Effects of 5'-fluoro-2-deoxyuridine on mitochondrial biology in Caenorhabditis elegans.

5-Fluoro-2'-deoxyuridine (FUdR) is a DNA synthesis inhibitor commonly used to sterilize Caenorhabditis elegans in order to maintain a synchronized aging population of nematodes, without contamination by their progeny, in lifespan experiments. All somatic cells in the adult nematode are post-mitotic and therefore do not require nuclear DNA synthesis. However, mitochondrial DNA (mtDNA) replicates independently of the cell cycle and thus represents a potential target for FUdR toxicity. Inhibition of mtDNA synthesis can lead to mtDNA depletion, which is linked to a number of diseases in humans. Furthermore, alterations in mitochondrial biology can affect lifespan in C. elegans. We characterized the effects of FUdR exposure on mtDNA and nuclear DNA (nucDNA) copy numbers, DNA damage, steady state ATP levels, nematode size, mitochondrial morphology, and lifespan in the germ line deficient JK1107 glp-1(q244) and PE255 glp-4(bn2) strains. Lifespan was increased very slightly by 25 µM FUdR, but was reduced by 400 µM. Both concentrations reduced mtDNA and nucDNA copy numbers, but did not change their ratio. There was no detectable effect of FUdR on mitochondrial morphology. Although both concentrations of FUdR resulted in smaller sized animals, changes to steady-state ATP levels were either not detected or restricted to the higher dose and/or later timepoints, depending on the method employed and strain tested. Finally, we determined the half-life of mtDNA in somatic cells of adult C. elegans to be between 8 and 13 days; this long half-life very likely explains the small or undetectable impact of FUdR on mitochondrial endpoints in our experiments. We discuss the relative pitfalls associated with using FUdR and germline deficient mutant strains as tools for the experimental elimination of progeny.

Cerium oxide nanoparticles are more toxic than equimolar bulk cerium oxide in Caenorhabditis elegans.

Engineered cerium oxide nanoparticles (CeO2 NPs) are widely used in biomedical and engineering manufacturing industries. Previous research has shown the ability of CeO2 NPs to act as a redox catalyst, suggesting potential to both induce and alleviate oxidative stress in organisms. In this study, Caenorhabditis elegans and zebrafish (Danio rerio) were dosed with commercially available CeO2 NPs. Non-nano cerium oxide powder (CeO2) was used as a positive control for cerium toxicity. CeO2 NPs suspended in standard United States Environmental Protection Agency reconstituted moderately hard water, used to culture the C. elegans, quickly formed large polydisperse aggregates. Dosing solutions were renewed daily for 3 days. Exposure of wild-type nematodes resulted in dose-dependent growth inhibition detected for all 3 days (p < 0.0001). Non-nano CeO2 also caused significant growth inhibition (p < 0.0001), but the scale of inhibition was less at equivalent mass exposures compared with CeO2 NP exposure. Some metal and oxidative stress-sensitive mutant nematode strains showed mildly altered growth relative to the wild-type when dosed with 5 mg/L CeO2 NPs on days 2 and 3, thus providing weak evidence for a role for oxidative stress or metal sensitivity in CeO2 NP toxicity. Zebrafish microinjected with CeO2 NPs or CeO2 did not exhibit increased gross developmental defects compared with controls. Hyperspectral imaging showed that CeO2 NPs were ingested but not detectable inside the cells of C. elegans. Growth inhibition observed in C. elegans may be explained at least in part by a non-specific inhibition of feeding caused by CeO2 NPs aggregating around bacterial food and/or inside the gut tract.

Genetic diversity analysis using lowly polymorphic dominant markers: the example of AFLP in pigs.

