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目的：女子大学生の月経状況やセルフケア行動に関する実態を調査し、看護介入のための基礎資料とする。方法：A女子大学において、在籍する全員を対象としてウェブ調査を実施した。大学のウェブシステムを利用して、5,339人に調査依頼をした。本研究実施に際し、研究者の所属施設で研究倫理審査を受けた。結果：1,499名から回答が得られた（有効回答率28.1％）。月経に関しては、周期異常のあるもの381名（25.4％）、日常生活に支障があるもの411名（28.4％）であった。月経時の身体症状では下腹部痛が最も多く1,070名（71.3％）であった。月経時の対処方法は横になる・休むが最も多く798名（53.2％）であった。考察：月経異常を有するもの、月経症状が日常生活に支障のあるもの、いずれも4分の1程度存在していることから、月経状況を整えるためのセルフケアができる知識やケアの方法について教育的介入を行う必要性が示唆された。

Aims: To investigate rates of attention-deficit hyperactivity disorder (ADHD) in patients with chronic pain attending a pain clinic, the effects of a screening measure for ADHD in patients with chronic pain, and the effects of ADHD drugs on both pain and ADHD symptoms. Methods: We retrospectively surveyed 110 patients with chronic pain visiting the Anesthesiology and Pain Relief Center at the University of Tokyo in Japan, who had also consulted a psychiatrist, between April 2012 and July 2015. Results: Of the total of 110 patients with chronic pain, 35 (31.8%) were also diagnosed with ADHD, and the average Wender Utah Rating Scale (WURS) score among the ADHD patients was 39.0 ± 22.1 (n = 25). Only 36.0% of these patients exceeded the cutoff value, suggesting that 64.0% of the patients with ADHD were not identified by screening with the WURS. Twenty-six patients initiated treatment with ADHD medication, with dosage adjustment completed in 21. Of these 21 patients 20 (95.0%) had improved ADHD symptoms. Improved pain symptoms were observed in 14 patients (66.6%), with a reduction in the pain numerical rating scale of 64.7% ± 30.1%. Conclusions: This is the first study investigating the comorbidity of ADHD and chronic pain at pain clinics showing a high level of comorbidity and amelioration of pain and ADHD symptoms with treatment. Careful interpretation is required when the WURS is used to screen patients with chronic pain.

Antizyme (AZ) regulates cellular polyamines (i.e., putrescine, spermidine, and spermine) through binding to ornithine decarboxylase and subsequent ubiquitin-independent degradation of the enzyme protein by the 26S proteasome. Screening for AZ-binding proteins using a yeast two-hybrid system identified ATP citrate lyase (ACLY), a cytosolic enzyme which catalyzes the production of acetyl-CoA that is used for lipid anabolism or acetylation of cellular components. We confirmed that both AZ1 and AZ2 bind to ACLY and AZ colocalizes with ACLY to the cytoplasm. Unexpectedly, neither AZ1 nor AZ2 accelerated ACLY degradation. Additionally, purified AZ, particularly AZ1, increased the activity of purified ACLY in a dose-dependent manner in vitro, suggesting that AZ activates ACLY through protein-protein interaction. Polyamines themselves had no effect on the ACLY activity in vitro. Knockdown of AZ1 and/or AZ2 in human cancer cells significantly decreased the ACLY activity as well as cellular levels of acetyl-CoA and cholesterol. Our results are the first to show the crosstalk between polyamine and acetyl-CoA metabolism. We hypothesize that AZ may promote acetyl-CoA synthesis to downregulate spermidine and spermine through acetylation.

