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Biomedical subjects

G Magnusson

Publications and source records attributed to G Magnusson.

At least 73 records · Page 4Linked to original sources

Synthesis of the Forssman pentasaccharide and terminal tetra-, tri-, and di-saccharide fragments.

The 2-(trimethylsilyl)ethyl (TMSEt) beta-glycosides of the Forssman pentasaccharide [alpha-D-GalNAc-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Gal- (1-->4)-beta-D-Gal-(1-->4)-D-Glc] and the terminal tetrasaccharide, as well as the methyl glycosides 1 and 2 of the terminal di- and tri-saccharides, were synthesised by silver trifluoromethanesulfonate-promoted alpha-glycosylation of suitably protected mono-, di-, tri-, and tetrasaccharide alcohols with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosyl bromide, followed by removal of protecting groups. The anomeric TMSEt group of the Forssman pentasaccharide and terminal tetrasaccharide was removed with trifluoroacetic acid-dichloromethane, to give the corresponding hemiacetal sugars 4 and 6.

Carbohydrate Sequence↗

Apoptosis of Spodoptera frugiperda cells induced by okadaic acid is abrogated by baculovirus infection.

Okadaic at low concentrations specifically inhibits protein phosphatase 2A. Treatment of cells with this inhibitor leads to growth arrest and cell injury within 1 to 2 days. After testing several different types of cells we found that mouse 3T6 cells and insect Sf9 cells responded to okadaic acid by apoptosis, as defined by changes in morphology and degradation of chromatin to fragments of nucleosome size. Infection of insect cells by baculovirus prevented the okadaic acid-induced apoptosis. Analysis of phosphoprotein synthesis by polyacrylamide gel electrophoresis showed that okadaic acid treatment resulted in hyperphosphorylation of some cellular proteins, including histone H1. Concomitant baculovirus infection of the cells did not change the overall phosphorylation pattern, but appeared to mitigate the okadaic acid-induced hyperphosphorylation of histone H1.

Animals↗

Mouse polyomavirus late region mutants expressing a defective VP2 capsid protein exhibit an enhanced viral DNA replication.

We have constructed mouse polyomavirus mutants with deletions or insertions in the late region. These viral constructs exhibit two different phenotypes, characterized by a differential replication ability. The first type shows a twofold higher level of DNA synthesis with respect to wild type, while mutants of the second type are virtually unable to replicate. In this paper we investigate the replicative behavior of the first class of mutants, which expresses defective viral coat proteins. Our data raise the possibility that, of the three late gene viral products, VP2 is involved in the formation of an encapsidation intermediate that, in an indirect fashion, affects the rate of viral DNA synthesis.

Animals↗

Calculated conformations of sialyl-Le(x)- and sialyl-Le(a)-lactones.

The minimum energy conformations of the four sterically reasonable SLe(x) and SLe(a) lactones were calculated using the molecular mechanics force-field MM2(91). The tetrasaccharide lactone involving the 3- and 2-position of the Gal moiety was found to be more stable than the 3,4-lactone both for SLe(x) and SLe(a).

CA-19-9 Antigen↗

Cardiovascular responses during one- and two-legged exercise in middle-aged men.

Eight healthy and regularly physically active men, 44-69 years old, performed one- and two-legged dynamic knee extension exercise at increasing work intensities, including one leading to exhaustion. Leg blood flow increased linearly in relation to work rate, reaching a peak value of 5.1 +/- 0.4 l min-1. With a mean weight of quadriceps femoris of 2.2 +/- 0.1 kg, a peak perfusion of 2.3 +/- 0.1 l kg-1 min-1 was attained. The maximal leg oxygen uptake was 0.72 +/- 0.07 l min-1 (0.33 +/- 0.03 l kg-1 min-1). At submaximal work the elevation in limb oxygen uptake accounted for between 70 and 100% of the rise in pulmonary oxygen uptake. Comparing two- with one-legged knee extension the cardiac output was 1.5 l min-1 higher at each work level, reaching 13.7 +/- 0.7 and 12.3 +/- 1.0, respectively at exhaustion, leaving 3.5 and 7.2 l min-1 of blood flow to the remaining body (cardiac output--leg blood flow). The mean arterial pressure was 119 +/- 5 mmHg at rest and increased to 155 mmHg for both test modes at the maximal work rate. The femoral arterial and venous plasma concentrations of lactate, ammonia and noradrenaline were significantly higher for two-legged as compared with one-legged exercise at the maximal load performed. However, the rate of release per leg, for both lactate and ammonia, did not differ between the two test conditions. It is concluded that physically active middle-aged men, with a well-retained muscle mass, can maintain a high skeletal muscle perfusion, similar to that of young males. However, the blood flow is achieved with a higher mean arterial pressure and an elevated sympathetic activity, as reflected by noradrenaline in plasma and spillover from the exercising limb.

