Biomedical subjects

K Kristiansen

Publications and source records attributed to K Kristiansen.

At least 19 recordsLinked to original sources

PubMed2006

The effect of 6 months supplementation with conjugated linoleic acid on insulin resistance in overweight and obese.

BACKGROUND: Contradicting results have been published regarding the effect of conjugated linoleic acid (CLA) on insulin resistance. However, only a few studies have used the euglycemic hyperinsulinemic clamp method, which is considered the standard for measuring insulin resistance. OBJECTIVE: To evaluate if CLA as a mixture of the main isomers trans-10 cis-12 and cis-9 trans-11 affects the insulin resistance in healthy overweight and obese male and female adults. DESIGN: The main study was a randomized, double-blind, placebo-controlled trial with change in body composition as primary end point comprising 118 subjects receiving supplementation with either placebo (olive oil) or CLA (Clarinol) for 6 months. A sub-population of 49 subjects agreed additionally to participate in an euglycemic hyperinsulinemic clamp study at baseline and after 6 months of supplementation with study drug. The primary outcome was the change in glucose uptake (M) as measured by the hyperinsulinemic euglycemic glucose clamp method. Secondary outcomes were the correlates between insulin resistance and changes in body composition or blood chemistry parameters. Forty-one subjects completed the clamp test at both time points. RESULTS: The median M of the CLA group was 11.0 mg min(-1) lean body mass (lbm)(-1) (n=24) at baseline, 10.3 mg min(-1) lbm(-1) (n=24) after 6 months, and the median difference was +0.21 mg min(-1) lbm(-1) (n=24). The median M of placebo group was 8.4 mg min(-1) lbm(-1) at baseline and 9.3 mg min(-1) lbm(-1) after 6 months and the median difference was -0.22 mg min(-1) lbm(-1) (n=17). No significant (P<0.05) differences were found within groups or between groups. Likewise, the glucose uptake insulin concentration ratio during clamp (M/I) was independent of treatment and time. Homeostasis model assessment (HOMA) and quantitative insulin sensitivity check index derived from fasting glucose and insulin were also independent of treatment and time, and HOMA for the clamp population (n=49) corresponded well with HOMA for the per protocol population (n=83). Correlation analysis showed that changes in M were inversely correlated to changes in glucohemoglobin (P=0.002), but did not correlate with changes in either glucose, insulin, insulin c-peptide, leptin, adiponectin or percent body fat. CONCLUSIONS: CLA does not affect glucose metabolism or insulin sensitivity in a population of overweight or obese volunteers.

Adolescent↗

PubMed2006

Peroxisome proliferator-activated receptor alpha, delta, gamma1 and gamma2 expressions are present in human monocyte-derived dendritic cells and modulate dendritic cell maturation by addition of subtype-specific ligands.

It has recently been shown by Chang et al. (J Immunol 2000;165:3584-91) that the maturation of dendritic cells (DC) in the presence of long-chain fatty acids redirects DC into Th0/Th2-inducing cells suggesting the involvement of a receptor for long-chain fatty acids like members of the peroxisome proliferator-activated receptors (PPAR) superfamily. Here, we show that immature and mature monocyte-derived DC (Mo-DC) express PPARalpha, PPARdelta, PPARgamma1 and PPARgamma2 mRNA with the highest level of PPARgamma1 mRNA. We were only able to observe the expression of PPARgamma1 protein by Western blotting probably because the protein level of the other subtypes is below the detection limit. Synthetic ligands specific for PPARalpha, PPARdelta or PPARgamma added at day 0-6 have similar effect on the maturation of Mo-DC driving the maturation of Mo-DC with atypical phenotype, reduced expression of IL-10, IL-12 p35 and IL-12 p40 mRNA and with reduced stimulatory effects in mixed leucocyte reaction (MLR). Our data suggest that naturally occurring PPAR ligands like fatty acids and fatty acid derivates have anti-inflammatory effects by redirecting DC into a less stimulatory mode.

Cell Differentiation↗

PubMed2005

Characterization of two isoalleles and three mutations in both isoforms of purified recombinant human porphobilinogen deaminase.

