Photosynthetic efficiency and survival of Dactylis glomerata and Lolium perenne following low temperature stress

Resistance to low temperature is crucial for overwintering crops. In this work we compared the resistance to low temperature treatment of some varieties of two forage grass species Dactylis glomerata L. and Lolium perenne L. in order to elucidate the reason for the better resistance found in some species. The variety Amila of D. glomerata and Diament of L. perenne were more tolerant to low temperature stress during the emergence and tillering phases as compared to the varieties Amera and Gagat. The improved tolerance and ability for recovery after stress were associated with the better recovery of photosynthetic efficiency of these varieties and better survival of their shoots after low temperature stress.     

Resistance to low temperature photoinhibition is not associated with isolated thylakoid membranes of winter rye

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5°C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20°C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [¹⁴C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.     

Vaccinia Virus Protein Complex F12/E2 Interacts with Kinesin Light Chain Isoform 2 to Engage the Kinesin-1 Motor Complex

During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.     

Second-messenger regulation of receptor association with clathrin-coated pits: a novel and selective mechanism in the control of CD4 endocytosis.

CD4, a member of the immunoglobulin superfamily, is not only expressed in T4 helper lymphocytes but also in myeloid cells. Receptor-mediated endocytosis plays a crucial role in the regulation of surface expression of adhesion molecules such as CD4. In T lymphocytes p56lck, a CD4-associated tyrosine kinase, prevents CD4 internalization, but in myeloid cells p56lck is not expressed and CD4 is constitutively internalized. In this study, we have investigated the role of cyclic AMP (cAMP) in the regulation of CD4 endocytosis in the myeloid cell line HL-60. Elevations of cellular cAMP were elicited by 1) cholera toxin, 2) pertussis toxin, 3) forskolin and IBMX, 4) NaF, or 5) the physiological receptor agonist prostaglandin E1. All five interventions led to an inhibition of CD4 internalization. Increased cAMP levels did not inhibit endocytosis per se, because internalization of insulin receptors and transferrin receptors and fluid phase endocytosis were either unchanged or slightly enhanced. The mechanism of cAMP inhibition was further analyzed at the ultrastructural level. CD4 internalization, followed either by quantitative electron microscopy autoradiography or by immunogold labeling, showed a rapid and temperature-dependent association of CD4 with clathrin-coated pits in control cells. This association was markedly inhibited in cells with elevated cAMP levels. Thus these findings suggest a second-messenger regulation of CD4 internalization through an inhibition of CD4 association with clathrin-coated pits in p56lck-negative cells.     

Structural basis for the phototoxicity of the fluorescent protein KillerRed

The red fluorescent protein KillerRed, engineered from the hydrozoan chromoprotein anm2CP, has been reported to induce strong cytotoxicity through the chromophore assisted light inactivation (CALI) effect. Here, we present the X-ray structures of KillerRed in its native and bleached states. A long water-filled channel is revealed, connecting the methylene bridge of the chromophore to the solvent. This channel facilitates the transit of oxygen and of reactive oxygen species (ROS) formed by reaction with the excited chromophore. The functional roles of key mutations used to produce KillerRed are discussed, strong chromophore distortions in the bleached state are revealed, and mechanisms for ROS production and self protection are proposed. The presence of a partially mature, photo-resistant, green-emitting state is characterized, which accounts for enhanced CALI by “pre-bleached” KillerRed.     

Evaluation of a method for detection of cells with reduced drug retention in solid tumours.

