Nitric oxide mediates oxylipin production and grazing defense in diatoms

Diatom blooms are important features of productive marine ecosystems and are known to support higher trophic levels. However, when stressed or wounded, diatoms can produce oxylipin molecules known to inhibit the reproduction and development of copepods and decrease microzooplankton growth rates. Using oxylipin chemical treatments, lipidomic analysis and functional genomic approaches, we provide evidence that nitric oxide (NO) and oxylipin signalling pathways in diatoms respond to protist grazers, resulting in increased defence fitness and survival. Exposure of the diatom Phaeodactylum tricornutum to the dinoflagellate Oxyrrhis marina resulted in NO production by P. tricornutum and pronounced change in its dissolved oxylipin profile. Experimentally elevating levels of NO also resulted in increased oxylipin production, and lower overall grazing rates. Furthermore, O. marina preferentially grazed on P. tricornutum prey with lower levels of NO, suggesting that this molecule and its effect on oxylipin pathways play a key role in prey selection. Exposure of O. marina grazing on P. tricornutum to exogenous oxylipins also decreased grazing rates, which is consistent with a grazing deterrence role for these molecules. These results suggest that NO and oxylipin production help to structure diatom communities, in part by modulating interactions with microzooplankton predators.     

Immunohistochemical evaluation of hepatic oval cell activation and differentiation toward pancreatic beta-cell phenotype in streptozotocin-induced diabetic mice

Oval cell (OvCs) involvement in regeneration is a well known phenomenon in models of liver injury, however, the activation of these cells following streptozotocin (STZ)-induced diabetes has not been studied yet. Differentiation of liver cells toward insulin-producing cells in diabetes has been reported, but the cell phenotype is still unclear. The aim of the present study was to confirm by immunohistochemical analysis, the activation of OvCs and their ability to express pancreatic beta-cell phenotype in STZ-induced diabetic mice. Using specific anti-A6 antibodies for mouse OvCs, we found a three-fold increase in periportal number and two-fold higher density of OvCs in diabetic livers, when compared to controls. Unlike non-diabetic controls, double staining technique showed co-localization of A6 and proinsulin in the cytoplasm of OvCs of diabetic animals, but no insulin staining was detected, probably reflecting the premature character of OvCs differentiation toward beta-cell-like phenotype. These data add valuable information concerning the nature and the stage of functional maturity of liver cells undergoing differentiation toward beta-cell phenotype in STZ-induced diabetic animals.     

Protective and therapeutic effects of molsidomine on radiation induced neural injury in rats

We investigated the protective and therapeutic effects of molsidomine (MOL) in a rat model of whole brain radiotherapy (RT). Forty female rats were divided into five groups of eight: group 1, control; group 2, 15 Gy single dose RT (RT); group 3, 4 mg/kg MOL treated for 5 days (MOL); group 4, 4 mg/kg MOL for 5 days, 10 days after RT treatment (RT + MOL); group 5, 4 mg/kg MOL treatment for 5 days before RT treatment and for 5 days after RT treatment (MOL + RT). All rats were sacrificed on day 16. Neurodegenerative changes in the brain and tissue levels of oxidants and antioxidants were evaluated. The oxidative parameters were increased and antioxidant status was decreased in group RT compared to groups MOL + RT and RT + MOL. Histopathological examination showed that treatment with MOL after RT application and treatment with MOL before RT treatment decreased neuronal degeneration. No difference in neuronal appearance was found between groups RT + MOL and MOL + RT. MOL treatment protected the nervous system of rats and may be a treatment option for preventing RT induced neural injury.     

