Resistance to Disruption of Multilamellar Fragments of Central Nervous System Myelin

Abstract: Single-bilayer vesicles of myelin are desirable for studying myelin development and metabolism. Accordingly, our interest was drawn to a procedure for ves-iculating myelin (Steck et al., Biochim. Biophys. Acta 509, 397–408, 1978). We used X-ray diffraction analysis to examine these putative vesicle preparations because much larger amounts of material can be surveyed by this method than by electron microscopy. The sharpness (width) of the rings in the X-ray diffraction pattern varies inversely with the number of bilayers per multilayer structure. We therefore expected to see the diffuse diffraction pattern characteristic of single bilayers. Diffraction patterns were recorded from isolated rat brain myelin before and after the vesiculation procedure. Both patterns showed sharp rings, indicating numerous multilayered structures. Average values ranging from 7 to 10 bilayers per multilayer were calculated in both cases. This procedure did produce a small fraction of single-bilayer structures, which were isolated by differential centnfu gation; however, these accounted for only about 1% of the total myelin present. The diffraction pattern of this material showed the diffuse band typical of single-bilayer structures, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated it had the same protein composition as in normal myelin. Similar results were also obtained using either fresh or frozen bovine brain myelin. Variations of the published vesiculation procedure (incubation in 0.1 M NaCl or in buffers containing glycerol; disruption by sonication or use of a Tissumizer) also were not effective in breaking down the multilamellar fragments into thinner structures. We conclude that the multilamellar fragments of isolated CNS myelin resist disruption into single-bilayer structures.     

Mechanism of proton-pumping in the cytochrome b/f complex

Several models have been proposed to interpret the mechanism of proton-pumping associated with the electron transfer reactions in the cytochrome b/f complex. Energetics considerations suggest that the proton pump is coupled to the oxidation of cytochrome b by plastoquinone. Experiments performed in living cells under anaerobic conditions suggest that proton-pumping can occur through two independent mechanisms. When the two b cytochromes are reduced prior to a flash illumination i.e. after a long dark anaerobic incubation (>10 minutes), proton-pumping is very likely associated with the reduction of a semiquinone by cyt b which occurs at a site close to the inner face of the membrane. The electrogenic phase is associated with the tranfer of protons via a transmembrane channel. This process is not inhibited by 2-n-nonyl-4-hydroxyquinoline N-oxide (NQNO). Under repetitive-flash or under aerobic conditions, proton-pumping occurs according to a modified Q-cycle mechanism, which is inhibited by NQNO.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Sequences of initiator and elongator methionine tRNAs in bean mitochondria

Summary Two bean mitochondria methionine transfer RNAs, purified by RPC-5 chromatography and two-dimensional gel electrophoresis, have been sequenced usingin vitro post-labeling techniques.One of these tRNAs^Met has been identified by formylation using anE. coli enzyme as the mitochondrial tRNA_F ^Met. It displays strong structural homologies with prokaryotic and chloroplast tRNA_F ^Met sequences (70.1–83.1%) and with putative initiator tRNA_m ^Met genes described for wheat, maize andOenothera mitochondrial genomes (88.3–89.6%).The other tRNA^Met, which is the mitochondrial elongator tRNA_F ^Met, shows a high degree of sequence homology (93.3–96%& with chloroplast tRNA_m ^Met, but a weak homology (40.7%) with a sequenced maize mitochondrial putative elongator tRNA_m ^Met gene.Bean mitochondrial tRNA_F ^Met and tRNA_m ^Met were hybridized to Southern blots of the mitochondrial genomes of wheat and maize, whose maps have been recently published (15, 22), in order to locate the position of their genes.     

Properties of neurofilament protein kinase.

Neurofilament (NF) protein kinase, partially purified from NF preparations [Toru-Delbauffe & Pierre (1983) FEBS Lett. 162, 230-234], was found to be distinct from both the casein kinase present in NFs and the cyclic AMP-dependent protein kinase which is able to phosphorylate NFs. NF-kinase phosphorylated the three NF protein components. The amount of phosphate incorporated per molecule was higher for NF 200 than for NF 145 and NF 68. Other proteins present in the NF preparations were also used as NF-kinase substrates. Two of them might correspond to the myelin basic proteins with Mr values of 18,000 and 21,000. Four other substrates in the NF preparation were not identified (respective Mr values 53,000, 55,000, 65,000 and greater than 300,000). NF kinase also phosphorylated two additional brain-cell cytoskeletal elements: GFAp and vimentin. Casein, histones and phosvitin, currently used as substrates for protein kinase assays, were very poor phosphate acceptors. Half-maximal NF-kinase activity was obtained at an NF protein concentration of about 0.25 mg/ml in heated, salt-washed, NF preparations. The specific activity was about 5 pmol of 32P incorporated/min per microgram of NF kinase preparation protein. ATP was a phospho-group donor (Km 8 X 10(-5) M), but GTP was not. NF-kinase activity remained stable at 65 degrees C for more than 1 h. The enzyme was not degraded by storage at -20 degrees C for several months in a buffer containing 50% (w/v) sucrose. Maximal activity was obtained with 5 mM-Mg2+ (Mg2+ could be replaced by Co2+); Zn2+ and Cu2+ inhibited the reaction. NF-kinase was not dependent on cyclic AMP, cyclic GMP, Ca2+ or Ca2+ plus dioleoylglycerol and phosphatidylserine.     

