Laboratory of Molecular Nutrition and Toxicology (Himeno Lab.)

Metallomics (2012) in press HOT Article
The article entitled “Roles of ZIP8, ZIP14, and DMT1 in transport of cadmium and manganese in mouse kidney proximal tubule cells” published in Metallomics, a journal covering the research fields related to biometals, by Dr. Fujishiro et al. was selected as a HOT Article of the issue.

Cadmium (Cd) is known as an environmental toxicant that causes renal damage in animals and humans. Dr. Fujishiro et al. in the Laboratory of Molecular Nutrition and Toxicology have been studying the role of ZIP8, a zinc transporter, in the transport of Cd and Mn. In the above-mentioned paper published in Metallomics, they proposed a hypothesis that ZIP8 plays a significant role in the accumulation of Cd in S3 segment of the proximal tubule cells in the kidney based on the observation on cultured proximal tubule cells and in situ hybridization in mouse kidney.

Department of Physical Chemistry (Fukuyama Lab.)

Heterocycles (2010) 81; 1571-1602.
This review focuses on the structural diversity, biological activities and synthesis of vibsane-type diterpenoids. Vibsane-type diterpenoids are considered to be rarely occurring natural products because they have been found exclusively in a few Viburnum speacies such as V. awabuki, V. odoratissimum, and V. suspensum. These diterpenoids are futher classified into 11-membered ring, 7-membered ring, and reaaranged (neovibsanin) types, and therefore, their chemical diversity forms a unique chemical library. We describe the absolute atereochemistry of the typical 11-membered ring vibsanin B and F, a variety of vibsane-type diterpenoids, the chemical correlations between the three subtypes, their biological activities, and the synthesis of vibsanin F and neovibsanin B.

Department of Biochemistry (Kuzuhara Lab.)

J Biol Chem (2009) 284; 6855-6860
Because the influenza A virus has an RNA genome, its RNA-dependent RNA polymerase, comprising the PA, PB1, and PB2 subunits, is essential for viral transcription and replication. The binding of RNA primers/promoters to the polymerases is an initiation step in viral transcription. In our current study, we reveal the 2.7 A tertiary structure of the C-terminal RNA-binding domain of PB2 by x-ray crystallography. This domain incorporates lysine 627 of PB2, and this residue is associated with the high pathogenicity and host range restriction of influenza A virus. We found from our current analyses that this lysine is located in a unique f-shaped structure consisting of a helix and an encircled loop within the PB2 domain. By electrostatic analysis, we identified a highly basic groove along with this phi loop and found that lysine 627 is located in the phi loop. A PB2 domain mutant in which glutamic acid is substituted at position 627 shows significantly lower RNA binding activity. This is the first report to show a relationship between RNA binding activity and the pathogenicity-determinant lysine 627. Using the Matras program for protein three-dimensional structural comparisons, we further found that the helix bundles in the PB2 domain are similar to that of activator 1, the 40-kDa subunit of DNA replication clamp loader (replication factor C), which is also an RNA-binding protein. This suggests a functional and structural relationship between the RNA-binding mechanisms underlying both influenza A viral transcription and cellular DNA replication. Our present results thus provide important new information for developing novel drugs that target the primer/promoter RNA binding of viral RNA polymerases.

Institute of Pharmacognosy (Toyota Lab.)

Chem Pharm Bull (2011) 59; 480-483
The ether extract of the New Zealand liverwort Marsupidium epiphytum gave four new prenyl bibenzyl derivatives, along with a known prenyl bibenzyl derivative which has been isolated from the Ecuadorian liverwort Lethocolea glossophylla; their structures were determined by 2D NMR spectrum. The chemical constituents of Marsupidium epiphytum are very characteristic since it elaborates dihydrooxepin type and prenyl type bibenzyls. These structures are closely related to those found in Radula spp. (Radulaceae), although bibenzyls with two prenyl groups have not been isolated from the Radula spp. Although Marsupidium spp. are different from Radula spp. morphologically, the constituents are closely related. This is the first example of isolation of prenyl bibenzyl derivatives from M. epiphytum, a species which has not previously been investigated phytochemically.

