Somatic embryogenesis in leaves and leaf-derived protoplasts of Actinidia deliciosa var. deliciosa cv. Hayward (kiwifruit)

Summary The obtention of embryogenic competence in Actinidia deliciosa var. deliciosa cv. Hayward is reported. Axillary buds from shoots submitted to cold (4°C) and starvation for 1.5 months, developed leaves with embryogenic competence. These leaves, cultured in darkness for 1.5 months on a medium containing zeatin as a sole growth regulator, originated compact structures from which embryos developed. The plating orientation and sectioning of leaves strongly affected the expression of the embryogenic potential. A selected fraction of the protoplasts isolated from these leaves was able to develop in an embryogenic way. The germination of the embryos is still only occasional.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 6-dimethylallyl aminopurine - IAA indole-3-acetic acid - NOA naphthoxyacetic acid - SEM Scanning Electron Microscopy     

Essential fatty acid deficiency lowers the activity of the acetylated low density lipoprotein receptor of rat peritoneal macrophages.

We compared phospholipid fatty acid composition, cholesterol ester accumulation, and receptor-mediated binding, internalization, and degradation of acetylated low-density lipoprotein (acetyl-LDL) in cultured peritoneal macrophages from rats fed an essential fatty acid deficient or control diet. The deficient diet increased the 5,8,11-eicosatrienoic acid and decreased the omega 6 fatty acid content of macrophage phospholipid relative to control. The deficient diet did not affect macrophage uptake of [1-14C]oleate; however, it lowered the accumulation of intracellular labelled cholesteroyl oleate to 66% of the control. This effect was attributed to a diminution of the specific binding of acetyl-LDL, and not to acetyl-LDL internalization nor to degradation. The results demonstrate the sensitivity of the acetyl-LDL receptor to changes in its membrane environment, brought about through dietary means.     

Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.     

Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism.

The mechanism by which CD8+ T cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in acutely infected CD4+ T cells was investigated. Cytotoxicity was not involved, as the antiviral activity of the CD8+ cells did not correlate with the ability to lyse HIV-infected or uninfected CD4+ T cells. In addition, the frequency of HIV-infected CD4+ cells increased during coculture with CD8+ T cells even in the absence of detectable levels of virus replication. Moreover, separation of the CD4+ and CD8+ cells by a 0.4-micron-pore-size filter delayed HIV replication, indicating a role, at least in part, for a soluble factor. However, cell contact was required for optimal antiviral activity. These results extend further the observation on the mechanism of antiviral HIV activity by CD8+ cells from infected individuals. They support the conclusion that CD8+ cells can play a major role in preventing development of disease in HIV-infected individuals.     

Several CD4 domains can play a role in human immunodeficiency virus infection in cells.

The human immunodefiency virus (HIV) uses the human CD4 glycoprotein as a receptor for infection of susceptible cells. Cells expressing a series of mutated forms of the CD4 gene have shown a variability in their ability to support replication of three HIV type 1 (HIV-1) and three HIV-2 strains. Moreover, when different stages of virus production were examined by a variety of assays, a consistent delay was observed in all cell lines containing CD4 mutants compared with those with intact full-length CD4. Cells expressing the CD4.415 mutant (modified at the serine 415 corresponding to a phosphorylation site of the cytoplasmic domain) showed only a minimal effect on virus replication. Cells expressing CD4.403 and CD4.401 mutants (lacking the whole cytoplasmic domain) manifested a moderate delay in production of virus progeny. The most substantial effect on HIV replication was observed in cells expressing a chimeric hybrid containing sequences corresponding to the first 177 residues of the N-terminal CD4 fused to CD8 sequences encoding the hinge, transmembrane, and cytoplasmic domains of the human CD8. Furthermore, in a cell-to-cell contact assay, fusion was absent when the CD4 proximal membrane domain was replaced by the CD8 counterpart. In addition, a strong correlation between the down-modulation of the surface CD4 and HIV expression was observed. These observations suggest that in addition to the known binding region, other domains of CD4 could play an important role in regulating HIV entry of cells.     

Organisation and functions of the actV A region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor

Summary Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and-ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.     

A 2.6 kb intron separates the signal peptide coding sequence of an anther-specific protein from the rest of the gene in sunflower

Summary Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450_SU1 and P-450_SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450_SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450_SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450_SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450_SU2 in the absence of P-450_SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.     

