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J Knudsen

Publications and source records attributed to J Knudsen.

At least 19 recordsLinked to original sources
PubMed2006

Revisiting the structure of the p(4 x 4) surface oxide on Ag(111).

Scanning tunneling microscopy (STM) and density-functional theory are used to reexamine the structure of the renowned p(4 x 4)-O/Ag(111) surface oxide. The accepted structural model [C. I. Carlisle, Phys. Rev. Lett. 84, 3899 (2000)10.1103/PhysRevLett.84.3899] is incompatible with the enhanced resolution of the current STM measurements. An "Ag6 model" is proposed that is more stable than its predecessor and accounts for the coexistence of the p(4 x 4) and a novel c(3 x 5log3)rect phase. This coexistence is an indication of the dynamic complexity of the system that until now has not been appreciated.

Journal Article↗
PubMed2003

Acyl-coenzyme A organizes laterally in membranes and is recognized specifically by acyl-coenzyme A binding protein.

Long chain acyl-coenzyme A (acyl-CoA) is a biochemically important amphiphilic molecule that is known to partition strongly into membranes by insertion of the acyl chain. At present, microscopically resolved evidence is lacking on how acyl-CoA influences and organizes laterally in membranes. By atomic force microscopy (AFM) imaging of membranes exposed to acyl-CoA in microM concentrations, it is shown that aggregate formation takes place within the membrane upon long-time exposure. It is known that acyl-CoA is bound by acyl-CoA binding protein (ACBP) with high affinity and specificity and that ACBP may bind and desorb membrane-bound acyl-CoA via a partly unknown mechanism. Following incubation with acyl-CoA, it is shown that ACBP is able to reverse the formation of acyl-CoA aggregates and to associate peripherally with acyl-CoA on the membrane surface. Our microscopic results point to the role of ACBP as an intermembrane transporter of acyl-CoA and demonstrate the ability of AFM to reveal the remodelling of membranes by surfactants and proteins.

Acyl Coenzyme A↗
PubMed2002

Imiquimod 5% cream in the treatment of anogenital warts in female patients.

OBJECTIVE: To investigate the efficacy and safety of imiquimod 5% cream in the treatment of anogenital warts in a female population. METHODS: In two open-label studies, female patients with anogenital warts applied imiquimod 5% cream three times a week for up to 16 weeks. Patients who cleared their warts were monitored for a 6-month follow-up period. Patients could be re-treated with imiquimod 5% cream for up to an additional 16 weeks if their warts recurred or new warts developed during the follow-up period. The treatment period could also be extended for up to an additional 16 weeks if patients only experienced partial clearance during the initial 16-week treatment period. RESULTS: Of the female patients who applied imiquimod 5% cream, 75% (449/600) experienced complete clearance of their warts (treatment failure analysis). This includes 46 patients who experienced total clearance when they applied imiquimod for longer than 16 weeks as their warts had only partially cleared in the initial 16 weeks of therapy. During the 6 months of follow-up after the initial treatment period, 15% of patients had recurrent warts. Thirty-nine (75%) of those patients experienced total clearance again after they re-applied imiquimod for up to an additional 16 weeks. The most frequently observed local skin reaction was erythema. CONCLUSION: In these studies, imiquimod 5% cream was an effective and well-tolerated treatment for anogenital warts in females and continued to be safe and effective in the small proportion of patients who needed to re-apply imiquimod after wart recurrence.

Adult↗
PubMed2001

The Acyl-CoA synthetases encoded within FAA1 and FAA4 in Saccharomyces cerevisiae function as components of the fatty acid transport system linking import, activation, and intracellular Utilization.

