Biomedical subjects

K Kristiansen

Publications and source records attributed to K Kristiansen.

At least 19 recordsLinked to original sources

PubMed1995

Transcription in vitro of Tetrahymena class II and class III genes.

A method for preparation of transcriptionally active nuclear extracts from the ciliated protozoan Tetrahymena thermophila is described. Cells were lysed in the presence of gum arabic, and nuclei were further purified in the presence of Ficoll 400. Highly concentrated nuclear extracts were prepared by ultracentrifugation of nuclei in a buffer containing potassium glutamate and spermidine. These extracts supported accurate transcription initiation of T. thermophila class II and III genes. Using the histone H3-II gene as a template, we demonstrated that physiologically induced changes in transcriptional activity in vivo were reflected in the transcriptional activity of the nuclear extract in vitro. By electrophoretic mobility shift assays, five conserved sequence elements in the upstream region of the histone H3-II gene were shown specifically to bind proteins in extracts from exponentially growing as well as from starved cells, and by UV cross-linking we further characterized the specific binding of two proteins to an oligonucleotide containing a conserved CCAAT box motif. Transcription competition experiments showed that addition of this oligonucleotide decreased transcription significantly. Competition with oligonucleotides corresponding to the two proximal conserved sequence elements almost completely abolished transcription of the H3-II gene suggesting that binding of transacting factors to these elements is crucial for initiation of transcription.

Acetates↗

PubMed1995

The phosphorylated ribosomal protein S7 in Tetrahymena is homologous with mammalian S4 and the phosphorylated residues are located in the C-terminal region. Structural characterization of proteins separated by two-dimensional polyacrylamide gel electrophoresis.

A single basic ribosomal protein, protein S7, can be multiply phosphorylated in the ciliated protozoan Tetrahymena. Induction of phosphorylation is highly regulated, and the phosphorylation proceeds in a strictly sequential manner. The first site to be phosphorylated is a serine residue and the second a threonine. In this paper we report the complete primary structure of Tetrahymena thermophila ribosomal protein S7 including identification of the phosphorylated serine and threonine residues. Most of the sequence information was obtained from peptides generated by in situ digestion of S7 in two-dimensional gels using an approach that combined traditional protein chemistry with mass spectrometry. T. thermophila ribosomal protein S7 has a molecular mass of 29,459 Da and contains 259 amino acid residues. Phosphorylation takes place on Ser258 and Thr248 in the C-terminal region of the protein. Alignment of T. thermophila ribosomal protein S7 with known ribosomal proteins yielded the surprising result that T. thermophila S7 is homologous, not with mammalian ribosomal protein S6, but with mammalian ribosomal protein S4. These findings clearly distinguish the pattern of phosphorylation of ribosomal proteins in Tetrahymena from all other eukaryotes analyzed to date.

Amino Acid Sequence↗

PubMed1995

Genetic diagnosis with the denaturing gradient gel electrophoresis technique improves diagnostic precision in familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant inherited disorder of lipid metabolism caused by mutations in the LDL receptor gene. FH is characterized clinically by elevated LDL cholesterol level and premature coronary disease. Diagnosing FH on clinical grounds may be difficult, and previous genetic methods are too cumbersome for routine use except in the few populations with FH-founder mutations. A simple mutation screening technique based on denaturing gradient gel electrophoresis (DGGE) has been highly useful in detecting mutations in other genes, and in the present study we evaluated the diagnostic potential of this method for the diagnosis of FH. METHODS AND RESULTS: Conditions for screening exon 3 of the LDL receptor gene using the DGGE technique were established and 14 Danish FH families were examined. An index patient from 1 family had an abnormal DGGE pattern; consequently, an examination of exon 3 of the LDL receptor gene in 21 members of this patient's family was done. The DGGE pattern was seen only in patients with a definite clinical diagnosis of FH. Subsequent sequencing of exon 3 of the LDL receptor gene in these individuals revealed the presence of the French-Canadian type 4 Trp66-Gly mutation. However, in 4 of 11 cases in which a definite clinical diagnosis of FH had been made, the inheritance of the French-Canadian type 4 mutation could be rejected on the basis of genetic analysis. CONCLUSIONS: Introduction of a simple genetic analysis based on DGGE may improve the precision of diagnosis in FH families.

Adolescent↗

PubMed1995

7-Deaza purine bases offer a higher ion stability in the analysis of DNA by matrix-assisted laser desorption/ionization mass spectrometry.

