Characterization of three capsular polysacharides produced by Burkholderia pseudomallei

Three kinds of capsular polysaccharide (CP) were found to be produced by Burkholderia pseudomallei. When the bacterium was grown with the medium without glycerol, CP-1a and CP-1b were produced. CP-1a was mainly 1.4-linked glucan and CP-1b was identified as a polymer composed of galactose and 3-deoxy-D-manno-octulosonic acid, whose chemical structure was recently reported by other laboratories. When the bacterium was grown with the medium containing 5" glycerol. CP-2 was synthesized. CP-2 contained galactose, rhamnose, mannose, glucose and a uronic acid in a ratio of approximately 3:1:0.3:1:1. Methylation analysis of the purified polysaccharides demonstrated that the two acidic polysaccharides. CP-1b and CP-2 shared no common structure, indicating that CP-2 was an acidic capsular polysaccharide whose chemical characters were not reported previously.     

Ac as a tool for the functional genomics of rice

To examine whether the maize autonomous transposable element Ac can be used for the functional analysis of the rice genome, we used Southern blot analysis to analyze the behaviour of Ac in 559 rice plants of four transgenic families through three successive generations. All families showed highly active transposition of Ac, and 103 plants (18.4%) contained newly transposed Ac insertions. In nine of the 12 independent transpositions analyzed, their germinal transmission was detected. Partial sequencing of 99 Ac-flanking sequences revealed that 21 clones exhibited significant similarities with protein-coding genes in databases and four of them matched rice cDNA sequences. These results indicate preferential Ac transposition into protein-coding rice genes. To examine the feasibility of PCR-based screening of gene knockouts in rice Ac plants, we prepared bulked genomic DNA from the leaves of approximately 6000 rice Ac plants and pooled the DNA according to a three-dimensional matrix. Of 14 randomly selected genes, two gene knockouts were identified, and one encoding a rice cytochrome P450 (CYP86) gene was shown to be stably inherited to the progeny. Together, these results suggest that Ac can be efficiently used for the functional analysis of the rice genome.     

Glutamate induces Cl− and K+ currents in the olfactory interneurons of a terrestrial slug

Glutamate-induced responses in the procerebral neurons of the terrestrial slug Limax marginatus were examined using the nystatin-perforated patch recording technique applied in the voltage-clamp mode and local application of drugs. The procerebrum contains two types of interneurons with different spontaneous activities, bursting and nonbursting neurons. In the bursting neurons, a puff of glutamate evoked a rapidly desensitizing current followed by a smaller sustained current. The reversal potential of the early component showed that the current was mediated by Cl⁻ ions, while the late component was presumed to be mediated by K⁺ ions. In the nonbursting neurons, glutamate evoked a sustained current with a strong outward rectification, and the current was mediated by K⁺ ions. Ibotenate selectively evoked the rapidly desensitizing response in the bursting neurons, whereas quisqualate evoked a non-desensitizing K⁺ current both in the bursting and nonbursting neurons. The glutamate-induced K⁺ current had similar characteristics with the spontaneous synaptic activities in the procerebrum neurons, suggesting the possibility that glutamate receptors are involved in the spontaneous oscillatory activity. Accepted: 10 February 1999     

Erratum to: Phylogeny and biogeography of the genus Stevia (Asteraceae: Eupatorieae): an example of diversification in the Asteraceae in the new world

The genus Stevia comprises approximately 200 species, which are distributed in North and South America, and are representative of the species diversity of the Asteraceae in the New World. We reconstructed the phylogenetic relationships using sequences of ITS and cpDNA and estimated the divergence times of the major clade of this genus. Our results suggested that Stevia originated in Mexico 7.0–7.3 million years ago (Mya). Two large clades, one with shrub species and another with herb species, were separated at about 6.6 Mya. The phylogenetic reconstruction suggested that an ancestor of Stevia was a small shrub in temperate pine–oak forests and the evolutionary change from a shrub state to a herb state occurred only once. A Brazilian clade was nested in a Mexican herb clade, and its origin was estimated to be 5.2 Mya, suggesting that the migration from North America to South America occurred after the formation of the Isthmus of Panama. The species diversity in Mexico appears to reflect the habitat diversity within the temperate pine–oak forest zone. The presence of many conspecific diploid–polyploid clades in the phylogenetic tree reflects the high frequency of polyploidization among the perennial Stevia species.

     

B-RAF regulation of Rnd3 participates in actin cytoskeletal and focal adhesion organization

The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.     

Lycoparins A-C, new alkaloids from Lycopodium casuarinoides inhibiting acetylcholinesterase

Three new Lycopodium alkaloids, lycoparins A-C (1-3), have been isolated from the club moss Lycopodium casuarinoides. Structures and stereochemistry of 1-3 were elucidated on the basis of 2D NMR correlations. Lycoparins C (3) exhibited an inhibitory activity against acetylcholinesterase, while lycoparins A (1) and B (2) did not show activity.     

Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs

In many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G0/G1 phase derived from miniature pigs. CB (7.5 microg/ml) and CD (2.5 microg/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 microg/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 microg/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 microg/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 microg/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 microg/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.     

Carbachol-induced Suppression of Contraction Rhythm in Spontaneously Beating Cultured Cardiac Myocytes From Neonatal Rats

Nitric oxide (NO) and reactive oxygen species (ROS) are known to play various functional and pathophysiological roles as an intracellular messenger in the heart. In this study, we investigated whether the increased production of NO and/or ROS was involved in the cholinergic regulation of rhythmic contraction in spontaneously beating cultured cardiac myocytes from neonatal rats. Exposure of cultures to carbachol, an agonist of muscarinic acetylcholine receptors (mAchR), produced a dose-dependent decrease in the beat rate of cultured cardiac myocytes, and such a effect was significantly attenuated by pre-treatment with an NOS inhibitor, as well as an NO scavenger. In addition, exposure to an NO donor (SNAP) also decreased the beat rate dose-dependently. Carbachol- or SNAP-induced suppression of the contraction rhythm was significantly attenuated by co-treatment with 5-hydroxydecanoate (5-HD). In contrast, treatment with diazoxide decreased the beat rate dose-dependently. Carbachol treatment increased the intensity of 2',7'-dichlorodihydrofluorescein fluorescence, suggesting that the production of ROS was enhanced by the treatment. In addition, the carbachol- or diazoxide-induced suppression of contraction rhythm was attenuated by co-treatment with 2-mercaptopropionyl glycine, a scavenger of ROS. The present study has suggested that the mAchR-NO-mitoK ATP -ROS pathway is a factor responsible for carbachol-induced suppression of contraction rhythm in cultured cardiac myocytes.