No expression and distribution patterns of polyamines (PAs), spermine, spermidine, and their precursor putrescine in mammalian hair follicle are available, although polyamines are known to correlate well with hair growth and epidermal tumor genesis. Immunohistochemistry (IHC) using our original two monoclonal antibodies (mAbs) ASPM-29 specific for spermine or spermidine, and APUT-32 specific for putrescine allowed us to detect immunoreactivity for polyamines in hair follicles from normal adult rats. A wide range of immunoreactivity for the total spermine and spermidine was observed in the compartments of hair follicle: The highest degree of immunoreactivity for polyamines was observed in the matrix, in the Huxley’s layer, in the deeper Henle’s layer, and in the cuticle of the inner root sheath/the hair cuticle, while moderate immunoreactivity existed in the lower-to-mid cortex and the companion layer, followed by lower immunoreactivity in the outer root sheath, including the bulge region and in the deeper medulla, in which the immunoreactivity was also evident in their nuclei. In addition, somewhat surprisingly, with IHC by APUT-32 mAb, we detected significant levels of putrescine in the compartments, in which the immunostaining pattern was the closely similar to that of the total spermine and spermidine. Thus, among these compartments, the cell types of the matrix, the Huxley’s layer, the deeper Henle’s layer, and the cuticle of the inner root sheath/the hair cuticle seem to have the biologically higher potential in compartments of anagen hair follicle, maybe suggesting that they are involved more critically in the biological event of hair growth. In addition, we noted sharp differences of immunostaining by IHCs between ASPM-29 mAb and APUT-32 mAb in the epidermis cells and fibroblast. ASPM-29 mAb resulted in strong staining in both the cell types, but APUT-32 mAb showed only very light staining in both types. Consequently, the use of the two IHCs could be extremely useful in further studies on hair cycle and epidermal tumor genesis experimentally or clinically.

The proto-oncogene c-Myc encodes a short-lived protein c-Myc that regulates various cellular processes including cell growth, differentiation and apoptosis. Degradation of c-Myc is catalyzed by the proteasome and requires phosphorylation of Thr-58 for ubiquitination by E3 ubiquitin ligase, Fbxw7/ FBW7. Here we show that a polyamine regulatory protein, antizyme 2 (AZ2), interacts with c-Myc in the nucleus and nucleolus, to accelerate proteasome-mediated c-Myc degradation without ubiquitination or Thr-58 phosphorylation. Polyamines, the inducer of AZ2, also destabilize c-Myc in an AZ2-dependent manner. Knockdown of AZ2 by small interfering RNA (siRNA) increases nucleolar c-Myc and also cellular pre-rRNA whose synthesis is promoted by c-Myc. AZ2-dependent c-Myc degradation likely operates under specific conditions such as glucose deprivation or hypoxia. These findings reveal the targeting mechanism for nucleolar ubiquitin-independent c-Myc degradation.

PurposeThis study aimed to investigate whether changes in psychosocial factors and pain severity were associated with reduction in disability due to pain among patients with chronic pain. We hypothesized that increased self-efficacy would reduce disability.Patients and methodsThis longitudinal observational study included 72 patients. Patients' psychological and physical variables were assessed before and after 3 months of treatment. Demographic and clinical information were collected, including the Pain Disability Assessment Scale (PDAS), the Pain Self-Efficacy Questionnaire (PSEQ), the Hospital Depression and Anxiety Scale, and the Numeric Rating Scale (NRS) to assess pain intensity. First, univariate regression analyses were conducted to clarify associations between change in PDAS and sex, age, pain duration, changes in psychosocial factors (self-efficacy, anxiety, and depression) and change in pain intensity. Second, multivariate regression was conducted using the variables identified in the univariate analyses (PSEQ and NRS) to detect the most relevant factor for reducing disability.ResultsUnivariate regression analyses clarified that changes in PSEQ (β = -0.31; 95% CI: -0.54--0.08, p = 0.008) and NRS (β = 0.24; 95% confidence interval [CI]: 0.01-0.47, p = 0.04) were associated with reduction in PDAS. Multivariate regression analysis demonstrated that change in PSEQ (β = 0.26; 95% CI: -0.50--0.02; p = 0.01) was associated with a reduction in disability, independent of change in NRS.ConclusionThese findings suggest improved self-efficacy is associated with reduced disability in patients with chronic pain, independent of reduction in pain intensity. Focusing on improvement in self-efficacy may be an effective strategy in chronic pain treatment in addition to pain relief.

