Use of degenerate oligonucleotide primed PCR (DOP-PCR) for the genotyping of low-concentration DNA samples

Degenerate oligonucleotide primed amplification (DOP-PCR) is an efficient method for performing whole genome amplification. We analysed the yield of DNA using this technique starting with various quantities of material. We used DOP-PCR products to genotype single nucleotide polymorphisms and insertion/deletion polymorphisms. DOP-PCR also proved usable for SSCP analysis.     

The role of size, sequence and haplotype in the stability of FRAXA and FRAXE alleles during transmission

Factors involved in the stability of trinucleotide repeats during transmission were studied in 139 families in which a full mutation, premutation or intermediate allele at either FRAXA or FRAXE was segregating. The transmission of alleles at FRAXA, FRAXE and four microsatellite loci were recorded for all individuals. Instability within the minimal and common ranges (0-40 repeats for FRAXA, 0-30 repeats for FRAXE) was extremely rare; only one example was observed, an increased in size at FRAXA from 29 to 39 repeats. Four FRAXA and three FRAXE alleles in the intermediate range (41-60) repeats for FRAXA, 31-60 for FRAXE) were unstably transmitted. Instability was more frequent for FRAXA intermediate alleles that had a tract of pure CGG greater than 37 although instability only occurred in two of 13 such transmissions: the changes observed were limited to only one or two repeats. Premutation FRAXA alleles over 100 repeats expanded to a full mutation during female transmission in 100% of cases, in agreement with other published series. There was no clear correlation between haplotype and probability of expansion of FRAXA premutations. Instability at FRAXA or FRAXE was more often observed in conjunction with a second instability at an independent locus suggesting genomic instability as a possible mechanism by which at least some FRAXA and FRAXE mutations arise.      

Quantitation by electron microscopy of the binding of highly specific antibodies to benzo[a]pyrene-DNA adducts

Highly specific antibodies bound to carcinogen adducts in DNAmodified with (+ / - )7ß, 8-dihydroxy-9, 10-epoxy-7,8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE I) were quantitatedby electron microscopy (EM) visualization and these observationswere compared with quantitation of adducts by enzyme-linkedimmunosorbent assay (ELISA). The antiserum, elicited in rabbitsfollowing inoculation with BPDE I-modified DNA, has been foundto be highly specific in its recognition of BPDE I-deoxyguanosinemoieties. Parallel DNA samples prepared for analysis by ELISAand EM quantitation were randomized, encoded, and analyzed todetermine extents of carcinogen modification in double-blindstudies. After levels of modification were determined by immunoassays,DNA samples were prepared for EM analysis by incubation withamounts of anti-BPdG-DNA serum in excess of that necessary forcomplete binding of antibody to antigenic sites. At equilibrium,samples were enzymatically digested with papain in order tocleave anti-BPdG-DNA IgG molecules into Fab fragments in situ.Following column exclusion chromatography, BPdG-DNA-Fab complexeswere incubated with ferritin-labeled Fab‘ fragments ofgoat [anti-rabbit F(ab’)2] IgG in amounts in excess ofthose necessary for complete binding. When DNA samples weremodified to between 0 and 40 fmol adduct/ µg DNA, excellentagreement was obtained between ELISA quantitation and visualizationby EM of antibodies bound to adducts.     