<Emphasis Type="Italic">Aloe vera</Emphasis> transformation: the role of Amberlite XAD-4 resin and antioxidants during selection and regeneration

A system for genetic transformation and subsequent plant regeneration via indirect organogenesis from callus was developed for Aloe vera. Young seedlings served as primary explants. Callus cultures were established on Murashige and Skoog (1962) medium supplemented with 3 mg l⁻¹ benzylaminopurine and 2 mg l⁻¹ indole acetic acid. A protocol was developed to switch from the differentiated stage, using in vitro shoots or young regenerated plants, back to the de-differentiated stage of the callus and vice versa. Long-term maintenance of this callus paved the way for genetic manipulation of Aloe vera. Calluses were bombarded with a plasmid containing uidA and hpt genes, both under the control of the 35S promoter. Dithiothreitol and gibberellic acid were found to play a major role in reducing tissue necrosis following bombardment. Transformed shoots were regenerated under stepwise selection in hygromycin-containing liquid medium supplemented with different antioxidants. Amberlite XAD-4 resin was embedded into alginate beads and added to the selection medium. Amberlite was best for adsorbing different phenolic compounds and blocking explant necrosis. Shoot initiation occurred after transfer of the transformed cells to Murashige and Skoog medium supplemented with 2.0 mg l⁻¹ thidiazuron and 0.1 mg l⁻¹ indole butyric acid. Murashige and Skoog medium supplemented with 1 mg l⁻¹ zeatin riboside promoted shoot elongation. Rooting and plant development were obtained on Murashige and Skoog basal medium supplemented with 15 mg l⁻¹ hygromycin lacking growth regulators. The transgenic nature of the regenerated plants was verified by histochemical GUS assay and Southern blot hybridization.     

Suppression of somatic embryogenesis in Citrus cell cultures by extracellular proteins

Nucellar-derived cell cultures of sour orange (Citrus aurantium L.) proliferate as proembryogenic masses. By a change in the carbon source of the medium from sucrose to glycerol they are induced to undergo synchronous embryogenesis forming embryo initials that develop into globular embryos. The proembryogenic masses released glycoproteins to the medium. Exogenous addition of the glycoproteins to cells in glycerol-containing medium modified the course of embryo development in a dose-dependent manner. Addition of 20 g · ml^–1 of glycoproteins blocked embryogenesis and resulted in an accumulation of embryo initials. When glycoproteins were added to cultures containing advanced globularstage embryos further development was suppressed. The inhibitory component of the glycoproteins was found to be a family of polypeptides with apparent molecular masses of 53–57 kDa. While these proteins normally accumulated only in cultures of proembryogenic masses, they could be induced to accumulate in glycerol-containing medium by the addition of the glycoproteins. Thus, their accumulation was not a direct consequence of the type of growth medium used or the developmental state of the cultures. The results indicate that the 53-to 57 kDa glycoproteins could play a regulatory role in in-vitro embryogenesis in sour orange. The normal progression of embryo development appears to depend, in an obligatory manner, on the absence of these glycosylated extracellular proteins from the medium.Abbreviations kDa kilodalton - PEM proembryogenic masses - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - 2D-PAGE Two-dimensional polyacrylamide gel electrophoresis We thank Dr. S. Satoh (Institute of Biological Sciences, Tsukuba, Japan) for sending protein samples of the purified 57-kDa glycoprotein. This research was supported by a grant from the Charles H. Revson Foundation for Basic Research in the Life Sciences of the Israel Academy of Sciences. R.F. is a recipient of the Jack and Florence Goodman Career Development Chair.     

Regeneration of <Emphasis Type="Italic">Aloe arborescens</Emphasis> via somatic organogenesis from young inflorescences

A system for in vitro regeneration of Aloe arborescens was developed using young inflorescences as explants. Different phytohormone combinations of N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ), benzyladenine (BA), 6-(γ,γ-dimethylallyl-amino)purine riboside (2iPR), zeatin ribozide (ZR), N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) and kinetin (K), with or without ancymidol, were examined in order to induce plant regeneration. Efficient shoot regeneration was initiated on Murashige and Skoog (MS) medium supplemented with BA or TDZ. MS medium enriched with 19.6, 22.2 μM BA and 3.92 μM ancymidol (MSBA5/1 medium), promoted organogenesis enabling 87.3% of the explants to regenerate 6.04 ± 1.79 shoots/explant. Subsequent shoot elongation and plant regeneration were strongly affected by the medium composition used for shoot induction. Optimal elongation (three to four shoots per explant) was obtained when shoots, initiated on MSBA5/1 medium, were subsequently transferred onto MS containing only 4.4 μM BA. Rooting was performed on MS media lacking growth regulators. Histological analysis revealed that the initiated shoots originated from the receptacle tissue surrounding the residual vascular tissue of the flower buds.     

