Gene conversion is a likely cause of mutation in PKD1

Approximately 70% of the gene responsible for the most common form of autosomal dominant polycystic kidney disease ( PKD1 ) is replicated in several highly homologous copies located more proximally on chromosome 16. We recently have described a novel technique for mutation detection in the duplicated region of PKD1 that circumvents the difficulties posed by these homologs. We have used this method to identify two patients with a nearly identical cluster of base pair substitutions in exon 23. Since pseudogenes are known to be reservoirs for mutation via gene conversion events for a number of other diseases, we decided to test whether these sequence differences in PKD1 could have arisen as a result of this mechanism. Using changes in restriction digest patterns, we were able to show that these sequence substitutions are also present in N23HA, a rodent-human somatic cell hybrid that contains only the PKD1 homologs. Moreover, these changes were also detected in total DNA from several affected and unaffected individuals that did not harbor this mutation in their PKD1 gene copy. This is the first example of gene conversion in PKD1 , and our findings highlight the importance of using gene-specific reagents in defining PKD1 mutations.      

Dynamic and static phantoms for evaluation of digital subtraction angiographic systems

Two types of phantoms were developed with which to evaluate the overall performance of digital subtraction angiography (DSA) systems. A dynamic phantom, called a "fish bone" phantom, consists of polyethylene tubes that simulate blood vessels with various lesions, such as stenoses, ulcers, and aneurysms. With this phantom, washout curves were obtained representing the relationship between iodine content and time. It will be useful for qualitative assessment of DSA images, evaluation of different image-processing schemes, and studies of blood flow analysis. A static phantom, called a "C-D" phantom, can be used for measurement of quantitative contrast-detail (C-D) diagrams and for daily monitoring of DSA systems. This was constructed of tubes of seven different diameters (2.15-0.28 mm) and 14 different concentrations of contrast medium (100%-1.1% Renografin-76 [meglumine and sodium diatrizoate]). The C-D diagrams were determined from an observer performance study using C-D phantom images obtained at four different DSA settings.     

Polymyxin B,a selective inhibitor of protein kinase C,diminishes the release of noradrenaline and the enhancement of release caused by phorbol 12,13-dibutyrate

Summary Slices of the rabbit hippocampus were labelled with ³H-noradrenaline, superfused continuously with a modified Krebs-Henseleit medium containing the uptake inhibitor cocaine and stimulated electrically (2 ms, 3 Hz, 24 mA, 5 V/cm). Phorbol 12,13-dibutyrate (PDB), a potent activator of protein kinase C (PKC), strongly enhanced the electrically-evoked overflow of tritium. In contrast, polymyxin B, a relatively selective inhibitor of PKC, diminished the evoked tritium overflow in a time-and concentration-dependent manner. The enhancement of the evoked overflow of tritium caused by PDB was strongly reduced in the presence of polymyxin B (100 mol/l). These results suggest 1. that PKC may be involved in the physiological mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals and 2. that the PDB-induced enhancement of noradrenaline release may be due to a direct activation of PKC.Abbreviations PKC protein kinase C - PDB phorbol 12,13-dibutyrate - TPA 12-O-tetradecanoyl 13-acetate     

Modulation of electrically evoked [3H]-noradrenaline release from cultured chick sympathetic neurons

In the present study we attempted a comprehensive characterization of modulation of noradrenaline release from chick sympathetic neurons. To this purpose sympathetic neurons derived from chick lumbosacral paravertebral ganglia and kept in culture for 7 days were loaded with 0.05 mol/l [³H]-noradrenaline and subjected to electrical field stimulation (36 pulses/3 Hz). Since the released transmitter was partially recaptured, superfusion was usually performed in the presence of (+)-oxaprotiline, an inhibitor of noradrenaline re-uptake. [³H]-Noradrenaline was released in a manner which was dependent on extracellular Ca²⁺ and sensitive to tetrodotoxin (TTX). -Conotoxin (-CTX; 100 nmol/l) abolished [³H]-noradrenaline release indicating that influx through -CTX-sensitive Ca²⁺-channels was essential for transmitter release. 1,4-dihydro-2,6-dimethyl-5-nitro4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester ((±)Bay K 8644) and 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3-nitro-5-pyridinecarboxylic acid isopropyl ester ((±)-202-791), agonists at L-type voltage sensitive Ca²⁺-channels (VSCCs), increased noradrenaline release and induced, in addition, an overflow of tritium which was Ca²⁺-dependent and prevented by the presence of TTX. The L-type VSCC antagonists (–)-202-791 and (+)-4-(4-benzofurazanyl)-1,4-dihydro2,6-dimethyl-3,5-pyridinedicar boxylic acid methyl, isopropyl ester ((+)-PN 200–110) diminished [³H]-noradrenaline release. These data suggest that L-type VSCCs, probably located on the cell body of the neuron, play an additional role in modulation of release. The full ₂-adrenoceptor agonists 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline ( UK-14,304) and noradrenaline significantly inhibited noradrenaline release, whereas clonidine, a partial a2-agonist, produced only a slight inhibition even at 10 mol/l. The facilitation of noradrenaline release observed in the presence of the ₂-adrenoceptor antagonist rauwolscine was very low in comparison to that obtained with brain slices and isolated smooth muscle tissues. These results corroborate the observation that noradrenaline release from chick sympathetic neurons is regulated by an ₂-adrenoceptor which needs further subtype characterization. The experiments were mostly performed at 25°C, since a rise in temperature to 37°C increased the resting outflow, but not the evoked overflow of tritium, approximately 4-fold. In the presence of pargyline to block monoamine oxidase, however, the temperature-dependent enhancement was diminshed and the release showed properties comparable to those observed at 25°C (with respect to TTX-sensitivity, Ca²⁺ dependence and modulation via ₂-adrenoceptors). In addition to the ₂-adrenoceptors, we detected inhibitory -adrenoceptors, opioid and receptors, and P₂ purinoceptors as well as facilitatory prostaglandin (PG) E receptors. No indication was found for a functional relevance of 5-hydroxytryptamine (5-HT), opioid , PGD, adenosine A₁ or glutamate receptors. In conclusion, electrically evoked noradrenaline release from cultured chick sympathetic neurons shows the properties of action-potential-induced transmitter release and is bidirectionally regulated by various substances. Therefore, sympathetic neurons in culture offer the possibility to investigate directly the mechanisms bringing about receptor-coupled modulation of transmitter release.Abbreviations ATP adenosine 5-triphosphate - Bay K 8644 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester - DAGO (d-Ala²,N-methyl-Phe⁴,Gly-ol⁵)-enkephalin - DPDPE (d-Pen ^2,5)-enkephalin - 5-HT 5-hydroxytryptamine - -CTX -conotoxin - KRBB modified Krebs-Ringer bicarbonate buffer - NMDA N-methyl-d-aspartic acid - PG prostaglandin - PN 200-110 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxy lic acid methyl, isopropyl ester - R-PIA R(–)-N⁶-(2-phenyl-isopropyl)-adenosine - TTX tetrodotoxin - U-50,488H trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzene acetamide - UK-14,304 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline - VSCC voltage sensitive Ca²⁺-channel - 202-791 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3-nitro-5-pyridinecarboxylic acid isopropyl ester Correspondence to: C. Allgaier at the above address