The concept of cerebral lacunae from 1838 to the present

The term lacunae was first used by Dechambre (1838) referring to small cavities developed during the process of resorption within cerebral softenings. Some years later, lacunae was applied by Durand-Fardel (1843) to small cavities located in the basal ganglia and hypothetically attributed to old, healed cerebral softenings. Durand-Fardel (1842, 1854) described "l'état criblé" as many round small holes ("criblures") always containing a patient blood vessel and located in the hemispheric white matter. He believed that "état criblé" was caused by mechanical compression of cerebral tissue related to the dilatation of the blood cerebral vessels during repetitive cerebral congestion. Chronic dementia or delirium were considered as the clinical counterpart of "état criblé". In the second half of the XIXth century, the few reports about lacunae were confusing due to the imprecise use of the terms "lacunae" and "état criblé". Furthermore, some authors described lacunae as sequelae of haemorrhage, others as old softenings or both. In the beginning of the XXth century, the masterly work of P. Marie (1901) established a clear distinction between the "foyers lacunaires de désintégration" and "état criblé" de Durand-Fardel or single perivascular dilatation of one of the lenticulo-striate arteries at its entrance into the lenticular nucleus or post-mortem charges (cerebral porosity, "état de fromage de gruyère"). He described lacunae as small cerebral softenings caused by occlusion of the blood vessels by a "local arteriosclerotic process". However, he also stated that some lacunae containing a patent blood vessel were due to a perivascular space dilatation destroying the adjacent brain parenchyma by a process of "destructive vaginalitis". Marie observed that lacunae were frequently clinically asymptomatic, but "that the hemiplegia of the old people was more often due to cerebral lacunae than to cerebral haemorrhage or softening". During the first half of the XXth century, all the papers devoted to cerebral lacunae were in accordance with the work of P. Marie, developing his own's contradictions. Many authors emphasized the "destructive vaginalitis way" and claimed that lacunae were dilatations of the perivascular spaces. Many other authors developed the "softening way" masterfully illustrated by Fisher who considered lacunae as small deep infarcts caused by a specific pathological process of lipohyalinosis due to arteriolar wall modification by hypertensive disease.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expanding cerebellar lacunes due to dilatation of the perivascular space associated with Binswanger's subcortical arteriosclerotic encephalopathy

An 80-year-old hypertensive woman developed right hemiplegia and died 24 hours after admission. Neuropathologic examination revealed multiple cerebral infarcts of various ages and diffuse subcortical arteriosclerotic encephalopathy. Clusters of asymptomatic "expanding" lacunes, due to dilatation of the perivascular spaces, were found in both dentate nuclei. These cavities, which presented as space-occupying lesions, were surrounded by a single layer of flattened cells and contained 1 or more sections of normal-looking arterioles. Such a topographic grouping of lacunes in the dentate nucleus has not been described previously. The mechanism of widening of the perivascular compartment remains unclear; its occurrence in a hypertensive patient and its association with typical Binswanger's subcortical arteriosclerotic encephalopathy and severe atherosclerosis with multiple infarcts suggest a common pathophysiologic mechanism possibly including an alteration of the blood-brain barrier.     

Immunohistochemical localization of DNA adducts in rat liver tissue and phenotypically altered foci during oral administration of 2-acetylaminofluorene

Histological studies using paired immunofluorescence stainingand peroxidase-anti-peroxidase staining were performed on sectionsof rat livers with an antiserum specific for the 2-acetylaminofluorene(AAF)-DNA adduct N-deoxyguanosin-(8-yl)-aminofluorene (dG-8-AF).This is the predominant adduct in rat liver DNA at 5 (80%) and28 (100%) days of AAF feeding. Nuclear staining was observedin livers of male Fischer rats fed 0.02% AAF for these timeperiods, and was not present in livers of animals fed controldiet or detected when specific antiserum, first absorbed withthe immunogen adduct, was utilized. In addition, nuclear stainingwas unchanged after incubation with RNase and abolished afterincubation with DNase. Adducts were not readily detectable whenwhole-liver adduct concentrations were less than an averageof 10⁵ adducts per cell (30–50 fmol/µg DNA). Theoverall pattern of adduct distribution in livers of AAF-fedanimals was distinctly non-uniform. A predominance of nuclearstaining was found in the periportal areas by both immunofluorescenceand immunoperoxidase procedures. In contrast, staining was veryweak in the centrilobular areas. When animals were fed AAF for28 days and control diet subsequently for 7, 14, 21 or 28 days,the overall intensity of the immunohistochemical staining decreasedwith time on control diet. However, the pattern of localizationremained the same as in livers of rats fed AAF for 28 days,with the predominance of adducts being in the periportal areas.In male rats fed 0.02% AAF for 8 weeks, foci positive for -glutamyltranspeptidase(GGT) became apparent, and the nuclei in these areas showedno immunofluorescence, indicating the absence of detectablelevels of the dG-8-AF adduct. Twenty adduct-negative areas inthe median lobes of three rat livers were positive for GGT,which suggests that loss of ability to form adducts in theseregions occurs concomitantly with early phenotypic changes.     