Defects in the enzyme porphobilinogen deaminase (PBG-D) are associated with acute intermittent porphyria (AIP). Human PBG-D is transcribed into a housekeeping or an erythroid form as a result of differential promoter usage and splicing. In addition, three pairs of isoallelic forms have been described. However, whether the enzymatic properties of housekeeping and erythroid forms differ is unknown. In this study the two isoallelic forms, K210 and E210, were cloned and expressed in Escherichia coli together with three mutations associated with a clinical AIP phenotype. The mutations were introduced in the K210 isoallelic background and expressed as both the housekeeping and the erythroid form. The proteins were expressed as GST fusions and purified to homogeneity. Initial experiments revealed that the GST-PBG-D fusions and the purified PBG-D obtained by proteolytic removal of the GST moiety had enzymatic properties that were indistinguishable. Consequently, all analyses with mutant PBG-D were performed on the GST-fusion proteins. Comparison of the wild-type proteins revealed a significant difference in Km between isoalleles with a Km of 9 microM for K210 and 7 microM for E210, whereas no significant difference in activity or kinetics between the housekeeping and the erythroid isoforms was observed. The mutant proteins showed 0.3-1.0% wild-type activity, depending on mutation. There was a clear correlation between yield of recombinant protein and CRIM status of patients. Furthermore, co-expression of the mutant proteins with the bacterial chaperone GroESL did not affect protein yield or function to any significant extent, supporting the view that the investigated mutations primarily influence structure and function and not folding of the proteins.

Alleles↗

PubMed2005

The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin.

BACKGROUND: Alterations in specific signal transduction pathways may explain the hyperproliferation and abnormal differentiation of the keratinocytes as well as the increased expression of inflammatory cytokines seen in psoriasis. Major signalling pathways used by eukaryotic cells to transduce extracellular signals into cellular responses impinge on the mitogen-activated protein kinases (MAPKs). OBJECTIVES: To investigate the expression of the MAPK p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in psoriatic skin. METHODS: Keratome biopsies were taken from patients with plaque-type psoriasis. Western blot analysis was used to determine p38, ERK and JNK activity and protein levels, whereas kinase assays were used to examine the kinase activity of p38. RESULTS: We demonstrated increased levels of the phosphorylated forms of p38 and ERK1/2 in lesional psoriatic skin compared with nonlesional psoriatic skin. No abnormality was found in the activation and expression of JNK1/2. Ex vivo kinase assays confirmed the increased activation of p38, and furthermore demonstrated increased kinase activity of the p38 isoforms p38alpha, p38beta and p38delta in lesional compared with nonlesional psoriatic skin. p38gamma was not detected in the psoriatic skin. Clearance of the psoriatic lesions, induced by climatotherapy at the Dead Sea for 4 weeks, led to a normalization in the activity of both p38 and ERK1/2. CONCLUSIONS: Taken together, our results demonstrate that the activity of the MAPKs p38alpha, p38beta and p38delta and ERK1/2 are increased in lesional psoriatic skin compared with nonlesional psoriatic skin, and that clearance of psoriasis normalizes the p38 and ERK1/2 activity. Thus, p38 and ERK1/2 might be potential targets in the treatment of psoriasis.

Adult↗

PubMed2001

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate.

This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli. Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH. When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically. The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S-transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPARalpha LBD. Total yield of GST-PPARalpha LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPARalpha LBD, the only active form, increased with the length of induction period. Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 +/- 0.4 cell divisions. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific growth rate was observed when inducer was added in the lag-phase, and no soluble protein was produced. These results suggest that solely soluble GST-PPARalpha LBD acts as a growth inhibitor and that GST-PPARalpha LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction. Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth.

Animals↗

PubMed2001

Protein-binding elements in the proximal parotid secretory protein gene enhancer essential for salivary-gland-specific expression.

The murine parotid secretory protein (PSP) gene is expressed at high levels in the parotid gland and at lower levels in the sublingual gland. A proximal enhancer core necessary for tissue-specific expression was identified previously, and it was demonstrated that one element, parotid gland element I (PGE I), exhibited specific binding of parotid gland nuclear proteins. In the present study, we demonstrate that a related adjacent element, PGE II, which binds nuclear proteins in a much less tissue-restricted manner, is able to compete with PGE I for binding of parotid-gland-specific factors. The functional significance of PGE I and PGE II was examined in transgenic mice. Deletion of PGE II reduced transgene expression only in the parotid gland, whereas deletion of PGE I appeared to reduce expression in both of the PSP-expressing salivary glands. Combined deletion of PGE I and PGE II reduced expression below the limit of detection. Thus PGE I and PGE II are functionally important salivary-gland-specific binding elements that are necessary for the salivary-gland-specific expression of a PSP minigene in transgenic mice.

