Differentiation potential of an immortalized non-tumorigenic human liver epithelial cell line as liver progenitor cells.

We report the differentiation potential of an immortalized non-tumorigenic human liver epithelial cell line, THLE-5b. Under basic culture conditions THLE-5b showed undifferentiated phenotypes. When grown as cell aggregates, THLE-5b exhibited a hepatocyte-like ultrastructure, ammonia metabolic activity and several other indicators that suggest hepatocytic maturation, including up-regulation or induction of liver-specific genes such as albumin and tryptophane 2,3-dioxygenase, and down-regulation of biliary cell markers such as gamma-glutamyl transpeptidase (GGT). Under these conditions, transcriptional factors such as HNF-1 and HNF-4alpha were also up-regulated or induced. In Matrigel culture, expression of GGT was up-regulated. THLE-5b expressed both albumin and alpha 1-antitrypsin, but lost expression of CK19 in severe combined immunodeficient mice. Thus, THLE-5b can be aligned with progenitor cells, which are committed to the hepatocytic or biliary epithelial cell lineage. These results imply that bipotent progenitor cell populations similar to THLE-5b cells may exist in adult human liver.

Distribution, structure, organ-specific expression, and phylogenic analysis of the pathogenesis-related protein-3 chitinase gene family in rice (Oryza sativa L.).

Rice (Oryza sativa L.) pathogenesis-related (PR)-3 chitinases, like other PR proteins, are each coded by one of the genes of a multigene family in the plant genome. We assembled the database information about rice PR-3 chitinase sequences. A total of 12 PR-3 chitinase loci (Cht1 to Cht12) were found deployed in the rice genome. Some of the loci were occupied by 2 or more alleles. For all the loci expect Cht4, Cht5, Cht6, and Cht11, the amino acid sequence was polymorphic between japonica and indica varieties of rice, but glutamic acid acting as a catalytic residue was completely conserved in all the loci expect Cht7. All the genes except Cht7, which was not tested in this study, were transcripted in some organs (leaf, sheath, root, and meristem) of rice plants. These results suggest that chitinase proteins encoded by the genes at these loci have important biological effects, at least antifungal activities, on rice plants. We also proposed a new classification of rice PR-3 chitinases based on their domain structures. This classification was consistent with the results of phylogenetic analysis of rice chitinases.

Widespread collaboration of Isw2 and Sin3-Rpd3 chromatin remodeling complexes in transcriptional repression.

The yeast Isw2 chromatin remodeling complex functions in parallel with the Sin3-Rpd3 histone deacetylase complex to repress early meiotic genes upon recruitment by Ume6p. For many of these genes, the effect of an isw2 mutation is partially masked by a functional Sin3-Rpd3 complex. To identify the full range of genes repressed or activated by these factors and uncover hidden targets of Isw2-dependent regulation, we performed full genome expression analyses using cDNA microarrays. We find that the Isw2 complex functions mainly in repression of transcription in a parallel pathway with the Sin3-Rpd3 complex. In addition to Ume6 target genes, we find that many Ume6-independent genes are derepressed in mutants lacking functional Isw2 and Sin3-Rpd3 complexes. Conversely, we find that ume6 mutants, but not isw2 sin3 or isw2 rpd3 double mutants, have reduced fidelity of mitotic chromosome segregation, suggesting that one or more functions of Ume6p are independent of Sin3-Rpd3 and Isw2 complexes. Chromatin structure analyses of two nonmeiotic genes reveals increased DNase I sensitivity within their regulatory regions in an isw2 mutant, as seen previously for one meiotic locus. These data suggest that the Isw2 complex functions at Ume6-dependent and -independent loci to create DNase I-inaccessible chromatin structure by regulating the positioning or placement of nucleosomes.

Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates.

To facilitate the biochemical characterization of chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae, we have developed a system to assemble nucleosomal arrays on immobilized templates using recombinant yeast core histones. This system enabled us to analyze the interaction of Isw2 ATP-dependent chromatin remodeling complex with nucleosomal arrays. We found that Isw2 complex interacts efficiently with both naked DNA and nucleosomal arrays in an ATP-independent manner, suggesting that ATP is required at steps subsequent to this physical interaction. We identified the second subunit of Isw2 complex, encoded by open reading frame YGL 133w (herein named ITC1), and found that both subunits of the complex, Isw2p and Itc1p, are essential for efficient interaction with DNA and nucleosomal arrays. Both subunits are also required for nucleosome-stimulated ATPase activity and chromatin remodeling activity of the complex. Finally, we found that ITC1 is essential for function of Isw2 complex in vivo, since isw2 and itc1 deletion mutants exhibit virtually identical phenotypes. These results demonstrate the utility of our in vitro system in studying interactions between chromatin-associated proteins and nucleosomal arrays.

