sephadex G-200. Two protein bands with similar molecular weight, 34,000 and 36,000, were obtained when analyzing the pure protein G on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield using this purification scheme was 27% of the protein G solubilized from the cells or 70 micrograms/ml packed bacteria. The Stokes radius and frictional ratio of protein G were determined to 3.53 nm and 1.64, respectively, suggesting an elongated fibrous molecule. The protein did not contain any intrachain disulfide bonds. The amino acid composition of protein G was determined and was found to be different from that of protein A, the well known staphylococcal IgG-binding protein. The equilibrium constants of the reactions between protein G and human, rabbit, mouse, and goat polyclonal IgG, determined by Scatchard plots, ranged between 1 X 10(10) and 7 X 10(10), for rat polyclonal IgG 1.4 X 10(9), and human monoclonal IgG1, IgG2, IgG3, and IgG4 between 2 X 10(9) and 6 X 10(9). These affinity constants were always greater than the corresponding values for protein A. The binding between protein G and various polyclonal and monoclonal IgG was pH dependent between 2.8 and 10, strongest at pH 4 and 5, and weakest at pH 10.

Animals↗
PubMed1986

Detection and purification of rat and goat immunoglobulin G antibodies using protein G-based solid-phase radioimmunoassays.

Using the newly described streptococcal surface protein, protein G, which has powerful immunoglobulin G binding properties, solid-phase radioimmunoassays were developed for the quantitation of goat and rat immunoglobulin G bound to the plastic surface of microtiter plates. The binding of goat immunoglobulin G to the surface via a specific antigen (guinea pig alpha 1-microglobulin) permitted the determination of antigen-specific antibodies with a detection limit of 50-100 ng. Optimum assay conditions were established and the whole assay procedure could be brought to completion at room temperature in less than a working day. The value of the assays was illustrated by monitoring rat and goat immunoglobulin G antibodies during their purification from whole sera by classical chromatographic procedures.

Alpha-Globulins↗
PubMed1986

Alpha 1-microglobulin is mitogenic for guinea pig lymphocytes.

Guinea pig alpha 1-microglobulin (alpha 1-m) was found to exert mitogenic effects on guinea pig lymphocytes, i.e. peritoneal exudate lymphocytes (PEL) or T lymphocytes from regional or mesenteric lymph nodes (LNL). On the other hand, spleen T lymphocytes did not respond to alpha 1-m. Stimulation of LNL required the presence of accessory cells. The stimulatory effect was strain-specific: alpha 1-m clearly induced proliferation of lymphocytes from Strain 2 guinea pigs, whereas Strain 13 lymphocytes responded poorly or not at all. Moreover, alpha 1-m inhibited antigen-driven proliferation of guinea pig lymphocytes. None of the effects described were species-specific, e.g. human alpha 1-m exerted similar effects on the guinea pig lymphocytes. These data indicate that alpha 1-m may be involved in the regulation of lymphocyte activation.

Alpha-Globulins↗
PubMed1985

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins.

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.

Amino Acid Sequence↗
PubMed1985

Immunological analysis of alpha 1-microglobulin in different mammalian and chicken serum. alpha 1-Microglobulin is 5-8 kilodaltons larger in primates.

Heterologous radioimmunoassays for a semiquantitative analysis of alpha 1-microglobulin were developed, exploiting the binding between polyclonal rabbit or goat antisera against human, guinea pig, or rat alpha 1-microglobulin and 125I-labeled human, guinea pig, or rat alpha 1-microglobulin. Homologues of this protein were detected in human, guinea pig, Rhesus monkey, rat, mouse, rabbit, goat, horse, and cow serum by inhibition of a set of heterologous radioimmunoassays. Serum proteins were separated by gel chromatography, and fractions were pooled, concentrated, and radiolabeled with 125I. By immunoprecipitation of the radioiodinated serum pools with heterologous anti-alpha 1-microglobulin-sera, and separating the precipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analogues of alpha 1-microglobulin were isolated from serum of man, guinea pig, Rhesus monkey, rat, mouse, horse, and chicken. The apparent molecular weight of alpha 1-microglobulin was 31,000-32,000 in human and monkey serum and 24,000-26,000 in guinea pig, rat, mouse, horse, and chicken serum. The possibility of an addition of a 5,000-8,000-Da peptide in primate alpha 1-microglobulin is discussed.

