Shin-ichiro Niwa

Regulation of CD8+ T Cell Development by Thymus-Specific Proteasomes

Proteasomes are responsible for generating peptides presented by the class I major histocompatibility complex (MHC) molecules of the immune system. Here, we report the identification of a previously unrecognized catalytic subunit called β5t. β5t is expressed exclusively in cortical thymic epithelial cells, which are responsible for the positive selection of developing thymocytes. Although the chymotrypsin-like activity of proteasomes is considered to be important for the production of peptides with high affinities for MHC class I clefts, incorporation of β5t into proteasomes in place of β5 or β5i selectively reduces this activity. We also found that β5t-deficient mice displayed defective development of CD8+ T cells in the thymus. Our results suggest a key role for β5t in generating the MHC class I–restricted CD8+ T cell repertoire during thymic selection.

Tumor Necrosis Factor-α Regulates Transforming Growth Factor-β-dependent Epithelial-Mesenchymal Transition by Promoting Hyaluronan-CD44-Moesin Interaction

Aberrant epithelial-mesenchymal transition (EMT) is involved in development of fibrotic disorders and cancer invasion. Alterations of cell-extracellular matrix interaction also contribute to those pathological conditions. However, the functional interplay between EMT and cell-extracellular matrix interactions remains poorly understood. We now show that the inflammatory mediator tumor necrosis factor-α (TNF-α) induces the formation of fibrotic foci by cultured retinal pigment epithelial cells through activation of transforming growth factor-β (TGF-β) signaling in a manner dependent on hyaluronan-CD44-moesin interaction. TNF-α promoted CD44 expression and moesin phosphorylation by protein kinase C, leading to the pericellular interaction of hyaluronan and CD44. Formation of the hyaluronan-CD44-moesin complex resulted in both cell-cell dissociation and increased cellular motility through actin remodeling. Furthermore, this complex was found to be associated with TGF-β receptor II and clathrin at actin microdomains, leading to activation of TGF-β signaling. We established an in vivo model of TNF-α-induced fibrosis in the mouse eye, and such ocular fibrosis was attenuated in CD44-null mice. The production of hyaluronan and its interaction with CD44, thus, play an essential role in TNF-α-induced EMT and are potential therapeutic targets in fibrotic disorders.

Crystal structure of a chaperone complex that contributes to the assembly of yeast 20S proteasomes

Eukaryotic 20S proteasomes are composed of two α-rings and two β-rings, which form an αββα stacked structure. Here we describe a proteasome-specific chaperone complex, designated Dmp1–Dmp2, in budding yeast. Dmp1–Dmp2 directly bound to the α5 subunit to facilitate α-ring formation. In Δdmp1 cells, α-rings lacking α4 and decreased formation of 20S proteasomes were observed. Dmp1–Dmp2 interacted with proteasome precursors early during proteasome assembly and dissociated from the precursors before the formation of half-proteasomes. Notably, the crystallographic structures of Dmp1 and Dmp2 closely resemble that of PAC3—a mammalian proteasome-assembling chaperone; nonetheless, neither Dmp1 nor Dmp2 showed obvious sequence similarity to PAC3. The structure of the Dmp1–Dmp2–α5 complex reveals how this chaperone functions in proteasome assembly and why it dissociates from proteasome precursors before the β-rings are assembled.

Double‐cleavage production of the CTL epitope by proteasomes and PA28: role of the flanking region

Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens, and the γ‐interferon‐inducible proteasome activator PA28 has been thought to play an important role in the generation of immunodominant MHC ligands by proteasomes. Several attempts have been made to show that proteasomes have the ability to yield cytotoxic T lymphocyte (CTL) epitopes effectively from model polypeptides derived from viral and intracellular proteins in vitro, but their antigen processing mechanism is poorly understood.

A mammalian ortholog of Drosophila timeless, highly expressed in SCN and retina, forms a complex with mPER1.

It is now becoming clear that the circadian rhythm of behaviours and hormones arises from a rhythm at the level of gene expression, and that mammals and Drosophila essentially use homologous genes as molecular gears in the control of circadian oscillation. In Drosophila, the period and timeless genes form a functional unit of the clock and its autoregulatory feedback loop for circadian rhythm. However, in mammals, the counterpart of timeless has not been found.