Animals↗

PubMed2001

Acyl-CoA esters antagonize the effects of ligands on peroxisome proliferator-activated receptor alpha conformation, DNA binding, and interaction with Co-factors.

The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARalpha. Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, antagonizes the effects of agonists on PPARalpha conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPARalpha-retinoid X receptor alpha heterodimer to the acyl-CoA oxidase peroxisome proliferator response element. PPARalpha bound specifically to immobilized palmitoyl-CoA and Wy14643, but not BRL49653, abolished binding. S-Hexadecyl-CoA increased in a dose-dependent and reversible manner the sensitivity of PPARalpha to chymotrypsin digestion, and the S-hexadecyl-CoA-induced sensitivity required a functional PPARalpha ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPARalpha but increased recruitment of the nuclear receptor co-repressor NCoR. In cells, the concentration of free acyl-CoA esters is kept in the low nanomolar range due to the buffering effect of high affinity acyl-CoA-binding proteins, especially the acyl-CoA-binding protein. By using PPARalpha expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecyl-CoA was able to increase the mobility of the PPARalpha-containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo.

Acyl Coenzyme A↗

PubMed2001

Cell death in Tetrahymena thermophila: new observations on culture conditions.

We previously suggested that the cell fate of the protozoan ciliate, Tetrahymena thermophila, effectively relates to a quorum-sensing mechanism where cell-released factors support cell survival and proliferation. The cells have to be present above a critical initial density in a chemically defined nutrient medium in order to release a sufficient level of these factors to allow a new colony to flourish. At a relatively high rate of metabolism and/or macromolecular synthesis and below this critical density, cells began to die abruptly within 30 min of inoculation, and this death took the form of an explosive disintegration lasting less than 50 milliseconds. The cells died at any location in the culture, and the frequency of cell death was always lower in well-filled vials than those with medium/air interface. Cell death was inhibited by the addition of Actinomycin D or through modifications of the culture conditions either by reducing the oxygen tension or by decreasing the temperature of the growth medium. In addition, plastic caps in well-filled vials release substances, which promote cell survival. The fate of low-density cultures is related to certain 'physical' conditions, in addition to the availability of oxygen within closed culture systems.

Animals↗

PubMed2001

Optimization of methods and treatment conditions for studying effects of fatty acids on cell growth.

Antiproliferative properties of molecular regulators of lipid metabolism have been increasingly studied during recent years. Discussion is ongoing concerning optimal treatment conditions and assays used for monitoring proliferation and cytotoxicity. The objective of the present work was to optimize methods and treatment conditions used for studying antiproliferative effects of fatty acids and analogs, represented by palmitic acid (PA) and the beta-oxidation-restricted fatty acid analog tetradecylthioacetic acid (TTA), in rat (BT4Cn) and human (D54Mg and GaMg) glioma cell lines. Changes in [3H]thymidine incorporation preceded changes in cell number in TTA-treated glioma cell cultures, and the growth inhibition was more significantly expressed by [3H]thymidine incorporation than cell number. Addition of bovine serum albumin decreased cellular fatty acid uptake and reduced the effects of TTA and PA on [3H]thymidine incorporation. Determination of the antiproliferative effect of TTA in BT4Cn cells by MTT conversion and [3H]thymidine incorporation yielded concordant results. TTA-mediated reduction in cell number corresponded to reduction in cellular protein and total DNA content in BT4Cn cells. Reduced growth potential in TTA-treated multicellular D54Mg and GaMg spheroids supported the findings from monolayer cultures. In conclusion, cell density, treatment period, fatty acid administration, and methods for growth determination may profoundly influence the outcome of cell growth experiments. Thus, experimental conditions should be carefully controlled when performing cell growth experiments, and effects on cell growth should preferably be confirmed by different methods.

Animals↗

PubMed2001

Bioinformatics and receptor mechanisms of psychotropic drugs.

