Sanae Miyake

Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe.

Loss of function of the Schizosaccharomyces pombe gap1 gene results in the same phenotypes as those caused by an activated ras1 mutation, i.e., hypersensitivity to the mating factor and inability to perform efficient mating. Sequence analysis of gap1 indicates that it encodes a homolog of the mammalian Ras GTPase-activating protein (GAP). The predicted gap1 gene product has 766 amino acids with relatively short N- and C-terminal regions flanking the conserved core sequence of GAP. Genetic analysis suggests that S. pombe Gap1 functions primarily as a negative regulator of Ras1, like S. cerevisiae GAP homologs encoded by IRA1 and IRA2, but is unlikely to be a downstream effector of the Ras protein, a role proposed for mammalian GAP. Thus, Gap1 and Ste6, a putative GDP-GTP-exchanging protein for Ras1 previously identified, appear to play antagonistic roles in the Ras-GTPase cycle in S. pombe. Furthermore, we suggest that this Ras-GTPase cycle involves the ra12 gene product, another positive regulator of Ras1 whose homologs have not been identified in other organisms, which could function either as a second GDP-GTP-exchanging protein or as a factor that negatively regulates Gap1 activity.

Induction of antibodies against structural proteins of hepatitis C virus in mice using recombinant adenovirus

Replication-deficient recombinant adenoviruses expressing structural proteins of hepatitis C virus (HCV) were constructed. Each recombinant lacks adenoviral E1A and E3 genes and bears expression units for HCV structural proteins. The expression units contain HCV cDNAs coding for either the protein or core, one of two envelopes (E1 and E2) or all of these structural proteins (core, E1 and E2) under the control of the SR alpha promoter. In HeLa or HepG2 cells, the recombinants can express efficiently HCV genes after infection without replication of the recombinants. We detected 22-kDa core, 35-kDa E1 and 58-kDa E2 proteins of HCV in these cells. The recombinant expressing all three HCV structural proteins was inoculated into mice. Antibodies to each of the three HCV proteins were detected in all of the ten mice tested. The results indicate that the recombinant adenoviruses efficiently express HCV genes and induce specific antibody against the expressed HCV proteins in animals.

Identification of sna41 gene, which is the suppressor of nda4 mutation and is involved in DNA replication in Schizosaccharomyces pombe

The replication licensing factor limits DNA replication to once in a cell cycle and is thought to contain MCM proteins as its component parts. Six MCM subtypes have been identified in various species. These MCM proteins are thought to bind each other to make a heteromeric complex. The Nda4 protein of Schizosaccharomyces pombe is one of the MCM proteins and is involved in DNA replication.

Characterization of the Schizosaccharomyces pombe ral2 gene implicated in activation of the ras1 gene product.

Mutations in the Schizosaccharomyces pombe ral2 gene cause a phenotype indistinguishable from that of the ras1-defective mutant. Using cloned ral2 DNA, we disrupted the chromosomal gene. The disruptants showed the same phenotype as the original ral2 isolates, i.e., they had spherical cells, had no detectable mating activity, and exhibited no response to the mating pheromone, but their vegetative growth was apparently normal. Sequence analysis of the ral2 gene suggests that it encodes a polypeptide of 611 amino acid residues whose predicted amino acid sequence shows no strong homology to any known protein. Either multiple copies or even a single copy of the ras1Val-17 allele, which is an activated form of ras1, restored rodlike cell morphology and ability to respond to the mating factor to ral2 mutants. These results suggest that the ral2 and ras1 gene products interact intimately and that the ral2 gene product is involved in activation of the ras1 protein in S. pombe.

Adenovirus vector technology: An efficient method for constructing recombinant adenovirus and on/off switching of gene expression

An efficient method of constructing recombinant adenoviruses (Ad) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique SwaI site of the full‐length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected to 293 cells together with the Ad DNA‐terminal protein complex digested at several sites with EcoT22I. The use of the parent Ad DNA‐terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the restriction digestion drastically reduced regeneration of the parent virus. This method may facilitate the application of recombinant Ad and should be useful for further improvement of Ad vectors. Also a recombinant adenovirus expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off switching reporter unit, where a LacZ‐expression unit can be activated by the Cre‐mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co‐infection experiments together with the Cre‐expressing and the reporter recombinant Ad showed that the Cre‐mediated switching of gene expression was detected in nearly 100% of cultured CV1 cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in future gene therapy.

IDENTIFICATION OF TWO XENOPUS LAEVIS GENES, XMCM2 AND XCDC46, WITH SEQUENCE HOMOLOGY TO MCM GENES INVOLVED IN DNA REPLICATION

The Schizosaccharomyces pombe genes, nda1 and nda4, are essential for the normal regulation of DNA replication and belong to the MCM gene family. This gene family includes Saccharomyces cerevisiae MCM2, MCM3, MCM5/CDC46 and CDC47, S. pombe nda1, nda4, cdc21 and mis5, and genes encoding human BM28, P1MCM3 and P1.1MCM3 and mouse P1MCM3, most of which are considered to be required for the initiation of DNA replication. We isolated two homologues of the MCM genes, xMCM2 and xCDC46, from a Xenopus laevis cDNA library using the polymerase chain reaction (PCR) method. The predicted amino acid (aa) sequences of xMCM2 and xCDC46 are most similar to those of human BM28 (78% identity) and S. pombe Nda4 (48% identity), respectively. By Western blot analysis using anti-xMCM2 and anti-xCDC46 polyclonal antibodies (Ab) raised against glutathione S-transferase (GST)::xMCM2 or GST::xCDC46 fusion proteins, xMCM2 and xCDC46 were identified as 120- and 95-kDa proteins, respectively. When either xMCM2 or xCDC46 was immunoprecipitated with the specific Ab, the other was also co-precipitated. These results suggest that xMCM2 and xCDC46 physically interact with each other.