Expression of protective and cardiac tissue cross-reactive epitopes of type 5 streptococcal M protein in Escherichia coli.

The immunochemical properties of type 5 M protein antigens that were expressed in Escherichia coli K-12 by recombinant lambda bacteriophages isolated from a gene bank of serotype 5 Streptococcus pyogenes have been analyzed in detail. M proteins from partially purified bacteriophage lysates displayed precipitin lines of identity with a purified peptic extract of type 5 M protein (pep M5) in immunodiffusion assays. Immunoblot analyses of the M protein-positive lysates demonstrated that the cloned M protein component resided in five polypeptides with relative molecular weights of 57,900 (57.9K), 55.4K, 52.9K, 40.0K, and 32.6K. The hybrid lambda phage (lambda M5)-produced M protein contained immunoprotective epitopes; lambda M5 protein inhibited opsonization of type 5 streptococci by pep M5 antibodies, and antiserum raised against lambda M5 lysates opsonized type 5 streptococci. Each of the five antigenic polypeptides of the recombinant phage M protein also shared epitopes with human heart tissue, as demonstrated by the reactivity of immunoblots of lambda M5 antigens separated on sodium dodecyl sulfate gels with anti-pep M5 antibodies absorbed to and eluted from human heart sarcolemmal membranes. Moreover, antiserum raised against the lambda M5 lysates reacted with sarcolemmal membrane proteins with relative molecular weights of 200K, 59K, 55K, 53K, and 27K as determined by immunoblot analyses. These results demonstrate that the structural gene coding for type 5 streptococcal M protein which was inserted into lambda DNA expresses immunoprotective epitopes, some of which are shared with human heart tissue.     

The normal vascularization of the intradural filum terminale in man

Summary The arterial and venous blood-supply of the intradural filum terminale was studied microscopically in 18 fresh cadavers after removing the dorsolumbar spinal cord in one piece, with the roots and the filum in their dural sheath. The arteries were examined after manual injection of the artery of the lumbar enlargement, while study of the veins was made without injection since their bluish-black color made them easily identifiable. After gross examination, each specimen was fixed and then sectioned at 12 different levels from the medullary conus to the bottom of the dural sac for histologic study. The distribution of the vascularization the filum terminale appeared constant. A single artery, the artery of the filum, arises from the termination of the anterior spinal axis, either by trifurcation or from the proximal part of one of the 2 branches of the anastomotic ansa of the conus. The artery travels in front of the filum, with rapidly diminishing caliber; rarely, it can be followed into the sacral canal. The vein of the filum travels in front of that structure but behind the artery, as in the cord; its caliber is uniform but varies from subject to subject. It traverses the dura below and continuous with the anterior spinal vein above. No vessels were found on the dorsal aspect of the filum. While the artery of the filum is of a caliber proportional to that of the filum and appears to be a nutrient vessel, the vein has a caliber unrelated to that of the filum and appears rather as an intradural drainage route continuous with the anterior spinal vein. Several cases of disease of the filum terminale confirm this anatomic appearance and also show that, because of the existing hyperpressure in the vein of the filum, the posterior spinal vein also shares in the drainage of the latter and that entire system may function in both ascending and descending directions.

