Selenoprotein synthesis and regulation in Archaea

Background

The major biological form of selenium is that of the co-translationally inserted amino acid selenocysteine (Sec). In Archaea, the majority of proteins containing Sec, selenoproteins, are involved in methanogenesis. However, the function of this residue is often not known because selenium-independent homologs of the selenoproteins can be employed, sometimes even in one organism.

Effects of freezing on the structure of chloroplast membranes

Electron spin resonance (ESR) spectra of stearic acid spin labels incorporated into spinach thylakoids can be used to monitor membrane changes during freezing. Changes in the ESR parameters can be directly correlated to the extent of functional freeze damage. Freeze-induced changes in the ESR parameters strongly depend on the osmotic conditions of the incubation medium. Similar changes as on freezing can be observed by transferring thylakoids from an isotonic to a hypotonic medium, i.e., by swelling osmotically flattened thylakoids. This and computer simulations of spin label ESR spectra, which allow for variation of vesicle shape, lead to the conclusion that freeze-induced ESR spectral changes are due to swelling of the thylakoids. Indeed, van't Hoff plots of thylakoid packed volume indicate a freeze-induced increase in the apparent number of osmotically active molecules within the intrathylakoid lumen. During freezing, salt and/or sugar leak into the lumen. Simultaneously, proton channels are irreversibly opened. As the structural alterations obtained upon freezing are not accompanied by a change in bulk fluidity, these data are interpreted in terms of a local action of cryotoxic agents on critical microstructures, possibly at the rims of the thylakoid membranes.     

Crystal and molecular structure of the sulfhydryl protease calotropin DI at 3·2 Å resolution

The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3·2 Å resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0·857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of α-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin.Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.     

The reduction kinetics of chlorophyll a1 as an indicator for proton uptake between the light reactions in chloroplasts

The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll a₁ (P-700) after far-red preilluination have been studied with high time resolution in spinach chloroplasts.

1. 1. The kinetics of chlorophyll a₁ exhibits a pronounced lag phase of 2–3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II.

2. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction.

3. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values.

4. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll a₁. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules.

5. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylakoid membrane.

6. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll a₁ reduction, is estimated as > 10¹¹ M⁻¹ · s⁻¹. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution.

Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.     

pH-Abhängigkeit der 13C-15N-kopplungskonstanten 15N-hochmarkierter aminosäuren,gewonnen aus algenmassenkulturenpH dependence of 13C-15N coupling constants of highly 15N-enriched amino acid isolated from mass cultivation of algae

The alga Ankistrodesmus braunii was grown with [¹⁵N]nitrate under the optimized conditions of a large-scale mass cultivation. 19.7% of the dried algae were isolated as a mixture of amino acids. The ¹⁵N-labelled amino acids (¹⁵N content up to 98%) were separated by ion exchange chromatography using pyridine acetate gradients. The ¹⁵N content of the analytically pure amino acids was determined by combined gas-liquid chromatography-mass spectrometry of the trifluoroacetylated methylesters and by emission spectroscopy in the ¹⁵N analysator. Using pulse Fourier transform ¹³C nuclear magnetic resonance, the pH dependence of the ¹³C-¹⁵N coupling constants of Asp, Pro, Ser, Glu, Gly, Ala, Val, Ile and Leu was determined in aqueous solutions. Increasing coupling constants were found with pH and decreasing electron density, respectively. The relation of Binsch et al. (Binsch, G., Lambert, J.B., Roberts, B.W. and Roberts, J.D. (1964) J. Am. Chem. Soc. 86, 5564–5570) between the coupling constant and the product of the S-part of the ¹³C and ¹⁵N hybridization $\text{S}{}_{\mathrm{C}}\text{·}\text{S}{}_{\mathrm{N}}\text{= 80 · J (}{}^{13}\text{C}\text{-}{}^{15}\text{X}\text{)}$ fits best in acidic medium. The magnitude of the coupling constants correlates well with the electron densities calculated by Del Re et al. (Del Re, G., Pullman, B. and Yonezawa, T. (1963) Biochim. Biophys. Acta 75, 153–182). The recording of ¹³C nuclear magnetic resonance spectra over the entire pH range revealed no change in the sign of the ¹³C-¹⁵N coupling constants of the amino acids.     

Increase in histone acetylation and transitions in histone variants during Friend cell differentiation

Histone acetylation of Murine Erythroleukemia Cells (MELC) has been re-examined. It is demonstrated that sodium butyrate causes hyperacetylation of core histones in inducible as well as non-inducible MELC strains. This indicates that histone hyperacetylation per se is not sufficient to activate genes. However, [3H]acetate incorporation into core histones of the inducible MELC line F4N increases after induction of differentiation with dimethylsulfoxide (DMSO), in contrast to the non-inducible variant F4+. Thus histone acetylation may play a role as an auxiliary mechanism for gene activation (and inactivation). In addition, the appearance of a histone H3 variant during differentiation of MELC is reported.     

