Biomedical subjects

R Nielsen

Publications and source records attributed to R Nielsen.

At least 37 records · Page 2Linked to original sources

PubMed2000

Evidence for P2Y-type ATP receptors on the serosal membrane of frog skin epithelium.

The present study presents the first evidence for P2Y-type adenosine 5'-triphosphate (ATP) receptors on the basolateral membranes of frog skin epithelial cells. Cytosolic calcium ([Ca2+]i) was measured with fura-2 and Calcium-Green-1 using epifluorescence microscopy and confocal laser scanning microscopy respectively. In the presence of Ca2+ in the solutions ATP increased [Ca2+]i. The increase in [Ca2+]i was due to the agonist activity of ATP and not to the activity of the potential products of ATP metabolism, i.e. adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) or adenosine, as shown by a comparison of the magnitude of the increases in [Ca2+]i caused by the various compounds. The rise in [Ca2+]i was predominantly monophasic at low ATP concentrations (below 100 microM). At higher concentrations the initial spike was followed by a plateau phase. In the absence of Ca2+ in the extracellular solution ATP caused Ca2+ release from intracellular stores. This could be inhibited by pre-treatment of the tissue with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. The nucleotide uridine 5'-triphosphate (UTP) had similar effects on [Ca2+]i although the plateau level of the [Ca2+]i response was higher with this P2Y agonist. Confocal laser scanning microscopy showed that all cell layers of the epithelium responded to ATP. Our data indicates that serosal ATP acts on serosal P2Y-type receptors in frog skin epithelium. This is the first evidence of a phospholipase C-coupled receptor in this tissue.

Adenosine Monophosphate↗

PubMed2000

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae.

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III. The toxins are important virulence factors. In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs. The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively. A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation. However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest. The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.

Actinobacillus Infections↗

PubMed2000

Effect of alpha1-adrenergic stimulation of Cl- secretion and signal transduction in exocrine glands (Rana esculenta).

In the present work, the effect of stimulation of alpha-adrenergic receptors on Cl- secretion via exocrine frog skin glands was investigated. The alpha-adrenergic stimulation was performed by addition of the adrenergic agonist noradrenaline in the presence of the beta-adrenergic antagonist propranolol. In the presence of propranolol, noradrenaline had no effect on the cellular cAMP content. The Cl- secretion was measured as the amiloride-insensitive short circuit current (ISC). Addition of noradrenaline induced a biphasic increase in the ISC. The increase in ISC coincided with an increase in the net 36Cl- secretion. The noradrenaline-induced increase in ISC was dose-dependent with an EC50 of 13 +/- 0.3 microM. Epifluorescence microscopic measurements of isolated, fura-2-loaded frog skin gland acini were used to characterize the intracellular calcium ([Ca2+]i) response. Application of noradrenaline induced a biphasic [Ca2+]i response, which was dose-dependent with an EC50 of 11 +/- 6 microM. The Ca2+ plateau unlike the peak-response was sensitive to removal of Ca2+ from the extracellular medium. The noradrenaline-induced increase in the Cl- secretion as well as in [Ca2+]i was sensitive to the alpha1-adrenergic antagonist prazosine. Ryanodine and caffeine had no effect on [Ca2+]i indicating that the release was independent of ryanodine-sensitive Ca2+ stores. Noradrenaline mediated a significant increase in the cellular inositol 1,4,5-trisphosphate (IP3) content suggesting that the signal transduction pathway leading to the noradrenaline-induced increase in Ca2+ involved IP3 and a release of Ca2+ from IP3-sensitive stores.

Adrenergic alpha-Agonists↗

PubMed2000

Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney.

