Identification of ortho-hydroxy anilide as a novel scaffold for lysine demethylase 5 inhibitors

Fe(II)/α-ketoglutarate-dependent lysine demethylases (KDMs) are attractive drug targets for several diseases including cancer. In this study, we designed and screened ortho-substituted anilides that are expected to function as Fe(II) chelators, and identified ortho-hydroxy anilide as a novel scaffold for KDM5A inhibitors. Treatment of human lung cancer A549 cells with a prodrug form of 4-carboxy-2-hydroxy-formanilide (9c) increased trimethylated lysine 4 on histone H3 level, suggesting KDM5 inhibition in the cells.     

Crystal structure of intracellular family 1 beta-glucosidase BGL1A from the basidiomycete Phanerochaete chrysosporium

The white-rot fungus Phanerochaete chrysosporium has two intracellular beta-glucosidases (BGL1A and BGL1B) belonging to glycoside hydrolase (GH) family 1. BGL1B effectively hydrolyzes cellobiose and cellobionolactone, but BGL1A does not. We have determined the crystal structure of BGL1A in substrate-free and gluconolactone complexed forms. The overall structure and the characteristic of subsite -1 (glycone site) were similar to those of other known GH1 enzymes. The loop regions covering on the (beta/alpha)(8) barrel was significantly deviated, and they form a unique subsite +1 (aglycone site) of BGL1A.     

Functional implication of nucleolin in the mouse first molar development

We examined the functional implication of nucleolin in the mouse first molar development. Both the nucleolin mRNA and protein expressions were demonstrated in the odontogenic epithelial cells in the early stage and in the inner enamel epithelial layer in the late stage. The expression pattern of nucleolin corresponded to the proliferating cells in the tooth germ, thus showing that nucleolin could possibly be related to cell proliferation. No in situ signal of nucleolin was found in the primary enamel knot (PEK). Furthermore, nucleolin protein was demonstrated in the PEK by immunohistochemistry. The existence of nucleolin protein in the PEK may possibly be related to the apoptosis in the PEK cells. An inhibition assay using the hemagglutinating virus of Japan-liposome containing nucleolin antisense phosphorothioated oligonucleotide (AS S-ODN) in cultured mouse mandibles at embryonic day (E) 11.0 showed a marked growth inhibition of tooth germ. Moreover, no developmental arrest was found in the cultured tooth germ at E15.0 treated with nucleolin AS S-ODN. Real time PCR was performed to examine the mRNA expression of nucleolin-related genes, and a significant reduction in the midkine mRNA expression was thus observed in the mouse mandible after being treated with nucleolin AS S-ODN. This inhibition assay indicated that nucleolin could thus be involved in the early stage of tooth germ initiation and morphogenesis, possibly by regulating the midkine expression.     

Role of MAPK phosphatase-1 in the induction of monocyte chemoattractant protein-1 during the course of adipocyte hypertrophy

Monocyte chemoattractant protein-1 (MCP-1), an important chemokine whose expression is increased during the course of obesity, plays a role in macrophage infiltration into obese adipose tissue. This study was designed to elucidate the role of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the induction of MCP-1 during the course of adipocyte hypertrophy. We examined the time course of MKP-1 and MCP-1 mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation in the adipose tissue from mice rendered mildly obese by a short term high fat diet. We also studied the role of MKP-1 in the induction of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. MCP-1 mRNA expression was increased, followed by ERK activation and down-regulation of MKP-1, an inducible dual specificity phosphatase to inactivate ERK, in the adipose tissue at the early stage of obesity induced by a short term high fat diet, when macrophages are not infiltrated. Down-regulation of MKP-1 preceded ERK activation and increased production of MCP-1 in 3T3-L1 adipocytes in vitro during the course of adipocyte hypertrophy. Adenovirus-mediated restoration of MKP-1 in hypertrophied 3T3-L1 adipocytes reduced the otherwise increased ERK phosphorylation, thereby leading to the significant reduction of MCP-1 mRNA expression. This study provides evidence that the down-regulation of MKP-1 is critical for increased production of MCP-1 during the course of adipocyte hypertrophy.     

Localization of activated caspase-3-positive and apoptotic cells in the developing tooth germ of the mouse lower first molar

This study examined the immunohistochemical detection of activated caspase-3, and its association with apoptosis, during tooth morphogenesis of the mouse lower first molar. The distribution of cells positive for caspase-3 closely corresponded with the localization of the terminal deoxynucleotidyl transferase-mediated deoxyuridine-5-triphosphate-biotin nick end labelling (TUNEL)-positive apoptotic cells through the developmental course of tooth germs from embryo day 12 (E12) to E19, thus showing that the apoptosis occurring in the developing odontogenic tissue was induced by the activation of the caspase family. The specific distribution pattern of apoptotic cells in the developing odontogenic epithelial tissue from the initiation (E12) of tooth germ to the completion of tooth crown morphology (E19) also suggests that apoptotic events are related not only to a deletion of functionally suspended cells, but also participate in initiation and the completion of tooth morphogenesis. Electron microscopic examination revealed that apoptotic cells were present in the primary enamel knot, and these apoptotic cells were phagocytized by neighbouring odontogenic epithelial cells, thus indicating the prompt disposal of any dead cells by epithelial cells.     

