Corticosterone acutely prolonged N-methyl-d-aspartate receptor-mediated Ca2+ elevation in cultured rat hippocampal neurons

This work reports the first demonstration that corticosterone (CORT) has a rapid and transient effect on NMDA receptor-mediated Ca2+ signaling in cultured rat hippocampal neurons. Using single cell Ca2+ imaging, CORT and agonists of glucocorticoid receptors were observed to modulate the NMDA receptor-mediated Ca2+ signals in a completely different fashion from pregnenolone sulfate. In the absence of steroids, 100 micro m NMDA induced a transient Ca2+ signal that lasted for 30-70 s in 86.1% of the neurons prepared from postnatal rats (3-5 days old). After pre-treatment with 0.1-100 micro m CORT for 10-20 min, NMDA induced extremely prolonged Ca2+ elevation. This prolonged Ca2+ elevation was terminated by the application of MK-801 and followed by washing out of CORT. The proportion of CORT-modulated neurons within the NMDA-responsive cells increased from 25.1 to 95.5% when the concentration of CORT was raised from 0.1 to 50 micro m. Substitution of BSA-conjugated CORT produced essentially the same results. When hippocampal neurons were preincubated with 10 micro m cortisol and 1 micro m dexamethasone for 20 min, a very prolonged Ca2+ elevation was also observed upon NMDA stimulation. The CORT-prolonged Ca2+ elevation caused a long-lasting depolarization of the mitochondrial membrane, as observed with rhodamine 123. In contrast, incubation with 100 micro m pregnenolone sulfate did not considerably alter the time duration of NMDA-induced transient Ca2+ elevation, but caused a significant increase in the peak amplitude of Ca2+ elevation in hippocampal neurons. These results imply that high levels of CORT induce a rapid and non-genomic prolongation of NMDA receptor-mediated Ca2+ elevation, probably via putative membrane surface receptors for CORT in the hippocampal neurons.     

Phylogenetic analysis of bacteria preserved in a permafrost ice wedge for 25,000 years

Phylogenetic analysis of bacteria preserved within an ice wedge from the Fox permafrost tunnel was undertaken by cultivation and molecular techniques. The radiocarbon age of the ice wedge was determined. Our results suggest that the bacteria in the ice wedge adapted to the frozen conditions have survived for 25,000 years.     

Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds

Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First, VSR1 functioned as a sorting receptor for beta-conglycinin by recognizing the vacuolar targeting signal. Second, when green fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing GFP-CT24. From approximately 3,000,000 lines of M2 seeds, we obtained >100 fluorescent seeds and designated them green fluorescent seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1, gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage proteins.     

Arabidopsis KAM2/GRV2 is required for proper endosome formation and functions in vacuolar sorting and determination of the embryo growth axis

We isolated an Arabidopsis thaliana mutant, katamari2 (kam2), that has a defect in the organization of endomembranes. This mutant had deformed endosomes and formed abnormally large aggregates with various organelles. Map-based cloning revealed that kam2 is allelic to gravitropism defective 2 (grv2). The KAM2/GRV2 gene encodes a homolog of a DnaJ domain-containing RECEPTOR-MEDIATED ENDOCYTOSIS-8, which is considered to play a vital role in the endocytotic pathway from the plasma membrane to lysosomes in animal cells. Immunofluorescent staining showed that KAM2/GRV2 protein localizes on punctate structures, which did not merge with any markers for Golgi, trans-Golgi network, endosomes, or prevacuolar compartments. KAM2/GRV2, which does not have a predicted transmembrane domain, was peripherally associated with the membrane surface of uncharacterized compartments. KAM2/GRV2 was expressed at the early to middle stages of seed maturation. We found kam2 mis-sorted seed storage proteins by secreting them from cells, indicating that KAM2/GRV2 is involved in the transport of the proteins into protein storage vacuoles. kam2 had another defect in embryogenesis. Half of the developing kam2-1 cotyledons grew into the opposite space of the seeds before the walking stick-shaped embryo stage. Our findings suggest that KAM2/GRV2 is required for proper formation of the endosomes involving protein trafficking to the vacuoles and determination of growth axis of the embryo.     

Plasma carotenoid concentrations before and after supplementation with astaxanthin in middle-aged and senior subjects

A randomized, double-blind human trial was conducted to assess the effect on the plasma carotenoid concentration of 4- or 12-week astaxanthin supplementation (1 or 3 mg/d) of 20 Japanese middle-aged and senior subjects. The plasma carotenoid concentration was significantly higher after the astaxanthin supplementation than that before in both the 1 mg/d (10 subjects) and 3 mg/d (10 subjects) groups.