Department of Structure and Function Research (Setsu Lab.)

Exp Ther Med (2011) 2; 793-797
Lymph node metastasis is considered a factor in determining the prognosis of squamous cell carcinoma (SCC). Both oral and cervical SCC tumor cells prefer lymph vessels as the route of metastasis. D2-40 is a specific marker of lymphatic endothelial cells. This study clarifies the distribution and characteristics of lymphatic vessels in oral and cervical SCCs. Immunohistochemistry was performed in 20 oral and 20 cervical SCCs (10 non-metastatic and 10 metastatic to lymph nodes) using D2-40, CD31, CD34, CD105 and double staining with D2-40 and keratin. Lymphatic vessel density (LVD) was also determined morphologically. Results showed that lymphatic vessels in both types of SCCs were distributed mainly at the superficial region beneath the epithelium. The LVD in each tumor was significantly higher compared to the corresponding normal mucosa. Moreover, the LVD in lymph node metastasis in each tumor was significantly higher compared to their non-metastatic counterparts. Cancer cell invasion was observed in the lymphatic vessels suggesting the existence of lymph node involvement during metastasis. The new lymphatic vessels that proliferated around the cancer nests in both SCCs have endothelial cell characteristics inferred to be associated with early lymphatic development and initial dissemination of cancer cells.

Department of Pharmaceutics (Sakurai Lab.)

Biol Trace Elem Res (2011) 142: 713-722
The aim of this study was to examine enhancing effect of L-histidine into cultured rat lung microvascular endothelial cells (LMECs), which constitute the gas-blood barrier. Uptake of L-histidine into LMECs markedly increased with the addition of ZnSO4 (0.1 mmol/L), and this enhanced uptake of L-histidine was drastically reduced in the presence of the Na+-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH). However, the uptake of L-histidine together with ZnSO4 was not reduced by the addition of metabolic inhibitor, 2,4-dinitrophenol (DNP) or by sodium ion replacement. Moreover, the addition of the system N-substrate, L-glutamic acid g-monohydroxamate did not significantly decrease the uptake of L-histidine with 143 mmol/L Na + + 1 mmol/L BCH. These results indicated that system-N transporter does not play a role in the uptake of L-histidine in the presence of ZnSO4, suggesting that only system-L transporter is involved in the uptake of L-histidine, although L-histidine in the absence of ZnSO4 was taken up by at least two pathways of Na+-dependent system-N and Na+-independent system-L processes into rat LMECs. The uptake of L-histidine into rat LMECs in the presence of ZnSO4 was also found to be unaffected by pH (5.0-7.4), indicating that uptake of L-histidine into LMECs by the addition of zinc may not be involved in the H+-coupled transporters.

Department of Clinical pharmacy (Kyotani Lab.)

J Nutr sci Vitaminol (2010) 56; 145-149
We examined the immunomodulatory effect of Eriobotrya japonica seed extract (ESE) on rat allergic dermatitis elicited by repeated dinitrofluorobenzene (DNFB) application on the ear. Oral administration of ESE significantly inhibited development of allergic dermatitis based on lower ear thickness and serum immunoglobulin E (IgE) levels. Th1 cytokine interferon-γ (IFN-γ) and interleukin-2 (IL-2), Th2 cytokine interleukin-4 (IL-4) and interleukin-10 (IL-10) in the lesional skin were determined. Oral administration of ESE significantly decreased IL-4 while significantly increasing IL-10 in lesional skin, and the lower levels of IFN-γ and IL-2 were reversed by oral administration of ESE. The infiltration of eosinophils in the lesional skin was decreased by oral administration of ESE. These results suggested that ESE exerts anti-allergic actions by improving the balance of Th1/Th2 in allergic dermatitis.

Department of Clinical pharmacy (Okano Lab.)

