PubMed Health⌕ Search

Biomedical subjects

N Gregersen

Publications and source records attributed to N Gregersen.

At least 91 records · Page 5Linked to original sources
PubMed1995

Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene.

We identified three overlapping human expressed sequence tags with significant homology to the E. coli ClpP amino sequence by screening the EMBL nucleotide database. With this sequence information we applied 5' and 3'-rapid amplification of cDNA ends (RACE) to amplify and sequence human clpP cDNA in two overlapping fragments. The open reading frame encodes a 277 amino acid long precursor polypeptide. Two ClpP specific motifs surrounding the active site residues are present and extensive homology to ClpP's from other organisms was observed. Northern blotting showed high relative expression levels of clpP mRNA in skeletal muscle, intermediate levels in heart, liver and pancreas, and low levels in brain, placenta, lung and kidney. By analysis of human/rodent cell hybrids the human clpP gene was assigned to chromosome 19.

Adenosine Triphosphatases↗
PubMed1995

Serotonin depletion decreases serotonin transporter mRNA levels in rat brain.

In order to study the impact of serotonin depletion on gene expression of the serotonin transporter (5-HTt) we measured 5-HTt mRNA levels by Northern blot in rats treated with p-chlorophenylalanine methyl ester (PCPA) for 10 days. Six rats received PCPA i.p. only, and another 6 rats receiving 0.9% NaCl served as controls. An additional group of 6 rats received both PCPA i.p. and imipramine, 5 mg/kg/day by osmotic minipumps. 5-HTt mRNA levels decreased to 81.1% (P = 0.05) and 76.0% (P = 0.05) of the control level for PCPA treated animals without and with concomitant imipramine treatment, respectively. The average level of the PCPA treated groups was 78.6% (P = 0.03). The isolated effect of 21 days of imipramine treatment was a 5-HTt mRNA level of 89.4%, which was not significantly different from the control level. In conclusion, 5-HTt gene expression is suppressed in the serotonin depleted state. A decreased synaptic reuptake of 5-HT may be interpreted as a compensatory mechanism aiming at preserving adequate synaptic 5-HT levels in a generally deficient state.

Animals↗
PubMed1995

One short well conserved region of Alu-sequences is involved in human gene rearrangements and has homology with prokaryotic chi.

Alu elements have repeatedly been found involved in gene rearrangements in humans. Although these elements have been suggested to stimulate gene rearrangements, sparse information is available for the possible mechanism(s) of these events. Here we present a compilation of Alu elements that have been involved in recombinational events leading to gene rearrangements, indicating the presence of a common 26 bp core sequence at or close to the sites of recombination. Besides the obvious possibility of retrotransposition, gene rearrangements may be induced by sequences that stimulate genetic recombination. We suggest that the core sequence stimulates recombination and may thereby cause the frequent involvement of these elements in gene rearrangements. Curiously, the core sequence contains the pentanucleotide motif CCAGC, which is also part of chi, an 8 bp sequence known to stimulate recBC mediated recombination in Escherichia coli.

Base Sequence↗
PubMed1995

Comparison between medium-chain acyl-CoA dehydrogenase mutant proteins overexpressed in bacterial and mammalian cells.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a potentially lethal inherited defect in the beta-oxidation of fatty acids. By comparing the behaviour of five missense MCAD mutant proteins expressed in COS cells and in Escherichia coli, we can define some of these as "pure folding mutants." Upon expression in E. coli, these mutant proteins produce activity levels in the range of the wild-type enzyme only if the chaperonins GroESL are co-overproduced. When overexpressed in COS cells, the pure folding mutants display enzyme activities comparable to the wild-type enzyme. The results suggest that the MCAD mutations can be modulated by chaperones, a phenomenon that may influence the manifestation of the MCAD disease.