Depletion of myeloid cells exacerbates hepatitis and induces an aberrant increase in histone H3 in mouse serum

Tissue‐resident macrophages and bone marrow (BM)‐derived monocytes play a crucial role in the maintenance of tissue homeostasis; however, their contribution to recovery from acute tissue injury is not fully understood. To address this issue, we generated an acute murine liver injury model using hepatocyte‐specific Cflar‐deficient (CflarHep‐low) mice. Cellular FLICE‐inhibitory protein expression was down‐regulated in Cflar‐deficient hepatocytes, which thereby increased susceptibility of hepatocytes to death receptor–induced apoptosis. CflarHep‐low mice developed acute hepatitis and recovered with clearance of apoptotic hepatocytes at 24 hours after injection of low doses of tumor necrosis factor α (TNFα), which could not induce hepatitis in wild‐type (WT) mice. Depletion of Kupffer cells (KCs) by clodronate liposomes did not impair clearance of dying hepatocytes or exacerbate hepatitis in CflarHep‐low mice. To elucidate the roles of BM‐derived monocytes and neutrophils in clearance of apoptotic hepatocytes, we examined the effect of depletion of these cells on TNFα‐induced hepatitis in CflarHep‐low mice. We reconstituted CflarHep‐low mice with BM cells from transgenic mice in which human diphtheria toxin receptor (DTR) was expressed under control of the lysozyme M (LysM) promoter. TNFα‐induced infiltration of myeloid cells, including monocytes and neutrophils, was completely ablated in LysM‐DTR BM‐reconstituted CflarHep‐low mice pretreated with diphtheria toxin, whereas KCs remained present in the livers. Under these experimental conditions, LysM‐DTR BM‐reconstituted CflarHep‐low mice rapidly developed severe hepatitis and succumbed within several hours of TNFα injection. We found that serum interleukin‐6 (IL‐6), TNFα, and histone H3 were aberrantly increased in LysM‐DTR BM‐reconstituted, but not in WT BM‐reconstituted, CflarHep‐low mice following TNFα injection. Conclusion: These findings indicate an unexpected role of myeloid cells in decreasing serum IL‐6, TNFα, and histone H3 levels via the suppression of TNFα‐induced hepatocyte apoptosis. (Hepatology 2017;65:237‐252).

Mapping of four ras superfamily genes by physical and genetic means in Schizosaccharomyces pombe

SummaryFour ras superfamily genes, namely ypt1, ypt2, ypt3 and ryh1, have been located on the S. pombe linkage map. This was achieved by constructing strains carrying a new NotI cutting site and the S. cerevisiae LEU2 gene integrated next to the respective gene. The physical location of these genes of the chromosomes was then determined by NotI restriction analysis of the DNA prepared from each strain. Fine genetic mapping was carried out by conventional tetrad analysis using the integrated LEU2 gene as a marker. The results indicated that ypt1 is tightly linked to top1 on the right arm of chromosome II; that ypt2 is 2.5 cM apart from ura2 on the right arm of chromosome I; that ypt3 is tightly linked to arg3 on the left arm of chromosome I; and that rhy1 is located approximately 20 cM from ade3 on the left arm of chromosome I.

Blockade of TNF receptor superfamily 1 (TNFR1)–dependent and TNFR1-independent cell death is crucial for normal epidermal differentiation

Background: A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD‐like ICE)‐inhibitory protein plays a crucial role in prevention of keratinocytes from TNF‐&agr;–dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE‐inhibitory protein in TNF‐&agr;–independent cell death remains unclear. Objective: We investigated contribution of TNF‐&agr;–dependent and TNF‐&agr;–independent signals to the development of dermatitis in epidermis‐specific Cflar‐deficient (CflarE‐KO) mice. Methods: We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of CflarE‐KO;Tnfrsf1a+/− and CflarE‐KO;Tnfrsf1a−/− mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF‐related apoptosis‐inducing ligand to block TNF‐&agr;–independent cell death of CflarE‐KO;Tnfrsf1a−/− mice. Results: CflarE‐KO;Tnfrsf1a−/− mice were born but experienced severe dermatitis and succumbed soon after birth. CflarE‐KO;Tnfrsf1a+/− mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from CflarE‐KO;Tnfrsf1a−/− mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF‐related apoptosis‐inducing ligand substantially prolonged survival of CflarE‐KO;Tnfrsf1a−/− mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of CflarE‐KO;Tnfrsf1a−/− mice. Treatment of primary keratinocytes with IL‐6 and, to a lesser extent, IL‐17A suppressed expression of epidermal differentiation markers. Conclusion: TNF receptor superfamily 1 (TNFR1)–dependent or TNFR1‐independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1‐dependent and TNFR1‐independent cell death might be an alternative strategy to treat skin diseases when treatment with anti–TNF‐&agr; antibody alone is not sufficient. GRAPHICAL ABSTRACT Figure. No caption available.