BACKGROUND: Reabsorption of albumin from the glomerular filtrate occurs via receptor-mediated endocytosis in the proximal tubule. This process is initiated by binding of albumin in apical clathrin-coated pits, followed by endocytosis and degradation in lysosomes. Although binding sites have been characterized by kinetic studies, the receptors responsible for the binding of albumin have not been fully identified. Two giant glycoproteins, cubilin and megalin, constitute important endocytic receptors localized to the kidney proximal tubule. METHODS: In the present study, we examined the colocalization of cubilin and megalin in the endocytic pathway and the relationship between the uptake of albumin and the expression of cubilin and megalin in opossum kidney (OK) proximal tubule cells by immunocytochemistry and immunoblotting. RESULTS: OK cells expressed both cubilin and megalin. The light microscope labeling patterns for cubilin and megalin were almost identical and were mainly located at the surface area of the cells. Cubilin and megalin were also shown to colocalize on cell surface microvilli, in coated pits, and in endocytic compartments at the electron microscope level. Endocytosed bovine serum albumin (BSA) was identified exclusively in cells expressing megalin and cubilin. Uptake of BSA-FITC was saturable and inhibited by receptor-associated protein (RAP) and by intrinsic factor-vitamin B12 complex (IF-B12) at high concentrations. Significant inhibition was also observed by specific antibodies to cubilin, and megalin and cubilin antisense oligonucleotides likewise significantly reduced albumin uptake. Egg albumin did not affect the uptake of BSA. CONCLUSION: The present observations suggest that the two receptors cubilin and megalin are both involved in the endocytic uptake of albumin in renal proximal tubule cells.

Albumins↗

PubMed2000

Estimation of population parameters and recombination rates from single nucleotide polymorphisms.

Some general likelihood and Bayesian methods for analyzing single nucleotide polymorphisms (SNPs) are presented. First, an efficient method for estimating demographic parameters from SNPs in linkage equilibrium is derived. The method is applied in the estimation of growth rates of a human population based on 37 SNP loci. It is demonstrated how ascertainment biases, due to biased sampling of loci, can be avoided, at least in some cases, by appropriate conditioning when calculating the likelihood function. Second, a Markov chain Monte Carlo (MCMC) method for analyzing linked SNPs is developed. This method can be used for Bayesian and likelihood inference on linked SNPs. The utility of the method is illustrated by estimating recombination rates in a human data set containing 17 SNPs and 60 individuals. Both methods are based on assumptions of low mutation rates.

Genetic Linkage↗

PubMed2000

Codon-substitution models for heterogeneous selection pressure at amino acid sites.

Comparison of relative fixation rates of synonymous (silent) and nonsynonymous (amino acid-altering) mutations provides a means for understanding the mechanisms of molecular sequence evolution. The nonsynonymous/synonymous rate ratio (omega = d(N)d(S)) is an important indicator of selective pressure at the protein level, with omega = 1 meaning neutral mutations, omega < 1 purifying selection, and omega > 1 diversifying positive selection. Amino acid sites in a protein are expected to be under different selective pressures and have different underlying omega ratios. We develop models that account for heterogeneous omega ratios among amino acid sites and apply them to phylogenetic analyses of protein-coding DNA sequences. These models are useful for testing for adaptive molecular evolution and identifying amino acid sites under diversifying selection. Ten data sets of genes from nuclear, mitochondrial, and viral genomes are analyzed to estimate the distributions of omega among sites. In all data sets analyzed, the selective pressure indicated by the omega ratio is found to be highly heterogeneous among sites. Previously unsuspected Darwinian selection is detected in several genes in which the average omega ratio across sites is <1, but in which some sites are clearly under diversifying selection with omega > 1. Genes undergoing positive selection include the beta-globin gene from vertebrates, mitochondrial protein-coding genes from hominoids, the hemagglutinin (HA) gene from human influenza virus A, and HIV-1 env, vif, and pol genes. Tests for the presence of positively selected sites and their subsequent identification appear quite robust to the specific distributional form assumed for omega and can be achieved using any of several models we implement. However, we encountered difficulties in estimating the precise distribution of omega among sites from real data sets.

Amino Acid Substitution↗

PubMed2000

Estimating synonymous and nonsynonymous substitution rates under realistic evolutionary models.

Approximate methods for estimating the numbers of synonymous and nonsynonymous substitutions between two DNA sequences involve three steps: counting of synonymous and nonsynonymous sites in the two sequences, counting of synonymous and nonsynonymous differences between the two sequences, and correcting for multiple substitutions at the same site. We examine complexities involved in those steps and propose a new approximate method that takes into account two major features of DNA sequence evolution: transition/transversion rate bias and base/codon frequency bias. We compare the new method with maximum likelihood, as well as several other approximate methods, by examining infinitely long sequences, performing computer simulations, and analyzing a real data set. The results suggest that when there are transition/transversion rate biases and base/codon frequency biases, previously described approximate methods for estimating the nonsynonymous/synonymous rate ratio may involve serious biases, and the bias can be both positive and negative. The new method is, in general, superior to earlier approximate methods and may be useful for analyzing large data sets, although maximum likelihood appears to always be the method of choice.

