Tomiyasu Murata

Identification of nuclear import and export signals within the structure of the zinc finger protein TIS11.

TIS11, a member of the CCCH zinc finger protein family, functions as a positive transcriptional regulator. TIS11 was localized in both the cytoplasm and nucleus when transiently expressed in COS-7 cells. Upon treatment with leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, a marked nuclear accumulation of TIS11 was observed, indicating that TIS11 shuttles between the nucleus and the cytoplasm. By deletion studies using a green fluorescent protein fusion system, we have mapped a functional nuclear localization signal (NLS) to a region containing two tandem repeats of the zinc finger motif of TIS11. A site-directed mutagenesis analysis of TIS11 NLS has revealed the critical importance of two arginine residues (Arg127 and Arg131 in the rat TIS11). Furthermore, we demonstrated that the N-terminal Leu-rich region of TIS11 serves as an LMB-sensitive nuclear export signal (NES), indicating that TIS11 follows a CRM1-mediated export pathway. These results suggest that TIS11 is subject to constant nucleocytoplasmic shuttling due to its NLS and NES.

Involvement of regucalcin in lipid metabolism and diabetes

Regucalcin (RGN/SMP30) was originally discovered in 1978 as a unique calcium-binding protein that does not contain the EF-hand motif of calcium-binding domain. The regucalcin gene (rgn) is localized on the X chromosome and is identified in over 15 species consisting the regucalcin family. Regucalcin has been shown to play a multifunctional role in cell regulation; maintaining of intracellular calcium homeostasis and suppressing of signal transduction, translational protein synthesis, nuclear deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis, proliferation, and apoptosis in many cell types. Moreover, regucalcin may play a pathophysiological role in metabolic disorder. The expression of regucalcin is stimulated through the action of insulin in liver cells in vitro and in vivo and it is decreased in the liver of rats with type I diabetes induced by streptozotocin administration in vivo. Overexpression of endogenous regucalcin stimulates glucose utilization and lipid production in liver cells with glucose supplementation in vitro. Regucalcin reveals insulin resistance in liver cells. Deficiency of regucalcin induces an impairment of glucose tolerance and lipid accumulation in the liver of mice in vivo. Overexpression of endogenous regucalcin has been shown to decrease triglyceride, total cholesterol and glycogen contents in the liver of rats, inducing hyperlipidemia. Leptin and adiponectin mRNA expressions in the liver tissues are decreased in regucalcin transgenic rats. Decrease in hepatic regucalcin is associated with the development and progression of nonalcoholic fatty liver disease and fibrosis in human patients. Regucalcin may be a key molecule in lipid metabolic disorder and diabetes.

Methyl galbanate, a novel inhibitor of nitric oxide production in mouse macrophage RAW264.7 cells

It is well known that inflammation is associated with various neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease. An inflammatory mediator, nitric oxide (NO), is produced by inducible NO synthase (iNOS) in microglia and seems to be one of the possible causes of neurodegeneration. Several natural and synthetic compounds which exert anti-inflammatory effects by inhibiting NO production have been reported to date. The aim of this work was to investigate whether any of the 6 terpenoid coumarins (methyl galbanate, galbanic acid, farnesiferol A, badrakemone, umbelliprenin, and aurapten) isolated from Ferula szowitsiana DC. have inhibitory activity against NO production in RAW264.7 mouse macrophage cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Of the 6 terpenoid coumarins tested, methyl galbanate significantly decreased NO production in LPS/IFN-γ-stimulated RAW264.7 cells. In the presence of methyl galbanate, LPS/IFN-γ-induced iNOS mRNA expression was significantly decreased to 52% of the level found with LPS/IFN-γ stimulation alone. Methyl galbanate slightly attenuated COX-2 mRNA expression. Using the RAW264.7-tsAM5NE co-culture system, we showed that methyl galbanate protected neuronally differentiated tsAM5NE cells from NO-induced cell death by inhibiting the production of NO. Our finding suggests that methyl galbanate may be useful for developing a new drug against neurodegenerative diseases.

Apoptosis inducing activity of 4-substituted coumarins from Calophyllum brasiliense in human leukaemia HL-60 cells.

