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Showing papers by "University of Southern Denmark published in 1999"


Journal ArticleDOI
TL;DR: A generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag and Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein.
Abstract: We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein The TAP method has been tested in yeast but should be applicable to other cells or organisms

2,866 citations


Journal ArticleDOI
01 Apr 1999-Gut
TL;DR: This report summarises conclusions from an evidence-based workshop which evaluated major clinical strategies for the management of the full spectrum of gastro-oesophageal reflux disease, with an emphasis on medical management.
Abstract: This report summarises conclusions from an evidence-based workshop which evaluated major clinical strategies for the management of the full spectrum of gastro-oesophageal reflux disease, with an emphasis on medical management. The disease was defined by the presence of oesophageal mucosal breaks or by the occurrence of reflux induced symptoms severe enough to impair quality of life. Endoscopy negative patients were recognised as the most common subgroup; most of these patients can be diagnosed by a well structured symptom analysis. There is a consistent hierarchy of effectiveness of available initial and long term therapies that applies for all patient subgroups. Lifestyle measures were judged to be of such low efficacy that they were rejected as a primary therapy for all patient subgroups. Proton pump inhibitor therapy was considered the initial medical treatment of choice because of its clearly superior efficacy which results in the most prompt achievement of desirable outcomes at the lowest overall medical cost. It was acknowledged that most of patients require long term management and that any maintenance therapy should be chosen by step down to the regimen that is still effective, but least costly. Endoscopic monitoring of routine long term therapy was considered inappropriate, on the basis that control of symptoms is an acceptably reliable indicator of healing in patients with oesophagitis. Laparoscopic antireflux surgery was recognised as a significant therapeutic advance, the results of which, however, depend substantially on the experience of the surgeon. There are currently no published direct comparisons of cost and efficacy outcomes of optimal medical and surgical therapies for reflux disease. To a significant degree, the choice between medical and surgical therapy should depend on informed patient preference. Substantial advances have occurred recently in the understanding and treatment of reflux disease. By contrast, there has been relatively little research into the best …

904 citations


Journal ArticleDOI
09 Sep 1999-Nature
TL;DR: It is shown that endophilin I is essential for the formation of synaptic-like microvesicles (SLMVs) from the plasma membrane and proposed that, through this action, endophILin I works with dynamin to mediate synaptic vesicle invagination from the Plasma membrane and fission.
Abstract: Endophilin I is a presynaptic protein of unknown function that binds to dynamin, a GTPase that is implicated in endocytosis and recycling of synaptic vesicles. Here we show that endophilin I is essential for the formation of synaptic-like microvesicles (SLMVs) from the plasma membrane. Endophilin I exhibits lysophosphatidic acid acyl transferase (LPAAT) activity, and endophilin-I-mediated SLMV formation requires the transfer of the unsaturated fatty acid arachidonate to lysophosphatidic acid, converting it to phosphatidic acid. A deletion mutant lacking the SH3 domain through which endophilin I interacts with dynamin still exhibits LPAAT activity but no longer mediates SLMV formation. These results indicate that endophilin I may induce negative membrane curvature by converting an inverted-cone-shaped lipid to a cone-shaped lipid in the cytoplasmic leaflet of the bilayer. We propose that, through this action, endophilin I works with dynamin to mediate synaptic vesicle invagination from the plasma membrane and fission.

538 citations


Journal ArticleDOI
TL;DR: Binding of angiostatin to the alpha/beta-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.
Abstract: Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin bound in a concentration-dependent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiostatin binding site. This finding was confirmed by ligand blot analysis of isolated human umbilical vein endothelial cell plasma membrane fractions, which demonstrated that plasminogen bound to a 44-kDa protein, whereas angiostatin bound to a 55-kDa species. Amino-terminal sequencing coupled with peptide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin II and the angiostatin binding protein as the α/β-subunits of ATP synthase. The presence of this protein on the cell surface was confirmed by flow cytometry and immunofluorescence analysis. Angiostatin also bound to the recombinant α-subunit of human ATP synthase, and this binding was not inhibited by a 2,500-fold molar excess of plasminogen. Angiostatin’s antiproliferative effect on endothelial cells was inhibited by as much as 90% in the presence of anti-α-subunit ATP synthase antibody. Binding of angiostatin to the α/β-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.

