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P A Pedersen

Publications and source records attributed to P A Pedersen.

At least 37 records · Page 2Linked to original sources
PubMed1998

Structure-function relationships based on ATP binding and cation occlusion at equilibrium in Na,K-ATPase.

This work evaluates the results of measurements of equilibrium binding of ATP and cations in lethal or partially active mutations of Na,K-ATPase that were expressed at high yield in yeast cells. ATP binding studies allowed estimation of the expense in free energy required to position the gamma-phosphate in proximity of the carboxylate groups of the phosphorylated residue Asp369 and the role of this residue in governing long range E1-E2 transitions. An arginine residue (Arg546) appearing to be involved in ATP binding has been identified. Wild type yeast enzyme was capable of occluding two T1(+)-ions per ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(T1+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. The substitutions to Glu327(Gln,Asp), Asp804(Asn,Glu), Asp808(Asn,Glu) and Glu779(Asp) completely abolished occlusion or severely reduced the affinity for T1+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to one T1+ ion per alpha 1 beta 1 unit with a 3-fold decrease of the apparent affinity for the ion (Kd(T1+) = 24 +/- 8 mM). These carboxylate groups in transmembrane segments 4, 5, and 6 therefore appear to be essential for high affinity occlusion of K(+)-ions.

Adenosine Triphosphate↗
PubMed1997

Identification of Asp804 and Asp808 as Na+ and K+ coordinating residues in alpha-subunit of renal Na,K-ATPase.

Mutations to Asp804 and Asp808 in the alpha-subunit almost abolish Na,K-ATPase activity, but high-affinity binding of [3H]ATP or [3H]ouabain at equilibrium and E1-E2 transitions are preserved. Titration of K+-ion displacement of [3H]ATP or [3H]ouabain shows that the mutations interfere with occlusion of K+ in the E2[2K] conformation. Reduced phosphorylation levels or affinities for Na+ in presence of oligomycin indicate that Asp804 and Asp808 also contribute to coordination of Na+ in the E1P[3Na] form. Demonstration of alternate interactions of Na+ or K+ with Asp804 and Asp808 support the notion of cation binding in a ping-pong sequence in catalytic models of Na,K-pumping.

Adenosine Triphosphate↗
PubMed1997

The Danish National Health Service Register. A tool for primary health care research.

The purpose of the article is to describe the Danish National Health Service Register and its value in primary health care research, using mainly general practice as an example. The Danish National Health Service Register is a data system available for counties and municipalities to manage the National Health Insurance covering primary health care providers. The counties use the register for administrative purposes, especially for the settling of accounts with providers. The register contains data on all citizens, providers, and health care services reimbursed by the health authorities, but holds no data regarding health status. The accuracy and degree of completeness of persons and variables in the National Health Service Register rests on the fact that most primary health care services in Denmark are reimbursed and, therefore, included in the data base. This tie to the economy of both health care authorities and providers is supposed to confer the final register with a high degree of completeness. It is a clear advantage that almost all citizens in Denmark are assigned to only one general practice, and that the attitude towards research is positive among Danish patients and doctors. The register has so far only been used occasionally for research purposes. To take advantage of the register for research purposes within clinical and health services research, however, one must possess not only a detailed knowledge of the Danish society, including the structure of the Danish health care system, but also an intimate acquaintance with rather complex agreement system and the actual interpretation of this.

Denmark↗
PubMed1996

Consequences of mutations to the phosphorylation site of the alpha-subunit of Na, K-ATPase for ATP binding and E1-E2 conformational equilibrium.

Expression of Na, K-ATPase in yeast allowed targeting of alpha beta-units with lethal substitutions at the phosphorylation site alpha 1 (D369N) beta 1 and alpha 1 (D369A) beta 1 at the cell surface at the same concentration of alpha-subunit and [3H] ouabain binding sites as for wild type Na, K-ATPase. Phosphorylation and reaction with vanadate were abolished, and the mutations had no Na, K-ATPase or K-phosphatase activity. Binding of [3H]-ATP at equilibrium revealed an intrinsic high affinity of the D369A mutation for ATP (KD = 2.8 nM) that was 39-fold higher than for wild type Na, K-ATPase (KD = 109 nM). The affinities for ADP were unaffected, indicating that the negative charge at residue 369 determines the contribution of the gamma-phosphate to the free energy of ATP binding. Analysis of the K(+)-ATP antagonism showed that the reduction of charge and hydrophobic substitution at Asp369 of the alpha-subunit caused a large shift in conformational equilibrium toward the E2-form. This was accompanied by a large increase in affinity for [3H] ouabain in Mg2+ medium with KD = 4.9 nM for D369A compared to KD = 51 nM for D369N and KD = 133 nM for wild type, and [3H] ouabain binding (KD = 153 nM) to D369A was detectable even in absence of Mg2+. In addition to its function as receptor of the gamma-phosphate of ATP, Asp369 has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E1-E2 transitions which are coupled to reorientation of cation sites and changes in affinity for digitalis glycosides.

