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Biomedical subjects

J Vuust

Publications and source records attributed to J Vuust.

At least 19 recordsLinked to original sources
PubMed1999

High-throughput single-strand conformation polymorphism analysis by automated capillary electrophoresis: robust multiplex analysis and pattern-based identification of allelic variants.

Genetic diagnosis of an inherited disease or cancer often involves analysis for unknown point mutations in several genes; therefore, rapid and automated techniques that can process a large number of samples are needed. We describe a method for high-throughput single-strand conformation polymorphism (SSCP) analysis using automated capillary electrophoresis. The operating temperature of a commercially available capillary electrophoresis instrument (ABI PRISM 310) was expanded by installation of a cheap in-house designed cooling system, thereby allowing us to perform automated SSCP analysis at 14-45 degrees C. We have used the method for detection of point mutations associated with the inherited cardiac disorders long QT syndrome (LQTS) and hypertrophic cardiomyopathy (HCM). The sensitivity of the method was 100% when 34 different point mutations were analyzed, including two previously unpublished LQTS-associated mutations (F157C in KVLQT1 and G572R in HERG), as well as eight novel normal variants in HERG and MYH7. The analyzed polymerase chain reaction (PCR) fragments ranged in size from 166 to 1,223 bp. Seventeen different sequence contexts were analyzed. Three different electrophoresis temperatures were used to obtain 100% sensitivity. Two mutants could not be detected at temperatures greater than 20 degrees C. The method has a high resolution and good reproducibility and is very robust, making multiplex SSCP analysis and pattern-based identification of known allelic variants as single nucleotide polymorphisms (SNPs) possible. These possibilities, combined with automation and short analysis time, make the method suitable for high-throughput tasks, such as genetic screening.

Alleles↗
PubMed1999

Familial hypertrophic cardiomyopathy associated with a novel missense mutation affecting the ATP-binding region of the cardiac beta-myosin heavy chain.

Mutations in the cardiac beta -myosin heavy chain gene (MYH7), and other genes encoding cardiac sarcomere proteins may cause familial hypertrophic cardiomyopathy (F-HCM), an autosomal dominant disease, characterized by myocardial hypertrophy. We analysed the MYH7 gene in three generations of a family with one borderline and four clinically verified cases of hypertrophic cardiomyopathy, and identified a mutation in exon 7 changing the 190 arginine residue into a threonine residue. The mutation is located in the ATP-binding region of the myosin head and alters the charge in the F-helix close to the phosphate-binding P-loop. The mutation may thus interfere with the coupling between ATP-hydrolysis and the transition into mechanical energy. In conclusion, the novel Arg190Thr mutation in exon 7 of the MYH7 gene is associated with the development of symptomatic myocardial hypertrophy in adults.

Adenosine Triphosphate↗
PubMed1999

A single strand conformation polymorphism/heteroduplex (SSCP/HD) method for detection of mutations in 15 exons of the KVLQT1 gene, associated with long QT syndrome.

Congenital long QT syndrome (LQTS) is characterised by prolongation of the QT interval on ECG and cardiac arrhythmias, syncopes and sudden death. A rapid and reliable genetic diagnosis of the disease may be of great importance for diagnosis and treatment of LQTS. Mutations in the KVLQT1 gene, encoding a potassium-channel subunit of importance for the depolarisation of cardiac myocytes, is believed to be associated with 50% of all LQTS cases. Our data confirms that KvLQT1 isoform 1 is encoded by 16 exons, and not 15, as reported previously. We have used genomic DNA sequences to design intronic PCR primers for amplification of 15 exons of KVLQT1 and optimised a non-radioactive single stranded conformation polymorphism/heteroduplex (SSCP/HD) method for detection of mutations in KVLQT1. The sensitivity of the method was 100% when it was tested on 15 in vitro constructed mutants. By multiplexing the PCR amplification of KVLQT1, it is possible to cover all 15 exons in four PCR reactions.