Oligodeoxynucleotides which contain 7-deaza analogues of the normal purine nucleotides have been synthesized both enzymatically and chemically. When subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis, the modified samples offer both higher stability and increased sensitivity compared to otherwise identical unmodified oligodeoxynucleotides. In view of these observations, models for the fragmentation of oligodeoxynucleotides in MALDI-MS with positive ion detection mode are presented. Additionally, the potential use of 7-deaza purine nucleotides in the MALDI-MS analysis of DNA sequencing reactions is discussed.

Base Sequence↗

PubMed1995

A simple HPLC method for the isolation and quantification of the allergens tuliposide A and tulipalin A in Alstroemeria.

A practical, rapid, reliable and sensitive method for the isolation and determination of the allergens tuliposide A and alpha-methylene-gamma-butyrolactone (tulipalin A), by reversed-phase high-performance liquid chromatography (RP-HPLC), has been developed in order to select Alstroemeria species for breeding purposes. From the aqueous extracts of flowers, stems and leaves, of several Alstroemeria species, the contents of 6-tuliposide A and tulipalin A were determined by isocratic RP-HPLC, using distilled water as mobile phase. The compounds were detected by an UV detector at 208 nm. Differences in 6-tuliposide A and tulipalin A content were found among the species investigated, with the highest concentrations in stems and flowers. The absence of other tuliposides (e.g., 1-tuliposide A, 1- and 6-tuliposide B) in extracts was proven by TLC, RP-HPLC, 1H- and 13C-NMR. 6-Tuliposide A and tulipalin A were identified by 1H- and 13C-NMR and comparison with authentic material, respectively. With this HPLC method, it is possible to investigate a large number of plants for their contents of tuliposide A and tulipalin A, within a minimum of time, and to isolate them directly from aqueous extracts.

4-Butyrolactone↗

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↗

PubMed1994

Matrix assisted laser desorption/ionization mass spectrometry of enzymatically synthesized RNA up to 150 kDa.

Enzymatically synthesized RNA samples (in vitro transcripts) were analysed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Spectra of RNA up to 150 kDA (461 nucleotides) are shown. Polymerase generated sample heterogeneity and its contribution to mass resolution are discussed. A time course exonuclease digest of a 55 nt in vitro transcript was analyzed to investigate the performance of MALDI-MS on complex mixtures. Based on these data, the analysis by MALDI-MS of DNA sequencing reactions, produced by the action of an RNA polymerase, is discussed.

Base Sequence↗

PubMed1994

Yeast acyl-CoA-binding protein: acyl-CoA-binding affinity and effect on intracellular acyl-CoA pool size.

Acyl-CoA-binding protein (ACBP) is a 10 kDa protein characterized in vertebrates. We have isolated two ACBP homologues from the yeast Saccharomyces carlsbergensis, named yeast ACBP types 1 and 2. Both proteins contain 86 amino acid residues and are identical except for four conservative substitutions. In comparison with human ACBP, yeast ACBPs exhibit 48% (type 1) and 49% (type 2) conservation of amino acid residues. The amino acid sequence of S. carlsbergensis ACBP type 1 was found to be identical with the one ACBP present in Saccharomyces cerevisiae. A recombinant form of this protein was expressed in Escherichia coli and S. cerevisiae, purified, and its acyl-CoA-binding properties were characterized by isoelectric focusing and microcalorimetric analyses. The yeast ACBP was found to bind acyl-CoA esters with high affinity (Kd 0.55 x 10(-10) M). Overexpression of yeast ACBP in S. cerevisiae resulted in a significant expansion of the intracellular acyl-CoA pool. Finally, Southern-blotting analysis of the two genes encoding ACBP types 1 and 2 in S. carlsbergensis strongly indicated that this species is a hybrid between S. cerevisiae and Saccharomyces monacensis.

Acyl Coenzyme A↗

PubMed1994

Comparison of IR- and UV-matrix-assisted laser desorption/ionization mass spectrometry of oligodeoxynucleotides.

UV-matrix assisted laser desorption/ionization mass spectrometry (UV-MALDI-MS) with 3-hydroxypicolinic acid as matrix and IR-MALDI-MS with succinic acid as matrix have proved their feasibility for highly accurate and sensitive mass determination of nucleic acids (DNA and RNA). In this work, a detailed comparison of these two MALDI-methods and between positive- and negative ion mass spectra for the analysis of oligodeoxynucleotides is undertaken. Mass spectra of DNA sequences with up to 40 nucleotides are shown. Both linear and reflectron time-of-flight mass analyzers were used within this study and are compared for their potential in the MALDI analysis of oligodeoxynucleotides. The role of molecule-ion fragmentation is also discussed.

