Carbonic anhydrase activity in acetate grown Methanosarcina barkeri

Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K _i=0.5 mM), by azide (apparent K _i=1 mM), and by cyanide (apparent K _i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H₂/CO₂ grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO₂ and CH₄. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO₂ with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein).     

The haplotype distribution of the ΔF508 mutation in cystic fibrosis families in Scotland

Summary The gene defective in cystic fibrosis (CF) has recently been isolated and the major mutation identified. The haplotype distribution of this mutation (ΔF508) has been determined for 215 CF chromosomes in the Scottish population. ΔF508 represents 73% of all CF mutations in this group. There remains considerable linkage disequilibrium between XV2c and KM19 and other mutations in the CF gene.     

Induction of reversible changes in cell-surface glycoconjugates and lung colonization potential by 13-cis retinoic acid

Murine squamous carcinoma cells (KLN205) grown in a medium supplemented with the retinoid, 13-cis retinoic acid (RA), had dose-dependent, selective increases in the expression of certain lectin receptors, which correlated with a dramatic decrease in the ability to form pulmonary colonies (P ?.0003) (Couch MJ, Pauli BU, Weinstein RS, Coon JS: JNCI, 78:971 ?977, 1987). These findings suggest a possible relationship between the RA-induced glycoconjugate alterations and the decreased experimental metastatic behavior. We further define the mechanism of RA's action. The finding that RA treatment (5 × 10^?6 M, 5 × 10^?7 M) did not perturb the cell cycle of KLN205 cells provides further proof that the decreased metastatic behavior is not attributable to any inhibition in the rate of growth or to alterations in the cell cycle. Furthermore, since stable subpopulations with variable lectin binding could not be detected, the mechanism of RA's action does not appear to be due to selection of variant tumor-cell subpopulations. Finally, in a scries of experiments designed to determine the reversibility of the RA treatment, the RA-induced decrease in metastatic behavior reverted back to a more metastatic state in the same time frame (3 days) as the reversion of the RA-induced changes in cell-surface glycoconjugate expression. This reversion provides further evidence for a close relationship between the RA-induced modulation of tumor cell-surface glycoconjugate expression and the decreased metastatic behavior; it suggests that transient, reversible modulation of the tumor cell surface may play a role in determining metastatic behavior.     

Cadmium-2-acetylaminofluorene interaction in isolated rat hepatocytes

Cadmium (Cd) is a non-essential, highly toxic heavy metal and a ubiquitous environmental contaminant. Evidence exists that Cd can affect parameters which are of great importance in the response towards xenobiotics. However, there is a lack of information about the mechanisms that take place at the cellular and molecular levels upon dual exposure to Cd and other toxins. The purpose of the present work was therefore to examine the biochemical interactions between Cd and a well-known genotoxic hepatocarcinogen, 2-acetylaminofluorene (AAF) in isolated rat hepatocytes. The cells were incubated for 10 hr with a sub-cytotoxic concentration (0.22 M) of ¹⁰⁹Cd. This was followed by a 10 hr exposure to 1 M [³H]AAF. Cellular distribution of Cd and ³H was determined. Sephadex G-75 elution profiles of the cytosol showed that Cd was almost entirely associated with the intermediate molecular weight (IMW) fractions containing metallothionein (MT) (>80%), and with high molecular weight proteins. In parallel, the highest proportion of ³H was found in the low molecular weight components. Further analysis of IMW fractions by DEAE A-25 anion-exchange chromatography revealed that, in addition to Cd, there was some ³H which coeluted along with MT-I and MT-II isoforms, but preferentially with MT-I. Moreover, Cd pretreatment caused a 1.6-fold increase in MT level, as measured by the silver-saturation assay. Under these conditions, there was a 17% lower binding of ³H to the DNA. This reduced binding was neither accompanied by diminished AAF uptake nor by inhibition of cytochrome P-450 activity. Taken together, these results suggest that Cd exposure has a protective effect against the genotoxicity of AAF. MT, whose synthesis is induced, could play a role in the Cd-AAF interaction through scavenging of reactive metabolites.Abbreviations AAF 2-acetylaminofluorene - Cd cadmium - DMSO dimethyl sulfoxide - HBSS Hank's balanced salt solution - LDH lactate dehydrogenase - MT metallothionein - UDS unscheduled DNA synthesis     

Genetic and physical mapping of the patatin genes in potato and tomato

Summary Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10–15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5 promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5 promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.     

