Cloning and sequencing of a rat CuZn superoxide dismutase cDNA. Correlation between CuZn superoxide dismutase mRNA level and enzyme activity in rat and mouse tissues

The molecular cloning and nucleotide sequence of a cDNA clone (pR SOD) for rat CuZn superoxide dismutase (CuZnSOD) is reported. Nucleotide sequence homology with human superoxide dismutase is 86% for the coding region and 71% for the 3' untranslated region. The deduced amino acid sequence is given and the homologies with the sequences reported for other species are presented. Northern blot analysis of total RNA from various rat and mouse tissues and from two mouse cell lines show that pR SOD hybridizes with one mRNA species of about 0.7 kb. The amount of CuZnSOD mRNA in each tissue, measured by densitometry of the Northern blot autoradiograms, correlates with the enzymatic activity based on protein content. These results indicate that the control of CuZnSOD activity in mammalian tissues is largely dependent on the regulation of CuZnSOD mRNA levels. In human liver, fibroblasts and FG2 hepatoma cells, two CuZnSOD mRNAs (0.7 kb and 0.9 kb) are observed. The level of CuZnSOD mRNA in FG2 is 25% that of the liver and four times more abundant than in fibroblasts.     

Circumnutation in Phaseolus vulgaris. I. Growth, osmotic potential and cell ultrastructure in the free-moving part of the shoot

Circumnutation of Phaseolus vulgaris L. cv. Blanc de Juillet twining shoots was examined at the cellular level. Cell growth was monitored inside the free-moving part of the shoot as a function of time and position with respect to the terminal bud. Complete elongation of epidermal cells required >7 days. A 60% increase in length of the cells in the bending zone was observed. Scanning and transmission electron microscopy studies showed the elasticity of cell walls, the extent of plasmodesmata and endoplasmic reticulum of epidermal cells. The osmotic potential in epidermal cells of the bending zone displayed significant differences only between the concave and convex sides. The pattern of cell growth in twining shoots is discussed. Circumnutation could be seen as a consequence of periodic turgor changes inducing periodic growth.     

New chromosome 21 DNA markers isolated by pulsed field gel electrophoresis from an ETS2-containing Down syndrome chromosomal region

To generate new chromosome 21 markers in a region that is critical for the pathogenesis of Down syndrome (D21S55-MX1), we used pulsed field gel electrophoresis (PFGE) to isolate a 600-kb NruI DNA fragment from the WA17 hybrid cell line, which has retained chromosome 21 as the only human material. This fragment, which contains the oncogene ETS2, was used to construct a partial genomic library. Among the 14 unique sequences that were isolated, 3 were polymorphic markers and contained sequences that are conserved in mammals. Five of these markers mapped on the ETS2-containing NruI fragment and allowed us to define an 800-kb high-resolution PFGE map.     

Two Down syndrome patients with rec(21),dupq,inv(21)(p11;q2109) from a familial pericentric inversion

Aneusomie de recombinaison arose from a familial pericentric inversion of a chromosome 21. Two female patients had a typical Down syndrome; one of them had slight psychomotor retardation. There was partial trisomy 21q2109----qter in these two patients but ZnCu SOD activity was normal.     

Human prostatic steroid 5α-reductase isoforms—A comparative study of selective inhibitors

The present study describes the independent expression of the type 1 and 2 isoforms of human 5α-reductase in the baculovirus-directed insect cell expression system and the selectivity of their inhibition. The catalytic properties and kinetic parameters of the recombinant isozymes were consistent with published data. The type 1 isoform displayed a neutral (range 6–8) pH optimum and the type 2 isoform an acidic (5–6) pH optimum. The type 2 isoform had higher affinity for testosterone than did the type 1 isoform (K_m = 0.5 and 2.9 μM, respectively). Finasteride and turosteride were selective inhibitors of the type 2 isoform (K_i (type 2) = 7.3 and 21.7 nM compared to K_i (type 1) = 108 and 330 nM, respectively). 4-MA and the lipido-sterol extract of Serenoa repens (LSESr) markedly inhibited both isozymes (K_i (type 1) = 8.4 nM and 7.2 μg/ml, respectively; K_i (type 2) = 7.4 nM and 4.9 μg/ml, respectively). The three azasteroids were competitive inhibitors vs substrate, whereas LSESr displayed non-competitive inhibition of the type 1 isozyme and uncompetitive inhibition of the type 2 isozyme. These observations suggest that the lipid component of LSESr might be responsible for its inhibitory effect by modulating the membrane environment of 5α-reductase. Partially purified recombinant 5α-reductase type 1 activity was preserved by the presence of lipids indicating that lipids can exert either stimulatory or inhibitory effects on human 5α-reductase.     

Oxygen consumption during the fenton-type reaction between hydrogen peroxide and a ferrous-chelate (Fe2+-DTPA)

The Fenton-type reaction between ferrous diethylenetriamine pentaacetic acid (Fe²⁺-DTPA, 50–200 μM) and H₂O₂ (20–1000 μM) in phosphate buffer at pH 7.0 results in consumption of dissolved oxygen. This observation differs from many prior reports that oxygen is liberated when more concentrated solutions of H₂O₂ are decomposed by iron salts. The rate and total quantity of oxygen consumed were dependent upon the concentrations of ferrous chelate, H₂O₂, and excess DTPA. Evidence is provided that both the ferrous-DTPA chelate and free DTPA can participate in the oxygen-consuming reactions. Oxygen was also consumed during the Fenton reaction between ferrous ions and H₂O₂ when DTPA and phosphate buffer were omitted. Under these conditions, oxygen evolution was observed at higher H₂O₂ concentrations (e.g., 400 μM). The consumption of oxygen during the Fenton-type reaction of an iron chelate at neutral pH may be relavant to events that take place in biologic systems.     

Thermal selection of PGM allozymes in newly founded populations of the thermotolerant vent polychaete Alvinella pompejana

Alvinella pompejana lives on the top of chimneys at deep-sea hydrothermal vents of the East Pacific Rise. It is thought to be one of the most thermotolerant and eurythermal metazoans. Our experimental approach combines methods of population genetics and biochemistry, considering temperature as a potential selective factor. Phosphoglucomutase (Pgm-1 locus) is one of the most polymorphic loci of A. pompejana and exhibits four alleles, from which alleles 90 and 100 dominate with frequencies of approximately 0.5 in populations. Results from previous studies suggested that allele 90 might be more thermostable than allele 100. Significant genetic differentiation was found by comparing contrasted microhabitats, especially the young, still hot, versus older and colder chimneys, with allele 90 being at highest frequency on young chimneys. Moreover the frequency of allele 90 was positively correlated with mean temperature at the opening of Alvinella tubes. In parallel, thermostability and thermal optimum experiments demonstrated that allele 90 is more thermostable and more active at higher temperatures than allele 100. This dataset supports an additive model of diversifying selection in which allele 90 is favoured on young hot chimneys but counterbalanced over the whole metapopulation by the dynamics of the vent ecosystem.     

Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.