Biomedical subjects
Publications and source records attributed to Nobuhiro Takahashi.
A chemostat culture was constructed with phenol and forest soil as an inoculum. We report the complete genome sequence of Rhodococcus qingshengii strain A3-8, which was isolated from the culture. The genome consists of a chromosome (6,436,695 bp) and a linear plasmid pA38 (257,365 bp).
A disintegrin and metalloproteinase with thrombospondin motif (adamalysin-thrombospondins, ADAMTS) degrades aggrecan, one of the major extracellular matrix (ECM) components in cartilage. Mandibular condylar cartilage differs from primary cartilage, such as articular and growth plate cartilage, in its metabolism of ECM, proliferation, and differentiation. Mandibular condylar cartilage acts as both articular and growth plate cartilage in the growing period, while it remains as articular cartilage after growth. We hypothesized that functional and ECM differences between condylar and primary cartilages give rise to differences in gene expression patterns and levels of aggrecan and ADAMTS-1, -4, and -5 during growth and aging. We employed in situ hybridization and semiquantitative RT-PCR to identify mRNA expression for these molecules in condylar cartilage and primary cartilages during growth and aging. All of the ADAMTSs presented characteristic, age-dependent expression patterns and levels among the cartilages tested in this study. ADAMTS-5 mainly contributed to ECM metabolism in growth plate and condylar cartilage during growth. ADAMTS-1 and ADAMTS-4 may be involved in ECM turn over in articular cartilage. The results of the present study reveal that ECM metabolism and expression of related proteolytic enzymes in primary and secondary cartilages may be differentially regulated during growth and aging.
Although interactions between cell surface proteins and extracellular ligands are key to initiating embryonic stem cell differentiation to specific cell lineages, the plasma membrane protein components of these cells are largely unknown. We describe here a group of proteins expressed on the surface of the undifferentiated mouse embryonic stem cell line D3. These proteins were identified using a combination of cell surface labeling with biotin, subcellular fractionation of plasma membranes, and mass spectrometry-based protein identification technology. From 965 unique peptides carrying biotin labels, we assigned 324 proteins including 235 proteins that have putative signal sequences and/or transmembrane segments. Receptors, transporters, and cell adhesion molecules were the major classes of proteins identified. Besides known cell surface markers of embryonic stem cells, such as alkaline phosphatase, the analysis identified 59 clusters of differentiation-related molecules and more than 80 components of multiple cell signaling pathways that are characteristic of a number of different cell lineages. We identified receptors for leukemia-inhibitory factor, interleukin 6, and bone morphogenetic protein, which play critical roles in the maintenance of undifferentiated mouse embryonic stem cells. We also identified receptors for growth factors/cytokines, such as fibroblast growth factor, platelet-derived growth factor, ephrin, Hedgehog, and Wnt, which transduce signals for cell differentiation and embryonic development. Finally we identified a variety of integrins, cell adhesion molecules, and matrix metalloproteases. These results suggest that D3 cells express diverse cell surface proteins that function to maintain pluripotency, enabling cells to respond to various external signals that initiate differentiation into a variety of cell types.
Zebrafish has become a favorite model organism not only in genetics and developmental biology, but also for the study of cancer, neuroscience and metabolism. However, strategies for reverse genetics in zebrafish are mostly limited to the use of antisense oligonucleotides, and therefore the development of other targeting methods is highly desirable. Here, we report an approach to gene targeting in this system in which single-stranded oligonucleotides and zebrafish Rad52 protein are employed. It has been proposed that a single-stranded oligonucleotide containing a mutation can be incorporated into the genome by annealing to the single-stranded region of the lagging strand of the replication fork. Rad52 is expected to accelerate the annealing step. In vitro experiments using purified truncated Rad52 proteins and replication protein A (RPA) showed that annealing of oligonucleotides is accelerated by Rad52 in the presence of RPA. We developed a simple and sensitive PCR-based method to detect point mutations in the genome. In exploratory experiments, we found that microinjection of single-stranded oligonucleotide targeted to a specific gene together with truncated Rad52 into zebrafish embryos resulted in a low level of recombinant copies in 3 of the 80 embryos tested under these conditions.
In this study, we screened for protein disulfide isomerase (PDI)-binding proteins in bovine liver microsomes under strict salt concentrations, using affinity column chromatography. One main band observed using SDS-PAGE was identified as ERp57 (one of the PDI family proteins) by LC-MS/MS analysis. The K(D) value of PDI binding to ERp57 was calculated as 5.46x10(-6)M with the BIACORE system. The interactions between PDI and ERp57 occurred specifically at their a and b domains, respectively. Interestingly, low concentrations of ERp57 enhanced the chaperone activity of PDI, while high concentrations interfered with chaperone activity. On the other hand, ERp57 did not affect the isomerase activity of PDI. Additionally, following pre-incubation of ERp57 with calreticulin (CRT), decreased interactions were observed between ERp57 and PDI, and vice versa. Based on the data, we propose that once ERp57 binds to PDI or CRT, the resultant complex inhibits further interactions. Therefore, ERp57 selectively forms a protein-folding complex with PDI or CRT in ER.