Adenosine Triphosphate↗
PubMed1996

Expression in high yield of pig alpha 1 beta 1 Na,K-ATPase and inactive mutants D369N and D807N in Saccharomyces cerevisiae.

Studies of structure-function relationships in Na,K-ATPase require high yield expression of inactive mutations in cells without endogenous Na,K-ATPase activity. In this work we developed a host/vector system for expression of fully active pig Na,K-ATPase as well as the inactive mutations D369N and D807N at high levels in Saccharomyces cerevisiae. The alpha 1- and beta 1-subunit cDNAs were inserted into a single 2-microns-based plasmid with a high and regulatable copy number and strong galactose-inducible promoters allowing for stoichiometric alterations of gene dosage. The protease-deficient host strain was engineered to express high levels of GAL4 transactivating protein, thereby causing a 10-fold increase in expression to 32,500 +/- 3,000 [3H]ouabain sites/cell. In one bioreactor run 150-200 g of yeast were produced with 54 +/- 5 micrograms of Na,K-pump protein/g of cells. Through purification in membrane bound form the activity of the recombinant Na,K-ATPase was increased to 42-50 pmol/mg of protein. The Na,K dependence of ATP hydrolysis and the molar activity (4,500-7,000 min-1) were close to those of native pig kidney Na,K-ATPase. Mutations to the phosphorylation site (D369N) or presumptive cation sites (D807N), both devoid of Na,K-ATPase activity, were expressed in the yeast membrane at the same alpha-subunit concentration and [3H]ouabain binding capacity as the wild type Na,K-ATPase. The high yield and absence of endogenous activity allowed assay of [3H]ATP binding at equilibrium, demonstrating a remarkable 18-fold increase in affinity for ATP in consequence of reducing the negative charge at the phosphorylation site (D369N).

Adenosine Triphosphate↗
PubMed1996

Na-K-ATPase isoform (alpha 3, alpha 2, alpha 1) abundance in rat kidney estimated by competitive RT-PCR and ouabain binding.

For understanding the regulation of sodium reabsorption, it is important to know whether the alpha 2- or alpha 3-isoform of Na-K-adenosinetriphosphatase (Na-K-ATPase) is expressed in mammalian kidney in addition to the predominant alpha 1 beta 1-isozyme. Here we applied competitive polymerase chain reaction (PCR) for estimation of mRNA in parenchymal zones of rat kidney for comparison to high-affinity [3H]ouabain binding. The alpha 3-isoform mRNA was demonstrated to form 0.04-0.05% of the amount of alpha 1-isoform mRNA in the cortex, medulla, and papilla of rat kidney. The alpha 2-mRNA was demonstrated in all three zones and constituted 0.03% of the amount of alpha 1-mRNA in cortex. The abundance of the alpha 1 truncated mRNA was 0.1-0.8% of that of the alpha 1-mRNA. The upper limit for expression of Na-K-ATPase isozyme with high ouabain affinity (dissociation constant, 69-141 nM) was 0.096-0.14% of the concentration of alpha 1 beta 1-Na-K-ATPase. Thus a small but well-defined pool of alpha 2- and alpha 3-isoforms constitutes < or = 0.1% of the amount of alpha 1-isoform at both the mRNA and protein level in rat kidney.

Animals↗
PubMed1994

Self-referral and self-payment in Danish primary care.

This study aims at characterizing the group of people who want to have the right to consult any general practitioner or practising specialist without referral on condition of part self-payment, as opposed to the group of people who choose to be registered with a general practice that offers free services but controls further access to the health care system. All adults or a 10% sample of those listed in nationwide Danish registers were examined cross-sectionally for social and demographic factors and utilization of primary and secondary health care. A minority, which totals 3% of the population, chose free choice of doctor and part self-payment. On average, this group is older and has a higher income. Its mortality and its utilization of general practice and hospital services are lower, and its use of practising specialists is higher, than the majority. Among the persons who chose free choice and self-payment, the pattern of utilization is more likely to be due to a wish for free choice and for specialized medical care than to high morbidity. Dissatisfaction caused by restrictions on self-referral to specialists can be met by offering an option of a parallel system of free choice of doctor on condition of part self-payment.