Amino Acid Sequence↗
PubMed1999

Recessive Romano-Ward syndrome associated with compound heterozygosity for two mutations in the KVLQT1 gene.

We describe a Swedish family with the proband and his brother suffering from severe Romano-Ward syndome (RWS) associated with compound heterozygosity for two mutations in the KVLQT1 (also known as KCNQ1 and KCNA9) gene (R518X and A525T). The mutations were found to segregate as heterozygotes in the maternal and the paternal lineage, respectively. None of the heterozygotes exhibited clinical long QT syndrome (LQTS). No hearing defects were found in the proband. The data strongly indicates that the compound heterozygosity for R518X and A525T is the cause of an autosomal recessive form of RWS in this family. Our findings support the implication of a higher frequency of gene carriers than previously expected. We suggest that relatives of 'sporadic RWS' patients should be considered potential carriers, at risk of dying suddenly from drug-induced LQTS.

Child, Preschool↗
PubMed1998

Structure and tissue-specific expression of genes encoding bovine copper amine oxidases.

A cDNA coding for an enzyme belonging to the family of copper amine oxidases was cloned from a bovine lung cDNA library using a PCR approach. The nucleotide sequence of this cDNA was found to be different from that of the previously published liver cDNA encoding bovine serum amine oxidase, another copper amine oxidase. Analyses using reverse transcription followed by PCR of RNA extracted from different bovine tissues confirmed that the copper amine oxidase gene expressed in bovine liver is closely related to, but different from, the copper amine oxidase gene expressed in bovine lung, kidney, spleen and heart. Northern blotting data showed that the level of copper amine oxidase expression in liver is considerably higher than in the other tissues tested. Southern blotting analyses of bovine chromosomal DNA suggested the existence of at least three copper amine oxidase genes. Two of these genes are apparently expressed in a tissue-specific manner as outlined above. A fragment of a third copper amine oxidase gene is identified. The exon-intron organization of the bovine copper amine oxidase genes analyzed is similar to that of the related human diamine oxidase gene, except that no intron in the position equivalent to that of the third intron in the human gene is found. In the third gene, a complete replacement of the third intron of the bovine copper amine oxidase gene (equivalent to the fourth intron of the human gene) has occurred.

Amine Oxidase (Copper-Containing)↗
PubMed1998

Novel donor splice site mutation in the KVLQT1 gene is associated with long QT syndrome.

INTRODUCTION: Inherited long QT syndrome (LQTS) recently has been associated with mutations in genes coding for potassium (KVLQT1, KCNE1, and HERG) or sodium (SCN5A) ion channels involved in regulating either sodium inward or potassium outward currents of heart cells, resulting in prolongation of the repolarization period. We describe a new mutation, a -1 donor splice site mutation in a kindred with two affected members (QTc = 0.61 and 0.54 sec). METHODS AND RESULTS: Single stranded conformation polymorphism (SSCP) analyses were performed on DNA fragments amplified by polymerase chain reaction from DNA extracted from whole blood. Aberrant conformers were analyzed by DNA sequencing. SSCP analysis of the KVLQT1 gene revealed an aberrant conformer in the affected family members. DNA sequencing confirmed the presence of a G-->A change in the last nucleotide of codon 344. This mutation does not cause an amino acid change, but a change of the splice site characteristics at the 3' end of exon 6. The mutation may affect, through deficient splicing, the putative sixth transmembrane segment of the K+ channel, and this type of mutation has not previously been described in KVLQT1. CONCLUSION: The clinical course of LQTS in the affected family members, in whom no deaths occurred despite 20 to 30 syncopes, can be explained by the ability of the cellular machinery to perform partial correct splicing in the mutant allele. This type of mutation may be misinterpreted as a normal variant, since it is a point mutation causing neither an amino acid change nor the introduction of a stop codon.

Aged↗
PubMed1998

The glutamate-rich protein (GLURP) of Plasmodium falciparum is a target for antibody-dependent monocyte-mediated inhibition of parasite growth in vitro.