A variant form of the 1,25-dihydroxyvitamin D3 receptor with low apparent hormone affinity in cultured monkey kidney cells (LLC-MK2). A model for tissue resistance to vitamin D

Chandler et al. (Chandler, J.S., Chandler, S.K., Pike, J.W., and Haussler, M.R. (1984) J. Biol. Chem. 259, 2214-2222) previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) caused the induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) in a rhesus monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity 1,25-(OH)2D3 receptor. We have re-examined this phenomenon and report here that 24-hydroxylase induction is mediated by a receptor variant in LLC-MK2 cells with low hormone affinity. Dose response analysis showed that in contrast to LLC-PK1 (a typical receptor-positive cell line), the LLC-MK2 line was less sensitive to 1,25-(OH)2D3 by 2 orders of magnitude. Employing optimal concentrations of 1,25-(OH)2D3 for 24-hydroxylase induction in each cell type, the early time courses of this bioresponse were identical in LLC-MK2 and LLC-PK1 and were consistent with a nuclear action of hormone-receptor complexes. Moreover, the rank order of potency of vitamin D3 congeners as inducers of 24-hydroxylase activity in LLC-MK2 cells agreed well with their relative affinity for the 1,25-(OH)2D3 receptor. An examination of 1,25-(OH)2D3 receptor content via DNA-cellulose chromatography in LLC-MK2 cells incubated at ligand concentrations 10-25-fold higher than the normal 2 nM revealed a minimum of 1600 receptor-like molecules/LLC-MK2 cell. These results show that LLC-MK2 cells possess a variant receptor form with apparent low affinity for 1,25-(OH)2D3. This system should serve as a model for clinical syndromes characterized by the requirement for massive doses of vitamin D to prevent rickets.     

The effect of histone H1 on the compaction of oligonucleosomes. A quasielastic light scattering study

The structural properties of H1-depleted oligonucleosomes are investigated by the use of quasielastic laser light scattering, thermal denaturation and circular dichroism and compared to those of H1-containing oligomers. To obtain information on the role of histone H1 in compaction of nucleosomes, translational diffusion coefficients (D) are determined for mono-to octanucleosomes over a range of ionic strength. The linear dependences of D on the number of nucleosomes show that the conformation of stripped oligomers is very extended and does not change drastically with increasing the ionic strength while the rigidness of the chain decreases due to the folding of linker DNA. The results prove that the salt-induced condensation is much smaller for H1-depleted than for H1-containing oligomers and that histone H1 is necessary for the formation of a supercoiled structure of oligonucleosomes, already present at low ionic strength.     

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.     

The presence of LHRH-like receptors in Dunning R3327H prostate tumors

Quantitative analyses of LH-RH-like membrane receptors were performed in five tumors from the transplantable Dunning R3372H rat prostatic adenocarcinoma. The binding of D-Trp⁶-LH-RH, an agonist of LH-RH, was observed in all 5 tumors. The antagonist [Ac-Dp-Cl-Phe^1,2,D-Trp³,D-Lys⁶,D-Ala¹⁰]-LH-RH was bound to 4 tumors. The apparent equilibrium dissociation constant (K_d) for D-Trp⁶-LH-RH receptor was from 2.6–3.9 × 10^?10 M. The apparent equilibrium B_max values (maximum number of binding sites) were from 17.2–86.0 fmol/mg membrane protein for D-Trp⁶-LH-RH receptor. The K_d for the antagonist was from 2.4–2.7 × 10^?10 M and the B_max values were from 35.5–66.0 fmol/mg membrane protein. Similar binding studies performed in 6 normal rat prostates showed no binding capacities.