Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae

Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology-based PCR method. The ORFs of full-length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of beta-amyrin and lupeol, suggesting that these genes encode beta-amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, beta-amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, beta-amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78-93%) to beta-amyrin synthases, and were located in the same branch as beta-amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the beta-amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to beta-amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves.     

Dual-tagging gene trap of novel genes in Drosophila melanogaster

A gene-trap system is established for Drosophila. Unlike the conventional enhancer-trap system, the gene-trap system allows the recovery only of fly lines whose genes are inactivated by a P-element insertion, i.e., mutants. In the gene-trap system, the reporter gene expression reflects precisely the spatial and temporal expression pattern of the trapped gene. Flies in which gene trap occurred are identified by a two-step screening process using two independent markers, mini-w and Gal4, each indicating the integration of the vector downstream of the promoter of a gene (dual tagging). mini-w has its own promoter but lacks a polyadenylation signal. Therefore, mini-w mRNA is transcribed from its own promoter regardless of the vector integration site in the genome. However, the eyes of flies are not orange or red unless the vector is incorporated into a gene enabling mini-w to be spliced to a downstream exon of the host gene and polyadenylated at the 3' end. The promoter-less Gal4 reporter is expressed as a fusion mRNA only when it is integrated downstream of the promoter of a host gene. The exons of trapped genes can be readily cloned by vectorette RT-PCR, followed by RACE and PCR using cDNA libraries. Thus, the dual-tagging gene-trap system provides a means for (i) efficient mutagenesis, (ii) unequivocal identification of genes responsible for mutant phenotypes, (iii) precise detection of expression patterns of trapped genes, and (iv) rapid cloning of trapped genes.     

Effects of Chronic Administration of Interferon α A/D on Serotonergic Receptors in Rat Brain

The effects of chronic administration of interferon (IFN; recombinant human IFN -A/D) on serotonergic binding sites in rat brain were investigated. IFN was injected daily for 2 weeks at a dose of 100000 I.U./kg, (i.p.) in male Wistar rats. IFN did not alter either [³H]ketanserin binding to 5-HT_2A receptors or [³H]paroxetine binding to 5-HT transporters. Scatchard analysis of [³H]8-hydroxy-dipropylaminotetraline (8-OH-DPAT) binding to 5-HT_1A receptors demonstrated the presence of high- and low-affinity binding sites in both treatment and control groups. IFN significantly increased both Kd and Bmax measures of [³H]8-OH-DPAT binding at low-affinity binding sites, but not at the high-affinity sites. These results suggest that IFN affects the low-affinity 5-HT_1A receptors sites and may be involved in the development of IFN-induced psychiatric disturbances.     

Progression of flagellar stages during artificially delayed motility initiation in sea urchin sperm

Transition from immotile to motile flagella may involve a series of states, in which some of regulatory mechanisms underlying normal flagellar movement are working with others being still suppressed. To address ourselves to the study of starting transients of flagella, we analyzed flagellar movement of sea urchin sperm whose motility initiation had been retarded in an experimental solution, so that we could capture the instance at which individual spermatozoa began their flagellar beating. Initially straight and immotile flagella began to shiver at low amplitude, then propagated exclusively the principal bend (P bend), and finally started stable flagellar beating. The site of generation of the P bend in the P-bend propagating stage varied in position in the basal region up to 10 microm from the base, indicating that the ability of autonomous bend generation is not exclusively possessed by the very basal region but can be unmasked throughout a wider region when the reverse bend (R bend) is suppressed. The rate of change in the shear angle, the curvature of the R bend and the frequency and regularity of beating substantially increased upon transition from P-bend propagating to full-beating, while the propagation velocity of bends remained unchanged. These findings indicate that artificially delayed motility initiation may accompany sequential modification of the motile system and that mechanisms underlying flagellar motility can be analyzed separately under experimentally retarded conditions.     

Synthesis and antinematodal activity of 3-n-alkylphenols

Several 3-alkylphenols including 3-undecylphenol, which was isolated from a Sumatran rainforest plant, were synthesized to investigate their antinematodal activity against the phytopathogenic nematodes, Bursapherencus xylophilus. A three-step synthesis involving the treatment of 2-cyclohexen-1-one with the Grignard reagent, oxidation of the resulting 1-alkyl-2-cyclohexen-1-ol and subsequent aromatization of 3-alkyl-2-cyclohexen-1-one successfully afforded such phenols. Among the 3-alkylphenols, 3-nonylphenol showed the highest activity, while 3-decylphenol and 3-undecylphenol also showed high activity.     

