Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly⁻⁸² to Gly³¹⁶), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca²⁺ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.     

Prospectively Isolated Cancer-Associated CD10+ Fibroblasts Have Stronger Interactions with CD133+ Colon Cancer Cells than with CD133? Cancer Cells

Although CD133 has been reported to be a promising colon cancer stem cell marker, the biological functions of CD133⁺ colon cancer cells remain controversial. In the present study, we investigated the biological differences between CD133⁺ and CD133⁻ colon cancer cells, with a particular focus on their interactions with cancer-associated fibroblasts, especially CD10⁺ fibroblasts. We used 19 primary colon cancer tissues, 30 primary cultures of fibroblasts derived from colon cancer tissues and 6 colon cancer cell lines. We isolated CD133⁺ and CD133⁻ subpopulations from the colon cancer tissues and cultured cells. In vitro analyses revealed that the two populations showed similar biological behaviors in their proliferation and chemosensitivity. In vivo analyses revealed that CD133⁺ cells showed significantly greater tumor growth than CD133⁻ cells (P = 0.007). Moreover, in cocultures with primary fibroblasts derived from colon cancer tissues, CD133⁺ cells exhibited significantly more invasive behaviors than CD133⁻ cells (P<0.001), especially in cocultures with CD10⁺ fibroblasts (P<0.0001). Further in vivo analyses revealed that CD10⁺ fibroblasts enhanced the tumor growth of CD133⁺ cells significantly more than CD10⁻ fibroblasts (P<0.05). These data demonstrate that the in vitro invasive properties and in vivo tumor growth of CD133⁺ colon cancer cells are enhanced in the presence of specific cancer-associated fibroblasts, CD10⁺ fibroblasts, suggesting that the interactions between these specific cell populations have important roles in cancer progression. Therefore, these specific interactions may be promising targets for new colon cancer therapies.     

The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.     

Introduction of amino acid residues at the N-terminus of the zeocin-resistance protein increases its expression in Saccharomyces cerevisiae

Nucleotide sequences proximal to the initiation codon of a gene are known to affect the expression efficiency of that gene. We screened 10-bp random sequences upstream of the initiation codon of the zeocin-resistance gene to identify sequences that could enhance its expression in Saccharomyces cerevisiae. Of the isolated sequences, 20 sequences exhibited a common feature, i.e. ATG at the position −9 through −7, which resulted in the incorporation of three amino acids at the N-terminus of the protein. The introduction of these sequences upstream of the initiation codon increased the expression levels of zeocin-resistance protein by 2.2–6.5-fold. One of these sequences increased the expression levels of three out of four human proteins, thereby suggesting that this sequence may also enhance the expression efficiency of mammalian proteins in yeast.     

IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^−/−) mice and RAG-2 and RORγt double deficient (RAG-2^−/− × RORγt^−/−) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^−/− mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^−/− × RORγt^−/− mice that lacked T and NKT cells. Surprisingly, RAG-2^−/− × RORγt^−/− mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^−/− mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^−/− mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.     

Potential stocks of reef fish-based ecosystem services in the Kuroshio Current region: Their relationship with latitude and biodiversity

The latitudinal decline of species richness is a general spatial pattern of biodiversity, and it applies to marine species as well. Based on a latitudinal gradient of marine species richness, potential stocks of marine ecosystem services are expected to be higher in lower latitudes through increment in biodiversity. However, little is known about the relationships of the marine ecosystem services with latitude and biodiversity. We estimated the latitudinal patterns and relationships with the biodiversity of potential stocks of three major reef fish-based ecosystem services (fisheries production, aquarium fish production, and recreational diving) at ten coral habitats from tropical to temperate zones in the Kuroshio Current region (8°37′N–33°24′N) using field survey data and information from relevant websites and administrative statistics. We found a latitudinal declin from south to north in potential stocks of aquarium fish production and diving in this region, whereas the peaks of fisheries production were found around both tropical and sub-tropical zones. Our results also showed strong positive effects of biodiversity on potential stocks of the three ecosystem services, highlighting the importance of conserving diverse fish species to sustain multiple services at high levels. Broad spatial patterns of the reef fish-based ecosystem services are useful as baselines for future evaluation of their changes. As the effects of climate change on reef fishes are predicted to vary among different latitude zones, our estimates of the ecosystem services infer specific management and economic actions for the respective zones against climate change.     

Live cell imaging of X chromosome reactivation during somatic cell reprogramming

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.     

Distribution and Morphological Features of Microglia in the Developing Cerebral Cortex of Gyrencephalic Mammals

Microglia have been attracting much attention because of their fundamental importance in both the mature brain and the developing brain. Though important roles of microglia in the developing cerebral cortex of mice have been uncovered, their distribution and roles in the developing cerebral cortex in gyrencephalic higher mammals have remained elusive. Here we examined the distribution and morphology of microglia in the developing cerebral cortex of gyrencephalic carnivore ferrets. We found that a number of microglia were accumulated in the germinal zones (GZs), especially in the outer subventricular zone (OSVZ), which is a GZ found in higher mammals. Furthermore, we uncovered that microglia extended their processes tangentially along inner fiber layer (IFL)-like fibers in the developing ferret cortex. The OSVZ and the IFL are the prominent features of the cerebral cortex of higher mammals. Our findings indicate that microglia may play important roles in the OSVZ and the IFL in the developing cerebral cortex of higher mammals.     

Triterpenes from periandra dulcis roots

Two new triterpenes from the root of Periandra dulcis were found to be 3,25-dioxoolean-12(13)-en-30-oic acid and 3,25-dioxoolean-18(19)-en-30-oic acid.