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.     

Toxicokinetics of soman: species variation and stereospecificity in elimination pathways

Blood levels of the four stereoisomers of the nerve agent C(+/-)P(+/-)-soman were analyzed gas chromatographically in anaesthetized, artificially respirated and atropinized rats, guinea pigs and marmosets at intravenous doses of C(+/-)P(+/-)-soman corresponding with 1-6 LD50. The relatively nontoxic C(+/-)P(+/-)-isomers disappear within a few minutes from the blood stream of all three species, whereas the levels of the highly toxic C(+/-)P(-)-isomers remain toxicologically relevant for periods of 50-100 min in three species of doses of 2-3 LD50. Elimination pathways were quantified using 14C-labeled soman stereoisomers. Whereas the C(+/-)P(+)-isomers are largely eliminated by way of enzymatic hydrolysis, the major elimination pathway for the C(+/-)P(-)-isomers is binding to various proteins, in competition with binding to target acetylcholinesterase. Intraspecies nonlinearity with dose in the toxicokinetics of the C(+/-)P(-)-isomers is related to heterogeneous reactivity of the binding sites. Interspecies nonlinearity is probably due to decreasing amounts of binding sites in the order rats greater than guinea pigs greater than primates, leading to increasing "toxico-availability" in the reversed order.     

Reactivation of acetylcholinesterase inhibited by methamidophos and analogous (di)methylphosphoramidates

Reactivation of electric eel acetylcholinesterase (AChE) inhibited by MeO(NH₂)P(O)SMe (methamidophos) and by MeS(NH₂)P(O)SMe was studied at pH 7.5 and 25° C. The former inhibited enzyme shows a rather rapid spontaneous reactivation (t_1/2=3.7 h); this reactivation is accelerated by 1 M of the bispyridinium oximes TMB4 and obidoxime, and, to a lesser extent, by the monopyridinium oximes P2S and its 1-benzyl analogue (benzyl-P2A). The latter inhibited enzyme shows rapid aging (t_1/2=0.6 h). Reactivation with 1 mM of the bispyridinium oximes is incomplete and reactivation with 1 mM of the monopyridinium oximes proceeds very slowly. These large differences between the properties of the two inhibited enzymes indicate that the methylthio group is the leaving group during inhibition of AChE by methamidophos. Additional support is afforded by the observation of induced aging of the former inhibited enzyme by thiourea.Upon comparison of the reactivation of AChE inhibited by methamidophos with that of AChE inhibited by an N-methyl analogue, cruf ornate, and an N,N-dimethyl analogue, tabun, it appears that the rate of spontaneous reactivation decreases with increasing alkylation of the P-NH₂ group. Whereas benzyl-P2A is somewhat less active than P2S for reactivation of AChE inhibited by methamidophos, it is superior to P2S for reactivation of AChE inhibited by crufomate and also superior to P2S and to the bispyridinium oximes for AChE inhibited by tabun.