Base Sequence↗

PubMed1994

Acyl-CoA-binding protein (ACBP) can mediate intermembrane acyl-CoA transport and donate acyl-CoA for beta-oxidation and glycerolipid synthesis.

The dissociation constants for octanoyl-CoA, dodecanoyl-CoA and hexadecanoyl-CoA binding to acyl-CoA-binding protein (ACBP) were determined by using titration microcalorimetry. The KD values obtained, (0.24 +/- 0.02) x 10(-6) M, (0.65 +/- 0.2) x 10(-8) M and (0.45 +/- 0.2) x 10(-13) M respectively, were much lower than expected. ACBP was able to extract hexadecanoyl-CoA from phosphatidylcholine membranes immobilized on a nitrocellulose membrane. The acyl-CoA/ACBP complex formed was able to transport acyl-CoA to mitochondria or microsomes in suspension, or to microsomes immobilized on a nitrocellulose membrane, and to donate them to beta-oxidation or glycerolipid synthesis in mitochondria or microsomes, respectively.

Acyl Coenzyme A↗

PubMed1994

Turnover of acyl-CoA-binding protein in four different cell lines measured by using two-dimensional polyacrylamide-gel electrophoresis.

Acyl-CoA-binding protein (ACBP), also named diazepam-binding inhibitor or endozepine, is a 10 kDa protein for which a surprisingly large number of biological activities has been suggested. Some of these would seem to require a rapid intracellular turnover of the protein. In this paper we report on the turnover of ACBP in cell lines derived from mouse, rat and man. ACBP was identified in two-dimensional gels by using specific antibodies. Cells were labelled with [35S]methionine and chased for various periods of time. Total protein was extracted, subjected to two-dimensional PAGE, and radioactivity in the spot containing ACBP was determined by liquid-scintillation counting. ACBP half-life was determined, and varied from 25 to 53 h depending on the cell line and the growth conditions. In all cases, radioactivity in ACBP was lost slightly faster than radioactivity in total protein. These results are discussed in relation to the possible function suggested for ACBP.

3T3 Cells↗

PubMed1994

Detection of a single base deletion in codon 424 of the low density lipoprotein receptor gene in a Danish family with familial hypercholesterolemia.

We performed a screening of exon 9 of the low density lipoprotein receptor (LDLR) gene in 14 Danish families with familial hypercholesterolemia (FH) using the denaturing gradient gel electrophoresis (DGGE) technique. In one of the probands from these families an abnormal band pattern in the gradient gel was detected. Subsequent DGGE analysis of the family of this index patient revealed that the DGGE pattern cosegregated with the disease in this family. Sequencing of the exon showed a deletion of a C in codon 424 of the LDLR gene resulting in a frame shift with the introduction of a stop codon 5 codons further downstream. The mutation is referred to as FH-Odense. The predicted truncated receptor protein consists of the 428 amino terminal amino acids. Consequently, the cytosolic and membrane spanning parts of the mature LDL receptor, which normally secure the receptor in the plasma membrane, are missing. The FH-Odense mutation results in severe premature coronary atherosclerosis as shown by the clinical expression in 5 generations of the affected family.

Adolescent↗

PubMed1993

[Jan Evangelista Purkinje (1787-1869)].

The author reviews the life and work of the physiologist Jan Evangelista Purkinje. In addition to his remarkable achievements as a scientist, Purkinje played a distinctive role in the struggle to establish national and cultural independence for the Czech people.

Austria↗

PubMed1993

Ion stability of nucleic acids in infrared matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with infrared laser light of a wavelength of 2.94 microns has been used for the analysis of nucleic acids. Spectra of oligodeoxynucleotides up to 26 nucleotides, oligothymidylic acids up to 100 nucleotides as well as different synthetic RNA oligomers and RNA transcripts up to 104 nucleotides are presented. A main problem in the analysis of oligodeoxynucleotides was found to be related to the loss of bases. The stability of oligothymidylic acids as opposed to oligodeoxynucleotides containing all four bases indicates that the loss of bases is correlated with A, C and G protonation which decreases the stability of the N-glycosidic bond. Experiments indicate that the breakage of the N-glycosidic bond probably occurs during the desorption process due to proton transfer from the phosphodiester groups to the ionizable bases. RNA displayed a significantly higher stability in MALDI-MS due to the presence of a 2'-OH group. Consequently, signals of RNA transcripts with a length of up to 142 nucleotides could be detected by MALDI-MS. Technical details of the method, including the distribution of positive counterions on the phosphodiester backbone, the upper mass limit and mass accuracy are discussed along with a number of potential analytical applications.

