Specificity of leaf mitochondrial and chloroplast processing systems for nuclear-encoded precursor proteins

The specificity of the mitochondrial and chloroplast processing enzymes for the nuclear-encoded precursor proteins was investigated. Mitochondrial precursor proteins of the Nicotiana plumbaginifolia and the Neurospora crassa subunits of F₁-ATPase and the Neurospora Rieske FeS precursor protein were processed to the correct mature size by matrix extracts isolated from spinach leaves, yeast, rat liver and beef heart. The mitochondrial extracts failed to process chloroplast precursor proteins of the stromal small subunit of ribulose 1,5-bisphosphate carboxylase and the thylakoid 33 kDa protein of the oxygen-evolving complex. Both mitochondrial F₁ precursors were specifically processed by a soluble stromal extract from chloroplasts. However, no processing of the Rieske FeS precursor protein was observed under the same conditions with the chloroplast extract. The cleavage of the mitochondrial F₁ precursors by the chloroplast extract was shown to be sensitive to the metal chelators EDTA and ortho-phenanthroline. The cleavage site of the mitochondrial F₁ precursor by the chloroplast soluble extract appears to be located at the N-terminus.Abbreviations ATPase adenosine triphosphatase - Rieske FeS non-heme iron sulphur protein of the ubiquinol cytochrome c oxidoreductase complex - Rubisco ribulose 1,5-bisphosphate carboxygenase/oxygenase - RMSF phenylmethylsulphonylfluoride - EDTA ethylenediaminetetraacetic acid     

Chloride permeability in human red cells: Influence of membrane protein rearrangement resulting from ATP depletion and calcium accumulation

Summary As 15% of band 3 protein, the assumed chloride channel, is associated with spectrin, the major peripheral protein of a lattice located at the red cell membrane-cytosol interface, the present study was undertaken to evaluate whether a rearrangement of the lattice modifies the functional property of band 3 protein. Such a rearrangement was modulated by depletion of cell ATP and/or by accumulation of Ca²⁺ ions within the cell.ATP depletion induces an inhibition of the electroneutral one-for-one chloride exchanges. Neither the modification of red cell morphology due to ATP depletion (discocyte-echinocyte transformation) nor a direct effect of the decrease in internal ATP level can account for this inhibition. On the other hand, it seems reasonable to consider that inhibition is related to the changes in membrane protein organization (formation of heteropolymers) induced by the decrease in ATP level. But it does not appear that the degree of inhibition is modified when this altered assembly of membrane protein is stabilized by disulfide linkages.Accumulation of Ca²⁺ ions in the cell at a relatively low concentration (10m range) inhibits chloride exchange without apparent modification of the assembly of membrane proteins. This effect of calcium on chloride exchanges is speculatively denoted as a direct effect of calcium.Calcium loading of fresh red cells at higher concentrations (500 to 1000 m) obtained by use of the ionophore A23187 induces a very strong inhibition of chloride exchanges. In this case, inhibition can be reasonably accounted for by two simultaneous effects of calcium: a direct effect which explains half of the inhibition and an indirect effect due to the formation of membrane protein complexes stabilized by covalent crosslinkages (activation by Ca²⁺ ions of a transglutaminase).It is interesting to note that intracellular calcium, whatever the level, inhibits electroneutral exchanges of chloride but increases net chloride movements.     

Passive ionic properties of frog retinal pigment epithelium

Summary The isolated pigment epithelium and choroid of frog was mounted in a chamber so that the apical surfaces of the epithelial cells and the choroid were exposed to separate solutions. The apical membrane of these cells was penetrated with microelectrodes and the mean apical membrane potential was –88 mV. The basal membrane potential was depolarized by the amount of the transepithelial potential (8–20mV). Changes in apical and basal cell membrane voltage were produced by changing ion concentrations on one or both sides of the tissue. Although these voltage changes were altered by shunting and changes in membrane resistance, it was possible to estimate apical and basal cell membrane and shunt resistance, and the relative ionic conductanceT _i of each membrane. For the apical membrane:T _K0.52,T _{HCO 3}=0.39 andT _Na=0.05, and its specific resistance was estimated to be 6000–7000 cm². From the basalT _K=0.90 and its specific resistance was estimated to be 400–1200 cm². From the basal potassium voltage responses the intracellular potassium concentration was estimated at 110mm. The shunt resistance consisted of two pathways: a paracellular one, due to the junctional complexes and another, around the edge of the tissue, due to the imperfect nature of the mechanical seal. In well-sealed tissues, the specific resistance of the shunt was about ten times the apical plus basal membrane specific resistances. This epithelium, therefore, should be considered tight. The shunt pathway did not distinguish between anions (HCO₃ ^–, Cl^–, methylsulfate, isethionate) but did distinguish between Na⁺ and K⁺.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Hydrogen isotope discrimination in higher plants: Correlations with photosynthetic pathway and environment

