Cadherin activity is required for activity-induced spine remodeling

Neural activity induces the remodeling of pre- and postsynaptic membranes, which maintain their apposition through cell adhesion molecules. Among them, N-cadherin is redistributed, undergoes activity-dependent conformational changes, and is required for synaptic plasticity. Here, we show that depolarization induces the enlargement of the width of spine head, and that cadherin activity is essential for this synaptic rearrangement. Dendritic spines visualized with green fluorescent protein in hippocampal neurons showed an expansion by the activation of AMPA receptor, so that the synaptic apposition zone may be expanded. N-cadherin-venus fusion protein laterally dispersed along the expanding spine head. Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion. Inhibition of actin polymerization with cytochalasin D abolished the spine expansion. Together, our data suggest that cadherin-based adhesion machinery coupled with the actin-cytoskeleton is critical for the remodeling of synaptic apposition zone.     

Fast-track DRESSA: a bioassay for fast, sensitive, and selective detection of halogenated and polycyclic aromatic hydrocarbons

Dioxin and related chemicals cause a variety of toxic and biological effects via the aryl hydrocarbon receptor (AhR). We recently reported a mammalian cell-based bioassay system (dioxin-responsive-element-based sensing via secreted alkaline phosphatase; DRESSA) that can detect dioxin and dioxin-like chemicals with high sensitivity. In this report, we describe an advanced method (designated "fast-track DRESSA") that achieves fast, selective, and sensitive detection of dioxin and other toxic compounds. By optimization of assay conditions on cell number and serum concentration, the fast-track DRESSA enabled detection of 0.5 pM 2,3,7,8-tetrachlorodibenzo-p-dioxin within 6 h. It also enabled detection of 10 pM 3-methylcholanthrene, 100 pM benzo[a]pyrene, and 100 pM beta-naphthoflavone within 6-16 h. By combination with the AhR antagonist alpha-naphthoflavone, nonspecific, false-positive responses could be eliminated. Because of its time-saving property and easiness, sensitiveness, and specificity, the fast-track DRESSA would be advantageous for high-throughput screening of dioxin and dioxin-like compounds in environmental samples.     

Molecular Characterization of Mn-superoxide Dismutase and Gene Expression Studies in Dietary Restricted <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> Rotifers

Superoxide dismutases (SODs) promote a conversion of harmful reactive oxygen species (ROS) to relatively moderate forms, resulting in the extension of lifespan in the nematode Caenorhabditis elegans under caloric restriction. The lifespan of the rotifer Brachionus plicatilis is also markedly extended by caloric restriction. We, therefore, cloned cDNA encoding SOD activated with Mn (Mn SOD) from B. plicatilis and examined its expression pattern in rotifers raised with energy restricted diet. The full length deduced amino acid sequence of the rotifer Mn SOD showed 61% identity with the C. elegans ortholog. Four amino acid residues that are essential to the binding of this enzyme to Mn were conserved in the rotifer Mn SOD. Subsequently we examined the mRNA expression patterns of Mn SOD using highly sensitive quantitative real-time PCR for various rotifer populations that are likely to differ in their lifespans in experiments on calorie restricted diets. The accumulated mRNA levels of Mn SOD were found to increase in supposedly long-lived rotifers. These results suggest that Mn SOD is possibly related to the aging of B. plicatilis.     

A 90-kDa glycoprotein exhibited stage-specific expression in meiotic germ cells in rat testes

We produced a monoclonal antibody, designated MC301, against the extract of testicular cells from 12-day-old rats. This age corresponds to the onset of meiosis during testis development. MC301 specifically recognized a 90-kDa glycoprotein, GP90-MC301, which was ubiquitously expressed in various tissues and localized predominantly in the Golgi area of epithelial cells. In adult testes, stage-specific intense expression of GP90-MC301 was shown in the cytoplasm of meiotic spermatogenic cells from the preleptotene to mid-pachytene stages. Immunoelectron microscopy demonstrated that the glycoprotein was localized in spermatocytes on protein synthesis-related organelles such as the Golgi apparatus, endoplasmic reticulum, and nuclear envelope. The plasma membrane of spermatocytes and the intercellular space surrounding the cells were also immunoreactive. No specific immunoreactivity was found on the organelles in other testicular cells. A considerable amount of the glycoprotein was detected in the extracellular fluid of the testes. These results suggest that GP90-MC301 is produced mainly by spermatocytes in the testis and secreted into the surrounding intercellular space. The evidence for developmentally regulated expression of GP90-MC301 in the meiotic spermatogenic cells suggests a possible role for the glycoprotein in male germ cell meiosis.     