519 citations


Journal ArticleDOI
TL;DR: The aim of this study was to compare results obtained by eight different short-term assays of estrogenlike actions of chemicals conducted in 10 different laboratories in five countries.
Abstract: The aim of this study was to compare results obtained by eight different short-term assays of estrogenlike actions of chemicals conducted in 10 different laboratories in five countries. Twenty chemicals were selected to represent direct-acting estrogens, compounds with estrogenic metabolites, estrogenic antagonists, and a known cytotoxic agent. Also included in the test panel were 17beta++-estradiol as a positive control and ethanol as solvent control. The test compounds were coded before distribution. Test methods included direct binding to the estrogen receptor (ER), proliferation of MCF-7 cells, transient reporter gene expression in MCF-7 cells, reporter gene expression in yeast strains stably transfected with the human ER and an estrogen-responsive reporter gene, and vitellogenin production in juvenile rainbow trout. 17beta-Estradiol, 17alpha-ethynyl estradiol, and diethylstilbestrol induced a strong estrogenic response in all test systems. Colchicine caused cytotoxicity only. Bisphenol A induced an estrogenic response in all assays. The results obtained for the remaining test compounds--tamoxifen, ICI 182.780, testosterone, bisphenol A dimethacrylate, 4-n-octylphenol, 4-n-nonylphenol, nonylphenol dodecylethoxylate, butylbenzylphthalate, dibutylphthalate, methoxychlor, o,p'-DDT, p,p'-DDE, endosulfan, chlomequat chloride, and ethanol--varied among the assays. The results demonstrate that careful standardization is necessary to obtain a reasonable degree of reproducibility. Also, similar methods vary in their sensitivity to estrogenic compounds. Thus, short-term tests are useful for screening purposes, but the methods must be further validated by additional interlaboratory and interassay comparisons to document the reliability of the methods.

437 citations


Journal ArticleDOI
TL;DR: The novel technique electron capture dissociation of electrospray generated [M + nH]n+ polypeptide cations produces rapid cleavage of the backbone NH-Ca bond to form c and z ions, which supports the previously proposed non-ergodic mechanism, and the low internal energy increment of fragments.
Abstract: The novel technique electron capture dissociation (ECD) of electrospray generated [M + nH]n+ polypeptide cations produces rapid cleavage of the backbone NH−Ca bond to form c and z· ions (in the modified notation of Roepstorff and Fohlman). The potential of the Fourier transform mass spectrometry equipped with ECD in structure analysis of O-glycosylated peptides in the 3 kDa range has been investigated. Totally, 85% of the available interresidue bonds were cleaved in five glycopeptides; more stable c ions accounted for 62% of the observed fragmentation. The c series provided direct evidence on the glycosylation sites in every case studied, with no glycan (GalNAc and dimannose) losses observed from these species. Less stable z· ions supported the glycan site assignment, with minor glycan detachments. These losses, as well as the observed formation of even-electron z ions, are attributed to radical-site-initiated reactions. In favorable cases, complete sequence and glycan position information is obtained fro...

378 citations


Journal ArticleDOI
TL;DR: The extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein, which is in practice irreversible in the presence of fusicoccin.

311 citations


Journal ArticleDOI
TL;DR: Gemin3 binds SMN via its unique COOH-terminal domain, and the presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.
Abstract: The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.