---

La vascularisation normale du filum terminale intradural chez l'homme

Résumé La vascularisation artérielle et veineuse du filum terminale intradural a été étudiée sur 18 cadavres frais, sous microscope, après prélèvement en monobloc de la moelle épinière dorso-lombaire, des racines et du filum dans leur étui dural. L'examen des artères a été fait après injection manuelle de l'artère du renflement lombaire, tandis que l'étude des veines s'est faite sans injection compte tenu d'une coloration bleu-noir spontanée qui les rendent aisément identifiables. Après étude macroscopique, chaque pièce a été fixée, puis coupée à 12 niveaux différents depuis le cône médullaire jusqu'au fond du cul-de-sac dure-mérien, pour étude histologique. La distribution de la vascularisation du FT apparaît constante. Une artère unique, l'artère du FT, naît de la terminaison de l'axe spinal antérieur, soit par trifurcation, soit de la partie proximale d'une des 2 branches de l'anse anastomotique du cône. L'artère chemine devant le FT; son calibre diminue rapidement; rarement, elle a pu être suivie jusque dans le canal sacré. Une veine, la veine du FT, chemine en avant du FT mais en arrière de l'artère, comme au niveau médullaire. Son calibre est uniforme mais variable d'un sujet à l'autre. Elle traverse la dure-mère en bas; elle se continue avec la veine spinale antérieure en haut. Aucun vaisseau n'a été retrouvé à la face dorsale du FT. Si l'artère du FT a un calibre qui est proportionnel à celui du filum et apparaît comme un vaisseau nourricier, la veine a un calibre sans aucun rapport avec le volume de celui-ci et apparaît davantage comme une voie de drainage intradural en continuité avec la veine spinale antérieure. Quelques cas de pathologie du FT confirment cet aspect anatomique et montrent aussi qu'en raison de l'hyperpression veineuse régnant dans la veine du FT, la veine spinale postérieure participe également au drainage de celle-ci et que l'ensemble peut fonctionner dans les 2 sens ascendant ou descendant.

     

Influence of CYP2D6-dependent metabolism on the steady-state pharmacokinetics and pharmacodynamics of metoprolol and nicardipine,alone and in combination

1 The metabolism of metoprolol depends in part on the genetically determined activity of the CYP2D6 isoenzyme. In vitro studies have shown that nicardipine is a potent inhibitor of CYP2D6 activity. Since the combination of metoprolol and nicardipine is likely to be used for the treatment of hypertension, we examined the interaction between these two drugs at steady-state. 2 Fourteen healthy volunteers, seven extensive and seven poor metabolisers of dextromethorphan were studied in a double-blind, randomised cross-over four-period protocol. Subjects received nicardipine 50 mg every 12 h, metoprolol 100 mg every 12 h, a combination of both drugs and placebo during 5.5 days. Steady-state pharmacokinetics of nicardipine and metoprolol were analyzed. Beta-adrenoceptor blockade was assessed as the reduction of exercise-induced tachycardia. 3 During treatment with metoprolol, alone or in combination with nicardipine, its steady-state plasma concentrations were higher in subjects of the poor metaboliser phenotype than in extensive metabolisers. Beta-adrenoceptor blockade was also more pronounced in poor metabolisers than in extensive metabolisers of dextromethorphan during treatment with metoprolol alone or in combination with nicardipine (24.0 +/- 2.4% vs 17.1 +/- 3.5% and 24.1 +/- 2.5% vs 15.4 +/- 2.7% reduction in exercise trachycardia, respectively, P < 0.01 in each case). 4 Nicardipine produced a small increase in plasma metoprolol concentration in extensive metabolisers from 35.9 +/- 16.6 to 45.8 +/- 15.4 ng ml(-1) (P < 0.02), but had no significant effect in poor metabolisers. However, nicardipine did not alter the R/S metoprolol ratio in plasma 3 h after dosing, the plasma concentration of S-(-)-metoprolol 3 h after dosing or the beta-adrenoceptor blockade produced by metoprolol in subjects of both phenotypes. The partial metabolic clearance of metoprolol to alpha-hydroxy-metoprolol was not altered significantly in extensive metabolisers. Plasma nicardipine concentration and beta-adrenoceptor blocking effects did not differ between the phenotypes and were not influenced by metoprolol. We conclude that beta-adrenoceptor blockade during repeated dosing with metoprolol is more pronounced in poor than in extensive metaboliser subjects, that nicardipine decreases a CYP2D6-independent route of metoprolol elimination but does not increase beta-adrenoceptor blockade during repeated dosing with metoprolol.     

Drug interaction microcomputer software evaluation: the Medical Letter Drug Interactions Program

The Medical Letter Drug Interactions Program was evaluated using general and specific criteria. The installation process, ease of learning, and use were rated excellent. The user documentation and technical support were good. The quality of the clinical documentation was excellent. The scope of coverage and overall clinical performance were good. The frequency of updates is fair. The primary advantages of the program are low costs and fast, reliable screening tool for drug interactions.