Protective effects of differentiation inducers on natural killer sensitivity of K 562 cells: Analysis at a single-cell level

K 562 cells induced to differentiate by sodium butyrate (SB) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were studied for their capacities to be bound and killed by large granular lymphocytes (LGL) in a single-cell cytotoxicity assay in agarose. After SB treatment, K 562 cells were less efficient in binding to LGL, whereas the frequency of killer cells among bound LGL was unaffected. When TPA was used to induce K 562 differentiation, the binding of LGL to their target and the lytic efficiency of the bound LGL were both diminished when compared to control K 562 cells. It has been demonstrated that the expression of structures involved in the binding of natural killer (NK) effectors to their targets could be correlated with the target-differentiation stage. It is shown that phorbol-ester treatment can also affect NK target structures involved in the killing step.     

Importance deVernonia guineensis Benth. dans l'alimentation de quelques Fourmis de savane

Résumé La ComposéeVernonia guineensis Benth., abondante dans les savanes préforestières de Côte d'Ivoire, fleurit quelques semaines après le passage des feux de brousse. Plusieurs espèces de Fourmis sont récoltées régulièrement sur la plante: ce sont surtoutCamponotus acvapimensis et unCremastogaster du genreAcroclia.Les Fourmis se nourrissent de sécrétions sucrées qui suintent au niveau des bractées des jeunes capitules, mais pratiquent également l'élevage d'Homoptères.Divers types de constructions sont décrits et, en particulier, les loges édifiées parC. acvapimensis pour abriter un Jassidæ:Selenocephalus sp.L'influence de l'environnement végétal sur la faune myrmécologique exploitantV. guineensis est mise en évidence.

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Summary Vernonia guineensis Benth. (Compositæ). common in the forest-savanna mosaic of Ivory Coast, blooms a few weeks after the fires. Several species of Ants are collected then on the forb; especiallyCamponotus acvapimensis and a species ofCremastogaster (Acroclia).These ants feed on seepings from the young flower heads, and search for exsudates of Homopterans.Various buildings are described, particularly shelters made byC. acvapimensis for the leaf—hopperSelenocephalus sp.Influence of botanical environment on the ants exploitingV. guineensis is shown.

     

Fluorescence polarization of polypeptides having a fluorescent endgroup

The polarization of fluorescence of a chromophore chemically bound on the NH₂ end of a poly(L -benzyl glutamate) molecule has been studied as a function of temperature, viscosity, and solvent. The relaxation times depend on both the overall rotation of the helix and the local rotation of the endgroup. In m–cresol the endgroup is rigidly bound and the rotational diffusion constant of the molecule is in good agreement with the values obtained by Kerr effect and dielectric relaxation. In other helicogenic solvents (DMF, DCE, etc.) the local rotation is nearly free. In m-cresol-DMF mixtures a sharp decrease of the polarization around a composition of 40% DMF can be interpreted as a change in the freedom of rotation of the endgroup. No discontinuity in the optical rotation is observed in the solvent mixture. The question of how a rapidly rotating endgroup could show an extrinsic Cotton effect as observed by Bloutand Yamaoka for the system Acridine Orange–PBLG in chloroform is then raised. Polarization of fluorescence measurements on this system show a nearly complete freedom of rotation of the dye and OH D measurements show no detectable Cotton effect in the dye absorption band.     

Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori _fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described.     

Adaptive surface antigen variation in Mycoplasma bovis to the host immune response

Abstract The variability of predominant Mycoplasma bovis surface antigens in the presence of specific immune pressure was analyzed in an in vitro assay to determine if M. bovis could escape immune destruction. We have shown that serum antibodies from immunized or experimentally infected calves and monoclonal antibodies which specifically react with previously characterized or as yet undefined major M. bovis membrane surface proteins cause repression of expression or shortening of the target protein, or induce switching to expression of an antigenically distinct variant protein. We have further demonstrated that removal of the inducing antibody results in reversion to the original phenotype. These results suggest that the level of expression and the length of M. bovis surface antigens in the host is modulated by cognate antibodies. According to the surface antigenic variation systems, random selection of preexisting variants resistant to antibody-mediated inhibition or direct regulation of gene expression may be means by which this organism evades host immune defences.     

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

Dynamics of single-stranded DNA in polyacrylamide gels during pulsed field gel electrophoresis. A birefringence study

The study of the orientation of single-stranded DNA in polyacrylamide gels in denaturing conditions has been undertaken by electric birefringence in order to determine the mechanism involved in the electrophoretic transport. The presence of an overshoot in the birefringence signal, when applying the electric field, and the study of the influences of the electric field and of the gel concentration on the dynamics show that a mechanism of reptation with elongation of the molecule occurs in polyacrylamide gels with low T values. Therefore it is suggested that the use of pulsed fields in sequencing electrophoresis is possible and can lead to a large increase of the length of the fragments that can be sequenced in one single run.     

Developmental and pathogen-induced activation of an msr gene,str246C,from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5′ flanking DNA sequence from the str246C gene fused to the β-glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5′ deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.