Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells.

ATP is released from cells in response to various stimuli. Our previous studies on pancreas indicated that pancreatic acini could be major stores of secreted ATP. In the present study, our aim was to establish the role of the vesicular nucleotide transporter (VNUT), SLC17A9, in storage and release of ATP. Freshly prepared acini from mice and AR42J rat acinar cells were used in this study. We illustrate that in AR42J cells, quinacrine (an ATP store marker) and Bodipy ATP (a fluorescent ATP analog) co-localized with VNUT-mCherry to vesicles/granules. Furthermore, in acini and AR42J cells, a marker of the zymogen granule membranes, Rab3D, and VNUT co-localized. Dexamethasone treatment of AR42J cells promoted formation of acinar structures, paralleled by increased amylase and VNUT expression, and increased ATP release in response to cholinergic stimulation. Mechanical stimulus (pressure) and cell swelling also induced ATP release, but this was not influenced by dexamethasone, most likely indicating different non-zymogen-related release mechanism. In conclusion, we propose that VNUT-dependent ATP release pathway is associated with agonist-induced secretion process and downstream purinergic signalling in pancreatic ducts.

Early predictors of the long-term outcome of low back pain--results of a 22-year prospective cohort study.

BACKGROUND: poor outcome of low back pain in patients seen in general practice is related to the pain history, physical impairment and working conditions, at least in short-term follow-ups. We do not know if these findings hold, seen from the perspective of decades. OBJECTIVES: to show if patients consulting the GP for the first time regarding an episode of low back pain have excess poor outcome 22 years later and, if so, whether the best predictors are data based on the symptoms, clinical signs or work history. The design of the study is a 22-year follow-up of an inception cohort of 78 patients with low back pain. The setting of the study is a single general practice in a suburb of Copenhagen, Denmark. METHODS: selected predictors were separated into pain characteristics, clinical signs and indicators related to the work history. Outcome measures were the 1-year period prevalence of low back pain, use of painkillers for low back pain, use of health care providers, impairments due to low back pain and unfitness for work caused by low back pain. The influence of the predictors was assessed by relative risks. RESULTS: after 22 years, four out of five patients still experienced low back pain. The perception of poor working conditions correlates with recurrent low back pain, intake of painkillers and limitations to daily life. CONCLUSION: compared with pain history and clinical findings, the perception of workload is a better predictor of the long-term outcome of low back pain.

Identification and partial characterization of Taastrup virus: a newly identified member species of the Mononegavirales.

We present a 8904-nt sequence of the central part of the RNA genome of a novel virus with a filovirus-like, nonidentical morphology named Taastrup virus (TV) detected in the leafhopper Psammotettix alienus. Sequence analysis identified five potential open reading frames (ORFs) and a complex pattern of homologies to various members of the Mononegavirales suggests a genome organization with the following order of genes: 3'-N-P-M-G-L-5'. Sequence analyses reveal an unusually large glycoprotein (G) containing both potential O-linked (14) and N-linked (9) glycosylation sites-a feature shared with the glycoproteins of Filoviridae and Pneumovirinae, and a nucleoprotein (N) with homology to the nucleoprotein of viral hemorrhagic septicemia virus (VHSV), a member of the Rhabdoviridae. Highly conserved domains were identified in the RNA-dependent RNA polymerase (L) between TV and other viruses within the order of Mononegavirales, and homology was found in particular with members of the Rhabdoviridae. The sequence similarities and the unique filovirus-like but nonidentical morphology unambiguously refer this newly identified virus to the order of Mononegavirales but to no family more than any, to other within the order.

Cloning and characterisation of a glucoamylase gene (GlaM) from the dimorphic zygomycete Mucor circinelloides.