281 citations


Journal ArticleDOI
TL;DR: The findings suggest that CDKN2A/p16 inactivation occurs during the malignant transformation of NFs in NF1 patients and raises the possibility that p16 immunohistochemistry may provide ancillary information in the distinction of NF from MPNST.
Abstract: Patients with neurofibromatosis 1 (NF1) are predisposed to develop multiple neurofibromas (NFs) and are at risk for transformation of NFs to malignant peripheral nerve sheath tumors (MPNSTs). Little is known, however, about the biological events involved in the malignant transformation of NFs. We examined the CDKN2A/p16 gene and p16 protein in NFs and MPNSTs from patients with NF1. On immunohistochemical analysis, all NFs expressed p16 protein. The MPNSTs, however, were essentially immunonegative for p16, with striking transitions in cases that contained both benign and malignant elements. None of the benign tumors had CDKN2A/p16 deletions, whereas three of six MPNSTs appeared to have homozygous CDKN2A/p16 deletions. Methylation analysis and mutation analysis of CDKN2A/p16 in MPNSTs did not reveal any abnormalities. These results show that malignant transformation of NF is associated with loss of p16 expression, which is often secondary to homozygous deletion of the CDKN2A/p16 gene. The findings suggest that CDKN2A/p16 inactivation occurs during the malignant transformation of NFs in NF1 patients and raises the possibility that p16 immunohistochemistry may provide ancillary information in the distinction of NF from MPNST.

232 citations


Journal ArticleDOI
TL;DR: In this article, a supramolecular system that switches reversibly, via three different states, through electrochemical adjustment of the guest properties of tetrathiafulvalene (TTF) has been developed.
Abstract: A supramolecular system that switches reversibly, via three different states, through electrochemical adjustment of the guest properties of tetrathiafulvalene (TTF) has been developed. 1H NMR, lumi...

231 citations


Journal ArticleDOI
TL;DR: Reverse transcription-PCR analysis showed that the main sites of synthesis of gp-340 are lung, trachea, salivary gland, small intestine, and stomach, and the distribution ofgp-340 in macrophages is compatible with a role as an opsonin receptor for SP-D.
Abstract: Surfactant protein D (SP-D) is an oligomeric C type lectin that promotes phagocytosis by binding to microbial surface carbohydrates. A 340-kDa glycoprotein (gp-340) has been shown to bind SP-D in the presence of calcium but does so independently of carbohydrate recognition. This protein exists both in a soluble form and in association with the membranes of alveolar macrophages. The primary structure of gp-340 has been established by molecular cloning, which yielded a 7,686-bp cDNA sequence encoding a polypeptide chain of 2,413 amino acids. The domain organization features 13 scavenger receptor cysteine-rich (SRCR) domains, each separated by an SRCR-interspersed domain, except for SRCRs 4 and 5, which are contiguous. The 13 SRCR domains are followed by two C1r/C1s Uegf Bmp1 domains separated by a 14th SRCR domain and a zona pellucida domain. gp-340 seems to be an alternative spliced form of DMBT1. Reverse transcription–PCR analysis showed that the main sites of synthesis of gp-340 are lung, trachea, salivary gland, small intestine, and stomach. Immunohistochemistry revealed strong staining for gp-340 in alveolar and other tissue macrophages. Immunostaining of the macrophage membrane was either uniform or focal in a way that suggested capping, whereas other macrophages showed strong intracellular staining within the phagosome/phagolysosome compartments. In some macrophages, SP-D and gp-340 were located in the same cellular compartment. Immunoreactive gp-340 was also found in epithelial cells of the small intestine and in the ducts of salivary glands. The distribution of gp-340 in macrophages is compatible with a role as an opsonin receptor for SP-D.

Journal ArticleDOI
13 Aug 1999-Science
TL;DR: It is shown that protein phosphatase 1 (PP1) is essential for bilayer mixing, the last step of membrane fusion, which is crucial for the biogenesis and maintenance of cellular compartments.
Abstract: Intracellular membrane fusion is crucial for the biogenesis and maintenance of cellular compartments, for vesicular traffic between them, and for exo- and endocytosis. Parts of the molecular machinery underlying this process have been identified, but most of these components operate in mutual recognition of the membranes. Here it is shown that protein phosphatase 1 (PP1) is essential for bilayer mixing, the last step of membrane fusion. PP1 was also identified in a complex that contained calmodulin, the second known factor implicated in the regulation of bilayer mixing. The PP1-calmodulin complex was required at multiple sites of intracellular trafficking; hence, PP1 may be a general factor controlling membrane bilayer mixing.