Exogenous long-chain fatty acids are activated to coenzyme A derivatives prior to metabolic utilization. In the yeast Saccharomyces cerevisiae, the activation of these compounds prior to metabolic utilization proceeds through the fatty acyl-CoA synthetases Faa1p and Faa4p. Faa1p or Faa4p are essential for long-chain fatty acid import, suggesting that one or both of these enzymes are components of the fatty acid transport system, which also includes Fat1p. By monitoring the intracellular accumulation of the fluorescent long-chain fatty acid analogue 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid, long-chain fatty acid transport was shown to be severely restricted in a faa1 Delta faa4 Delta strain. These data established for the first time a mechanistic linkage between the import and activation of exogenous fatty acids in yeast. To investigate this linkage further, oleoyl CoA levels were defined following incubation of wild type and mutant cells with limiting concentrations of exogenous oleate. These studies demonstrated oleoyl CoA levels were reduced to less than 10% wild-type levels in faa1 Delta and faa1 Delta faa4 Delta strains. Defects in metabolic utilization and intracellular trafficking were also found in the fatty acyl-CoA synthetase-deficient strains. The faa1 Delta faa4 Delta strain had a marked reduction in endogenous acyl-CoA pools, suggesting these enzymes play a role in maintenance of endogenous acyl-CoA pools, metabolism and trafficking. In addition, this strain had levels of in vivo beta-oxidation of exogenous oleate reduced 3-fold when compared with the isogenic parent. Northern analyses demonstrated an additional defect in fatty acid trafficking as FAA1 or FAA4 were required for the transcriptional regulation of the genes encoding the peroxisomal enzymes acyl-CoA oxidase (POX1) and medium-chain acyl-CoA synthetase (FAA2). These data support the hypothesis that fatty acyl-CoA synthetase (Faa1p or Faa4p) functions as a component of the fatty acid import system by linking import and activation of exogenous fatty acids to intracellular utilization and signaling.

Acyl Coenzyme A↗
PubMed2001

Binding site differences revealed by crystal structures of Plasmodium falciparum and bovine acyl-CoA binding protein.

Acyl-CoA binding protein (ACBP) maintains a pool of fatty acyl-CoA molecules in the cell and plays a role in fatty acid metabolism. The biochemical properties of Plasmodium falciparum ACBP are described together with the 2.0 A resolution crystal structures of a P. falciparum ACBP-acyl-CoA complex and of bovine ACBP in two crystal forms. Overall, the bovine ACBP crystal structures are similar to the NMR structures published previously; however, the bovine and parasite ACBP structures are less similar. The parasite ACBP is shown to have a different ligand-binding pocket, leading to an acyl-CoA binding specificity different from that of bovine ACBP. Several non-conservative differences in residues that interact with the ligand were identified between the mammalian and parasite ACBPs. These, together with measured binding-specificity differences, suggest that there is a potential for the design of molecules that might selectively block the acyl-CoA binding site.

Amino Acid Sequence↗
PubMed2001

The structural basis of acyl coenzyme A-dependent regulation of the transcription factor FadR.

FadR is an acyl-CoA-responsive transcription factor, regulating fatty acid biosynthetic and degradation genes in Escherichia coli. The apo-protein binds DNA as a homodimer, an interaction that is disrupted by binding of acyl-COA: The recently described structure of apo-FadR shows a DNA binding domain coupled to an acyl-CoA binding domain with a novel fold, but does not explain how binding of the acyl-CoA effector molecule > 30 A away from the DNA binding site affects transcriptional regulation. Here, we describe the structures of the FadR-operator and FadR- myristoyl-CoA binary complexes. The FadR-DNA complex reveals a novel winged helix-turn-helix protein-DNA interaction, involving sequence-specific contacts from the wing to the minor groove. Binding of acyl-CoA results in dramatic conformational changes throughout the protein, with backbone shifts up to 4.5 A. The net effect is a rearrangement of the DNA binding domains in the dimer, resulting in a change of 7.2 A in separation of the DNA recognition helices and the loss of DNA binding, revealing the molecular basis of acyl-CoA-responsive regulation.

Acyl Coenzyme A↗
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

Acyl-CoA-binding protein in the armadillo Harderian gland: its primary structure and possible role in lipid secretion.

