Search PubMed⌕ Search

Biomedical subjects

K Kristiansen

Publications and source records attributed to K Kristiansen.

At least 37 records · Page 2Linked to original sources
PubMed2000

Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm.

Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty acids are activated to CoA esters, which bind with high affinity to the acyl-CoA-binding protein (ACBP). Thus, the availability of known and potential PPAR ligands may be regulated by lipid-binding proteins. In this report we show by transient transfection of CV-1 cells that coexpression of ACBP and adipocyte lipid-binding protein (ALBP) exerts a ligand- and PPAR subtype-specific attenuation of PPAR-mediated trans-activation, suggesting that lipid-binding proteins, when expressed at high levels, may function as negative regulators of PPAR activation by certain ligands. Expression of ACBP, ALBP, and keratinocyte lipid-binding protein (KLBP) is induced during adipocyte differentiation, a process during which PPARgamma plays a prominent role. We present evidence that endogenous ACBP, ALBP, and KLBP not only localize to the cytoplasm but also exhibit a prominent nuclear localization in 3T3-L1 adipocytes. In addition, forced expression of ACBP, ALBP, and KLBP in CV-1 cells resulted in a substantial accumulation of all three proteins in the nucleus. These results suggest that lipid-binding proteins, contrary to the general assumption, may exert their action in the nucleus as well as in the cytoplasm.

3T3 Cells↗
PubMed1999

Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein.

In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importance of these 26 sequence positions for structure, function, stability, and folding has been analyzed using single-site mutations. A total of 28 mutant proteins were analyzed which covered 17 conserved sequence positions and three nonconserved positions. As a first step, the influence of the mutations on the protein folding reaction has been probed, revealing a folding nucleus of eight hydrophobic residues formed between the N- and C-terminal helices [Kragelund, B. B., et al. (1999) Nat. Struct. Biol. (In press)]. To fully analyze the role of the conserved residues, the function and the stability have been measured for the same set of mutant proteins. Effects on function were measured by the extent of binding of the ligand dodecanoyl-CoA using isothermal titration calorimetry, and effects on protein stability were measured with chemical denaturation followed by intrinsic tryptophan and tyrosine fluorescence. The sequence sites that have been conserved for direct functional purposes have been identified. These are Phe5, Tyr28, Tyr31, Lys32, Lys54, and Tyr73. Binding site residues are mainly polar or charged residues, and together, four of these contribute approximately 8 kcal mol-1 of the total free energy of binding of 11 kcal mol-1. The sequence sites conserved for stability of the structure have likewise been identified and are Phe5, Ala9, Val12, Leu15, Leu25, Tyr28, Lys32, Gln33, Tyr73, Val77, and Leu80. Essentially, all of the conserved residues that maintain the stability are hydrophobic residues at the interface of the helices. Only one conserved polar residue, Gln33, is involved in stability. The results indicate that conservation of residues in homologous proteins may result from a summed optimization of an effective folding reaction, a stable native protein, and a fully active binding site. This is important in protein design strategies, where optimization of one of these parameters, typically function or stability, may influence any of the others markedly.

Acyl Coenzyme A↗
PubMed1999

Activation of peroxisome proliferator-activated receptor gamma bypasses the function of the retinoblastoma protein in adipocyte differentiation.

The retinoblastoma protein (pRB) is an important regulator of development, proliferation, and cellular differentiation. pRB was recently shown to play a pivotal role in adipocyte differentiation, to interact physically with adipogenic CCAAT/enhancer-binding proteins (C/EBPs), and to positively regulate transactivation by C/EBPbeta. We show that PPARgamma-mediated transactivation is pRB-independent, and that ligand-induced transactivation by PPARgamma1 present in RB+/+ and RB-/- mouse embryo fibroblasts is sufficient to bypass the differentiation block imposed by the absence of pRB. The differentiated RB-/- cells accumulate lipid and express adipocyte markers, including C/EBPalpha and PPARgamma2. Interestingly, adipose conversion of pRB-deficient cells occurs in the absence of compensatory up-regulations of the other pRB family members p107 and p130. RB+/+ as well as RB-/- cells efficiently exit from the cell cycle after completion of clonal expansion following stimulation with adipogenic inducers. We conclude that ligand-induced activation of endogenous PPARgamma1 in mouse embryo fibroblasts is sufficient to initiate a transcriptional cascade resulting in induction of PPARgamma2 and C/EBPalpha expression, withdrawal from the cell cycle, and terminal differentiation in the absence of a functional pRB.

