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Irinotecan (CPT11) is widely prescribed for treatment of various intractable cancers such as advanced and metastatic colon cancer cells, but its continuous treatment promotes the resistance development. In this study, we established CPT11-resistant variants of three human colon cancer (DLD1, RKO and LoVo) cell lines, and found that gain of the resistance elicited an up-regulation of aldo-keto reductase (AKR) 1C3 in the cells. Additionally, the sensitivity to CPT11 toxicity was decreased and increased by overexpression and knockdown, respectively, of the enzyme. Moreover, the resistant cells suppressed formation of reactive 4-hydroxy-2-nonenal by CPT11 treatment, and the suppressive effect was almost completely abolished by addition of an AKR1C3 inhibitor. These results suggest that up-regulated AKR1C3 contributes to promotion of the chemoresistance by detoxifying the reactive aldehyde. Western blot and real-time polymerase-chain reaction analyses and ATP-binding cassette (ABC) B1-functional assay revealed that, among three ABC transporters, ABCB1 was the most highly up-regulated by development of the CPT11 resistance, inferring a significant contribution of pregnane-X receptor-dependent signaling to the ABCB1 up-regulation. The combined treatment with inhibitors of AKR1C3 and ABCB1 potently sensitized the resistant cells to CPT11 and its active metabolite SN38. Taken together, our results suggest that combination of AKR1C3 and ABCB1 inhibitors is effective as adjuvant therapy to enhance CPT11 sensitivity of intractable colon cancer cells.

We investigated the amyloidogenic potential of inactivated vaccines and the localized production of serum amyloid A (SAA) at the injection site in white layer chickens. Hens in the treated group were injected intramuscularly three times with high doses of inactivated oil-emulsion Salmonella Enteritidis vaccine and multivalent viral and bacterial inactivated oil-emulsion vaccines at two-week intervals. Chickens in the control group did not receive any inoculum. In the treated group, emaciation and granulomas were present, while several chickens died between 4 and 6 weeks after the first injection. Hepatomegaly was seen at necropsy, and the liver parenchyma showed inconsistent discolouration with patchy green to yellowish-brown areas, or sometimes red-brown areas with haemorrhage. Amyloid deposition in the liver, spleen, duodenum, and at injection sites was demonstrated using haematoxylin and eosin staining, Congo red, and immunohistochemistry. The incidence of chicken amyloid A (AA) amyloidosis was 47% (28 of 60) in the treated group. In addition, RT-PCR was used to identify chicken SAA mRNA expression in the liver and at the injection sites. Furthermore, SAA mRNA was detected by in situ hybridization in fibroblasts at the injection sites, and also in hepatocytes. We believe that this is the first report of the experimental induction of systemic AA amyloidosis in white layer chickens following repeated inoculation with inactivated vaccines without the administration of amyloid fibrils or other amyloid-enhancing factors.

Overdose administration of sibutramine, a serotonin-noradrenalin reuptake inhibitor, is considered to elicit severe side effects including hypertension, whose pathogenic mechanism remains unclear. Here, we found that 48-h incubation with >10μM sibutramine provokes apoptosis of human aortic endothelial (HAE) cells. Treatment with the lethal concentration of sibutramine facilitated production of reactive oxygen species (ROS), altered expression of endoplasmic reticulum stress response genes (heat shock protein 70 and C/EBP homologous protein), and inactivated 26S proteasome-based proteolysis. The treatment also decreased cellular level of nitric oxide (NO) through lowering of expression and activity of endothelial NO synthase. These results suggest that ROS production and depletion of NO are crucial events in the apoptotic mechanism and may be linked to the pathogenesis of vasoconstriction elicited by the drug. Compared to sibutramine, its metabolites (N-desmethylsibutramine and N-didesmethylsibutramine) were much less cytotoxic to HAE cells, which hardly metabolized sibutramine. In contrast, both the drug and metabolites showed low cytotoxicity to hepatic HepG2 cells with high metabolic potency and expression of cytochrome P450 (CYP) 3A4. The cytotoxicity of sibutramine to HepG2 and Chang Liver cells was remarkably augmented by inhibition and knockdown of CYP3A4. This study also suggests an inverse relationship between sibutramine cytotoxicity and CYP3A4-mediated metabolism into the N-desmethyl metabolites.

Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most effective chemotherapeutic drugs that are used for treatment of patients with gastrointestinal cancer cells, but its continuous administration often evokes the development of chemoresistance. In this study, we investigated alterations in antioxidant molecules and functions using a newly established CDDP-resistant variant of gastric cancer MKN45 cells, and found that aldo-keto reductase 1B10 (AKR1B10) is significantly up-regulated with acquisition of the CDDP resistance. In the nonresistant MKN45 cells, the sensitivity to cytotoxic effect of CDDP was decreased and increased by overexpression and silencing of AKR1B10, respectively. In addition, the AKR1B10 overexpression markedly suppressed accumulation and cytotoxicity of 4-hydroxy-2-nonenal that is produced during lipid peroxidation by CDDP treatment, suggesting that the enzyme acts as a crucial factor for facilitation of the CDDP resistance through inhibiting induction of oxidative stress by the drug. Transient exposure to CDDP and induction of the CDDP resistance decreased expression of peroxisome proliferator-activated receptor-γ (PPARγ) in MKN45 and colon cancer LoVo cells. Additionally, overexpression of PPARγ in the cells elevated the sensitivity to the CDDP toxicity, which was further augmented by concomitant treatment with a PPARγ ligand rosiglitazone. Intriguingly, overexpression of AKR1B10 in the cells resulted in a decrease in PPARγ expression, which was recovered by addition of an AKR1B10 inhibitor oleanolic acid, inferring that PPARγ is a downstream target of AKR1B10-dependent mechanism underlying the CDDP resistance. Combined treatment with the AKR1B10 inhibitor and PPARγ ligand elevated the CDDP sensitivity, which was almost the same level as that in the parental cells. These results suggest that combined treatment with the AKR1B10 inhibitor and PPARγ ligand is an effective adjuvant therapy for overcoming CDDP resistance of gastrointestinal cancer cells.

Tobacco smoke and radioactive radon gas impose a high risk for lung cancer. The radon-derived ionizing radiation and some components of cigarette smoke induce oxidative stress by generating reactive oxygen species (ROS). Respiratory lung cells are subject to the ROS that causes DNA breaks, which subsequently bring about DNA mutagenesis and are intimately linked with carcinogenesis. The damaged cells by oxidative stress are often destroyed through the active apoptotic pathway. However, the ROS also perform critical signaling functions in stress responses, cell survival, and cell proliferation. Some molecules enhance radiation-induced tumor cell killing via the reduction in DNA repair levels. Hence the DNA repair levels may be a novel therapeutic modality in overcoming drug resistance in lung cancer. Either survival or apoptosis, which is determined by the balance between DNA damage and DNA repair levels, may lender the major problems in cancer therapy. The purpose of this paper is to take a closer look at risk factor and at therapy modulation factor in lung cancer relevant to the ROS.

The Japanese giant salamander (Andrias japonicus) is a near threatened species endemic to western Japan and is strictly protected by law. However, available information regarding the genetic diversity and genetic structure in this species, essential for its effective conservation, has been limited. We developed four microsatellite markers from A. japonicus and characterized these markers for two populations of this species, as well as for some captive Chinese giant salamanders (A. davidianus) of unknown original locality or localities. These markers, showing expected heterozygosities of 0.00–0.50 in the former and 0.63–0.89 in the latter, will be useful in documenting population genetic properties for each of the two species.

In the present study, we aimed to determine the effects of herbs or spices on the expression of presenilin 1, a molecule involved in γ-secretase activity and the generation of amyloid-β peptide in Alzheimer's disease. Western blot analysis revealed that presenilin 1 protein expression was down-regulated by stimulation with turmeric or cinnamon extracts in vitro, while the effects on presenilin 1 gene expression examined by reverse transcriptase-polymerase chain reaction were unaltered. Our results showed that curcumin, a component of turmeric, induced the down-regulation of presenilin 1 protein in Jurkat and K562 cell lines.

