Emi Murayama

Characterization of Prismalin-14, a novel matrix protein from the prismatic layer of the Japanese pearl oyster (Pinctada fucata)

The mollusc shell is a hard tissue consisting of calcium carbonate and organic matrices. The organic matrices are believed to play important roles in shell formation. In the present study, we extracted and purified a novel matrix protein, named Prismalin-14, from the acid-insoluble fraction of the prismatic layer of the shell of the Japanese pearl oyster (Pinctada fucata), and determined its whole amino acid sequence by a combination of amino acid sequence analysis and MS analysis of the intact protein and its enzymic digests. Prismalin-14 consisted of 105 amino acid residues, including PIYR repeats, a Gly/Tyr-rich region and N- and C-terminal Asp-rich regions. Prismalin-14 showed inhibitory activity on calcium carbonate precipitation and calcium-binding activity in vitro. The scanning electron microscopy images revealed that Prismalin-14 affected the crystallization of calcium carbonate in vitro. A cDNA encoding Prismalin-14 was cloned and its expression was analysed. The amino acid sequence deduced from the nucleotide sequence of Prismalin-14 cDNA was identical with that determined by peptide sequencing. Northern-blot analysis showed that a Prismalin-14 mRNA was expressed only at the mantle edge. In situ hybridization demonstrated that a Prismalin-14 mRNA was expressed strongly in the inner side of the outer fold of the mantle. These results suggest that Prismalin-14 is a framework protein that plays an important role in the regulation of calcification of the prismatic layer of the shell.

Molecular cloning of a cDNA encoding a soluble protein in the coral exoskeleton

Organic substances were extracted from the calcified exoskeleton of the reef coral Galaxea fascicularis. In an SDS-PAGE analysis of the extract, a protein with an apparent molecular mass of 53 kDa was detected as well as two other weaker bands. A Ca2+ overlay analysis failed to find a Ca2+-binding protein in the extract. Periodic acid Schiff staining indicated that the 53 kDa protein was glycosylated. A cDNA containing the entire open reading frame for this protein was obtained. Analysis of the deduced protein sequence suggests that the protein, named galaxin, is synthesized as a precursor consisting of a signal peptide, a propeptide sequence, and a mature protein of 298 amino acids. Galaxin exhibits a novel amino acid sequence which is characterized by a tandem repeat structure. Galaxin transcripts were detected in the adult coral, but not in planktonic larvae.

Molecular cloning and expression of an otolith matrix protein cDNA from the rainbow trout, Oncorhynchus mykiss

The fish otolith is a hard tissue consisting of calcium carbonate and organic matrices. The matrix proteins play important roles in otolith formation, but little is known about the nature of these proteins. In this study, matrix proteins were extracted from the otoliths of rainbow trout, Oncorhynchus mykiss, and chum salmon, Oncorhynchus keta. EDTA-soluble matrix proteins were separated by SDS-PAGE, revealing two major components in the otoliths of both species with apparent molecular masses of 55 and 43 kDa. N-terminal and some internal amino acid sequences of the 55-kDa otolith matrix protein were determined. A cDNA fragment encoding this protein of O. mykiss was amplified by reverse transcription PCR using two degenerate primers designed from the amino acid sequences. A cDNA encoding this protein was obtained by screening a saccular cDNA library using the amplified cDNA fragment as a probe. Nucleotide sequence analysis revealed that the cDNA clone has a sequence of 2.5 kb and the open reading frame encoding 344 amino acid residues. Northern blot analysis showed that mRNA of this protein is expressed specifically in the sacculus, and consistently during the day.

Immunohistochemical localization of two otolith matrix proteins in the otolith and inner ear of the rainbow trout, Oncorhynchus mykiss: comparative aspects between the adult inner ear and embryonic otocysts.

The fish otolith consists mainly of calcium carbonate and organic matrices, the latter of which may play important roles in the process of otolith formation. We previously identified two otolith matrix proteins, named otolith matrix protein-1 (OMP-1) and otolin-1, from the rainbow trout, Oncorhynchus mykiss, and the chum salmon, O. keta. In this study, recombinant proteins corresponding to OMP-1 and otolin-1 were synthesized using yeast and bacterial expression systems, respectively, to produce specific antibodies against each protein. Immunohistochemical analysis using these antisera revealed that in the otoliths of adult fish, OMP-1 and otolin-1 were colocalized along the daily rings possibly formed by alternate deposition of calcium carbonate and organic matrices. In the adult inner ear, OMP-1 was produced at most of the saccular epithelium, while otolin-1 was produced at a limited part of cylindrical cells located at the marginal zone of the sensory epithelium. In the embryonic inner ear, these proteins had already existed in the otolith primordia when calcification had commenced. In addition, otolin-1 was localized in the fibrous materials connecting otolith primordia and sensory epithelium at this stage. These results indicate that these proteins are required as essential components for otolith formation and calcification.

Repressive domain of unliganded human estrogen receptor alpha associates with Hsc70.