Polyamine depletion increases cellular ribonucleotide levels

Summary Depletion of the putrescine and spermidine content of Ehrlich ascites tumor cells by -difluoromethylornithine (DFMO) treatment results in at least a 1500-fold increase in the decarboxylated S-adenosylmethionine (deSAM) content. The accumulation of this adenine nucleoside occurs because of the absence of putrescine and spermidine to act as aminopropyl group acceptors in the spermidine and spermine synthase reactions and because of an increase in S-adenosylmethionine decarboxylase activity. The fact that the synthesis of deSAM continues in DFMO-treated cells makes the pathway an adenine trap. This prompted a study of the adenine nucleotide pools. High-performance liquid chromatographic analysis showed that the total adenine nucleotide pool increased, rather than decreased, as a result of DFMO treatment; the major contributors to the increase being ATP and ADP, which increased 2.6 and 1.9 times, respectively. The cellular content of other ribonucleotides increased as well, particularly that of UTP and CTP. When putrescine was added together with DFMO, the increases in cellular ribonucleotide contents were prevented, showing that they were indeed caused by polyamine depletion.     

Cloning of Micrococcus luteus 3-methyladenine-DNA glycosylase genes in Escherichia coli

The 3-methyladenine-DNA glycosylase (m3ADG) excises 3-methyladenine (m3A) residues formed in DNA after treatment with alkylating agents. In Escherichia coli, the repair of this type of damage depends on the products of the genes tagA and/or alkA, which code for m3ADG I (20 kDa) and II (30 kDa), respectively. The tagA- and alkA--single mutants are sensitive to alkylating agents, the double mutant much more so. We have cloned two genes of Micrococcus luteus that can partly substitute the function of the E. coli tagA- and alkA- genes. An M. luteus genome bank was made by shotgun cloning of EcoRI + BamHI-digested DNA into pBR322. Two hybrid plasmids were identified that confer methylmethane sulfonate (MMS) resistance to the tagA- ada+ mutant and a capacity to reactivate MMS-treated bacteriophage lambda. Each hybrid plasmid directed the synthesis of 21-kDa m3ADG in E. coli tagA- ada-, which were not inhibited by 4 mM m3A. However, the restriction maps of the two cloned genes were different, and they showed no sequence homology as judged by the lack of cross hybridization.     

Catastrophic impact of hurricanes on atoll outer reef slopes in the Tuamotu (French Polynesia)

Underwater effects on coral reefs of the six hurricanes which ravaged French Polynesia between December 82 and April 83 were observed by SCUBA diving around high islands and atolls during September and October 1983. Special attention was paid to Tikehau atoll reef formations (Tuamotu archipelago) where quantitative studies on scleractinians, cryptofauna and fishes were conducted in 1982 immediatly prior to the hurricanes. On outer reef slopes coral destruction, varying from 50 to 100%, was a function of depth. Upper slope coral communities composed of small colonies well adapted to high energy level environments, suffered less than deeper formations. However, there is a narrow erosional trough in this zone at a depth of 6 m that was probably the result of storm-wave action (plunge point). Coral destruction was spectacular at depths greater than 12 m: 60 to 80% between 12 m and 30 m and 100% beyond 35 m, whereas earlier living coral coverage ranged from 60 to 75% in these zones. The outer slope was transformed into a scree zone covered with coarse sand and dead coral rubble. Dives on different sites around steep outer slopes (>45°) of the atolls and more gentle slopes (<25°) of some parts of the high islands permitted the formulation of an explanatory hypothesis: direct coral destruction by hurricane-induced waves occurred between the surface and 18–20 m; on low-angle slopes broken colonies were thrown up on reef flats and beaches; on steep slopes avalanches destroyed much of the living corals and left scree slopes of rubble and sand.     

Studies with purified immature porcine Leydig cells in primary culture

The steroidogenic capacity of purified immature porcine Leydig cells in culture was studied over several days. The cells were obtained by fractionating crude testicular interstitial cell suspensions on a discontinuous Percoll gradient (d = 1.037, 1.042, 1.052, 1.098 g/ml), and characterized by specific binding of 125I-human chorionic gonadotropin (hCG), testosterone (T) and cyclic adenosine 3':5'-monophosphate (cAMP) production in response to hCG, and the enzymatic determination of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. The Leydig cells were recovered in a density band between 1.052-1.068 g/ml and grown in a chemically defined medium (Mather et al., 1981). In the absence of hCG, T production was low throughout the 6 days of culture. However, in response to hCG (10 mIU/ml), the cultured Leydig cells showed a progressive increase in T synthesis, which reached a maximum at Days 3-4. 8-Br-cAMP (1 mM) induced a comparable rise in T production to that obtained with hCG throughout the culture period. In contrast, 8-Br-cAMP induced a near maximal increase in dehydroepiandrosterone (DHEA) production from Day 1. This paper demonstrates that purified immature porcine Leydig cells in primary culture are a valuable model to study the ontogeny of Leydig cell function.     

Theoretical conformational analysis of phospholipids II. Role of hydration in the gel to liquid crystal transition of phospholipids

To obtain a satisfactory agreement between computed transition temperatures and those determined experimentally, we introduce explicitly water molecules which hydrate the polar headgroup of dipalmitoylphosphatidylethanolamine molecules. The calculated free energy curves as a function of the intermolecular interchain distance and the degree of hydration of the polar groups permit the determination of the transition of the phospholipid system from the gel to the liquid crystalline phase. The detailed structure of the hydration shell is defined using the supermolecular approach.