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Biomedical subjects

P Bellemann

Publications and source records attributed to P Bellemann.

16 recordsLinked to original sources
PubMed1992

Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells.

The suicide plasmid pfdA31-Tn5 was constructed to mutagenize Erwinia amylovora and Escherichia coli strains by electorporation. This vector carries the bacteriophage fd replication origin, a beta-lactamase gene and the transposon Tn5. For propagation the plasmid depends on host cells producing fd gene-2 protein. Electroporation of E.amylovora or E.coli cells with plasmid pfdA31-Tn5 yielded more than 10(4) transposition events per micrograms DNA. We have produced and characterized transposon mutants of E.amylovora affecting either galactose metabolism or the synthesis of the phytotoxin (L)-2,5-dihydrophenylalanine. A Tn5-insertion in a gene, involved in exopolysaccharide synthesis of E.amylovora strain Ea7/74, was subcloned into vector pfdA31 and used to mutagenize E.amylovora strain Ea1/79 by site-directed recombination.

Blotting, Southern↗
PubMed1992

Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization.

Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.

Chromosome Mapping↗
PubMed1992

Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis.

Detection and identification of the fire blight pathogen, Erwinia amylovora, can be accurately done by polymerase chain reaction (PCR) analysis in less than 6 h. Two oligomers derived from a 29-kb plasmid which is common to all strains of E. amylovora were used to amplify a 0.9-kb fragment of the plasmid. By separation of the PCR products on agarose gel, this fragment wa specifically detected when E. amylovora DNA was present in the amplification assay. It was not found when DNA from other plant-pathogenic bacteria was used for the assay. A visible band specific to the 0.9-kb fragment was produced with DNA from fewer than 100 E. amylovora cells. A signal of similar strength was also obtained from E. amylovora cell lysates in the presence of the mild detergent Tween 20. Signals were weaker when bacteria were added to the PCR mixture without the detergent. As with results obtained from hybridization experiments using pEA29 DNA< the PCR signal was obtained with E. amylovora isolates from various geographic regions. This technique could also be used for detection of the fire blight pathogen in extracts of tissue obtained from infected plant material.

Base Sequence↗
PubMed1988

Modulation of ANP receptor-mediated cGMP accumulation by atrial natriuretic peptides and vasopressin in A10 vascular smooth muscle cells.

ANP receptor binding and desensitization were demonstrated in the A10 vascular smooth muscle cell (VSMC) line. Concomitantly, the ANP receptor coupled guanylate cyclase activity was reduced by the receptor down-regulation with ANP. The ANP stimulated cGMP accumulation is modulated by arginine-vasopressin, while the arginine-vasopressin mediated cAMP system remained unaffected by ANP. Results suggest negative coupling of arginine-vasopressin receptors to the guanylate cyclase activity, and indicate that the vasorelaxant activity of ANP might be regulated in part by arginine-vasopressin via specific receptor sites.

Animals↗
PubMed1987

Effects of salt loading and nitrendipine on dihydropyridine receptors in hypertensive rats.

It has already been emphasized that salt-dependent hypertension might be especially sensitive to calcium antagonists, and it has recently been shown that binding at different receptor types might be altered by dietary sodium chloride. Therefore, it was of interest to find the extent to which the specific membrane-associated dihydropyridine (DHP) receptors are influenced by salt loading and/or nitrendipine treatment. Groups of 10-12 salt-sensitive (S/JR) or salt-resistant (R/JR) Dahl rats and spontaneously hypertensive rats (SHRSP) or normotensive Wistar-Kyoto rats (WKY) received diets containing low (0.4%) or high (8%) NaCl for 21 days; treated animals received 300 ppm nitrendipine additionally with the diet. Blood pressure of R/JR and WKY did not differ markedly on the different diets, whereas in hypertensive rat strains, salt loading increased blood pressure after 3 weeks. High salt resulted in an increased number of cardiac DHP receptors in SHRSP; the addition of nitrendipine enhanced DHP receptor capacity in the heart and brain membranes of both strains (SHRSP and S/JR). No major changes in affinity were observed. Augmentation in the number of DHP receptors as analyzed in a follow-up interim observation study on SHR occurred more rapidly in heart than in brain tissue. It can be concluded that an altered number of the DHP receptor sites, and not changes in affinity, might be essential during treatment with calcium antagonists. The DHP receptor sites, and not changes in affinity, might be essential during treatment with calcium antagonists. The DHP receptor density can be modified by high salt intake, as shown in salt-loaded SHRSP.

Animals↗
PubMed1986

Receptor binding, cGMP stimulation and receptor desensitization by atrial natriuretic peptides in cultured A10 vascular smooth muscle cells.

Receptor characteristics for atrial natriuretic peptides (ANP) were demonstrated in the permanent tissue culture system of vascular smooth muscle cell (VSMC) line. 125I-ANP exhibited reversible and saturable binding to A10 rat VSMC, and the equilibrium dissociation constant, Kd was 157 pM and maximal binding capacity, Bmax amounted to 115 fmol/mg of protein. Binding of the 125I-ligand was highly specific for certain potently displacing ANP analogues with inhibition constants (Ki values) in the nano- or even subnanomolar concentration range. Pretreatment of VSMC with ANP yielded receptor desensitization to one half of the ANP receptor density, and supports the conclusion of receptor autoregulation by ANP in A10 VSMC. The receptor coupled intracellular cGMP system was stimulated, and thus demonstrates the application of A10 cells as a model for the study of ANP receptor interaction involved in vascular smooth muscle relaxation.

