Kenichi Mitsui

RNA-binding Protein TLS Is a Major Nuclear Aggregate-interacting Protein in Huntingtin Exon 1 with Expanded Polyglutamine-expressing Cells

Formation of intracellular aggregates is the hallmark of polyglutamine (polyQ) diseases. We analyzed the components of purified nuclear polyQ aggregates by mass spectrometry. As a result, we found that the RNA-binding protein translocated in liposarcoma (TLS) was one of the major components of nuclear polyQ aggregate-interacting proteins in a Huntington disease cell model and was also associated with neuronal intranuclear inclusions of R6/2 mice. In vitro study revealed that TLS could directly bind to truncated N-terminal huntingtin (tNhtt) aggregates but could not bind to monomer GST-tNhtt with 18, 42, or 62Q, indicating that the tNhtt protein acquired the ability to sequester TLS after forming aggregates. Thioflavin T assay and electron microscopic study further supported the idea that TLS bound to tNhtt-42Q aggregates at the early stage of tNhtt-42Q amyloid formation. Immunohistochemistry showed that TLS was associated with neuronal intranuclear inclusions of Huntington disease human brain. Because TLS has a variety of functional roles, the sequestration of TLS to polyQ aggregates may play a role in diverse pathological changes in the brains of patients with polyQ diseases.

Cell cycle arrest and apoptosis of leukemia cells induced by L -asparaginase

Apoptotic cell death of murine leukemia cells induced by E. coli L-asparaginase was studied. Deprivation of L-asparagine from the culture of L5178Y cells by L-asparaginase caused the fragmentation of chromosomal DNA of the leukemia cells within 24 h. Prior to the degradation of DNA, cell cycles of L5178Y cells were found to be arrested in G1 phase, and evidence of the DNA strand breaks was initially observed in G1 phase cells as early as 8 h after the asparaginase treatment. Therefore, apoptosis of leukemia cells induced by L-asparaginase is an event that is associated with the cell cycle arrest in G1 phase.

Proteomics of polyglutamine aggregates

In nine members of polyglutamine (polyQ) diseases, CAG repeat expansions of their responsible genes are observed. The disease is considered to be caused by the formation of polyQ aggregates that sequester proteins essential for cell viability. To understand the pathological process of polyQ diseases, a proteomic approach was used to identify aggregate interacting proteins (AIPs). Constructs were designed to express EGFP-fused, CAG-expanded (150 Q) huntingtin exon1 under the control of an ecdysone-inducible promoter and either lacking or containing a nuclear localization signal (NLS). After induction of a stably transfected Neuro 2A cell line with ecdysone, aggregates form in either the cytoplasm or the nucleus. The aggregates in these two different compartments were isolated with different methods. Cytoplasmic aggregate particles were purified using a fluorescence-activated cell sorter (FACS) by monitoring EGFP fluorescence, whereas nuclear aggregates were purified by using the detergent insoluble nature of aggregates. The resulting highly pure aggregates were subjected to SDS-PAGE followed by Coomassie blue staining. Bands containing AIP candidates were excised, and, after in-gel digestion with trypsin, were analyzed by mass spectrometry to identify the proteins. Novel candidates were confirmed as AIPs by immunocytological analysis to observe colocalization with polyQ aggregates. This chapter describes methods for the establishment of stable mutant cells, the purification of polyQ aggregates, and sample preparation for mass spectrometry analysis in detail.

Phenylalanine ammonia-lyase modified with polyethylene glycol: Potential therapeutic agent for phenylketonuria

Summary.Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the defects in the phenylalanine hydroxylase (PAH) gene. Individuals homozygous for defective PAH alleles show elevated levels of systemic phenylalanine and should be under strict dietary control to reduce the risk of neuronal damage associated with high levels of plasma phenylalanine. Researchers predict that plant phenylalanine ammonia-lyase (PAL), which converts phenylalanine to nontoxic t-cinnamic acid, will be an effective therapeutic enzyme for the treatment of PKU. The problems of this potential enzyme therapy have been the low stability in the circulation and the antigenicity of the plant enzyme. Recombinant PAL originated from parsley (Petroselinum crispum) chemically conjugated with activated PEG2 [2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine] showed greatly enhanced stability in the circulation and was effective in reducing the plasma concentration of phenylalanine in the circulation of mice. PEG-PAL conjugate will be an effective therapeutic enzyme for the treatment of PKU.

Susceptibility loci for the defective foreign protein-induced tolerance in New Zealand Black mice: implication of epistatic effects of Fcgr2b and Slam family genes.

In contrast to normal mice, autoimmune‐prone New Zealand Black (NZB) mice are defective in susceptibility to tolerance induced by deaggregated bovine γ globulin (DBGG). To examine whether this defect is related to the loss of self‐tolerance in autoimmunity, susceptibility loci for this defect were examined by genome‐wide analysis using the F2 intercross of nonautoimmune C57BL/6 (B6) and NZB mice. One NZB locus on the telomeric chromosome 1, designated Dit (Defective immune tolerance)‐1, showed a highly significant linkage. This locus overlapped with a locus containing susceptibility genes for autoimmune disease, namely Fcgr2b and Slam family genes. To investigate the involvement of these genes in the defective tolerance to DBGG, we took advantage of two lines of Fcgr2b‐deficient B6 congenic mice: one carries autoimmune‐type, and the other carries B6‐type, Slam family genes. Defective tolerance was observed only in Fcgr2b‐deficient mice with autoimmune‐type Slam family genes, indicating that epistatic effects of both genes are involved. Thus, common genetic mechanisms may underlie the defect in foreign protein antigen‐induced tolerance and the loss of self‐tolerance in NZB mouse‐related autoimmune diseases.