Acyl-CoA Dehydrogenase↗
PubMed1995

Prenatal diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in a family with a previous fatal case of sudden unexpected death in childhood.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a potentially fatal inherited disease with a carrier frequency of approximately 1:100 in most Caucasian populations. The disease is implicated in sudden unexpected death in childhood. A prevalent disease-causing point mutation (A985G) in the MCAD gene has been characterized, thus rendering diagnosis easy in the majority of cases. Since the clinical spectrum of MCAD deficiency ranges from death in the first days of life to an asymptomatic life, there are probably other genetic factors--in addition to MCAD mutations--involved in the expression of the disease. Thus, families who have experienced the death of a child from MCAD deficiency might have an increased risk of a seriously affected subsequent child. In such a family we have therefore performed a prenatal diagnosis on a chorionic villus sample by a highly specific and sensitive polymerase chain reaction (PCR) assay for the G985 mutation. The analysis was positive and resulted in abortion. We verified the diagnosis by direct analysis on blood spots and other tissue material from the aborted fetus and from family members.

Acyl-CoA Dehydrogenase↗
PubMed1995

A polymerase chain reaction-based method for the semiquantitative study of interleukin-8 mRNA in human basophil leukocytes.

We examined a potential method for quantitative analysis of cytokine expression patterns in purified human basophil leukocyte preparations. Basophil mRNA was reverse transcribed and cytokine cDNA levels determined by competitive polymerase chain reaction (PCR) with internal standard cDNAs constructed by site-directed mutagenesis. Co-reverse transcribed glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cDNA levels were used as an internal control to correct for unequal efficiencies in RNA isolation and reverse transcription. The method was subjected to a statistical validation giving the within series precision of the analysis. This method was used to examine interleukin-8 expression patterns in basophils from different donors. The results from this study revealed increased interleukin-8 mRNA levels after in vitro challenge with a variety of stimuli.

Base Sequence↗
PubMed1995

Complexity of molecular genetics of dyslipidemia in a family highly susceptible to ischemic heart disease.

In a Danish family highly susceptible to ischemic heart disease, hyperlipidemia did not simply cosegregate with a previously undescribed 10 bp deletion in the LDL receptor gene causing heterozygous familial hypercholesterolemia (FH). This mutation, designated as FH DK-4, deletes 10 nucleotides from exon 4 coding for the third cysteine-rich repeat of the ligand-binding domain. The resulting translational frameshift and stop codon corresponding to amino acid position 181 in the LDL receptor cDNA is predicted to result in a truncated LDL receptor protein. Several family members had hyperlipidemia and early onset of ischemic heart disease not due to the 10 bp deletion, and several family members had unexpectedly high serum lipoprotein(a) contributing to high concentrations of serum LDL cholesterol. The study illustrates important limitations and possibilities of molecular genetic diagnosis.

Base Sequence↗
PubMed1995

Improved RNase protection assay for quantifying LDL-receptor mRNA; estimation of analytical imprecision and biological variance in peripheral blood mononuclear cells.

We have improved the protocol for RNA quantification by using RNase protection. Instead of precipitation and extraction with phenol and chloroform, we use a faster and more reliable precipitation based on guanidinium thiocyanate (GdSCN). The internal standard is produced by in vitro transcription of a DNA template constructed so as to allow simultaneous detection of the in vitro transcript and the low-density lipoprotein receptor (LDLR) mRNA by use of the same probe and hybridization. Addition of this internal standard at the step for RNA isolation reduced the analytical imprecision from 40.8% to 19.3%. Estimates of the within- and between-subject biological variations of the LDLR mRNA content in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers were 21.5% and 13.6%, respectively, and the analytical imprecision was 22.6%. The mean content of LDLR mRNA in PBMCs from healthy individuals was 0.78 copies per cell.