Adipocytes↗
PubMed1999

Cloning and characterization of the gene encoding the highly expressed ribosomal protein l3 of the ciliated protozoan Tetrahymena thermophila. Evidence for differential codon usage in highly expressed genes.

We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features characteristic of ribosomal protein genes in Tetrahymena. The codon usage of the L3 gene is highly biased. A thorough analysis of codon usage in Tetrahymena genes revealed that genes could be categorized into two classes according to codon usage bias. Class A comprises r-protein genes and a number of other highly expressed genes. Class B comprises weakly expressed genes such as the conjugation induced CnjB and CnjC genes, but surprisingly, this class also contains abundantly expressed genes such as the genes encoding the surface antigens SerH3 and SerH1. Codon usage is slightly more restricted in class A than in class B, but both classes exhibit distinct and different codon usage biases. Class A genes preferentially use C and U in the silent third codon positions, whereas class B genes preferentially use A and U in the silent third codon positions. The analysis suggests that two different strategies have been employed for optimization of codon usage in the A+T-rich genome of Tetrahymena.

Animals↗
PubMed1999

Effects of insulin-like growth factor-I and cortisol on Na+, K+-ATPase expression in osmoregulatory tissues of brown trout (Salmo trutta).

The effect of recombinant bovine IGF-I (rbIGF-I) on hypo-osmoregulatory ability and the effect of rbIGF-I and cortisol (F) alone and in combination on Na+,K+-ATPase expression in fresh water (FW) acclimated brown trout (Salmo trutta) were examined in two experiments. In Experiment 1, fish were given three injections of saline or 0.01 or 0.1 microgram rbIGF-I/g, respectively, and subjected to a 24-h 25 ppt seawater (SW) challenge test 24 h after the last injection. Fish treated with 0.01 and 0.1 microgram rbIGF-I/g had better hypo-osmoregulatory ability than control fish as judged by their higher level of muscle water content and lower plasma osmolality after 24 h exposure to 25 ppt SW. Compared with control fish, gill Na+,K+-ATPase activity was unchanged 24 h after the first injection at either dose but significantly stimulated after three injections of either dose of rbIGF-I. In Experiment 2, fish were given three injections of saline, 0.1 microgram rbIGF-I/g, 4 microgram F/g, or 0.1 microgram rbIGF-I + 4 microgram F/g and sampled in FW 24 h after the last injection. IGF-I and F had additive stimulatory effects on Na+,K+-ATPase activity and alpha-subunit Na+,K+-ATPase mRNA levels in the gill. Injections of IGF-I and F alone and in combination increased Na+,K+-ATPase-immunoreactive (NKIR) cell number in the primary gill filament but had no effect on secondary lamellar NKIR cell number. NKIR cells were abundant in kidney tubules, pyloric ceca, and posterior intestine, but Na+,K+-ATPase enzyme activity was unaffected by treatment with F and/or IGF-I in these tissues. F but not rbIGF-I increased in vitro fluid transport capacity in the posterior intestine. In addition to confirming an overall SW-adaptive effect of rbIGF-I and F in FW-acclimated S. trutta, the study suggests the effect to be associated with stimulation of chloride cell development and Na+,K+-ATPase expression in the gill. The study indicates that the stimulatory effects of the two hormones on Na+,K+-ATPase expression are additive, highly organ specific, and restricted to the primary filament epithelium of the gill.

Animals↗
PubMed1999

Rapid identification of DNA-binding proteins by mass spectrometry.

We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.

Animals↗
PubMed1999

The formation of a native-like structure containing eight conserved hydrophobic residues is rate limiting in two-state protein folding of ACBP.