The small GTPase Rab5 controls the fusogenic properties of early endosomes through activation of effector proteins. Expression of a GTPase-defective mutant, Rab5(Q79L), is known to cause formation of enlarged early endosomes. To identify proteins interacting with this GTP-bound form of active Rab5 1, we used a two-hybrid system to screen a human fetal cDNA library with a human Rab5A protein bearing a mutation of Q79L as bait. The positive clone cDNAs were reconfirmed to be interaction-positive by using an independent yeast clone containing the bait. Then, more than 3 positive preys were identified, and subsequent sequence analysis revealed one of the preys to be RUFY3: RUN and FYVE domain containing 3. We performed double immunefluorescent staining for human RUFY3 and human Rab5A(Q79L) in 3Y1 cells. Analysis with confocal microscopy clearly showed that the Rab5(Q79L) and the RUFY3 colocalized in large vesicle structures in 3Y1 cells (data not shown). This indicated an interaction between RUFY3 and Rab5(Q79L), which may be altered by the nucleotide-bound state of Rab5. We checked this possibility using other Rab5 mutant. The Rab5(S34N) mutant preferentially binds GDP, whereas the Rab5(Q79L) mutant is unable to hydrolyze bound GTP. As shown in Figure ​Figure1,1, the superimposed image by confocal microscopy again confirmed the cytoplasmic vesicular localization of RUFY3 with constitutively active Rab5(Q79L) well colocalized in almost all vesicles, whereas not with dominant negative Rab5(S34N). These data clearly indicated that RUFY3 was localized in enlarged vesicles with the active GTP-bound form of Rab5, where Rab5(Q79L) sequester RUFY3. However, there is an another possibility for the phenomenon seen in Figure ​Figure1,1, that Rab5(Q79L) mutant inhibited endosome traffic and resulted in RUFY3 accumulation in Rab5(Q79L) compartment. Figure 1 Colocalization of RUFY3 and active Rab5(Q79L). The 3Y1 cells were transiently transfected with either GFP-tagged-Rab5(S34N) and pcDNA3.1-RUFY3 (left panels), or GFP-tagged-Rab5(Q79L) and pcDNA3.1-RUFY3 (right panels). The cells were then fixed and incubated ... We next determined whether Rab5 was associated with RUFY3 by glutathione S-transferase (GST) pull-down assays. We found that carboxyl terminal domain of RUFY3 was associated with Rab5 in v-SRC transformed SRD cells (Fig. ​(Fig.2A),2A), but not in normal 3Y1 cells and 293T cells (data not shown). The RUN domain of RUFY3 as well as control GST protein was not able to bind Rab5 (Fig. ​(Fig.2A,2A, upper panel). In this assay, we found that Rap2 was weakly bound to the RUN domain of RUFY3 (Figure ​(Figure2A,2A, middle panel), which was consistent with the previous reports 2. All the other small GTPases including Ran (Figure ​(Figure2A,2A, lower panel) 3, Rab2, Rab7, Rho, Ras did not bind to RUFY3 (data not shown). These data suggested that RUFY3 could bind to Rab5 and Rap2. As a complementary approach, we also used a pull-down assay with immobilized GST-Rab5 fusion proteins 4 preloaded with GTP (GST-Rab5(Q79L)) but not with GDP (GST-Rab5(S34N)) could bind to RUFY3 (Figure ​(Figure2B).2B). Finally, we examined the biochemical in vivo endogenous interactions between Rab5 and RUFY3 (Figure ​(Figure2C).2C). U937 (monocyte-derived resident macrophage) cells were subjected to immunoprecipitation using either anti-Rab5 or anti-RUFY3 antibodies. As shown in Figure ​Figure2C,2C, Rab5, but not control, was coprecipitated with RUFY3. Reciprocally, RUFY3 was coprecipitated with Rab5, but not with the mock antibody. Figure 2 (A) Interaction between the full-length RUFY3 and various small GTPase proteins were analyzed in the GST pull down assay with GST only, GST fusion of RUFY3 C terminal region (370-469) or of RUFY3 RUN domain (93-232). These GST fusion proteins were immobilized ... The two-hybrid assay also revealed that the carboxyl terminal region of RUFY3 was necessary for this interaction, since a truncated protein consisting of the first 390 residues of RUFY3 did not interact with Rab5(Q79L) (data not shown). The carboxyl terminal region of the RUFY3 is predicted to be mainly alpha-helical and contains coiled-coil domains, as determined according to the Lupas algorithm. The RUFY gene family (RUFY1, RUFY2), which is most similar structure to RUFY3, contains an amino terminal RUN domain and a carboxyl terminal FYVE domain 5 with two coiled-coil domains in-between 6. The RUFY proteins are localized predominantly to endosomes as evidenced by their colocalization with early endosome antigen marker (EEA1) 6. However, both RUFY1 and RUFY2 have not been shown to associate with active Rab5. The endosomal localization of EEA1 was also determined by its ability to bind Rab5 7. To our knowledge, the region has not been identified as a general binding motif for active Rab5. The GTP-bound active form of Rab5 localized on the cytoplasmic face of early endosomes, and promotes membrane trafficking through interaction with specific effector molecules such as EEA1 8.The mode of action of Rab5 in phagosomal maturation remain obscure. However, it has been reported that oxidized low density lipoprotein interferes with normal phagosomal maturation, possibly via blocking membrane fusion events such as proper activation of the Rab5 9.