Antizyme inhibitor 1 (Azin1), a positive regulator of cellular polyamines, is induced by various proliferative stimuli and repressed by polyamines. It has been reported that the translational repression of Azin1 by polyamines involves an upstream open reading frame on the mRNA, but little has been known about polyamine effect on its transcription or splicing. We found multiple forms of Azin1 transcripts formed by alternative splicing and initiation of transcription from putative alternative start sites. One of the novel splice variants, Azin1-X, has a premature termination codon on 5′ extension of exon 7, encodes a C-terminal truncated form of protein (Azin1ΔC), and is subject to nonsense-mediated mRNA decay. 2-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, increased both transcription from the canonical transcription start site and the ratio of the full-length mRNA to Azin1-X mRNA, whereas polyamines show the opposite effect. Thus, polyamines regulate two novel steps of Azin1 expression, namely the transcription and a particular splicing pattern, both of which may affect the level of mRNA encoding the full-length active Azin1 protein.

Cytokeratin (CK) 18 M30 antigen has been proposed as a diagnostic marker of nonalcoholic fatty liver disease (NAFLD). We studied serum CK18 M30 antigen level and examined the correlations among CK18 and biological data, dietary intake, and plasma fatty acid composition in middle-aged Japanese males with (NAFLD; n=42) and without NAFLD (control; n=35). NAFLD was diagnosed if subjects showed fatty liver on abdominal ultrasonography and their alcohol consumption was

Ornithine decarboxylase (ODC), antizyme (AZ), and antizyme inhibitor (AIn) play a key role in regulation of intracellular polyamine levels by forming a regulatory circuit through their interactions. To gain insight into their functional importance in cell growth and differentiation, we systematically examined the changes of their expression, cellular polyamine contents, expression of genes related to polyamine metabolism, and β-casein gene expression during murine mammary gland development. The activity of ODC and AZ1 as well as putrescine level were low in the virgin and involuting stages, but they increased markedly during late pregnancy and early lactation when mammary cells proliferate extensively and begin to augment their differentiated function. The level of spermidine and expression of genes encoding spermidine synthase and AIn increased in a closely parallel manner with that of casein gene expression during pregnancy and lactation. On the other hand, the level of spermidine/spermine N 1-acetyltransferase (SSAT) mRNA and AZ2 mRNA decreased during those periods. Immunohistochemical analysis showed the translocation of ODC and AIn between the nucleus and cytoplasm and the continuous presence of AZ in the nucleus during gland development. Reduction of AIn by RNA interference inhibited expression of β-casein gene stimulated by lactogenic hormones in HC11 cells. In contrast, reduction of AZ by AZsiRNA resulted in the small increase of β-casein gene expression. These results suggested that AIn plays an important role in the mammary gland development by changing its expression, subcellular localization, and functional interplay with AZ.

Antizymes (AZs) are polyamine-induced proteins that negatively regulate cellular polyamine synthesis and uptake. Three antizyme isoforms are conserved among mammals. AZ1 and AZ2 have a broad tissue distribution, while AZ3 is testis specific. Both AZ1 and AZ2 inhibit ornithine decarboxylase (ODC) activity by binding to ODC monomer and target it to the 26S proteasome at least in vivo. Both also inhibit extra-cellular polyamine uptake. Despite their being indistinguishable by these criteria, we show here using enhanced green fluorescent protein (EGFP)-AZ2 fusion protein that in mammalian cells, the subcellular location of AZ2 is mainly in the nucleus, and is different from that of AZ1. The C-terminal part of AZ2 is necessary for the nuclear distribution. Within a few hours, a shift in the distribution of EGFP-AZ2 fusion protein from cytoplasm to the nucleus or from nucleus to cytoplasm is observable in NIH3T3 cells. In addition, we found that in cells a majority of AZ2, but not AZ1, is phosphorylated at Ser-186, likely by protein kinase CK2. There may be a specific function of AZ2 in the nucleus. J. Cell. Biochem. 108: 1012–1021, 2009. © 2009 Wiley-Liss, Inc.