Journal ArticleDOI
TL;DR: It is demonstrated that enzymatic removal of N-linked glycans with simultaneous partial (50%) 18O-labeling of glycosylated asparagine residues prior to proteolysis and MALDI peptide mass mapping can overcome problems and increase the specificity of subsequent database searches.
Abstract: Peptide mass mapping using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in conjunction with interrogation of sequence databases is a powerful tool for the identification of proteins. Glycosylated proteins often yield poor MALDI peptide maps due to shielding of proteolytic cleavage sites and the presence of modified peptides. Here we demonstrate that enzymatic removal of N-linked glycans with simultaneous partial (50%) 18O-labeling of glycosylated asparagine residues prior to proteolysis and MALDI peptide mass mapping can overcome these problems. As a result, more peptides are observed in MALDI spectra which, in turn, increases the specificity of subsequent database searches. Furthermore, the detection of a labeled peptide directly translates into partial sequence information as N-linked carbohydrates are exclusively attached to asparagine residues that form part of the NXS/T sequence. The mass of the formerly glycosylated peptide together with the NXS/T sequence pattern represents...

Journal ArticleDOI
01 Jan 1999-Allergy
TL;DR: This work aims to provide abridge between the clinic and the clinician, and demonstrates the importance of knowing the individual risks and benefits to the individual patient before and during the course of treatment.
Abstract: Authors' af®liations: C. Ortolani, M. Ispano, Department of Allergology and Clinical Immunology, Niguarda Ca Granda Hospital, Milan, Italy C. Bruijnzeel-Koomen, Department of Dermatology, Academic Hospital, Utrecht, The Netherlands U. Bengtsson, Allergy Centre, Sahlgrenska University Hospital, GoÈ teborg, Sweden C. Bindslev-Jensen, Department of Dermatology, Odense University Hospital, Odense, Denmark B. BjoÈ rksteÂn, Department of Paediatrics, University Hospital, LinkoÈping, Sweden A. Hùst, Department of Paediatrics, Odense University Hospital, Odense, Denmark R. Jarish, Dermatologic and Pediatric Allergy Clinic, Vienna, Austria C. Madsen, Institute of Toxicology, National Food Agency of Denmark K. Nekam, Department of Allergy and Clinical Immunology, National Institute of Rheumatology and Physiotherapy, Budapest, Hungary R. Paganelli, Department of Clinical Medicine, University La Sapienza, Rome, Italy L.K. Poulsen, Laboratory of Medical Allergology, National University Hospital, Copenhagen, Denmark B. WuÈ thrich, Allergy Unit, Department of Dermatology, University Hospital, Zurich, Switzerland

Journal ArticleDOI
TL;DR: The identification of 92 novel protein spots on the yeast 2‐D protein map are reported, extending the number of protein spots identified on the authors' yeast reference map to 401 and correspond to the products of 279 different genes.
Abstract: By proving the opportunity to visualize several hundred proteins at a time, two-dimensional (2-D) gel electrophoresis is an important tool for proteome research. In order to take advantage of the full potential of this technique for yeast studies, we have undertaken a systematic identification of yeast proteins resolved by this technique. We report here the identification of 92 novel protein spots on the yeast 2-D protein map. These identifications extend the number of protein spots identified on our yeast reference map to 401. These spots correspond to the products of 279 different genes. They have been essentially identified by three methods: gene overexpression, amino acid composition and mass spectrometry. These data can be accessed on the Yeast Protein Map server (htpp://www.ibgc.u-bordeaux2.fr/YPM).

Journal ArticleDOI
TL;DR: This review discusses the properties of the FDA-approved NNRTI drugs and focuses on the recent efforts being made to produce second generation inhibitors that circumvent this resistance problem.
Abstract: Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are promising drugs for the treatment of HIV when used in combination with other anti-HIV drugs such as nucleoside reverse transcriptase (RT) inhibitors and protease inhibitors. The first generation of NNRTIs have, however, suffered from the rapid development of resistance. This review discusses the properties of the FDA-approved NNRTI drugs and focuses on the recent efforts being made to produce second generation inhibitors that circumvent this resistance problem.