J Physiol Sci (2011) 61; 403~406
Although the ability of zinc to retard oxidative process has been recognized for insights to elucidate the role of intracellular Zn2+ in cells suffering from oxidative stress, the effects of N-ethylmaleimide (NEM) and ZnCl2 on cellular thiol content and intracellular Zn2+ concentration were studied by use of 5-chloromethylfluorescein diacetate (5-CMF-DA) and FluoZin-3 pentaacetoxymethyl ester (FluoZin-3-AM) in rat thymocytes. The treatment of cells with NEM attenuated 5-CMF fluorescence and augmented FluoZin-3 fluorescence in a dose-dependent manner. These NEM-induced phenomena were observed under external Zn -free condition. Results suggest that NEM decreases cellular thiol content and induces intracellular Zn2+ release. Micromolar ZnCl2 dose-dependently augmented both FluoZin-3 and 5-CMF fluorescences, suggesting that the elevation of intracellular Zn2+ concentration increases cellular thiol content. Taken together, it is hypothesized that intracellular Zn2+ release during oxidative stress is a trigger to restore cellular thiol content that is decreased by oxidative stress.

Department of Pharmacology (Akagi Lab.)

Neurosci Lett (2012) in press
Trans-3-(3’4’-dimethoxyphenyl)-4-[(E)-3”,4”-dimethoxystyryl]cyclohex-1-ene (Comp.1) and cis-3-(3’4’-dimethoxyphenyl)-4-[(E)-3”,4”-dimethoxystyryl]cyclohex-1-ene (Comp.2), phenylbutenoid dimers, have been isolated as neurotrophic molecules from an Indonesian medicinal plant, Zingiber purpureum. The aim of this study was to explore the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. Comp.1 or Comp.2 significantly induced neurite sprouting in PC12 cells. Comp.1 or Comp.2 significantly increased the neurite length and number of neurites in primary cultured rat cortical neurons. Comp.1 and Comp.2 also provided significant protection against cell death caused by deprivation of serum. The in vivo effects of both Comp.1 and Comp.2 were evaluated on hippocampal neurogenesis in olfactory bulbectomized (OBX) mice, an experimental depression and dementia animal model. Comp.1, Comp.2, or fluoxetine, an antidepressant, were administrated once a day on days 15 to 28 after OBX. Neurogenesis was assessed by analysis of cells expressing NeuN, a neuronal marker, and 5-bromo-2’-deoxyuridine (BrdU) uptake. Immunohistochemical analysis showed that the number of BrdU/NeuN double-labeled cells in the dentate gyrus was significantly decreased 30 days after OBX. Chronic treatment with Comp.1, Comp.2 or fluoxetine significantly increased the number of BrdU/NeuN double-labeled cells. These results indicate that Comp.1 and Comp.2 have neurotrophic effects, and have the potential for disease modification in depression and dementia.

Department of Public Health (Suzuki Lab.)

FASEB J (2010) 24; 2375-2384.
Oxidative stress accelerates adipocyte differentiation and lipid accumulation, leading to endoplasmic reticulum (ER) stress, which causes insulin resistance. Because metallothionein (MT) has a role in prevention of oxidative and ER stress, we examined the effects of MT on the development of obesity induced by 27 wk of a high-fat diet (HFD) in female MT-I- and MT-II-null (MT(-/-)) and wild-type (MT(+/+)) mice. Body weight, fat mass, and plasma cholesterol increased at a greater rate in MT(-/-) mice fed an HFD than in MT(-/-) mice fed a control diet (CD) and MT(+/+) mice fed an HFD, indicating that MT(-/-) mice fed an HFD became obese and hypercholesterolemic and that MT could prevent HFD-induced obesity. The observed increases in the levels of plasma leptin and leptin mRNA in the white adipose tissue of MT(-/-) mice fed the HFD suggested a leptin-resistant state. Enhanced expression of a mesoderm-specific transcript, which regulates the enlargement of fat cells, was accompanied by enlarged adipocytes in the white adipose tissue of young MT(-/-) mice before obesity developed after 3 and 8 wk of feeding the HFD. Thus, MT may have a preventive role against HFD-induced obesity by regulating adipocyte enlargement and leptin signaling.