This article reports a novel strategy for the cloning of glucoamylase genes using conserved sequences and semi-nested PCR and its application in cloning the GlaM glucoamylase gene and cDNA from the dimorphic zygomycete Mucor circinelloides. The deduced 609-amino-acid enzyme (including signal peptide) is 63% identical to the Rhizopus oryzae raw starch-degrading glucoamylase and is the third glucoamylase reported to have the putative starch-binding domain placed N-terminally. The C-terminal catalytic domain is separated from the starch-binding domain by a serine/threonine-rich linker. An alignment of the cloned gene and cDNA sequences showed that the gene contains three introns. The transcriptional start site and the site of polyadenylation were defined by primer extension and 3'RACE, respectively. The atypical Kozak sequence is identical to the one used in R. oryzae in positions -1 to -4. Northern slot blots revealed that glucoamylase transcription is induced during growth on starch and repressed by glucose. In silico analysis of the 1.9-kb promoter sequence cloned by inverse PCR revealed the presence of several putative regulatory elements, most notably a 19-bp sequence containing six overlapping copies of the Saccharomyces cerevisiae Nrg1p binding sequence.

[Randomized controlled trial of structured, individualized treatment of type 2 diabetes mellitus. The project of Diabetes Care in General Practice]. / Randomiseret kontrolleret undersøgelse af struktureret, individualiseret behandling af type 2-diabetes mellitus. Projektet Diabetesomsorg i almen praksis.

INTRODUCTION: We assessed the effect of a multifaceted intervention directed at general practitioners to improve type 2 diabetes care. MATERIALS AND METHODS: Three hundred and eleven Danish practices with 474 general practitioners were randomised to structured personal care (intervention group) or routine care (comparison group). Of 970 surviving patients (aged 40+ years) diagnosed with diabetes in 1989-1991, 874 (90.1%) were assessed after 6 years. Intervention comprised regular follow-up and individualized goal-setting, supported by reminders to doctors, clinical guidelines, feed-back, and continuing medical education. RESULTS: Predefined non-fatal outcomes and mortality were the same in both groups. The following risk factor levels were lower in the intervention patients than in the comparison patients: fasting plasma glucose (7.9 vs 8.7 mmol/l, medians, P = 0.0007), haemoglobin A1c (8.5 vs 9.0%, P < 0.0001, normal range 5.4-7.4%), systolic blood pressure (145 vs 150 mmHg, P = 0.0004), and cholesterols (6.0 vs 6.1 mmol/l, P = 0.029, baseline-adjusted). Both groups had sustained a weight loss since diagnosis (2.6 vs 2.0 kg). Metformin was the only drug used more frequently in the intervention group (24 vs 15%). Intervention doctors arranged more follow-up consultations, referred fewer patients to diabetes clinics, and were more optimistic in their goal-setting. DISCUSSION: In primary care, individualized goal-setting with educational and surveillance support may for at least six years bring risk factors of patients with type 2 diabetes to a level that in other trials has been shown to reduce diabetic complications, but without adverse weight gain.

Randomised controlled trial of structured personal care of type 2 diabetes mellitus.

OBJECTIVE: To assess the effect of a multifaceted intervention directed at general practitioners on six year mortality, morbidity, and risk factors of patients with newly diagnosed type 2 diabetes. DESIGN: Pragmatic, open, controlled trial with randomisation of practices to structured personal care or routine care; analysis after 6 years. SETTING: 311 Danish practices with 474 general practitioners (243 in intervention group and 231 in comparison group). PARTICIPANTS: 874 (90.1%) of 970 patients aged >/=40 years who had diabetes diagnosed in 1989-91 and survived until six year follow up. INTERVENTION: Regular follow up and individualised goal setting supported by prompting of doctors, clinical guidelines, feedback, and continuing medical education. MAIN OUTCOME MEASURES: Predefined clinical non-fatal outcomes, overall mortality, risk factors, and weight. RESULTS: Predefined non-fatal outcomes and mortality were the same in both groups. The following risk factor levels were lower for intervention patients than for comparison patients (median values): fasting plasma glucose concentration (7.9 v 8.7 mmol/l, P=0.0007), glycated haemoglobin (8.5% v 9.0%, P<0.0001; reference range 5.4-7.4%), systolic blood pressure (145 v 150 mm Hg, P=0.0004), and cholesterol concentration (6.0 v 6.1 mmol/l, P=0.029, adjusted for baseline concentration). Both groups had lost weight since diagnosis (2.6 v 2.0 kg). Metformin was the only drug used more frequently in the intervention group (24% (110/459) v 15% (61/415)). Intervention doctors arranged more follow up consultations, referred fewer patients to diabetes clinics, and set more optimistic goals. CONCLUSIONS: In primary care, individualised goals with educational and surveillance support may for at least six years bring risk factors of patients with type 2 diabetes to a level that has been shown to reduce diabetic complications but without weight gain.

