Kiyomi Hikita

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.

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.

Establishment and characterization of a noradrenergic adrenal chromaffin cell line, tsAM5NE, immortalized with the temperature-sensitive SV40 T-antigen.

We established a clonal adrenal medullary cell line, named tsAM5NE, from transgenic mice harbouring the temperature‐sensitive Simian virus 40 large T‐antigen gene, under the control of the tyrosine hydroxylase promoter. tsAM5NE cells conditionally grew at a permissive temperature of 33°C and exhibited the noradrenergic chromaffin cell phenotype. To understand the characteristics of tsAM5NE cells, we first examined the responsiveness of the cells to ligands of the GDNF (glial cell line‐derived neurotrophic factor) family. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not artemin or persephin. At the non‐permissive temperature of 39°C, GDNF or neurturin caused tsAM5NE cells to differentiate into neuron‐like cells; however, the differentiated cells died in a time‐dependent manner. Interestingly, LIF (leukaemia inhibitory factor) did not affect the GDNF‐mediated cell proliferation at 33°C, but promoted the survival and differentiation of GDNF‐treated cells at 39°C. In the presence of GDNF plus LIF, the morphological change induced by the temperature shift was associated with up‐regulated expression of neuronal markers, indicating that the cells had indeed undergone neuronal differentiation. Thus, we demonstrated that tsAM5NE cells had the capacity to terminally differentiate into neuron‐like cells in response to GDNF plus LIF when the oncogene was inactivated by the temperature shift. Thus, this cell line provides a useful model system for studying the mechanisms regulating neuronal differentiation.

Temperature-dependent, neurotrophic factor-elicited, neuronal differentiation in adrenal chromaffin cell line immortalized with temperature-sensitive SV40 T-antigen.

We established adrenal medullary cell lines from transgenic mice expressing an oncogene, the temperature‐sensitive simian virus 40 large T‐antigen, under the control of the tyrosine hydroxylase promoter. A clonal cell line, named tsAM5D, conditionally grew at a permissive temperature of 33°C and exhibited the dopaminergic chromaffin cell phenotype as exemplified by the expression pattern of mRNA for catecholamine‐synthesizing enzymes and secretory vesicle‐associated proteins. tsAM5D cells proliferated at the permissive temperature in response to basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). At a non‐permissive temperature of 39°C, bFGF and CNTF acted synergistically to differentiate tsAM5D cells into neuron‐like cells. In addition, tsAM5D cells caused to differentiate by bFGF plus CNTF at 39°C became dependent solely on nerve growth factor for their survival and showed markedly enhanced neurite outgrowth. In the presence of bFGF and CNTF, the morphological change induced by the temperature shift was associated with up‐regulated expression of neuronal marker genes including neuron‐specific enolase, growth‐associated protein‐43, microtubule‐associated protein 2, neurofilament, and p75 neurotrophin receptor, indicating that the cells underwent neuronal differentiation. Thus, we demonstrated that tsAM5D cells could proliferate at permissive 33°C, and also had the capacity to terminally differentiate into neuron‐like cells in response to bFGF and CNTF when the oncogene was inactivated by shifting the temperature to non‐permissive 39°C. These results suggest that tsAM5D cells should be a good tool to allow a detailed study of mechanisms regulating neuronal differentiation.

Inhibitory effect of carbazolequinone derivatives on lipopolysaccharide and interferon‐γ‐induced nitric oxide production in mouse macrophage RAW264.7 cells

The aim of this study was to examine the mechanism underlying the inhibitory effect of our synthesized carbazolequinone derivatives on nitric oxide (NO) production in activated macrophages.

Neuronal differentiation elicited by glial cell line-derived neurotrophic factor and ciliary neurotrophic factor in adrenal chromaffin cell line tsAM5D immortalized with temperature-sensitive SV40 T-antigen

To understand the characteristics of tsAM5D cells immortalized with the temperature‐sensitive simian virus 40 large T‐antigen, we first examined the responsiveness of the cells to ligands of the glial cell line‐derived neurotrophic factor (GDNF) family. tsAM5D cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not persephin or artemin. At the nonpermissive temperature of 39°C, GDNF or neurturin caused tsAM5D cells to differentiate into neuron‐like cells; however, the differentiated cells died in a time‐dependent manner. Interestingly, ciliary neurotrophic factor (CNTF) did not affect the GDNF‐mediated cell proliferation at 33°C but promoted the survival and differentiation of GDNF‐treated cells at 39°C. In the presence of GDNF plus CNTF, the morphological change induced by the temperature shift was associated with up‐regulated expression of various neuronal marker genes, indicating that the cells had undergone neuronal differentiation. In addition, tsAM5D cells caused to differentiate by GDNF plus CNTF at 39°C became dependent solely on nerve growth factor (NGF) for their survival and neurite outgrowth. Moreover, upon treatment with GDNF plus CNTF, the dopaminergic phenotype was suppressed by the temperature shift. Thus, we demonstrated that tsAM5D cells had the capacity to differentiate terminally into neuron‐like cells in response to GDNF plus CNTF when the oncogene was inactivated by the temperature shift. This cell line provides a useful model system for studying the role of a variety of signaling molecules for GDNF/CNTF‐induced neuronal differentiation.