Summary The ratio of deuterium to hydrogen (expressed as D) in hydrogen released as water during the combustion of dried plant material was examined. The D value (metabolic hydrogen) determined on plant materials grown under controlled conditions is correlated with pathways of photosynthetic carbon metabolism. C₃ plants show mean D values of-132 for shoots and -117 for roots; C₄ plants show mean D values of -91 for shoots and-77 for roots and CAM plants a D value of-75 for roots and shoots. The difference between the D value of shoot material from C₃ and C₄ plants was confirmed in species growing under a range of glasshouse conditions. This difference in D value between C₃ and C₄ species does not appear to be due to differences in the D value (tissue water) in the plants as a result of physical fractionation of hydrogen isotopes during transpiration. In C₃ and C₄ plants the hydrogen isotope discrimination is in the same direction as the carbon isotope discrimination and factors contributing to the difference in D values are discussed. In CAM plants grown in the laboratory or collected from the field D values range from-75 to +50 and are correlated with ¹³C values. When deprived of water, the D value (metabolic hydrogen) in both soluble and insoluble material in leaves of Kalanchoe daigremontiana Hamet et Perr., becomes less negative. These changes may reflect the deuterium enrichment of tissue water during transpiration, or in field conditions, may reflect the different D value of available water in areas of increasing aridity. Whatever the origin of the variable D value in CAM plants, this parameter may be a useful index of the water relations of these plants under natural conditions.     

Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.     

Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis

Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain -, -, and -zein and -, -, and -coixin. The -coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa -zeins. Like the -zeins, the C1 and C2 -coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to -coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of -zein and represents 15% of the total coixin. The -zein fraction was composed of a major 17 kDa protein band, while the -coixin fraction consisted of a mixture of - and -coixins.Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa -zein, as did C4 and C5 antisera. The antiserum against -coixin showed strong cross-reaction with -zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa -zeins as well as the 28 and 16 kDa -zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa -zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone. The cDNA clone representing the 28 kDa -zein cross-hybridized with only one band of Coix genomic DNA, in contrast to the three bands observed in maize. This same Coix sequence also cross-hybridized with the cDNA clone representing the 16 kDa -zein. The relevance of these findings are discussed in the context of the origin of zein and coixin genes.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.     

Structure and physical chemistry of membranes

Summary With the concept of the unit membrane as a central theme, a number of current problems for the theory of membrane structures are discussed from the point of view of the physical chemist. In view of the wide variety of chemical compositions of membranes, some current opinions of the forces maintaining the integrity of membranes are of limited generality. The thermodynamic status of the membrane is discussed, and it is suggested that the new small-systems thermodynamics can be usefully employed as a conceptual basis for discussing fluctuations and phase-changes in membranes. Arguments are given to suggest that the lipid interior of membranes is more ordered and crystalline than is currently supposed. The role of water in membrane structures remains an enigma.     

Effect of busulphan treatment and elevated temperature on the expression of the β-pol gene in rat testis

Changes in the expression pattern of the DNA polymerase gene during inhibition of spermatogenesis by busulphan and by temperature (artificial cryptorchidism) have been studied. Transient arrest of spermatogenesis in two-month-old rats after injection of a single dose of busulphan (10 mg/kg) resulted in parallel but transient decrease in the 1.4 kb of -pol mRNA level to an undetectable value, followed by its reappearance after resumption of spermatogenesis. An artificial cryptorchidism also caused a drastic decrease of -pol mRNA level. Both results as well as morphological examination of testis after busulphan injection and artificial cryptochidism revealed that spermatocytes and spermatids represent the testicular cell fraction containing the elevated amount of -pol mRNA. Involvement of DNA polymerase in meiotic recombination is discussed.     

