Detection of brain-specific gene expression in brain cells in primary culture: a novel promoter assay based on the use of a retrovirus vector.

Promoter activities of the brain-specific genes for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were investigated in brain cells in primary culture with the use of a novel retrovirus vector, pIP200. With this vector, promoter activity can be expressed in terms of beta-galactosidase activity. Differentiation of the primary brain cells to mature glial cells was not affected by treatment with the pIP200 virus vector. The 256-bp 5'-flanking region of the GFAP gene directed astrocyte-specific expression of lacZ. It was silent in fibroblasts, even in multiple copies. The 1.3-kb 5'-flanking region of the MBP gene exhibited strict tissue (oligodendrocyte) specificity under the present assay method but showed some leakiness when integrated into the chromosome in multiple copies. Promoter regions conferring cell type specificity in brain were effectively identified by the present method.     

Establishment and characterization of Roberts syndrome and SC phocomelia model medaka (Oryzias latipes)

Roberts syndrome and SC phocomelia (RBS/SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/SC by reverse genetics. The RBS/SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80S mutant. The R80S mutant is the animal model for RBS/SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/SC.     

Structural insight into receptor-selectivity for lurasidone

Lurasidone is a novel antipsychotic agent with high affinity for dopamine D₂, 5-hydroxyltryptamine 5-HT_2A, and 5-HT₇ receptors. Lurasidone has negligible affinity for histamine H₁ and muscarinic M₁ receptors, which are thought to contribute to side effects such as weight gain, sedation, and worsening of cognitive deficits. Our interests focus on why lurasidone has such high selectivity for only a part of these aminergic G-protein coupled receptors (GPCRs) and the different binding profile from ziprasidone, which has the same benzisothiazolylpiperazine moiety as lurasidone. In order to address these issues, we constructed structural models of lurasidone–GPCR complexes by homology modeling of receptors, exhaustive docking of ligand, and molecular dynamics simulation-based refinement of complexes. This computational study gave reliable structural models for D₂, 5-HT_2A, and 5-HT₇, which had overall structural complementarities with a salt bridge anchor at the center of the lurasidone molecule, but not for H₁ and M₁ owing to steric hindrance between the norbornane-2,3-dicarboximide and/or cyclohexane part of lurasidone and both receptors. By comparison with the structural models of olanzapine–GPCRs and ziprasidone–GPCRs constructed using the same computational protocols, it was suggested that the bulkiness of the norbornane-2,3-dicarboximide part and the rigidity and the bulkiness of the cyclohexyl linker gave lurasidone high selectivity for the desired aminergic GPCRs. Finally, this structural insight was validated by a binding experiment of the novel benzisothiazolylpiperazine derivatives. This knowledge on the structural mechanism behind the receptor selectivity should help to design new antipsychotic agents with preferable binding profiles, and the established computational protocols realize virtual screening and structure-based drug design for other central nervous system drugs with desired selectivity for multiple targets.     

Control of potato aphids by the addition of barley strips in potato fields: a successful example of vegetation management

The densities of barley and potato aphids, their natural enemies and hyperparasitoids were assessed in three experimental potato fields as a case study to investigate the effectiveness of the addition of barley strips in potato fields for conservation biological control. These fields were located in a low plant-diversity landscape, but common aphid species and their natural enemies were present. The barley strips in the potato fields were found to support different species of aphids of potato, but these different sets of aphids shared a common set of natural enemies. The amount of time between peak aphid densities and peaks of their natural enemies' populations was shorter in the potato fields than in the barley strips. The levels of winged aphids in a potato monoculture field were significantly higher than those in a field with barley strips. The wingless and winged aphid populations in the field without barley strips was almost three times higher than in the fields with the barley strips, as measured at the peak aphid density. This result is one of few examples of the application of the conservation effect of greenhouse banker plants on outdoor crops.     

Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure

The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca(2+) ([Ca(2+)](ext))-induced [Ca(2+)](cyt) transients and stomatal closure in Arabidopsis. CAS (Ca(2+) sensing receptor) is a plant-specific putative Ca(2+)-binding protein that was originally proposed to be a plasma membrane-localized external Ca(2+) sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N-terminus acidic Ca(2+)-binding region is likely exposed to the stromal side of the membrane. The phenotype of T-DNA insertion CAS knockout mutants and cDNA mutant-complemented plants revealed that CAS is essential for stomatal closure induced by external Ca(2+). In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca(2+). Furthermore, using the transgenic aequorin system, we showed that [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane-localized CAS is essential for [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients and stomatal closure.