With the objective of identifying anti‐tumour‐promoting agents, we carried out a primary screening of ten 4‐substituted coumarins isolated from Calophyllum brasiliense Camb. (Guttiferae), to determine the ability of these compounds to inhibit proliferation of the human leukaemia cell line HL‐60. Among the 4‐substituted coumarins isolated, calophyllolide (2) and mammea B/BB (3) showed significant cytotoxicity against HL‐60 cells. Fluorescence microscopy with Hoechst 33342 staining revealed that the percentage of apoptotic cells with fragmented nuclei and condensed chromatin increased in a time‐dependent manner after treatment with calophyllolide (2) or mammea B/BB (3). In addition, the activity of caspase‐9 and caspase‐3 was also enhanced in a time‐dependent manner upon treatment with the 4‐substituted coumarins 2 and 3. Caspase‐9 and caspase‐3 inhibitors suppressed apoptosis induced by 4‐substituted coumarins 2 and 3. These results suggest that calophyllolide (2) and mammea B/BB (3) induced apoptosis in HL‐60 cells through activation of the caspase‐9/caspase‐3 pathway, which is triggered by mitochondrial dysfunction.

Alternatively spliced variants of the regucalcin gene in various human normal and tumor tissues.

Regucalcin plays a pivotal role as a suppressor protein in signal transduction in various cell types. The regucalcin gene, which is localized on the X chromosome, consists of 7 exons and 6 introns. Decreased liver regucalcin gene expression has been suggested to play a suppressive role in the development of hepatocellular carcinogenesis in animal models. This study was undertaken to determine the changes in regucalcin gene expression in various human normal and tumor tissues, including liver, kidney, brain and lung tissues. The full-length and alternatively spliced variants of regucalcin mRNA were found to be expressed in various human tissues. This expression was suppressed in tumor tissues of hepatocellular carcinoma, kidney transitional cell carcinoma, brain malignant meningioma and lung non-small cell carcinoma. The full-length regucalcin protein was found to be highly expressed in normal human liver and kidney tissues; its expression was suppressed, however, in the liver and kidney tumor tissues. The spliced variant proteins were found to be expressed in the normal liver and kidney tissues, and decreased in the tumor tissues. Such alternative variants were not observed in the liver and kidneys of rats and mice. The alternatively spliced variants of the regucalcin gene were found to be expressed in various human normal and tumor tissues.

Basic fibroblast growth factor promotes glial cell-derived neurotrophic factor gene expression mediated by activation of ERK5 in rat C6 glioma cells

Extracellular signal-regulated kinases (ERKs) play important physiological roles including proliferation, differentiation and gene expression. ERK5 contains kinase domain that shares homology with ERK1/2 and the T-E-Y activation motif at amino-terminal half, whereas the extended carboxy-terminal half is unique. Because the physiological role of ERK5 in glial cells remains unclear, we examined the involvement of ERK5 in expression of neurotrophic factors and cytokines in rat C6 glioma cells, comparing it with ERK1/2. Basic fibroblast growth factor (bFGF) induced both ERK5 and ERK1/2 phosphorylation in a time- and concentration-dependent manner. Among the neurotrophic factors and cytokines, bFGF induced significant gene expression of glial cell-derived neurotrophic factor (GDNF). The GDNF gene expression and protein synthesis induced by bFGF were blocked by BIX02189 and PD98059 that selectively inhibit ERK5 and ERK1/2 signaling, respectively. The effect was also blocked by overexpression of a dominant-negative MEK5 mutant, indicating that GDNF expression induced by bFGF requires both ERK5 and ERK1/2. Because GDNF gene expression is regulated by various transcription factors, we examined the activity of these factors. We demonstrated that phosphorylation of cAMP-response element-binding protein at Ser 133 was induced by bFGF, which was blocked by BIX02189 and PD98059. Expression of c-fos, a major component of activator protein-1, and early growth response-1 was enhanced by bFGF, and expression of these genes was blocked by BIX02189, PD98059 and overexpression of dominant-negative MEK5. Taking these results together, bFGF promotes GDNF expression accompanied by the activation of ERK5, ERK1/2 and their downstream transcription factors in C6 glioma cells.

Inhibition of Glucose-Induced Insulin Secretion Through Inactivation of Glucokinase by Glyceraldehyde