One important aspect in biotechnology is gene discovery and target validation for drug discovery. Information from the human genome (HUGO) project may be used to deduce the amino acid sequence of all proteins produced in the human body. However, knowing the amino acid sequence of a protein is not the same as knowing its function. Identification of novel molecular targets for discovery of new, safer and more efficient therapeutic drugs from the human genome sequences requires multidisciplinary research efforts, including proteomics, structural biology and bioinformatics. In addition to possible effects on gene expression, most of the currently used therapeutic drugs either have enzymes or membrane proteins as their molecular targets of action. These membrane proteins include transporters of small molecules across cell membranes, ion channels, or receptors that convey signals from one side of a membrane to the other. Our research group as well as others have used computational techniques, along with biotechnology, molecular biology and other experimental techniques, to construct detailed 3-dimensional models of transporter proteins and G-protein coupled receptors (GPCRs), which are the molecular targets of action of psychotropic drugs. The models have been used to simulate the molecular dynamics and study the ligand binding and signal transduction mechanisms of these receptors. The use of bioinformatics, as exemplified in our modelling of GPCRs, is only one of the key factors for success in post-genomic research for new targets for therapeutic drugs.

Animals↗

PubMed2001

Modulation of keratinocyte gene expression and differentiation by PPAR-selective ligands and tetradecylthioacetic acid.

Peroxisome proliferator-activated receptors (PPARs) are pleiotropic regulators of growth and differentiation of many cell types. We have performed a comprehensive analysis of the expression of PPARs, transcriptional cofactors, and marker genes during differentiation of normal human keratinocytes using a combination of reverse transcriptase polymerase chain reaction, Northern and Western blotting, and immunohistochemistry. PPARdelta was the predominant PPAR subtype in human keratinocytes and highly expressed in basal cells and suprabasal cells. Induction of PPARalpha and PPARgamma expression was linked to differentiation, and accordingly, expression of PPARalpha and PPARgamma was in essence confined to suprabasal cells. Differentiation was not accompanied by significant changes in the expression of the coactivators CREB-binding protein, p300, steroid receptor coactivator 1, or the corepressors nuclear receptor corepressor and silence mediator for retinoid and thyroid hormone receptors. We critically evaluated the effects of selective PPAR ligands and a synthetic fatty acid analog, tetradecylthioacetic acid. Tetradecylthioacetic acid activated all human PPAR subtypes in the ranking order PPARdelta >> PPARalpha > PPARgamma. All selective PPAR ligands marginally induced transglutaminase-1 expression with the PPARdelta-selective ligand L165041 being the most potent. The PPARalpha- and PPARgamma-selective ligands Wy14643 and BRL49653 had negligible effect on involucrin expression, whereas a dose-dependent induction was observed with L165041. Simultaneous addition of L165041 and BRL49653 synergistically induced strong involucrin expression. Additionally, L165041 potently induced CD36 mRNA expression. Administration of tetradecylthioacetic acid resulted in a dramatic decrease in proliferation and a robust upregulation of the expression of involucrin and transglutaminase. Our results indicate that tetradecylthioacetic acid may affect keratinocyte gene expression and differentiation via PPAR-dependent and PPAR-independent pathways, and that the latter play an important role.

Adult↗

PubMed2001

Tetradecylthioacetic acid inhibits growth of rat glioma cells ex vivo and in vivo via PPAR-dependent and PPAR-independent pathways.

The peroxisome proliferator-activated receptors (PPARs) are transcription factors involved in fatty acid metabolism and energy homeostasis. The PPARs also play crucial roles in the control of cellular growth and differentiation. Especially, the recently emerged concept of ligand-dependent PPARgamma-mediated inhibition of cancer cell proliferation through induction of G(1)-phase arrest and differentiation is of clinical interest to cancer therapy. Tetradecylthioacetic acid (TTA) is a sulphur-substituted saturated fatty acid analog with unique biochemical properties. In this study, we investigated the effects of TTA-administration on cell proliferation in glioma cancer models. The rat glioma cell line BT4Cn, whether grown in culture or implanted in rats, expressed significant levels of PPARgamma and PPARdelta, with PPARgamma being the predominant PPAR subtype. In BT4Cn cells, TTA activated all PPAR subtypes in a dose-dependent manner. In cell culture experiments, the PPARgamma-selective ligand BRL49653 moderately inhibited growth of BT4Cn cells, whereas administration of TTA resulted in a marked growth inhibition. Administration of the PPARgamma-selective antagonist GW9662 abolished BRL49653-induced growth inhibition, but only marginally reduced the effect of TTA. TTA reduced tumor growth and increased the survival time of rats with implanted BT4Cn tumor. TTA-induced apoptosis in BT4Cn cells, and the administration of TTA led to cytochrome c release from mitochondria and increased the glutathione content in glioma cells. In conclusion, our results indicate that TTA inhibits proliferation of glioma cancer cells through both PPARgamma-dependent and PPARgamma-independent pathways, of which the latter appears to predominate.

