Metaphase and anaphase analysis of V79 cells exposed to erionite, UICC chrysotile and UICC crocidolite

The cytogenetic effects of erionite treatment of V79 cells werecompared with those of UICC crocidolite and UICC chrysotiletreatment. A significant reduction in diploid cells with anaccompanying increase in aneuploid and polyploid cells was observedwith all three treatments. In the erionite-treated cultures,an increase in aneuploid was observedy at all dose levels rangingfrom 10 to 100 µg/ml, whereas in the crocidolite- andchrysotile-treated cultures, significant increases in aneuploidywere observed at all dose levels except the low dose, 10 µg/ml.Chromatid aberrations were observed in cultures treated withcrocidolite and chrysotile and were especially pronounced atdose 100 µg/ml of chrysotile. The clastogenic effect oferionite was weaker but statistically significant at dose 100µg/ml. An extrapolation of these cytogenetic changes overdose in number of fibers suggests that erionite was more reactivethan the other two minerals in producing aneuploidy. The numberof fibers required to produce a similar degree of cytogeneticeffects was several orders of magnitude higher for chrysotileand crocidolite than erionite. These results correlate withthe higher tumorigenic potency of erionite. In general, fewercells treated with erionite entered anaphase than those treatedwith the other two minerals. As a result, abnormal anaphasesrepresenting chromosomal mis-segregation were observed onlyin the chrysotile- and crocidolite-treated cultures. To ourknowledge, this is the first report on cytogenetic effects oferionite.     

Abnormality of type IX collagen in a patient with diastrophic dysplasia

There is growing evidence that a spectrum of chondrodysplasias are caused by mutations in the gene coding for type II collagen. The basic molecular defect in diastrophic dysplasia has not been defined, but it appears not to be in collagen type II. Cartilage contains other tissue-specific collagens, types IX, X, and XI, but no mutations have yet been found in their genes in clinical disease. Type IX collagen is hypothesized to play a role in the regulation of type II collagen fibril organization and structure in cartilage extracellular matrix. In this study, we have examined iliac crest growth cartilage from a patient with diastrophic dysplasia. Although collagen fibrils were markedly increased in diameter on transmission electron microscopy, type II collagen appeared to be normal biochemically. Type XI collagen was also normal. However, type IX collagen appeared abnormal on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a pronounced excess of the COL1 domain of the molecule in pepsin extracts. The findings point to an abnormality in structure or metabolism of type IX collagen in diastrophic dysplasia. © 1994 Wiley-Liss, Inc.     

Polymorphisms of the equine major histocompatibility complex class II DRA locus

The full extent of the polymorphism of ELA-DRA in Equidae is not yet known. Given the apparent differences in DRA polymorphisms between Equidae and other species, the aims of this study were to more fully characterize ELA-DRA, determine the extent of gene polymorphism and establish the allele-frequency distribution. An allele reference panel for the second exon of ELA-DRA was established by sequence-based typing of 69 equine DNA samples consisting of various breeds of domestic horse (Equus caballus), together with donkeys (Equus asinus), Grant's zebras (Equus boehmi) and one onager (Equus hemionus). Five of the six previously reported alleles detected using single-strand conformation polymorphism were found: ELA-DRA*0101, ELA-DRA*0201, ELA-DRA*0301, ELA-DRA*0501 (Albright-Fraser DG et al. Polymorphism of DRA among equids. Immunogenetics 1996: 43: 315-7) and ELA-DRA*0601 (GenBank accession number AF5419361). In addition to the previously reported alleles, five novel ELA-DRA alleles were detected within the ELA-DRA allele reference panel. One of these was identified in E. caballus (ELA-DRA*JBH11), one in E. boehmi and E. hemionus (ELA-DRA*JBZ185) and three in E. asinus (ELA-DRA*JBD3, ELA-DRA*JBD17 and ELA-DRA*JBH45). A total of 565 equine DNA samples were screened using reference-strand-mediated conformation analysis, a double-stranded conformation-based mutation detection system that can be used to type existing ELA-DRA alleles and identify new variants. Based on our findings, at least 11 ELA-DRA alleles are now known to exist, and this level of polymorphism at the DRA locus appears to be unique to the genus Equus. Both the previously reported alleles and the new alleles displayed a species-specific distribution.     

The kyphoscoliotic type of Ehlers-Danlos syndrome (type VI): differential effects on the hydroxylation of lysine in collagens I and II revealed by analysis of cross-linked telopeptides from urine

The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS type VIA) (OMIM 225400) is an autosomal recessive connective tissue disorder that results from mutations in the lysyl hydroxylase 1 gene (PLOD1) causing underhydroxylation of lysine residues in tissue collagens, particularly of skin. Previous studies have shown that the pool of collagen cross-linking amino acids, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) excreted in urine has an abnormally low HP/LP ratio, which is diagnostic of the condition. Here we isolated cross-linked peptides containing these residues from the urine of a child with EDS VIA homozygous for a mutation that results in a stop codon and effective null expression of PLOD1 enzyme activity. Peptides that had originated from bone type I collagen and cartilage type II collagen were identified. A cross-linked N-telopeptide fraction that is derived from bone type I collagen contained only LP, no HP, which means that the helical lysines at residues 930 of alpha 1(I) and 933 of alpha 2(I) of the collagen triple-helix had not been hydroxylated. The equivalent peptide fraction from a normal child's urine gave a ratio of HP to LP of 1.5:1 typical for normal bone collagen. A second cross-linked peptide that is derived from the C-telopeptide domain of cartilage type II collagen showed both HP and LP in a 2:1 ratio, compared with 18:1 for the equivalent peptide from a normal child's urine. The results show that in EDS VIA, bone type I collagen is more markedly underhydroxylated than cartilage type II collagen, at least at those helical sites that form cross-links. The residual fraction of HP found in the urine of EDS VI patients therefore appears to be contributed in significant part by the degradation products of cartilage. Since PLOD1 is null, other PLOD genes must be responsible for the helical hydroxylation activity that results in HP. The presented approach of analyzing urinary cross-linked C-telopeptide fragments of type II collagen may allow the detection of chondrodysplasias due to genetic defects in lysyl hydroxylase isoforms active in cartilage.