Chemical Precipitation↗
PubMed1995

Molecular diagnosis and characterization of medium-chain acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common defect in mitochondrial beta-oxidation in humans. It is an autosomal recessive disorder which usually presents in infancy. The disease manifests itself in periods of metabolic stress to the beta-oxidation system and may be fatal. Four years ago we identified a prevalent disease-causing mutation (G985) which causes an amino acid change (K304E) in the mature MCAD protein. Using a Polymerase Chain Reaction (PCR) based assay for this mutation we have demonstrated: 1. that the G985 mutation is present in 90% of the disease alleles from patients from all over the world; 2. that the allele frequency of G985 in the general population from most European countries is very high (the carrier frequency ranges from 1/68 to 1/333); 3. that MCAD deficiency is not, as has previously been suggested, related to Sudden Infant Death Syndrome (SIDS). Moreover, investigation by Restriction Fragment Length Polymorphism (RFLP) analysis of several families with diagnosed MCAD deficiency revealed that the G985 mutation is only present in chromosomes of a particular RFLP haplotype, suggesting a common chromosomal background for this mutation. The other mutations in the MCAD gene are distributed to all known MCAD RFLP haplotypes. Because 80% of the patients are homozygous for the G985 mutation, DNA based diagnosis of most patients is now fast and easy. In order to make DNA based diagnosis possible for the remaining 20% of patients we have set up PCR/solid-phase based semi-automated sequencing of all 12 exons of the MCAD gene. We have so far identified the mutation in 33 of 45 non-G985 homozygous families with verified MCAD deficiency, thereby bringing the number of known mutations in the MCAD gene up to 26. In order to investigate in detail the molecular defects of the mutant MCAD proteins we overexpressed them in COS-7 and in an E. coli based expression system with and without co-overexpression of the molecular chaperones GroES and GroEL. The expression studies revealed that the primary effect of all the identified mutations is on formation of correct enzyme structure, and does not directly affect the catalytically active regions of the enzyme. We find that our diagnostic set up, consisting of an initial testing by the G985 assay, followed by semi-automated sequencing of DNA from those patients who were indicated to be compound heterozygous, is an important improvement to the diagnosis of MCAD deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyl-CoA Dehydrogenase↗
PubMed1994

Genotyping compared with protein phenotyping of the common apolipoprotein E polymorphism.

Apolipoprotein E (apo E) genotypes have been determined in 460 Danish men, with the use of the polymerase chain reaction (PCR) to amplify a 244 base pair fragment spanning the first-base polymorphic sites in the codons of amino acids 112 and 158 followed by restriction endonuclease cleavage. The results were compared with the apo E phenotypes previously determined by isoelectric focusing (IEF) of delipidated plasma, not pretreated with neuraminidase, followed by apo E specific immunoblotting. Conflicting results were found in only 9 cases (2.0%) and in each case only with respect to one allele. Five of the discrepancies can be explained, post hoc, by technical difficulties with the IEF method ('faint bands'). A possible cause of the other 4 discrepancies is the presence of rare mutations. Our findings in this large study are reassuring, since, if appreciable and systematic misclassification of genotypes do occur by using IEF, as has been reported from some laboratories, it may influence the validity of genetic epidemiological studies.

Adult↗
PubMed1994

Characterization of wild-type human medium-chain acyl-CoA dehydrogenase (MCAD) and mutant enzymes present in MCAD-deficient patients by two-dimensional gel electrophoresis: evidence for post-translational modification of the enzyme.

Two-dimensional gel electrophoresis was used to study and compare wild-type medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) and mis-sense mutant enzyme found in patients with MCAD deficiency. By comparing the patterns for wild-type and mutant MCAD expressed in Escherichia coli or in eukaryotic COS-7 cells we demonstrate that variants with point mutations changing the net charge of the protein can be readily resolved from the wild-type protein. After expression of the cDNA in eukaryotic cells two spots representing mature MCAD can be distinguished, one with an isoelectric point (pI) corresponding to that obtained for the mature protein expressed in E. coli and another one shifted to lower pI. This demonstrates that MCAD protein is partially modified after transport into the mitochondria and removal of the transit peptide. The observed pI shift would be compatible with phosphorylation of one aspartic acid residue per monomer. Comparison of pulse labeling and steady-state amounts of MCAD protein in overexpressing COS-7 cells confirms that K304E MCAD is synthesized and transported into mitochondria in amounts similar to the wild-type protein, but is degraded much more readily. For wild-type MCAD, the spot representing the nonmodified form predominates after pulse labeling while that representing the modified form is relatively stronger in steady state, demonstrating that the modification occurs in mitochondria after the transit peptide has been removed. For K304E mutant MCAD, the nonmodified spot is relatively stronger both in pulse labeling and in steady state, indicating that either the efficiency of modification or the stability of the modified form is affected by the K304E mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl-CoA Dehydrogenase↗
PubMed1994

