Hirotaka Naitou

Anaplasma phagocytophilum-infected ticks, Japan.

We report Anaplasma phagocytophilum infection of Ixodes persulcatus and I. ovatus ticks in Japan. Unique p44/msp2 paralogs (and/or 16S rRNA genes) were detected in tick tissues, salivary glands, and spleens of experimentally infected mice. These findings indicate the public health threat of anaplasmosis in Japan.

Cancer antineovascular therapy with liposome drug delivery systems targeted to BiP/GRP78

Angiogenesis is crucial for tumor growth and hematogenous metastasis. Specifically expressed and functional protein molecules in angiogenic endothelial cells, especially on the plasma membrane, may be molecular targets for antiangiogenic drugs and drug delivery systems (DDS) in cancer therapy. To discover such target molecules, we performed subcellular proteome analysis of human umbilical vein endothelial cells (HUVECs) treated with or without vascular endothelial growth factor (VEGF) using 2‐dimensional difference in‐gel electrophoresis (2D‐DIGE) and matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry (MALDI‐TOF/TOF‐MS). Among the identified proteins, BiP/GRP78, a molecular chaperone, was highly expressed in the membrane/organelle fraction of HUVECs after VEGF treatment. The involvement of BiP in VEGF‐induced angiogenesis was examined by RNA interference. BiP knockdown significantly suppressed VEGF‐induced endothelial cell proliferation and VEGF‐induced phosphorylation of extracellular‐regulated kinase 1/2, phospholipase C‐γ, and VEGF receptor‐2 in HUVECs. Cell surface biotinylation analysis revealed that the cell surface expression of BiP was elevated in VEGF‐activated HUVECs. Aiming to apply BiP to a target molecule in liposomal DDS, we developed liposomes modified with the WIFPWIQL peptide, which has been shown to bind to BiP, and investigated its potential for cancer therapy. The WIFPWIQL‐modified liposomes (WIFPWIQL liposomes) were significantly taken up by VEGF‐activated HUVECs as compared to peptide‐unmodified liposomes. WIFPWIQL liposomes appeared to accumulate in tumor endothelial cells in vivo. WIFPWIQL liposomes containing doxorubicin significantly suppressed tumor growth and prolonged the survival of colon26 NL‐17 carcinoma cell‐bearing mice. In summary, BiP may regulate VEGF‐induced endothelial cell proliferation through VEGFR‐2‐mediated signaling and be an effective target molecule for cancer antineovascular therapy.

Characterization of Ehrlichia species from Ixodes ovatus ticks at the foot of Mt. Fuji, Japan.

A total of 390 adult ticks (288 Ixodes ovatus and 102 I. persulcatus) collected at the foot of Mt. Fuji and two near cities in Shizuoka prefecture, Japan, were examined for Ehrlichia infection by isolation with laboratory mice from whole tick tissues. Ehrlichial DNAs were detected from the spleens of mice inoculated with tissues from I. ovatus, but not I. persulcatus. The prevalence of ehrlichiae in the ticks was estimated to be ca. 3%. The 16S rDNA analysis revealed that the sequences of 8 ehrlichial isolates (termed “Shizuoka” isolates) obtained were identical, and they were very similar, but not identical, to those of two Ehrlichia species strain variants recently isolated in Japan, followed by Ehrlichia chaffeensis in the US. Analysis of parts of the omp‐1 multigene family specific for monocytic ehrlichiosis agents showed that the Shizuoka isolates were distinct from other ehrlichial organisms. The Shizuoka isolates caused death in immunocompetent laboratory mice, suggesting that they are highly pathogenic in mice. The data show that the Shizuoka isolates are likely to be a new strain variant of Ehrlichia species in Japan. Further characterization and surveillance will be required in Japan due to the presence of these human ehrlichiosis agent‐like organisms.

