Construction of a linkage map of Lentinula edodes (shiitake) with the HEGS (high-efficiency genome scanning) system: use of versatile AFLP and PCR-based gene markers

An improved linkage map of Lentinula edodes (shiitake) was constructed with an HEGS (high-efficiency genome scanning) system. Two hundred twenty-one HEGS-derived amplified fragment length polymorphism (AFLP-H) markers and 21 gene markers were developed and combined with 203 previously developed sequencer-derived AFLP markers (AFLP-S markers) and 3 mating factor loci (A, Bα, and Bβ) to construct a comprehensive linkage analysis. As a result, a novel linkage map with 166 markers including 2 mating factors (A and B), 10 HEGS-derived gene markers, 72 AFLP-H markers, and 82 AFLP-S markers was obtained. Of the total 448 markers, 273 could not be located on a linear map and thus were assigned to linkage groups as accessory markers. The map covers a total length of 1398.4 centimorgans (cM) with an average marker interval distance of 8.4 cM. The map consists of 11 linkage groups (LGs) in agreement with our previous map, and 7 LGs among them were found to contain branched linkages, which may be the result of reciprocal translocations representing dynamic reorganization of the shiitake genome. The previously reported linkage map was improved in terms of number of markers, marker density, linear order of markers, and total map length. Contribution no. 384 of the Tottori Mycological Institute     

Vasotocin/isotocin-immunoreactive neurons in the medaka fish brain are sexually dimorphic and their numbers decrease after spawning in the female

In teleosts, the distribution of neurons in the preoptic-hypothalamic region and their associated neurohypophysial hormones, such as vasotocin (VT), appears to be different among species. This differential distribution is thought to reflect the social and/or sexual status of individuals within a species. In the present study, we analyzed the number, size and the distribution of vasotocin/isotocin (VT/IT) neurons in the brains of both male and female medaka (Oryzias latipes) using immunohistochemistry. VT/IT neurons were similarly located in an inverted L-shape in the nucleus preopticus in both gender, as has been already reported in salmonids. However, computer-assisted image analysis revealed sexual dimorphism in the number of VT/IT-immunoreactive (ir) neurons, with greater numbers found in males as compared to females. Further, in the female brain, the number of VT/IT-ir neurons decreased significantly after spawning. In pre-spawning compared to post-spawning females, the small-sized VT/IT-ir neurons dominated. Sexual differentiation of the medaka is fully dependent upon the steroid status during the early developmental stages and steroids are also known to trigger gender-specific behavior in the adult medaka. Our findings strongly suggest that VT and/or IT neurons may be functionally related to ovulation and/or the reproductive axes through connections to their steroidal status.     

A RecA-mediated exon profiling method

We have developed a RecA-mediated simple, rapid and scalable method for identifying novel alternatively spliced full-length cDNA candidates. This method is based on the principle that RecA proteins allow to carry radioisotope-labeled probe DNAs to their homologous sequences, resulting in forming triplexes. The resulting complex is easily detected by mobility difference on electrophoresis. We applied this exon profiling method to four selected mouse genes as a feasibility study. To design probes for detection, the information on known exonic regions was extracted from public database, RefSeq. Concerning the potentially transcribed novel exonic regions, RNA mapping experiment using Affymetrix tiling array was performed. As a result, we were able to identify alternative splice variants of Thioredoxin domain containing 5, Interleukin1β, Interleukin 1 family 6 and glutamine-rich hypothetical protein. In addition, full-length sequencing demonstrated that our method could profile exon structures with >90% accuracy. This reliable method can allow us to screen novel splice variants from a huge number of cDNA clone set effectively.     

A new motif necessary and sufficient for stable localization of the delta2 glutamate receptors at postsynaptic spines

The number of each subclass of ionotropic glutamate receptors (iGluRs) at the spines is differentially regulated either constitutively or in a neuronal activity-dependent manner. The delta2 glutamate receptor (GluRdelta2) is abundantly expressed at the spines of Purkinje cell dendrites and controls synaptic plasticity in the cerebellum. To obtain clues to the trafficking mechanism of the iGluRs, we expressed wild-type or mutant GluRdelta2 in cultured hippocampal and Purkinje neurons and analyzed their intracellular localization using immunocytochemical techniques. Quantitative analysis revealed that deletion of the 20 amino acids at the center of the C terminus (region E) significantly reduced the amount of GluRdelta2 protein at the spines in both types of neurons. This effect was partially antagonized by the inhibition of endocytosis by high dose sucrose treatment or coexpression of dominant negative dynamin. In addition, mutant GluRdelta2 lacking the E region (GluRdelta2DeltaE), but not wild-type GluRdelta2, was found to colocalize with the endosomal markers Rab4 and Rab7. Moreover, the antibody-feeding assay revealed that GluRdelta2DeltaE was internalized more rapidly than GluRdelta2wt. These results indicate that the E region (more specifically, a 12-amino-acid-long segment of the E2 region) is necessary for rendering GluRdelta2 resistant to endocytosis from the cell surface at the spines. Furthermore, insertion of the E2 region alone into the C terminus of the GluR1 subtype of iGluRs was sufficient to increase the amount of GluR1 proteins in the spines. Therefore, we propose that the E2 region of GluRdelta2 is necessary, and also sufficient, to inhibit endocytosis of the receptor from postsynaptic membranes.     