Subcellular localization of VDAC in mitochondria and ER in the cerebellum

The voltage-dependent anion channel (VDAC) provides passage for adenine nucleotides, Ca2+ and other metabolites into and from mitochondria. Here, the intracellular localization and oligomeric organization of VDAC in brain mitochondria and ER are demonstrated. Immunohistochemical staining of VDAC in rat cerebellum showed high labeling of the Purkinje neurons. Immunogold labeling and EM analysis of the cerebellar molecular layer showed specific VDAC immunostaining of the mitochondrial outer membrane, highly enhanced in contact sites between mitochondria or between mitochondria and associated ER. Purified ER membranes contain VDAC, but not other mitochondrial proteins. Chemical cross-linking of isolated mitochondria, ER or purified VDAC demonstrated the existence of VDAC in oligomeric form. Based on the enrichment of VDAC in the junctional face of closely associated mitochondrial and ER membranes and the existence of VDAC oligomers, we propose an involvement of VDAC in specialized intermembrane communication between mitochondria or between ER and mitochondria, serving to complement the tight structural and functional coupling observed between these organelles.     

Is the Phytophthora citrophthora culture filtrate a reliable tool for the in vitro selection of resistant Citrus variants?

Summary Nucellar calli from four Citrus cultivars with known resistance to the Phytophthora citrophthora pathogen were chosen as experimental material to test the pathogen's response to culture filtrate (CF). Sensitivity of the four calli to CF of the fungus was in reverse order to what is known on the susceptibility of the cultivars in vivo. Sensitivity of protoplasts derived from the same four calli to 2,4-dichlorophenoxyacetic acid (2,4-D) was in the same order as that of calli to CF. Protoplasts derived from calli selected for tolerance to CF showed a higher plating efficiency with increasing concentration of CF in the medium. TLC and GLC determinations showed the presence of indole acetic acid in the culture filtrate. Results indicate that CF of P. citrophthora cannot be used as a selection tool in vitro.Contribution No. 1655-E, 1986 series, from the Agricultural Research Organization, Bet-Dagan, Israel     

A novel device for islet transplantation providing immune protection and oxygen supply

Islet transplantation as a biological β-cell replacement therapy has emerged as a promising option for achieving restoration of metabolic control in type 1 diabetes patients. However, partial or complete loss of islet graft function occurs in relatively short time (months to few years) after implantation. The high rate of early transplant dysfunction has been attributed to poorly viable and/or functional islets and is mediated by innate inflammatory response at the intravascular (hepatic) transplant site and critical lack of initial nutrient/oxygen supply prior to islet engraftment. In addition, the diabetogenic effect of mandatory immunosuppressive agents, limited control of alloimmunity, and the recurrence of autoimmunity limit the long-term success of islet transplantation. In order to abrogate instant blood-mediated inflammatory reaction and to provide oxygen supply for the islet graft, we have developed an extravascular (subcutaneous) transplant macrochamber (the 'βAir' device). This device contains islets immobilized in alginate, protected from the immune system by a thin hydrophilized teflon membrane impregnated with alginate and supplied with oxygen by daily refueling with oxygen-CO (2) mixture. We have demonstrated successful utilization of the oxygen-refueling macrochamber for sustained islet viability and function as well as immunoprotection after allogeneic subcutaneous transplantation in healthy minipigs. Considering the current limitations of intraportal islet engraftment and the restricted indication for islet transplantation mainly due to necessary immunosuppressive therapy, this work could very likely lead to remarkable improvements in the procedure and moreover opens up further strategies for porcine islet cell xenotransplantation.