Parendozoicomonas haliclonae gen. nov. sp. nov. isolated from a marine sponge of the genus Haliclona and description of the family Endozoicomonadaceae fam. nov. comprising the genera Endozoicomonas,Parendozoicomonas, and Kistimonas

Two Gram-stain-negative, facultative anaerobic, motile, rod-shaped strains, S-B4-1U^T and JOB-63a, forming small whitish transparent colonies on marine agar, were isolated from a sponge of the genus Haliclona. The strains shared 99.7% 16S rRNA gene sequence identity and a DNA-DNA hybridization value of 100%, but were differentiated by genomic fingerprinting using rep-PCRs. 16S rRNA gene sequence phylogeny placed the strains as a sister branch to the monophyletic genus Endozoicomonas (Oceanospirillales; Gammaproteobacteria) with 92.3–94.3% 16S rRNA gene sequence similarity to Endozoicomonas spp., 91.9 and 92.1% to Candidatus Endonucleobacter bathymodiolin, and 91.9 to 92.1% to the type strains of Kistimonas spp. Core genome based phylogeny of strain S-B4-1U^T confirmed the phylogenetic placement. Major fatty acids were summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) and 8 (C_18:1 ω7c/C_18:1 ω6c) followed by C_10:0 3-OH, C_16:0, and C_18:0. The G + C content was 50.1–51.4 mol%. The peptidoglycan diamino acid of strain S-B4-1U^T was meso-diaminopimelic acid, the predominant polyamine spermidine, the major respiratory quinone ubiquinone Q-9; phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine were major polar lipids. Based on the clear phylogenetic distinction, the genus Parendozoicomonas gen. nov. is proposed, with Parendozoicomonas haliclonae sp. nov. as type species and strain S-B4-1U^T (= CCM 8713^T = DSM 103671^T = LMG 29769^T) as type strain and JOB-63a as a second strain of the species. Based on the 16S rRNA gene sequence phylogeny of the Oceanospirillales within the Gammaproteobacteria, the Endozoicomonaceae fam. nov. is proposed including the genera Endozoicomonas, Parendozoicomonas, and Kistimonas as well as the Candidatus genus Endonucleobacter.     

Fluctuations in S-adenosyl-L-methionine (AdoMet) during mitotic cycle of the myxomycete Physarum polycephalum

A sensitive radioisotope dilution method was used to measure the S-adenosyl-L-methionine (AdoMet) content in macroplasmodia of the slime mold Physarum polycephalum during the mitotic cycle. The AdoMet pool had two maxima, one during mitosis, the other in the middle of G2 phase.     

The murine bone marrow macrophage,a sensitive indicator cell for murine migration inhibitory factor and a new method for their harvest

Murine bone marrow macrophages grown on Teflon-coated petri dishes for a period of 8–16 days can be removed with a yield of 90–95% and a viability greater than 95% following incubation in 1 mM EDTA. Bone marrow cells cultured on Teflon-coated dishes did not differ in their replication rate, peroxidase and nonspecific esterase content, pinocytosis, secretion of lysozyme and neutral proteinases from bone marrow cells cultured on plastic dishes. Murine bone marrow macrophages were found to be sensitive indicator cells for mouse migration inhibitory factor (MIF). Large numbers of cells for the MIF assay can be obtained, since their yield is 10–15 times higher than the yield of oil-induced peritoneal exudate macrophages from the same number of mice.     

Oxygen dependence of nuclear DNA replication in Ehrlich ascites cells

Oxygen was excluded from cultured Ehrlich ascites cells for 5-7 h and then readmitted. During the anaerobic period and for about 1 h following reoxygenation the DNA synthesis of the cells was studied by determining the DNA synthesis rate from [3H] thymidine incorporation data, by evaluation of the thymidine (pulse labelling) index, by DNA fibre autoradiography, and by alkaline sucrose gradients in order to follow the maturation of the daughter chains. The DNA synthesis rate was found to decay to a few percent of the initial value within 5-7 h after deoxygenation. Immediately after reoxygenation it increased to exceed the control level within 0.5-1 h. The only partial process of the genome replication definitely responding to deoxygenation/reoxygenation was the initiation of new replicon units, while progress of the replication forks and maturation of the new daughter chains were not significantly affected. The coordination of replicon initiation within groups or clusters was maintained throughout. The interruption of replication at the level of initiation of clusters upon deoxygenation was interpreted as a regulatory response of the cells to ensure basic viability under unfavourable conditions.     

Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence. Cytoplasmic microtubules, vinblastine-induced paracrystals, and mitotic figures.

Antibody against tubulin from the outer doublets of sea urchin sperm flagella reacts with tubulin-containing structures in mammalian cells. Thus cytoplasmic microtubules, vinblastine-induced paracrystals and the full spectrum of mitotic figures can be visualized by immunofluorescence. These results show that the tubulin structure has been highly conserved during evolution.     

Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system

The electrical properties of the clonal muscle cell line L6 can be revealed by the measurement of ion fluxes. Under many circumstances, this technique provides a useful alternative to electro-physiology. In myoblasts, sodium uptake through voltage-dependent ionophores can be stimulated by veratridine and inhibited by tetrodotoxin. In myotubes which result from fusion of myoblasts, these voltage-dependent sodium channels appear to increase in number, paralleling the development of the action potential. Furthermore, in myotubes (but not myoblasts) carbamylcholine is able to stimulate a sodium influx through ionophores which are inhibitable by curare (dTC) but not tetrodotoxin (TTX). This demonstrates the presence of acetylcholine receptors on the fused cells. The cells also have a manganese-inhibitable calcium channel which appears to be voltage dependent and may be responsible for the calcium-dependent component of the action potential. Depolarizing concentrations of potassium in the medium stimulate calcium uptake both in the presence and absence of sodium. Veratridine and carbamylcholine also stimulate calcium influx, but both require the presence of sodium. This indicates that the depolarization necessary for opening the calcium channel is dependent upon sodium influx in these latter cases. Myoblasts and myotubes appear to have these channels in about equal numbers.     

Light inhibition of respiration is due to a dual function of the cytochrome b6-f complex and the plastocyanin/cytochrome c-553 pool in Aphanocapsa

Studies of the respiratory electron transport pathway in the blue-green alga, Aphanocapsa, demonstrated the presence of cytochrome oxidase and a cytochrome complex. The use of antimycin A showed only the occurrence of a plastidal type of cytochrome complex (the cytochrome b6-f complex), which is insensitive to this inhibitor. Determination of the extent of photooxidation of cytochromes c-553 and f-556 under conditions of high and low cytochrome oxidase activities indicated an electron flow through both cytochromes to cytochrome oxidase. Direct evidence for a common segment of photosynthetic and respiratory electron transport from plastoquinone via the cytochrome b6-f complex to the soluble plastocyanin/cytochrome c-553 pool, as well as a competition between cytochrome oxidase and Photosystem I for reductants in this pool in the light, was obtained by measurements of electron transport with suitable electron donors in this alga.     

Transport of mono- and divalent cations across chloroplast membranes mediated by the ionophore A23187

Effects of the ionophore A23187 on isolated broken and intact chloroplasts in the pH range of 6.2 to 7.6 have been studied. In both types of chloroplasts, uncoupling of photosynthetic electron transport by A23187 (6–10 μm) was mediated either by Mg²⁺ or—in the absence of divalent cations (i.e., when EDTA was added to the medium)—by high concentrations of Na⁺, but not of K⁺ ions. At increased concentrations of the ionophore (above about 10 μm) and high pH (7.2 to 7.6), uncoupling in broken chloroplasts was also mediated by K⁺ ions. The inhibition of the energy-dependent slow decline of chlorophyll fluorescence in intact chloroplasts by the ionophore (which denotes uncoupling) is reversed by EDTA in the presence of K⁺, but not of Na⁺ ions. In 3-(3′,4′-dichlorophenyl)1,1-dimethylurea-poisoned intact chloroplasts, the yield of variable chlorophyll fluorescence is lowered by A23187 + EDTA and increased again by addition of NaCl or KCl. Chlorophyll fluorescence spectra at 77 °K of intact chloroplasts incubated with A23187 + EDTA indicated that the distribution of excitation energy had changed in favor of photosystem I, as expected from a depletion of Mg²⁺. This change was reversed by MgCl²⁺, KCl, or NaCl. From a comparison of low-temperature fluorescence spectra of broken and intact chloroplasts at different levels of Mg²⁺ in the medium, the concentration of free Mg²⁺ in the stroma of the intact chloroplasts at pH 7.6 in the dark was estimated at 1 to 4 mm. The results show that in chloroplasts the specificity of A23187 for divalent cations is limited. In the presence of EDTA, the ionophore mediates fast $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange across thylakoid membranes, whereas K⁺ is transferred much less efficiently. Both Na⁺ and K⁺ ions seem to be transported readily across the chloroplast envelope by the action of the ionophore, leading to an exchange of Mg²⁺ for monovalent cations at the thylakoid membrane surfaces in intact chloroplasts.     