Similar to those of other species, the Harderian glands of armadillo produce an abundant lipid secretion, most of which is composed of 1-alkyl-2,3-diacylglycerol. Biosynthesis of this component is apparently performed with the participation of one cytosolic pool of acyl-CoA and another of free fatty acids. The acyl-CoA-binding protein (ACBP) is present at a concentration at least 7-fold that of the heart-type fatty acid-binding protein (H-FABP), though lower than that in other armadillo organs such as liver and brain. The ACBP complete amino acid sequence was determined by Edman degradation of peptides generated by cleavage of the protein with cyanogen bromide, endopeptidase Glu-C, and trypsin. ACBP consists of 86 residues and has a calculated molecular mass of 9783 Da, taking into account that an acetyl group is blocking the N-terminus. Identity percentages between armadillo Harderian gland ACBP and other known ACBPs show that the protein belongs to the liver-specific ACBP isoform (L-ACBP). The fact that the ACBP concentration is higher than that of FABP suggests that the Harderian gland is able to store acyl-CoA amounts in ACBP larger than those of fatty acids in H-FABP for 1-alkyl-2,3-diacylglycerol synthesis.

Amino Acid Sequence↗
PubMed2001

The interaction of acyl-CoA with acyl-CoA binding protein and carnitine palmitoyltransferase I.

The affinity of recombinant rat acyl-CoA binding protein (ACBP) towards acyl-CoAs was investigated using both fluorimetric analysis and isothermal titration microcalorimetry, neither of which requires the physical separation of bound and free ligand for determining the dissociation constants (K(d)). The displacement of 11-(dansylamino)undecanoyl-CoA (DAUDA-CoA) from ACBP yielded binding parameters for the competing acyl-CoAs that compared favourably with those obtained using ultra-sensitive microcalorimetric titration. The K(d) values of ACBP for oleoyl-CoA and docosahexaenoyl-CoA are 0.014 and 0.016 microM, respectively. Under identical experimental conditions, carnitine palmitoyltransferase I (CPT I) of purified rat liver mitochondria has K(d) values of 2.4 and 22.7 microM for oleoyl-CoA and docosahexaenoyl-CoA, respectively. Given that CPT I was not only present at a much lower concentration but also has an appreciably lower affinity for acyl-CoAs than ACBP, it is proposed that CPT I is capable of interacting directly with ACBP-acyl-CoA binary complexes. This is supported by the fact that the enzyme activity correlated with the concentration of ACBP-bound acyl-CoA but not the free acyl-CoA. A transfer of acyl-CoA from ACBP-acyl-CoA binary complexes to CPT I could be a result of the enzyme inducing a conformational alteration in the ACBP leading to the release of acyl-CoA.

Acyl Coenzyme A↗
PubMed2001

Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae.

Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:10(5). A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under control of the GAL1 promoter exhibited an altered acyl-CoA profile with a threefold increase in the relative content of C18:0-CoA, without affecting total acyl-CoA level as previously reported for an adapted acb1Delta strain. Depletion of Acb1p did not affect the general phospholipid pattern, the rate of phospholipid synthesis, or the turnover of individual phospholipid classes, indicating that Acb1p is not required for general glycerolipid synthesis. In contrast, cells depleted for Acb1p showed a dramatically reduced content of C26:0 in total fatty acids and the sphingolipid synthesis was reduced by 50-70%. The reduced incorporation of [(3)H]myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport. The plasma membrane of the Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids. Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures. The present results strongly suggest that Acb1p plays an important role in fatty acid elongation and membrane assembly and organization.

Carrier Proteins↗
PubMed2000

Affinity labeling fatty acyl-CoA synthetase with 9-p-azidophenoxy nonanoic acid and the identification of the fatty acid-binding site.

Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP-forming, EC ) catalyzes the esterification of fatty acids to CoA thioesters for further metabolism and is hypothesized to play a pivotal role in the coupled transport and activation of exogenous long-chain fatty acids in Escherichia coli. Previous work on the bacterial enzyme identified a highly conserved region (FACS signature motif) common to long- and medium-chain acyl-CoA synthetases, which appears to contribute to the fatty acid binding pocket. In an effort to further define the fatty acid-binding domain within this enzyme, we employed the affinity labeled long-chain fatty acid [(3)H]9-p-azidophenoxy nonanoic acid (APNA) to specifically modify the E. coli FACS. [(3)H]APNA labeling of the purified enzyme was saturable and specific for long-chain fatty acids as shown by the inhibition of modification with increasing concentrations of palmitate. The site of APNA modification was identified by digestion of [(3)H]APNA cross-linked FACS with trypsin and separation and purification of the resultant peptides using reverse phase high performance liquid chromatography. One specific (3)H-labeled peptide, T33, was identified and following purification subjected to NH(2)-terminal sequence analysis. This approach yielded the peptide sequence PDATDEIIK, which corresponded to residues 422 to 430 of FACS. This peptide is immediately adjacent to the region of the enzyme that contains the FACS signature motif (residues 431-455). This work represents the first direct identification of the carboxyl-containing substrate-binding domain within the adenylate-forming family of enzymes. The structural model for the E. coli FACS predicts this motif lies within a cleft separating two distinct domains of the enzyme and is adjacent to a region that contains the AMP/ATP signature motif, which together are likely to represent the catalytic core of the enzyme.