The acyl-coenzyme A-binding proteins (ACBPs) contain 26 highly conserved sequence positions. The majority of these have been mutated in the bovine protein, and their influence on the rate of two-state folding and unfolding has been measured. The results identify eight sequence positions, out of 24 probed, that are critical for fast productive folding. The residues are all hydrophobic and located in the interface between the N- and C-terminal helices. The results suggest that one specific site dominated by conserved hydrophobic residues forms the structure of the productive rate-determining folding step and that a sequential framework model can describe the protein folding reaction.

Amino Acid Sequence↗
PubMed1999

Isolation and quantification of tuliposides and tulipalins in tulips (Tulipa) by high-performance liquid chromatography.

The content of tuliposides and tulipalins were determined in Tulipa species and cultivars by reversed-phase high-performance liquid chromatography (RP-HPLC), using a water:methanol gradient as mobile phase. The compounds were detected by a diode array detector employed at 208 nm. The investigation revealed, in addition to 1- and 6-tuliposide A, tuliposide D and the lactonized aglycones tulipalin A and (-)-tulipalin B, the new tuliposide F and 6-tuliposide B, the latter being a new acyl derivative of the known 1-tuliposide B. All compounds were isolated by preparative RP-HPLC and identified by NMR and mass spectroscopy. The predominant compounds were 6-tuliposide A and B present in amounts up to 1.5% and 1.3% of fresh weight, respectively. 6-Tuliposide A and tulipalin A seem to be the major allergens in tulips, although tuliposide D and F may also contribute to the allergenic properties. Tulipalin A and (-)-tulipalin B occur in intact tulips and are not only produced in response to fungal attack or after excision of the plants. A few species were found to have very low allergen content and a relatively high level of tuliposide B, indicating it should be possible to breed non-allergenic and disease-resistant tulips.

4-Butyrolactone↗
PubMed1998

Osmoregulation and salinity effects on the expression and activity of Na+,K(+)-ATPase in the gills of European sea bass, Dicentrarchus labrax (L.).

The European sea bass, Dicentrarchus labrax, tolerates salinities ranging from freshwater (FW) to hypersaline conditions. In two experiments, we analysed changes in plasma ions, muscle water content (MWC), gill Na+,K(+)-ATPase activity, and alpha-subunit mRNA expression during the course of acclimation from 15 ppt salt water to FW or high salinity seawater (HSSW). In Experiment 1, fish (6.2 +/- 1.1 g) were acclimated from 15 ppt to either FW, 5, 15, 25, 50, or 60 ppt SW and sampled after 10 days. Gill Na+,K(+)-ATPase activity was stimulated in FW- and in 50 and 60 ppt SW-groups relative to the 15 ppt control group. In Experiment 2, subgroups of fish (89 +/- 7 g) were transferred from 15 ppt SW to FW or 50 ppt SW, and sampled 1, 2, 4, and 10 days later. Plasma osmolality, [Na+] and [Cl-] decreased in the FW-group and increased in the HSSW-group one day after transfer and lasting until day 10. This was accompanied by a pronounced increase in MWC in the FW-group and an insignificant decrease in the HSSW-group. The plasma [Na+]:[Cl-]-ratio increased markedly in the FW-group and decreased slightly in the HSSW-group, suggesting acid-base balance disturbances after transfer. Gill Na+,K(+)-ATPase activity was unchanged in 15 ppt SW but doubled in FW- and HSSW-groups after transfer. In both groups, this was preceded by a 2- to 5-fold elevation of the gill alpha-subunit Na+,K(+)-ATPase mRNA level. Thus increased expression of alpha-subunit mRNA is part of the molecular mechanism of both FW and SW acclimation in sea bass. Gill Na+,K(+)-ATPase Na(+)-, K(+)-, and ouabain-affinity were similar in fish acclimated to FW, 15 ppt, and HSSW, suggesting that identical isoforms of the catalytic subunit of the enzyme are expressed irrespective of salinity.

Adaptation, Physiological↗
PubMed1998

Inhibition of 3T3-L1 adipocyte differentiation by expression of acyl-CoA-binding protein antisense RNA.