Estrogen receptor (ER) is a hormone‐inducible transcription factor as a member of the nuclear receptor gene superfamily. Unliganded ER is transcriptionally silent and capable of DNA binding; however, it is unable to suppress the basal activity of the target gene promoters, unlike non‐steroid hormone receptors that associate with corepressors in the absence of their cognate ligands. To study the molecular basis of how unliganded human ERα is maintained silent in gene regulation upon the target gene promoters, we biochemically searched interactants for hERα, and identified heat shock protein 70 (Hsc70). Hsc70 appeared to associate with the N‐terminal hormone binding E domain, that also turned out a transcriptionally repressive domain. Competitive association of Hsc70 with a best known coactivator p300 was observed. Thus, these findings suggest that Hsc70 associates with unliganded hERα, and thereby deters hERα from recruiting transcriptional coregulators, presumably as a component of chaperone complexes.

Mycobacteria Counteract a TLR-Mediated Nitrosative Defense Mechanism in a Zebrafish Infection Model

Pulmonary tuberculosis (TB), caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb), is a major world health problem. The production of reactive nitrogen species (RNS) is a potent cytostatic and cytotoxic defense mechanism against intracellular pathogens. Nevertheless, the protective role of RNS during Mtb infection remains controversial. Here we use an anti-nitrotyrosine antibody as a readout to study nitration output by the zebrafish host during early mycobacterial pathogenesis. We found that recognition of Mycobacterium marinum, a close relative of Mtb, was sufficient to induce a nitrosative defense mechanism in a manner dependent on MyD88, the central adaptor protein in Toll like receptor (TLR) mediated pathogen recognition. However, this host response was attenuated by mycobacteria via a virulence mechanism independent of the well-characterized RD1 virulence locus. Our results indicate a mechanism of pathogenic mycobacteria to circumvent host defense in vivo. Shifting the balance of host-pathogen interactions in favor of the host by targeting this virulence mechanism may help to alleviate the problem of infection with Mtb strains that are resistant to multiple drug treatments.

NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation

The ontogeny of haematopoietic niches in vertebrates is essentially unknown. Here we show that the stromal cells of the caudal haematopoietic tissue (CHT), the first niche where definitive haematopoietic stem/progenitor cells (HSPCs) home in zebrafish development, derive from the caudal somites through an epithelial-mesenchymal transition (EMT). The resulting stromal cell progenitors accompany the formation of the caudal vein sinusoids, the other main component of the CHT niche, and mature into reticular cells lining and interconnecting sinusoids. We characterize a zebrafish mutant defective in definitive haematopoiesis due to a deficiency in the nascent polypeptide-associated complex alpha subunit (NACA). We demonstrate that the defect resides not in HSPCs but in the CHT niche. NACA-deficient stromal cell progenitors initially develop normally together with the sinusoids, and HSPCs home to the resulting niche, but stromal cell maturation is compromised, leading to a niche that is unable to support HSPC maintenance, expansion and differentiation.

Trim33 is essential for macrophage and neutrophil mobilization to developmental or inflammatory cues

ABSTRACT Macrophages infiltrate and establish in developing organs from an early stage, often before these have become vascularized. Similarly, leukocytes, in general, can quickly migrate through tissues to any site of wounding. This unique capacity is rooted in their characteristic amoeboid motility, the genetic basis of which is poorly understood. Trim33 (also known as Tif1-γ), a nuclear protein that associates with specific DNA-binding transcription factors to modulate gene expression, has been found to be mainly involved in hematopoiesis and gene regulation mediated by TGF-β. Here, we have discovered that in Trim33-deficient zebrafish embryos, primitive macrophages are unable to colonize the central nervous system to become microglia. Moreover, both macrophages and neutrophils of Trim33-deficient embryos display a reduced basal mobility within interstitial tissues, and a profound lack of a response to inflammatory recruitment signals, including local bacterial infections. Correlatively, Trim33-deficient mouse bone marrow-derived macrophages display a strongly reduced three-dimensional amoeboid mobility in fibrous collagen gels. The transcriptional regulator Trim33 is thus revealed as being essential for the navigation of macrophages and neutrophils towards developmental or inflammatory cues within vertebrate tissues. Highlighted Article: Trim33, a transcriptional regulator known to modulate TGF-β signalling and hematopoiesis, is essential for the mobilization of macrophages and neutrophils towards developmental or inflammatory cues.

Retraction: Repressive domain of unliganded human estrogen receptor α associates with Hsc70

Retraction: The above article in Genes to Cells (doi: 10.1111/j.1365‐2443.2005.00904.x), published online on 13 October 2005 in Wiley Online Library (http://onlinelibrary.wiley.com/), has been retracted by agreement between the authors, the journal Editor in Chief, Mitsuhiro Yanagida, and Wiley Publishing Asia Pty Ltd. The retraction has been agreed to due to lack of the gel images for the lanes 1, 2 and 3 of ‘αTRAP220’ and for the lanes 1, 2 and 4 of ‘αHsc70’ in Figure 4(A).