Molecular cloning and expression in Streptomyces lividans of a proteinous alpha-amylase inhibitor (HaimII) gene from Streptomyces griseosporeus

The gene encoding a proteinous alpha-amylase inhibitor (HaimII) of Streptomyces griseosporeus YM-25 has been cloned in Escherichia coli K12 using a deoxyinosine-containing synthetic oligonucleotide as the probe. A 1.6 kilobases BamHI fragment was confirmed to hybridize with the probe and subcloned in an E. coli-S. lividans shuttle vector. The plasmid clone was transferred into S. lividans by transformation. An appreciable amount of alpha-amylase inhibitor activity was found in the culture medium of S. lividans harboring the plasmid. As the specificity was indistinguishable from that of HaimII produced by the original S. griseosporeus strain, we concluded that the HaimII protein was synthesized in S. lividans and excreted into the medium.     

Role of interleukin-2 and interferon-γ in induction of activated natural killer cells from mice primed in vivo and subsequently challenged in vitro with the streptococcal preparation OK432

Summary The natural-killer(NK)-cell-mediated cytotoxicity to syngeneic tumor cells can be augmented by in vivo priming and subsequent in vitro challenge with the streptococcal preparation OK432. Supernatants of cocultures of spleen cells with OK432 contained interleukin-2 (IL-2) and interferon (IFN), mainly IFN-. As the anti-(mouse IFN-) monoclonal antibody but not anti-(mouse IFN-) antibody inhibited the induction of activated NK cells with OK432, the IFN- participated in this response. The enhancement of NK cell activity and production of IL-2 were partially inhibited by the pretreatment of spleen cells with mitomycin C or irradiation, and were completely abolished by pretreatment with actinomycin D. The IL-2 activity after treatment with various metabolic inhibitors ran parallel to the NK activity in a system augmented with OK432. The activity of incubated spleen cells with IL-2 receptors was increased by OK432 treatment, and the NK cell and IFN activities of supernatants were also abrogated by the treatment with anti-(mouse IL-2 receptor) monoclonal antibody, to block the interaction between IL-2 and these receptors of effector cells. The panning method clarified that the incubated spleen cells with IL-2 receptors are responsible for the production of IFN-. These results suggest that IL-2 plays a major role in inducing the activated NK cells from murine spleen cells primed in vivo and subsequently challenged in vitro with OK432, by the production of IFN-.     

Successful adoptive immunotherapy with OK432-inducible activated natural killer cells in tumor-bearing mice

We had demonstrated that the NK cell mediated cytotoxicity of murine spleen cells could be augmented byin vivo priming and subsequentin vitro challenge with a streptococcal preparation OK432, and the cell surface phenotype of induced killer cells was Thy-1⁺, asialo GM1⁺, suggesting that the activated cells were of NK lineage (OK-NK cell). We had also clarified that IL-2 played a major role in inducing the OK-NK cells via the production of IFN-. In this study, we examined the effect of adoptive transfer of OK-NK cells on syngeneic tumors in mice. Mice were implanted with SP2 myeloma cells intraperitoneally (i.p.), or C26 colon adenocarcinoma cells subcutaneously to make the models of peritonitis carcinomatosa or solid tumor, and the OK-NK cells were transferred i.p. or intratumorally, adoptively. By the adoptive transfer of OK-NK cells, 92% of mice bearing SP2-tumor had be cured. The tumor growth of C26-solid tumor was inhibited, and the survival rate of mice bearing C26-tumor was significantly increased. The intratumoral remnants of¹²⁵I-labelled OK-NK cells were 61, 27 and 8% at 4, 12 and 36h after intratumoral transfer, respectively. By multiple transfer of OK-NK cells, the antitumor effect was more effectively augmented than that of a single transfer. Results in this study suggested that OK-NK cells could be useful for the therapy of cancer patients.     

Purification and some properties of a Haim-sensitive alpha-amylase from newly isolated Bacillus sp. No. 195.