Stromal low temperature compartment derived from the inner membrane of the chloroplast envelope

Leaf discs of four dicotyledonous species, when incubated at temperatures of 4 to 18°C (optimum at 12°C) for 30 or 60 minutes, responded by accumulations of membranes in the chloroplast stroma in the space between the inner membrane of the envelope and the thylakoids. The accumulated membranes, here referred to as the low temperature compartment, were frequently continuous with the envelope membrane and exhibited kinetics of formation consistent with a derivation from the envelope. Results were similar for expanding leaves of garden pea (Pisum sativum), soybean (Glycine max), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum). We suggest that the stromal low temperature compartment may be analogous to the compartment induced to form between the transitional endoplasmic reticulum and the Golgi apparatus at low temperatures. The findings provide evidence for the possibility of a vesicular transfer of membrane constituents between the inner membrane of the chloroplast envelope and the thylakoids of mature chloroplasts in expanding leaves.     

Activation of Plasma Membrane NADH Oxidase Activity by Products of Phospholipase A

An auxin-stimulated NADH oxidase activity (NADH oxidase I) of plasma membrane vesicles, highly purified by aqueous two-phase partition from soybean (Glycine max Merr.) hypocotyls was activated by lysophospholipids and fatty acids, both products of phospholipase A action. The activation of NADH oxidase activity occurred slowly, suggesting a mechanism whereby the lipids acted to stabilize the enzyme in a more active configuration. In contrast to activation by lipids, the activation by auxin was rapid. The average K_m of the NADH oxidase after activation by lipids was four- to fivefold less than the K_m before activation. The V_max was unchanged by activation. The increases occurred in the presence of detergent and thus were not a result of exposure of latent active sites. Also, the activation did not result from activation of a peroxidase or lipoxygenase. Fatty acid esters, where growth promoting effects have been reported, also activated the auxin-stimulated oxidase. However, the auxin stimulation of NADH oxidase I did not appear to be obligatorily mediated by phospholipase A, nor did inhibitors of phospholipase A₂ block the stimulation of the oxidase by auxins.     

Floral morphogenesis in Anagallis: Scanning-electron-micrograph sequences from individual growing meristems before,during, and after the transition to flowering

Non-destructive scanning electron microscopy allows one to visualize changing patterns of individual cells during epidermal development in single meristems. Cell growth and division can be followed in parallel with morphogenesis. The method is applied here to the shoot apex of Anagallis arvensis L. before, during, and after floral transition. Phyllotaxis is decussate; photoperiodic induction of the plant leads to the production of a flower in the axil of each leaf. As seen from above, the recently formed oval vegetative dome is bounded on its slightly longer sides by creases of adjacent leaf bases. The rounded ends of the dome are bounded by connecting tissue, horizontal bands of node cells between the opposed leaf bases. The major growth axis runs parallel to the leaf bases. While slow-growing at the dome center, this axis extends at its periphery to form a new leaf above each band of connecting tissue. Connecting tissue then forms between the new leaves and a new dome is defined at 90° to the former. The growth axis then changes by 90°. This is the vegetative cycle. The first observed departure from vegetative growth is that the connecting tissue becomes longer relative to the leaf creases. Presumably because of this, the major growth axis does not change in the usual way. Extension on the dome continues between the older leaves until the axis typically buckles a second time, on each side, to form a second crease parallel to the new leaf-base crease. The tissue between these two creases becomes the flower primordium. The second crease also delimits the side of a new apical dome with the major axis and growth direction altered by 90°. During this inflorescence cycle the connecting tissue is relatively longer than before. Much activity is common to both cycles. It is concluded that the complex geometrical features of the inflorescence cycle may result from a change in a biophysical boundary condition involving dome geometry, rather than a comprehensive revision of apical morphogenesis.Abbreviation SEM scanning electron microscopy, micrograph Use of the SEM facility of Professor G. Goffinet, Institute of Zoology, University of Liège, is greatly appreciated. We thank Dr. R. Jacques, C.N.R.S., Le Phytotron, Gif-sur-Yvette, France, for providing the experimental material, and Mr. Philippe Ongena for expert photography. Support was from grants from the U.S. Department of Agriculture and National Science Foundation as well as from the Fonds National de la Recherche Scientifique, Fonds de la Recherche Fondamentale et Collective, and the Action de Recherche Concertée of Belgium.