Several lines of evidence have recently underscored the significance of fatty acids or fatty acid-derived metabolites as signaling molecules in adipocyte differentiation. The acyl-CoA-binding protein (ACBP), which functions as an intracellular acyl-CoA pool former and transporter, is induced during adipocyte differentiation. In this report we describe the effects of expression of high levels of ACBP antisense RNA on the differentiation of 3T3-L1 cells. Pools of 3T3-L1 cells transfected with vectors expressing ACBP antisense RNA showed significantly less lipid accumulation as compared with cells transfected with the control vector. When individual clones were analyzed the degree of differentiation at day 10 was inversely correlated with the level of ACBP antisense RNA expression at day 0. Furthermore, in the clones with the highest levels of ACBP antisense expression, the induction of expression of the adipogenic transcription factors peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha as well as several adipocyte-specific genes was significantly delayed and reduced. The adipogenic potential of antisense-expressing cells was partially restored by transfection with a vector expressing high levels of ACBP. Taken together, these results are strong evidence that inhibition of differentiation is causally related to the decreased expression of ACBP, indicating that ACBP plays an important role during adipocyte differentiation.

1-Methyl-3-isobutylxanthine↗
PubMed1998

DNA sequence analysis by MALDI mass spectrometry.

Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products. Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination. Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. In the present work the use of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented. The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated by sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.

Animals↗
PubMed1998

A human homologue of Escherichia coli ClpP caseinolytic protease: recombinant expression, intracellular processing and subcellular localization.

We have recently cloned a human cDNA (hClpP) with significant sequence similarity to the ATP-dependent Escherichia coli ClpP protease [Bross, Andresen, Knudsen, Kruse and Gregersen (1995) FEBS Lett. 377, 249-252]. In the present study, synthesis, intracellular processing and subcellular localization of hClpP have been analysed in intact cells and in a cell-free system. Using pulse-labelling/immunoprecipitation of Chang cells transfected with the hClpP cDNA, we observed two major bands with apparent molecular masses of approx. 39 and 37 kDa. A pulse-chase experiment showed that these bands were converted into one mature-enzyme band with a molecular mass of approx. 32 kDa that was stable for at least 24 h. The 37 kDa band co-migrated with a band produced upon expression of full-length hClpP in E. coli, and the 32 kDa band co-migrated with the product of E. coli-expressed hClpP in which the 56 N-terminal residues had been deleted, indicating that the 37 kDa moiety represents the precursor and that approx. 56 residues are cleaved off during maturation. The processing of hClpP in intact cells was dependent on mitochondrial membrane potential. These results were confirmed in an import assay system using in vitro transcription and translation directed by the hClpP cDNA and isolated rat liver mitochondria. No protease activity towards a series of fluorogenic peptides could be observed in extracts of Chang cells overexpressing hClpP, indicating that the protease may not be active without co-factors. Immunofluorescence studies using confocal-laser-scanning microscopy showed co-localization of the hClpP and the mitochondrially located Hsp60 (heat-shock protein 60). Taken together, the results reported here show that hClpP is localized inside mitochondria and that the trafficking and processing of hClpP resembles the typical biogenesis pathway for nuclear-encoded mitochondrial proteins.

Adenosine Triphosphatases↗
PubMed1998

Staurosporine-induced cell death in Tetrahymena thermophila has mixed characteristics of both apoptotic and autophagic degeneration.

Staurosporine blocks signal transduction associated with cell survival, proliferation and chemosensory behaviour in the ciliated protozoan, Tetrahymena thermophila. Staurosporine inhibits cell proliferation and in vivo protein phosphorylation induced by phorbol ester. It also reduces the in vitro phosphorylation of the PKC-specific substrate, myelin basic protein fragment 4-14. Our results show that cell death in the presence of staurosporine is associated with morphological and ultrastructural changes similar to both apoptosis and autophagic degeneration, but these in turn can be postponed or prevented by 8-bromo-cyclic GMP, protoporphyrin IX, hemin or actinomycin D, although phorbol ester and insulin were ineffective. The results support the notion that staurosporine-induced cell death is an active process, associated with and/or requiring de novo RNA synthesis.

