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Biomedical subjects

T Johansson

Publications and source records attributed to T Johansson.

At least 55 records · Page 3Linked to original sources
PubMed1998

Baculovirus-mediated large-scale expression and purification of a polyhistidine-tagged rubella virus capsid protein.

The capsid protein of rubella virus was produced in baculovirus-infected Spodoptera frugiperda insect cells, with a polyhistidine affinity tag at the carboxy terminus. The RV capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal-ion affinity chromatography. Immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight. The final yield was 5 mg of purified protein per liter of cell culture. Surface plasmon resonance was used to investigate the antigenic potential of the histidine tagged capsid protein in an antigen-antibody interaction study. A specific interaction between the two proteins was shown. Our results suggest that this strategy should be useful in interaction studies of other virus-specific proteins and antibodies.

Amino Acid Sequence↗
PubMed1998

Evaluation of pulsed-field gel electrophoresis of genomic restriction fragments in the discrimination of Yersinia enterocolitica O:3.

One hundred and six Yersinia enterocolitica serogroup O:3, biotype 4 isolated from human and porcine samples in 1984 and in the years 1993 5 were examined by pulsed-field gel electrophoresis (PFGE). The genomic profiles produced by the enzymes NotI and XbaI were studied. Sixteen (A-P) and 8 (1-8) different pulsotypes were obtained, respectively. By combining the pulsotypes produced by both NotI and XbaI 24 different types were distinguished. The two major types, designated as A1 and B1, comprised 36% of all strains tested. The proportions of pulsotypes A1 and B1 were, 35.9 and 25.6%, respectively, among strains isolated in 1984. The corresponding figures among the strains isolated in 1993-5 were 35.8 and 41.8%. Nine pulsotypes were found only in 1984 and nine only in 1993-5. The proportions of the major pulsotypes, A1 and B1, in human isolates were 42.9 and 35.7% and in porcine isolates 22.2 and 36.1% respectively. Six types were found among both human and porcine isolates, 8 only among human strains and 10 only among porcine strains.

Animals↗
PubMed1997

Behaviourally mediated sexual selection: characteristics of successful male black grouse

Recent studies of lekking animals suggest that a suite of characters may be favoured by sexual selection. Examples of such traits are high survival, increased androgen levels, territory features and morphological characters including exaggerated morphological ornaments. Here we argue that such selection is often mediated by behavioural differences and we use results from a field study of lekking black grouse, Tetrao tetrixas an example of our argument. In the absence of females, males display a range of stereotyped behaviours including vocalizations, visual displays and fighting. When females attend the lek, the behavioural repertoire of the males becomes more limited. Time budget analyses reveal that in the vast majority of cases, males in the presence of females perform only three types of behaviour: a vocal display called rookooing; a courtship behaviour called circling; and fighting. This suggests that female preference could be based on male courtship and fighting behaviour while females visit individual male territories and that displays not used in the presence of females could be ruled out as important for female preference. Sexual selection in black grouse, however, is a complex interplay between male-male competition and female choice and therefore female preference is not the only determinant of male success. Successful males were more often involved in fights in the absence of females on the lek and defended territories that were larger than expected. Therefore, we hypothesize that differences in male fighting ability result in some males occupying relatively large territories at the centre of the leks. Relatively large territories seem to be needed for successful courtship. Therefore behavioural differences mediate differences in male copulation success both through female preference and male-male competition.

Journal Article↗
PubMed1997

Recombinant human extracellular superoxide dismutase produced in milk of transgenic rabbits.

Expression of human extracellular superoxide dismutase (EC-SOD), a glycosylated, tetrameric metalloprotein, was targeted to the lactating mammary gland of transgenic rabbits. Efficient expression of the recombinant whey acidic protein/ec-sod gene was achieved and up to 3 mg ml-1 of the enzyme was secreted into the milk. Rabbit milk-produced recombinant EC-SOD was primarily found in the whey and purified by a two-step chromatographic method. To evaluate the rabbit milk-produced human EC-SOD, comparisons with native and Chinese hamster ovary cell (CHO)-produced EC-SOD were performed. All proteins were tetrameric and N-glycosylated. The behaviour on SDS-PAGE and size-exclusion chromatography indicated that the masses, and thereby the extent of post-translational modification of the proteins was similar. The monosaccharide composition of both recombinant EC-SOD variants was analysed and indicated similarities in the attached N-glycans on the two proteins. Furthermore, the peptide maps of the three EC-SOD variants revealed that all proteins had similar polypeptide backbones.

