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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.05.025 Byline: Kohji Ikenishi, Fumiko Nishiumi, Tohru Komiya Keywords: Antisense morpholino oligo; Xdsg mRNA; Inhibition of PGC formation; Xdsg protein; XVLG1 protein Abstract: In order to know the role of the Xdsg gene in presumptive PGCs (pPGCs) of Xenopus, we attempted to inhibit the translation of Xdsg mRNA in pPGCs by injecting antisense morpholino oligo (asMO), together with Fluorescein Dextran-Lysine (FDL), into single germ plasm-bearing cells of 32-cell embryos. Among three types of asMOs complementary to different parts of the 5'-untranslated region of Xdsg mRNA tested, only one asMO, designated as Xdsg-3, inhibited the translation of the mRNA in FDL-labeled pPGCs, resulting in the absence of labeled PGCs in experimental tadpoles. On the other hand, two other asMOs, Xdsg-1 and -2, did not inhibit the translation, so that a similar number of labeled PGCs found in FDL-injected but asMO-uninjected control tadpoles were observed in experimental tadpoles derived from asMO-injected embryos. Surprisingly, use of Xdsg-3 asMO resulted in the disappearance of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) from FDL-labeled pPGCs by inhibiting the translation of XVLG1 mRNA. However, the effect of Xdsg-3 asMO on the translation of Xdsg and XVLG1 mRNAs and PGC formation could be canceled by the coinjection with Xdsg mRNA. Consequently, the Xdsg protein in pPGCs may play an important role in the formation of PGCs by regulating the production of XVLG1 protein. Author Affiliation: Department of Biology, Graduate School of Science, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi, Osaka 558-8585, Japan Article History: Received 8 December 2005; Revised 8 May 2006; Accepted 23 May 2006

As a result of a high-throughput in situ hybridization screening for adult mouse testes, we found that the mRNA for Tmco5 is expressed in round and elongating spermatids. Tmco5 belongs to the Tmco (Transmembrane and coiled-coil domains) gene family and has a coiled-coil domain in the N-terminal and a transmembrane domain in the C-terminal region. A monoclonal antibody raised against recombinant TMCO5 revealed that the protein is expressed exclusively in the elongating spermatids of step 9 to 12 and is localized to the manchette, a transiently emerging construction, which predominantly consists of cytoskeleton microtubules and actin filaments. This structure serves in the transport of Golgi-derived non-acrosomal vesicles. Moreover, induced expression of TMCO5 in CHO cells resulted in the co-localization of TMCO5 with β-tubulin besides the reorganization of the Golgi apparatus. Judging from the results and considering the domain structure of TMCO5, we assume that Tmco5 may have a role in vesicle transport along the manchette. [ABSTRACT FROM AUTHOR]

Amyloid plaques and neurofibrillary tangles (NFTs) are the major pathological characteristics of Alzheimer's disease (AD). NFTs are composed of tubular filaments and paired helical filaments containing polymerized hyperphosphorylated tau protein. Another feature of AD is excessive generation of nitric oxide (NO). Protein disulfide isomerase (PDI) is a chaperon protein located in the endoplasmic reticulum (ER). It was recently reported that NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. In addition, we previously reported the presence of PDI-immunopositive NFTs in AD. Here, we found that protein disulfide isomerase P5 (P5), which is a member of the PDI protein family, was co-localized with tau in NFTs. To our knowledge, this is the first report of P5-immunopositive inclusion in AD. Furthermore, we showed that S-nitrosylated P5 was present and the expression level of P5 was decreased in AD brains compared with that of control brains. We also demonstrated that the knock-down of PDI or P5 by siRNA could affect the viability of SH-SY5Y cells under ER stress. Previously, the observation of S-nitrosylated PDI in AD was reported. NO may inhibit P5 by inducing S-nitrosylation in the same manner as PDI, which inhibits its enzymatic activity allowing protein misfolding to occur in AD. The accumulation of misfolded proteins induces ER stress and may cause apoptosis of neuronal cells through S-nitrosylation and down-regulation of PDI and P5 in AD. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondria perform multiple functions critical to the maintenance of cellular homeostasis. Here we report that the downregulation of histone deacetylase 6 (HDAC6) causes a reduction in the net activity of mitochondrial enzymes, including respiratory complex II and citrate synthase. HDAC6 deacetylase and ubiquitin-binding activities were both required for recovery of reduced mitochondrial metabolic activity due to the loss of HDAC6. Hsp90, a substrate of HDAC6, localizes to mitochondria and partly mediates the regulation of mitochondrial metabolic activity by HDAC6. Our finding suggests that HDAC6 regulates mitochondrial metabolism and might serve as a cellular homeostasis surveillance factor. [Copyright &y& Elsevier]

