Mutation analysis and prenatal diagnosis in a Lesch-Nyhan family showing non-random X-inactivation interfering with carrier detection tests

Summary A nonsense mutation at the CpG-site in the codon for Arg(169) in the gene for hypoxanthine phosphoribosyltransferase (hprt) was identified by genomic polymerase chain reaction (PCR) and DNA sequencing in cultured fibroblasts from two brothers with Lesch Nyhan's syndrome. The recurrence of mutation at this CpG-site in several unrelated Lesch-Nyhan families suggests that deamination of 5-methylcytosine is a possible mechanism for mutagenesis. The level of hprt-mRNA in the fibroblasts of the patients was similar to that in healthy controls, whereas hprt-enzyme activity was not detectable. The mutation in this family was also identified in five female relatives and prenatally in a male fetus. Unexpectedly, results from hair follicle analyses and fibroblast selection studies in 8-azaguanine and 6-thioguanine medium showed a non-carrier phenotype in three of the female heterozygotes, whereas X-inactivation mosaicism was demonstrated in one heterozygote. A possible explanation for the apparent non-random X-inactivation in this family is the co-existence of the hprt mutation with an undefined X-linked lethal mutation. This observation is of practical relevance for carrier detection in other Lesch-Nyhan families.     

A multivariate comparison between three NW. European populations ofArtemisia norvegica (Asteraceae) by means of chemometric and morphometric data

Specimens from Scotland, S. and C. Norway were grown in the botanical garden of Bergen, Norway. Some of the Scottish specimens came from a meristem tissue culture. The specimens were compared by a principal component analysis of lipids and related compounds, and of morphological characters from leaves and flowers. The populations differed from each other, but some overlap was found in leaf characters. The results are discussed in relation to distribution and immigration history, and it is argued that the differences among the populations may have evolved in postglacial time.     

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. XII. Enantioselectivity and temperature dependence in microsomes and reconstituted cytochrome P-450 systems from rat liver.

Formation of metabolic intermediate (MI) complexes was studied with the enantiomers of amphetamine, 1-phenyl-2-pentanamine, N-hydroxyamphetamine, and 2-nitroso-1-phenylpropane (the C-nitroso analogue of amphetamine). Three different enzyme systems were used; liver microsomes from phenobarbital pretreated rats and two reconstituted systems containing the P450 2B1 and P450 2C11 forms of cytochrome P-450. Enantioselective complex formation in microsomes was shown for the amines and the nitroso compound, but not for the hydroxylamine. The highly purified P450 2B1 system formed the MI complex with all substrates tested, and the enantioselectivity observed with the microsomal system was reproduced. In the P450 2C11 system the nitroso compounds were completely inactive, whereas the enantiomers of N-hydroxyamphetamine still produced the complex at a high rate. Changes in temperature were shown to affect (R)-2-nitroso-1-phenylpropane more than its enantiomer. Both enantiomers showed biphasic Arrhenius plots for MI complex formation in microsomes (breaks around 22 degrees C), but the activation energies of the (R)-isomer were about five times higher than those of the (S)-isomer. A theory is presented which suggests different modes of interaction with the active site of P-450 to account for the different behaviour of the various substrates.     

Drug Stimulated DNA Cleavage Mediated by Cauliflower Topoisomerase II

We have investigated cauliflower (Brassica oleracea) topoisomerase II with respect to its interaction with DNA and demonstrate that the enzyme shares the characteristics of topoisomerase II purified from a variety of phylogenetically remote organisms. In the presence of the 2-nitroimidazole Ro 15-0216, cauliflower topoisomerase II-mediated DNA cleavage is extensively stimulated (approximately 20-fold) only at a site recognized as a major cleavage site for the enzyme in the absence of drug. The conservation of the enzyme's DNA specificity in the presence of Ro 15-0216 is in contrast to the effect exerted by traditional topoisomerase II inhibitors, which cause enzyme-mediated cleavage to take place at a multiple number of DNA sites. Ro 15-0216 may therefore prove useful as a tool in the elucidation of the enzyme's DNA interaction sites and its involvement in nucleic acid metabolism in plant cells.     