Monocyte-dependent as well as direct inhibitory effects of antimalarial antibodies point toward antigens accessible at the time of merozoite release as targets for biologically active antibodies capable of mediating protection against Plasmodium falciparum. The glutamate-rich protein (GLURP), being an antigen associated with mature schizont-infected erythrocytes, was therefore the object of the present investigation, in which we analyzed whether anti-GLURP antibodies can either interfere directly with merozoite invasion or act indirectly by promoting a monocyte-dependent growth inhibition, antibody-dependent cellular inhibition. GLURP-specific human immunoglobulin G (IgG) antibodies, from pooled IgG of healthy Liberian adults who were clinically immune to malaria, were purified by affinity chromatography on columns containing R0 (N-terminal nonrepetitive region of GLURP) or R2 (C-terminal repetitive region of GLURP) recombinant protein or synthetic peptides as ligands. Analysis of the pattern of reactivity of highly purified anti-GLURP antibodies led to the definition of at least four B-cell epitopes. One epitope was specific for R0, two were specific for R2, and the fourth displayed cross-reactivity between R0 and R2. None of the purified IgG antibodies had direct invasion-inhibitory effects, even at high concentrations. In contrast, when allowed to cooperate with monocytes, all anti-GLURP IgG preparations mediated a strong monocyte-dependent parasite growth inhibition in a dose-dependent manner.

Animals↗
PubMed1998

Mutation Detection by Cleavase in Combination With Capillary Electrophoresis Analysis: Application to Mutations Causing Hypertrophic Cardiomyopathy and Long-QT Syndrome.

Background: Genetic screening requires methods for mutation detection that are sensitive, precise, and robust. In population screening for complex genetic diseases in which large genes and/or several genes may be affected, automation of the mutation detection analysis is desirable. Methods and Results: The combined use of Cleavase nuclease and analysis of the DNA cleavage pattern by capillary electrophoresis was evaluated with respect to sensitivity, reproducibility, and dependency of experimental conditions in detecting mutations in the human genes MYH7 and KVLQT1 (KCNA9). The cleavage patterns obtained by capillary electrophoresis were very reproducible and robust. Conclusions: The combination of a sensitive enzymatic mutation detection method capillary electrophoresis may prove to be a useful system for automated, large-scale genetic screening.

Journal Article↗
PubMed1998

Polyamine-stimulated binding of diamine oxidase to DNA.

Diamine oxidase is a Cu-containing enzyme which intracellularly participates in the regulation of the levels of putrescine, spermidine and spermine and in this process produces growth inhibitory amino aldehydes and hydrogen peroxide. Extracellularly, the enzyme participates in the inactivation of biogenic amines, notably histamine. Here we present evidence that in the presence of polyamines, diamine oxidase has the ability to bind DNA and to oxidise DNA-bound polyamines. The enzyme associates with chromosomal DNA since it can be released from human placental DNA by treatment with DNase I and it may be involved in the degradation of DNA. Thus, diamine oxidase may belong to a new class of DNA-binding proteins.

Amine Oxidase (Copper-Containing)↗
PubMed1997

New consensus features for tyrosine O-sulfation determined by mutational analysis.

Tyrosine sulfation is an ubiquitous modification of proteins synthesized along the secretory pathway. It enhances protein-protein interactions and may be necessary for the bioactivity of secreted proteins and peptides. To predict tyrosine sulfation, a consensus has been proposed based on sequence comparisons of known substrates and on in vitro studies using synthetic peptides. This consensus predicts the presence of acidic residues on the amino-terminal side of the target tyrosine as the key feature. Using site-directed mutagenesis, we have examined the role of residues neighboring the sulfation site of an intact protein, human progastrin, in vivo. The results show that the charge of the residue in the amino-terminal position (-1) of the tyrosine is critical and can be neutral or acidic, whereas a basic residue abolishes sulfation. In addition, the degree of sulfation is influenced by the residues in positions -2 and -3. Hence, surprisingly a basic residue in position -2 enhances sulfation. Our data suggest a considerably broader range of substrates for the tyrosylprotein sulfotransferase than hitherto assumed and that the tyrosylprotein sulfotransferase is cell-specifically expressed.