Molecular Identification of Ehrlichia Species and 'Candidatus Neoehrlichia mikurensis' from Ticks and Wild Rodents in Shizuoka and Nagano Prefectures, Japan

A total of 293 ticks and 111 wild rodents that were collected in Shizuoka and Nagano Prefectures, Japan, were examined for infection of Ehrlichia species and ‘Candidatus Neoehrlichia mikurensis.’ The 16S rDNA or the omp‐1 gene of these bacterial DNAs were detected from the spleens of tick‐inoculated mice (5 positive/total 29 mice) or from the spleens of wild rodents (25 positive/total 111 rodents) by PCR amplification. Sequencing of the 16S rDNA revealed Ehrlichia spp. from the 5 tick‐inoculated mice and 8 wild rodents, and ‘Candidatus N. mikurensis’ from 17 wild rodents. The data suggest the presence of additional genetic variants, and potential vectors and/or reservoirs for these bacteria in central Japan.

Proteomic analysis of hypoxia-induced tube breakdown of an in vitro capillary model composed of HUVECs: potential role of p38-regulated reduction of HSP27.

We recently reported that hypoxia could induce the breakdown of capillary‐like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia‐induced tube breakdown through p38‐regulated and caspase‐independent mechanisms. The involvement of adhesion proteins, integrins, VE‐cadherin, PECAM‐1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2‐D DIGE coupled with MALDI‐TOF/TOF‐MS to identify altered protein expression. The differential proteomic analysis of tube‐forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38‐regulated and caspase‐independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia‐induced tube breakdown. Taken together, it was shown that p38‐regulated and caspase‐independent reduction of HSP27 plays an important role in hypoxia‐induced tube breakdown.

Proteome Analysis of UV‐B‐Induced Anti‐apoptotic Regulatory Factors¶

Ultraviolet (UV) irradiation is well known to induce apoptosis, a hallmark event of which is the occurrence of sunburn cells in the epidermis. Keratinocytes in which DNA damaged by UV irradiation is not repaired undergo apoptosis as sunburn cells. However, we have previously reported that low‐dose UV‐B irradiation (∼0.1 J/cm2) suppressed the apoptosis induced by cell detachment and serum depletion. Dysregulation of apoptosis is important in tumor progression and malignancy and in promoting resistance to cancer therapy. To develop a better understanding of the antiapoptotic effect of UV irradiation, and to design the effective induction of apoptosis, we tried the proteome analysis of the molecules regulating apoptosis in low‐dose UV‐B‐irradiated NIH3T3 cells, using two‐dimensional difference gel electrophoresis (DIGE). Of a total of 3811 protein spots detected, 42 were found to be different between the cells undergoing apoptosis and cells after the irradiation. Of the spots selected, 25 were identified using MALDI‐TOF/TOF‐MS, some as structural proteins. Although typical apoptosis‐related molecules were not detected, possibly because proteins with low molecular weights were difficult to identify in the gel conditions used in this study, some of the proteins were considered to be involved in apoptosis. The DIGE system used in this experiment has advantages (including a high level of statistical confidence) for discovering new functional proteins related to the regulation of apoptosis.

Mechanism of Retinoid X Receptor Partial Agonistic Action of 1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic Acid and Structural Development To Increase Potency

We have reported that retinoid X receptor (RXR) partial agonist 1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid (CBt-PMN, 4a) shows a significant antidiabetes effect in the KK-A(y) type 2 diabetes model mice, with reduced side effects compared to RXR full agonists. To elucidate the mechanism of the RXR partial agonist activity of 4a, we synthesized derivatives of 4a, evaluated their RXR agonist activity, and performed structure-activity relationship analysis. Reporter gene assay revealed that though 6b, which possesses an amino group at the 2-position of 5-carboxybenzimidazole, showed RXR full-agonist activity, compounds 6d and 6e, which possess an oxygen atom and a sulfur atom at the corresponding position, respectively, showed weak RXR agonist activity. On the other hand, 6c, which has a trifluoromethyl group at the corresponding position, acts as an RXR partial agonist, having similar Emax (67 ± 2%) and lower EC50 (15 ± 0 nM) compared to those of 4a (Emax = 75 ± 4%, EC50 = 143 ± 2 nM). A fluorescence polarization assay of cofactor recruitment confirmed that fluorescein-labeled D22 coactivator peptide was less efficiently recruited to RXR by 4a and 6c than by LGD1069 (1), a known RXR full agonist. Electrostatic potential field calculations and computational docking studies suggested that full agonists show an electrostatic attraction, which stabilizes the holo structure and favors coactivator recruitment, between the side chain at the benzimidazole 2-position and the α-carbonyl oxygen of asparagine-306 in helix 4 (H4) of the RXR receptor. However, RXR partial agonists 4a and 6c lack this interaction. Like 4a, 6c showed a significant antidiabetes effect in KK-A(y) type 2 diabetes model mice with reduced levels of the side effects associated with RXR full agonists. These findings should aid the design of new RXR partial agonists as antitype 2 diabetes drug candidates.