The ubiquitin E3 protein ligase Nedd4-2 is a physiological regulator of the epithelial sodium channel ENaC, which is essential for transepithelial Na+ transport and is linked to Liddle's syndrome, an autosomal dominant disorder of human salt-sensitive hypertension. Nedd4-2 function is negatively regulated by phosphorylation via a serum- and glucocorticoid-inducible protein kinase (Sgk1), which serves as a mechanism to inhibit the ubiquitination-dependent degradation of ENaC. We report here that 14-3-3 proteins participate in this regulatory process through a direct interaction with a phosphorylated form of human Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2). The interaction is dependent on Sgk1-catalyzed phosphorylation of hNedd4-2 at Ser-468. We found that this interaction preserved the activity of the Sgk1-stimulated ENaC-dependent Na+ current while disrupting the interaction decreased ENaC density on the Xenopus laevis oocytes surface possibly by enhancing Nedd4-2-mediated ubiquitination that leads to ENaC degradation. Our findings suggest that 14-3-3 proteins modulate the cell surface density of ENaC cooperatively with Sgk1 kinase by maintaining hNedd4-2 in an inactive phosphorylated state.
A protein subset expressed in the mouse embryonic stem (ES) cell line, E14-1, was characterized by mass spectrometry-based protein identification technology and data analysis. In total, 1790 proteins including 365 potential nuclear and 260 membrane proteins were identified from tryptic digests of total cell lysates. The subset contained a variety of proteins in terms of physicochemical characteristics, subcellular localization, and biological function as defined by Gene Ontology annotation groups. In addition to many housekeeping proteins found in common with other cell types, the subset contained a group of regulatory proteins that may determine unique ES cell functions. We identified 39 transcription factors including Oct-3/4, Sox-2, and undifferentiated embryonic cell transcription factor I, which are characteristic of ES cells, 88 plasma membrane proteins including cell surface markers such as CD9 and CD81, 44 potential proteinaceous ligands for cell surface receptors including growth factors, cytokines, and hormones, and 100 cell signaling molecules. The subset also contained the products of 60 ES-specific and 41 stemness genes defined previously by the DNA microarray analysis of Ramalho-Santos et al. (Ramalho-Santos et al., Science 2002, 298, 597-600), as well as a number of components characteristic of differentiated cell types such as hematopoietic and neural cells. We also identified potential post-translational modifications in a number of ES cell proteins including five Lys acetylation sites and a single phosphorylation site. To our knowledge, this study provides the largest proteomic dataset characterized to date for a single mammalian cell species, and serves as a basic catalogue of a major proteomic subset that is expressed in mouse ES cells.
The aims of this study were to identify hydrogen sulfide (H2S)-producing bacteria among tongue biofilm microflora and to investigate the relationship between bacterial flora and H2S levels in mouth air. Oral malodour levels in 10 subjects (age 21-56 years) were assessed by gas chromatography, and Breathtron and organoleptic scores. Based on these assessments, subjects were divided into two groups: an odour group and a no/low odour group. Tongue coatings were sampled and spread onto Fastidious Anaerobe Agar plates containing 0.05% cysteine, 0.12% glutathione and 0.02% lead acetate, and were then incubated anaerobically at 37 degrees C for 2 weeks. Bacteria forming black or grey colonies were selected as H2S-producing phenotypes. The numbers of total bacteria (P<0.005) and H2S-producing bacteria (P<0.05) in the odour group were significantly larger than those in the no/low odour group. Bacteria forming black or grey colonies (126 isolates from the odour group; 242 isolates from the no/low odour group) were subcultured, confirmed as producing H2S and identified according to 16S rRNA gene sequencing. Species of Veillonella (38.1% in odour group; 46.3% in no/low odour group), Actinomyces (25.4%; 17.7%) and Prevotella (10.3%; 7.8%) were the predominant H2S-producing bacteria in both the odour and no/low odour groups. These results suggest that an increase in the number of H2S-producing bacteria in the tongue biofilm is responsible for oral malodour, although the bacterial composition of tongue biofilm was similar between the two groups.
The present study was undertaken to examine whether synthesis of type IV collagen and laminin around the epithelial rests of Malassez (ERM) requires direct contact between cells from ERM and periodontal ligament fibroblasts. Human periodontal ligament (HPDL) explants produced outgrowths containing both ERM cells and fibroblasts when cultured in a modified serum-free medium. The interface between ERM cells and fibroblasts was examined using phase-contrast microscopy (PCM) and scanning electron microscopy (SEM). Expression of type IV collagen and laminin was studied by immunohistochemistry and in situ hybridization. It was observed that ERM cells grew underneath fibroblasts or attached to them. At the interface, type IV collagen and laminin and their respective mRNAs were abundant in both ERM cells and fibroblasts, while these proteins and mRNAs showed little if any staining in cells further away from the interface. Hence, these findings indicate that synthesis of type IV collagen and laminin is induced by direct interaction between ERM cells and periodontal ligament fibroblasts.
