An autosomal dominant retinitis pigmentosa family with close linkage to D7S480 on 7q

Retinitis pigmentosa is the most prevalent inherited disorder of the retina. It can be autosomal dominant (adRP), autosomal recessive (arRP) or X-linked (XLRP). A form of adRP mapping to chromosome 7q was reported in a large Spanish pedigree. We have typed DNA from the members of another Spanish family for polymorphic markers from the known candidate genes. Positive lod scores were obtained only for the markers located on 7q31-35, giving a maximum lod score of 2.98 (3.01 by multipoint analysis) at = 0.00 for D7S480. A brief clinical evaluation is given.     

Superoxide dismutase mimetic activity of a cyclic tetrameric Schiff base N-coordinated Cu(II) complex

A pulse radiolytic study using the cyclic tetrameric Schiff base N-coordinated copper complex Cu(TAAB)²⁺ has been performed. The reaction of the Cu(TAAB)²⁺ complex with superoxide revealed pseudo first-order characteristics with the rate constant of k ₂ = (2.9 ± 0.5) × 10⁸ mol^–1 s^–1 dm³. The complex survive presence of competing serum albumin in physiological concentrations. The complex stability constant K = 1.15 × 10¹⁸ (log K = 18.06) is two orders of magnitude higher than that of Cu(II)-serum albumin (log K = 16.2). Transient changes of the stability during the oxidation/reduction process and in the presence of 600 /mol l^–1 albumin did not affect significantly either the electronic absorption of the complex or its catalytic activity.     

Junction formation in aggregated embryonal carcinoma cells

Under the action of supplemental calcium, H6 mouse embryonal carcinoma cell aggregates undergo compaction, a morphological phenomenon similar to mouse embryonic compaction. Formation of various types of cell junctions, especially gap junctions, is associated with compaction of the embryo and we sought to analyze the pattern of junction formation during aggregation and compaction of H6 cells. At 24 hr of aggregation, gap junctions were abundant in both uncompacted and compacted aggregates but quantitative analysis of freeze fracture replicas of these junctions showed a 20-fold increase in the size of the largest gap junctions in compacted aggregates. Such a difference in size could even be detected at 12 hr of aggregation. Tight junctions were not normally formed in 12 hr aggregates but initial stages of tight junction formation could be noticed in 12 hr compacted aggregates. More definitive tight junctions and desmosomes were evident only after 48 hr of aggregation. Thus we have observed that both uncompacted and compacted aggregates can form gap junctions at similar frequencies, suggesting that cell flattening, which contributes to the compacted morphology, is not a requisite for gap junctions. Likewise, generation of the compacted morphology seems to be independent of gap junction formation. This supports the idea that compaction in embryonal carcinoma cells results from calcium-induced cell flattening, probably through the mobilization of cytoskeletal elements. Calcium-dependent features of H6 cell aggregation and compaction enables the independent analysis of separate steps in compaction.     

The effects of diphosphonates on the growth and glycolysis of connective-tissue cells in culture.

1. The effects of two diphosphonates (compounds containing a P-C-P bond), disodium dichloromethanediphosphonate and disodium 1-hydroxyethane-1,1-diphosphonate, on the metabolism of cultured rat calvaria cells, rabbit ear cartilage cells and rat skin fibroblasts were investigated. 2. The diphosphonates had no effect on the growth of cartilage cells and on the exponential growth of the calvaria cells and the fibroblasts. However, dichloromethanediphosphonate stopped the growth of the calvaria cells and the fibroblasts after the beginning of confluence, whereas the untreated cells were still growing to a certain extent. This inhibition was dose-dependent. After the drug was withdrawn, the cells recovered slowly. 1-Hydroxyethane-1,1-diphosphonate had no detectable effect on the growth of any of the cell types studied. Both diphosphonates decreased the cloning efficiency of calvaria cells and fibroblasts. 3. The K+ content of cartilage, calvaria and skin cells was diminished only by the highest (0.25 mM) concentration of dichloromethanediphosphonate. 4. Radioactive dichloromethanediphosphonate and 1-hydroxyethane-1,1-diphosphonate were taken up linearly with time for at least 48 h by calvaria cells and fibroblasts. The diphosphonate concentration in the cells depended on its concentration in the medium. 5. Both diphosphonates, in a dose-dependent fashion, markedly inhibited glycolysis, dichloromethanediphosphonate being more effective than 1-hydroxyethane-1,1-diphosphonate, at drug doses that had no effect on cell growth or cellular K+ content. Calvaria cells were much more sensitive than cartilage cells. When cartilage cells were cultured in an N2 atmosphere, these effects on glucose and lactate metabolism disappeared. 6. As increased acid production appears to be associated with resorption of bone, this decrease in lactate may explain why diphosphonates are effective inhibitors of bone resorption in vivo.     

Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [³⁵S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of ³⁵S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.     

The contribution of fungal enzymes to the digestion of leaves by Gammarus fossarum Koch (Amphipoda)

Summary Gut extracts of Gammarus fossarum liberated reducing substances (at pH values 7) and amino acids (pH7) from freshly shed oak leaves only after removal of soluble leaf phenols. When carboxymethylcellulose was used at a concentration equal to that of leaf cellulose, release of reducing substances was considerably higher. Fungal enzymes extracted from decomposing leaves were active against structural carbohydrates but showed no proteolytic activity. At low pH values, they retained their full activity in the presence of gut enzymes of G. fossarum, at higher pH values they were inhibited. Conditioned leaves released larger amounts of reducing substances and amino acids when exposed to gut enzymes. The improvement varies with the fungal species used for conditioning and with the length of the conditioning period. The digestibility of leaf carbohydrates and proteins reached separate peaks and then declined. Fungal carbohydrases ingested by G. fossarum retained some activity for up to 4h.     

Microbial Oxidation of Methane and Methanol: Crystallization of Methanol Dehydrogenase and Properties of Holo- and Apo-Methanol Dehydrogenase from Methylomonas methanica

Procedures are described for the purification and crystallization of methanol dehydrogenase from the soluble fraction of the type I obligate methylotroph Methylomonas methanica strain S1. The crystallized enzyme is homogeneous as judged by acrylamide gel electrophoresis and ultracentrifugation. The enzyme had a high pH optimum (9.5) and required ammonium salt as an activator. In the presence of phenazine methosulfate as an electron acceptor, the enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight as well as subunit size of methanol dehydrogenase was 60,000, indicating that it is monomeric. The sedimentation constant (s(20,w)) was 3.1S. The amino acid composition of the crystallized enzyme is also presented. Antisera prepared against the crystalline enzyme were nonspecific; they cross-reacted with and inhibited the isofunctional enzyme from other obligate methylotrophic bacteria. The crystalline methanol dehydrogenase had an absorption peak at 350 nm in the visible region and weak fluorescence peaks at 440 and 470 nm due to the presence of a pteridine derivative as the prosthetic group. A procedure was developed for the preparation of apo-methanol dehydrogenase. The molecular weights, sedimentation constants, electrophoretic mobilities, and immunological properties of apo- and holo-methanol dehydrogenases are identical. Apo-methanol dehydrogenase lacked the absorption peak at 350 nm and the fluorescence peaks at 440 and 470 nm and was catalytically inactive. All attempts to reconstitute an active enzyme from apo-methanol dehydrogenase, using various pteridine derivatives, were unsuccessful.     

Vitamin D metabolism and expression in rats fed on low-calcium and low-phosphorus diets

1. Cholecalciferol, radioactively labelled with both (14)C and (3)H, was administered weekly for 7 weeks to rats that had been depleted of vitamin D for 4 weeks before repletion with the radioactive vitamin. This permitted measurement of the steady-state effect on vitamin D metabolism of low-calcium and low-phosphorus regimens, as compared with a normal mineral intake. These dietary manoeuvres were carried out during the last 3 weeks of repletion. Cholecalciferol, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol were determined in plasma, intestine, kidney and bone. Ca(2+)-binding-protein content was measured in intestine and kidneys of comparable animals. 2. In rats on the low-calcium diets, 1,25-dihydroxycholecalciferol concentration was elevated in plasma, bone, kidney and intestine, and intestinal Ca(2+)-binding protein was increased to over twice the concentration found in the control animals. 3. The low-phosphorus regimens led to a decrease in plasma phosphate and 1,25-dihydroxycholecalciferol in all tissues studied, for the latter to the point where it was undetectable in plasma and bone. Intestinal and renal concentrations of Ca(2+)-binding protein were unchanged in the low-phosphate-intake group and decreased in the very-low-phosphate-intake group. 4. It is concluded that in the rat, unlike in the chick, hypophosphataemia is not associated with a stimulation of the production of 1,25-dihydroxycholecalciferol or its expression in the synthesis of Ca(2+)-binding protein. Therefore the plasma phosphate concentration does not appear to be directly involved in the regulation of the functional metabolism of vitamin D.     

Microbial Oxidation of Hydrocarbons: Properties of a Soluble Methane Monooxygenase from a Facultative Methane-Utilizing Organism, Methylobacterium sp. Strain CRL-26

Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C₁ to C₈), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH₂ or NADPH₂ could act as an electron donor. Formate and NAD⁺ (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD⁺ and secondary alcohol dehydrogenase generated the NADH₂ required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.