Delayed treatment with cannabidiol has a cerebroprotective action via a cannabinoid receptor-independent myeloperoxidase-inhibiting mechanism

We examined the neuroprotective mechanism of cannabidiol, non-psychoactive component of marijuana, on the infarction in a 4 h mouse middle cerebral artery (MCA) occlusion model in comparison with Delta(9)-tetrahydrocannabinol (Delta(9)-THC). Release of glutamate in the cortex was measured at 2 h after MCA occlusion. Myeloperoxidase (MPO) and cerebral blood flow were measured at 1 h after reperfusion. In addition, infarct size and MPO were determined at 24 and 72 h after MCA occlusion. The neuroprotective effect of cannabidiol was not inhibited by either SR141716 or AM630. Both pre- and post-ischemic treatment with cannabidiol resulted in potent and long-lasting neuroprotection, whereas only pre-ischemic treatment with Delta(9)-THC reduced the infarction. Unlike Delta(9)-THC, cannabidiol did not affect the excess release of glutamate in the cortex after occlusion. Cannabidiol suppressed the decrease in cerebral blood flow by the failure of cerebral microcirculation after reperfusion and inhibited MPO activity in neutrophils. Furthermore, the number of MPO-immunopositive cells was reduced in the ipsilateral hemisphere in cannabidiol-treated group. Cannabidiol provides potent and long-lasting neuroprotection through an anti-inflammatory CB(1) receptor-independent mechanism, suggesting that cannabidiol will have a palliative action and open new therapeutic possibilities for treating cerebrovascular disorders.     

Evolutionary origin of a periodical mass‐flowering plant

The evolutionary origin of periodical mass‐flowering plants (shortly periodical plants), exhibiting periodical mass flowering and death immediately after flowering, has not been demonstrated. Within the genus Strobilanthes (Acanthaceae), which includes more than 50 periodical species, Strobilanthes flexicaulis on Okinawa Island, Japan, flowers gregariously every 6 years. We investigated the life history of S. flexicaulis in other regions and that of closely related species together with their molecular phylogeny to reveal the evolutionary origin of periodical mass flowering. S. flexicaulis on Taiwan Island was found to be a polycarpic perennial with no mass flowering and, in the Yaeyama Islands, Japan, a monocarpic perennial with no mass flowering. Molecular phylogenetic analyses indicated that a polycarpic perennial was the ancestral state in this whole group including S. flexicaulis and the closely related species. No distinctive genetic differentiation was found in S. flexicaulis among all three life histories (polycarpic perennial, monocarpic perennial, and periodical plant). These results suggest that among S. flexicaulis, the periodical mass flowering on Okinawa Island had evolved from the polycarpic perennial on Taiwan Island via the monocarpic perennial in the Yaeyama Islands. Thus, the evolution of life histories could have taken at the level of local populations within a species.     

Nerve growth factor stimulates regeneration of perivascular nerve,and induces the maturation of microvessels around the injured artery

An immature vasa vasorum in the adventitia of arteries has been implicated in induction of the formation of unstable atherosclerotic plaques. Normalization/maturation of the vasa vasorum may be an attractive therapeutic approach for arteriosclerotic diseases. Nerve growth factor (NGF) is a pleotropic molecule with angiogenic activity in addition to neural growth effects. However, whether NGF affects the formation of microvessels in addition to innervation during pathological angiogenesis is unclear. In the present study, we show a new role for NGF in neovessels around injured arterial walls using a novel in vivo angiogenesis assay.     

Heterotrimeric G proteins control stem cell proliferation through CLAVATA signaling in Arabidopsis

Cell-to-cell communication is a fundamental mechanism for coordinating developmental and physiological events in multicellular organisms. Heterotrimeric G proteins are key molecules that transmit extracellular signals; similarly, CLAVATA signaling is a crucial regulator in plant development. Here, we show that Arabidopsis thaliana Gβ mutants exhibit an enlarged stem cell region, which is similar to that of clavata mutants. Our genetic and cell biological analyses suggest that the G protein beta-subunit1 AGB1 and RPK2, one of the major CLV3 peptide hormone receptors, work synergistically in stem cell homeostasis through their physical interactions. We propose that AGB1 and RPK2 compose a signaling module to facilitate meristem development.     

Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining

A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the k_cat/K_m value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.     