Horizontally transferred genes are believed to play a critical role in the divergence of bacterial strains from a common ancestor, but whether all of these genes express functional proteins in the cell remains unknown. Here, we used an integrated LC-based protein identification technology to analyze the proteome of Escherichia coli strain K12 (JM109) and identified 1,480 expressed proteins, which are equivalent to approximately 35% of the total open reading frames predicted in the genome. This subset contained proteins with cellular abundance of several dozens to hundreds of thousands of copies, and included nearly all types of proteins in terms of chemical characteristics, subcellular distribution, and function. Interestingly, the subset also contained 138 of 164 gene products that are currently known to be essential for bacterial viability (84% coverage). However, the subset contained only a very small population (10%) of protein products from genes mapped within K-loops, which are "hot spots" for the integration of foreign DNAs within the K12 genome. On the other hand, these genes in K-loops appeared to be transcribed to RNAs almost as efficiently as the native genes in the bacterial cell as monitored by DNA microarray analysis, raising the possibility that most of the recently acquired foreign genes are inadequate for the translational machinery for the native genes and do not generate functional proteins within the cell.
Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale two-dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S-carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC-MS/MS system in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G-protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell-cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery.
BACKGROUND: To clarify the roles of the p53-MDM2-p14(ARF) cell cycle regulation system in oncogenesis and cytodifferentiation of odontogenic tumors, p53 gene status and expression of p53, MDM2, and p14(ARF) proteins was analyzed in ameloblastomas as well as tooth germs. METHODS: Paraffin sections of 16 tooth germs and 46 benign and 5 malignant ameloblastomas were examined immunohistochemically for the expression of p53, MDM2, and p14(ARF) proteins. Frozen tissue samples of 10 benign ameloblastomas and 1 malignant (metastasizing) ameloblastoma were analyzed by direct DNA sequencing to detect p53 gene alteration. RESULTS: Immunohistochemical reactivity for p53 was detected in 2 of 13 tooth germs, 13 of 29 ameloblastomas, and 5 of 5 malignant ameloblastomas, and the expression ratio of p53 in tooth germs was significantly lower than those in benign and malignant ameloblastomas. Direct DNA sequencing showed no alteration of p53 gene exons 5-8 in any sample of 10 benign ameloblastomas and 1 metastasizing ameloblastoma. Expression of MDM2 and p14(ARF) was detected in all samples of normal and neoplastic odontogenic epithelium, and the expression ratios in tooth germs tended to be lower than those in benign and malignant ameloblastomas. In ameloblastomas, expression of p53, MDM2, and p14(ARF) was significantly higher in plexiform cases than in follicular cases. Markedly decreased reactivity for p53, MDM2, and p14(ARF) was detected in keratinizing and granular cells in ameloblastoma subtypes. Basal cell ameloblastoma showed slightly higher reactivity for p53, MDM2, and p14(ARF) as compared with other subtypes. CONCLUSION: Elevated expression of p53, MDM2, and p14(ARF) in benign and malignant ameloblastomas suggests that alteration of the p53-MDM2-p14(ARF) cascade is involved in oncogenesis and/of malignant transformation of odontogenic epithelium. p53 gene status implied that p53 mutation might play a minor role in neoplastic changes of odontogenic epithelium. Immunoreactivity for p53, MDM2, and p14(ARF) in ameloblastoma variants suggests that these factors might be associated with tissue structuring and cytodifferentiation of ameloblastomas.
Calcineurin, a calcium/calmodulin-dependent serine/threonine protein phosphatase, is a key constituent of signaling pathways involved in antigen-dependent T-cell activation and development of the mammalian heart. In addition, calcineurin constitutes a part of the Wnt/calcium-signaling pathway that regulates early stages of dorsoventral axis formation in Xenopus embryos. Although some of the Wnt family members are involved in organ formation at relatively late stages of Xenopus development, the involvement of calcineurin in the development of those organs remains unclear. In the present study, we demonstrate that calcineurin inhibitors (cyclosporine A, FK506, and FK520), but not non-calcineurin inhibitors (rapamycin and GPI1046) that bind the same intracellular receptor as that for FK506, induce edema and gut coiling disruption and exhibit teratogenesis in the kidney, heart, gut, liver, and somitic tissue during Xenopus development. The same effects were observed by injecting the calcineurin inhibitors into the dorsal side, but not ventral side, of blastomeres at the 4-cell stage, although the inhibitors did not affect dorsoventral axis formation. These results suggest that calcineurin is involved in dorsal-side signaling that leads to the formation of the heart, kidney, liver, gut and somitic tissue during Xenopus embryogenesis.
