Somatic embryogenesis in wild cherry (Prunus avium)

Indirect somatic embryogenesis was obtained inPrunus avium L. from either somatic or zygotic embryos. An embryogenic line was established by reinduction of embryogenic calluses from somatic embryos. The line was maintained for more than 3 years through 6 generations of embryogenic cultures. In the last 2 generations, more than 50% of the explants were embryogenic. Embryos at different stages of development were produced. Among cotyledonary-stage embryos, 50% had two cotyledons and a distinct hypocotyl, 43% had one or more than 2 cotyledons and 7% had fused cotyledons. Most of the embryos were translucent and conversion into plantlets was very rare. Secondary embryos could be observed to occur with low frequency from cultured somatic embryos and from embryos emerging from calluses. Indirect somatic embryogenesis was also induced from immature zygotic embryos. From one donor tree, 51% of the explants were embryogenic when cultured on a medium containing 0.9 μM kinetin, 0.9 μM BA and 0.5 μM NAA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mesenchymal stem cells support expansion of in vitro irradiated CD34(+) cells in the presence of SCF, FLT3 ligand, TPO and IL3: potential application to autologous cell therapy in accidentally irradiated victims

Ex vivo expansion of residual autologous hematopoietic stem and progenitor cells collected from victims soon after accidental irradiation (autologous cell therapy) may represent an additional or alternative approach to cytokine therapy or allogeneic transplantation. Peripheral blood CD34+ cells could be a useful source of cells for this process provided that collection and ex vivo expansion of hematopoietic stem and progenitor cells could be optimized. Here we investigated whether mesenchymal stem cells could sustain culture of irradiated peripheral blood CD34+ cells. In vitro irradiated (4 Gy 60Co gamma rays) or nonirradiated mobilized peripheral blood CD34+ cells from baboons were cultured for 7 days in a serum-free medium supplemented with stem cell factor+thrombopoietin+interleukin 3+FLT3 ligand (50 ng/ml each) in the presence or absence of mesenchymal stem cells. In contrast to cultures without mesenchymal stem cells, irradiated CD34+ cells cultured with mesenchymal stem cells displayed cell amplification, i.e. CD34+ (4.9-fold), CD34++ (3.8-fold), CD34++/Thy-1+ (8.1-fold), CD41+ (12.4-fold) and MPO+ (50.6-fold), although at lower levels than in nonirradiated CD34+ cells. Fourteen times more clonogenic cells, especially BFU-E, were preserved when irradiated cells were cultured on mesenchymal stem cells. Moreover, we showed that the effect of mesenchymal stem cells is related mainly to the reduction of apoptosis and involves cell-cell contact rather than production of soluble factor(s). This experimental model suggests that mesenchymal stem cells could provide a crucial tool for autologous cell therapy applied to accidentally irradiated victims.     

Microfilamentous system and secretion of enzyme in the exocrine pancreas. Effect of cytochalasin B

The microfilaments in the acinar cell of the exocrine pancreas are essentially located in the apical part of the cell: thin microfilaments (50 A), cytochalasin B (CB)-sensitive, form the axis of the microvilli and a network lying beneath the apical membrane; thicker filaments (100 A), at least partly CB-insensitive, form bundles parallel to the plasma cell membrane and the desmosomal links. CB interaction with the acinar cell of the exocrine pancreas involves at least two sites: a membrane site involved in the inhibitory effect of CB on the monosaccharide transport and a less sensitive site at the filamentous level at least partly responsible for the inhibitory effect of CB in the secretion of the exportable enzyme from the pancreatic cell. CB did not alter the energy balance of the acinar cell nor the exchanges of 15-Ca between the extracellular medium and the pancreatic tissue. CB (2 times 10-minus 7 and 2 times 10-minus 6 M) has secretagogue properties whereas CB (2 times 10-minus 5 M) has inhibitory effect on stimulated secretion and secretagogue properties. The mechanism of these secretory effects is not yet explained. The analysis presented in this investigation affords strong evidence for the involvement of the microfilamentous network in the last steps of the secretory cycle in the acinar cell of the exocrine pancreas.     