Journal ArticleDOI
TL;DR: A background diet rich in olive oil may attenuate the acute procoagulant effects of fatty meals, which might contribute to the low incidence of IHD in Mediterranean areas.

Journal ArticleDOI
TL;DR: In this paper, a non-stationary state space model for multivariate longitudinal count data driven by a latent gamma Markov process is proposed, where the Poisson counts are assumed to be conditionally independent given the latent process.
Abstract: SUMMARY We propose a nonstationary state space model for multivariate longitudinal count data driven by a latent gamma Markov process. The Poisson counts are assumed to be conditionally independent given the latent process, both over time and across categories. We consider a regression model where time-varying covariates may enter via either the Poisson model or the latent gamma process. Estimation is based on the Kalman smoother, and we consider analysis of residuals from both the Poisson model and the latent process. A reanalysis of Zeger's (1988) polio data shows that the choice between a stationary and nonstationary model is crucial for the correct assessment of the evidence of a long-term decrease in the rate of U.S. polio infection.

Journal ArticleDOI
TL;DR: In this paper, the authors show that the standard symmetric two-period R&D model leads to an asymmetric equilibrium only, with endogeneous innovator and imitator roles.
Abstract: With one-way spillovers, the standard symmetric two-period R&D model leads to an asymmetric equilibrium only, with endogeneous innovator and imitator roles. We show how R&D decisions and measures of firm heterogeneity—market shares, R&D shares, and profits—depend on spillovers and on R&D costs. While a joint lab always improves on consumer welfare, it yields higher profits, cost reductions, and social welfare only under extra assumptions, beyond those required with multidirectional spillovers. Finally, the novel issue of optimal R&D cartels is addressed. We show an optimal R&D cartel may seek to minimize R&D spillovers between its members.

Journal ArticleDOI
TL;DR: It is shown that Doc1/Apc10 is a subunit of the yeast APC throughout the cell cycle, and an APC10 homology region is proposed to call the DOC domain, in several protein sequences that also contain either cullin or HECT domains.

Book
22 Dec 1999
TL;DR: Clinical examination of swine Microbiological sampling Diseases ofSwine Therapeutic treatment Pathological examination Anesthesia and analgesia Acclimatization Clinical Examination Prior to Anesthesia Preoperative Fasting Premedication Ear Vein Catheter Induction of Anesthesia Endotracheal Intubation Cricothyrotomy Artificial Ventilation Thermal Support Maintenance of Anesthetic Inhalation Anesthesia
Abstract: Important biological features Breeds The Landrace The Yorkshire, or Large White The Duroc The Hampshire The Pietrain Other Breeds Hybrid Breeds Miniature Breeds The Vietnamese Potbelly Pig The Ossabaw Island Hog Nomenclature Behavior Anatomical and physiological features Integument and Skeleton Digestive System Abdominal Organs Cardiovascular System Pulmonary System Lymphatic and Endocrine System Normative values Husbandry Housing Environmental conditions Temperature and Humidity Ventilation Illumination Noise Environmental enrichment Group Housing Individual Housing Nutrition Nutrient Requirements Feeding Levels Sanitation Frequency Methods Transportation Record keeping Securing welfare Management and quality assurance Microbiological monitoring Infections in Swine and Their Impact on Experiments Pathological Changes, Clinical Disease, and Mortality Immunomodulation Physiological Modulation Competition between Microorganisms within the Animal Interference with Reproduction Interference with Oncogenesis Contamination of Biological Products Impact of the Normal Flora Zoonotic Diseases Legal Regulations Precautions to Prevent Infections Rederivation Barrier Protection Health Monitoring Common Findings in Health Monitoring Genetic monitoring Accreditation AAALAC International ISO 9000 Veterinary care Clinical examination of swine Microbiological sampling Diseases of swine Therapeutic treatment Pathological examination Anesthesia and analgesia Acclimatization Clinical Examination Prior to Anesthesia Preoperative Fasting Premedication Ear Vein Catheter Induction of Anesthesia Endotracheal Intubation Cricothyrotomy Artificial Ventilation Thermal Support Maintenance of Anesthesia Inhalation Anesthesia Halothane Isoflurane Desflurane Sevoflurane Enflurane Nitrous Oxide Injectable Anesthesia Propofol Ketamine and Other Dissociatives Barbiturates Adrenoreceptor (alpha2) Agonists Benzodiazepines Alpha-Chloralose Medetomidine-Butorphanol-Ketamine (MBK) Supplementary Analgesia Fluid Infusion Depth of Anesthesia Monitoring during Anesthesia Neuromuscular Blocking Agents Postoperative Management Clinical Signs of Postoperative Pain Postoperative Analgesics Anesthetic Emergencies Laryngospasm and Edema Euthanasia Experimental techniques Restraint Sampling techniques Blood Sampling Urine and Feces Cerebrospinal Fluid Bile and Pancreatic Excretions Administration of compounds Basic surgical procedures Catheterization The Carotid Artery and the Internal Jugular Vein The Carotid Artery The Internal Jugular Vein The External Jugular Vein The Cephalic Vein The Femoral Artery and Vein Laparotomy Portal Vein Cannulation The Hepatic Vein The Pancreatic Duct Exteriorization of Catheters Catheter Sizes and Material Catheter Maintenance Safety testing of chemicals and drugs Dermal Toxicology Acute and Chronic Systemic Toxicology Necropsy Equipment Preservation Procedure Special issues regarding gene-modified swine Resources Associations Books Journals Internet resources Swine and diet, and equipment Miniature Swine Resources Diet Resources References Index