ATP-dependent nucleosome remodeling and histone hyperacetylation synergistically facilitate transcription of chromatin.

Drosophila nucleosome remodeling factor (NURF) is an ISWI-containing protein complex that facilitates nucleosome mobility and transcriptional activation in an ATP-dependent manner. Numerous studies have implicated histone acetylation in transcriptional activation. We investigated the relative contributions of these two chromatin modifications to transcription in vitro of a chromatinized adenovirus E4 minimal promoter that contains binding sites for the GAL4-VP16 activator. We found that NURF could remodel chromatin and stimulate transcription irrespective of the acetylation status of histones. In contrast, hyperacetylation of histones in the absence of NURF was unable to stimulate transcription, suggesting that NURF-dependent chromatin remodeling is an obligatory step in E4 promoter activation. When chromatin templates were first hyperacetylated and then incubated with NURF, significantly greater transcription stimulation was observed. The results suggest that changes in chromatin induced by acetylation of histones and the mobilization of nucleosomes by NURF combine synergistically to facilitate transcription. Experiments using single and multiple rounds of transcription indicate that these chromatin modifications stimulate transcription preinitiation as well as reinitiation.

Down-regulation of p27Kip1 expression is required for development and function of T cells.

The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27(Kip1) is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4(-)CD8(-) thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27(Kip1) expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27(Kip1) expression. The development of thymocytes in the transgenic strain in which p27(Kip1) is most abundant (p27-Tg(high) mice) appeared to be blocked at the CD4(-)CD8(-)CD25(+)CD44(low) stage. Peripheral T cells from p27-Tg(high) mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tg(high) mice. These results suggest that down-regulation of p27(Kip1) expression is required for the development, proliferation, and immunoresponsiveness of T cells.

The Isw2 chromatin remodeling complex represses early meiotic genes upon recruitment by Ume6p.

The ISWI class of chromatin remodeling factors exhibits potent chromatin remodeling activities in vitro. However, the in vivo functions of this class of factors are unknown at a molecular level. We have found that S. cerevisiae Isw2 complex represses transcription of early meiotic genes during mitotic growth in a parallel pathway to Rpd3-Sin3 histone deacetylase complex. This repressor function of lsw2 complex is largely dependent upon Ume6p, which recruits the complex to target genes. Nuclease digestion analyses revealed that lsw2 complex establishes nuclease-inaccessible chromatin structure near the Ume6p binding site in vivo. Based on these findings, we propose a model for the mechanism of transcriptional repression by two distinct chromatin remodeling complexes.

Development of dendritic epidermal T cells with a skewed diversity of gamma delta TCRs in V delta 1-deficient mice.

One of the most intriguing features of gammadelta T cells that reside in murine epithelia is the association of a specific Vgamma/Vdelta usage with each epithelial tissue. Dendritic epidermal T cells (DETCs) in the murine epidermis, are predominantly derived from the "first wave" Vgamma5+ fetal thymocytes and overwhelmingly express the canonical Vgamma5/Vdelta1-TCRs lacking junctional diversity. Targeted disruption of the Vdelta1 gene resulted in a markedly impaired development of Vgamma5+ fetal thymocytes as precursors of DETCs; however, gammadeltaTCR+ DETCs with a typical dendritic morphology were observed in Vdelta1-/- mice and their cell densities in the epidermis were slightly lower than those in Vdelta1+/- epidermis. Moreover, the Vdelta1-deficient DETCs were functionally competent in their ability to up-regulate cytokines and keratinocyte growth factor-expression in response to keratinocytes. Vgamma5+ DETCs were predominant in the Vdelta1-/- epidermis, though Vgamma5- gammadeltaTCR+ DETCs were also detected. The Vgamma5+ DETCs showed a typical dendritic shape, gammadeltaTCR(high), and age-associated expansion in epidermis as observed in conventional DETCs of normal mice, whereas the Vgamma5- gammadeltaTCR+ DETCs showed a less dendritic shape, gammadeltaTCR(low), and no expansion in the epidermis, consistent with their immaturity. These results suggest that optimal DETC development does not require a particular Vgamma/Vdelta-chain usage but requires expression of a limited diversity of gammadeltaTCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment.

New milbemycins from Streptomyces hygroscopicus subsp. aureolacrimosus: fermantation, isolation and strucutre elucidation.

