Sugars as signal molecules in plant seed development.

Higher plants as sessile organisms react very flexible to environmental changes and stresses and use metabolites like glucose, sucrose and nitrate not only as nutrients but also as signals as part of their life strategies. The role of metabolites as signal molecules has attracted considerable interest during recent years. Data reviewed here for developing plant seeds suggest a trigger function of especially sugars also in development in that metabolic regulatory control can override developmental regulation, i.e., the developmental programme only continues normally if a certain metabolic state is sensed at a given time point in a given cell or tissue. Several experimental strategies have provided mainly correlative evidence that certain sugar levels and/or the resulting changes in osmotic values are necessary within defined tissues or cells to maintain a distinct stage of differentiation or to proceed with the developmental programme. In young legume seeds, but certainly also in other tissues, a high hexose (probably mainly glucose) level seems to maintain the capacity of cells to divide whereas - later in seed development - a certain sucrose level is necessary to induce storage-associated cell differentiation. A major determinant of embryo hexose levels in young legume seeds is an apoplastic invertase preferentially expressed in the inner cell layers of the seed coat. The enzyme cleaves the incoming photoassimilate sucrose into glucose and fructose. During development the tissue harbouring the invertase is degraded in a very specific spatial and temporal pattern as part of the developmental programme and is thus creating steep glucose gradients within the cotyledons. These gradients can be measured at nearly cellular resolution and were found to be correlated positively with cell division rate and negatively with cell differentiation and storage activities. A hexose and a sucrose transporter accumulating only in the epidermal cell layer of the cotyledons seem to be essential in creating and maintaining these gradients. To gain further insights into the role of metabolites, especially sugars, as triggers of developmental processes we foremost have to identify receptor molecules already characterised in yeast, and to describe and understand the signal transduction networks involved.     

Congener Level PCB Desorption Kinetics of Field-Contaminated Sediments

The purpose of this research was to study congener level PCB desorption kinetics of field-contaminated sediments and develop a simple methodology to analyze the desorption behavior. Batch desorption kinetic studies were conducted using XAD-4 resin. Two-phase desorption kinetics were observed for most PCB congeners, consisting of an initial fast rate followed by an extended period of slow rate. A dual first-order rate model was fitted to the PCB desorption data to estimate PCB concentrations in the fast and slow desorbing pools. The fast and slow desorption rates were found to decrease with increasing chlorina-tion of PCB congeners, decreasing ortho chlorination, and decreasing temperature. Estimated first-order desorption rate constants for the fast pools were found to be two orders of magnitude higher than those for the corresponding slow pools. The log of first-order rate constants for the different PCB congeners were found to be linearly related to the log of octanol-water partition coefficients. Therefore, desorption rate constants of higher chlorinated PCB congeners, which typically require long-term desorption tests, could be extrapolated using measured rate constants of lower-chlorinated PCB congeners from short-term desorption tests. This finding may provide a process to significantly reduce the duration of experiments required for estimating desorption kinetics.     

Quantitative determination of free intracellular amino acids in single human polymorphonuclear leucocytes: Recent developments in sample preparation and high-performance liquid chromatography

The described procedure allows quantitative, highly precise and reproducible analysis of free amino acid concentrations in single polymorphonuclear leucocytes (PMLs). This method is superior to previously described procedures with regard to sample size, PML separation, sample preparation and stability, as well as the chosen fluorescence high-performance liquid chromatography procedure, and can satisfy the high demands for ultra-sensitive and comprehensive amino acid analysis, especially for the continuous surveillance of severe diseases and organ dysfunction.     

Suppression of the neoplastic phenotype by transfection of phospholipase C β 3 to neuroendocrine tumor cells

The expression of phospholipase C β 3 (PLCB3) is low or absent in several neuroendocrine neoplasias. To investigate the role of PLCB3 in the neuroendocrine tumorigenesis, we transfected a PLCB3 construct to three neuroendocrine tumor cell lines with a low PLCB3 expression. The growth rate and tumorigenicity were assessed in vitro by [³H]thymidine incorporation and cell counting, in vivo, by xenografting to nude mice. In vitro, PLCB3 expressing clones showed a significant growth inhibition. The tumor weight was reduced for one of the two xenografted PLCB3-transfected cell lines and in both, a reduced number of proliferating (Ki-67 positive) cells was observed. This study implies an essential role for PLCB3 in the neuroendocrine tumorigenesis.     

An analysis of polygenes affecting wing shape on chromosome 3 in Drosophila melanogaster.

Loci on the third chromosome of Drosophila melanogaster that affect an index of wing shape were mapped, using recombinant isogenic lines, with transposable elements as markers. Many genes with small subequal effects are dispersed along the whole chromosome. Their alleles act nearly additively in heterozygotes. They have small correlated effects on leg shape, but no detectable effects on halteres. Small negative net interactions occur over most of the chromosome. The data set of 519 recombinant isogenic lines can be explained reasonably well by two models. One model posits an indefinitely large number of loci with no interactions. The other model posits 11 loci with additive effects whose sum equals the total phenotypic range and with large positive and negative interactions that nearly cancel each other.     

Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals.

