Use of ATP bioluminescence measurements for the estimation of biomass during biological humification

Summary The development of the microflora during the humification of grape pulp has been investigated by the determination of ATP using the bioluminescence technique. Several extraction methods were tested including the use of dimethylsulphoxide, trichloroacetic acid, grinding and ultrasonification. Dimethylsulphoxide and ultrasonification for 15 sec appeared to be the most effective. The ATP extract was stabilized when it was mixed with 0.75 mM glycine, 4.4 mM Mg-EDTA, pH 7.5 and frozen. The relative error of the ATP assay by bioluminescence did not exceed 6.5%. This method allowed us to show that at least five distinct reproducible microbial phases exist during grape pulp humification. These results show that the microbial biomass changes noticeably and at distinct times during composting.     

Ultrastructure d'une cyanophycée aérienne calcifiée cavernicole: Geitleria calcarea Friedmann

Résumé L'ultrastructure de Geitleria calcarea Friedmann, cyanophycée filamenteuse aérienne, calcifiée et cavernicole, rarement signalée, est étudiée ici pour la première fois. La cytologie de cet organisme présente des originalités. Huit enveloppes cellulaires concentriques sont dénombrées: membrane plasmique, L I, L II, L III, L IV, enveloppe pariétale, gaine fibreuse et gaine carbonatée. La dernière est décrite à l'aide du M.E.B. Les thylacoïdes très nombreux ont une forme extrêmement contournée. Ils supportent des phycobilisomes cylindriques alternant avec des polysaccharides de même allure. L'organisation des synapses est détaillée. L'élongation est apicale et le mode de ramification est soit latéral, soit dichotomique.

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The ultrastructure of the atmophytic lime-encrusted filamentous blue-green alga Geitleria calcarea Friedmann, which grows in caves, is studied here for the first time. The cytology of this organism presents original features concerning the cellular sheath, envelopes, wall, thylacoïds and pit connections. Its filament growth is apical and its type of branching is lateral and dichotomous.

     

Comparison of basic peptides- and lipid-based strategies for the delivery of splice correcting oligonucleotides

Expression of alternatively spliced mRNA variants at specific stages of development or in specific cells and tissues contributes to the functional diversity of the human genome. Aberrations in alternative splicing were found as a cause or a contributing factor to the development, progression, or maintenance of numerous diseases. The use of antisense oligonucleotides (ON) to modify aberrant expression patterns of alternatively spliced mRNAs is a novel means of potentially controlling such diseases. Oligonucleotides can be designed to repair genetic mutations, to modify genomic sequences in order to compensate for gene deletions, or to modify RNA processing in order to improve the effects of the underlying gene mutation. Steric block ON approach have proven to be effective in experimental model for various diseases. Here, we describe our experience in investigating two strategies for ON delivery: ON conjugation with basic peptides and lipid-based particulate system (lipoplex). Basic peptides or Cell Penetrating Peptides (CPP) such as the TAT-derived peptide appear to circumvent many problems associated with ON and drug delivery. This strategy may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease. Lipoplexes result from the intimate interaction of ON with cationic lipids leading to ON carrying particles able to be taken up by cells and to release ON in the cytoplasm. We have used as an experimental model the correction of a splicing alteration of the mutated β-globin intron causing thalassemia. Data on cell penetration and efficacy of correction of specific steric block ON delivered either by basic peptides or lipoplex are described. A comparison of the properties of both delivery systems is made respective to the use of this new class of therapeutic molecules.     

The differential effects of the carcinogen dimethylnitrosamine on isocitrate and 6-phosphogluconate metabolism in single intact cells

A microspectrofluorimetric study is made of the influence of dimethylnitrosamine on NADP reduction, following sequential microinjections into the same L cell, of two substrates: (1) isocitrate, with activity of isocitrate dehydrogenase both in the extramitochondrial and intramitochondrial compartments, (2) 6-phosphogluconate, with activity of the dehydrogenase in the extramitochondrial compartment. In control L cells a two-step reduction of NAD(P) is obtained followed by relatively slow reoxidation. In the minutes which follow addition of carcinogen, e.g., dimethylnitrosamine, to the cell medium the isocitrate and 6-phosphogluconate-induced transient NADP reoxidation is decreased in magnitude compared to control, while the rate constant of NADPH reoxidation is considerably accelerated, possibly due to requirements at the level of the microsomal metabolizing system. Observations within the first hour of carcinogen addition suggest an interesting system for evaluating the immediate actions of carcinogens at extranuclear sites: i.e., a comparative study of NADP reduction-reoxidation rate constants via injection of substrates for extra- vs. intramitochondrial pathways.     