Role of phylogenetically conserved amino acids in folding of Na,K-ATPase.

This paper focuses on the amino acid sequence 708-TGDGVNDSPALKK in pig kidney Na,K-ATPase as one of the best conserved among P-type ATPases. In Ca-ATPase this sequence forms a strand-loop-helix structure as part of a Rossman fold next to the phosphorylation site. Substitution of polar residues in the investigated sequence interfered with high-level accumulation of mutant protein. Mutant alpha1-subunit protein only accumulated in membranes from yeast cells grown at 15 degrees C whereas wild-type protein accumulated at both 15 and 35 degrees C. A systematic screen for the molecular mechanism behind lack of accumulation of mutant protein at 35 degrees C showed that transcription and translation were unaffected by the mutations. To demonstrate in vivo protein folding problems, an unfolded protein response reporter system was constructed in yeast. In this strain, only expression of mutant Na,K-ATPase alpha1-subunit caused induction of the unfolded protein response at 35 degrees C, indicating folding problems in the ER. Lowering the expression temperature to 15 degrees C prevented induction of the unfolded protein response after mutant protein expression, indicating correct folding at this temperature. At the permissive temperature mutant proteins were able to escape the endoplasmic reticulum quality control, reach the plasma membrane, and bind ouabain with high affinity. Since mutants in the 708-TGDGVNDSPALKK segment had a thermo inactivation profile identical to that of wild type, they were classified as temperature-sensitive synthesis mutants. The results indicate that this segment contributes side chains of importance for overall folding and maturation of Na,K-ATPase and all other P-type ATPases.

Structure-function relationships of Na(+), K(+), ATP, or Mg(2+) binding and energy transduction in Na,K-ATPase.

The focus of this article is on progress in establishing structure-function relationships through site-directed mutagenesis and direct binding assay of Tl(+), Rb(+), K(+), Na(+), Mg(2+) or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E(2)[2K] or E(1)P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu(327) in transmembrane segment M4, Glu(779) in M5, Asp(804) and Asp(808) in M6 are essential for tight binding of K(+) and Na(+). Asn(324) and Glu(327) in M4, Thr(774), Asn(776), and Glu(779) in 771-YTLTSNIPEITP of M5 contribute to Na(+)/K(+) selectivity. Free ATP binding identifies Arg(544) as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp(710) or Asn(713) do not interfere with free ATP binding. Asp(710) is essential and Asn(713) is important for coordination of Mg(2+) in the E(1)P[3Na] complex, but they do not contribute to Mg(2+) binding in the E(2)P-ouabain complex. Transition to the E(2)P form involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713) and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369).

Role of conserved TGDGVND-loop in Mg2+ binding, phosphorylation, and energy transfer in Na,K-ATPase.

