Biomedical subjects

Jesper Christensen

Publications and source records attributed to Jesper Christensen.

13 recordsLinked to original sources

PubMed2006

Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV.

Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar K(i) values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.

Amino Acid Sequence↗

PubMed2006

The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3.

Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation and transcriptional repression in a variety of species. H3K9me3 has long been regarded as a 'permanent' epigenetic mark. In a search for proteins and complexes interacting with H3K9me3, we identified the protein GASC1 (gene amplified in squamous cell carcinoma 1), which belongs to the JMJD2 (jumonji domain containing 2) subfamily of the jumonji family, and is also known as JMJD2C. Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and alpha-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression of GASC1 or other JMJD2 members markedly decreases H3K9me3/me2 levels, increases H3K9me1 levels, delocalizes HP1 and reduces heterochromatin in vivo. Previously, GASC1 was found to be amplified in several cell lines derived from oesophageal squamous carcinomas, and in agreement with a contribution of GASC1 to tumour development, inhibition of GASC1 expression decreases cell proliferation. Thus, in addition to identifying GASC1 as a histone trimethyl demethylase, we suggest a model for how this enzyme might be involved in cancer development, and propose it as a target for anti-cancer therapy.

Cell Proliferation↗

PubMed2005

Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription.

The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family. Sequence analysis of E2F8 predicts the presence of two distinct E2F-related DNA binding domains suggesting that E2F8 and, the recently, identified E2F7 form a subgroup within the E2F family. We show that E2F transcription factors bind the E2F8 promoter in vivo and that expression of E2F8 is being induced at the G1/S transition. Purified recombinant E2F8 binds specifically to a consensus E2F-DNA-binding site indicating that E2F8, like E2F7, binds DNA without the requirement of co-factors such as DP1. E2F8 inhibits E2F-driven promoters suggesting that E2F8 is transcriptional repressor like E2F7. Ectopic expression of E2F8 in diploid human fibroblasts reduces expression of E2F-target genes and inhibits cell growth consistent with a role for repressing E2F transcriptional activity. Taken together, these data suggest that E2F8 has an important role in turning of the expression of E2F-target genes in the S-phase of the cell cycle.

Amino Acid Sequence↗

PubMed2005

Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage recognition complex, XPC-hHR23B, recognizes this lesion. Our results demonstrate that XPC-hHR23B recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are recognized by XPC-hHR23B alone, but also that XPA-RPA may interact cooperatively with XPC-hHR23B on damaged DNA, forming a multimeric complex. Since XPC-hHR23B and XPA-RPA participate in the recognition and verification of DNA damage, these results support the hypothesis that interplay between components of the global genome repair sub-pathway of NER is critical for the recognition of psoralen DNA ICLs in the mammalian genome.

Cross-Linking Reagents↗

PubMed2005

Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA.

Human high mobility group box (HMGB) 1 and -2 proteins are highly conserved and abundant chromosomal proteins that regulate chromatin structure and DNA metabolism. HMGB proteins bind preferentially to DNA that is bent or underwound and to DNA damaged by agents such as cisplatin, UVC radiation, and benzo[a]pyrenediol epoxide (BPDE). Binding of HMGB1 to DNA adducts is thought to inhibit nucleotide excision repair (NER), leading to cell death, but the biological roles of these proteins remain obscure. We have used psoralen-modified triplex-forming oligonucleotides (TFOs) to direct a psoralen-DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so, and likely modulates their repair by the NER machinery. The abundance of HMGB1 suggests that it may play an important role in determining the sensitivity of cells to DNA damage under physiological, experimental, and therapeutic conditions.

Base Sequence↗

PubMed2004

Arctic atmospheric contaminants in NE Greenland: levels, variations, origins, transport, transformations and trends 1990-2001.

This review is based on the results obtained from the Danish AMAP programme for the Arctic atmosphere during the 1990s. The purpose of the programme is to quantify the pollution, apportion source contributions, follow the trends, and identify midlatitude source areas and transport pathways. The project has been carried out in North Greenland as integrated monitoring, which is an interacting combination of field measurements and model calculations of atmospheric transport and transformation in the Northern Hemisphere. At the monitoring site at Station Nord the large and seasonally recurrent variations in the pollutant concentrations are testimony to the influence in this region of the phenomenon of Arctic Haze. These results can only be understood in terms of long range transport from distant pollution sources. The measurements also comprise a large number of particle-born elements. These results are used to build receptor models, which show that the ambient concentrations and their variations to a high degree can be explained by the influence of only four source types of both natural and anthropogenic nature. The challenging phenomena of atmospheric ozone and mercury depletion around Polar sunrise have been studied at Station Nord over several years. The results show that these two phenomena are closely connected, presumably through photochemical reactions with atmospheric halogens released from sea ice. A large-scale Eulerian model system for the Northern Hemisphere has been developed in this AMAP project. The validity of the model is illustrated by comparisons between measured and calculated air concentrations. The model has been used to calculate both the vertical distribution and the atmospheric depositions for several pollutants at various locations in Greenland and split into quantified contributions from different and geographically distant source areas. Mercury deposition estimates for the Northern Hemisphere are also presented. They show that the mercury depletion events are accompanied by very intense depositions to land and sea of reactive mercury that may seriously affect the Arctic ecosystems. Finally, measurement and model data are combined to demonstrate, despite considerable meteorological noise, that ambient air concentrations in North-eastern Greenland have decreased during the last decade. Quantified trends, attributable to emission reductions in distant source areas, are presented for several pollutants.