Journal ArticleDOI
TL;DR: The genotype frequencies in the centenarians and the blood donors were similar for all polymorphisms, and this study suggests that common variations in genes associated with cardiovascular risk do not contribute significantly to longevity.
Abstract: Do extremely old persons have a genetically favourable profile which has protected them from cardiovascular death? We have tried to answer this question by measuring DNA polymorphisms of selected cardiovascular risk indicators [factor VII, FVII (R/Q353, intron 7 (37bp)n, and -323ins10), beta fibrinogen (-455G/A), plasminogen activator inhibitor type 1, PAI-1 (-675(4G/5G)), tissue plasminogen activator, t-PA (intron 8 ins311), platelet receptor glycoprotein IIb/IIIa, GPIIb/IIIa (L/P33), prothrombin (20210G/A), methylene tetrahydrofolate reductase, MTHFR (A/V114), angiotensin converting enzyme, ACE (intron 16 ins287), and angiotensinogen (M/T235)] Blood was collected from 187 unselected Danish centenarians, and 201 healthy Danish blood donors, aged 20-64 years (mean age 42 years) Genomic DNA was amplified using PCR and the genotype was determined by RFLP methods or allele-specific amplification followed by agarose gel electrophoresis The frequencies of the high-risk alleles in centenarians were: for FVII R/Q353 091; for FVII intron 7 (37bp)n 067; for FVII-323 ins10 090; for fibrinogen 016; for PAI-1 052; for t-PA 059; for GPIIb/IIIa 016; for prothrombin 0008; for MTHFR 033; for ACE 052; and for angiotensinogen 036 Comparable frequencies were observed in the blood donors Subgroup analysis of men and women separately gave similar results The genotype frequencies in the centenarians and the blood donors were similar for all polymorphisms, and this study suggests that common variations in genes associated with cardiovascular risk do not contribute significantly to longevity

Journal ArticleDOI
TL;DR: The soundgenerating mechanism in the bird syrinx has been the subject of debate as mentioned in this paper, and endoscopic imaging of the syrinix during phonation provided evidence for vibrations of membranes and labia, bu
Abstract: The soundgenerating mechanism in the bird syrinx has been the subject of debate Recent endoscopic imaging of the syrinx during phonation provided evidence for vibrations of membranes and labia, bu

