Essentiality of a Newly Identified Carbohydrate-Binding Module for the Function of CelB (BH0603) from the Alkaliphilic Bacterium Bacillus halodurans

CelB (BH0603) from Bacillus halodurans is a modular glycoside hydrolase with a family 5 catalytic module, an immunoglobulin-like module, and module PfamB of unknown function. The recombinant PfamB module bound to Avicel and was essential for CelB hydrolytic function. We propose that module PfamB be designated a new carbohydrate-binding module.     

Dual-color system for simultaneously monitoring intracellular Ca(2+) and ATP dynamics

Although Ca(2+) regulates energy metabolism through diverse pathways, there have been no methods to monitor both Ca(2+) dynamics and metabolic activity simultaneously. Here we report a novel system for simultaneously monitoring intracellular Ca(2+) and ATP levels using a blue-emitting photoprotein and a red-emitting beetle luciferase. Using this system, we monitored the dynamic changes simultaneously in both intracellular Ca(2+) and ATP levels during chondrogenesis. We have found that both intracellular Ca(2+) and ATP levels oscillated and their oscillations have a nearly antiphase relationship with each other. The dual-color monitoring system is useful for studying the relationship between Ca(2+) dynamics and energy metabolic pathways.     

11-ketotestosterone synchronously induces oocyte development and silvering-related changes in the Japanese eel, Anguilla japonica

To evaluate the effects of sex steroids on silvering in the Japanese eel, Anguilla japonica, the development of oocytes, eye size, digestive tract, and swim bladder were studied in relation to observations of the profiles of plasma levels of sex steroids (estradiol 17β, E2; testosterone, T; 11-ketotestosterone; 11-KT) during silvering for each sex and by administrating 11-KT to yellow eels. All steroids examined in the study increased in female eels after silvering had begun, whereas in males, only 11-KT increased significantly, and no statistical differences were found in plasma levels of E2 and T between eels in both developmental stages. 11-KT appeared to induce the early stage of oocyte growth, enlargement of the eyes, degeneration of the digestive tract and the development of the swim bladder. This suggested that 11-KT synchronously accelerates early development of the ovaries and the morphological changes, possibly in adaption to oceanic migration, and that 11-KT is one of the most important factors in early stages of development in the Japanese eel, as it appears to be in other anguillid eels.     

Spectrochemistry and artificial color modulation of Cypridina luminescence: indirect evidence for chemiexcitation of a neutral dioxetanone and emission from a neutral amide

We demonstrate that the emission energy of the cypridinid oxyluciferin depends strongly on the polarity and, to a lesser extent, on the basicity of the medium. The emission color can be tuned by environment effects from violet to green. We also provide firm evidence that the natural system utilizes the neutral form of the emitter.     

Effect of pre-incubation irradiance on survival of cryopreserved gametophytes of Undaria pinnatifida (Phaeophyta) and morphology of sporophytes formed from the gametophytes

Gametophyte strains originating from indigenous sporophytes of Undaria pinnatifida (Harvey) Suringar in Iwate Prefecture, Northeast Japan, were maintained for 9–10 months at 45 μmol photons m⁻² s⁻¹. Before cryopreservation in liquid nitrogen for more than 12 h (1–14 days) using a two-step cooling method with a mixture of cryoprotectants (10% l-proline and 10% glycerol), these were pre-incubated for 2, 4 and 8 months at 15 μmol photons m⁻² s⁻¹. After 1 week of thawing, no surviving gametophytes were detected in the strains without pre-incubation, but both the female and male gametophytes, pre-incubated for more than 4 months, showed high survival rates (43–60% for females and 64–100% for males). This revealed the induction of freezing tolerance by incubation at low irradiance. Thereafter, sporophytes derived from cryopreserved gametophytes and subcultured gametophytes, stored under pre-incubation conditions, were formed from the strain, and a morphological comparison was conducted with 10 characters (stipe length, stipe wet weight, blade length, blade wet weight, blade width, incision depth, blade thickness, sporophyll length, sporophyll wet weight, and sporophyll width). The morphology of the sporophytes formed from the cryopreserved gametophytes corresponded well with that of the subcultured gametophytes from the same strain. The results suggest that the cryopreservation method is applicable for preserving culture stocks of U. pinnatifida to be used in mariculture.     