Optical measurement of age-related calcification in human blood vessels

Vascular calcification is commonly associated with aging. Quantification of calcium accumulation in vessel walls is important in understanding the mechanisms of vascular calcification. To elucidate age-related change of calcification, site dependence of calcification, and the effect of hemodynamic stress on calcification, we measured calcium contents in various blood vessels with atomic emission spectrometry and simulated blood flow in the vessels by computational fluid dynamics. The content of calcium in the arteries increased progressively with aging while there is no change in the veins. The higher accumulation of calcium occurred in the arteries of the lower limb in comparison to the arteries of the upper limb. In the arterial bifurcation, there was the correlation at hemodynamic stress distribution and calcium content. The results of this study quantitatively support clinical findings of nonuniform calcification, and suggest that hemodynamic stress affects vascular calcification.     

Mammalian Bax initiates plant cell death through organelle destruction

Mammalian Bax is known to cause cell death when expressed in plants. We examined transgenic plants expressing both Bax and organelle-targeted green fluorescent protein to determine the cellular changes that occur during Bax-induced cell death. The mitochondria changed morphologically from being bacilli-shaped to being round, eventually becoming swollen. Mitochondria streaming also stopped. The chloroplasts lost membrane function and their contents leaked out, followed by the disruption of the vacuole. Light was not essential for Bax-induced ion leakage or organelle disruption. These results indicate that Bax induces temporal and spatial cell death events at the organelle level in the plant. A heterologous system, using Bax, would therefor be available to investigate cell death, which is commonly conserved in animals and plantsElectronic Supplementary Material Supplementary material is available for this article at     

Structure of a cell polarity regulator, a complex between atypical PKC and Par6 PB1 domains

A complex of atypical PKC and Par6 is a common regulator for cell polarity-related processes, which is an essential clue to evolutionary conserved cell polarity regulation. Here, we determined the crystal structure of the complex of PKCiota and Par6alpha PB1 domains to a resolution of 1.5 A. Both PB1 domains adopt a ubiquitin fold. PKCiota PB1 presents an OPR, PC, and AID (OPCA) motif, 28 amino acid residues with acidic and hydrophobic residues, which interacts with the conserved lysine residue of Par6alpha PB1 in a front and back manner. On the interface, several salt bridges are formed including the conserved acidic residues on the OPCA motif of PKCiota PB1 and the conserved lysine residue on the Par6alpha PB1. Structural comparison of the PKCiota and Par6alpha PB1 complex with the p40phox and p67phox PB1 domain complex, subunits of neutrophil NADPH oxidase, reveals that the specific interaction is achieved by tilting the interface so that the insertion or extension in the sequence is engaged in the specificity determinant. The PB1 domain develops the interaction surface on the ubiquitin fold to increase the versatility of molecular interaction.     

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.     

ABCC13, an unusual truncated ABC transporter,is highly expressed in fetal human liver

In the present study, we have cloned the cDNA of ABCC13, a novel ABC transporter, from the cDNA library of adult human placenta. The ABCC13 gene spans approximately 70kb on human chromosome 21q11.2 and consists of 14 exons. The open reading frame of the ABCC13 cDNA encodes a peptide consisting of 325 amino acid residues. The amino acid sequence corresponding to putative membrane-spanning domains was remarkably similar to ABCC1, ABCC2, ABCC3, and ABCC6. The ABCC13 gene was expressed in the fetal liver at the highest level among the organs studied. While ABCC13 was expressed in the bone marrow, its expression in peripheral blood leukocytes of adult humans was much lower and no detectable levels were observed in differentiated hematopoietic cells. The expression of ABCC13 in K562 cells decreased during cell differentiation induced by TPA. These results suggest that the expression of human ABCC13 is related with hematopoiesis.