Amino Acid Sequence↗
PubMed1997

High-throughput analysis of fragile X (CGG)n alleles in the normal and premutation range by PCR amplification and automated capillary electrophoresis.

Fragile X syndrome is caused by expansion of a (CGG)n trinucleotide repeat within the 5' untranslated region of the FMR1 gene transcript. The disease is reliably diagnosed by Southern blotting, but this method constitutes a significant workload and requires large samples. Therefore, for large research or screening projects in which a large majority of the samples will be normal, a more rapid and less expensive method is needed. We present a method for accurate, high-throughput analysis of the FRAXA (CGG)n region in the normal and premutation range. The method is based on polymerase chain reaction (PCR) amplification of DNA extracted from whole blood or eluted from dried blood spots on filter-paper, followed by automated capillary electrophoresis and detection by multicolour fluorescence. This method allows a throughput of 144 samples in 48 h, with an intra-assay accuracy in size determination of 0.2-1.8 bp. We performed a blind reanalysis of samples from 30 patients, previously analysed by Southern blotting or PCR with radioactive labelling. In this study normal and premutation alleles, ranging from 28-121 (CGG)n repeats, were correctly determined with respect to number of (CGG)n repeats. All full-mutation alleles and one large premutation allele in a sample of a heterozygote failed to amplify. The method was used to determine the distribution of FRAXA (CGG)n repeats in the Danish population.

Alleles↗
PubMed1996

An efficient cellular system for mutational analysis of prohormone processing.

A novel system for heterologous expression of prohormones based on transient transfection of the HIT beta-cell line was established using human progastrin as a model. Progastrin was expressed at high levels compared to other gene transfer systems in endocrine cells, and the processing pattern was similar to that of normal antral gastrin cells. Thus, gastrin was partially tyrosine O-sulfated and carboxyamidated. Cell extracts contained mainly gastrin-17 and gastrin-34 and the corresponding glycine-extended forms. In contrast, the media contained more incompletely processed gastrin forms. This suggests that gastrin was directed to the regulated secretory pathway but that some progastrin products were constitutively secreted. Glucose increased both the level of gastrin gene expression and maturation to carboxyamidated peptides, indicating that glucose influences the activity of the amidation enzyme complex, peptidylglycine alpha-amidating mono-oxygenase (PAM), in insulin cells. Mutational analysis of tyrosine sulfation of gastrin demonstrated that substitution of the uncharged residue carboxy-terminal to the tyrosine with an acidic residue does not increase sulfation in contrast to previous results, where the amino-terminal residue was replaced with an acidic residue. The mutant peptides displayed sulfation-dependent processing, supporting our recent suggestion that tyrosine sulfation increases the proteolytic processing of prohormones.

Amino Acid Sequence↗
PubMed1996

Recombinant Toxoplasma gondii surface antigen 1 (P30) expressed in Escherichia coli is recognized by human Toxoplasma-specific immunoglobulin M (IgM) and IgG antibodies.

The immunodominant surface antigen of Toxoplasma gondii, surface antigen 1 (SAG1), was expressed in Escherichia coli as a fusion protein containing a majority of the SAG1 protein supplied with six histidyl residues in the N-terminal end. The recombinant protein was purified on a Ni-chelate column and then on a fast-performance liquid chromatography column and was in a nonreduced condition. It was recognized by T. gondii-specific human immunoglobulin G (IgG) and IgM antibodies as well as by a mouse monoclonal antibody (S13) recognizing only nonreduced native SAG1. Antibodies induced in mice by the recombinant SAG1 recognized native SAG1 from the T. gondii RH isolate in culture. Recombinant SAG1 is suitable for use in diagnostic systems for detecting anti-SAG1-specific IgG and IgM antibodies.