Animals↗
PubMed1996

Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: large scale production of the E1 protein.

The two envelope glycoproteins of rubella virus (RV), E1 of 58 kDa and E2 of 42-47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10-1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.

Animals↗
PubMed1996

Structure of the human kappa-casein gene.

The human kappa-casein-encoding gene, Kca, was cloned and sequenced. The structural gene consists of five exons ranging from 33 to 496 nucleotides (nt) separated by introns ranging from 1146 to 2942 nt, and extends over 8821 nt. All intron/exon splice junctions conform to the GT/AG rule. The gene organization is similar to that of the bovine gene. The 5'-flanking region contains an A + T-rich sequence; TTTAATT, close to where the TATA motif is found in most other genes, a CAAT box, and an AP-1 consensus sequence. In addition, one Alu repetitive element was found in the second intron.

Amino Acid Sequence↗
PubMed1996

Characterisation of the complex of plasminogen activator inhibitor type 1 with tissue-type plasminogen activator by mass spectrometry and size-exclusion chromatography.

Glycosylated human plasminogen activator inhibitor type 1 (PAI-1), produced in Chinese hamster ovary (CHO) cells, showed a variety of compounds with different molecular weights when subjected to electrospray mass spectrometry (ES-MS), owing to the heterogeneity of the carbohydrate chains. However, non-glycosylated human PAI-1, produced in E. coli, gave rise to a prominent species with a molecular weight of 42,774, consistent with the amino-acid sequence. A non-glycosylated mutant of the proteinase domain (B-chain) of tissue-type plasminogen activator (tPA) produced in C 127 cells, had a molecular weight of 28,168. Full-length, glycosylated, tPA showed a large heterogeneity in molecular mass. For a mass study, a tPA-PAI-1 complex was formed, composed of non-glycosylated PAI-1 and non-glycosylated B-chain. This complex was remarkably stable at room temperature in buffer with a neutral pH. The mass spectrum of the complex provided two main species, a peptide with a mass of 3803 and a dominating species of 67,133. These masses are consistent with a complex where PAI-1 is cleaved at the P1-P1' position. A trace of a species with a molecular mass of 70,942 was also found, corresponding to the complete, non-dissociated complex with PAI-1. Separation of the cleaved peptide, corresponding to the hydrophobic C-terminal 33 amino-acid residues of PAI-1, from the complex, was achieved by size-exclusion chromatography in the presence of 30% acetonitrile. Thus, in the complex between tPA and PAI-1, the proteins are held together by a tight covalent bond, but the C-terminal cleaved peptide of PAI-1 is only bound to the complex by hydrophobic forces. To assess whether this is specific to the tPA B-chain alone, experiments with the complex of full-length, glycosylated tPA and glycosylated PAI-1 were also performed, and it was possible to demonstrate the release of the C-terminal PAI-1 peptide by chromatography, mass spectrometry, as well as by SDS-PAGE.

Amino Acid Sequence↗
PubMed1996

A cluster of genes encoding major isozymes of lignin peroxidase and manganese peroxidase from the white-rot fungus Trametes versicolor.

A gene cluster from the white-rot basidiomycete Trametes (Coriolus) versicolor (Tv) PRL 572 containing three structural genes, LPGIII, LPGIV and MPGI, was characterized. The genes are arranged in the same transcriptional direction, within a 10-kb region, and found to encode quantitatively dominant isozymes of lignin peroxidase (LP) and manganese peroxidase (MP). The second gene in sequence, LPGIV, predicts a 346-amino-acid (aa) mature polypeptide (36.9 kDa, pI 4.31) which is identical with the partial aa sequence information available on the LP12 isozyme (43.1 kDa, pI 3.27). The first gene, LPGIII, encodes a 341-aa polypeptide (36.1 kDa, pI 3.93) which has not been identified at the protein level. However, the similarity of LPGIV would suggest that the predicted product is an LP-type enzyme. LPGIII and LPGIV are homologous to the tandemly arranged genes LPGII and LPGI, respectively, recently described by Jönsson and Nyman [Biochim. Biophys. Acta 1218 (1994) 408-412]. The homologous genes, LPGIII/LPGII and LPGIV/LPGI, are 99% and 96% identical in sequence, respectively, and are predicted to encode identical polypeptides, since base substitutions in the predicted exons are all synonymous. The third gene, MPGI, is different in intron-exon organization and predicted to be disrupted by five rather than six introns, as are the LP genes. The deduced polypeptide, 339 aa in size (35.9 kDa, pI 4.07), is identical with the partial aa sequence information available for isozyme MP2 (44.5 kDa, pI 3.09). The MPGI- and LPGIV-encoded polypeptides are 70% identical in sequence which suggests that MP and LP from Tv may be regarded as members of the same family within the plant peroxidase superfamily. Most importantly, this study identifies a gene encoding the MP2 isozyme, and further shows that genes encoding MP and LP can be closely linked on the chromosome and may be coordinately transcribed.