Δ5 Desaturase activity in rat kidney microsomes

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-¹⁴C]eicosa-8,11,14trienoic acid (20:3n-6), was separated from the product [1-¹⁴C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 M. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of 5 desaturase were 56 M and 60 pmoles·min^–1·mg^–1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.     

Pretraining but not Preexposure to the Task Apparatus Prevents the Memory Impairment Induced by Blockade of Protein Synthesis,PKA or MAP Kinase in Rats

Adult male Wistar rats were trained and tested in a step-down inhibitory avoidance task (0.4 mA footshock, 24 h training-test interval). Fifteen minutes before or 0, 1.5 or 3 hours after training, animals received a 0.8 l intrahippocampal infusion of the protein synthesis inhibitor anisomycin (80 g), the PKA inhibitor Rp-cAMP (0.05 g), the MAPK kinase inhibitor PD 098059 (50 M solution) or vehicle (phosphate buffer in saline, pH 7.4). Anisomycin, Rp-cAMP and PD 098059 impaired retention test performance in animals injected at different times, prior and after training. Pretraining with a low footshock intensity (0.2 mA) 24 h before training prevented the amnestic effect of all drugs studied. However, simple preexposure to the inhibitory avoidance apparatus did not alter the amnestic effects of all drugs. The results suggest that memory processing requires hippocampal mechanisms dependent on protein synthesis, PKA and MAPK kinase at different times after training. These findings suggest that weak training must be sufficient to produce some lasting cellular expression of the experience so that the enhancement of consolidation of a previously acquired memory is not dependent on protein synthesis, PKA or MAPK.     

Stimulation of proliferation and immunoglobulin production of human-human hybridoma by various types of caseins and their protease digests

We have studied the effect of such milk proteins as caseins, lactalbumin, and lactoglobulin, on proliferation and immunoglobulin production of human-human hybridoma HB4C5 cells. It was found that -, -, and -caseins stimulated both proliferation and IgM product ion of human-human hybridoma HB4C5 cells, while the activities of -lactalbumin and -lactoglobulin were negligible. To localize the active sites of these caseins, effect of protease treatments on the activities were examined. When caseins were digested with trypsin, casein digests stimulated proliferation of the hybridoma, but not their IgM production. When -casein was digested with chymosin and fractionated top--casein and glycomacropeptide, both fragments stimulated proliferation of the cells, but onlyp--casein fragment stimulated IgM production. These results indicate that -casein has at least two proliferation stimulating sites and an IgM production stimulating site in thep--casein region.     

Protein compositional analysis of inclusion bodies produced in recombinant Escherichia coli

Summary Culture conditions favouring the simulataneous formation of soluble protein and inclusion bodies (IBs) were chosen for producing the cytoplasmic protein -galactosidase or the periplasmic protein TEM--lactomase. Soluble and insoluble cell fractions of Escherichia coli producing either -galactosidase or TEM--lactomase were analyzed by one- and two-dimensional gel electrophoresis and subsequent silver staining or immunodection of the recombinant protein. The results show that truncated fragments of the recombinant protein were not present in the soluble cell fraction but accumulate in the IB fraction. The presense of other cellular, non-plasmid-encoded proteins in IB preparations such as the outer membrane proteins OmpF, OmpC, and OmpA or the ribosomal subunit proteins L7/L12 was attributed to co-precipitation of cell-debris-associated components. Protein-folding enzymes were not detected in IB preprations. The specificity of in-vivo protein association in the formation of IBs and its implication on protein purification is discussed. Correspondence to: J. E. Bailey     

Human platelet basic protein/connective tissue activating peptide-III maps in a gene cluster on chromosome 4q12–q13 along with other genes of the β-thromboglobulin superfamily

Summary Pro-platelet basic protein (pro-PBP) is the precursor of the two platelet -granule proteins, PBP and connective tissue activating peptide-III. Upon platelet activations they are released and further processed in plasma to -thromboglobulin and neutrophil-activating peptide-2. The gene encoding pro-PBP is mapped in this study to chromosome 4q12–q13. At least four other members of this family of small inducible cytokines, including NAP-1/Il-8 and platelet factor 4, reside within the same locus, indicating a gene cluster for the -thromboglobulin family.     