Biosynthesis of riboflavin 6,7-dimethyl-8-ribityllumazine 5′-phosphate is not a substrate for riboflavin synthase

Phosphotransferase from carrot is shown to catalyze the phosphorylation of 6,7-dimethyl-8-ribityllumazine specifically at position 5′ of the ribityl side chain. The lumazine 5′-phosphate is neither a substrate nor an inhibitor of riboflavin synthase from Bacillus subtilis and Escherichia coli. It follows that the obligatory product of riboflavin synthase is riboflavin and not FMN.     

Effects of long-term treatment of rats with antidepressants on adrenergic-receptor sensitivity in cerebral cortex: Structure activity study

The effects of 20 tricyclic and 12 chemically unrelated ‘atypical’ antidepressant drugs on the noradrenaline (NA) receptor coupled adenylate cyclase system were investigated in slices of the rat cerebral cortex. While no changes occurred after a single dose, 14 tricyclic compounds down-regulated the receptor system when administered for 9 days. Six tricyclic antidepressants (trimipramine, butriptyline, noxiptyline, doxepine, dosulepine, propizepine) failed to desensitize the NA sensitive adenylate cyclase although some were potent inhibitors of the neuronal uptake of NA. Using the two optically active enantiomers of oxaprotiline inhibition of NA uptake was observed with the (+)-enantiomer while the (?)-enantiomer had only weak inhibitory potency. However, in contrast to published data, both enantiomers and the racemate administered at 30 mg/kg for 9 days did down-regulate the NA receptor coupled adenylate cyclase. Therefore, the experiments were repeated with Sprague-Dawley rats from a different supplier. Now, data published earlier were reproducible, only the racemate and the (+)-enantiomer of oxaprotiline being significantly active on the desensitization of the NA sensitive adenylate cyclase. Using F-344, Long Evans and Wistar rats significant differences were found in the response of the adrenoceptor coupled adenylate cyclase to a 9 day treatment with 30 mg/kg imipramine. Although some of the atypical antidepressants are potent inhibitors of the biogenic amine uptake systems none of these compounds lead to statistically significant changes of the NA stimulated cAMP formation after a 9-day treatment period. Only with the NA uptake inhibitor tandamine and with the serotonin uptake inhibitors zimelidine and fluoxetine a trend toward adrenergic down-regulation was recognized. Using enantiomers of mianserin only the (?)-isomer which is a poor NA uptake inhibitor, was slightly active. It thus appears that the therapeutic action of antidepressant drugs cannot generally be related to postsynaptic adaptive changes in the sensitivity of the noradrenergic adenylate cyclase receptor system. Variabilities in pharmacokinetics and in NA sensitivity of the cAMP generating system in various rat strains and possibly in different animal species may be important factors determining whether β-receptor down-regulation will occur during chronic treatment with antidepressant drugs.     

Morphogenesis of bacteriophage lambda deletion mutants. I. Abnormal head-related structures produced in normal Escherichia coli.

Late in the morphogenesis of bacteriophage lambda, DNA condenses into the nascent head and is cut from a concatemeric replicative intermediate by a nucleolytic function, Ter, acting at specific sites, called cos. As a result of this process, heads of lambda deletion mutants contain less DNA than those of the wild-type phage. It has been reported that phage with very large deletions (22% of the genome or more) grow poorly but that normal growth can be restored by the non-specific addition of DNA to the genome. This finding implies that DNA content may exert a physical effect on some stage of head assembly.We have investigated the effects of two long deletions, b221 and tdel33, on head assembly. Bacteria infected with the mutants were lysed with non-ionic detergent under conditions favoring stabilization of labile structures containing condensed DNA. It has proved possible to isolate two aberrant head-related structures produced by the deletion mutants. One of these (“overfilled heads”) contains DNA which is longer than the deletion mutant genome and is about the same size as that found in wild-type heads. These structures appear to be unable to attach tails. The second type of structure (“incompletely filled heads”) contains a short piece of DNA, 40% of the length of the mutant genome. The incompletely filled heads are found both with and without attached tails. Both of these abnormal structures are initially attached to the replicating DNA but are released by treatment with DNAase. The nature of these abnormal structures indicates that very small genomes affect a late stage of head morphogenesis, after the DNA is complexed with a capsid of normal size. The results presented suggest that underfilling of the capsid interferes with the ability of the Ter function to properly cleave cos.