Base Sequence↗
PubMed1996

Recombinant human bile salt-stimulated lipase: an example of defective O-glycosylation of a protein produced in milk of transgenic mice.

The expression of recombinant human bile salt-stimulated lipase (bssl) was targeted to the lactating mammary gland of transgenic mice. Expression of recombinant genes comprising bsslcDNA, or alternatively genomic bssl DNA, under control of regulatory elements derived from the murine whey acidic protein (wap) gene was achieved and evaluated. Constructs containing genomic bssl sequences mediated high levels (0.5-1 mg ml-1) of recombinant human BSSL in the milk. The recombinant BSSL produced was purified, biochemically characterized and compared to native BSSL and recombinant BSSL produced in mouse C127 and hamster CHO cells. Recombinant BSSL derived from transgenic mice showed a different migration and distribution after SDS-PAGE electrophoresis, lower apparent molecular mass on size-exclusion chromatography and no detectable interactions with a panel of lectins. These results indicate a significantly lower degree of O-glycosylation of recombinant BSSL in milk from transgenic mice than was found for the native enzyme or recombinant CHO- or C127 cell-produced BSSL. Despite these differences, mouse-milk-derived recombinant BSSL exhibited similar lipase activity, the same stability to low pH and similar sensitivity to elevated temperatures as the native enzyme. The observation that mouse-C127-cell-produced recombinant BSSL is heavily O-glycosylated makes species-related restrictions less attractive as an explanation for the reduced O-glycosylation.

Animals↗
PubMed1995

The gene from the white-rot fungus Trametes versicolor encoding the lignin peroxidase isozyme LP7.

The structural gene LPGVI (1463 bp including 6 introns), characterized from the white-rot basidiomycete Trametes versicolor (PRL 572), encodes a 342-residue mature polypeptide of 36.7 kDa, preceded by a 26-residue signal/propeptide. LPGVI is identified as the gene encoding the 43 kDa lignin peroxidase isozyme LP7 as based on the partial amino acid sequence information available. The polypeptide deduced shares 72-74% identity in sequence with other lignin peroxidases and 70% with a manganese (II) peroxidase from T. versicolor.

Amino Acids↗
PubMed1995

Disulfide bonds and glycosylation in fungal peroxidases.

Four conserved disulfide bonds and N-linked and O-linked glycans of extracellular fungal peroxidases have been identified from studies of a lignin and a manganese peroxidase from Trametes versicolor, and from Coprinus cinereus peroxidase (CIP) and recombinant C. cinereus peroxidase (rCIP) expressed in Aspergillus oryzae. The eight cysteine residues are linked 1-3, 2-7, 4-5 and 6-8, and are located differently from the four conserved disulfide bridges present in the homologous plant peroxidases. CIP and rCIP were identical in their glycosylation pattern, although the extent of glycan chain heterogeneity depended on the fermentation batch. CIP and rCIP have one N-linked glycan composed only of GlcNAc and Man at residue Asn142, and two O-linked glycans near the C-terminus. The major glycoform consists of single Man residues at Thr331 and at Ser338. T. versicolor lignin isoperoxidase TvLP10 contains a single N-linked glycan composed of (GlcNAc)2Man5 bound to Asn103, whereas (GlcNAc)2Man3 was found in T. versicolor manganese isoperoxidase TvMP2 at the same position. In addition, mass spectrometry of the C-terminal peptide of TvMP2 indicated the presence of five Man residues in O-linked glycans. No phosphate was found in these fungal peroxidases.