Ovarian function and pregnancy rates in reindeer calves (Rangifer tarandus ) in Southern Norway

Reindeer calves (n = 632) were slaughtered in November/December (n = 476) or in January (n = 156). Dressed weights and amount of perirenal fat were recorded, and the reproductive organs were collected. A separate group of 130 reindeer calves were weighed at 7 months of age and were followed up with repeated weighings and pregnancy examinations up to 21 months. The onset of puberty and the pregnancy rate were significantly influenced by body weight and the amount of perirenal fat. Approximately 60 g of perirenal fat and 22 kg dressed weight were found at the lower limits for pregnancy. A total of 222 (35%) animals had reached puberty and 126 (20%) were pregnant when examined after slaughter. Animals which conceived during their first autumn showed only a moderate weight gain the following year, and the calf mortality rate in these animals was 47.4%. It was concluded that calf pregnancies are common among the reindeer of Southern Norway and that measures need to be taken to prevent them.     

Variations in the cytoplasmic region account for the heterogeneity of the chicken MHC class I (B-F) molecules

Molecular variation among major histocompatibility complex (MHC) class I (B-F) proteins from B-homozygous chickens is apparently caused by C-terminal variation. Analysis of the total B-F protein pool revealed substantial heterogeneity with two or three molecular mass constituents, each being comprised by several isoelectric focusing variants. This heterogeneity could not be reduced by enzymatic deglycosylation. By contrast, proteolytic removal of a small (M _r 1000–4000) fragment from the chain resulted in the generation of a M _r 36 000 fragment, common to all the molecular mass variants. Unlike the parent proteins, the M _r 36 000 fragment derived from isolated variants yielded identical, simple patterns in two-dimensional gel electrophoresis and identical finger prints in peptide mapping. This, together with N-terminal amino acid sequencing, as well as comparison of hydrophobicity properties of fragments obtained by gradual proteolytic digestion, indicated that the small peptide responsible for the major B-F heterogeneity was situated in the intracellular, C-terminal part.     

High-affinity folate binding in human liver membranes

High-affinity binding of³H-folate in Triton X-100 solubilized membranes of human liver displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. Ultrogel^® AcA 44 chromatography of solubilized membranes saturated with³H-folate revealed a major peak of 100 kDa and a minor peak of 25 kDa. The 100 kDa peak could represent a hydrophobic membrane associated molecular form of the protein. This notion was supported by the fact that the two peaks had identical molecular weights as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis with immunoblotting.     

Uptake and distribution in rat brain of organic and inorganic selenium

Polyunsaturated fatty acids (PUFAs) occur in relatively high amounts in phospholipids of the synapses. PUFAs may thus determine the fluidity of the synaptosomal membrane and, hereby, they may regulate the neuronal transmission. It was therefore tempting to suggest a system in the brain, that inhibits autooxidation of PUFAs. In order to trace such a protection system, Wistar rats were equally loaded with 4500 kBq of 75-Se either as selenite or as L-Se-methionine. By means of gradient ultracentrifugation, particulate fractions of the brains were isolated, and the radioactivity as well as the glutathione-transferase and -peroxidase activities were estimated. The distribution of the two selenium components among the particulate fractions was different. Thus, selenite gave higher radioactivity in myelin, then followed by the light synaptosomal and the vesicular fraction. L-Se-methionine was more equally incorporated in all particulate fractions, although highest activity was found in the mitochondrial fraction. Myelin and synaptic vesicles were devoid of transferase activity. On the other hand, the synaptosomal fraction showed highest specific transferase activity. The glutathione peroxidase activity was highest in the myelin fraction, followed by the vesicular and the synaptosomal fractions. The data obtained thus support the idea that the PUFAs of the synaptic compartment are protected against peroxidation, at least in part, by the selenium containing glutathione peroxidase.     

Algal Carotenoids 51. Secondary carotenoids 2.Haematococcus pluvialis aplanospores as a source of (3S, 3′S)-astaxanthin esters

Aplanospores ofHaematococcus pluvialis MUR 145 contained 0.7% carotenoids (dry wt. basis) consisting of β,β-carotene (5% of total carotenoid), echinenone (4%), canthaxanthin (4%), (3S,3′S)-astaxanthin diester (34%), (3S,3′S)-astaxanthin monoester (46%), (3S,3′S)-astaxanthin (1%) and (3R,3′R,6′R)-lutein (6%). The astaxanthin esters were examined by TLC and HPLC and VIS,¹H NMR and mass spectra recorded. Their chirality was determined by the camphanate method (Vecchi & Müller, 1979) after anaerobic hydrolysis. The tough cell wall of the aplanospores required enzymatic treatment prior to pigment extraction. The potential use of this microalga as a feed ingredient in aquaculture is discussed briefly.