Callus formation and plant regeneration in various <Emphasis Type="Italic">Lilium</Emphasis> species and cultivars

Summary In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.     

Local phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells

Neurite outgrowth is an important process in the formation of neuronal networks. Rac1 and Cdc42, members of the Rho-family GTPases, positively regulate neurite extension through reorganization of the actin cytoskeleton. Here, we examine the dynamic linkage between Rac1/Cdc42 and phosphatidylinositol 3-kinase (PI3-kinase) during nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Activity imaging using fluorescence resonance energy transfer probes showed that PI3-kinase as well as Rac1/Cdc42 was transiently activated in broad areas of the cell periphery immediately after NGF addition. Subsequently, local and repetitive activation of PI3-kinase and Rac1/Cdc42 was observed at the protruding sites. Depletion of Vav2 and Vav3 by RNA interference significantly inhibited both Rac1/Cdc42 activation and the formation of short processes leading to neurite outgrowth. At the NGF-induced protrusions, local phosphatidylinositol 3,4,5-trisphosphate accumulation recruited Vav2 and Vav3 to activate Rac1 and Cdc42, and conversely, Vav2 and Vav3 were required for the local activation of PI3-kinase. These observations demonstrated for the first time that Vav2 and Vav3 are essential constituents of the positive feedback loop that is comprised of PI3-kinase and Rac1/Cdc42 and cycles locally with morphological changes.     

Rosmarinic acid inhibits the formation of reactive oxygen and nitrogen species in RAW264.7 macrophages

Antioxidant action of Rosmarinic acid (Ros A), a natural phenolic ingredient in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, was analyzed in relation to the Ikappa-B activation in RAW264.7 macrophages. Ros A inhibited nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein synthesis induced by lipopolysaccharide (LPS), and also effectively suppressed phorbol 12-myristate 13-acetate (PMA)-induced superoxide production in RAW264.7 macrophages in a dose-dependent manner. Peroxynitrite-induced formation of 3-nitrotyrosine in bovine serum albumin and RAW264.7 macrophages were also inhibited by Ros A. Moreover, Western blot analysis demonstrated that LPS-induced phosphorylation of Ikappa-Balpha was abolished by Ros A. Ros A can act as an effective protector against peroxynitrite-mediated damage, and as a potent inhibitor of superoxide and NO synthesis; the inhibition of the formation of reactive oxygen and nitrogen species are partly based on its ability to inhibit the serine phosphorylation of Ikappa-Balpha.     

Gastric acidity in patients with follicular gastritis is significantly reduced, but can be normalized after eradication for Helicobacter pylori

BACKGROUND: Follicular gastritis is thought to be caused by Helicobacter pylori infection. However, the pathophysiology of it remains unclear. MATERIALS AND METHODS: We assessed gastric acidity in 15 patients with follicular gastritis, aged 20-37 years, using a 24-hour intragastric pH-metry, as well as by histologic and serologic evaluations; and compared it with that in other age-matched groups: 18 cases of H. pylori-positive antrum-predominant gastritis, 12 of pangastritis, and 24 H. pylori-negative normals. In eight cases with follicular gastritis, it was re-assessed 6 months after the eradication therapy for H. pylori. RESULTS: During nighttime, the percentage of time with intragastric pH above 3.0 in follicular gastritis was significantly higher than that in normals (p<.0001), and in antrum-predominant gastritis (p<.001), but was comparable with that in pangastritis. In the daytime period, this parameter in follicular gastritis was significantly higher than that in normal (p<.001), in antrum-predominant gastritis (p<.001), and in pangastritis (p<.05). Marked mononuclear cell and neutrophil infiltration but no apparent glandular atrophy were observed in both the antrum and corpus. Serum pepsinogen I/II ratio was significantly lower in follicular gastritis than that in normals (p<.0001) and in antrum-predominant gastritis (p<.001), whereas serum gastrin was significantly higher than that in normals (p<.0001), in antrum-predominant gastritis (p<.01) and in pangastritis (p<.05). After eradication for H. pylori, all of the parameters in follicular gastritis were altered to the same ranges as those in normals. CONCLUSIONS: In follicular gastritis, gastric acidity is significantly reduced, but can be normalized by eradication of H. pylori. It can thus be speculated that inflammatory cytokines or H. pylori-infection-induced prostaglandins might strongly inhibit gastric acid secretion in follicular gastritis.     