Age Distribution↗
PubMed1993

[Increasing asthma mortality in Denmark 1969-1988. Not because of changed coding practices].

We have studied asthma mortality in Denmark from 1969 to 1988. Age standardized mortality rates calculated in three age groups, 10-34, 35-59, and > 60 years, disclosed similar trends. Increasing mortality from asthma in the mid-1970s to 1988 was seen in all three age groups with higher mortality in 1979-1988 as compared with 1969-1978 of 95%, 55%, and 69%, respectively. Since the eighth revision of the International Classification of Diseases (ICD-8) was used in Denmark over the entire 20-year period, changes in coding practice due to change of classification system cannot explain the findings.

Adolescent↗
PubMed1992

Amoxicillin in treatment of acute uncomplicated exacerbations of chronic bronchitis. A double-blind, placebo-controlled multicentre study in general practice.

The aim of the study was to evaluate whether a broad-spectrum penicillin, amoxicillin, was superior to placebo in resolving symptoms of acute exacerbations of chronic bronchitis in patients from general practice. 131 general practitioners included 278 patients over a period of 30 months. The patients were randomly assigned to treatment with amoxicillin 750 mg b.i.d. or corresponding placebo for 7 days. Patients with pneumonia, a temperature above 38.5 degrees C or heart rate over 100 were excluded for safety reasons. The main effect parameter--the doctors' overall evaluation of the treatment--did not demonstrate any statistically significant difference between amoxicillin or placebo, 63% versus 64% of the patients. Resolution of symptoms was obtained by 19% (25/132) of the patients in the amoxicillin group compared with 10% (13/136) of the patients in the placebo group, P = 0.03. The present findings do not favour routine use of antibiotics in an attempt to improve the course of acute exacerbations as defined in this study in patients with chronic bronchitis.

Acute Disease↗
PubMed1992

[Occurrence of asthma and non-infectious rhinitis in Denmark].

Population surveys of asthma have shown total asthma prevalence rates of 3.8 (2-5) and 3.2 (3-4) in 1978-1979 and 1987. These figures do not fit with the general statements of an increased asthma prevalence during recent decades. Hospitalization has increased from 1 per 1,000 to 1.6 per 1,000 inhabitants from 1979 to 1987. The annual mortality from asthma has doubled during the last ten years, and is now 3 per 100,000 inhabitants. All epidemiological data about asthma are blurred because of uncertain definitions of asthma. Allergic and vasomotor rhinitis occurs among 10 per cent of the adult population and seems to be recorded by 50 per cent more in 1987 than in 1978-1979, especially among the younger population. Also in rhinitis diagnostic criteria seem to vary in a way that makes estimates of variation over a longer period of time uncertain.

Adolescent↗
PubMed1992

Increasing asthma mortality in Denmark 1969-88 not a result of a changed coding practice.

We have studied asthma mortality in Denmark from 1969 to 1988. Age standardized mortality rates calculated in three age groups, 10-34, 35-59, and greater than or equal to 60 years, disclosed similar trends. Increasing mortality from asthma in the mid-1970s to 1988 was seen in all three age groups with higher mortality in 1979-88 as compared with 1969-78 of 95%, 55%, and 69%, respectively. Since the eighth revision of the International Classification of Diseases (ICD8) was used in Denmark over the entire 20-year period, changes in coding practice due to change of classification system cannot explain the findings.

Adolescent↗
PubMed1991

Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli.

Small, defined in-frame deletions and in-frame duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The cellular localization and proteolytic stability of the different mutated fimbrial subunit proteins were determined, and compared with those of the wild-type protein. Based upon these results, we predict a functional role of specific structures in the K88ab fimbrial subunit protein in subunit-subunit interactions as well as in interactions between FaeG and the other proteins encoded by the K88ab operon. The results obtained were further compared with results obtained from operon deletions, linker insertion mutagenesis and the current model for biogenesis of K88 fimbriae. One of the mutated fimbrial subunit genes was used to construct a secreted in-frame fusion between FaeG and a characterized epitope (lacking cysteine) from the Hepatitis B pre-S2 protein. Such fusion proteins might be useful in the design of recombinant vaccines.

Amino Acid Sequence↗
PubMed1991

Structure-function analysis of the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli.

Several in-frame linker insertions have been made in various positions in the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The effects of the linker insertions have been investigated with regard to the ability of the mutated fimbrial subunits to be exported to the surface of the bacterial cell and assembled into a fimbrial structure. The structure/function relationship of the subunit protein is discussed in the light of the phenotypes of the mutations constructed, secondary structure predictions, and hydrophilicity plots.

Amino Acid Sequence↗