Adult↗

Synthesis of the 2"-hydroxy, 4"-deoxy and 4"-epi analogues of beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D-Gal, the terminal trisaccharide of globotetraose.

Radical deoxygenation of methyl 3,6-di-O-benzoyl-2-deoxy-4-O-imidazol-1-yl-thiocarbonyloxy-2-phtha limido-beta-D - glucopyranoside 5 gave methyl 3,6-di-O-benzoyl-2,4-dideoxy-2-phthalimido-beta-D-glucopyranoside 6, which was converted into the corresponding methyl thioglycoside donor 9. Methylsulfenyl trifluoromethane-sulfonate-promoted glycosylation of 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-(2,4,6-tri-O-benzyl-alpha-D-galactopyranosyl)-bet a-D- galactopyranoside 10, followed by removal of protecting groups gave the 4"-deoxy analogue 12 of the terminal trisaccharide of globotetraose. Silver trifluoromethanesulfonate-promoted glycosylation of the same disaccharide alcohol 10 with 3,4,6-tri-O-acetyl-2-deoxy-2- phthalimido-alpha/beta-D-glucopyranosyl bromide 13 and 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide 16, followed by deblocking, gave the 2"-hydroxy and 4"-epi analogues 15 and 18, respectively.

Carbohydrate Conformation↗

Synthesis of 3'- and 4'-deoxyfluorolactose and its Me3SiCH2CH2 and ceramide derivatives.

(2-Trimethylsilyl)ethyl (Me3SiCH2CH2)3'- and 4'-deoxyfluorolactosides (1 and 3) were synthesized by glycosylation of Me3SiCH2CH2 2,3,6-tri-O-benzyl-beta-D-glucopyranoside with 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-beta-D-galactopyranosyl bromide and 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-beta-D-galactopyranosyl bromide. Anomeric deblocking of the fully acetyled Me3SiCH2CH2 glycosides (12 and 13) gave the corresponding hemiacetals 14 and 15. Removal of the acetyl groups gave 3'- and 4'-deoxyfluorolactose (2 and 4). The deoxyfluorolactosylceramides 5 and 6 were synthesized via boron trifluoride etherate- or silver triflate-activation of the trichloroacetimidates prepared from 14 and 15. Silver triflate-mediated glycosylations showed lower reaction rates, and fewer byproducts were formed.

Carbohydrate Conformation↗

Glycopeptides bind MHC molecules and elicit specific T cell responses.

Carbohydrates are T cell independent antigens because they do not bind to MHC molecules. However, glycopeptides might potentially bind to MHC molecules via their peptide component for presentation to T cells. We have conjugated the disaccharide galabiose [Gal alpha (1-4)Gal beta] to the amino terminus of a T cell peptide determinant from hen egg-white lysozyme [HEL(52-61)]. The resulting glycopeptide (Gal2-52-61) and a nonglycosylated analogue containing tyrosine and glutamic acid at the amino-terminus (YE-52-61) bound equally well to purified I-Ak. T cell hybridomas were produced after immunization with Gal2-52-61. Many of the T cell hybridomas were glycopeptide-specific and responded to Gal2-52-61 but not to nonglycosylated synthetic peptides or to HEL presented by APC, indicating that the carbohydrate moiety influenced T cell recognition. Recognition was lost with the amino terminal attachment of the disaccharide to a peptide six amino acids longer at the amino terminus than HEL(52-61). Recognition also was lost with peptides containing only a single galactosyl residue or with galabiose bound to a different I-Ak binding peptide. T cells directed to Gal2-52-61 recognized glycopeptides having significant variation in the disaccharide structure, such as HEL(52-61) glycopeptides carrying lactose, cellobiose, or hepta-o-acetylated galabiose. Peptide residues were important features of the T cell epitope; Ala substitutions of two critical T cell contact residues of HEL(52-61) (Tyr53 and Leu56) abrogated T cell reactivity to the glycopeptides without affecting binding to I-Ak. In conclusion, we propose that these T cells recognize a peptide conformation specific to glycopeptide-I-Ak complexes and that this recognition does not involve specific interaction between the carbohydrate moiety and the T cell receptor.