Alpha-Globulins↗
PubMed1985

Rat alpha 1-microglobulin. Purification from urine and synthesis by hepatocyte monolayers.

Rat alpha 1-microglobulin was isolated from the urine of rats treated with sodium chromate, and was purified by the use of gel chromatography, affinity chromatography on concanavalin-A-Sepharose and ion-exchange chromatography. The protein was heterogeneous in charge, had a tendency to form dimers, and was associated with a brown-coloured chromophore. The size of the protein (25 kDa) was similar to guinea pig alpha 1-microglobulin but smaller than the human protein, when measured with sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunological cross-reaction with human and guinea pig alpha 1-microglobulin was demonstrated. The concentration of alpha 1-microglobulin in rat serum was 16.4 mg/l (SD = 8.5 mg/l, n = 13) and rat serum alpha 1-microglobulin was eluted from a gel chromatography column at two different positions corresponding to monomeric alpha 1-microglobulin and IgA. The latter alpha 1-microglobulin activity could be absorbed by anti-IgA serum. Rat alpha 1-microglobulin and albumin were continuously released into the medium of rat hepatocyte monolayers, and alpha 1-microglobulin was isolated from the medium by the use of immunoprecipitation with anti-(alpha 1-microglobulin). Tritiated leucine, added to the medium, was incorporated into the protein, suggesting a de novo synthesis of alpha 1-microglobulin by the hepatocytes. The size of hepatic alpha 1-microglobulin was similar to that of purified urinary rat alpha 1-microglobulin, when determined with sodium dodecyl sulfate/polyacrylamide gel electrophoresis.

Animals↗
PubMed1985

Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies.

Protein G is an immunoglobulin (IgG)-binding bacterial cell wall protein recently isolated from group G streptococci. We have investigated the avidity of protein G for various monoclonal and polyclonal Ig of the IgG class, and compared it with the binding properties of protein A, the staphylococcal Fc-binding protein. Radiolabeled Ig were mixed with Sepharose-coupled protein G or protein A, and the amounts of radioactivity bound to the matrix-coupled bacterial proteins were determined. The avidity was found to be greater for protein G than for protein A for all examined Ig. Protein G bound all tested monoclonal IgG from mouse IgG1, IgG2a, and IgG3, and rat IgG2a, IgG2b, and IgG2c. In addition, polyclonal IgG from man, cow, rabbit, goat, rat, and mouse bound to protein G, whereas chicken IgG did not. The binding property of protein G was additionally exploited in the Western blot assay, in which iodine-labeled protein G was used successfully for the detection of a rat monoclonal antibody against ovalbumin, and for the detection of rabbit and goat polyclonal whole antisera against human urinary proteins. In these experimental situations, protein G was found to be a powerful reagent for the detection of IgG, and consequently the antigen against which these antibodies are directed.

Antibodies, Bacterial↗
PubMed1984

Immunoreactive pancreatic colipase, lipase and phospholipase A2 in human plasma and urine from healthy individuals.

A radioimmunoassay for each of the human pancreatic proteins (colipase, lipase and phospholipase A2) is described. Determinations of the mean concentration of each protein in plasma and urine from healthy individuals were carried out with the radioimmunoassays. The values obtained in plasma were 0.5 nM (5.3 micrograms/l), 0.6 nM (32 micrograms/l) and 0.3 nM (4.3 micrograms/l) for colipase, lipase and phospholipase A2, respectively. In urine, the corresponding values were found to be 0.2 nM (2.4 micrograms/l), 0.09 nM (4.4 micrograms/l) and less than 0.017 nM (0.2 micrograms/l). No physical interaction between any of the three proteins and the lipid particles of plasma was demonstrated by centrifugation experiments or gel filtration. Gel filtration of plasma depleted of fat by centrifugation showed the proteins only in their monomeric form. The corresponding porcine proteins displayed a binding to antibodies against the human proteins, but with a lower affinity than the homologous interactions. The binding was weak but could differentiate between the porcine proforms and activated ones, i.e., procolipase and colipase87.

Adolescent↗
PubMed1984

Alpha 1-microglobulin glycopeptides inhibit antigen-specific stimulation of human peripheral blood leucocytes.