Immune tolerance induced by polyethylene glycol-conjugate of protein antigen: clonal deletion of antigen-specific Th-cells in the thymus.

Polyethylene glycol (PEG) conjugates of protein antigens induce antigen-specific immune tolerance of helper T (Th)-cells. However, the mechanism of this Th-cell tolerance has remained unelucidated. Using transgenic mice with ovalbumin (OVA)-specific T-cell receptor (TCR) genes, we examined the response of OVA-specific Th-cells towards tolerogenic PEG-conjugate of OVA in vitro and in vivo. When stimulated with PEG-OVA in vitro, transgenic OVA-specific Th-cells proliferated and produced interleukin 2, the levels of which were comparable to those induced by unmodified OVA. In contrast, PEG-OVAadministered into the circulation of transgenic mice induced unresponsiveness in peripheral OVA-specific Th-cells. Moreover, in the thymus of these transgenic mice, the frequency of immature CD4+CD8+ (double positive) thymocytes was reduced. A similar phenomenon was not observed in transgenic mice treated with unmodified OVA. As autoreactive T-cells are known to be clonally deleted at the immature double positive stage in the thymus, Th-cell tolerance induced by PEG-protein antigens is at least in part mediated by central tolerance in the thymus, and is likely caused by the markedly enhanced stability of PEG-protein conjugates in the circulatory system.

Apoptosis of leukemia cells induced by valine-deficient medium.

l-Asparaginase from Escherichia coli, which catalyzes the hydrolysis of l-asparagine to l-asparatic acid and ammonia, has been used as an effective therapeutic agent for the treatment of acute lymphoblastic leukemia and lymphosarcoma. However, only limited types of leukemia cells are sensitive to l-asparaginase treatment. Murine leukemia cells treated with l-asparaginase undergo apoptosis, which is closely associated with the cell cycle arrest in G1 phase. Apoptosis of leukemia cells is also induced by lacking l-asparagine from the culture medium.4 Apoptosis is closely associated with the activation of caspases, mitochondrial permeability transition, cell volume loss, chromatin condensation and nucleosomal DNA fragmentation. The present study deals with the fragmentation of chromosomal DNA and activation of caspase-3 (CPP32, Yama or apopain) in murine and human leukemia cells treated with amino acid-deficient medium. First, we examined the fragmentation of chromosomal DNA in murine leukemia cells (L5178Y and L1210) induced by treatment with one amino acid-deficient medium out of total 20 amino acids in the RPMI 1640 medium containing 10% dialyzed fetal bovine serum, penicillin (50 U/ml) and streptomycin (50 mg/ml) for 24 h. Among them, valineand isoleucine-deficient media induced the fragmentation of chromosomal DNA within 24 h incubation. Then, DNA fragmentation and caspase-3 activity of murine leukemia cells (L5178Y and L1210) treated with one of the amino acid-deficient media (lvaline, l-isoleucine and l-asparagine) was tested (Figure 1). The treatment of L5178Y or L1210 cells with valine-deficient medium for 8, 12, 16, 20 and 24 h causes the fragmentation of chromosomal DNA into the size equivalent to single and multiple nucleosomes (Figure 1a). A similar phenomenon is also observed for L5178Y and L1210 cells treated with isoleucine-deficient medium for 12, 16, 20 and 24 h incubation. DNA fragmentation was also observed in L5178Y cells treated with asparagine-deficient medium for 20 and 24 h incubation but was not observed in L1210 cells within 24 h incubation (data not shown). Figure 1b shows the caspase-3 activity of L5178Y and L1210 cells treated with one amino acid-deficient medium (valine, isoleucine or asparagine) during the course of incubation (0–24 h). In the case of L5178Y cells, the caspase-3 activity of valine-deficient medium increases with incubation time and becomes maximal for 12 h followed by decreasing activity (curve a). In the treatment with isoleucine-deficient medium, the activity appears at more than 12 h incubation and increases linearly with time (curve b). In asparaginedeficient medium, the caspase-3 activity was little observed (curve c). A similar result was obtained for L1210 cells as shown by the lower column of Figure 1b in which the cas-

Hyperspectral imaging of pathology samples

The object of the experiment described in this paper was to demonstrate that cells stained with multiple fluorophores could be identified and quantified simultaneously. Hyperspectral imaging was used to classify spleen cells of a Balb/c mouse, with the anti-mouse CD4 antibody conjugated with Alexa 488, 532, 546 and 568. It was found that the system was able to identify the specific fluorophore present and map their location in the cells. The system also provided relative signal strength data. Spectral libraries were constructed with color-coded spectra that enabled automatic spectral identification in subsequent acquisitions.

High-performance liquid chromatographic–fluorimetric assay for cathepsin A (lysosomal protective protein) activity

A rapid and sensitive assay for the determination of cathepsin A activity is reported. This method is based on fluorimetric detection of a dansylated peptide, 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe-L-Leu, after separation by high-performance liquid chromatography using a C18 reversed-phase column and isocratic elution. This method is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe at concentrations as low as 300 fmol, yields highly reproducible results and requires less than 7.0 min per sample for separation and quantitation. The optimum pH for cathepsin A activity was 4.5-5.0. The Km and Vmax values were respectively 14.9 microM and 27.91 pmol/microg/h with the use of enzyme extract obtained from mouse kidney. Cathepsin A activity was strongly inhibited by Ag+, Hg2+, diisopropylfluorophosphate and p-chloromercuriphenylsulphonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in kidney. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.