Animals↗

PubMed2000

Peroxisome proliferator-activated receptor delta (PPARdelta )-mediated regulation of preadipocyte proliferation and gene expression is dependent on cAMP signaling.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a key regulator of terminal adipocyte differentiation. PPARdelta is expressed in preadipocytes, but the importance of this PPAR subtype in adipogenesis has been a matter of debate. Here we present a critical evaluation of the role of PPARdelta in adipocyte differentiation. We demonstrate that treatment of NIH-3T3 fibroblasts overexpressing PPARdelta with standard adipogenic inducers led to induction of PPARgamma2 expression and terminal adipocyte differentiation in a manner that was strictly dependent on simultaneous administration of a PPARdelta ligand and methylisobutylxanthine (MIX) or other cAMP elevating agents. We further show that ligands and MIX synergistically stimulated PPARdelta-mediated transactivation. In 3T3-L1 preadipocytes, simultaneous administration of a PPARdelta-selective ligand and MIX significantly enhanced the early expression of PPARgamma and ALBP/aP2, but only modestly promoted terminal differentiation as determined by lipid accumulation. Finally, we provide evidence that synergistic activation of PPARdelta promotes mitotic clonal expansion in 3T3-L1 cells with or without forced expression of PPARdelta. In conclusion, our results suggest that PPARdelta may play a role in the proliferation of adipocyte precursor cells, whereas activation of endogenous PPARdelta in 3T3-L1 cells appears to have only minor impact on the processes leading to terminal adipocyte differentiation.

1-Methyl-3-isobutylxanthine↗

PubMed2000

The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum.

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed2000

Ribosome synthesis in Tetrahymena: a quantitative analysis.

We have performed a detailed quantitative analysis of the transcription and accumulation of ribosomal RNA and ribosomal protein mRNA in the ciliated protozoan Tetrahymena thermophila during changes in growth conditions, and found that: (1) nutritional downshifts lead to a rapid decrease in transcriptional activity whereas nutritional upshifts lead to rapid restoration of transcriptional activity, (2) starvation leads to decreased translation of ribosomal protein mRNA and (3) the rate of ribosomal protein mRNA degradation decreases after a nutritional upshift. We present evidence that the proximal promoters of two ribosomal protein genes and the ribosomal RNA gene compete for binding of nuclear factor(s) in vitro, suggesting that the coordinated regulation of these genes may involve a common set of transcriptional regulators.

Animals↗

PubMed2000

Differential modes of agonist binding to 5-hydroxytryptamine(2A) serotonin receptors revealed by mutation and molecular modeling of conserved residues in transmembrane region 5.