We have developed a novel method for microinjection into Xenopus eggs and embryos. Microinjection was performed into eggs or embryos that were placed in wells (ca. 2.5 mm x 2.5 mm x 0.8 mm for each well) at the bottom of a commercially available hybridoma dish, which was filled with 1.5% methylcellulose solution. Eggs or embryos, rotated to a desired orientation in the viscous methylcellulose solution with a hair loop, could remain in the orientation for more than twenty minutes. Accordingly, samples such as mRNAs, DNAs, proteins and antisense morpholino oligonucleotides could be easily and efficiently microinjected into any part (animal, vegetal, dorsal, lateral or ventral) of more than five hundred eggs and embryos in one day. In addition, methylcellulose did not interfere with the development of the injected eggs and embryos. [ABSTRACT FROM AUTHOR]

Mitochondrial precursor proteins with basic targeting signals may be transported across the outer membrane by sequential binding to acidic receptor sites of increasing affinity. To test this ‘acid chain’ hypothesis, we assayed the interaction of mitochondrial precursors with three acidic receptor domains: the cytosolic domain of Tom20 and the intermembrane space domain of Tom22 and Tim23. The apparent affinity and salt resistance of precursor binding increased in the order Tom20

Abstract: Using a large-scale in situ hybridization screening system, we found that mRNA coding for ME1, a basic helix-loop-helix (bHLH) transcription factor, was abundantly expressed in Paneth cells of adult small intestinal crypts. Other functionally related E-protein mRNAs, ME2, and E2A, however, could not be detected in the cells. ME1 mRNA was first detected in the jejunum and ileum two weeks after birth when the number of Paneth cells starts to increase. ME1 is the first identified bHLH transcription factor expressed in the Paneth cells and may be used as a molecular marker and a key molecule for analyzing transcriptional regulation in the Paneth cell. [Copyright &y& Elsevier]

Abstract: Directional migration of primordial germ cells (PGCs) toward future gonads is a common feature in many animals. In zebrafish, mouse and chicken, SDF-1/CXCR4 chemokine signaling has been shown to have an important role in PGC migration. In Xenopus, SDF-1 is expressed in several regions in embryos including dorsal mesoderm, the target region that PGCs migrate to. CXCR4 is known to be expressed in PGCs. This relationship is consistent with that of more well-known animals. Here, we present experiments that examine whether chemokine signaling is involved in PGC migration of Xenopus. We investigate: (1) Whether injection of antisense morpholino oligos (MOs) for CXCR4 mRNA into vegetal blastomere containing the germ plasm or the precursor of PGCs disturbs the migration of PGCs? (2) Whether injection of exogenous CXCR4 mRNA together with MOs can restore the knockdown phenotype? (3) Whether the migratory behavior of PGCs is disturbed by the specific expression of mutant CXCR4 mRNA or SDF-1 mRNA in PGCs? We find that the knockdown of CXCR4 or the expression of mutant CXCR4 in PGCs leads to a decrease in the PGC number of the genital ridges, and that the ectopic expression of SDF-1 in PGCs leads to a decrease in the PGC number of the genital ridges and an increase in the ectopic PGC number. These results suggest that SDF-1/CXCR4 chemokine signaling is involved in the migration and survival or in the differentiation of PGCs in Xenopus. [Copyright &y& Elsevier]

Abstract: Using a large-scale in situ hybridization screening, we found that the mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) was localized to the germ plasm of Xenopus laevis. The mRNA is maternally transcribed in oocytes and, during maturation, transported to the vegetal germ plasm through the late pathway where VegT and Vg1 mRNAs are transported. In the 3′-untranslated region (UTR) of the mRNA, there are clusters of E2 and VM1 localization motifs that were reported to exist in the mRNAs classified as the late pathway group. With in situ hybridization to the sections of embryos, the signal could be detected in the cytoplasm of migrating presumptive primordial germ cells (pPGCs) until stage 35. At stage 40, when the cells cease to migrate and reach the dorsal mesentery, the signal disappeared. A possible role of XGRIP2 in pPGCs of Xenopus will be discussed. [Copyright &y& Elsevier]

We performed high-throughput in situ hybridization screening of sections of mouse epidermis using an equalized skin cDNA library as probes and identified a novel gene giving rise to two splicing variants, both of which are expressed in the spinous layer. This gene was mapped between two genes encoding keratinocyte-related peptides, suprabasin and keratinocyte differentiation-associated protein (Kdap), on human chromosome 19q13.1. These gene products appeared to carry functional signal sequences. We then designated these two splicing variants as dermokine-α and -β. Northern blotting and quantitative RT-PCR revealed that dermokine-α/-β, suprabasin, and Kdap were highly expressed in stratified epithelia. In mouse embryonic development, dermokine-α/-β began to be expressed during the period of stratification. Also, in differentiating primary cultured human keratinocytes, transcription of dermokine-α/-β, suprabasin, and Kdap was induced. These findings indicated that dermokine-α/-β, suprabasin, and Kdap are secreted from the spinous layer of the stratified epithelia and that these genes form a novel gene complex on the chromosome. [Copyright &y& Elsevier]