Zebrafish serotonin-N-acetyltransferase-2 gene regulation: pineal-restrictive downstream module contains a functional E-box and three photoreceptor conserved elements

Pineal function is defined by a set of very narrowly expressed genes that encode proteins required for photoperiodic transduction and rhythmic melatonin secretion. One of these proteins is serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT), which controls the daily rhythm in melatonin production. Here, pineal-specific expression of the zebrafish aanat-2 (zfaanat-2) was studied using in vivo transient expression analyses of promoter-reporter constructs; this revealed that specificity is determined by two regions located 12 kb away from each other. One is the 5'-flanking region, and the other is a 257-bp sequence, located 6 kb downstream of the transcribed region. This 3'-sequence, designated pineal-restrictive downstream module (PRDM), has a dual function: enhancement of pineal expression and inhibition of extrapineal expression. The former is an autonomic property of PRDM whereas the later function requires interaction with the upstream regulatory region of zfaanat-2. Functional analyses of the PRDM sequence revealed that three photoreceptor conserved elements (TAATC) and a single perfect E-box (CACGTG) are crucial for the dual function of PRDM. These results indicate that pineal specificity of zfaanat-2 is determined by the dual functionality of the PRDM and the interaction between upstream regulatory region and downstream photoreceptor conserved elements and E-box element.     

Six1 controls patterning of the mouse otic vesicle

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.     

Sequences of symmetry-breaking in phyllotactic transitions

This paper studies the transition of phyllotactic patterns by a group-theoretic approach. Typical phyllotactic patterns are represented here as dotted patterns on a cylinder, where the cylinder is regarded as the stem of a plant and the dots are points where leaves branch from the stem. We can then classify the symmetries of the alternate and opposite phyllotaxis into four types of groups, and clarify sequences of symmetry-breaking among these groups. The sequences turn out to correspond to transition paths of phyllotactic patterns found in the wild. This result shows the usefulness of classification of phyllotactic patterns based on their group symmetries. Moreover, the breaking of reflection symmetry is found to be an important rule for real phyllotactic transitions.     

An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease

Various types of stress, such as disruption of calcium homeostasis, inhibition of protein glycosylation and reduction of disulfide bonds, result in accumulation of misfolded proteins in the endoplasmic reticulum (ER). The initial cellular response involves removal of such proteins by the ER, but excessive and/or long-term stress results in apoptosis. In this study, we used a randomized ribozyme library and ER stress-mediated apoptosis (tunicamycin-induced apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify factors involved in this process. We identified a double-stranded RNA-dependent protein kinase (PKR) as one of the participants in this process. The level of nuclear PKR was elevated, but the level of cytoplasmic PKR barely changed in tunicamycin-treated SK-N-SH cells. Furthermore, tunicamycin also raised levels of phosphorylated PKR in the nucleus. We also detected the accumulation of phosphorylated PKR in the nuclei of autopsied brain tissues in Alzheimer's disease. Thus, PKR might play a role in ER stress-induced apoptosis and in Alzheimer's disease.     

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.     

A novel 11-residual peptaibol-derived carrier peptide for in vitro oligodeoxynucleotide delivery into cell

Using a pore- and channel-forming peptide, TV-XIIa, which is an 11-residual peptaibol isolated from the fungus Trichoderma viride, we developed a vehicle for the cellular delivery of such polar biologically active agents as antisense oligodeoxynucleotides (ODNs). To function as an ODN carrier, basic amino acids, 10-mer of lysine, were conjugated to the C-terminus of TV-XIIa and the designed carrier peptide, Ac-U-N-I-I-U-P-L-L-U-P-I-K-K-K-K-K-K-K-K-K-K-OH (U: alpha-aminoisobutyric acid), was synthesized by the Fmoc-based solid-phase method. The complex between the carrier peptide and ODNs, which was electrostatically formed, was capable of crossing the membranes of NIH3T3 cells and the ODNs were accumulated in the cytoplasm and the nucleus. However, the complex was not taken up by A549 cells. The translocation of the complex occurred at both 4 and 37 degrees C in NIH3T3 cells and did not seem to involve an energy-dependent endocytic process.     

Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDnas identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse KIAA-homologous genes since 2001. As an extension of this project, we also started to accumulate mouse cDNA clones homologous to the human FLJ cDNA clones which are another long cDNA resource produced in our institute. We have isolated the cDNA clones from size-fractionated cDNA libraries derived from five different mouse tissues and natural killer T-cells. Although the human FLJ cDNA clones were originally derived from human spleen libraries, one-third of their mouse homologues were obtained from the brain library. We designated these homologues "mFLJ" plus a 5-digit number and herein characterized 110 mFLJ cDNA clones. We assigned an integrity of the CDSs from the comparison of the 110 cDNA clones with the corresponding human FLJ cDNA clones. The average size of the 110 mouse cDNA sequences was 3.8 kb and that of the deduced amino acid sequences from their longest CDS in each cDNA was 663 amino acid residues. Homology and/or motif search against public databases revealed new domains and/or motifs in 26 mFLJ gene products which provide additional speculation regarding the function of FLJ genes.     

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.