Photoperiodic response of serotonin- and galanin-immunoreactive neurons of the paraventricular organ and infundibular nucleus in Japanese quail, Coturnix coturnix japonica

We investigated the photoperiodic response of serotonin- and galanin (GA)- immunoreactive (ir) cells in the paraventricular organ (PVO) and infundibular nucleus (IF) of the Japanese quail and the interaction of these cells with gonadotropin-releasing hormone (GnRH)-ir neurons in the hypothalamus. Serotonin-ir cells were located in series from the PVO to the IF, and were connected with each other. The number of serotonin-ir cells differed significantly between light and dark phases on the short days (SD), but did not differ between light and dark phases on long days (LD). GA-ir cells were also found in the PVO and IF. The number of GA-ir cells under SD conditions was significantly greater than under LD conditions but did not change diurnally. Both serotonin-ir and GA-ir fibers ran along the GnRH-ir cells in the nucleus commissurae pallii. Serotonin-ir and GA-ir fibers were connected with the GnRH-ir fibers in the external layer of the median eminence (ME). We confirmed that GA-ir fibers were closely associated with serotonin-ir neurons in the PVO and IF. GA-ir neurons have at least 2 routes of regulating GnRH neurons directly, and indirectly via the serotonin-ir cells in the PVO and IF.     

Expression of DNase gamma during Fas-independent apoptotic DNA fragmentation in rodent hepatocytes

Endonuclease-induced DNA fragmentation is a hallmark of apoptosis. DNase gamma (DNase ) was recently identified as one of the endonucleases responsible for apoptotic DNA fragmentation. In this study, immunohistochemistry for DNase was performed on paraffin sections of rodent liver in well-defined models of hepatocyte apoptosis induced by Fas antibody (Fas) or cycloheximide (CHX), and necrosis induced by lipopolysaccharide (LPS) or carbon tetrachloride (CCl₄). DNase immunoreactivity was compared with TdT-mediated dUTP nick-end labeling (TUNEL) reactivity. Our results showed TUNEL reactivity in both apoptotic and necrotic hepatocytes. DNase immunoreactivity was not detected during LPS-induced or CCl₄-induced hepatocyte necrosis. In contrast, it was evident during CHX-induced, but not Fas-induced, apoptotic DNA fragmentation. These findings suggest that DNase plays an important role in Fas-independent apoptotic DNA fragmentation in hepatocytes.     

Chemical and physical defence in early and late leaves in three heterophyllous birch species native to northern Japan

BACKGROUND AND AIMS: Betula ermanii, B. maximowicziana and B. platyphylla var. japonica have heterophyllous leaves (i.e. early leaves and late leaves) and are typical pioneer species in northern Japan. Chemical and physical defences against herbivores in early and late leaves of these species were studied. METHODS: Two-year-old seedlings were grown under full sunlight in a single growing season. Three-week-old leaves of each seedling were harvested three times (May, July and October). Total phenolics and condensed tannin content were determined for chemical defence and leaf toughness and trichome density were assessed for physical defence. Defoliation of early leaves in May was also performed to study the contribution of early leaves to subsequent growth. KEY RESULTS: Chemical and physical defences were greater in early than late leaves in B. platyphylla and B. ermanii, whereas the reverse was true in B. maximowicziana. In contrast to its weak chemical defences, the trichome density in B. maximowicziana was very high. In B. platyphylla and B. ermanii, the relative growth rates (RGR) were greater early in the growing season. Negative effects on growth of removal of early leaves were significant only in B. platyphylla. CONCLUSIONS: B. platyphylla and B. ermanii invest in defence in early rather than late leaves, since early leaves are crucial to subsequent growth. In contrast, B. maximowicziana more strongly defends its late leaves, since its RGR is maintained at the same level throughout the growing season.     

Growth,net photosynthesis and leaf nutrient status of <Emphasis Type="Italic">Fagus crenata</Emphasis> seedlings grown in brown forest soil acidified with H<Subscript>2</Subscript>SO<Subscript>4</Subscript> or HNO<Subscript>3</Subscript> solution

To obtain basic information for evaluating critical loads of acid deposition for protecting Japanese beech forests, growth, net photosynthesis and leaf nutrient status of Fagus crenata seedlings grown for two growing seasons in brown forest soil acidified with H₂SO₄ or HNO₃ solution were investigated. The whole-plant dry mass of the seedlings grown in the soil acidified by the addition of H₂SO₄ or HNO₃ solution was significantly less than that of the seedlings grown in the control soil not supplemented with H⁺ as H₂SO₄ or HNO₃ solution. However, the degrees of reduction in the whole-plant dry mass and net photosynthetic rate of the seedlings grown in the soil acidified by the addition of H⁺ as H₂SO₄ solution at 100 mg l^–1 on the basis of air-dried soil volume (S-100 treatment) were greater than those of the seedlings grown in the soil acidified by the addition of H⁺ as HNO₃ solution at 100 mg l^–1 (N-100 treatment). The concentrations of Al and Mn in the leaves of the seedlings grown in the S-100 treatment were significantly higher than those in the N-100 treatment. A positive correlation was obtained between the molar ratio of (Ca+Mg+K)/(Al+Mn) in the soil solution and the relative whole-plant dry mass of the seedlings grown in the acidified soils to that of the seedlings grown in the control soil. Based on the results, we concluded that the negative effects of soil acidification due to sulfate deposition are greater than those of soil acidification due to nitrate deposition on growth, net photosynthesis and leaf nutrient status of F. crenata, and that the molar ratio of (Ca+Mg+K)/(Al+Mn) in soil solution is a suitable soil parameter for evaluating critical loads of acid deposition in efforts to protect F. crenata forests in Japan.