The GLABRA2 homeodomain protein directly regulates CESA5 and XTH17 gene expression in Arabidopsis roots

Arabidopsis root hair formation is determined by the patterning genes CAPRICE ( CPC ), GLABRA3 ( GL3 ), WEREWOLF ( WER ) and GLABRA2 ( GL2 ), but little is known about the later changes in cell wall material during root hair formation. A combined Fourier-transform infrared microspectroscopy–principal components analysis (FTIR-PCA) method was used to detect subtle differences in the cell wall material between wild-type and root hair mutants in Arabidopsis. Among several root hair mutants, only the gl2 mutation affected root cell wall polysaccharides. Five of the 10 genes encoding cellulose synthase ( CESA1 – 10 ) and 4 of 33 xyloglucan endotransglucosylase ( XTH1 – 33 ) genes in Arabidopsis are expressed in the root, but only CESA5 and XTH17 were affected by the gl2 mutation. The L1-box sequence located in the promoter region of these genes was recognized by the GL2 protein. These results indicate that GL2 directly regulates cell wall-related gene expression during root development.     

The nuclear receptor gene nhr-25 plays multiple roles in the Caenorhabditis elegans heterochronic gene network to control the larva-to-adult transition

Developmental timing in the nematode Caenorhabditis elegans is controlled by heterochronic genes, mutations in which cause changes in the relative timing of developmental events. One of the heterochronic genes, let-7, encodes a microRNA that is highly evolutionarily conserved, suggesting that similar genetic pathways control developmental timing across phyla. Here we report that the nuclear receptor nhr-25, which belongs to the evolutionarily conserved fushi tarazu-factor 1/nuclear receptor NR5A subfamily, interacts with heterochronic genes that regulate the larva-to-adult transition in C. elegans. We identified nhr-25 as a regulator of apl-1, a homolog of the Alzheimer's amyloid precursor protein-like gene that is downstream of let-7 family microRNAs. NHR-25 controls not only apl-1 expression but also regulates developmental progression in the larva-to-adult transition. NHR-25 negatively regulates the expression of the adult-specific collagen gene col-19 in lateral epidermal seam cells. In contrast, NHR-25 positively regulates the larva-to-adult transition for other timed events in seam cells, such as cell fusion, cell division and alae formation. The genetic relationships between nhr-25 and other heterochronic genes are strikingly varied among several adult developmental events. We propose that nhr-25 has multiple roles in both promoting and inhibiting the C. elegans heterochronic gene pathway controlling adult differentiation programs.     

Methods for detection, isolation and culture of mouse and human invariant NKT cells

This protocol describes methods to identify, purify and culture CD1d restricted invariant natural killer T (iNKT) cells from mouse tissue or human blood samples. The methods for identification and purification of iNKT cells are based on the interaction between iNKT cell receptor and its ligand. The iNKT cell receptor is composed of the invariant V alpha 14 J alpha 18/V beta 8.2 in mice or V alpha 24 J alpha 18/V beta 11 in humans and is expressed only on iNKT cells but not on conventional T cells. The iNKT cell antigen receptor in both species recognizes alpha-galactosylceramide (alpha-GalCer) presented by the MHC class I-like CD1d. Thus, alpha-GalCer-loaded CD1d dimer can be used for analysis and purification by fluorescence-activated cell sorting (FACS). Isolation of 1 x 10(6) purified iNKT cells from mouse thymus, spleen or liver requires 5-6 mice and takes 1-2 h for mononuclear cell preparation from mouse tissues, 1.5 h for enrichment by magnetic beads and 4 h for detection and purification of the iNKT cells by FACS. In the case of isolation of human peripheral blood mononuclear cells (PBMCs) from whole blood, it takes 2 h and requires 5 ml of blood to obtain 5 x 10(6) PBMCs, which contain 500-25,000 iNKT cells.