Amino Acid Sequence↗

Synthesis of the monodeoxy derivatives of 2-(trimethylsilyl)ethyl beta-lactoside.

Monodeoxy derivatives of 2-(trimethylsilyl)ethyl (Me3SiEt) beta-lactoside were synthesized, by deoxygenation at the disaccharide level, for the 2'-, 3'-, 4'-, and 6'-monodeoxylactosides. The 2-, 3-, and 6-deoxy derivatives were synthesized by beta-D-galactosylation of suitably protected monodeoxygenated Me3SiEt glucosides. Silver silicate was shown to be an efficient glycosylation promoter in the preparation of the 2- and 3-deoxylactosides.

Carbohydrate Sequence↗

Lactose repressor-operator DNA interactions: kinetic analysis by a surface plasmon resonance biosensor.

Lactose repressor binding to operator DNA and subsequent dissociation of the complex was monitored continuously by a biosensor, measuring surface plasmon resonance. In this analysis a synthetic, double-stranded oligonucleotide containing the operator site was immobilized on the sensor surface and repressor protein was passed over the surface. The formation of the repressor-operator complex was specific and could be inhibited by isopropyl-beta-D-thiogalactopyranoside inducer. From the association curve, the apparent kass was determined to be 1.8 x 10(6) M-1 s-1. Dissociation of the complex was, for the first time for the lac repressor, determined as an uncatalyzed reaction and the kdiss was determined to be 3.4 x 10(-4) s-1. As a reference, the repressor-operator interaction was analyzed by electrophoretic mobility shift assay under similar reaction conditions. With this method the equilibrium binding constant was calculated to be 2.4 (+/- 0.2) x 10(8) M-1. The corresponding value calculated from biosensor data was 5.1 x 10(9) M-1.

Base Sequence↗

Lytic functions of mutant polyomavirus large T-antigen with deletion of retinoblastoma protein-binding motif.

Polyomavirus large T-antigen has a binding site for the retinoblastoma protein. Mutant proteins without this binding site retain their activity in the initiation of viral DNA synthesis, but are not able to induce immortalization of rat embryo fibroblasts. To investigate the function of the retinoblastoma binding site in polyomavirus lytic infection, a set of mutants with small in-frame deletions was constructed. We found that the mutant large T-antigens had partial or complete defects in immortalization and had corresponding disabilities in trans-activation of the adenovirus E2 promoter in secondary rat embryo cells. However, one partially immortalization-defective mutant trans-activated the E2 promoter in HeLa cells. All mutants were normal in trans-activation of the polyomavirus late promoter. The mutant large T-antigens had a decreased activity in viral DNA replication. This phenotype was observed only after transfection of growth-arrested mouse cells. The replication of the mutants could be restored to nearly wild-type levels when middle and small T-antigens were coexpressed in the cells. The same effect was achieved with a 287-amino-acid N-terminal fragment of large T-antigen with an intact retinoblastoma protein-binding motif. This result suggests that the effect on viral DNA synthesis was indirect.

3T3 Cells↗

2-(Trimethylsilyl)ethyl glycosides. Transformation into the corresponding 1-O-acyl sugars.

(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside and (trimethylsilyl)ethyl 2,3,6-tri-O-acetyl-4-O-(2,3,6-tri-O-acetyl-4-O-[2-acetamido-4,6-di-O-ace tyl-2- deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-beta-D- galactopyranosyl]-beta-D-galactopyranosyl)-beta-D-glucopyranoside have been transformed in high yield into the corresponding 1-O-beta-acyl saccharides by treatment with various carboxylic anhydrides in the presence of boron trifluoride-diethyl ether in toluene. The carboxylic anhydrides (4-pentenoic, 2-cyclopentenylacetic, 2,4-dimethoxy-benzoic, and 2-methoxybenzoic anhydride) were synthesized from the corresponding carboxylic acids by treatment with N,N-bis[2-oxo-3-oxazolidinyl]phosphorodiamidic chloride.

Acylation↗

Anti-GM3-lactam monoclonal antibodies of the IgG type recognize natural GM3-ganglioside lactone but not GM3-ganglioside.