DNA markers are commonly used for large-scale evaluation of genetic diversity in farm animals, as a component of the management of animal genetic resources. AFLP markers are useful for such studies as they can be generated relatively simply; however, challenges in analysis arise from their dominant scoring and the low level of polymorphism of some markers. This paper describes the results obtained with a set of AFLP markers in a study of 59 pig breeds. AFLP fingerprints were generated using four primer combinations (PC), yielding a total of 148 marker loci, and average harmonic mean of breed sample size was 37.3. The average proportion of monomorphic populations was 63% (range across loci: 3%-98%). The moment-based method of Hill and Weir (2004, Mol Ecol 13:895-908) was applied to estimate gene frequencies, gene diversity (F(ST)), and Reynolds genetic distances. A highly significant average F(ST) of 0.11 was estimated, together with highly significant PC effects on gene diversity. The variance of F(ST) across loci also significantly exceeded the variance expected under the hypothesis of AFLP neutrality, strongly suggesting the sensitivity of AFLP to selection or other forces. Moment estimates were compared to estimates derived from the square root estimation of gene frequency, as currently applied for dominant markers, and the biases incurred in the latter method were evaluated. The paper discusses the hypotheses underlying the moment estimations and various issues relating to the biallelic, dominant, and lowly polymorphic nature of this set of AFLP markers and to their use as compared to microsatellites for measuring genetic diversity.

Genetic diversity within and between European pig breeds using microsatellite markers.

An important prerequisite for a conservation programme is a comprehensive description of genetic diversity. The aim of this study was to use anonymous genetic markers to assess the between- and the within-population components of genetic diversity for European pig breeds at the scale of the whole continent using microsatellites. Fifty-eight European pig breeds and lines were analysed including local breeds, national varieties of international breeds and commercial lines. A sample of the Chinese Meishan breed was also included. Eleven additional breeds from a previous project were added for some analyses. Approximately 50 individuals per breed were genotyped for a maximum of 50 microsatellite loci. Substantial within-breed variability was observed, with the average expected heterozygosity and observed number of alleles per locus being 0.56 [range 0.43-0.68] and 4.5 respectively. Genotypic frequencies departed from Hardy-Weinberg expectations (P < 0.01) in 15 European populations, with an excess of homozygotes in 12 of them. The European breeds were on average genetically very distinct, with a Wright F(ST) index value of 0.21. The Neighbour-Joining tree drawn from the Reynolds distances among the breeds showed that the national varieties of major breeds and the commercial lines were mostly clustered around their breeds of reference (Duroc, Hampshire, Landrace, Large White and Piétrain). In contrast, local breeds, with the exception of the Iberian breeds, exhibited a star-like topology. The results are discussed in the light of various forces, which may have driven the recent evolution of European pig breeds. This study has consequences for the interpretation of biodiversity results and will be of importance for future conservation programmes.

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers.

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.

DNA microsatellite analysis for parentage control in Austrian pigs.

We present an efficient parentage control for pigs based on ten polymorphic microsatellite markers analyzed in a single PCR reaction. Assuming one known parent ("paternity control"), combined exclusion probabilities (CEPs) ranged from 99.18% (Landrace), 99.74% (Piétrain) to 99.76% (Large White) for the most important Austrian breeds. Assuming a known parent-pair ("parentage control", e.g. a substituted offspring), the CEP of the 10-plex PCR increased to 99.97% (Landrace) and 99.99% (Piétrain and Large White). We developed an additional standby battery of 5 markers, which might be applied in those cases, where the CEP of the 10-plex PCR is not sufficient. Therefore an automated, cost and time reduced genotype analysis for pigs is available.

Breath hydrogen analysis in patients with ileoanal pouch anastomosis.

The possible influence on functional outcomes of hydrogen production in the ileoanal pouch after restorative proctocolectomy was investigated by means of lactulose H2 breath tests. Eight of 15 patients had significant increases in breath hydrogen after 10 g lactulose. One patient declined to participate in further investigations, the remaining seven responders had no evidence of small bowel bacterial overgrowth after glucose H2 breath tests. The ability to produce hydrogen by anaerobic fermentation of lactulose in the pouch was unrelated to the age of the patients or of the pouch. Seven of eight responders had successive breath tests after ingestion of lactulose 20 g and wheat starch 100 g. Five of seven had significant increases after lactulose but none after wheat starch. The overall function of the pouch continence, spontaneity of defecation, and 24 hour stool frequency was significantly better in responders than in non-responders. The absence of H2 production of 100 g wheat starch may indicate either increased absorption or defective fermentation.

[Popliteal artery entrapment syndrome]. / Arteria poplitea afklemningssyndrom.