Reduced hypothermic and hypnotic responses to ethanol in PACAP-deficient mice

Using pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice, we investigated whether PACAP is involved in the intoxicating effects of ethanol. The structure of PACAP is highly conserved during evolution, and in Drosophila, loss-of-function mutations in a PACAP-like neuropeptide gene, amnesiac, result in impairment of memory retention and increased sensitivity to ethanol. In mice, PACAP deficiency is associated with impaired memory performance and hippocampal long-term potentiation (LTP), however, sensitivity to ethanol has not been well investigated. Here, we addressed this issue in our recently developed PACAP-deficient mice. Sleep time (duration of the loss of righting reflex) was markedly shortened in PACAP-deficient mice compared with wild-type, although latency to the loss of righting reflex was not different between the two groups. Ethanol-induced hypothermia in wild-type control mice was significantly reduced in PACAP-deficient mice. Blood ethanol levels were not different between the two groups, excluding the possibility of increased ethanol metabolism. Thus, in contrast to that in Drosophila, PACAP deficiency in mammals caused a reduced sensitivity to ethanol. However, in both cases, PACAP or amnesiac products are likely to play significant roles in modifying the intoxicating effects of ethanol.     

Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities

An integrated study on cell growth, enzyme activities and carbon flux redistribution was made to investigate how the central metabolism of Escherichia coli changes with the knockout of genes in the oxidative pentose phosphate pathway (PPP). Mutants deficient in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were constructed by disrupting the zwf and gnd genes and were grown in minimal media with two different carbon sources, such as glucose or pyruvate. It was shown that the knockout of either gnd or zwf gene did not affect the cell growth rate significantly, but the cellular metabolism was changed. While the specific substrate uptake rate and the specific carbon dioxide evolution rate for either mutant grown on glucose were higher than those obtained for the parent strain, these two rates were markedly decreased in mutants grown on pyruvate. The measurement of enzyme activities implied a significant change in metabolism, when alternative pathways such as the Entner–Doudoroff pathway (EDP) and the malic enzyme pathway were activated in the gnd mutant grown on glucose. As compared with the parent strain, the activities of phosphoglucose isomerase were increased in mutants grown on glucose but decreased in mutants grown on pyruvate. The metabolic flux redistribution obtained based on ¹³C-labeling experiments further indicated that the direction of the flux through the non-oxidative PPP was reversed in response to the gene knockout. Moreover, the knockout of genes caused an increased flux through the tricarboxlic acid cycle in mutants grown on glucose but caused a decrease in the case of using pyruvate. There was also a negative correlation between the fluxes through malic enzyme and isocitrate dehydrogenase in the mutants; and a positive correlation was found between the fluxes through malic enzyme and phosphoenolpyruvate carboxylase.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Role of primary sensorimotor cortex and supplementary motor area in volitional swallowing: a movement-related cortical potential study

We investigated the role of the cerebral cortex, particularly the face/tongue area of the primary sensorimotor (SMI) cortex (face/tongue) and supplementary motor area (SMA), in volitional swallowing by recording movement-related cortical potentials (MRCPs). MRCPs with swallowing and tongue protrusion were recorded from scalp electrodes in eight normal right-handed subjects and from implanted subdural electrodes in six epilepsy patients. The experiment by scalp EEG in normal subjects revealed that premovement Bereitschaftspotentials (BP) activity for swallowing was largest at the vertex and lateralized to either hemisphere in the central area. The experiment by epicortical EEG in patients confirmed that face/tongue SMI and SMA were commonly involved in swallowing and tongue protrusion with overlapping distribution and interindividual variability. BP amplitude showed no difference between swallowing and tongue movements, either at face/tongue SMI or at SMA, whereas postmovement potential (PMP) was significantly larger in tongue protrusion than in swallowing only at face/tongue SMI. BP occurred earlier in swallowing than in tongue protrusion. Comparison between face/tongue SMI and SMA did not show any difference with regard to BP and PMP amplitude or BP onset time in either task. The preparatory role of the cerebral cortex in swallowing was similar to that in tongue movement, except for earlier activation in swallowing. Postmovement processing of swallowing was lesser than that of tongue movement in face/tongue SMI; probably suggesting that the cerebral cortex does not play a significant role in postmovement processing of swallowing. SMA plays a supplementary role to face/tongue SMI both in swallowing and tongue movements.     

An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease

Various types of stress, such as disruption of calcium homeostasis, inhibition of protein glycosylation and reduction of disulfide bonds, result in accumulation of misfolded proteins in the endoplasmic reticulum (ER). The initial cellular response involves removal of such proteins by the ER, but excessive and/or long-term stress results in apoptosis. In this study, we used a randomized ribozyme library and ER stress-mediated apoptosis (tunicamycin-induced apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify factors involved in this process. We identified a double-stranded RNA-dependent protein kinase (PKR) as one of the participants in this process. The level of nuclear PKR was elevated, but the level of cytoplasmic PKR barely changed in tunicamycin-treated SK-N-SH cells. Furthermore, tunicamycin also raised levels of phosphorylated PKR in the nucleus. We also detected the accumulation of phosphorylated PKR in the nuclei of autopsied brain tissues in Alzheimer's disease. Thus, PKR might play a role in ER stress-induced apoptosis and in Alzheimer's disease.