To achieve the effective intracellular delivery of siRNA and silence specific genes, various types of conjugates between cell-penetrating peptides (CPPs; Transportan, Penetratin, Tat) and cationic peptides were developed. Uptake, intracellular localization, cytotoxicity, and biological activity of siRNA were significantly dependent on the kind of CPP used and the length of the cationic peptides in the conjugate. Transportan-based conjugates yielded both high internalization of siRNA and strong gene silencing activity, while Penetratin- and Tat-based conjugates did not. These different properties of CPPs emphasize the importance of careful peptide selection and design when attempting the application of CPP technology.

Pneumocystis pneumonia (PcP) is the most common opportunistic disease in immunocompromised patients. Alveolar macrophages are responsible for the clearance of Pneumocystis organisms; however, they undergo a high rate of apoptosis during PcP due to increased intracellular polyamine levels. In this study, the sources of polyamines and mechanisms of polyamine increase and polyamine-induced apoptosis were investigated. The level of ornithine decarboxylase (ODC) was elevated in alveolar macrophages, and the number of alveolar macrophages that took up exogenous polyamines was increased 20-fold during PcP. Monocytes, B lymphocytes, and CD8+ T lymphocytes that were recruited into the lung during PcP expressed high levels of ornithine decarboxylase, suggesting that these cells are sources of polyamines. Both protein and mRNA levels of antizyme inhibitor (AZI) were increased in alveolar macrophages during PcP. This AZI overexpression correlated with increased polyamine uptake by alveolar macrophages, because AZI expression knockdown decreased the polyamine uptake ability of these cells. AZI expression knockdown also decreased the apoptosis rate of alveolar macrophages. Pneumocystis organisms and zymosan A were found to induce AZI overexpression in alveolar macrophages, suggesting that β-glucan, which is the major component of the Pneumocystis cell wall, induces AZI overexpression. The levels of mRNA, protein, and activity of polyamine oxidase were increased in alveolar macrophages during PcP, indicating that the H2O2 generated during polyamine catabolism caused alveolar macrophages to undergo apoptosis. Taken together, results of this study indicate that Pneumocystis organisms induce AZI overexpression in alveolar macrophages, leading to increased polyamine synthesis and uptake and apoptosis rate of these cells.

Eicosapentaenoic acid (EPA) (1.8 g/day) was administered to 12 chronic hepatitis C patients receiving combination therapy of pegylated interferon (PEG-IFN) α-2b and ribavirin for 48 weeks (EPA group). Twelve patients were not administered EPA (control group). All patients also received vitamin E and C (300, 600 mg/day, respectively) during the therapy. Serum alanine aminotransferase improved to a normal level in 8 of 12 patients from the EPA group and 6 of 12 patients from the control group after 12 weeks. Lymphocyte counts decreased significantly after 8 weeks in the control group, but not the EPA group. T-helper (Th) 1 decreased after 4 weeks in the control group, but not in the EPA group (two-way ANOVA; P

The objective structured clinical examination (OSCE) has recently been introduced to assess the clinical skills of medical trainees. This study develops scales and assesses the problems with such a system for evaluating the skill of a student at a registered dietitian school. All 34 undergraduates in the third year of a 4-year dietitian course were enrolled to examine the reliability of the scales for evaluating skills and to assess the test reliability. Significant correlations were found between two observers, and an acceptable Cronbach alpha level for the evaluation sheet was obtained. However, the result for the total correlation among collected data revealed several items decreasing the internal consistency. These items need to be investigated thoroughly and improved in order to increase reliability. We conclude that OSCE is a useful addition to the dietetic curriculum, offering an objective means of identifying weaknesses and strengths in specific areas of clinical competence.

〔目的〕地域在住高齢者45名（65-91歳）を対象に転倒恐怖感を測定し，その関連因子を包括的に検討した。〔方法〕転倒恐怖感の測定はModified Falls Efficacy Scale（以下，MFES）得点を用い，MFES得点139点以下を転倒恐怖感有とした。関連因子として，身体機能，転倒経験，ADL－20，身体活動セルフ・エフィカシー，外出自粛について検討した。〔結果〕対象者の68.9％に転倒恐怖感がみられ，MFES得点はADL－20得点，外出自粛と有意な相関を示した。転倒恐怖感に関連する因子を多重ロジスティクモデルにて検討した結果，ADL－20得点のみが抽出された。〔結語〕転倒恐怖感の軽減には，運動指導に加えADL向上を目指す生活指導も重要と考えられる。