Affinity Labels↗
PubMed2000

Crystal structure of FadR, a fatty acid-responsive transcription factor with a novel acyl coenzyme A-binding fold.

FadR is a dimeric acyl coenzyme A (acyl CoA)-binding protein and transcription factor that regulates the expression of genes encoding fatty acid biosynthetic and degrading enzymes in Escherichia coli. Here, the 2.0 A crystal structure of full-length FadR is described, determined using multi-wavelength anomalous dispersion. The structure reveals a dimer and a two-domain fold, with DNA-binding and acyl-CoA-binding sites located in an N-terminal and C-terminal domain, respectively. The N-terminal domain contains a winged helix-turn-helix prokaryotic DNA-binding fold. Comparison with known structures and analysis of mutagenesis data delineated the site of interaction with DNA. The C-terminal domain has a novel fold, consisting of a seven-helical bundle with a crossover topology. Careful analysis of the structure, together with mutational and biophysical data, revealed a putative hydrophobic acyl-CoA-binding site, buried in the core of the seven-helical bundle. This structure aids in understanding FadR function at a molecular level, provides the first structural scaffold for the large GntR family of transcription factors, which are keys in the control of metabolism in bacterial pathogens, and could thus be a possible target for novel chemotherapeutic agents.

Acyl Coenzyme A↗
PubMed2000

Formation of hydrogen bonds precedes the rate-limiting formation of persistent structure in the folding of ACBP.

A burst phase in the early folding of the four-helix two-state folder protein acyl-coenzyme A binding protein (ACBP) has been detected using quenched-flow in combination with site-specific NMR-detected hydrogen exchange. Several of the burst phase structures coincide with a structure consisting of eight conserved hydrophobic residues at the interface between the two N and C-terminal helices. Previous mutation studies have shown that the formation of this structure is rate limiting for the final folding of ACBP. The burst phase structures observed in ACBP are different from the previously reported collapsed types of burst phase intermediates observed in the folding of other proteins.

Amides↗
PubMed2000

Isolation and characterization of two acyl-CoA-binding proteins from proembryogenic masses of Digitalis lanata Ehrh.

Two acyl-CoA-binding-protein (ACBP) isoforms were isolated from proembryogenic masses of Digitalis lanata Ehrh. by column chromatography and preparative HPLC. The ACBPs had molecular masses of 9926 and 9997 Da, respectively. Partial sequence data indicated high similarity to each other and to ACBPs of other plant species such as Ricinus communis, Brassica napus and Arabidopsis thaliana. The isolated ACBPs bound palmitoyl-CoA with high affinity as determined by isoelectric-point shift.

Acyl Coenzyme A↗
PubMed2000

Murine FATP alleviates growth and biochemical deficiencies of yeast fat1Delta strains.

