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Biomedical subjects

H Söderlund

Publications and source records attributed to H Söderlund.

At least 19 recordsLinked to original sources
PubMed1996

Acetyl xylan esterase from Trichoderma reesei contains an active-site serine residue and a cellulose-binding domain.

The axe1 gene encoding acetyl xylan esterase was isolated from an expression library of the filamentous fungus Trichoderma reesei using antibodies raised against the purified enzyme. Apparently axe1 codes for the two forms, pI 7 and pI 6.8, of acetyl xylan esterase previously characterized. The axe1 encodes 302 amino acids including a signal sequence and a putative propeptide. The catalytic domain has no amino acid similarity with the reported acetyl xylan esterases but has a clear similarity, especially in the active site, with fungal cutinases which are serine esterases. Similarly to serine esterases, the axe1 product was inactivated with phenylmethylsulfonyl fluoride. At its C-terminus it carries a cellulose binding domain of fungal type, which is separated from the catalytic domain by a region rich in serine, glycine, threonine and proline. The binding domain can be separated from the catalytic domain by limited proteolysis without affecting the activity of the enzyme towards acetylated xylan, but abolishing its capability to bind cellulose.

Acetylesterase↗
PubMed1996

Yeast protein translocation complex: isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61 beta subunit.

A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion-defective sec15-1 mutation. This gene encodes a protein homologous to the beta subunit of the mammalian Sec61 protein complex functioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane-spanning region at the C-terminus but no N-terminal signal sequence. Seb1p shows 30% identity to the mammalian Sec61 beta subunit and 34% identity to the Arabidopsis thaliana Sec61 beta subunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61-2 and sec61-3 mutants. Immunofluorescence and immunoelectron microscopy indicated that Seb1p resides in the ER membranes with the hydrophilic N-terminus exposed to the cytoplasm. The in vitro translated Seb1p was post-translationally inserted into microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterologous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a protein 53% identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature-sensitive phenotype. This study further emphasizes the evolutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis.

Amino Acid Sequence↗
PubMed1995

Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment.

Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are ready targets for labelling by an isothiocyanate derivative of the europium chelate (Eu3+). Here the labelling efficiency of a recombinant anti-human alpha-fetoprotein (hAFP) Fab fragment has been improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues, R154, R187 and R210, which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment and its labelling efficiency was found to be approximately 40% higher than that of the wild-type Fab fragment. With low degree of labelling, the affinities of the two Fab fragments were identical and comparable with that of the original monoclonal anti-hAFP IgG. With a higher degree of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. Electrostatic adsorption and covalent immobilization of the Fab fragments were characterized by BIAcore and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.

Amino Acid Sequence↗
PubMed1995

Properties of a single-chain antibody containing different linker peptides.

Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM. The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases. Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity. The fragments with linkers longer than 11 residues remained monomeric.

Amino Acid Sequence↗
PubMed1994

Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells.

Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.

Animals↗
PubMed1993

Quantification of mitochondrial DNA carrying the tRNA(8344Lys) point mutation in myoclonus epilepsy and ragged-red-fiber disease.

Myoclonus epilepsy and ragged-red-fiber syndrome (MERRF) is caused by a point mutation at nucleotide 8344 in the tRNA(Lys) gene of mitochondrial DNA. We analyzed leukocyte DNA from nine members of a large MERRF family using a new technique, solid-phase minisequencing. Quantitative analysis of the tRNA(8344Lys) mutation showed that the mutated mtDNA comprised from 9 to 72% of the total mtDNA in the leukocytes of these individuals. The minisequencing method is a promising tool for the diagnosis of MERRF. In addition to the identification of the tRNA(8344Lys) mutation, the relative amount of mutated mtDNA can be simultaneously determined in the same assay from one blood sample.

Age Factors↗
PubMed1993

Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates.

Present methods for quantification of hepatitis B virus (HBV) particles from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DNA in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers, one of which is biotinylated. The two biotinylated products can be quantified by hybridization on microplates coated with streptavidin, because their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantification of the original amount of HBV without tedious titrations of each sample with competitor. The lower limit for quantitative analysis with radioactive probes was between 4 and 40 virus particles in a 10-microliters serum samples.

Bacterial Proteins↗
PubMed1992

N-ras gene mutations in acute myeloid leukemia: accurate detection by solid-phase minisequencing.