The use of mammalian cultured cells loaded with a fluorescent dye shows specific membrane penetration of undissociated acetic acid

Acetic acid induces unique physiological responses in mammalian cells. Our previous study found that fura-2-loaded human embryonic kidney (HEK) 293T cells showed a robust intracellular fluorescence response immediately after stimulation with acetic acid, and no such response in the case of citric acid. In the present study, we aimed to identify the unique characteristics of acetic acid responsible for this phenomenon. We found that one such feature is its hydrophobicity. We also discovered that acetic acid induces cell responses by intracellular acidification. Of the components of acetic acid in solution (protons, acetate ions, and undissociated acetic acid), undissociated acetic acid might be the functional unit that penetrates the lipid bilayer of cell membranes to acidify the intracellular environment, thereby inducing cell responses. The method used in this study might be convenient in evaluating the intracellular acidification of cultured cells by acids in the external environment.     

DNA damage signaling triggers degradation of histone methyltransferases through APC/C(Cdh1) in senescent cells

Both the DNA damage response (DDR) and epigenetic mechanisms play key roles in the implementation of senescent phenotypes, but very little is known about how these two mechanisms are integrated to establish senescence-associated gene expression. Here we show that, in senescent cells, the DDR induces proteasomal degradation of G9a and GLP, major histone H3K9 mono- and dimethyltransferases, through Cdc14B- and p21(Waf1/Cip1)-dependent activation of APC/C(Cdh1) ubiquitin ligase, thereby causing a global decrease in H3K9 dimethylation, an epigenetic mark for euchromatic gene silencing. Interestingly, induction of IL-6 and IL-8, major players of the senescence-associated secretory phenotype (SASP), correlated with a decline of H3K9 dimethylation around the respective gene promoters and knockdown of Cdh1 abolished IL-6/IL-8 expression in senescent cells, suggesting that the APC/C(Cdh1)-G9a/GLP axis plays crucial roles in aspects of senescent phenotype. These findings establish a role for APC/C(Cdh1) and reveal how the DDR integrates with epigenetic processes to induce senescence-associated gene expression.     

Development and fibronectin signaling requirements of the zebrafish interrenal vessel

Background

The early morphogenetic steps of zebrafish interrenal tissue, the teleostean counterpart of the mammalian adrenal gland, are modulated by the peri-interrenal angioblasts and blood vessels. While an organized distribution of intra-adrenal vessels and extracellular matrix is essential for the fetal adrenal cortex remodeling, whether and how an intra-interrenal buildup of vasculature and extracellular matrix forms and functions during interrenal organogenesis in teleosts remains unclear.

Methodology and Principal Findings

We characterized the process of interrenal gland vascularization by identifying the interrenal vessel (IRV); which develops from the axial artery through angiogenesis and is associated with highly enriched Fibronectin (Fn) accumulation at its microenvironment. The loss of Fn1 by either antisense morpholino (MO) knockdown or genetic mutation inhibited endothelial invasion and migration of the steroidogenic tissue. The accumulation of peri-IRV Fn requires Integrin α5 (Itga5), with its knockdown leading to interrenal and IRV morphologies phenocopying those in the fn1 morphant and mutant. fn1b, another known fn gene in zebrafish, is however not involved in the IRV formation. The distribution pattern of peri-IRV Fn could be modulated by the blood flow, while a lack of which altered angiogenic direction of the IRV as well as its ability to integrate with the steroidogenic tissue. The administration of Fn antagonist through microangiography exerted reducing effects on both interrenal vessel angiogenesis and steroidogenic cell migration.

Conclusions and Significance

This work is the first to identify the zebrafish IRV and to characterize how its integration into the developing interrenal gland requires the Fn-enriched microenvironment, which leads to the possibility of using the IRV formation as a platform for exploring organ-specific angiogenesis. In the context of other developmental endocrinology studies, our results indicate a highly dynamic interrenal-vessel interaction immediately before the onset of stress response in the zebrafish embryo.     

Amoebal endosymbiont Protochlamydia induces apoptosis to human immortal HEp-2 cells

Protochlamydia, an environmental chlamydia and obligate amoebal endosymbiotic bacterium, evolved to survive within protist hosts, such as Acanthamobae, 700 million years ago. However, these bacteria do not live in vertebrates, including humans. This raises the possibility that interactions between Protochlamydia and human cells could induce a novel cytopathic effect, leading to new insights into host-parasite relationships. Therefore, we studied the effect of Protochlamydia on the survival of human immortal cell line, HEp-2 cells and primary peripheral blood mononuclear cells (PBMC). Using mainly 4',6-diamidino-2-phenylindole staining, fluorescent in situ hybridization, transmission electron microscopy, and also TUNEL and Transwell assays, we demonstrated that the Protochlamydia induced apoptosis in HEp-2 cells. The attachment of viable bacterial cells, but not an increase of bacterial infectious progenies within the cells, was required for the apoptosis. Other chlamydiae [Parachlamydia acanthamoebae and Chlamydia trachomatis (serovars D and L2)] did not induce the same phenomena, indicating that the observed apoptosis may be specific to the Protochlamydia. Furthermore, the bacteria had no effect on the survival of primary PBMCs collected from five volunteers, regardless of activation. We concluded that Protochlamydia induces apoptosis in human-immortal HEp-2 cells and that this endosymbiont could potentially be used as a biological tool for the elucidation of novel host-parasite relationships.