In the P-domain, the 369-DKTGTLT and the 709-GDGVNDSPALKK segment are highly conserved during evolution of P-type E1-E2-ATPase pumps irrespective of their cation specificities. The focus of this article is on evaluation of the role of the amino acid residues in the P domain of the alpha subunit of Na,K-ATPase for the E1P[3Na]--> E2P[2Na] conversion, the K+-activated dephosphorylation, and the transmission of these changes to and from the cation binding sites. Mutations of residues in the TGDGVND loop show that Asp710 is essential, and Asn713 is important, for Mg2+ binding and formation of the high-energy MgE1P[3Na] intermediate. In contrast Asp710 and Asp713 do not contribute to Mg2+ binding in the E2P-ouabain complex. Transition to E2P thus involves a shift of Mg2+ coordination away from Asp710 and Asn713 and the two residues become more important for K+-activated hydrolysis of the acyl phosphate bond at Asp369. Transmission of structural changes between the P-domain and cation sites in the membrane domain is evaluated in light of the protein structure, and the information from proteolytic or metal-catalyzed cleavage and mutagenesis studies.

Importance of conserved alpha -subunit segment 709GDGVND for Mg2+ binding, phosphorylation, and energy transduction in Na,K-ATPase.

The segment (708)TGDGVNDSPALKK(720) in the alpha-subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg(2+) or ATP and energy transduction. Here, 11 mutations of polar residues are expressed at reduced temperature in yeast with preserved capacities for high affinity binding of ouabain and ATP, whereas the Thr(708) --> Ser mutation and alterations of Asp(714) abolish all catalytic reactions. In mutations of Asp(710) and Asn(713), ATP affinity is preserved or increased, whereas Na,K-ATPase activity is severely reduced. Assay of phosphorylation from ATP in the presence of oligomycin shows that Asp(710) contributes to coordination of Mg(2+) during transfer of gamma-phosphate to Asp(369) in the high energy Mg.E(1)P[3Na] intermediate and that Asn(713) is involved in these processes. In contrast, Asp(710) and Asp(713) do not contribute to Mg(2+) binding in the E(2)P.ouabain complex. Transition to E(2)P thus involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713), and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369). The Asp(710) --> Ala mutation blocks interaction with vanadate, whereas Asn(713) --> Ala interferes with phosphorylation from P(i) of the E(2).ouabain complex, showing that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction. The Asp(710) --> Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of Tl(+) 2- to 3-fold, suggesting a role in transmission of K(+) stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.

Container-grown tree seedling responses to sodium chloride applications in different substrates.

Seedlings of Norway maple (Acer platanoides), silver birch (Betula pendula), Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) were grown in selected sodium chloride (NaCl) concentrations, soil types and under different watering regimes. Plants were raised from seeds, except for Scots pine plants which were obtained from a commercial source. Among the plant species tested, Scots pine was the most tolerant to soil salinity, while Norway spruce was the most susceptible. For both Norway maple and Norway spruce some half-sib families were more tolerant than others. No significant correlation was found between the tolerance of different half-sib families and the tolerance of mother trees observed in the field. The extent of leaf necrosis correlated significantly with the leaf concentrations of sodium (Na) and chloride (Cl). Among half-sib families within the species no such correlation was found. On the other hand, the least injured progeny of Norway maples had the highest concentrations of NaCl. The extent of salt-induced leaf necrosis varied with soil type, and a significant interaction between species and soil type was observed. Seedlings of Norway spruce grown in sand showed more severe necrosis and significantly higher concentrations of Na and Cl than seedlings grown in loam, silt loam, and peat. The severity of salt-induced leaf injury varied with the watering regime. Silver birch was the most affected species by drought and autumn watering treatments. Plants of silver birch subjected to drought showed increased leaf necrosis compared to the non-treated plants, and autumn watering treatment reduced the severity of leaf necrosis.

[Individual registration of children in the national health service. New possibilities for epidemiological research in primary health care]. / Eget sygesikringsbevis til børn. Nye muligheder for epidemiologisk forskning i den primaere sundhedssektor.

Since January 1st 1996 all Danish citizens, children included, have been recorded individually in The National Health Service Register (SSR). Services rendered to children are no longer registered with an adult person. This article describes the implementation of this new arrangement. The part of health services to children recorded under an adult personal identification number is getting asymptotically closer to a minimum of about four percent, which is determined by the average time of naming of children. After the introduction of individual registration of children the SSR has improved considerably as a basis for epidemiological studies in Danish primary care.

Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was examined as host for heterologous expression of the G protein-coupled VPAC1 receptor. Rat VPAC1 receptor cDNA and two chimeric constructs encoding the yeast mating factor pre-pro alpha-leader peptide fused in-frame to rat VPAC1 receptor were expressed in yeast cells under control of a galactose inducible promoter. The rat VPAC1 receptor was fused to the HSV tag epitope to ensure proper immunological detection of the receptor. Crucial conditions for high-level expression of active rat VPAC1 receptor included growth in amino acid supplemented minimal medium, fusion to the yeast alpha-leader peptide and a temperature shift from 30 degrees C to 15 degrees C before promoter induction. Western blotting showed that the expressed receptor was highly glycosylated and a band of 47 kDa was obtained upon endoglycosidase H treatment. Binding with radioiodinated vasoactive intestinal polypeptide revealed a KD of 2.5 nM and an IC50 of 15 nM when displacing with unlabeled vasoactive intestinal polypeptide. VPAC1 receptor density quantified by Western blotting was 510 pmol/mg membrane protein of which only 66 pmol/mg were able to bind vasoactive intestinal polypeptide.

Allergological examination in general practice and in the specialist field of allergology. A comparative study of the concordance between a group of general practitioners and the allergological specialised health care services to which they refer.

OBJECTIVE: To study the concordance between specially trained general practitioners (GPs) and specialist doctors working as consultants to GPs, with regard to diagnosis of allergic illnesses, evaluation of indications for hyposensitisation, and referrals from GPs to specialists. DESIGN: Thirty-four GPs and five specialists practising privately and eight allergologic outpatient clinics participated. The patients included had symptoms indicating allergologic examination. An identical diagnostic procedure was used by the GPs and by the specialists/outpatient clinics. SETTING: General practices, specialist practices, outpatient clinics. SUBJECTS: One-hundred and forty-eight patients. MAIN OUTCOME MEASURES: With regard to skin-prick test there was concordance between the GPs and the specialists in 82.5% of 1322 paired comparisons, in 74.0% with regard to anamnesis + skin-prick test, and in 66.5% with regard to the statement allergy. There was concordance with regard to indication for hyposensitisation in 88.3%, and for referral in 54.1%. There was symmetry concerning prick test and anamnesis + prick test, and asymmetry concerning the statement allergy, and indication for hyposensitisation and for referral. CONCLUSIONS: Specially trained GPs diagnosed specific allergy in concordance with specialists. There was asymmetry between GPs and specialists concerning the statement allergy, indication for hyposensitisation and for referral.

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.

Structure-function relationships of E1-E2 transitions and cation binding in Na,K-pump protein.

Fully active Na,K-ATPase and lethal mutations can be expressed in yeast cells in yields allowing for equilibrium ATP binding, occlusion of T1+, K+ displacement of ATP, and Na(+)-dependent phosphorylation with determinations of affinity constants for binding and constants for the conformational equilibria. Removal of the charge and hydrophobic substitution of the phosphorylated residue (Asp369Ala) reveals an intrinsic high affinity for ATP binding (Kd 2.8 vs. 100 nM for wild type) and causes a shift of conformational equilibrium towards the E2 form. Substitution of Glu327, Glu779, Asp804 or Asp808 in transmembrane segments 4, 5, and 6 shows that each of these residues are essential for high-affinity occlusion of K+ and for binding of Na+. Substitution of other residues in segment 5 shows that the carboxamide group of Asn776 is important for binding of both K+ and Na+. Differential effects of the relevant mutations identify Thr774 as specific determinant of Na+ binding in the E1P[3Na] form, whereas Ser775 is a specific participant of high-affinity binding of the E2[2K] form, suggesting that these residues engage in formation of a molecular Na+/K+ switch. The position of the switch may be controlled by rotating or tilting the helix during the E1-E2 transition.

Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase.

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.