Animals↗
PubMed1998

An improved PCR-based method for site directed mutagenesis using megaprimers.

An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduction of unwanted secondary mutations. The essential features of this protocol include an optimization of the template-primer amounts and ratio that allows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases.

DNA Primers↗
PubMed1998

5-Hydroxytryptamine2-family receptors (5-hydroxytryptamine2A, 5-hydroxytryptamine2B, 5-hydroxytryptamine2C): where structure meets function.

5-Hydroxytryptamine2 (serotonin2, 5-HT2)-family receptors are important for mediating many physiological functions, including vascular and nonvascular smooth muscle contraction, platelet aggregation, modulation of perception, mood, anxiety, and feeding behavior. A large number of psychopharmaceuticals, including atypical antipsychotic drugs, antidepressants, anxiolytics, and hallucinogens, mediate their actions, at least in part, via interactions with various 5-HT2-family receptors. This review article summarizes information about structure-function aspects of 5-HT2-family receptors. Evidence is presented that implies that conserved aromatic and charged residues are essential for ligand binding to 5-HT2A receptors. Additionally, findings are reviewed that are consistent with the hypothesis that residues located in intracellular loops 2 and 3 (i2 and i3) mediate coupling to specific G(alpha)-subunits such as G(alpha q). Studies are reviewed that suggest that 5-HT2-family receptors may be down-regulated by both agonists and antagonists, and usually this down-regulation is due to post-transcriptional mechanisms. Finally, a model for regulation of 5-HT2-family receptors by receptor-mediated endocytosis is advanced, and the particular structural features responsible for the various endocytotic pathways are emphasized. Taken together, these results suggest that discrete domains of the receptor structure are important for ligand binding, G-protein coupling, and internalization.

Amino Acid Sequence↗
PubMed1998

Serotonin 5-HT2A receptors: molecular biology and mechanisms of regulation.

Serotonin 5-HT2A receptors are essential for a large number of physiological functions in the central nervous system and periphery. This review article summarizes our current knowledge of the molecular biology and mechanisms of regulation of 5-HT2A receptors. The mode of drug binding using data derived from molecular modeling and site-directed mutagenesis is described. The cellular and subcellular localization of 5-HT2A receptors is described, and the concentration of 5-HT2A receptors on apical dendrites of pyramidal neurons is emphasized. Various modes of regulation of 5-HT2A receptors are also summarized, including transcriptional, post-translational and mRNA editing processes. Finally, an integrated model of 5-HT2A receptor regulation that involves various protein kinases (protein kinase C, G-protein receptor kinases), arrestins, clathrin-coated vesicles, endosomes and lysosomes. The relevance of these pathways for antidepressant and antipsychotic drug actions is emphasized.

Animals↗
PubMed1998

Molecular modeling of the endogenous peptide Leu-Ser-Ala-Leu, pindolol, 5-hydroxytryptamine and their interactions with the human 5-hydroxytryptamine1B (5-HT1B) receptor.

Molecular modeling techniques were used to build a three-dimensional model of the human 5-HT1B receptor. The receptor model was used to examine receptor interactions of 5-hydroxytryptamine (serotonin), (S)pindolol and of the tetrapeptide Leu-Ser-Ala-Leu (LSAL), which recently has been shown to interact specifically with the 5-HT1B receptor. We have assumed that the NH3(+)-LSAL-COO- form of the tetrapeptide is the biologically active, and propose that a negatively charged residue conserved among various species homologues of the 5-HT1B receptor may act as a counter-ion for the positively charged N-terminus of the tetrapeptide. The strongest LSAL-receptor interactions were obtained after molecular dynamics simulations that were started with the N-terminus of LSAL positioned close to Asp352 in transmembrane helix 7. The model suggests that Asp352 in transmembrane helix 7 may act as a counter-ion for the positively charged N-terminus, and that the side chains of Tyr109 (transmembrane helix 2) and Trp125 (transmembrane helix 3) may form hydrogen bonds with the negatively charged C-terminus of LSAL.

Amino Acid Sequence↗