Twelve new milbemycins have been isolated and characterized from some strains derived from Streptomyces hygroscopicus subsp. aureolacrimosus SANK 60286 and SANK 60526. The metabolites 1 approximately 4 and 9 approximately 11 were produced by strain RM28D-688 SANK 60797 as minor products. The metabolites 5 approximaetly 8 were obtained from a broth of strain 57-338 SANK 61796. Strain MK-1391 SANK 62896 was used for the production of metabolite 12. The new metabolites, eight alpha-class and four beta-class compounds, have new structural features. For example, milbemycins alpha26 and alpha27, have the 26-hydroxy moiety, and other derivatives (milbemycins alpha20 approximately 23) have different side chains at the C-26 position from those of milbemycins alpha11 and alpha14. In addition, 5-hydroxylmilbemycin beta7 (beta12), involved in the major biosynthetic pathway of 25-methyl and 25-ethyl milbemycins, was discovered.

Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse.

A principal pathway of 2-methoxyethanol (ME) metabolism is to the toxic oxidative product, methoxyacetaldehyde (MALD). To assess the role of aldehyde dehydrogenase (ALDH) in MALD metabolism, in vitro MALD oxidation was examined with liver subcellular fractions from Japanese subjects who carried three different ALDH2 genotypes and Aldh2 knockout mice, which were generated in this study. The activity was distributed in mitochondrial fractions of ALDH2*1/*1 and wild type (Aldh2+/+) mice but not ALDH2*1/*2, *2/*2 subjects or Aldh2 homozygous mutant (Aldh2-/-) mice. These data suggest that ALDH2 is a key enzyme for MALD oxidation and ME susceptibility may be influenced by the ALDH2 genotype.

The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo.

Drosophila ISWI, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF, CHRAC, and ACF. To clarify the biological functions of ISWI, we generated and characterized null and dominant-negative ISWI mutations. We found that ISWI mutations affect both cell viability and gene expression during Drosophila development. ISWI mutations also cause striking alterations in the structure of the male X chromosome. The ISWI protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for ISWI in transcriptional repression. These findings reveal novel functions for the ISWI ATPase and underscore its importance in chromatin remodeling in vivo.

Aberrant cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice.

The recent discovery of checkpoint kinases has suggested the conservation of checkpoint mechanisms between yeast and mammals. In yeast, the protein kinase Chk1 is thought to mediate signaling associated with the DNA damage checkpoint of the cell cycle. However, the function of Chk1 in mammals has remained unknown. Targeted disruption of Chk1 in mice showed that Chk1(-/-) embryos exhibit gross morphologic abnormalities in nuclei as early as the blastocyst stage. In culture, Chk1(-/-) blastocysts showed a severe defect in outgrowth of the inner cell mass and died of apoptosis. DNA replication block and DNA damage failed to arrest the cell cycle before initiation of mitosis in Chk1(-/-) embryos. These results may indicate that Chk1 is indispensable for cell proliferation and survival through maintaining the G(2) checkpoint in mammals.

The congenital anastomoses between hepatic arteries: angiographic appearance.

The purpose of this study was to evaluate congenital anastomoses between hepatic arteries demonstrated on angiography in ten patients and to correlate the anastomosis with types of hepatic arterial anatomy. We evaluated the types of the hepatic arterial anatomy based on Michels' classification for 720 patients and compared the anatomic types between the patients with the anastomoses (ten patients) and without the anastomoses (710 patients). The diameter of the anastomoses ranged from 1.5 to 3.0 mm (mean, 2.4 mm). Five anastomoses were classified as tortuous type and five as straight type. Based on Michels' classification for types of hepatic arterial anatomy, eight (80%) of ten patients with the congenital anastomoses were classified as type III (replaced right hepatic artery from superior mesenteric artery). The remaining two patients were classified as type IV (replaced right hepatic artery from superior mesenteric artery and replaced left hepatic artery from left gastric artery) and type VIIIa (replaced right hepatic artery from superior mesenteric artery and accessory left hepatic artery from left gastric artery). Eight (16%) of 48 patients who were classified as type III have the anastomoses. In conclusion, the congenital anastomoses were observed especially in patients with replaced right hepatic artery from superior mesenteric artery.

Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication.