Animals↗

PubMed2000

Likelihood analysis of ongoing gene flow and historical association.

We develop a Monte Carlo-based likelihood method for estimating migration rates and population divergence times from data at unlinked loci at which mutation rates are sufficiently low that, in the recent past, the effects of mutation can be ignored. The method is applicable to restriction fragment length polymorphisms (RFLPs) and single nucleotide polymorphisms (SNPs) sampled from a subdivided population. The method produces joint maximum-likelihood estimates of the migration rate and the time of population divergence, both scaled by population size, and provides a framework in which to test either for no ongoing gene flow or for population divergence in the distant past. We show the method performs well and provides reasonably accurate estimates of parameters even when the assumptions under which those estimates are obtained are not completely satisfied. Furthermore, we show that, provided that the number of polymorphic loci is sufficiently large, there is some power to distinguish between ongoing gene flow and historical association as causes of genetic similarity between pairs of populations.

Alleles↗

PubMed1999

Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance.

The C2 domain of cytosolic phospholipase A2 (C2cPLA2) plays an important role in calcium-dependent transfer of the protein from the cytosol to internal cellular membranes as a prelude for arachidonate release from membrane phospholipids. By using a recently developed electron paramagnetic resonance approach together with 13 site-specifically nitroxide spin labeled C2cPLA2s and membrane-permeant and -impermeant spin relaxants, we have determined the orientation of C2cPLA2 with respect to the surface of vesicles of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphomethanol. The structure reveals that the two calcium-binding regions on C2cPLA2 that display hydrophobic residues, CBR1 and CBR3, are partially inserted into the core of the membrane. CBR2 that contains predominantly hydrophilic residues is close to the membrane but not inserted. The long axis of the cylindrical C2cPLA2 molecule is tilted with respect to the bilayer normal, which brings a cluster of basic protein residues close to the phospholipid headgroups. Such an orientation places the two bound calcium ions close to the membrane surface. All together, the results provide structural support for previous proposals that binding of C2cPLA2 to the membrane interface is driven in part by insertion of hydrophobic surface loops into the membrane core. The results are contrasted with previous studies of the interfacial binding of the first C2 domain of synaptotagmin I, which has shorter surface loops that display basic residues for electrostatic interaction with the bilayer surface.

Biological Transport↗

PubMed1999

Megalin antagonizes activation of the parathyroid hormone receptor.

Parathyroid hormone (PTH) is predominantly cleared from the circulation by glomerular filtration and degradation in the renal proximal tubules. Here, we demonstrate that megalin, a multifunctional endocytic receptor in the proximal tubular epithelium, mediates the uptake and degradation of PTH. Megalin was purified from kidney membranes as the major PTH-binding protein and shown in BIAcore analysis to specifically bind full-length PTH and amino-terminal PTH fragments (Kd 0.5 microM). Absence of the receptor in megalin knockout mice resulted in 4-fold increased levels of amino-terminal PTH fragments in the urine. In F9 cells expressing both megalin and the PTH/PTH-related peptide receptor (PTH/PTHrP receptor), uptake and lysosomal degradation of the hormone was mediated through megalin. Blocking megalin-mediated clearance of PTH resulted in 3-fold increased stimulation of the PTH/PTHrP receptor. These data provide evidence that megalin is involved in the renal catabolism of PTH and potentially antagonizes PTH/PTHrP receptor activity in the proximal tubular epithelium.

Animals↗

PubMed1999

Single-locus tests of microsatellite evolution: multi-step mutations and constraints on allele size.

We evaluate some common simulation procedures as well as a recently developed likelihood method used for testing hypotheses regarding microsatellite evolution. Results from simulated data revealed that the tests for the detection of multi-step mutations in general have some power, whereas tests for the presence of constraints on the repeat number have only very limited power. The tests were applied to population data obtained from nine different baleen whale populations. High agreement was found between results obtained using the simulation-based approach and results obtained using a likelihood ratio test. In four of the nine population samples the tests rejected the one-step mutation model. In two instances the significant deviation was due to excess of heterozygosity and in two instances to a reduced level of heterozygosity relative to the expectations under the stepwise mutation model. The former significant deviation was consistent with occasional multi-step mutations, whereas the latter may indicate the presence of constraints on the number of repeats.