Mutations in the N-ras gene are found in one-third of patients with acute myeloid leukemia. The N-ras mutations could serve as markers for residual cells, if a highly sensitive method for detecting the mutations was available. We applied a new method, solid-phase minisequencing, to analyze bone-marrow cells from 16 patients with acute myeloid leukemia for mutations in codon 12, 13 and 61 of the N-ras gene. In the solid-phase minisequencing technique the mutations are identified by a primer extension reaction, in which a single labelled nucleoside triphosphate is incorporated into an immobilized DNA fragment previously amplified by the polymerase chain reaction. We identified N-ras mutations in 5 of the patients (30%). In one patient, we observed 2 mutations that were shown to be located in different alleles. With the solid-phase minisequencing method, we were able to determine the proportion of mutated cells in the samples. We found that in 4 of the samples only a fraction (7-64%) of the blasts carried an N-ras mutation, and in one sample practically all blast cells were mutated. The method was highly sensitive, allowing us to identify N-ras mutations even when the sample consisted of 99.7% normal cells and only 0.3% mutated blasts.

Base Sequence↗
PubMed1992

Detection of Plasmodium falciparum DNA in a microtitration plate-based hybridization test.

A rapid DNA-test, depending on the affinity based hybrid collection principle, was developed for the detection of Plasmodium falciparum DNA from clinical specimens. In this method, hybridization takes place in solution and the hybrids are collected onto a solid phase for measurement. Two probes are used, one labelled with an affinity tag (biotin) and the other with a detectable label (32P). In the present test a single oligonucleotide complementary to a 21-base pair sequence which is highly repeated in the parasite genome served both as capture and detector probe. The test is a 2-h hybridization performed in streptavidin coated microtitration plate wells, onto which the labelled hybrids simultaneously bind. The sensitivity of the assay with a crude erythrocyte lysate specimen was 1.6 x 10(9) repeat units corresponding to about 160 parasites in one microliter blood. The results allowed quantification of the repeat sequences and thus estimation of the degree of parasitemia in clinical specimens.

Animals↗
PubMed1992

Convenient and quantitative determination of the frequency of a mutant allele using solid-phase minisequencing: application to aspartylglucosaminuria in Finland.

Aspartylglucosaminuria (AGU) is a recessively inherited lysosomal disease caused by inadequate aspartylglucosaminidase (AGA) activity. The disease is prevalent in the genetically isolated Finnish population. We have used a new method, solid-phase minisequencing, to determine the frequency of two missense mutations in the AGA gene in this population. In samples from 70% of the Finnish AGU families, we found that the two nucleotide changes were always associated, and they were identified in 98% of the AGU alleles analyzed. Thus, the high prevalence of AGU in the Finnish population is the consequence of a founder effect of one ancient mutation. The identification of asymptomatic carriers by the minisequencing test proved to be unequivocal. The method also allowed quantification of a mutated nucleotide sequence present in less than 1% of a sample. The frequency of AGU carriers in this population was 1/36 when estimated by quantifying the mutated AGU allele in a pooled leukocyte sample from 1350 normal Finnish individuals.

Acetylglucosamine↗
PubMed1992

Screening for defined cystic fibrosis mutations by solid-phase minisequencing.

We have developed a rapid method for the quantitative detection of point mutations and deletions. In this minisequencing method, enzymatically amplified DNA, 5'-biotinylated in one strand, is bound to a solid phase and denatured. A detection primer, constructed to end immediately before the mutation, is annealed to the immobilized single-stranded template and elongated with a single, labeled deoxynucleoside residue. We have applied the solid-phase minisequencing method to the detection of the major mutation, delta F508, causing cystic fibrosis (CF). In the presence of the allele with the delta F508 mutation, [3H]dTTP is incorporated; with the nonmutated allele, [3H]dCTP is incorporated. Thus, samples from heterozygous individuals allow the incorporation of both labels. The method was evaluated by analyzing 59 coded DNA specimens collected from 20 Finnish CF patients and their parents. The ratio of [3H]C to [3H]T gave unambiguously the allele combination. The solid-phase minisequencing method was also applicable to the analysis of three CF mutations simultaneously, i.e., delta F508, G542X, and G551D. We conclude that the microtiter-plate-based minisequencing test is an accurate method for the screening of defined sequence alterations in the CF gene.

Base Sequence↗
PubMed1991

Rapid detection of a plant virus by solution hybridization using oligonucleotide probes.