In order to investigate how glucocorticoids affect the expression of secretory leukocyte peptidase inhibitor (SLPI), which is overexpressed in a variety of cancers, clobetasol was added to cell culture medium of U937 cells and the SLPI mRNA levels were examined. The in vitro effect of the treatment on SLPI expression was detected by reverse transcriptase-polymerase chain reaction. Clobetasol treatment of U937 cells induced an up- and down-regulation of SLPI expression in a dose-dependent manner. Western blotting confirmed the down-regulation of SLPI protein expression. We hypothesized a loop formation in the SLPI genome domain, in which the glucocorticoid receptor regulates bi-directional transcriptional activity.

Cip-interacting zinc finger protein 1 (Ciz1) stimulates DNA replication and has been implicated in the tumorigenesis of breast cancer cells. In order to investigate the possibility of using medicinal glucocorticoids against breast cancer, we studied whether certain glucocorticoids affect the expression of Ciz1. The in vitro effect of clobetasol treatment on the reduction of Ciz1 expression was detected by reverse transcriptase-polymerase chain reaction. Western blotting also confirmed the down-regulation of the protein in a dose-dependent manner upon clobetasol treatment in U937 monocytoid cells. Furthermore, we found that Ciz1 protein expression was decreased after pre-treatment of the cells with clobetasol and genistein. An extract of Lens culinaris also had a synergistic effect on the repression of Ciz1 protein expression.

The aim of this study was to determine the effects of several food ingredients and chemical inhibitors on the expression of presenilin, a molecule involved in γ-secretase activity and the generation of amyloid-β peptide in Alzheimer's disease. Western blotting revealed the downregulation of presenilin 1 protein expression by stimulation with genistein in vitro, while the effects on presenilin 1 gene expression examined by reverse transcriptase-polymerase chain reaction (RT-PCR) were unaltered in Daudi cells. Genistein likely downregulates presenilin via the inhibition of ubiquilin 1 expression in lymphoid cells. Our findings provide new insights that may help to establish preventive strategies against Alzheimer's disease.

Alternative splicing is a major contributor to transcriptome and proteome diversity, which can lead to the deregulation of crucial cellular processes and have been associated with a variety of human diseases including cancer. As p53, BRCA1, and PTEN proteins have a key role in preventing breast cancer formation, cancer-associated splicing variants of these tumor suppressor genes are potential molecular markers and may contribute to the development of diagnostic and prognostic methods. In the present review, we summarize these tumor suppressor genes at a viewpoint of alternative splicing involved in breast cancer.

Protein kinase AKT mediates cell proliferation and survival signals, and also contributes to cancer progression. Increased expression and/or activation of AKT is involved in a variety of human cancers. In cells treated with sage or rosemary extract, mRNA and protein expression levels of AKT1 were reduced compared with those of the control cells 48 h after the herbal treatments. We found that terpinolene, a common component of sage and rosemary, markedly reduced the protein expression of AKT1 in K562 cells and inhibited cell proliferation.

RUN domain is present in several proteins related to the functions of Rap and Rab family GTPases. Accumulating evidence supports the hypothesis that RUN domain-containing proteins act as a component of vesicle traffic and might be responsible for an interaction with a filamentous network linked to actin cytoskeleton or microtubules. That is to say, on one hand, RUN domains associate with Rab or Rap family proteins, on the other hand, they also might interact with motor proteins such as kinesin or myosin via intervention molecules. In this review, we summarize the background and current status of RUN domain research with an emphasis on the interaction between RUN domain and motor proteins with respect to the vesicle traffic on filamentous network.

SIRT1 is a mammalian candidate molecule involved in longevity and diverse metabolic processes. The present study aimed to determine the effects of certain herbs and spices on SIRT1 expression. Human cell lines Daudi, Jurkat, U937 and K562 were cultured in RPMI-1640. Herb and spice powders were prepared and the supernatants were collected. RT-PCR was used to quantify the expression level of the gene. Protein samples were then analyzed by Western blotting. Western blotting revealed the down-regulation of SIRT1 protein expression in Daudi cells treated with extracts of black pepper or turmeric. On the other hand, the effect on the SIRT1 gene expression examined by reverse transcription polymerase chain reaction was unaltered. In conclusion, component(s) of certain herbs and spices may induce the down-regulation of SIRT1 protein.

Dicer is an RNase III enzyme with two catalytic subunits, which catalyzes the cleavage of double-stranded RNA to small interfering RNAs and micro-RNAs, which are mainly involved in invasive nucleic acid defense and endogenous genes regulation. Dicer is abundantly expressed in embryos, indicating the importance of the protein in early embryonic development. In addition, Dicer is thought to be involved in defense mechanism against foreign nucleic acids such as viruses. This paper will mainly focus on the recent progress of Dicer-related research and discuss potential RNA interference pathways in aquatic species.