Polyamines are small cationic molecules required for cellular proliferation. Agmatine is a biogenic amine unique in its capacity to arrest proliferation in cell lines by depleting intracellular polyamine levels. We previously demonstrated that agmatine enters mammalian cells via the polyamine transport system. As polyamine transport is positively correlated with the rate of cellular proliferation, the current study examines the antiproliferative effects of agmatine on cells with varying proliferative kinetics. Herein, we evaluate agmatine transport, intracellular accumulation, and its effects on antizyme expression and cellular proliferation in nontransformed cell lines and their transformed variants. H-ras- and Src-transformed murine NIH/3T3 cells (Ras/3T3 and Src/3T3, respectively) that were exposed to exogenous agmatine exhibit increased uptake and intracellular accumulation relative to the parental NIH/3T3 cell line. Similar increases were obtained for human primary foreskin fibroblasts relative to a human fibrosarcoma cell line, HT1080. Agmatine increases expression of antizyme, a protein that inhibits polyamine biosynthesis and transport. Ras/3T3 and Src/3T3 cells demonstrated augmented increases in antizyme protein expression relative to NIH/3T3 in response to agmatine. All transformed cell lines were significantly more sensitive to the antiproliferative effects of agmatine than nontransformed lines. These effects were attenuated in the presence of exogenous polyamines or inhibitors of polyamine transport. In conclusion, the antiproliferative effects of agmatine preferentially target transformed cell lines due to the increased agmatine uptake exhibited by cells with short cycling times.

Autoimmune hepatitis (AIH) serum contains autoantibodies against solubl el iver antigen/liver and pancreas (SLA/LP), a protein that interacts with the selenocysteine (Sec)-tRNA Sec .W e investigated the infl uence of AIH serum on Sec production. Two (#3 and #4) of four AIH sera inhibited its synthesis, with serum #4 showing the strongest inhibition. This did not parallel the level of antibodies to SLA/LP (anti-SLA/LP) in sera, because the titer in serum #3 was higher than that in serum #4. Thus, w ef ound that AIH inhibited Sec synthesis but we could not conclude that this inhibition depended on an autoantibody against SLA/LP. To clarify the function of SLA/LP, we prepared an antibody against an SLA/LP peptide. Inhibition of Sec synthesis by this anti-peptide antibody was incomplete and sometimes did not occur. Absorption of autoantibodies in the AIH serum with the SLA/LP protein did not have any effects on Sec synthesis by Sec synthase (SecS) which converts Seryl (Ser)-tRNA Sec to Sec-tRNA Sec .W e then used SLA/L Pi nstead of active bovine SecS but we could not detect SecS activity in SLA/LP itself. Next, we studied the distribution of SLA/LP in HEK293 cells transfected with an SLA/LP expression vector. Immunofluorescence microscopy detected SLA/LP expression in the cytoplasm, but not the nucleus of HEK293 cells. Higher levels of SLA/LP in the cytosol did not correlate with higher Sec production, which remained identical to mock-transfected cells. Interestingly, we found that S LA/LP formed a complex in the cytosol of HEK293 cells. This complex was purified and the components analy zed using Nano-LC and MS/MS, which we identified as HSP7 0a nd tubulin. Altogether, our data suggest that SLA/LP may play an indirect role to produce Sec in the selenoprotein synthesis machinery.