Mitogen-activated protein kinase (MAPK) cascades are activated by diverse extracellular signals and participate in the regulation of an array of cellular programs. In this study, we investigated the roles of MAPKs in the induction of phase II detoxifying enzymes by chemicals. Treatment of human hepatoma (HepG2) and murine hepatoma (Hepa1c1c7) cells with tert-butylhydroquinone (tBHQ) or sulforaphane (SUL), two potent phase II enzyme inducers, stimulated the activity of extracellular signal-regulated protein kinase 2 (ERK2) but not c-Jun N-terminal kinase 1. tBHQ and SUL also activated MAPK kinase. Inhibition of MAPK kinase with its inhibitor, PD98059, abolished ERK2 activation and impaired the induction of quinone reductase, a phase II detoxifying enzyme, and antioxidant response element (ARE)-linked reporter gene by tBHQ and SUL. Overexpression of a dominant-negative mutant of ERK2 also attenuated tBHQ and SUL induction of ARE reporter gene activity. Interestingly, although expression of Ras and its mutant forms showed distinct effects on basal ARE reporter gene activity, they did not affect the activation of reporter gene by the inducers. Furthermore, a dominant-negative mutant of Ras had little effect on ERK2 activation by tBHQ and SUL, implicating a Ras-independent mechanism. Indeed, both tBHQ and SUL were able to stimulate Raf-1 kinase activity in vivo as well as in vitro. Thus, our results indicate that the induction of ARE-dependent phase II detoxifying enzymes is mediated by a MAPK pathway, which may involve direct activation of Raf-1 by the inducers.     

Germination and growth responses of hybridizing Carpobrotus species (Aizoaceae) from coastal California to soil salinity

Germination, growth, and physiological responses of hybridizing Carpobrotus from coastal California to soil salinity were studied. Hybrids are presumably the result of hybridization and introgression between the exotic Carpobrotus edulis, a succulent perennial invading coastal habitats, and the native or long-naturalized C. chilensis. Germination responses were investigated at 0, 10, 20, and 50% seawater. Seedling growth and physiology were compared by irrigating seedlings with solutions of the same seawater concentrations and in low and high nutrients. Germination was inhibited in the presence of salt, but recovered after transferring the seeds to fresh water. Seeds exposed to salt had higher final germination rates than control. Growth of Carpobrotus was slightly enhanced by low seawater concentrations but reduced at high salinity at both nutrient regimes. Leaf cell sap osmolarity increased with increasing soil salinity, and taxa did not differ significantly in this physiological adjustment. Leaf carbon isotope ratios (∂¹³C) ranged from −28 to −22‰ and became less negative at higher salinities, indicating an improved water use efficiency in the seedlings at high salt concentrations. In addition, ∂¹³C values were generally less negative at high than at low nutrients. Differences among taxa were generally small. The results show that salinity affects both establishment and growth of hybridizing Carpobrotus. The overall weak species differences in salt tolerance indicate that the exotic C. edulis can occupy the same sites as C. chilensis in terms of salinity. The similarity of hybrids in their response to salinity suggests that they may contribute to the invasion by Carpobrotus.     

Specific visualization of the distribution of the calcium dependent regulatory protein of cyclic nucleotide phosphodiesterase (modulator protein) in tissue culture cells by immunofluorescence microscopy: mitosis and intercellular bridge.

Monospecific antibodies against the homogeneous Ca++ dependent regulatory protein of cyclic nucleotide phosphodiesterase (CDR protein) from bovine brain were used in indirect immunofluorescence microscopy to visualize the cytoplasmic organization of this key regulatory protein in growing tissue culture cells. Although cells during interphase reveal only a weak general cytoplasmic fluorescence, a dramatic reorganization of CDR protein occurs with the onset of mitosis. Throughout the different mitotic stages CDR protein is strongly concentrated in the two polar parts of each half spindle. After completion of telophase (CDR protein appears at both cytoplasmic ends of the intercellular bridge which still connects the two daughter cells. Parallel use of monospecific antibodies against CDR protein and tubulin emphasizes the spatial restriction in the localization of CDR protein during mitosis and early G1 phase of the cell cycle.     

Cathepsin D targeted by acid sphingomyelinase-derived ceramide.

Ceramide has been recognized as a common intracellular second messenger for various cytokines, growth factors and other stimuli, such as CD95, chemotherapeutic drugs and stress factors. To understand the role of ceramide during apoptosis and other cellular responses, it is critically important to characterize direct targets of ceramide action. In this paper, we show that ceramide specifically binds to and activates the endosomal acidic aspartate protease cathepsin D. Direct interaction of ceramide with cathepsin D results in autocatalytic proteolysis of the 52 kDa pre-pro cathepsin D to form the enzymatically active 48/32 kDa isoforms of cathepsin D. Acid sphingomyelinase (A-SMase)-deficient cells show decreased cathepsin D activity, which could be reconstituted by transfection with A-SMase cDNA. The results of our study identify cathepsin D as the first endosomal ceramide target that colocalizes with and may mediate downstream signaling effects of A-SMase.     

DAip1, a Dictyostelium homologue of the yeast actin-interacting protein 1, is involved in endocytosis, cytokinesis, and motility.

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes.We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions.Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.