Animals↗
PubMed1995

Tyrosine O-sulfation promotes proteolytic processing of progastrin.

Tyrosine O-sulfation is a common post-translational modification of secretory and membrane proteins. The biological function of sulfation is known in only a few proteins, where it appears to enhance protein-protein interactions. Based on known sequences around sulfated tyrosines, a consensus sequence for prediction of target tyrosines has been proposed. However, some proteins are tyrosine sulfated at sites that deviate from the proposed consensus. Among these is progastrin. It is possible that the deviation explains the incomplete sulfation characteristic for bioactive gastrin peptides. In order to test this hypothesis, we have performed site-directed mutagenesis of the gastrin gene followed by heterologous expression in an endocrine cell line. The results show that substitution of the alanyl residue immediately N-terminal to the sulfated tyrosine with an acidic amino acid promotes the sulfation of gastrin peptides. Hence, the study supports the proposed consensus sequence for tyrosine sulfation. Importantly, however, the results also reveal that complete sulfation increases the endoproteolytic maturation of progastrin. Thus, our study suggests an additional function for tyrosine sulfation of possible general significance.

Amino Acid Sequence↗
PubMed1995

Development of a PCR-based technique for detection of Helicobacter pylori.

A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori, using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.

Adult↗
PubMed1995

Prevalence of erm gene classes in erythromycin-resistant Staphylococcus aureus strains isolated between 1959 and 1988.

The epidemiology of the two common erythromycin resistance methylase (erm) genes ermA and ermC was analyzed by Southern blotting in 428 erythromycin-resistant Staphylococcus aureus strains isolated from blood between 1959 and 1988 in Denmark. ermA and/or ermC was present in 98% of the erythromycin-resistant strains tested. ermA was found only as a chromosomal insert and was solely responsible for erythromycin resistance in these strains until about 1971. ermA was the only erm gene found in 337 strains and was a single insert in 61% of these strains, two inserts were seen in 37%, and three inserts were found in 2%. Thirteen different ermA EcoRI restriction fragment length polymorphisms were identified. ermA was not found in strains of phage type patterns group II and type 95, which are very common today. ermC was found on a plasmid in 77 strains. ermC was first seen in 1971 and spread rapidly in the S. aureus population, with a 5- to 10-fold increase every 5 years, and in 1984 to 1988, it was responsible for erythromycin resistance in 72% of the strains. The predominant plasmid carrying ermC was 2.5 kb, while four plasmids were smaller and three were larger. ermC has been found in all phage type patterns. Eight strains contained combinations of ermA and ermC, and no erm gene was detected in six strains.

Base Sequence↗
PubMed1995

Antigenicity and immunogenicity of recombinant glutamate-rich protein of Plasmodium falciparum expressed in Escherichia coli.

A recombinant Plasmodium falciparum glutamate-rich protein (GLURP) was produced in Escherichia coli as a nearly full-length protein. In order to map immunodominant regions on GLURP, the nonrepetitive amino-terminal region (R0) as well as the central repeat region (R1) and the carboxy-terminal repeat region (R2) were also produced as separate products. All four purified gene products reacted specifically with serum samples from adults living in an area of Liberia where malaria is holoendemic. It appears that the human immune response against GLURP is primarily directed against the R2 region because 94% of the serum samples reacted with this region in an immunoassay. Antibody reactivity against the R0 region was also observed in 75% of the serum samples, while the R1 region showed only weak antibody-binding activity. When the nearly full-length GLURP molecule was adsorbed to Al(OH)3 it was found to be immunogenic in mice. In these experiments, the antibody response was almost exclusively directed against the R2 region. When anti-GLURP sera were obtained from rabbits immunized with the three regions, R0, R1, and R2, respectively, they recognized in immunoprecipitation experiments authentic GLURP from P. falciparum grown in vitro. These results demonstrate that GLURP produced in E. coli can induce a humoral immune response against GLURP derived from blood-stage parasites.

Amino Acid Sequence↗