The ubiquitin-proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an F-box protein and substrate recognition component of an Skp1-Cullin-F-box protein (SCF) ubiquitin ligase, were generated. Although Skp2(-/-) animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show a reduced growth rate and increased apoptosis. Skp2(-/-) cells also exhibit increased accumulation of both cyclin E and p27(Kip1). The elimination of cyclin E during S and G(2) phases is impaired in Skp2(-/-) cells, resulting in loss of cyclin E periodicity. Biochemical studies showed that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro. These results suggest that specific degradation of cyclin E and p27(Kip1) is mediated by the SCF(Skp2) ubiquitin ligase complex, and that Skp2 may control chromosome replication and centrosome duplication by determining the abundance of cell cycle regulators.

Bioconversion of milbemycin-related compounds: isolation and utilization of non-producer, strain RNBC-5-51.

A non-producing strain, the so-called strain RNBC-5-51 SANK 60198, was isolated during a screening program of strain improvement in the milbemycin production. Strain RNBC-5-51 indicated almost the same characteristics as those in the parent strain, that is, the abundant spore formation on agar media and the good growth in liquid media. But it does not produce any kind of milbemycins. In addition, strain RNBC-5-51 accumulated precursor-like compounds of milbemycin-polyketide, the production of which were inhibited by the addition of cerulenin. In the bioconversion experiments, strain RNBC-5-51 converted milbemycin beta6 and A4 to milbemycin alpha14, and milbemycin beta7 and A3 to milbemycin alpha11, respectively. This strain also converted milbemycin D and avermectin B1a, to 26-(3-methyl-2-butenoyloxy)milbemycin D and 26-(3-methyl-2-butenoyloxy)avermectin B1a, respectively. These results suggest that milbemycin alpha11 is biosynthesized through the same route as milbemycin alpha14, and the mutated step in strain RNBC-5-51 might be in the polyketide synthetic pathway of milbemycins. Strain RNBC-5-51 loses the ability for de novo synthesis of milbemycins, but it retains the ability to bioconvert the milbemycin skeleton. This strain might be useful for C-26 modification of milbemycin-related compounds.

Balloon-occluded retrograde transvenous obliteration of gastric varix draining via the left inferior phrenic vein into the left hepatic vein.

We encountered a patient with gastric varix draining not via the usual left suprarenal vein but via the left inferior phrenic vein joining the left hepatic vein. Transfemoral balloon-occluded retrograde transvenous obliteration (BRTO) of the varix was performed under balloon occlusion of the left inferior phrenic vein via the left hepatic vein and retrograde injection of the sclerosing agent (5% of ethanolamine oleate) into the gastric varix. Disappearance of the gastric varix was confirmed on endoscopic examination 2 months later.

Establishment of an embryonic stem (ES) cell line derived from a non-obese diabetic (NOD) mouse: in vivo differentiation into lymphocytes and potential for germ line transmission.

A non-obese diabetic (NOD) mouse-derived embryonic stem (ES) cell line has been stably maintained in an undifferentiated state with a characteristic ES cell-like morphology, expressing the stem cell marker alkaline phosphatase, and displaying a normal diploid karyotype. After injecting the NOD-ES cells into blastocysts, chimeric mice were obtained. Small but significant numbers of lymphocytes expressed the NOD-derived MHC allele. When a chimeric mouse was mated with C57BL/6 mice, an agouti mouse was obtained, having the NOD-derived H-2 I-A(beta)g7 haplotype. Thus, an NOD-ES cell line which can differentiate into lymphocytes with potential for germ line transmission was successfully established.

Characterization of the imitation switch subfamily of ATP-dependent chromatin-remodeling factors in Saccharomyces cerevisiae.

We have identified and characterized two Imitation Switch genes in Saccharomyces cerevisiae, ISW1 and ISW2, which are highly related to Drosophila ISWI, encoding the putative ATPase subunit of three ATP-dependent chromatin remodeling factors. Purification of ISW1p reveals a four-subunit complex with nucleosome-stimulated ATPase activity, as well as ATP-dependent nucleosome disruption and spacing activities. Purification of ISW2p reveals a two-subunit complex also with nucleosome-stimulated ATPase and ATP-dependent nucleosome spacing activities but no detectable nucleosome disruption activity. Null mutations of ISW1, ISW2, and CHD1 genes cause synthetic lethality in various stress conditions in yeast cells, revealing the first in vivo functions of the ISWI subfamily of chromatin-remodeling complexes and demonstrating their genetic interactions. A single point mutation within the ATPase domain of both ISW1p and ISW2p inactivated all ATP-dependent biochemical activities of the complexes, as well as the ability of the genes to rescue the mutant phenotypes. This demonstrates that the ATP-dependent chromatin-remodeling activities are essential for the in vivo functions of both ISW1 and ISW2 complexes.