A novel solution hybridization method for the diagnosis of a plant virus was evaluated. Synthetic oligonucleotide probes were used for the detection of potato virus X (PVX) in crude leaf sap extracts by hybridization in solution. Three 40-nucleotide-long oligonucleotide probes complementary to RNA sequences of potato virus X near the 3' end were synthesized. Two probes were 32P-labelled and one biotinylated. The three probes were allowed to form hybrids with the target viral nucleic acid in solution, and the formed hybrids were isolated with the aid of the biotinylated capture probe using avidin polystyrene beads after the reaction. Alternatively, hybrids were captured from the poly(A) tail of the viral RNA on oligo(dT) cellulose. The maximum signal was obtained after 4 h hybridization. About 70% of the maximum signal was obtained after 2 h hybridization. Sensitivity with the two 32P-labelled oligonucleotide probes was 1-5 x 10(7) molecules of PVX RNA. This corresponds to 0.6-3 ng of the virus. Crude leaf sap did not interfere with the detection of the virus. These results suggest that this solution hybridization method permits rapid detection of a plant virus in crude plant sap without sample pretreatment and may thus open new avenues for the development of a nucleic-acid-based ELISA-like diagnostic test for the detection of plant viruses.

Enzyme-Linked Immunosorbent Assay↗
PubMed1991

Genetic analysis of the polymorphism of the human apolipoprotein E using automated solid-phase sequencing.

A direct sequencing approach has been used to analyze the polymorphism in the human apolipoprotein E gene. A method is described, in which the DNA is amplified by the polymerase chain reaction, immobilized, and sequenced by a semi-automatic procedure adaptable to clinical diagnosis. The three alleles of the apolipoprotein E gene, which differ from each other by two nucleotide substitutions and which influence serum cholesterol levels, were analyzed. The solid-phase method was able to resolve the correct nucleotide sequence in samples from both homozygous and heterozygous individuals. No cloning steps are needed and the immobilization and separation of the DNA is accomplished using magnetic beads.

Apolipoproteins E↗
PubMed1990

The Semliki-Forest-virus-specific nonstructural protein nsP4 is an autoproteinase.

The Semliki-Forest-virus-specific nonstructural proteins are translated as a large polyprotein (2431 amino acid residues), from which the mature polymerase components nsP1, nsP2, and nsP4 are released by proteolytic cleavages. The complete ns polyprotein (P1234) can be cleaved in two alternative ways yielding either P123 (with sequences of nsP1, nsP2 and nsP3) and nsP4 or P12 (nsP1 plus nsP2) and P34 (nsP3 plus nsP4). We studied the possible autoproteolytic role of nsP4 involved in the cleavage between nsP3 and nsP4 in an in vitro transcription-translation system. cDNAs encoding P34 precursor and shorter precursor protein segments covering the nsP3-nsP4 cleavage region, were cloned under the T7 RNA polymerase promoter. The mRNAs synthesized in vitro were capped and translated in rabbit reticulocyte lysates. The translational products were analyzed by SDS/PAGE. The precursor proteins containing nsP4 sequences were cleaved yielding the products with expected sizes, indicating that the cleavage took place at the nsP3-nsP4 junction. By deleting and truncating the cDNA coding for nsP4, the proteolytic activity was mapped within the 102 amino-terminal amino acids of nsP4. The cleavage between nsP3 and nsP4 can be inhibited by pepstatin A and probably takes place in cis, since exogenously added nsP4 was unable to mediate it.

Amino Acid Sequence↗
PubMed1990

A solution hybridization method for quantification of mRNAs: determining the amount and stability of oncogene mRNA.

A solution hybridization method for the quantification of specific mRNAs is described. This assay utilizes complementary RNA probes prepared by in vitro transcription, sandwich hybridization in solution, and affinity-based hybrid collection. The possibility of using this method for crude biological samples without purifying mRNAs makes it ideal when accurate quantification of multiple samples is needed. Human N-myc oncogene transcript was used as a model and as little as 0.24 pg (2 X 10(5) molecules) of N-myc mRNA could be detected. Using this assay it was shown that human neuroblastoma IMR-32 cells contain approximately 500 N-myc mRNA molecules per cell having a half-life of approximately 35 min.

DNA Probes↗
PubMed1990

A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E.

We describe a new technique by which single base changes in human genes can be conveniently detected. In this method the DNA fragment of interest is first amplified using the polymerase chain reaction with an oligonucleotide primer biotinylated at its 5'-end. The amplified 5'-biotinylated DNA is immobilized on an avidin matrix and rendered single-stranded. The variable nucleotide in the immobilized DNA is identified by a one-step primer extension reaction directed by a detection step primer, which anneals to the DNA immediately upstream of the site of variation. In this reaction a single labeled nucleoside triphosphate complementary to the nucleotide at the variable site is incorporated. The method is highly sensitive, allowing the use of nucleoside triphosphates labeled with radioisotopes of low specific activity (3H) as well as nonradioactive markers (digoxigenin). The procedure consists of few and simple operations and is thus applicable to the analysis of large numbers of samples. Here we applied it to the analysis of the three-allelic polymorphism of the human apolipoprotein E gene. We were able to correctly identify all possible combinations of the three apo E alleles.

Apolipoproteins E↗