Sequence analysis of the α-galactosidase <Emphasis Type="Italic">MEL</Emphasis> gene governing the efficient production of ethanol from raffinose-rich molasses in the yeast <Emphasis Type="Italic">Lachancea thermotolerans</Emphasis>

The yeast Lachancea thermotolerans, formerly Kluyveromyces thermotolerans, was tested for the ethanol fermentation of raffinose-rich molasses. Two melibiose-fermenting strains, NBRC 10066 and NBRC 10067, produced more ethanol than eight other strains. The concentration of ethanol synthesized by NBRC 10066 was slightly higher than that by NBRC 10067, probably on the basis of the expression of α-galactosidase. The regions corresponding to the α-galactosidase MEL1 gene of Saccharomyces cerevisiae were amplified. The nucleotide sequences of the two genes designated as MELth1 and MELth2 revealed single open reading frames of 1,416 bp encoding 472 amino acids but differed from each other in one base that converted the amino acid composition. The sequences of the 5′-upstream region from −1 to −515 of the two genes are identical except for one base.     

Fluorescent-labeled single-strand ATP aptamer DNA: chemo- and enantio-selectivity in sensing adenosine

One of the intriguing applications of aptamers is sensing molecules. In principle, an aptamer can specifically recognize and bind to a unique ligand, leading to a structural change of an aptamer. By acquiring information for the structural change, the detection of the ligand can be achieved. To design and explore an aptamer molecule to detect adenosine, we have synthesized some ATP aptamer variants labeled with donor and acceptor fluorophores. Although the fluorescent response of the aptamer variants was highly dependent on experimental temperature, we have found one of the variants showing suitable fluorescent response by titration with adenosine. The aptamer variant showed remarkable selectivity for adenosine over the other ribonucleosides. On the other hand, the enantio-specificity of the aptamer variant in the ligand recognition was not enough to selectively detect d-adenosine over l-adenosine.     

Adjusting estimative prediction limits

This note presents a direct adjustment of the estimative predictionlimit to reduce the coverage error from a target value to third-orderaccuracy. The adjustment is asymptotically equivalent to thoseof Barndorff-Nielsen & Cox (1994, 1996) and Vidoni (1998).It has a simpler form with a plug-in estimator of the coverageprobability of the estimative limit at the target value.     

Killing activity of human umbilical cord blood-derived TCRValpha24<Superscript>+</Superscript> NKT cells against normal and malignant hematological cells in vitro: a comparative study with NK cells or OKT3 activated T lymphocytes or with adult peripheral blood NKT cells

PURPOSE: We aimed to determine the effects of human umbilical cord blood (UCB)-derived natural killer T (NKT) cells as immunological effectors against hematological malignancies, as well as auto- or allo-dendritic cells (DCs) or EB transformed cell lines (EBCLs). MATERIALS: TCRValpha24(+) Vbeta11(+) UCB- or PB-NKT cells were isolated by sorting and activated by alpha-galactosylceramide-pulsed autologous DCs. UCB-NK cells were induced from CD34(+) cells by stem cell factor plus IL-15. UCB-T cells were primarily activated by anti-CD3 monoclonal antibody. All those effectors were cultured with IL-2 (100 U/ml), and their cytotoxic activities were evaluated by (51)Cr-release assay. UCB-NKT cells were cultured with IL-12, IL-18 or higher dose of IL-2 (1000 U/ml), and again tested for the cytotoxicity against selected targets. RESULTS: UCB-NKT cells exhibited a pattern of killing activity against various hematological malignancies similar to that of UCB-NK cells, but could not kill K562, which was a vulnerable target for NK cells. The level of activity was quite similar to that of PB-NKT cells. In contrast, OKT-3-activated UCB-T lymphocytes showed a stronger and wider spectrum of killing compared with UCB-NK or NKT cells. IL-12, IL-18 or a higher dose of IL-2 upregulated the activity; however several targets, including fresh leukemic cells, still remained resistant. NKT cells killed auto- or allo-DCs at a level similar to that of T cells, but could not kill allo-EBCLs, which were efficiently killed by T cells. While NK cells showed only marginal or no killing against DC or EBCLs. DISCUSSION: The anti-cancer activity of human NKT cells depends on the concentrations or the combination of Th1-cytokines. Basically, those cells might not be contributing to the immune surveillance of hematological malignancies, as shown by a relatively low cytotoxicity against malignant cells, together with the quite strong killing against auto-DCs.     

Cell‐autonomous signal transduction in the Xenopus egg Wnt/β‐catenin pathway

Wnt proteins are thought to bind to their receptors on the cell surfaces of neighboring cells. Wnt8 likely substitutes for the dorsal determinants in Xenopus embryos to dorsalize early embryos via the Wnt/β‐catenin pathway. Here, we show that Wnt8 can dorsalize Xenopus embryos working cell autonomously. Wnt8 mRNA was injected into a cleavage‐stage blastomere, and the subcellular distribution of Wnt8 protein was analyzed. Wnt8 protein was predominantly found in the endoplasmic reticulum (ER) and resided at the periphery of the cells; however, this protein was restricted to the mRNA‐injected cellular region as shown by lineage tracing. A mutant Wnt8 that contained an ER retention signal (Wnt8‐KDEL) could dorsalize Xenopus embryos. Finally, Wnt8‐induced dorsalization occurred only in cells injected with Wnt8 mRNA. These experiments suggest that the Wnt8 protein acts within the cell, likely in the ER or on the cell surface in an autocrine manner for dorsalization.     