Pacific oyster hemocytes undergo apoptosis following cell-adhesion mediated by integrin-like molecules

Previously, we demonstrated that in the Pacific oyster (Crassostrea gigas, a bivalve mollusc) apoptosis could be induced in hemocytes by treatment with Arg-Gly-Asp (RGD) peptides which are known to function as an integrin ligand. However, it is unclear where the RGD peptides are binding to the C. gigas hemocytes, or what mechanism or molecules are involved, e.g., integrin-ligand interactions. Therefore, this study was undertaken to investigate the binding interactions in C. gigas hemocytes. Initially, to confirm the presence of RGD-recognizing integrin-like molecule(s) on the hemocytes, we assessed the enhancement of spreading ability, and found that spreading ability was enhanced by immobilized human fibronectin, a fibronectin fragment containing the RGD motif, and C. gigas plasma in the presence of divalent cations. Interestingly, viability of the spreading hemocytes dramatically decreased 24 h later and DNA fragmentation with oligonucleosomal laddering of 180-200 bp in length was detected in the dead hemocytes by electrophoresis and TUNEL assay. These results indicated that hemocyte adhesion mediated by integrin-like molecules triggered apoptosis and suggested that integrin-activation contributes to the induction of apoptosis. This is the first report showing the possibility of an integrin functioning in the induction of apoptosis in invertebrate hemocytes.     

Matching base-pair number dependence of the kinetics of DNA-DNA hybridization studied by surface plasmon fluorescence spectroscopy

Two single-stranded DNA oligonucleotides consisting of complementary base-pairs can form double strands. This phenomenon is well studied in solutions, however, in order to clarify the physical mechanism of the hybridization occurring at a solid/solution interface, we studied the kinetics by surface plasmon fluorescence spectroscopy (SPFS): one single-stranded oligo-DNA (probe-DNA) was immobilized on the substrate, the other one (target-DNA) labelled with a fluorescent probe was added to the flow cell. After hybridization, the chromophores could be excited by the surface plasmon mode and their fluorescence detected with high sensitivity. The dependence of the k(on) and k(off) rate constants on the length of the hybridizing oligonucleotides was investigated by using a MM0 series (no mismatch) and the kinetics was found to be well described by a Langmuir adsorption model. From these measurements we found that also in the case of surface hybridization the affinity of the duplexes decreases as the number of matching base-pairs decreases from 15 to 10. In order to show that SPFS is the powerful technique with high sensitivity, the hybridization process for mixed target-oligos was measured by SPFS and analyzed by an expanded Langmuir model in which two components of target-oligo can bind to probe-DNA at the sensor surface competitively. Two sets of the k(on) and k(off) obtained from the experiment are successfully consistent with the k(on) and k(off) obtained from experiments for single (pure) target-DNA.     

Identification of putative metabolites of docosahexaenoic acid as potent PPARgamma agonists and antidiabetic agents

We found that putative metabolites of docosahexaenoic acid (DHA) are strong PPARgamma activators and potential antidiabetic agents. We designed DHA derivatives based on the crystal structure of PPARgamma, synthesized them and evaluated their activities in vitro and in vivo. The efficacy of 5E-4-hydroxy-DHA 2a as a PPARgamma activator was about fourfold stronger than that of pioglitazone. Furthermore, the 4-keto derivative (10b) showed antidiabetic activity in animal models without producing undesirable effects such as obesity and hepatotoxicity.