D-Glyceraldehyde irreversibly inhibited rat liver glucokinase in a concentration-dependent manner. The inactivation of glucokinase by glyceraldehyde was blocked by the presence of its substrates such as glucose and mannose. Glucokinase was highly sensitive to glyceraldehyde compared with some other glycolytic enzymes (from animal tissues) including hexokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. The amino acid analysis of untreated and glyceraldehyde-treated glucokinase suggested that glyceraldehyde-induced inactivation of glucokinase is caused by glycation of Lys residues of the enzyme by the triose. Treatment of pancreatic islets with 6 mM glyceraldehyde for 1 h at 37°C caused both inactivation of glucokinase and inhibition of glucose-induced insulin secretion. Another glucose-phosphorylating enzyme (hexokinase) in pancreatic islets, however, was little affected by glyceraldehyde. In addition, glyceraldehyde did not affect the insulin secretory responses of islets to nonglucose secretagogues such as glyceraldehyde and Leu. When pancreatic islets were cultured with a lower concentration (1 mM) of glyceraldehyde for a longer time (17 h) in the presence of 10 mM glucose to mimic the in vivo conditions, both glucokinase activity and glucose-induced insulin secretion were again decreased. This study demonstrates that glucose-induced insulin secretion is impaired by glyceraldehyde through the inactivation of glucokinase. The implication of this finding in the pathophysiology of type II diabetes is discussed.

Alpha- and beta-anomeric preference of glucose-induced insulin secretion at physiological and higher glucose concentrations, respectively

We determined the anomeric preference of glucose phosphorylation by islet glucokinase, glucose utilization by pancreatic islets, and insulin secretion induced by glucose over a wide range of glucose concentrations. alpha-D-Glucose was phosphorylated faster than beta-D-glucose by islet glucokinase at lower glucose concentrations (5 and 10 mM), whereas the opposite anomeric preference was observed at higher glucose concentrations (40 and 60 mM). At 20 mM, there was no significant difference in phosphorylation rate between the two anomers. Similar patterns of anomeric preference were observed both in islet glucose utilization and in glucose-induced insulin secretion. The present study affords strong evidence that glucokinase is responsible for the anomeric preference of glucose-stimulated insulin secretion through anomeric discrimination in islet glucose utilization.

Induction of apoptosis in human leukaemia HL‐60 cells by furanone‐coumarins from Murraya siamensis

To identify potential anti‐tumour agents, we screened five furanone‐coumarins isolated from Murraya siamensis Craib (Rutaceae) for their ability to inhibit the growth of human leukaemia HL‐60 cells. Among the furanone‐coumarins tested, murrayacoumarin B (compound 2) showed significant cytotoxicity against HL‐60 cells. Fluorescence microscopy with Hoechst 33342 staining revealed that the percentage of apoptotic cells with fragmented nuclei and condensed chromatin increased in a time‐dependent manner after treatment with murrayacoumarin B. Interestingly, this furanone‐coumarin induced the loss of the mitochondrial membrane potential. In addition, treatment with murrayacoumarin B stimulated the activities of caspase‐9 and caspase‐3, and caspase‐9 and caspase‐3 inhibitors suppressed the apoptosis induced by murrayacoumarin B. These results suggest that murrayacoumarin B induced apoptosis in HL‐60 cells through activation of the caspase‐9/caspase‐3 pathway triggered by mitochondrial dysfunction.

Protective Effects of Neurotrophic Factors on Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-mediated Apoptosis of Murine Adrenal Chromaffin Cell Line tsAM5D

We previously established the murine adrenal chromaffin cell line tsAM5D, which was immortalized with the temperature-sensitive simian virus 40 large T-antigen. tsAM5D cells have the capacity to differentiate into neuron-like cells in response to neurotrophic factors when the culture temperature is shifted from 33 to 39 °C. In this model system, the temperature shift in the absence of neurotrophic factors led to cell death. Hoechst staining analysis revealed that typical apoptotic nuclei appeared in a time-dependent manner after the temperature shift. Upon shifting to 39 °C, the degradation of T-antigen was accompanied by the transcriptional activation of p53 protein. Among the p53 target genes, death receptor 5 (DR5), which is the receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), showed the highest level of induction. Interestingly, TRAIL-neutralizing antibody protected tsAM5D cells from the temperature shift-induced apoptotic cell death by blocking the activation of caspase-8 and -3, indicating the involvement of TRAIL-mediated death signaling in the temperature shift-induced apoptosis. Glial cell line-derived neurotrophic factor (GDNF) inhibited the TRAIL-mediated activation of caspase-8 in tsAM5D cells exposed to 39 °C and cooperated with basic fibroblast growth factor and ciliary neurotrophic factor. Interestingly, the temperature shift induced oligomerization of DR5, which is the earliest process necessary for transduction of the death signal. This oligomerization was inhibited by treatment with GDNF plus ciliary neurotrophic factor but not by that with GDNF alone or GDNF plus basic fibroblast growth factor. These results are discussed with respect to the intracellular mechanism underlying the protective function of neurotrophic factors against TRAIL-mediated death signaling.