Phenolic constituents from stem bark of Erythrina poeppigiana and their inhibitory activity on human glyoxalase I

A novel isoflavone, erythgianin A (1), along with nine known compounds 2–10, was isolated from the stem bark of Erythrinapoeppigiana (Leguminosae). The unusual isoflavone structure of 1, possessing a highly oxidized 3″,4″-dihydroxy-2″-hydroxymethyl-2″-methyl-2″,3″-dihydropyrano substituent, was determined on the basis of spectroscopic analyses. All of the isolated compounds were evaluated for their in vitro inhibitory activity toward human glyoxalase I. Among the isolates, isolupalbigenin (10) with two prenyl groups showed the highest inhibitory activity.

Autocrine TGF-β signaling is required for the GDNF/CNTF-induced neuronal differentiation of adrenal chromaffin tsAM5D cells expressing temperature-sensitive SV40 T-antigen

We recently established adrenal medullary cell line tsAM5D, which was immortalized by use of a temperature-sensitive mutant of the oncogene simian virus 40 large T-antigen. In the present study, when co-treated with glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), tsAM5D cells proliferated at the permissive temperature (33 degrees C) for the T-antigen expression and differentiated into neuron-like cells at the nonpermissive temperature (39 degrees C). Interestingly, in GDNF/CNTF-treated cultures, the addition of pan-specific transforming growth factor (TGF)-beta-neutralizing antibody did not affect the cell proliferation at 33 degrees C, but significantly reduced the survival of neuronally differentiated cells at 39 degrees C. Using real-time RT-PCR for analysis of GDNF/CNTF-treated cells, we found that the expression of mRNAs for TGF-beta1, TGF-beta2, and TGF-beta3 was up-regulated by the temperature shift. These results suggest that autocrine TGF-beta signaling is necessary for the survival of GDNF/CNTF-differentiated tsAM5D cells upon the temperature shift.

Regucalcin confers resistance to amyloid‐β toxicity in neuronally differentiated PC12 cells

Amyloid‐β (Aβ), a primary component of amyloid plaques, has been widely associated with the pathogenesis of Alzheimers disease. The Ca2+‐binding protein regucalcin (RGN) plays multiple roles in maintaining cell functions by regulating intracellular calcium homeostasis, various signaling pathways, and gene expression systems. Here, we investigated the functional role of RGN against Aβ‐induced cytotoxicity in neuronally differentiated PC12 cells. Overexpression of RGN reduced Aβ‐induced apoptosis by reducing mitochondrial dysfunction and caspase activation. It also attenuated Aβ‐induced reactive oxygen species production and oxidative damage and decreased Aβ‐induced nitric oxide (NO) overproduction, upregulation of inducible NO synthase by nuclear factor‐κB, and nitrosative damage. Interestingly, the genetic disruption of RGN increased the susceptibility of neuronally differentiated PC12 cells to Aβ toxicity. Thus, RGN possesses antioxidant activity against Aβ‐induced oxidative and nitrosative stress and may play protective roles against Aβ‐induced neurotoxicity in Alzheimers disease.

Identification of Active Site Amino Acid Residues of Phenylethanolamine N-Methyltransferase

Phenylethanolamine N-methyltransferase (PNMT; EC2.1.1.28) is an enzyme which catalyzes the formation of epinephrine from norepinephrine, the last step of the catecholamine biosynthesis pathway (Axelrod, 1962). PNMT is a monomeric enzyme, and similarly to other mammalian small-molecule N-methyltransferase, S-adenosyl-L-methionine is required for the enzyme reaction as a methyl donor. In addition to its major role in producing adrenomedullary hormones, PNMT has some important roles in blood pressure and neuroendocrine regulation in the central nervous system. Studies using PNMT inhibitors that act in vivo suggested involvement of epinephrine neurons in blood pressure regulation. However, PNMT inhibitors, more or less, possesses a2-adrenoceptor blocking activity. Therefore, information regarding the structure of the active site may be useful for development of selective and potent inhibitors for possible use as antihypertensive agents (Fuller, 1982). We have previously isolated a full-length cDNA encoding human PNMT, and revealed the primary structure of the enzyme (Kaneda et al., 1988). Human PNMT consists of 282 amino acid residues with a predicted molecular weight of 30,853 including the initial methionine. Using PCR-based site-directed mutagenesis, we investigated amino acid residues located at the active site of the enzyme.