Molecular genetic characterization and urinary excretion pattern of metabolites in two families with MCAD deficiency due to compound heterozygosity with a 13 base pair insertion in one allele.

Two families with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency due to compound heterozygosity are described. All patients have a 13 bp insertion in exon 11 of one allele at the MCAD gene locus. In the other allele patients in one of the families harbour the prevalent G985 mutation, and the other family possess an unidentified mutation causing reduced levels of MCAD mRNA. We demonstrate that the disease in these families is inherited as an autosomal recessive trait. Individuals heterozygous for the mutations show heterozygous/control levels of beta-oxidation activities in cultured fibroblasts (9.1-16.3 pmol/min per mg protein; control 10-17 pmol/min per mg protein), and in the excretion of the 'beta-oxidation metabolites', hexanoylglycine (< 2 mumol/mmol creatinine), suberylglycine (< 2 mumol/mmol creatinine) and phenylpropionylglycine (< 2 mumol/mmol creatinine). This shows that there is no 'negative dominance' from the mutant monomeric protein onto the normal ones, in accordance with the finding of low levels of MCAD mRNA from the allele harbouring the 13 bp insertion as well as the allele with the unidentified mutation, and the low steady-state level of enzyme protein expressed from the G985-bearing allele. In the family possessing the G985 and the 13 bp insertion mutations, two asymptomatic compound heterozygous individuals were detected. They exhibited elevated excretion of hexanoylglycine (5-15 mumol/mmol creatinine) and suberylglycine (4-13 mumol/mmol creatinine), together with beta-oxidation activity in fibroblasts in the homozygous range (2.9 pmol/min per mg protein), showing a lack of correlation between the genotype, some biochemical parameters and the clinical phenotype.

Acyl-CoA Dehydrogenase↗
PubMed1994

Apolipoprotein B gene polymorphisms in ischemic heart disease and hypercholesterolemia: effects of age and sex.

The association of polymorphic alleles of the apolipoprotein B gene (Insertion/Deletion-, XbaI-, MspI-, EcoRI-, and 3'-VNTR polymorphisms) with variation in lipid concentrations (total cholesterol (T-C), HDL cholesterol (HDL-C), and log-triglycerides (TG)) in plasma was studied in 259 men and 59 women with moderate hypercholesterolemia (T-C 5.5-8.0 mmol/l and TG < 2.5 mmol/l) and ischemic heart disease, especially in relation to the effect of sex and age. The XbaI and the Ins/Del polymorphic alleles were associated with variation in T-C, but only in patients below the 75th percentile for age. The XbaI and Ins/Del polymorphic alleles were synergistically associated with variation in T-C: the X+ and the Del alleles were associated with higher cholesterol concentrations. Younger male patients had the highest frequency of haplotypes including both the X+ and the Del alleles, but the most striking difference was a significantly higher frequency of haplotypes including both the X- and the Ins alleles in female and in older male patients. The heterogeneity of association of polymorphic alleles in the apolipoprotein B gene to complex traits like hypercholesterolemia and ischemic heart disease in this study could explain why in most studies the X+ allele has been associated with higher cholesterol levels, whereas the X- allele has been associated with symptomatic atherosclerosis. The results of our study emphasize the importance of age and sex in measured genotype association studies.

Adult↗