Newly isolated Bacillus sp. No. 195 produced an extracellular alpha-amylase sensitive to Haim which was found to inhibit specifically animal alpha-amylases. The enzyme was purified easily by two steps of starch adsorption and gel filtration using Sephacryl S-200. The purified enzyme, which showed a single band on native-PAGE or SDS-PAGE, had a molecular weight of 60,000 as judged on SDS-PAGE. The optimum pH value for activity and the isoelectric point were around 7.0 and 4.5, respectively. The sensitivity of the amylase to Haim was similar to that of animal amylase rather than bacterial amylase. It was suggested that a Haim-amylase complex might be formed at the molar ratio of 1:1. The amino acid sequence F-S-W similar to the triplet F-E-W highly conserved among alpha-amylases sensitive to proteinaceous inhibitors, such as Hoe 467-A or Haim, was found in the amino-terminal part of the No. 195 amylase.     

Autonomous muscle cell differentiation in partial ascidian embryos according to the newly verified cell lineages

Recent analysis of cell lineages in ascidian embryos by the intracellular injection of a tracer enzyme has clearly demonstrated that muscle cells are derived not only from the B4.1-cell pair of the eight-cell stage embryo, as has hitherto been believed, but also from both the b4.2- and A4.1-cell pairs (H. Nishida and N. Satoh, 1983, Dev. Biol.99, 382–394). In order to reexamine the developmental autonomy in muscle lineage cells, the B4.1 pair was isolated from the eight-cell stage embryo. The progeny cells of the B4.1 pair, as well as those of the six other blastomeres, were then allowed to develop in isolation into partial embryos. Autonomous muscle cell differentiation not only in partial embryos originating from the B4.1 cells but also in those from the six other blastomeres was substantiated by (a) occurrence of localized histospecific muscle acetylcholinesterase and (b) development of myofibrils. These results support the validity of the recent cell lineage study and confirmed the self-differentiation potency of muscle lineage cells in ascidian embryos according to the newly verified cell lineages.     

Endemic impact of human T cell leukemia virus type 1 screening in bone allografts

Allograft bone is a widely used as a convenient tool for reconstructing massive bone defects in orthopedic surgery. However, allografts are associated with the risk of viral disease transmission. One of the viruses transmitted in this manner is human T-lymphotropic virus type 1 (HTLV-1), which is found worldwide but is unevenly distributed. The southwestern parts of Japan are a highly endemic for HTLV-1. We investigated the HTLV-1 seroprevalence in candidate allograft donors at the regional bone bank in Kagoshima, Japan during its first 5 years of service. Between 2008 and 2012, we collected 282 femoral heads at the Kagoshima regional bone bank from living donors with osteoarthritis of the hip joint. Among the 282 candidate donors, 32 donors (11.3 %) were seropositive for anti-HTLV-1 antibody; notably, this prevalence is higher than that reported for blood donors in this area. Additionally, to determine if HTLV-1 genes are detectable after processing, we examined the bone marrow of the femoral heads from seropositive donors by conducting PCR assays. Our results confirm the existence of viral genes following the heat treatment processing of the femoral heads. Therefore, it is important to inactivate a virus completely by heat-treatment. Together, our findings highlight the importance of HTLV-1 screening at bone banks, particularly in HTLV-1-endemic areas such as southwest Japan.     

Functionalized silk assembled from a recombinant spider silk fusion protein (Z‐4RepCT) produced in the methylotrophic yeast Pichia pastoris

Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk‐based materials has been realized. Recently, a recombinant spider silk fusion protein, Z‐4RepCT, was produced intracellularly in Escherichia coli and could after purification self‐assemble into silk‐like fibers with ability to bind antibodies via the IgG‐binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z‐4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z‐4RepCT retrieved from the extracellular fraction. Purification of secreted Z‐4RepCT resulted in a mixture of full‐length and degraded silk proteins that failed to self‐assemble into fibers. A position in the C‐terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C‐terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z‐4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk‐like fibers after enzymatic deglycosylation.