Base Sequence↗

PubMed1992

[La pierre de la folie. The stone of madness].

During the 16th and 17th centuries the expression "to have a stone in the head" was synonymous with peculiar behaviour or mental symptoms. Many quacksalvers earned their living by treating such patients with a scalp incision, pretending to remove a stone. Some of the great painters from this period have used the subjects as models for their works of art. A series of paintings of this kind have been traced in different museums, catalogues and publications, and are described, followed by a short survey of popular beliefs and superstitions connected with stones.

History, 15th Century↗

PubMed1992

Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles.

The F-antigen is a prominent liver protein which has been extensively used in studies on natural and induced immunological tolerance. However, its intracellular localization and biological function have remained elusive. It has generally been assumed that the F-antigen is confined phylogenetically to vertebrates. Now we have cloned and characterized a gene from the ciliated protozoan Tetrahymena thermophila encoding a protein which clearly is homologous with the rat F-antigen. The coding region of the Tetrahymena F-antigen (TF-ag) gene specifies a 46,051 M(r) protein and is interrupted by three introns. In accordance with the predicted molecular mass of the TF-ag protein, antibodies raised against a cro-lacZ'-TF-ag fusion protein specifically recognized a 45,000 M(r) protein in Western blots of total T. thermophila protein. Immunoelectron microscopy demonstrated that the TF-ag is associated with membranes of the Golgi apparatus and transport vesicles pointing to a role of TF-ag in membrane trafficking. Transcription of the TF-ag gene, as determined by run-on analyses, was only detectable in growing cells, and following transfer to starvation condition pre-existing TF-ag mRNA was rapidly degraded. The abundance of the TF-ag protein, however, declined only moderately during prolonged periods of starvation demonstrating that extensive release of the TF-ag did not take place. In combination these results suggest that the TF-ag protein is a recycled constituent of the intracellular membrane network in T. thermophila.

Amino Acid Sequence↗

PubMed1992

Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes. A typical housekeeping gene family.

Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous modulator of the GABAA receptor in brain membranes. ACBP/DBI, or proteolytically derived polypeptides of ACBP/DBI, have also been implicated in the control of steroidogenesis in mitochondria and glucose-stimulated insulin secretion. Thus, it appears that ACBP/DBI is a remarkable, versatile protein. Now we have molecularly cloned and characterized the ACBP/DBI gene family in rat. The rat ACBP/DBI gene family comprises one expressed gene and four processed pseudogenes of which one was shown to exist in two allelic forms. The expressed gene is organized into four exons and three introns. There is a remarkable correspondence between the structural modules of ACBP/DBI as determined by 1H nuclear magnetic resonance spectroscopy and the exon-intron architecture of the ACBP/DBI gene. Detailed analyses of transcription of the ACBP/DBI gene in brain and liver were performed to map transcription initiation sites and to examine if transcripts from the ACBP/DBI gene were subject to alternative processing. In both brain and liver, transcription is initiated from two major and multiple minor initiation sites. No evidence for alternative splicing was obtained. The promoter region of the ACBP/DBI gene is located in a CpG island and lacks a canonical TATA box. Thus, the ACDB/DBI gene exhibits all the hallmarks of a typical housekeeping gene.

Animals↗

PubMed1992

Chromatin structure and conserved sequence elements in genes encoding ribosomal proteins in Tetrahymena thermophila.

The chromatin structure of the macronuclear genes encoding ribosomal proteins S25 and L1 in the ciliated protozoan Tetrahymena thermophila was analyzed. Using the indirect end-labelling technique, DNase-I-hypersensitive regions were located in the promoter regions as well as in the 3' regions of the genes. The DNase-I-hypersensitive regions were present in chromatin of exponentially growing cells, where the rate of ribosomal-protein gene transcription is high, and in chromatin from starved cells, where transcription of ribosomal-protein genes is severely depressed. Micrococcalnuclease-digestion experiments revealed that the promoter regions of the S25 gene and the L1 gene are devoid of nucleosomes in exponentially growing cells. In starved cells, no nucleosomal organisation of the promoter region of the L1 gene could be detected, whereas nucleosomal structures were discernible in the promoter region of the S25 gene. A conspicuous polypurine sequence motif, AARGGGAAA, is present within or adjacent to the DNase-I-hypersensitive regions in the promoter of the S25 and the L1 gene, and interestingly, the same motif is found also in the promoter regions of the genes encoding ribosomal proteins L21 and L37.

Animals↗