A method for detection of cells with reduced drug retention was evaluated in solid tumours. After a 1 h incubation with daunorubicin (DNR), the right angle scatter (RAS), forward angle scatter (FAS), and specific fluorescence (Fluo) were measured in sensitive and resistant cells; only Fluo was related qualitatively, but not quantitatively, to resistance. Various incubation conditions were examined. When the pH of the incubation medium increased, the DNR retention increased in sensitive and resistant cells. In contrast, when the cell concentration increased, the DNR retention decreased. Using sensitive and resistant cell lines, a proportion of resistant cells lower than 10% can be detected in a mixture. To analyse cells from solid tumours, the cells were dissociated by repeated fine needle aspirations. Tumours from 22 patients have been processed with this technique; 8 samples were classified as S (sensitive); 2 as R (resistant); and 12 as I (intermediate). Further experiments were run to study and improve the method. Another method of detection of dead cells was tested. The intra-assay variability of the technique was found to be less than 10%. When the study was performed with different fragments of the same tumour, the variation, corresponding to the tumour heterogeneity, rose to 21 to 36%. The inter-assay reproducibility was too bad, so a variant of this technique has been adapted, using verapamil or cyclosporin A, which is able to block DNR efflux; this new method allows tumour cells to be used as their own controls.     

The osmotic integrity of the yeast cell requires a functional PKC1 gene product.

Seven temperature-sensitive cell lysis (cly) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY15 gene was isolated by complementation of the cly15 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990). Subsequent genetic analysis showed that CLY15 and PKC1 represent identical loci in the yeast genome. A truncated PKC1 gene encoding only the predicted catalytic domain of Pkc1p was able to complement pkc1 mutant strains. Similar to what has been reported recently (D. E. Levin and E. Bartlett-Heubusch, J. Cell Biol. 116:1221-1229, 1992), we observed that cells deleted for the PKC1 gene are viable when grown on osmotically stabilized medium but are osmotically fragile and lyse rapidly after a shift to hypotonic medium. As shown by light and electron microscopic examinations, the delta pkc1 strain exhibits many cells with a strongly elongated bud or chains of incompletely budded cells when grown on solid medium.     

Transformation of skeletal muscle by progressive sequential stimulation with a view toward its use as a myocardial substitute

Progressive sequential stimulation of a skeletal muscle using trains of 30 Hz impulses with increasing frequencies from 20/min. to 80/min. within 3 months, allowed us to obtain in goats a transformation of the fast twitch glycolytic muscular fibers into fatigue resistant slow twitch oxidative muscular fibers. The conditioned muscle can be used in the treatment of various myocardial lesions or to reinforce cardiac contractility in severe cardiac insufficiencies. The first clinical case successfully operated upon is reported.     

Structural effects of point mutations in proteins

A structural database of 11 families of chains differing by a single amino acid substitution has been built. Another structural dataset of 5 families with identical sequences has been used for comparison. The RMSD computed after a global superimposition of the mutated protein on each native one is smaller than the RMSD calculated among proteins of identical sequences. The effect of the perturbation is very local, and not necessarily the highest at the position of the mutation. A RMSD between mutated and native proteins is computed over a 3‐residue or a 7‐residue window at each position. To separate the effects of structural fluctuations due to point mutations from other sources, pair RMSD have been translated into P values which themselves are included in a score called P‐RANK. This score allows highlighting small backbone distortions by comparing these RMSD between mutated and native positions to the RMSD at the same positions in the absence of a mutation. It results from the P‐RANK that 38% of all mutations produce a significant effect on the displacement. When compared with a random distribution of RMSD at un‐mutated positions, we show that, even if the RMSD is greater when the mutation is in loops than in regular secondary structure, the relative effect is more important for regular secondary structures and for buried positions. We confirm the absence of correlation between RMSD and the predicted variation of free energy of folding but we found a small correlation between high RMSD and the error in the prediction of ΔΔG.     

The digestive gland of Viviparus ater (Mollusca, Gastropoda, Prosobranchia): an ultrastructural and histochemical study

The digestive gland of Viviparus ater was studied using histochemical and ultrastructural methods. Only one cell type was observed in the tubule epithelium of the gland. The cells are involved in an endocytotic process mediated by clathrin-coated vesicles and in the intracellular digestion of food materials (thus they can be regarded as digestive cells). The different stages of digestion and exocytotic extrusion of residual bodies into the tubule lumen were shown by electron microscopy. Very few, small mucocytes are scattered among the digestive cells. Calcium concretions, glycogen-containing cells and endocrine cells are scattered in the area of connective tissue present among the digestive tubules.