Photoaffinity spin-labeling of the Ca2+ ATPase in sarcoplasmic reticulum : Evidence for oligomeric structure

A spin labeled fatty acid (16-doxylstearic acid) was linked to a photochemical reacting group (azido derivative). When the molecule is introduced, at a low concentration, into rabbit sarcoplasmic reticulum membranes, the spectrum before illumination is identical to the spectrum obtained with the corresponding spin labeled fatty acid. After illumination, a large immobilized components is seen. It corresponds to about 70% of the ESR signal of the effectively bound label, at room temperature. The fraction of immobilized component varies with temperature, from 100% at 0°C to 50% at 35°C. Addition of a small amount of detergent (dodecyl octaethylene glycol monoether), under non solubilizing conditions, decreases the fraction of signal due to a strongly immobilized probe. A possible interpretation is that the immobilized signal reflects protein bound spin labels trapped in Ca²⁺ ATPase oligomers, which are partially dissociated by detergent addition or temperature increase.     

Production of gallic acid from tara tannin by bacterial strains

Summary Bacterial strains identified as Klebsiella pneumonia and Corynebacterium sp. degraded in few hours a great part of pure tara (Caesalpinia spinosa) tannin or crude water extract of tara pods powder. Large amounts of gallic acid were recovered during some experiments reaching 55 % of the theoretical yield in the best case. The feasibility of gallic acid production by a biotechnological process is discussed.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.     

Distribution and microhabitats of native and non-native gammarids (Amphipoda, Crustacea) in Brittany, with particular reference to the endangered endemic sub-species Gammarus duebeni celticus

Aim  To assess temporal changes in gammarid distribution in Brittany and microhabitat-use overlap between the endangered endemic Gammarus duebeni celticus Stock & Pinkster, 1970 , the expanding natives G. pulex (Linnaeus, 1758) and Echinogammarus berilloni (Catta, 1878), and the introduced G. tigrinus Sexton, 1939.
Location  Brittany and adjacent regions in western France.
Methods  The spatial and temporal patterns in distribution of gammarids at the scale of Brittany were studied using 351 sites. Longitudinal distributions (from the source to the estuary of the river) and microhabitat-use (substratum type and water velocity) were also considered in selected rivers.
Results  At the regional scale, all species occurred together less often than expected statistically, with significant deviations from expected for G. pulex vs. both G. duebeni celticus and G. tigrinus , and for E. berilloni vs. both G. duebeni celticus and G. tigrinus . However, at the microhabitat scale, E. berilloni occurred significantly more often than expected with the endemic G. duebeni celticus , and this appears to be due to similar substratum and water velocity preferences, although at both the regional and microhabitat scales E. berilloni prefers wider streams than G. duebeni celticus . This study reveals a decline in the endangered G. duebeni celticus since 1970.
Main conclusions  The longitudinal and local distributions of G. duebeni celticus , and the higher-than-expected co-occurrence of the species with G. pulex , suggest that the decline of the endemic species may be due to changes in the environment and/or interference from native G. pulex , which is expanding its range in Brittany. The results are discussed as regards to the consequences for regional biodiversity.     

The N-terminal domain I of human lactotransferrin binds specifically to phytohemagglutinin-stimulated peripheral blood human lymphocyte receptors

Human lactotransferrin receptors have been recently characterized on mitogen-stimulated human lymphocytes [(1989) Eur. J. Biochem. 179, 481-487]. In order to define the lactotransferrin recognition site by these receptors, the binding to lymphocytes of several tryptic fragments, isolated from human lactotransferrin by mild tryptic hydrolysis [(1984) Biochim. Biophys. Acta 787, 90-96], has been investigated. The 30 kDa N-tryptic fragment (residues 4-281) and the re-associated N,C-tryptic complex bind to lactotansferrin lymphocyte receptor with a dissociation constant of 44 nM and 39 nM, respectively, similar to the value obtained for the native lactotransferrin (Kd = 46 nM). However, neither the N-terminal domain II (residues 91-257) nor the 50 kDa C-tryptic fragment (residues 282-703) are recognized. These results suggest that the binding site of human lactotransferrin by the lymphocyte receptor is located in the N-terminal lobe and more precisely in the N-terminal domain I (residues 4-90 and/or 258-281).