Journal ArticleDOI
04 Nov 1999-Oncogene
TL;DR: The data suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and S RCR interspersed domains, and the major part of the gene harbours locus specific repeats, which may point to the D MBT1 locus as a region susceptible to chromosomal instability.
Abstract: Increasing evidence has accumulated for an involvement of the inactivation of tumour suppressor genes at chromosome 10q in the carcinogenesis of brain tumours, melanomas, and carcinomas of the lung, the prostate, the pancreas, and the endometrium. The gene DMBT1 (Deleted in Malignant Brain Tumours 1) is located at chromosome 10q25.3 – q26.1, within one of the putative intervals for tumour suppressor genes. DMBT1 is a member of the scavenger-receptor cysteine-rich (SRCR) superfamily and displays homozygous deletions or lack of expression in glioblastoma multiforme, medulloblastoma, and in gastrointestinal and lung cancers. Based on these properties, DMBT1 has been proposed to be a candidate tumour suppressor gene. We have determined the genomic sequence of DMBT1 to allow analyses of mutations. The gene has at least 54 exons that span a genomic region of about 80 kb. We have identified a putative exon with coding potential for a transmembrane domain. Our data further suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and SRCR interspersed domains. The major part of the gene harbours locus specific repeats. These repeats may point to the DMBT1 locus as a region susceptible to chromosomal instability.


Journal ArticleDOI
TL;DR: Two-dimensional 1H NMR spectroscopy obtained at 750 and 500 MHz is used to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand and showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site.
Abstract: LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2'-O, 4'-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional 1H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32A. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3 A. The average twist over the sequence are 35.9 degrees +/- 0.3 degrees. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by 1H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5'-CT(L)AG-3' site than in the unmodified 5'-CTAG-3' site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.

Journal ArticleDOI
TL;DR: It is proposed that C-signal instructs cells to move with high speed and low stop and reversal frequencies into aggregation centers during development and it is shown that appropriately starved individual cells change behavior during fruiting body formation.
Abstract: Fruiting body formation in Myxococcus xanthus depends on ordered changes in cell movements from swarming to aggregation in response to starvation. We show that appropriately starved individual cells change behavior during fruiting body formation. Specifically, from the time of initiation of aggregation, individual wild-type cells began to move with increased gliding speeds, the duration of the mean gliding interval increased, and the stop frequency decreased whereas the duration of the mean stop interval and the reversal frequency remained unchanged. Mutants lacking the cell surface-associated, intercellular C-signal (csgA mutants) failed to aggregate. Likewise, appropriately starved individual csgA cells did not change their behavior during development. In the absence of other cell–cell interactions, the motility defect of individual csgA cells was corrected in a time- and concentration-dependent manner after C-signaling was reestablished by exogenous MalE-CsgA protein. The C-signal-induced stimulation of motility depended on the cytoplasmic Frz signal transduction system. We propose that C-signal instructs cells to move with high speed and low stop and reversal frequencies into aggregation centers during development.

Journal ArticleDOI
TL;DR: In this article, an individualized cutoff for the screening instrument resulted in detection of a substantial number of cases with very mild dementia, which subsequently resulted in higher incidence rates than those reported in most other studies.
Abstract: Objective: Calculation of incidence of dementia and AD, including cases in the earliest phases of the diseases. Background: Establishment of incidence estimates is important for the future planning of the health care system, and incidence studies can offer insights into risk factors. Methods: A total of 5,237 persons age 65 to 84 years were randomly drawn among people living in the municipality of Odense, Denmark. Of this sample 3,086 persons were eligible for the incidence study. All participants were examined with CAMCOG, the cognitive section of The Cambridge Examination for Mental Disorders of the Elderly (CAMDEX), and the follow-up period was 2 years. Using multiple linear regression, the CAMCOG cutoff score was individualized to detect even minor cognitive decline with optimal precision. Possibly demented persons were further examined with the remaining part of the CAMDEX and neuropsychological tests. AD was diagnosed according to National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer’s Disease and Related Disorders Association criteria for probable AD, and vascular dementia and dementia of other types were diagnosed according to Diagnostic and Statistical Manual of Mental Disorders (3rd ed., revised) criteria for dementia. Finally, the severity of dementia was determined according to the Clinical Dementia Rating scale. Results: The incidence rate for very mild to severe dementia was 29.5 per 1,000 person-years and 20.9 for AD, and the rates were similar for men and women. Conclusion: Application of an individualized cutoff for the screening instrument resulted in detection of a substantial number of cases with very mild dementia, which subsequently resulted in higher incidence rates than those reported in most other studies.