Structure et fonctionnement des organes de Kölliker chez les jeunes octopodes (Mollusca,Cephalopoda)

Summary The present study is a reinvestigation of the peculiar integumental differentiations described for the first time by Kölliker in 1844. These transitory organs of microscopic size are composed of two parts; one arises from the ectoderm and forms a sac adhering to the epidermis and enclosing a bundle of chitinous setae that are secreted by the microvilli of a specialized basal cell; the other, arising from the mesoderm, is a muscular apparatus that allows evagination and spreading of the bundle. This functioning was observed in fresh skin preparations of newly-hatched Eledone moschata. The Kölliker organs are present in the young of all ineirrate octopods known so far, except for two species of Octopus (O. briareus, O. maya).

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Abréviations C chromatophore - CB cellule basale - CG cellule glandulaire - CM cellule murale - CS cloison sous-cutanée - D dictyosome - E épiderme - F faisceau de soies - FC fibres conjonctives - FP fibre profonde - G golgi - I interdigitations - K organe de Kölliker - M muscle - MB membrane basale - MV microvillus - N noyau - NU nucléole - R réticulum endoplasmique - S soies du faisceau - VS vaisseau sanguin     

Light adaptation of bacteriorhodopsin in the presence of valinomycin and potassium. pH-dependence

In the presence of valinomycin and K⁺, bacteriorhodopsin undergoes (i) a decrease of its maximum absorbance, (ii) a blue shift of the maximum wavelength of both the light and the dark adapted forms. However (iii) a normal light adaptation is maintained and (iv) the retinal-retinal interactions are not perturbed. The role of valinomycin as a K⁺-carrier allowing a H⁺-K⁺ competition as well as the stabilization of the deprotonated Schiff-base (linking retinal to the apo-opsin) is shown and discussed.Abbreviations bR bacteriorhodopsin - CD circular dichroism - DA dark-adapted - LA light-adapted - M-412 Meta-intermediate of the bacteriorhodopsin photocycle     

Coupling Chemical Energy by the hsp70/tim44 Complex to Drive Protein Translocation into Mitochondria

A dynamic complex between the mitochondrial cognate of hsp70 (mthsp70) and the inner membrane protein tim44 couples energy derived from ATP hydrolysis to drive multiple steps in the mitochondrial protein import pathway: (1) The dependent import step and the mthsp70/tim44 complex cooperate to facilitate the unidirectional transfer of the mitochondrial targeting signal across the inner membrane. (2) The mthsp70/tim44 complex helps to unfold domains on precursors proteins that arrive at the import apparatus in a folded conformation on the cis side of the outer membrane. (3) Completion of import is then driven by the mthsp70/tim44 complex in a manner that is independent of . Mechanisms proposed to explain how the mthsp70/tim44 complex harvests chemical energy to drive these aspects of the import process are discussed.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field-induced absorption changes of bacteriorhodopsin were studied with different samples of purple membranes which were prepared as randomly oriented and electrically oriented films of purple as well as cation-depleted blue bacteriorhodopsin. The absorption changes were proportional to the square of the field strength up to 300 kV/cm. The electric field from the intracellular side to the extracellular side of the purple bacteriorhodopsin induces a spectrum change, resulting in a spectrum similar to that of the cation-depleted blue bacteriorhodopsin. When the field was removed, the purple state was regenerated. The blue state was mainly affected by an electric field in the opposite direction, suggesting a reversible interaction with the Schiff's base bond of the retinal. Since the field-induced reaction of bacteriorhodopsin was observed in the presence of a concomitant steady ion flux, it is assumed that the generation of a local diffusion potential may play an important role in these spectral reactions. Although the fragments were fixed in the dried film, electric dichroism was observed. The dichroic contribution of the total absorbance change was about 15%. The angular displacement of the retinal transition moment was calculated to be 1.5° toward the membrane normal.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.