Amino Acid Sequence↗
PubMed1995

Intra- and postoperative cerebral complications of open-heart surgery.

A consecutive series of 1400 patients who had undergone open-heart surgery was retrospectively reviewed concerning postoperative cerebral dysfunction. The 30-day mortality was 1.6%. Forty-one patients (2.9%) showed signs of cerebral dysfunction, which proved fatal in seven cases. Neurologic symptoms were observed immediately after surgery in 14 patients, suggesting intraoperative damage. In 20 others there was an interval between surgery and the onset of cerebral symptoms, which in 12 cases were preceded by supraventricular tachycardia. Computed tomographic scans were performed on 27 patients and showed recent brain infarction in 22. No bleeding was found. At follow-up 34 of the 41 patients were alive, 21 of them with neurologic sequelae and 13 reporting complete recovery. Nineteen of the 34 survivors experienced no diminution of quality of life. Since half of the cerebral complications occurred postoperatively, more aggressive prevention and management of supraventricular tachyarrhythmia and anticoagulation therapy should be considered.

Adolescent↗
PubMed1994

Structure of the human beta-casein encoding gene.

The entire human beta-casein-encoding gene, Bca, was cloned and sequenced. The gene consists of eight exons ranging from 21 to 531 nucleotides (nt) in length and extending over 10,466 nt. Exon-2 contains the translational start, the entire signal sequence and the codons for the two first amino acids of the mature protein. This corresponds to the organization found in other species. The translational stop is localized to exon-7. Exon/intron boundaries are in accordance with the AG/GT rule and conform to suggested consensus sequences. Splice junctions are located between coding triplets. In all other species analyzed, Bca has been found to consist of nine exons; however, within intron-2 of the human gene, a sequence omitted from human mRNA, but corresponding to exon-3 of other known Bca genes, was revealed.

Amino Acid Sequence↗
PubMed1994

Expression and characterization of biologically active human extracellular superoxide dismutase in milk of transgenic mice.

We have targeted the expression of recombinant human extracellular superoxide dismutase, a glycosylated tetrameric metalloprotein, to the mammary gland of transgenic mice. This was achieved by using regulatory elements from either the murine whey acidic protein gene or the ovine beta-lactoglobulin gene to control expression of human extracellular superoxide dismutase cDNA. Whey acidic protein regulatory sequences directed high level mammary gland-specific expression of the recombinant gene and secretion of biologically active extracellular superoxide dismutase into the milk. The produced recombinant protein was fully active, it was in tetrameric form, it showed heparin affinity, and its mass was similar to that of the native enzyme. In addition, the in vivo plasma clearance in a rabbit model was similar to the previously studied native and recombinant forms. To our knowledge, this is the first example of efficient production of a tetrameric, protease-susceptible metalloprotein in milk of transgenic animals. Production at equivalent levels in transgenic farm animals would yield sufficient extracellular superoxide dismutase for therapeutic purposes.

Animals↗
PubMed1994

Separation of active and inactive forms of recombinant human plasminogen activator inhibitor type 1 (PAI-1) expressed in Chinese hamster ovary cells: comparison with native human PAI-1.

Human plasminogen activator inhibitor type 1, PAI-1, was expressed in Chinese hamster ovary cells. A production level of 10-15 mg latent PAI-1 per liter of media was achieved after methotrexate amplification. Latent recombinant PAI-1 was purified by two chromatographic steps, cation exchange chromatography on CM-Sepharose and affinity chromatography on heparin-Sepharose. The obtained latent PAI-1 was approximately 90-95% pure showing one homogenous peak upon size-exclusion chromatography. However, four different isoforms due to different degrees of sialylation could be seen upon isoelectric focusing. Purified latent PAI-1 was activated by incubation in 6 M guanidine-HCl. By this method, 40-60% of PAI-1 was converted to an active form after removing the denaturant. The active fraction of PAI-1 was separated from inactive material by size exclusion chromatography on Superdex 200. Active PAI-1 migrated as expected for a 43-kDa large protein, while inactive PAI-1 migrated as larger protein complexes, suggesting that the remaining inactive PAI-1 was in the form of aggregates. This method for the separation of active and inactive PAI-1 could also be used for activated native PAI-1 prepared from human endothelial cells. Active recombinant PAI-1 was remarkably stable at pH 5.5, both when stored on ice and when stored at room temperature.

Amino Acids↗