Alleles↗

PubMed1999

Variation in the pattern of nucleotide substitution across sites.

A model of nucleotide substitution that allows the transition/transversion rate bias to vary across sites was constructed. We examined the fit of this model using likelihood-ratio tests by analyzing 13 protein coding genes and 1 pseudogene. Likelihood-ratio testing indicated that a model that allows variation in the transition/transversion rate bias across sites provided a significant improvement in fit for most protein coding genes but not for the pseudogene. When the analysis was repeated with parameters estimated separately for first, second, and third codon positions, strong heterogeneity was uncovered for the first and second codon positions; the variation in the transition/transversion rate was generally weaker at the third codon position. The transition rate bias and branch lengths are underestimated when variation in the transition/transversion rate was not accommodated, suggesting that it may be important to accommodate variation in the pattern of nucleotide substitution for accurate estimation of evolutionary parameters.

Animals↗

PubMed1999

Evidence for a Na+/Ca2+ exchange mechanism in frog skin epithelium.

In the present study we investigated the possible existence of a Na+/Ca2+ exchange mechanism in the basolateral membrane of the frog skin epithelium and whether such a mechanism plays a role in the regulation of transepithelial Na+ transport. Cytosolic calcium ([Ca2+]i) was measured with the probe fura-2 in a set-up in which pieces of tissue were mounted on the stage of an epifluorescence microscope. Na+ transport was measured as the amiloride-sensitive short-circuit current (Isc) using a conventional voltage clamp. Basal [Ca2+]i was 65+/-6 nM (n=15). Removal of Na+ from the mucosal solution had no effect on [Ca2+]i. When Na+ was removed from the serosal solution, [Ca2+]i increased biphasically to a peak of 220+/-38 nM (n=8, P=0.006). Readdition of Na+ to the serosal solution returned [Ca2+]i to control level. The serosal Na+ gradient and changes in [Ca2+]i were closely correlated; stepwise changes in serosal Na+ were followed by stepwise changes in [Ca2+]i. These observations indicate the existence of a Na+/Ca2+ exchange mechanism in the basolateral membrane of the frog skin epithelium. The transepithelial Na+ transport decreased from 13.2+/-1.8 to 9.2+/-1.5 microA cm-2 (n=8, P=0.049) when Na+ was omitted from the serosal solution. When this protocol was repeated in the absence of serosal Ca2+, Na+ transport decreased similarly from 16.7+/-1.7 to 11.6 +/-1. 8 microA cm-2 (n=6, P=0.004). We conclude that it is unlikely that the observed decrease in Isc after removal of serosal Na+ is due to an increase in [Ca2+]i per se.

Algorithms↗

PubMed1999

EP3 receptors inhibit antidiuretic-hormone-dependent sodium transport across frog skin epithelium.

We examined the effect of prostaglandin E2 (PGE2) on antidiuretic hormone (ADH)-dependent Na+ transport and cAMP production in isolated frog skin epithelium. ADH caused an increase in transepithelial Na+ transport and a decrease in cellular potential, indicating an increase in apical Na+ permeability. Subsequent addition of PGE2 decreased Na+ transport and repolarised the cells. The PGE2 receptor EP1/3-selective analogue sulprostone and the PGE2 receptor EP2/3-selective analogue misoprostol were able to mimic the effect of PGE2. ADH increased cellular cAMP levels, whereas PGE2, sulprostone and misoprostol were able to reduce the ADH-dependent cAMP production. Measurements of intracellular Ca2+ concentration ([Ca2+]i) revealed that it was unaffected by both PGE2 and sulprostone. The inhibitory effect of PGE2 on ADH-dependent Na+ transport was also observed in Ca2+-depleted epithelia. We conclude that ADH stimulates transepithelial Na+ transport by increasing cellular cAMP levels, whereas PGE2 inhibits ADH-dependent Na+ transport by activating EP3-type receptors, which decrease cellular cAMP levels. We have found no evidence that [Ca2+]i is involved in the regulation of ADH-dependent Na+ transport by PGE2.

Algorithms↗

PubMed1999

Effect of carbachol and prostaglandin E2 on chloride secretion and signal transduction in the exocrine glands of frog skin (Rana esculenta).