Animals↗
PubMed1977

Effect of insulin on glycogen and protein synthesis in monolayer cultures of hepatocytes from normal and alloxan diabetic rats.

The effects of insulin on net glycogen synthesis and amino acid incorporation into protein were studied in cultured hepatocytes from adult normal and alloxan diabetic rats. Insulin stimulated glycogen synthesis in monolayer cells throughout a four day culture period and enhanced leucine incorporation into protein more effectively in normal cells with high glycogen levels than in cultured diabetic cells. These differences correlate well with the observed cellular ultrastructures which were maintained much better in the presence of insulin. Restoration of the morphological changes of alloxan diabetic hepatocytes to normal liver cell structures can be observed at any time during the culture period by giving insulin continuously.

Animals↗
PubMed1976

Effect of theophylline on calcium exchangeability in ventricular myocardium.

The effects of theophylline on contractile force and myocardial calcium exchangeability were studied in isolated, electrically driven Langendorff perfused guinea-pig hearts. Following a 30-min exposure to 45Ca, total cellular calcium and 45Ca activity were measured in right ventricular samples. "Nontoxic" theophylline concentrations (5 x 10(-5) -10(-3) g/ml) which augmented contractile force without producing arrhythmias or contractures had no effect on total tissue calcium and did not alter the size of the fraction of cellular calcium exchangeable under steady-state conditions. A "toxic" concentration of theophylline (2 x 10(-3) g/ml) induced contractures and increased the amount of exchangeable cellular calcium. The latter effect was due to an increase in total calcium; the unlabelled cellular calcium fraction remained unchanged under the influence of all theophylline concentrations studied. The results suggest that theophylline increases the steady-state calcium exchangeability in ventricular myocardium only when the total calcium concentration is also increased.

Animals↗
PubMed1975

Dissociation of theophylline uptake and inotropic effect in myocardial tissue: influence of temperature, pH and calcium.

1. The myocardial uptake and the positive inotropic effect of theophylline (100 mug/ml; 0.56 mM) were studied in isolated electrically driven guinea-pig hearts perfused by the Langendorff technique under various extracellular conditions. [3H]-theophylline was used. 2. Variations in temperature, hydrogen ion and calcium ion concentrations of the perfusion media changed the time course and magnitude of the effect of theophylline on myocardial twitch tension but did not affect the time course and amount of theophylline uptake. 3. Under all conditions, the build-up of the positive inotropic effect of theophylline was about three times faster than the uptake of the drug into the heart. 4. Since no relationship could be found between theophylline uptake and inotropic effect, the cardiac positive inotropic response to theophylline is thought unlikely to be due mainly to an interaction of the drug with intracellular receptor sites but is assumed to occur via an effect of the drug on the sarcolemma, that is at a site which the drug reaches before it enters the intracellular space.

Animals↗
PubMed1974

Relationship between theophylline uptake and inotropic effect in the guinea-pig heart.

1 The time course of the positive inotropic effect of theophylline was compared with the time course of the uptake and release of [(3)H]-theophylline in guinea-pig isolated, electrically driven hearts perfused by the Langendorff method.2 Formation of theophylline metabolites could not be detected under the experimental conditions used.3 Theophylline entered myocardial tissue very rapidly in two different phases. The first process (half-time 21 s) amounted to 93% and the second (half-time 5 min 50 s) to 7% of the total uptake. The development of the positive inotropic effect of theophylline was about four times faster than even the rapid component of the uptake of the drug into the myocardium.4 The amount of theophylline accumulated in myocardial tissue (after 10 min perfusion) incerased proportionally with theophylline concentrations in the perfusion media and no signs of saturation were detected. The tissue-medium ratio did not exceed 1. The water content of the myocardial tissue amounted to about 80% at all theophylline concentrations examined.5 The uptake of theophylline (3 mg/ml) was diminished by 9.2% after pretreatment of the hearts with caffeine (1 mg/ml). Theophylline uptake was also decreased by 13.5% when caffeine-pretreated hearts were perfused with a solution containing theophylline (300 mug/ml) plus caffeine (1 mg/ml).6 Theophylline release from the hearts was also very rapid. The efflux curve was composed of three components (half-times: 24 s; 1 min 24 s; 6 min 18 seconds). The intercepts of the linear portions of the efflux curve occurred at 61%, 38% and 1%, respectively. Contractile force and theophylline content in myocardial tissue declined in a similar manner.7 It is concluded that theophylline enters myocardial tissue very rapidly by passive diffusion. Theophylline distributes itself in the heart as freely as in the perfusion medium. A very small amount may be bound within the cell in a relatively specific way.8 It seems possible that the positive inotropic effect of theophylline is partly due to an action of the drug on intracellular calcium binding or storage sites. However, the principal action of theophylline is assumed to be on the sarcolemma where it increases calcium influx from the extracellular space. This conclusion is based on the fact that the time courses of the increase in contractile force and of theophylline uptake into the cell were dissimilar.

Animals↗