A numerical study of the blocking of migraine by Rolando sulcus

Migraine with aura is a complex phenomena, which remains still not completely understood. A striking fact is that its clinical manifestations may change from one patient to another. Migraine with aura may only consist in visual hallucinations, but may as well go on to temporary aphasy. However, for all the patients it always stops before it goes from area 3 to area 4, thus just before crossing Rolando sulcus. In this paper, we give arguments showing that the detailed geometry of Rolando sulcus in human cortex may by itself explain that migraine attack never crosses Rolando sulcus.     

Phosphorylation at tyrosine-524 influences nuclear accumulation of HSP72 with heat stress

Nuclear accumulation of heat shock protein (HSP) 72 occurs after cardiac ischemia. This nuclear accumulation of HSP72 with stress occurs in other tissues and species. We postulated that nuclear accumulation of HSP72 was important for the protective effect of HSP72 and that phosphorylation of a single tyrosine (Y(524)) regulated nuclear accumulation of HSP72. Western blots of immunoprecipitated HSP72 from Cos-1 cells demonstrated that tyrosine becomes phosphorylated after heat shock. Treatment with the tyrosine kinase inhibitor geldanamycin blocked nuclear accumulation of HSP72 with heat shock. Two epitope-tagged constructs were made: M17 converting Y(524) to aspartic acid (pseudophosphorylation) and M18 converting Y(524) to phenylalanine. When transfected into Cos-1 cells, M17 accumulates more rapidly and M18 less rapidly than wild-type (WT) HSP72 in the nucleus following heat shock. Cells expressing M18 had less viability after heat shock at 43.5 degrees C than other constructs. After heat shock at 45 degrees C, cells expressing M17 had superior survival compared with WT and M18. These data suggest that phosphorylation at Y(524) facilitates nuclear accumulation of HSP72 following heat stress, and substitution of aspartic acid at Y(524) enhances resistance to heat-shock injury.     

Intra-species DNA polymorphism in the tobacco cyst-nematode complex (Globodera tabacum) using AFLP.

Amplified fragment length polymorphism (AFLP) was used to obtain information on the within-species genetic variability of the tobacco cyst-nematode (TCN) complex. AFLP was found to be well suited to this type of study. The current classification of TCN was confirmed. Results indicate that the Globodera tabacum solanacearum group, believed to be restricted to the U.S.A., also occurs in Mexico. The within-species variability of TCN is considerable. Populations from Mexico may form a new subgroup. AFLP group-specific markers were identified for two of the TCN subgroups: Globodera tabacum tabacum and Globodera tabacum solanacearum.     

Peculiar inhibition of human mitochondrial aspartyl-tRNA synthetaseby adenylate analogs

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), the enzymes which esterify tRNAs with the cognate specific amino acid, form mainly a different set of proteins than those involved in the cytosolic translation machinery. Many of the mt-aaRSs are of bacterial-type in regard of sequence and modular structural organization. However, the few enzymes investigated so far do have peculiar biochemical and enzymological properties such as decreased solubility, decreased specific activity and enlarged spectra of substrate tRNAs (of same specificity but from various organisms and kingdoms), as compared to bacterial aaRSs. Here the sensitivity of human mitochondrial aspartyl-tRNA synthetase (AspRS) to small substrate analogs (non-hydrolysable adenylates) known as inhibitors of Escherichia coli and Pseudomonas aeruginosa AspRSs is evaluated and compared to the sensitivity of eukaryal cytosolic human and bovine AspRSs. l-aspartol-adenylate (aspartol-AMP) is a competitive inhibitor of aspartylation by mitochondrial as well as cytosolic mammalian AspRSs, with K_i values in the micromolar range (4–27 μM for human mt- and mammalian cyt-AspRSs). 5′-O-[N-(l-aspartyl)sulfamoyl]adenosine (Asp-AMS) is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10 nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300 nM). The higher sensitivity of human mt-AspRS for both inhibitors as compared to either bacterial or mammalian cytosolic enzymes, is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic events. In the scope of new antibacterial strategies directed against aaRSs, possible side effects of such drugs on the mitochondrial human aaRSs should thus be considered.