Contribution of Proline Residue for Efficient Production of MHC Class I Ligands by Proteasomes

Proteasomes are processing enzymes capable of generating major histocompatibility complex (MHC) class I ligands, but the mechanism of how they excise ligands without destroying them is largely unknown. Previously, we reported that most products of ornithine decarboxylase degraded in vitro by the 26 S ATP-dependent proteasome, which contained one or two Pro residues (Tokunaga, F., Goto, T., Koide, T., Murakami, Y., Hayashi, S., Tamura, T., Tanaka, K., and Ichihara, A. (1994) J. Biol. Chem. 269,17382–17385), which implied that the Pro residue has a role in the escape from random cleavage by proteasomes. Here, we examine the role of the Pro residue in producing MHC class I ligands in vitro. Proteasomes generated two cytotoxic T lymphocyte-epitopic precursor peptides, SIIPGLPLSL and DMYPHFMPTNL, from the 29-mer and 25-mer peptides harboring these sequences, which are derived from the c-akt proto-oncogene and the pp89 protein of mouse cytomagalovirus, respectively. Replacement of the first or second Pro residue within these epitopes by Ala resulted in a marked reduction of this epitope-derived production or their random cleavage by proteasomes, irrespective of the presence of PA28, which greatly accelerates the generation of unmodified ligands. Moreover, replacement of a single amino acid residue other than Pro in both epitopic and flanking regions by Ala or Leu had no or little appreciable effect on the SIIPGLPLSL or its derivative production. Thus, Pro residue(s) within these epitopic sequences presumably contributes to efficient production of MHC class I ligands through prevention of their random cleavage by proteasomes.

Development of a noninvasive monitoring instrument for serum Indocyanine Green dye concentration.

A noninvasive instrument to monitor serum Indocyanine Green (ICG) dye removal by the liver, which uses a new analytical method and an optical sensor, has been developed. The principal elements of the optical sensor are two light-emitting diodes at 810- and 940-nm wavelengths and a photodiode to detect light transmittance through the fingertip. A new analytical calibration method to compensate for the effect of blood volume variations has been developed. Thereby, only ICG concentration could be extracted. In addition, through clinical evaluation of hepatic disease patients, the correlation between serum ICG disappearance rates by this method and the conventional blood sampling method has been confirmed as evidence of its usefulness.

Electronic structures of silicon-containing polymers with unsaturated bonds

Abstract The electronic structures in the optimized geometries of polysilacetylene (PS), polysemisilacetylene (PSS), polysilacene (PSA) and polysemisilacene (PSSA), being silicon-substituted versions of polyacetylene and polyacene, are studied on the basis of the one-dimensional tight-binding self-consistent crystal orbital method. It is found that these polymers have band gap values similar to those of ordinary organic conductive polymers. Furthermore, the carbon-silicon bonds have large electron deviation, resulting in considerable degrees of polarization between these atoms. This feature is also discussed in connection with the possible manifestation of ferroelectric properties in PSS and PSSA.

Automatic Selection System of Monoclonal Antibody Producing Cells

Recently monoclonal antibodies are being applied to many areas of life science. Their importance is increasing more and more. But, the selection of cells that produce monoclonal antibodies is a labor-intensive operation. So the authors succeeded in developing the automatic system. Cells which have fused and distributed on culture plates are put in this system. This system cultivates these cells with a 12 channel multipipet and optical pH sensor (HAT selection). After that, this system detects the production of antibody of cells with ELIS A apparatus, and then does cloning cells with an image-processed cell counting unit (limiting dilution). Finally, cloned cells which produce monoclonal antibodies are put in a 24-well culture plate and taken out from this system. At any time in these processes, users can input or rearrange schedules and procedures, and can easily get a cell-count, ELIS A results, and past records of each culture well.

An instrument for liver function testing using an optical sensor

As a medical application of optoelectronic technology, an instrument to measure liver function was developed. For the estimation of liver function, Indocyanine green (ICG) is widely used as a tracer clinically. A description of an instrument for noninvasive measurement of ICG clearance and the results of the clinical evaluation of about 470 patients are presented. In in vivo measurement, to solve the problem of changing blood volume, the authors propose a novel calibration method before ICG injection. Using this instrument, called an ICG clearance meter, the value of ICG tests correlated well with the values calculated from the conventional blood sampling methods.<<ETX>>