Shigeki Kushida

Isolation of the poly(ADP-ribose) polymerase-encoding cDNA from Xenopus laevis: phylogenetic conservation of the functional domains

The complete nucleotide (nt) sequence of the Xenopus laevis poly(ADP-ribose) polymerase (PARP)-encoding cDNA was determined. The putative X. laevis PARP protein consists of 1008 amino acids (aa) with a molecular weight of 113 kDa. X. laevis PARP shares 74, 83, 73, 78 and 42% aa sequence homology with the human, bovine, mouse, chicken and Drosophila melanogaster PARPs, respectively. Comparison of the PARP aa sequences among these species showed conservation of two zinc-finger motifs in the DNA-binding domain, and an NAD-binding motif and a Rossmann fold in the catalytic domain. The first Leu of the putative leucine zipper of D. melanogaster PARP is substituted to Lys in X. laevis PARP. All the Glu residues in the leucine zipper are conserved in these six species.

Age-dependent paraparesis in WKA rats: evaluation of MHC k-haplotype and HTLV-1 infection.

Human T-cell leukemia virus type 1 (HTLV-1) infection is shown to be closely associated with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the occurrence of HAM/TSP was reported to be associated with MHC class II, the mechanism is still unclear. The WKA(RT1k) strain of rats was reported to develop HAM/TSP-like paraparesis after HTLV-1 infection, and was suggested to be an animal model of HAM/TSP. We asked whether MHC k-haplotype is specifically involved in the pathogenesis of paraparesis of WKA(RT1k) rats. We injected the HTLV-1 producing human T cells (MT-2 cells) intravenously into WKA(RT1k) rats and MHC congenic WKA.1L(RT1l) rats which have MHC l-haplotype of LEW rats on the WKA background. Positive antibody response to HTLV-1 antigens and presence of provirus in peripheral blood mononuclear cells confirmed that MT-2 cell-injected rats were infected with HTLV-1. Two of 13 MT-2 cell-injected WKA(RT1k) rats and five of 13 MT-2 cell-injected WKA.1L(RT1l) rats developed HAM/TSP-like hindlimb paraparesis between 16 and 26 months old. Interestingly, three of 14 MT-2 cell-uninjected WKA(RT1k) rats and four of 13 MT-2 cell-uninjected WKA.1L(RT1l) rats showed similar paraparesis between 15 and 26 months old. MHC k-haplotype is not specific to the development of paraparesis in WKA(RT1k) rats. The role of aging, genetic background, HTLV-1 infection and other factors on the development of HAM/TSP-like paraparesis in rats are discussed.

A neuropathological study of paraparetic rats injected with HTLV-I-producing T cells

In order to clarify the pathogenesis of HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP), we injected HTLV-I-producing rabbit or human T cells intravenously into WKA and F344 rats. Infection was confirmed from increase in the anti-HTLV-I antibody titer and from the presence of HTLV-I proviral DNA. Only WKA rats developed hindlimb paraparesis 78-124 weeks after the injection. Neuropathological examination of 5 rats showed degeneration of the anterolateral and posterior funiculi as well as the peripheral nerves, and this degeneration was characterized by prominent vacuolation and macrophage infiltration. The myelopathy and neuropathy were grossly similar to those in human HAM/TSP. Although pathological changes of the spinal cord were very mild in 2 paretic rats, and similar lesions were found in the spinal cords and peripheral nerves of 2 control WKA rats, the myelopathy, radiculoneuropathy, or both in the paretic rats showed greater severity than in the controls. The contribution of the aging process to the lesions of the spinal cord and peripheral nerve is discussed. It appears possible that HTLV-I may accelerate the aging process and give rise to paraparesis. The precise role of HTLV-I in the pathogenesis of rat paraparesis remains to be elucidated taking the role of the aging process of the spinal cord and peripheral nerve into account.

Artificial cytokine storm combined with hyperthermia induces significant anti-tumor effect in mice inoculated with Lewis lung carcinoma and B16 melanoma cells

In cancer immunotherapies combined with hyperthermia, one or two cytokines have been tested to augment the anti-tumor effect. However, the therapies have not shown sufficient improvement. The aim of this study is to find a new potent tumor immunotherapy in order to augment antitumor effect of hyperthermia by the cytokine cocktails in vivo. We used a combination therapy of local hyperthermia (LH) and various cytokine cocktails composed of IFNs (IFN-α, -β, and -γ), Th1 cytokines (IL-2, -12, -15, and -18), a Th2 cytokine (IL-4), inflammatory cytokines (IL-1α and TNF-α), and dendritic cell-inducible cytokines (IL-3 and GM-CSF). These cytokines in a proper combination augmented the anti-tumor effect of LH and prolonged survival time in Lewis lung carcinoma or B16 melanoma significantly. Moreover, the 12-cytokine cocktail suppressed B16 metastasis to the lung and lymph nodes, and complete regression of the tumors without regrowth occurred in 3 of 5 mice. In the cured three B16 mice, there was hyperplasia of lymphatic organs with many CD3-positive T lymphocytes. The most effective cytokine combination should be able to augment the anti-tumor effect of other therapies besides hyperthermia that induce the necrosis of tumor cells.

Cloning of Poly(ADP-ribose) Polymerase cDNA from Lower Eukaryotes

Poly(ADP-ribose) polymerase (EC 2.4.4.30) catalyzes DNA-dependent ADP-ribosylation of nuclear proteins with NAD as a substrate (1-8). Poly(ADP-ribose) polymerase activity was reported from various eukaryotic cells but not from prokaryotic cells (1-8).