Air Movements↗

PubMed2004

Tyrosine 547 constitutes an essential part of the catalytic mechanism of dipeptidyl peptidase IV.

Human dipeptidyl peptidase IV (DPP-IV) is a ubiquitously expressed type II transmembrane serine protease. It cleaves the penultimate positioned prolyl bonds at the N terminus of physiologically important peptides such as the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide. In this study, we have characterized different active site mutants. The Y547F mutant as well as the catalytic triad mutants S630A, D708A, and H740L showed less than 1% wild type activity. X-ray crystal structure analysis of the Y547F mutant revealed no overall changes compared with wild type apoDPP-IV, except the ablation of the hydroxyl group of Tyr(547) and a water molecule positioned in close proximity to Tyr(547). To elucidate further the reaction mechanism, we determined the crystal structure of DPP-IV in complex with diisopropyl fluorophosphate, mimicking the tetrahedral intermediate. The kinetic and structural findings of the tyrosine residue are discussed in relation to the catalytic mechanism of DPP-IV and to the inhibitory mechanism of the 2-cyanopyrrolidine class of potent DPP-IV inhibitors, proposing an explanation for the specificity of this class of inhibitors for the S9b family among serine proteases.

Amino Acid Motifs↗

PubMed2004

Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes.

Parasite-encoded variant surface antigens (VSAs) like the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in nonimmune patients tend to express a restricted subset of VSA (VSA(SM)) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSA(UM)). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSA(UM) to in vitro-selected sublines expressing VSA(SM) to identify PfEMP1 responsible for the VSA(SM) phenotype. Expression of VSA(SM) was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var genes, such as PFD1235w/MAL7P1.1, appear to be involved in the pathogenesis of severe disease and are thus attractive candidates for a vaccine against life-threatening P. falciparum malaria.

Amino Acid Sequence↗

PubMed2002

Rational combinatorial chemistry-based selection, synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII.

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca(2+)-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (approximately 1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability.

Amino Acid Sequence↗

PubMed2002

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions.

Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.

Base Sequence↗

PubMed2002

Detection of parvovirus B19 NS1-specific antibodies by ELISA and western blotting employing recombinant NS1 protein as antigen.

Human parvovirus B19 (B19) encodes a number of nonstructural proteins, including the major protein, NS1, and two structural proteins, VP1 and VP2. The use of denatured NS1 in enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) assay has provided an opportunity to study some of the immunologic properties of NS1, but the results have been equivocal and the diagnostic sensitivity poor, probably because of the absence of conformational epitopes. Various viral isolates and baculovirus vectors were employed to produce recombinant B19 NS1 under nondenaturing conditions for the first time. To assess the antigenicity of purified B19 NS1, the reaction patterns of 252 samples were compared by B19 NS1 and VP2 ELISA. In sera from individuals with past infection (VP2 IgG-positive), the use of this new antigen increased significantly the sensitivity of ELISA compared with WB (78% vs. 33%, P = 0.001), contradicting perpetuated claims that B19 NS1 IgG is detected primarily in patients with arthralgia or chronic infection. Previous reports of the absence of NS1 IgG during the initial phase of infection (< 6 weeks) were proved incorrect by the detection of NS1 IgG in 60% of samples from patients recently infected by B19. Including conformational epitopes in the ELISA increases the diagnostic sensitivity, although immunologically, a temporal (years) attenuation of NS1 antibodies appears to take place. This novel diagnostic tool may be useful as a supplement in case of borderline results by VP2 ELISA and for monitoring the efficacy of future capsid-based B19 vaccines.

Animals↗

PubMed2002

Baculovirus expression of erythrovirus V9 capsids and screening by ELISA: serologic cross-reactivity with erythrovirus B19.

Diagnosis of erythrovirus B19 (B19) relies on serology and the detection of viral DNA. Recently, a distinct erythrovirus isolate termed V9, markedly different from erythrovirus B19 (> 11% nucleotide disparity), was isolated. Standard B19 PCR assays were inconclusive and serologic tests failed to categorize V9 as an acute B19-like infection. Sequencing, combined with PCR studies, have since demonstrated the need for specific and differentiated techniques when examining samples for possible B19 or V9 viremia. The antigenic properties of the V9 capsid proteins have not been characterized previously. To address this question, V9 VP1 and VP2 open reading frames were cloned and expressed in insect cells using a baculovirus vector. Large quantities of purified recombinant V9 capsid protein were produced and electron micrographs revealed self-assembly of V9 VP1/VP2 and VP2 capsids into empty icosahedral erythrovirus-like particles with a diameter of approximately 23 nm. Screening of a panel of 270 clinical samples for the presence of V9 IgM and IgG antibodies in ELISA showed 100% serologic cross-reactivity between B19 and V9 when comparing V9 VP2 capsids to a commercial B19 VP2 assay. This suggests that both a V9 and a B19 antibody response may be diagnosed equally well by ELISA using either V9 or B19 recombinant capsids as antigen source. Retrospectively, translation of the V9 sequence indicates that despite a significant genetic variation on the DNA level, the majority of the discrepant DNA sequence represents silent mutations leading to an amino acid sequence very similar to the known B19 strains (96-97% homology).

Adolescent↗

PubMed2002

Parvovirus initiator protein NS1 and RPA coordinate replication fork progression in a reconstituted DNA replication system.

We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5' end and displacing the other strand while progressing in a 3'-to-5' direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5' end of the nick in the process and providing a 3' OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO(4) oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of Escherichia coli single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase delta, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with E. coli SSB or other proteins included as controls.

Animals↗