Carbachol (CCH) increased the short-circuit current across frog skin glands in a biphasic manner, which coincided with an increase in the transepithelial Cl- net flux. CCH also induced a biphasic increase in [Ca2+]i. Both these responses were mediated via muscarinic receptors. The plateau phase of the CCH-induced Cl- secretion was modestly inhibited by indomethacin and unaffected by tetrodotoxin or tetrodotoxin plus indomethacin, indicating that CCH can increase Cl- secretion directly via receptors on the secretory cells. Prostaglandin E2 (PGE2) increased secretion and cAMP production, indicating expression of EP2 and/or EP4 receptors. PGE2 failed to increase [Ca2+]i ruling out involvement of EP1 receptors. The secretory response to CCH was potentiated by prestimulation with PGE2, and it was investigated whether this potentiation is caused by interaction at the level of the messengers involved. Stimulation by CCH plus PGE2 failed to stimulate cAMP production further than PGE2 alone. Addition of PGE2 during the CCH-elevated [Ca2+]i plateau phase in most cases reduced the level of [Ca2+]i. These data show that the synergy between CCH and PGE2 is not based on interactions at the intracellular messenger level.

Animals↗

PubMed1999

Pain after primary inguinal herniorrhaphy: influence of surgical technique.

BACKGROUND: Pain is an important problem after ambulatory hernia repair. To assess the influence of the surgical technique on postoperative pain, two separate randomized, patient-blinded, controlled trials were performed in men with an indirect inguinal hernia. STUDY DESIGN: In study A, 48 patients with an internal inguinal ring smaller than 1.5 cm were randomly allocated to either simple extirpation of the hernial sac or extirpation plus annulorrhaphy. In study B, 84 patients with an internal inguinal ring wider than 1.5 cm were randomly allocated to extirpation plus annulorrhaphy or extirpation plus Lichtenstein mesh repair (modified). All operations were performed under unmonitored local anesthesia with standardized perioperative analgesia using methadone and tenoxicam. Pain was scored daily for the first postoperative week and after 4 weeks on a four-point verbal-rank scale (no, light, moderate, or severe pain) during rest, while coughing, and during mobilization (rising to the sitting position). Use of supplementary analgesics (paracetamol) was recorded. Cumulative daily pain scores for the first postoperative week and the number of patients who used supplementary analgesics were the main outcome measures. RESULTS: There were no significant differences in cumulative pain scores or use of supplementary analgesics between the treatment groups in either study. Cumulative pain scores were significantly higher during coughing and mobilization than during rest in both studies. CONCLUSIONS: Choice of surgical technique for open repair of a primary indirect inguinal hernia has no influence on postoperative pain.

Adolescent↗

PubMed1999

Effect of nonindependent substitution on phylogenetic accuracy.

All current phylogenetic methods assume that DNA substitutions are independent among sites. However, ample empirical evidence suggests that the process of substitution is not independent but is, in fact, temporally and spatially correlated. The robustness of several commonly used phylogenetic methods to the assumption of independent substitution is examined. A compound Poisson process is used to model DNA substitution. This model assumes that substitution events are Poisson-distributed in time and that the number of substitutions associated with each event is geometrically distributed. The asymptotic properties of phylogenetic methods do not appear to change under a compound Poisson process of DNA substitution. Moreover, the rank order of the performance of different methods does not change. However, all phylogenetic methods become less efficient when substitution follows a compound Poisson process.

Base Composition↗

PubMed1999

The age of nonsynonymous and synonymous mutations in animal mtDNA and implications for the mildly deleterious theory.

McDonald/Kreitman tests performed on animal mtDNA consistently reveal significant deviations from strict neutrality in the direction of an excess number of polymorphic nonsynonymous sites, which is consistent with purifying selection acting on nonsynonymous sites. We show that under models of recurrent neutral and deleterious mutations, the mean age of segregating neutral mutations is greater than the mean age of segregating selected mutations, even in the absence of recombination. We develop a test of the hypothesis that the mean age of segregating synonymous mutations equals the mean age of segregating nonsynonymous mutations in a sample of DNA sequences. The power of this age-of-mutation test and the power of the McDonald/Kreitman test are explored by computer simulations. We apply the new test to 25 previously published mitochondrial data sets and find weak evidence for selection against nonsynonymous mutations.

Alleles↗