Jun Kuwahara

Specific induction of macrophage inflammatory protein 1‐α in glial cells of Sandhoff disease model mice associated with accumulation of N‐acetylhexosaminyl glycoconjugates

Sandhoff disease is a lysosomal storage disease caused by simultaneous deficiencies of β‐hexosaminidase A (HexA; αβ) and B (HexB; ββ), due to a primary defect of the β‐subunit gene (HEXB) associated with excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N‐acetylhexosamine residues at their non‐reducing termini, and with neurosomatic manifestations. To elucidate the neuroinflammatory mechanisms involved in its pathogenesis, we analyzed the expression of chemokines in Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex β‐subunit gene allele (Hexb–/–). We demonstrated that chemokine macrophage inflammatory protein‐1 α (MIP‐1α) was induced in brain regions, including the cerebral cortex, brain stem and cerebellum, of SD mice from an early stage of the pathogenesis but not in other systemic organs. On the other hand, little changes in other chemokine mRNAs, including those of RANTES (regulated upon activation, normal T expressed and secreted), MCP‐1 (monocyte chemotactic protein‐1), SLC (secondary lymphoid‐tissue chemokine), fractalkine and SDF‐1 (stromal derived factor‐1), were detected. Significant up‐regulation of MIP‐1α mRNA and protein in the above‐mentioned brain regions was observed in parallel with the accumulation of natural substrates of HexA and HexB. Immunohistochemical analysis revealed that MIP‐1α‐immunoreactivity (IR) in the above‐mentioned brain regions of SD mice was co‐localized in Iba1‐IR‐positive microglial cells and partly in glial fibrillary acidic protein (GFAP)‐IR‐positive astrocytes, in which marked accumulation of N‐acetylglucosaminyl (GlcNAc)‐oligosaccharides was observed from the presymptomatic stage of the disease. In contrast, little MIP‐1α‐IR was observed in neurons in which GM2 accumulated predominantly. These results suggest that specific induction of MIP‐1α might coincide with the accumulation of GlcNAc‐oligosaccharides due to a HexB deficiency in resident microglia and astrocytes in the brains of SD mice causing their activation and acceleration of the progressive neurodegeneration in SD mice.

Role of zinc finger structure in nuclear localization of transcription factor Sp1.

Transcription factor Sp1 is localized in the nucleus and regulates gene expression. Our previous study demonstrated that the carboxyl terminal region of Sp1 containing 3-zinc finger region as DNA binding domain can also serve as nuclear localization signal (NLS). However, the nuclear transport mechanism of Sp1 has not been well understood. In this study, we performed a gene expression study on mutant Sp1 genes causing a set of amino acid substitutions in zinc finger domains to elucidate nuclear import activity. Nuclear localization of the GFP-fused mutant Sp1 proteins bearing concomitant substitutions in the first and third zinc fingers was highly inhibited. These mutant Sp1 proteins had also lost the binding ability as to the GC box sequence. The results suggest that the overall tertiary structure formed by the three zinc fingers is essential for nuclear localization of Sp1 as well as dispersed basic amino acids within the zinc fingers region.

Gene of rat Ca2+/calmodulin-dependent protein kinase II α isoform — its cloning and whole structure

The gene encoding the α isoform of rat Ca2+/ calmodulin‐dependent protein kinase II was cloned, and its exon‐intron organization was analyzed. The coding region of cDNA consists of 18 exons spanning more than 50 kilobase pairs. Each of the discrete functional units, such as the ATP‐binding site, the autophosphorylation site responsible for Ca2+‐independent activity, the calmodulin‐binding site, and link structure is encoded by a single exon. The largest and smallest exons consist of 229 and 41 base pairs, respectively. All splice junction sequences flanking the introns conform to the consensus splice junction sequence and the GT‐AG splice rule.

Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

Transcription factor Sp1 is localized in the nucleus and regulates the expression of many cellular genes, but the nuclear transport mechanism of Sp1 is not well understood. In this study, we revealed that GST-fused Sp1 protein bound to endogenous importin α in HeLa cells via the Sp1 zinc finger domains, which comprise the DNA binding domain of Sp1. It was found that the Sp1 zinc finger domains directly interacted with a wide range of importin α including the armadillo (arm) repeat domain and the C-terminal acidic domain. Furthermore, it turned out that all three zinc fingers of Sp1 are essential for binding to importin α. Taken together, these results suggest that the Sp1 zinc finger domains play an essential role as a NLS and Sp1 can be transported into the nucleus in an importin-dependent manner even though it possesses no classical NLSs.

Apoptosis-Inducing activity of synthetic intermediates of Halichlorine

Synthetic intermediates of alkaloid halichlorine with the azaspiro core structure have been found to induce apoptosis of cultured human cells including an acute monocytic leukemia cell line (THP-1) at micromolar concentrations. The novel biological activity of the intermediates was suggested to depend on the skeletal structure and silyloxymethyl functionality on the five-membered ring.

Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.