Fungal Mn oxides supporting Mn(II) oxidase activity as effective Mn(II) sequestering materials

We examined the Mn(II)-oxidizing ability of the biogenic Mn oxide (BMO) formed in cultures of a Mn(II)-oxidizing fungus, Acremonium strictum strain KR21-2. The newly formed BMO effectively sequestered dissolved Mn(II) mainly by oxidizing Mn(II) to insoluble Mn under air-equilibrated conditions, and this ability lasted for at least 8 days. Deaerating the BMOs, poisoning them with NaN3, or heating them all readily weakened their Mn(II) oxidation ability, indicating the involvement of enzymatic Mn(II) oxidation. There was no Mn(II)-oxidizing ability observed for mycelia cultivated without Mn(II) or for residual mycelia after the BMO phase was dissolved, suggesting the need for the oxide phase. A sodium dodecyl sulphate-polyacrylamide gel electrophoresis assay demonstrated that the oxide phase embeds the Mn(II) oxidase and thereby maintains the enzymatic activity in BMOs. Freezing at−80°C preserved the Mn(II)-oxidizing ability in BMOs for at least 4 weeks, while lyophilization caused a complete loss of this ability. Based on these results, we propose that fungal Mn oxides supporting Mn(II) oxidase activity are an effective Mn(II)-sequestering material capable of oxidizing Mn(II) continuously from solutions containing no additional nutrients to maintain biological activity.

Fluorescent retinoid X receptor ligands for fluorescence polarization assay.

Retinoid X receptor (RXR) agonists are candidate agents for the treatment of metabolic syndrome and type 2 diabetes via activation of peroxisome proliferator-activated receptor (PPAR)/RXR or liver X receptor (LXR)/RXR-heterodimers, which control lipid and glucose metabolism. Reporter gene assays or binding assays with radiolabeled compounds are available for RXR ligand screening, but are unsuitable for high-throughput screening. Therefore, as a first step towards stabilizing a fluorescence polarization (FP) assay system for high-throughput RXR ligand screening, we synthesized fluorescent RXR ligands by modification of the lipophilic domain of RXR ligands with a carbostyril fluorophore, and selected the fluorescent RXR agonist 6-[ethyl(1-isobutyl-2-oxo-4-trifluoromethyl-1,2-dihydroquinolin-7-yl)amino]nicotinic acid 8d for further characterization. Compound 8d showed FP in the presence of RXR and the FP was decreased in the presence of the RXR agonist LGD1069 (2). This compound should be a lead compound for use in high-throughput assay systems for screening RXR ligands.

Functional analyses of neutrophil-like differentiated cell lines under a hyperglycemic condition

Diabetic patients are prone to severe bacterial infections. The functional alterations of neutrophils by hyperglycemia are thought to be partially responsible for such infections. In this study, we investigated the functional changes of neutrophil-like differentiated cell lines (dHL-60, dTHP-1, and dNB-4) by treatment with 5.5 mM, 11 mM, or 35 mM of glucose. In dHL-60 cells, the incubation with high glucose (35 mM) resulted in the enhancement of cell aggregation, the suppression of cellular fragility, the induction of reactive-oxygen species (ROS) production by phorbol myristate acetate (PMA) stimulation, and the impairment of phagocytosis. In dTHP-1 cells, the treatment with higher glucose generated the suppression of cellular fragility and extremely impaired phagocytosis (by 35 mM), and induced ROS production due to PMA stimulation (by 11 mM). Furthermore, the higher glucose exposure to dNB-4 cells enlarged intracellular vacuoles (by 35 mM) and induced ROS production due to PMA stimulation (by 11 mM). Since the ROS generation of those cells was enhanced only after PMA stimulation under the higher glucose conditions, glucose may have a priming effect rather than a triggering effect. These extraordinary sensitivities caused by the higher glucose treatments may reflect the dysfunction or overactivation of neutrophils.