Journal ArticleDOI
TL;DR: In a population-based study of dementia, the cost of care for 245 demented elderly and 490 controls matched by age and gender was estimated and except for very mild dementia the cost did not differ between elderly who suffer from Alzheimer’s disease and those with other types of dementia.
Abstract: In a population-based study of dementia, the cost of care for 245 demented elderly and 490 controls matched by age and gender was estimated. Dementia of Alzheimer's type was diagnosed according to the NINCDS-ADRDA criteria, and vascular dementia and other types of dementia were diagnosed according to the DSM-IIIR criteria. Severity of dementia was determined by the Clinical Dementia Rating scale. The annual cost of medical care, domestic care, home help, nursing home and special equipment for nondemented patients was DKK 22,000 per person while the cost for very mildly, mildly, moderately and severely demented patients was DKK 49,000, DKK 93,000, DKK 138,000 and DKK 206,000, respectively. Except for very mild dementia the cost did not differ between elderly who suffer from Alzheimer's disease and those with other types of dementia. The net cost of dementia is the difference in cost between those with dementia and the matched controls and amounts on average to DKK 77,000 per person per year. However, priority setting cannot be based on the cost of dementia per se, but only on the cost of a specific dementia intervention compared to its health benefit.

Journal ArticleDOI
TL;DR: An important role for CYP3A4 in the oxidation of quinidine in vivo is confirmed, and this applies particularly to the formation of 3-hydroxyquinidine.
Abstract: Aims In vitro studies suggest that the oxidation of quinidine to 3-hydroxyquinidine is a specific marker reaction for CYP3A4 activity. To assess the possible use of this reaction as an in vivo marker of CYP3A4 activity, we studied the involvement of cytochromes CYP2C9, CYP2E1 and CYP3A4 in the in vivo oxidative metabolism of quinidine. Methods An open study of 30 healthy young male volunteers was performed. The pharmacokinetics of a 200 mg single oral dose of quinidine was studied before and during daily administration of 100 mg diclofenac, a CYP2C9 substrate (n=6); 200 mg disulfiram, an inhibitor of CYP2E1 (n=6); 100 mg itraconazole, an inhibitor of CYP3A4 (n=6); 250 ml single strength grapefruit juice twice daily, an inhibitor of CYP3A4 (n=6); 250 mg of erythromycin 4 times daily, an inhibitor of CYP3A4 (n=6). Probes of other enzyme activities, caffeine (CYP1A2), sparteine (CYP2D6), mephenytoin (CYP2C19), tolbutamide (CYP2C9) and cortisol (CYP3A4) were also studied. Results Concomitant administration of diclofenac reduced the partial clearance of quinidine by N-oxidation by 27%, while no effect was found for other pharmacokinetic parameters of quinidine. Concomitant administration of disulfiram did not alter any of the pharmacokinetic parameters of quinidine. Concomitant administration of itraconazole reduced quinidine total clearance, partial clearance by 3-hydroxylation and partial clearance by N-oxidation by 61, 84 and 73%, respectively. The renal clerance was reduced by 60% and the elimination half-life increased by 35%. Concomitant administration of grapefruit juice reduced the total clearance of quinidine and its partial clearance by 3-hydroxylation and N-oxidation by 15, 19 and 27%, respectively. The elimination half-life of quinidine was increased by 19%. The caffeine metabolic index was reduced by 25%. Concomitant administration of erythromycin reduced the total clearance of quinidine and its partial clearance by 3-hydroxylation and N-oxidation by 34, 50 and 33%, respectively. Cmax was increased by 39%. Conclusions The results confirm an important role for CYP3A4 in the oxidation of quinidine in vivo, and this applies particularly to the formation of 3-hydroxyquinidine. While a minor contribution of CYP2C9 to the N-oxidation of quinidine is possible, a major involvement of the CYP2C9 or CYP2E1 enzymes in the oxidation of quinidine in vivo is unlikely.