Popliteal artery entrapment syndrome (PAES) should be suspected in young persons without predisposing factors to arteriosclerosis, who present with unilateral claudication in the calf and foot. This entity results from a developmental abnormal relation between the popliteal artery and the medial head of the gastrocnemius muscle. PAES is mostly found in young sportsmen or young soldiers with well-developed muscles. The onset of the symptoms is often sudden, occurring during an episode of intense lower extremity activity. The diagnosis is best established by a thorough clinical examination combined with functional tests followed by a CT-scan and/or biplanar arteriography. The treatment is surgical with decompression of the popliteal artery and in cases of arterial thrombosis also interposition of an autologous graft. Prophylactic decompression should be considered in asymptomatic cases.

Molecular characterization of the porcine MHC class I region.

A porcine cosmid library was screened with a human MHC class I cDNA. Four positive clones were isolated and mapped with different restriction endonucleases. Altogether nine SLA class I genes were identified and their positions located within restriction maps. Sizes of class I homologous DNA sequences varied between 3600 and 5800 bp. The distances between these regions ranged from 11,900 to 22,200 bp.

[Cystic adventitial degeneration. A rare cause of intermittent claudication]. / Cystisk adventitiel degeneration. En sjoelden årsag til claudicatio intermittens.

Cystic adventitial disease of the popliteal artery is an unusual condition of uncertain etiology in which a mucin-containing cyst forms in the wall of the popliteal artery and causes symptoms of intermittent claudication. The patient is typically a young non-smoking male, whose arteries otherwise are normal. Arteriography may show a smooth-walled narrowing or a non-specific complete occlusion, but may also be normal. Non-invasive imaging techniques may be helpful. We report two cases of cystic adventitial disease. Both were successfully operated on with resection of the diseased arterial segment and vein bypass interposition.

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.

Associations between marker genotypes, halothane reaction, creatine kinase activity and meat quality characters in a sample of German Landrace pigs.

Routine blood typing of German Landrace pedigree populations and an earlier study revealed very low frequencies of the favourable alleles at the marker loci Phi, Pgd and H. The hypothesis was that in this population the whole linkage group of favourable alleles at the halothane and neighbouring marker loci may have been lost as a consequence of intense selection for leanness and type. The present study of 1050 German Landrace pigs at the Relliehausen experimental station, where some effort has been made to maintain a higher frequency of the favourable alleles PhiA (0.48), H- (0.43) and PgdA (0.70) gave quite different results. The frequency of halothane-positive pigs found by using a severe test was only 30%. Only 5.4%, 8.8%, 13.4% and 13.9% of animals with PhiA/A, H-/-, PgdA/A and PhiA/B genotypes respecitively were halothane-positive. Forty to sixty per cent of pigs with these marker genotypes could therefore be expected to be homozygous halothane-negative (N/N) animals. Creatine kinase activity and three selected meat quality characters showed highly significant differences between the A/A and the B/B genotypes for the marker loci Phi and Pgd, with the heterozygotes being intermediate. These differences are greater than those observed between halothane-negative and halothane-positive phenotypes. The only other consistently superior marker genotype in this population was the H blood group genotype H-/-. In contrast to findings from Sweden and Switzerland, the postalbumin locus Po2 and the suppressor locus S for the A-O blood groups did not exhibit useful marker qualities.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood-brain barrier, aging, brain blood flow, and sleep.

We have shown that significant ultrastructural changes occur with increasing age in the BBB in the nonhuman primate. It is probable that similar changes occur in aging humans. Clearly, morphophysiological changes, i.e., structural changes in cerebral capillaries, may alter the BBB mechanism as well as capillary perfusion which, in turn, may affect cerebral energy metabolism and neuronal function. Thus neurological function may be affected and sensitive indices of function such as sleep patterns altered.

Distribution of the A blood group activity on lipid and nonlipid fractions of pig organs.

Lipids and protein-containing nonlipids of pig erythrocytes, serum and several organs were tested for A blood group activity. Both lipids and nonlipids of brain, myocardium, skeletal muscle and adipose tissue show no A activity. The way of distribution of A activity on lipids and nonlipids of other tissues and serum differs among individual pigs. With respect to the acquisition of A activity of erythrocytes in a postnatal period, it seems likely that the lipidic A substance Tor part of it containing the A determinant) is transferred from plasma to the erythrocyte membrane.

Distribution of the A blood-group activity in porcine serum.

The A blood-group activity of pig serum is bound to a lipid, in some cases also to a nonlipid fraction. The major lipidic A activity (roughly 50%) is carried with the HDL class, while the VLDL and LDL classes contain roughly 25% each.