BACKGROUND: To clarify the roles of rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK) signaling pathway in oncogenesis and cytodifferentiation of odontogenic tumors, K-Ras gene status and expression of Ras, Raf1, MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)1, and ERK1/2 proteins were analyzed in ameloblastomas as well as in tooth germs. METHODS: Paraffin sections of 10 tooth germs and 46 benign and 6 malignant ameloblastomas were examined immunohistochemically for the expression of K-Ras, Raf1, MEK1, and ERK1/2. Frozen tissue samples of 22 benign ameloblastomas and 1 malignant (metastasizing) ameloblastoma were analyzed by direct DNA sequencing to detect K-Ras gene alteration. RESULTS: Immunohistochemical reactivity for K-Ras, Raf1, MEK1, and ERK1/2 was detected in both normal and neoplastic odontogenic epithelium, and these molecules were reactive chiefly with odontogenic epithelial cells neighboring the basement membrane. Plexiform ameloblastomas showed slightly stronger expression of these Ras/MAPK signaling molecules than follicular ameloblastomas. Keratinizing cells and granular cells showed decreased reactivity for the signaling molecules. Basal cell ameloblastomas showed slightly stronger reactivity for the signaling molecules than did the other subtypes. K-Ras immunoreactivity in malignant ameloblastomas was lower than that in dental lamina of tooth germs. Direct DNA sequencing showed a GGT to GCT point mutation at codon 12 of K-Ras gene in one ameloblastoma. CONCLUSION: Expression of K-Ras, Raf1, MEK1, and ERK1/2 in tooth germs and ameloblastomas suggests that Ras/MAPK signaling pathway functions to regulate cell proliferation and differentiation in both normal and neoplastic odontogenic epithelium. K-Ras gene status implied that K-Ras mutations might play a minor role in oncogenesis of odontogenic epithelium.
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Fibrillarin (FIB, Nop1p in yeast) is an RNA methyltransferase found not only in the fibrillar region of the nucleolus but also in Cajal bodies. FIB is essential for efficient processing of preribosomal RNA during ribosome biogenesis, although its precise function in this process and its role in Cajal bodies remain uncertain. Here, we demonstrate that the human FIB N-terminal glycine- and arginine-rich domain (residues 1-77) and its spacer region 1 (78-132) interact with splicing factor 2-associated p32 (SF2A-p32) and that the FIB methyltransferase-like domain (133-321) interacts with protein-arginine methyltransferase 5 (PRMT5, Janus kinase-binding protein 1). We also show that these proteins associate with several additional proteins, including PRMT1, tubulin alpha 3, and tubulin beta 1 to form a sub-complex that is principally independent of the association of FIB with preribosomal ribonucleoprotein complexes that co-immunoprecipitate with the sub-complex in human cells expressing FLAG-tagged FIB. Based on the physical association of FIB with SF2A-p32 and PRMTs, as well as the other reported results, we propose that FIB may coordinate both RNA and protein methylation during the processes of ribosome biogenesis in the nucleolus and RNA editing such as small nuclear (nucleolar) ribonucleoprotein biogenesis in Cajal bodies.
Nop56p is a component of the box C/D small nucleolar ribonucleoprotein complexes that direct 2'-O-methylation of pre-rRNA during its maturation. Genetic analyses in yeast have shown that Nop56p plays important roles in the early steps of pre-rRNA processing. However, its precise function remains elusive, especially in higher eukaryotes. Here we describe the proteomic characterization of human Nop56p (hNop56p)-associated pre-ribosomal ribonucleoprotein complexes. Mass spectrometric analysis of purified pre-ribosomal ribonucleoprotein complexes identified 61 ribosomal proteins, 16 trans-acting factors probably involved in ribosome biogenesis, and 29 proteins whose function in ribosome biogenesis is unknown. Identification of pre-rRNA species within hNop56p-associated pre-ribosomal ribonucleoprotein complexes, coupled with the known functions of yeast orthologs of the probable trans-acting factors identified in human, demonstrated that hNop56p functions in the early to middle stages of 60 S subunit synthesis in human cells. Interestingly, the nucleolar phosphoprotein treacle, which is responsible for the craniofacial disorder associated with Treacher Collins syndrome, was found to be a constituent of hNop56p-associated pre-rRNP complexes. The association of hNop56p and treacle within the complexes was independent of rRNA integrity, indicating a direct interaction. In addition, the protein compositions of the treacle-associated and hNop56p-associated pre-ribosomal ribonucleoprotein complexes were very similar, suggesting functional similarities between these two complexes with respect to ribosome biogenesis in human cells.
We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.