Saccharomyces cerevisiae is an ideal model eukaryote for studying fatty-acid transport. Yeast are auxotrophic for unsaturated fatty acids when grown under hypoxic conditions or when the fatty-acid synthase inhibitor cerulenin is included in the growth media. The FAT1 gene encodes a protein, Fat1p, which is required for maximal levels of fatty-acid import and has an acyl CoA synthetase activity specific for very-long-chain fatty acids suggesting this protein plays a pivotal role in fatty-acid trafficking. In the present work, we present evidence that Fat1p and the murine fatty-acid transport protein (FATP) are functional homologues. FAT1 is essential for growth under hypoxic conditions and when cerulenin was included in the culture media in the presence or absence of unsaturated fatty acids. FAT1 disruptants (fat1Delta) fail to accumulate the fluorescent long-chain fatty acid fatty-acid analogue 4, 4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-do decanoic acid (C1-BODIPY-C12), have a greatly diminished capacity to transport exogenous long-chain fatty acids, and have very long-chain acyl CoA synthetase activities that were 40% wild-type. The depression in very long-chain acyl CoA synthetase activities were not apparent in cells grown in the presence of oleate. Additionally, beta-oxidation of exogenous long-chain fatty acids is depressed to 30% wild-type levels. The reduction of beta-oxidation was correlated with a depression of intracellular oleoyl CoA levels in the fat1Delta strain following incubation of the cells with exogenous oleate. Expression of either Fat1p or murine FATP from a plasmid in a fat1Delta strain restored these phenotypic and biochemical deficiencies. Fat1p and FATP restored growth of fat1Delta cells in the presence of cerulenin and under hypoxic conditions. Furthermore, fatty-acid transport was restored and was found to be chain length specific: octanoate, a medium-chain fatty acid was transported in a Fat1p- and FATP-independent manner while the long-chain fatty acids myristate, palmitate, and oleate required either Fat1p or FATP for maximal levels of transport. Lignoceryl CoA synthetase activities were restored to wild-type levels in fat1Delta strains expressing either Fat1p or FATP. Fat1p or FATP also restored wild-type levels of beta-oxidation of exogenous long-chain fatty acids. These data show that Fat1p and FATP are functionally equivalent when expressed in yeast and play a central role in fatty-acid trafficking.

Animals↗
PubMed2000

Structure and expression of the mouse gene encoding the endozepine-like peptide from haploid male germ cells.

The endozepine-like peptide (ELP) represents a testis-specific isoform of the ubiquitous acyl-CoA binding protein (ACBP) and is highly expressed in late haploid stages of male germ cell development. The genomic sequence of the functional ELP gene as well as that of a pseudogene were analysed from independent bacteriophage clones of a 129sv mouse genomic library. Unlike the ACBP gene, which comprises four exons, the ELP gene has only a single intron within the region of the 5' untranslated region, suggesting that, like some other haploid expressed genes, the ELP gene might have evolved by retroposon-mediated gene duplication. Primer extension analysis was used to define the start site for transcription and hence the 5' promoter region. Electrophoretic mobility shift analysis was carried out on this region comparing nuclear extracts from adult mouse testis with those from mouse liver. Several testis-specific DNA-protein complexes were observed throughout 700 bp upstream of the transcription start site. One of these could be identified as corresponding to a steroidogenic factor-1 (SF-1) binding element. Further analysis using pure transcription factors showed that this element at position -340 was able to bind specifically to both SF-1 and to the germ cell nuclear factor (GCNF). Immunohistochemical analysis using an ELP-specific antibody showed that expression was very restricted within the testis to the postmeiotic germ cells, and in the ovary to interstitial/luteal cells, cell-types known to express GCNF and SF-1, respectively. Testes of CREM-tau knockout mice, lacking all spermatogenic stages later than round spermatids, were devoid of ELP immunoreactivity, whereas in RAD6 knockout mice the few remaining elongated spermatids were clearly defined by this excellent late haploid marker product. The ELP gene and its product thus offer an ideal system with which to investigate the differentiation of late haploid stages of spermatogenesis.

Animals↗
PubMed2000

Role of acyl-CoA binding protein in acyl-CoA metabolism and acyl-CoA-mediated cell signaling.

Long-chain acyl-CoA esters (LCA) act both as substrates and intermediates in metabolism and as regulators of various intracellular functions. Acyl-CoA binding protein (ACBP) binds LCA with high affinity and is believed to play an important role in intracellular acyl-CoA transport and pool formation and therefore also for the function of LCA as metabolites and regulators of cellular functions . The free concentration of cytosolic LCA is efficiently buffered to low nanomole concentration by ACBP and fatty acid binding protein (FABP). An additional important factor is the activity of acyl-CoA hydrolases. The estimated cellular free LCA concentration is two to four orders of magnitude lower than the concentrations reported to be necessary to regulate most LCA-affected cellular functions. Preliminary evidence indicates that the regulatory effect of LCA might be mediated by the LCA/ACBP complex.

Acyl Coenzyme A↗