Amyloplasts and vacuolar membrane dynamics in the living graviperceptive cell of the Arabidopsis inflorescence stem

We developed an adequate method for the in vivo analysis of organelle dynamics in the gravity-perceptive cell (endodermis) of the Arabidopsis thaliana inflorescence stem, revealing behavior of amyloplasts and vacuolar membranes in those cells. Amyloplasts in the endodermis showed saltatory movements even before gravistimulation by reorientation, and these movements were confirmed as microfilament dependent. From our quantitative analysis in the wild type, the gravity-oriented movement of amyloplasts mainly occurred during 0 to 3 min after gravistimulation by reorientation, supporting findings from our previous physiological study. Even after microfilament disruption, the gravity-oriented movement of amyloplasts remained. By contrast, in zig/sgr4 mutants, where a SNARE molecule functioning in vacuole biogenesis has been disrupted, the movement of amyloplasts in the endodermis is severely restricted both before and after gravistimulation by reorientation. Here, we describe vacuolar membrane behavior in these cells in the wild-type, actin filament-disrupted, and zig/sgr4 mutants and discuss its putatively important features for the perception of gravity. We also discuss the data on the two kinds of movements of amyloplasts that may play an important role in gravitropism: (1) the leading edge amyloplasts and (2) the en mass movement of amyloplasts.     

Accurate Determination of S-Phase Fraction in Proliferative Cells by Dual Fluorescence and Peroxidase Immunohistochemistry with 5-Bromo-2��-Deoxyuridine (BrdU) and Ki67 Antibodies

To ensure the maintenance of tissues in mammals, cell loss must be balanced with cell production, the proliferative activity being different from tissue to tissue. In this article, the authors propose a new method for the quantification of the proliferative activity, defined as the S-phase fraction of actively cycling cells, by dual labeling with fluorescence and peroxidase immunohistochemistry using BrdU (marker of S-phase) and Ki67 antibodies (marker of G₁-, S-, G₂-, and M-phases) after a one-step antigen retrieval. In the generative cell zones of fundic and pyloric glandular stomachs, where the majority of cells were cycling, the authors measured a proliferative activity of 31%. In the epithelium of the forestomach and the skin, where cycling cells are intermingled with G₀ and differentiated cells, proliferative activities were 21% and 13%, respectively. In the adrenal cortex, in which cycling cells were sparsely distributed, the proliferative activity reached 32%. During the regenerative process in the skin after a lesion, the proliferative activity increased in proximity to the wound. The present one-step dual-labeling method has revealed that the proliferative activity is different between tissues and depends on the physiological or pathological state.     

RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER AND 5.8S REGIONS IN JAPANESE ALEXANDRIUM SPECIES (DINOPHYCEAE)1

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment-length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra-specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS-1. Some restriction fragments from WKS-1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS-1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.     

Environment�CKHV�Ccarp�Chuman linkage as a model for environmental diseases

To predict outbreaks of infectious disease and to prevent epidemics, it is essential not only to conduct pathological studies but also to understand the interactions between the environment, pathogen, host and humans that cause and spread infectious diseases. Outbreaks of mass mortality in carp caused by Cyprinid herpesvirus 3 (CyHV-3), formerly known as koi herpesvirus (KHV), disease have occurred worldwide since the late 1990s. We proposed an environment?CKHV?Ccarp?Chuman linkage as a conceptual model for ??environmental diseases?? and specify research subjects that might be necessary to construct and shape this linkage.     

Daily spawning and development of sensitivity to gonadotropin and maturation-inducing steroid in the oocytes of the bambooleaf wrasse, Pseudolabrus japonicus

The cycle of oocyte development of the bambooleaf wrasse, Pseudolabrus japonicus, was studied to elucidate the endocrinological mechanism of oocyte maturation in a marine teleost. A single female reared with two males spawned every day for 17 days in captivity, indicating that this species is a daily spawner. Ovarian histology revealed that germinal vesicle migration of the largest oocytes progressed from 12:00 to 3:00 h, and germinal vesicle breakdown (GVBD) was completed at 6:00 h. Ovulation and spawning occurred between 6:00 and 9:00 h. The effectiveness of human chorionic gonadotropin (HCG) and 17,20-dihydroxy-4-pregnen-3-one (17,20-P), which is one of the most potent steroidal inducers of GVBD in bambooleaf wrasse oocytes, in inducing final oocyte maturation was examined at eight different times of the day. The responsiveness of the oocyte to HCG and steroid differed at different times of the day. The GVBD could be induced by HCG but not 17,20-P at 9:00 h. Between 12:00 and 18:00 h, not only HCG but also 17,20-P induced GVBD. Both GVBD and ovulation spontaneously occurred between 0:00 and 6:00 h without any hormonal treatment. These results clearly showed that the oocyte of the bambooleaf wrasse possessed a diurnal maturation cycle. Responsiveness of oocytes to HCG appeared earlier than responsiveness to 17,20-P. This suggests that sensitivity to 17,20 -P is induced by gonadotropic hormone (GTH).