Site-directed mutagenesis and molecular modeling were used to investigate the molecular interactions involved in ligand binding to, and activation of, the rat 5-hydroxytryptamine(2A) (5-HT(2A)) serotonin (5-HT) receptor. Based on previous modeling studies utilizing molecular mechanics energy calculations and molecular dynamics simulations, four sites (S239[5.43], F240[5.44], F243[5.47], and F244[5.48]) in transmembrane region V were selected, each predicted to contribute to agonist and/or antagonist binding. The F243A mutation increased the affinity of (+/-)4-iodo-2, 5-dimethoxyphenylisopropylamine, decreased the binding of alpha-methyl-5HT, N-omega-methyl-5HT, ketanserin, ritanserin, and spiperone and had no effect on the binding of 5-HT and 5-methyl-N, N-dimethyltryptamine. The F240A mutant had no effect on the binding of any of the ligands tested, whereas F244A caused an agonist-specific decrease in binding affinity (3- to 10-fold). S239A caused a 6- to 13-fold decrease in tryptamine-binding affinity and a 5-fold increase in affinity of 4-iodo-2, 5-dimethoxyphenylisopropylamine. A subset of the agonists used in binding studies were used to determine the efficacies and potencies of these mutants to activate phosphoinositide hydrolysis. The F243A and F244A mutations reduced agonist stimulated phosphoinositide hydrolysis, whereas the S239A and F240A mutations had no effect. There was little correlation between agonist binding and second messenger production. Furthermore, molecular dynamics simulations, considering these data, produced ligand-bound structures utilizing substantially different bonding interactions even among structurally similar ligands (differing by as little as one methyl group). Taken together, these results suggest that relatively minor changes in either receptor or ligand structure can produce drastic and unpredictable changes in both binding interactions and 5-HT(2A) receptor activation. Thus, our finding may have major implications for the future and feasibility of receptor structure-based drug design.

DOM 2,5-Dimethoxy-4-Methylamphetamine↗

PubMed2000

Long-chain acyl-CoA esters and acyl-CoA binding protein are present in the nucleus of rat liver cells.

A detailed analysis of the subcellular distribution of acyl-CoA esters in rat liver revealed that significant amounts of long-chain acyl-CoA esters are present in highly purified nuclei. No contamination of microsomal or mitochondrial marker enzymes was detectable in the nuclear fraction. C16:1 and C18:3-CoA esters were the most abundant species, and thus, the composition of acyl-CoA esters in the nuclear fraction deviates notably from the overall composition of acyl-CoA esters in the cell. After intravenous administration of the non-beta-oxidizable [(14)C]tetradecylthioacetic acid (TTA), the TTA-CoA ester could be recovered from the nuclear fraction. Acyl-CoA esters bind with high affinity to the ubiquitously expressed acyl-CoA binding protein (ACBP), and several lines of evidence suggest that ACBP functions as a pool former and transporter of acyl-CoA esters in the cytoplasm. By using immunohistochemistry, immunofluorescence microscopy, and immunoelectron microscopy we demonstrate that ACBP localizes to the nucleus as well as the cytoplasm of rat liver cell and rat hepatoma cells, suggesting that ACBP may also be involved in regulation of acyl-CoA-dependent processes in the nucleus.

Acyl Coenzyme A↗

PubMed2000

A highly conserved aspartic acid (Asp-155) anchors the terminal amine moiety of tryptamines and is involved in membrane targeting of the 5-HT(2A) serotonin receptor but does not participate in activation via a "salt-bridge disruption" mechanism.

Discovering the molecular and atomic mechanism(s) by which G-protein-coupled receptors (GPCRs) are activated by agonists remains an elusive goal. Recently, studies examining two representative GPCRs (rhodopsin and alpha(1b)-adrenergic receptors) have suggested that the disruption of a putative "salt-bridge" between highly conserved residues in transmembrane (TM) helix III, involving aspartate or glutamate, and helix VII, involving a basic residue, results in receptor activation. We have tested whether this is a general mechanism for GPCR activation by constructing a model of the 5-hydroxytryptamine (5-HT)(2A) receptor and characterizing several mutations at the homologous residues (Asp-155 and Asn-363) of the 5-HT(2A) serotonin receptor. All of the mutants (D155A, D155N, D155E, D155Q, and S363A) resulted in receptors with reduced basal activity; in no case was evidence for constitutive activity revealed. Structure-function studies with tryptamine analogs and various Asp-155 mutants demonstrated that Asp-155 interacts with the terminal, and not indole, amine moiety of 5-HT(2A) agonists. Interestingly, the D155E mutation interfered with the membrane targeting of the 5-HT(2A) receptor, and an inverse relationship was discovered when comparing receptor activation and targeting for a series of Asp-155 mutants. This represents the first known instance in which a charged residue located in a putative TM helix alters the membrane targeting of a GPCR. Thus, for 5-HT(2A) receptors, the TMIII aspartic acid (Asp-155) is involved in anchoring the terminal amine moiety of indole agonists and in membrane targeting and not in receptor activation by salt-bridge disruption.

Animals↗