Expression and complementation analyses of a chloroplast-localized homolog of bacterial RecA in the moss Physcomitrella patens

RecA protein is widespread in bacteria, and it plays a crucial role in homologous recombination. We have identified two bacterial-type recA gene homologs (PprecA1, PprecA2) in the cDNA library of the moss Physcomitrella patens. N-terminal fusion of the putative organellar targeting sequence of PpRecA2 to the green fluorescent protein (GFP) caused a targeting of PpRecA2 to the chloroplasts. Mutational analysis showed that the first AUG codon acts as initiation codon. Fusion of the full-length PpRecA2 to GFP caused the formation of foci that were colocalized with chloroplast nucleoids. The amounts of PprecA2 mRNA and protein in the cells were increased by treatment with DNA damaging agents. PprecA2 partially complemented the recA mutation in Escherichia coli. These results suggest the involvement of PpRecA2 in the repair of chloroplast DNA.     

Discovery of orally bioavailable cyclohexanol-based NR2B-selective NMDA receptor antagonists with analgesic activity utilizing a scaffold hopping approach

NR2B subunit containing N-methyl-d-aspartate (NMDA) receptor is an attractive target for chronic pain due to its involvement in disease states and its limited distribution in the central nervous system. Cyclohexanol-based leads 6a and 6c were identified as potent NR2B-selective NMDA antagonists utilizing a scaffold hopping approach. Further optimization of this series through replacement of the amide in the leads with an isoxazole and efforts to optimize the pharmacokinetic profiles led to the discovery of orally available brain penetrants 7k and 7l, which demonstrated analgesic activity in the mouse formalin test at early and late phases.     

Isolation of the <Emphasis Type="Italic">B</Emphasis> incompatibility factor mutants in <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

 B incompatibility factor mutants (Bmut) in Pleurotus ostreatus were recovered from common-B mating heterokaryons resulted from matings between wild-type monokaryons with different A but the same B factors (A1B2 and A2B2) after NTG mutagenesis. The mutant monokaryons such as A1B2mut and A2B2mut were observed to have regularly uninucleated hyphal cells and to be compatible with each other. Matings between A1B2mut and A2B2mut monokaryons produced stable heterokaryons (A1B2mut + A2B2mut) that had binucleated hyphal cells with true clamp connections and formed normal fruit-bodies. Mating tests using basidiospore progeny from each of these heterokaryons revealed the bipolar mating pattern. Genetic analysis suggested that the mutation of B factor in P. ostreatus might occur in the B incompatibility factor genes. Received: August 3, 2001 / Accepted: January 18, 2002     

Establishment of gene-trap and enhancer-trap systems in the moss Physcomitrella patens

Because of its simple body plan and ease of gene knockout and allele replacement, the moss Physcomitrella patens is often used as a model system for studies in plant physiology and developmental biology. Gene-trap and enhancer-trap systems are useful techniques for cloning genes and enhancers that function in specific tissues or cells. Additionally, these systems are convenient for obtaining molecular markers specific for certain developmental processes. Elements for gene-trap and enhancer-trap systems were constructed using the uidA reporter gene with either a splice acceptor or a minimal promoter. Through a high rate of transformation conferred by a method utilizing homologous recombination, 235 gene-trap and 1073 enhancer-trap lines were obtained from 5637 and 3726 transgenic lines, respectively. The expression patterns of these trap lines in the moss gametophyte varied. The candidate gene trapped in a gene-trap line YH209, which shows rhizoid-specific expression, was obtained by 5' and 3' RACE. This gene was named PpGLU, and forms a clade with plant acidic alpha-glucosidase genes. Thus, these gene-trap and enhancer-trap systems should prove useful to identify tissue- and cell-specific genes in Physcomitrella.     

Electron Microscope Observations on the Fine Structure of the Chloroplasts of Algae. II. The Chloroplasts of Caulerpa (Chlorophyceae)

The plastid ultrastructure of 12 species of Caulerpa (Chlorophyceae) was investigated by the electron microscope. A simpler pyrenoid structure was demonstrated in one species. The conspicuous concentric lamellar bodies, which are characteristic of algae with heteroplasty, were present both in the chloroplasts and the amyloplasts of all the species examined. The possible function and taxonomical value of the bodies were discussed.