Establishment of cell lines representative of honeybee character would greatly assist in their analysis. Here, we show that immortalized cell line, designated as MYN9, has been generated from honeybee embryo by the gene transfer of human c-myc proto-oncogene. The morphology of the cell is characteristic of embryonic stem cell, although the cell is stable and does not spontaneously differentiate. Polymerase chain reaction analyses show that the cell is originated from authentic honeybee cell. It is proposed that the integration of human c-myc gene into honeybee precursor populations results in the establishment of stable cell line suitable for cellular and molecular studies.

Dugongs (Dugong dugon) produce different types of vocalization such as chirp, trill, and barks. Previous reports showed that dugongs have two kinds of phonemes: long duration calls (trill) and short duration calls (chirp-squeaks hereinafter called chirp). Especially, the chirp and trill calls were widely reported in different populations. However, characteristics of call patterns in dugongs have never been reported. Moreover, the function of these calls was not revealed. The objective of this study is to classify the vocalization patterns of dugong calls and discuss the stability call sequences within and across local populations of dugongs. We recorded the underwater sound at the off Talibong Island, Trang, Thailand in 2004 and 2005 for 120 hours by an automatic underwater sound recording system (AUSOMS-D, System Intech, Tokyo). The AUSOMS-D is the water resistant stand-alone recording system and developed for passive acoustical monitoring targeting human audible range. The AUSOMS-D consisted of a pair of hydrophones located 2 m apart for calculating the bearings of the sound sources. The electric circuits and batteries were housed in a pressure-resistant case, and the hydrophones were connected to a stereo preamplifier. The sound signals were fed into a 1-kHz high pass filter to eliminate low frequency background noise. Digitized signals were recorded on an 80 GB removable hard disk drive by uncompressed format with a time stamp. The power supply system with batteries and DC/DC converter were also housed inside the case. The sampling frequency of the A/D converter was 44.1 kHz and the dynamic range was 74-120 dB (re 1 muPa) with a 16-bit resolution. Each hydrophone had flat frequency responses within 2 dB between 1 and 10 kHz. For the comparison purpose, we made recording of a captive dugong in Toba Aquarium, Japan, which was introduced from Philippine waters. We analyzed the underwater sound data set that was obtained in both animals in different environment and populations. Short duration calls with less than 300 milliseconds were defined as chirp and trill was defined as a call lasting over 300 milliseconds. The end of a call sequence was defined at the silence over 3 seconds. Total of 1174 audible calls were detected from total of 12 hours data set (from 3:50 to 6:50 1-4 March 2004). Chirp were observed more than trill calls (567 chirps and 67 trills). Chirp-to-chirp transitions were most frequency observed (81.68%), whereas trill-to-trill transitions were the least (4.27%). Transitions between the two types of calls were also observed (6.98%, 7.07%). Trill appeared in the middle and the end of a call sequence. The position of the trill did not differ between wild individual in Thailand and a captive individual from off the Philippines that are considered to be separated populations. The stability of the sequence of each type of calls in a call sequence is investigated. Unlike the song of birds or baleen whales, the call sequence pattern of the dugong suggests small differences across the populations. The call sequence analysis as well as the behavioral context observation will provide the key to interpret the function of dugong calls.

The objectives of the present study were to evaluate the anatomic localization of porcine reproductive and respiratory syndrome virus (PRRSV) in naturally infected pigs and to determine whether oral fluid could be used to detect the virus in infected animals. Two sows, seven 2-month-old grower pigs, and 70 6-month-old gilts were included in this study. PRRSV in sera and oral fluid were identified by nested reverse transcription PCR (nRT-PCR) while lung, tonsil, and tissue associated with oral cavity were subjected to nRT-PCR, immunohistochemistry, and in situ hybridization. In sows, PRRSV was identified in oral fluid and tonsils. PRRSV was also detected in oral fluid, tonsils, salivary glands, oral mucosa, and lungs of all seven grower pigs. However, viremia was observed in only two grower pigs. Double staining revealed that PRRSV was distributed in macrophages within and adjacent to the tonsillar crypt epithelium. In gilts, the North American type PRRSV field strain was detected 3 to 8 weeks after introducing these animals onto the farm. These results confirm previous findings that PRRSV primarily replicates in tonsils and is then shed into oral fluid. Therefore, oral fluid sampling may be effective for the surveillance of PRRSV in breeding herds.

2016年度, 平成28年度, PDF