Immunization of mice with a synthetic GM3-lactam-BSA (bovine serum albumin) conjugate (designed to emulate the corresponding natural GM3-lactone conjugate), followed by fusion of splenocytes with myeloma cells, gave rise to more than 300 monoclonal hybridomas producing antibodies to GM3-lactam-BSA, which did not react with Glc-BSA and BSA. Eight antibody clones were randomly chosen from the positive 300 hybridomas. The eight clones, all belonging to the IgG class, were unreactive against GM3-ganglioside, whereas two antibodies (P5-1 and P5-3, both IgG1, kappa) reacted with GM3-ganglioside lactone. Binding of these two antibodies to the GM3-lactam-BSA conjugate was inhibited by soluble glycosides of GM2-, GM3-, and GM4-lactam and by GM3- and GM4-lactam, respectively, but not by Gb3 or asialo-GM1 and GM2-saccharides. A third antibody (P3; IgG2b, kappa) was inhibited by GM2-, GM3-, and GM4-lactam, but did not recognize GM3-ganglioside lactone.

Animals↗

A bovine rotavirus serotype 1: serologic characterization of the virus and nucleotide sequence determination of the structural glycoprotein VP7 gene.

Bovine rotavirus T449 was isolated from feces of a calf with diarrhea. Serological characterization by serotype-specific monoclonal antibodies showed that the T449 virus belonged to serotype 1. This is the first report of a bovine rotavirus that does not belong to serotype 6, 8, or 10. The serotype 1 designation was confirmed by using an immunoperoxidase focus neutralization assay. The gene encoding the major neutralization antigen (VP7) was cloned and its nucleotide sequence was determined. The sequence obtained was 1062 bp in length and contained an open reading frame corresponding to 326 amino acid residues. Comparative analysis of the deduced amino acid sequence with the corresponding sequence of the human serotype 1 rotavirus strain, Wa, revealed a 90% identity. When compared to the predicted amino acid sequence of VP7 protein of the other serotypes an overall divergence of 20 to 25% was detected. These data show that the serological typing agrees with the result of the genetic analysis.

Amino Acid Sequence↗

Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.

Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.

Amino Acid Sequence↗

2-(Trimethylsilyl)ethyl glycosides. Synthesis of the asialo-GM1-tetrasaccharide, spacer glycoside, and BSA and Sepharose glycoconjugates.

The tri and tetra-saccharide glycosides beta-D-GalNAc-(1-4)-beta-D-Gal-(1-4)-beta-D-Glc-1-OTMSEt and beta-D-Gal-(1-3)-beta-D-GalNAc-(1-4)-beta-D-Gal-(1-4)-beta-D-Glc-1-OT MSEt (asialo-GM1) have been synthesized by sequential glycosylations of a suitably protected 2-(trimethylsilyl)ethyl (TMSEt) lactoside with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-galactopyranosyl chloride and 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl bromide. The tetrasaccharide glycoside was transformed into the corresponding hemiacetal sugar as well as the 1-chloro sugar. The latter was used for glycosylation of 2-bromoethanol. The resulting glycoside was used to alkylate methyl 3-mercaptopropionate and the resulting spacer glycoside was used for the preparation of the title glycoconjugates.

Carbohydrate Sequence↗

Prevalence of genital papilloma virus infections in asymptomatic and symptomatic women, studied with a combined dot-blot and Southern blot procedure.

A technique for the detection and typing of genital infections with human papilloma virus (HPV) is described. Following a non-invasive sampling and a simplified preparation procedure, the analysis was performed as a combined dot-blot and Southern blot analysis, where the former test was used to exclude cases without demonstrable content of HPV DNA. The subgenomic probes used in the Southern blot analyses have a higher specificity than can be achieved with genomic ones, and only one band is obtained for each of HPV types 6, 11, 16, 18, 31, 33 and 35. Extra bands indicating the presence of cross-reacting HPV of undetermined type occurred in only 0.7% of the 1,268 cases tested. HPV was demonstrated in 7.8% of the 612 health control samples and in 16% of 634 consecutive samples from patients with symptoms. All but type 18 occurred more commonly in symptomatic than in asymptomatic patients. Presence of HPV also correlated to cervical intraepithelial neoplasia (CIN) in simultaneously collected smears. The HPV was demonstrated in 13% of patients with normal cytology, in 45% with CIN and in 82% of patients with invasive carcinoma. All types of HPV occurred more commonly in CIN patients, while only types 16, 18, 31 and 33 were found in the patients with cancer.

Blotting, Southern↗