Glycopeptides were prepared from proteolytically digested human and guinea pig alpha 1-microglobulin (alpha 1-m) by gel chromatography on Sephadex G-100 and affinity chromatography on concanavalin A-Sepharose. Amino acid analysis showed that the average glycopeptide was a tripeptide containing the amino acids aspartic acid/asparagine, isoleucine, and serine and/or threonine. It has earlier been shown that human alpha 1-m carries two or three N-glycosidically linked oligosaccharides of the high-mannose type. These preparations of glycopeptides inhibited the proliferative response of peripheral human blood leucocytes to the antigen purified protein derivative. The dose-response relationship of the inhibitions showed a greater specific activity of the glycopeptides than of whole alpha 1-m. Mild alkali treatment of human alpha 1-m did not affect its specific inhibitory activity, suggesting that the peptide backbone is of little importance for the immunosuppressive effect of alpha 1-m. No difference in inhibitory activity was seen between human and guinea pig native alpha 1-m or alpha 1-m glycopeptides. Human asialo alpha 1-m exerted a suppressive effect on the antigen-specific leucocyte proliferation similar to that of native alpha 1-m.

Alpha-Globulins↗
PubMed1983

Synthesis of alpha 1-microglobulin by guinea-pig liver.

Explants from perfused guinea-pig livers were found to release alpha 1-microglobulin into the culture medium. Tritiated leucine in the medium was incorporated into the protein, suggesting a de novo synthesis of alpha 1-microglobulin by the liver tissue. The size of the protein could not be distinguished from that of purified urinary alpha 1-microglobulin when tested with sodium dodecyl sulfate/polyacrylamide gel electrophoresis. After intravenous injections of tritiated leucine into guinea-pigs, the 105 000 X g pellet of homogenized liver rapidly increased its content of radioactive alpha 1-microglobulin, with a maximum after 20 min. 3H-Labelled alpha 1-microglobulin appeared in serum after a lag phase of 20 min, and by comparing the rate of accumulation with albumin, the synthesis of guinea-pig alpha 1-microglobulin could be estimated to 20 micrograms g liver-1 h-1.

Alpha-Globulins↗
PubMed1981

Immunosuppressive properties of alpha 1-microglobulin.

alpha 1-Microglobulin (alpha 1m), a serum glycoprotein (26,000 d), was found to impede the proliferative response of human lymphocytes to purified protein derivative (PPD) and tetanus toxoid. The data suggest that alpha 1m operates through an unstable suppressor mechanism, which no longer can function after 24 h of preculturing. This effect of alpha 1m on antigen stimulation did not seem to be due to binding of alpha 1m to PPD or cells, to altered kinetics of the PPD response, or to non-specific cytotoxicity. In contrast, PPD-induced leucocyte migration was not reversed by alpha 1m. alpha 1m did not cause significant inhibition in experiments in which lymphocytes were stimulated by the mitogens phytohaemagglutinin or concanavalin A. Finally, alpha 1m had its own leucocyte migration inhibitory effect.

Alpha-Globulins↗
PubMed1981

Chronic threshold of stimulating electrodes: comparison of activated vitreous carbon with conventional platinum-iridium electrodes in animal tests.

Electrodes made of vitreous carbon are inert, corrosion-resistant, inactive to electrocatalytic reactions and are especially biocompatible. Upon activation, they attain a capacitance of 20 to 40 mF and become "non-polarizable". Therefore, they should be particularly suitable as stimulating and sensing electrodes for cardiac pacemakers. The connective tissue layer that develops around the electrode because of the foreign-body reaction is less than 100 micron thick. The threshold rise, through lower than that of conventional Pt-Ir or ELGILOY electrodes, cannot be attributed exclusively to the connective tissue layer that is formed. Under favourable conditions, the threshold in animal experiments remains below 525 mV. Blood-spaces adjoining the electrodes are found at higher chronic threshold values.

Animals↗
PubMed1980

alpha 1-Microglobulin.

A new low-molecular weight plasma protein was discovered and isolated from human urine by Ingemar Beggård. He named it alpha 1-microglobulin. The protein has a molecular weight of 26,000, contains about 20% carbohydrate, is electrophoretically heterogenous, carries a brown-coloured, unidentified substance, and is present in serum in free, as well as in high molecular weight complexes. alpha 1-Microglobulin seems to interfere with the in vitro response of lymphocytes to some antigens. The site of synthesis of alpha 1-microglobulin is unclear.

Alpha-Globulins↗