Background: Oxidative damage of the erythrocyte membrane plays an important role in ribavirin-induced anemia. The purpose of the present paper was to assess whether supplementation of α-tocopherol and ascorbic acid (vitamins) causes changes in the erythrocyte membrane fatty acid composition during interferon and ribavirin combination therapy for chronic hepatitis C patients. Methods: Fatty acid compositions in erythrocyte membrane phospholipids were determined by gas chromatography at 0, 2, 4, 8 weeks, and at the end of combination therapy (26 weeks) for interferon with ribavirin in 32 patients with chronic hepatitis C who were randomized to receive vitamins or not (controls). Results: Good compliance with orally administered vitamins and ribavirin were confirmed by their concentrations in erythrocytes or plasma. The hemoglobin level was negatively correlated with the ribavirin concentration at 8 weeks (r = 0.59, P = 0.01) after initiation of therapy in controls, but not in the vitamin group. Among the 26 kinds of fatty acids analyzed, only eicosapentaenoic acid (EPA) significantly decreased at 8 weeks after initiation of therapy (P = 0.03) and at the end of therapy (P = 0.004) in controls. Vitamins did not inhibit ribavirin-induced anemia, but attenuated the decrease of EPA in erythrocytes. The EPA level was negatively correlated with the drop in hemoglobin levels at 8 weeks after initiation of therapy in controls (r = 0.58, P = 0.015), but not in the vitamin group. Conclusions: Supplementation of α-tocopherol and ascorbic acid attenuates the ribavirin-induced decrease of EPA in erythrocyte membrane phospholipids in chronic hepatitis C patients.

Objective We investigated the effects of vitamin E and C supplementation on the fatty acid composition of mononuclear cells and on the clinical observations in patients who had chronic hepatitis C and received interferon-α-2b (IFN-α-2b) and ribavirin combination therapy. Methods Patients were randomly allocated to receive daily 500 mg of vitamin E and 750 mg of vitamin C (vitamin group, n = 14) or no supplement (non-vitamin group, n = 16) in addition to IFN-α-2b and ribavirin therapy. The fatty acid composition of mononuclear cell phospholipids was analyzed before and at 2, 4, and 8 wk after treatment. Results After vitamin supplementation, plasma and red blood cell α-tocopherol and plasma ascorbic acid levels increased in the vitamin group. Serum levels of alanine aminotransferase decreased significantly after 2 wk of treatment in both groups. At the start of treatment, a lower level of eicosapentaenoic acid (EPA) and a higher level of the molar ratio of arachidonic acid to EPA in mononuclear cells were observed in the present patients compared with healthy volunteers, and a significant correlation between the molar ratio and serum alanine aminotransferase level was found. The EPA level of mononuclear cells was maintained in the vitamin group during treatment, whereas a significant decrease was observed in the non-vitamin group at 4 and 8 wk after treatment. Conclusions Antioxidant vitamin supplementation during IFN-α-2b and ribavirin therapy prevented a decrease in EPA of mononuclear cell phospholipids. If a further decrease in the ratio of arachidonic acid to EPA can be achieved by using oral EPA supplementation, the efficacy of IFN-α-2b and ribavirin therapy may be improved.

The degradation of mammalian ornithine decarboxylase (ODC) (EC 4.1.1.17) by 26 S proteasome, is accelerated by the ODC antizyme (AZ), a trigger protein involved in the specific degradation of eukaryotic ODC. In prokaryotes, AZ has not been found. Previously, we found that in Selenomonas ruminantium, a strictly anaerobic and Gram-negative bacterium, a drastic degradation of lysine decarboxylase (LDC; EC 4.1.1.18), which has decarboxylase activities toward both L-lysine and L-ornithine with similar K(m) values, occurs upon entry into the stationary phase of cell growth by protease together with a protein of 22 kDa (P22). Here, we show that P22 is a direct counterpart of eukaryotic AZ by the following evidence. (i) P22 synthesis is induced by putrescine but not cadaverine. (ii) P22 enhances the degradation of both mouse ODC and S. ruminantium LDC by a 26 S proteasome. (iii) S. ruminantium LDC degradation is also enhanced by mouse AZ replacing P22 in a cell-free extract from S. ruminantium. (iv) Both P22 and mouse AZ bind to S. ruminantium LDC but not to the LDC mutated in its binding site for P22 and AZ. In this report, we also show that P22 is a ribosomal protein of S. ruminantium.

To elucidate the efficacy of dietary therapy for ulcerative colitis (UC), we analyzed the dietary fatty acid intakes, fatty acid compositions of phospholipids in the plasma and blood cells, and serum cytokines of 12 patients with UC. Correlations among fatty acid compositions in the diet, blood cell phospholipids and serum cytokines were also investigated in UC patients. Although the fat energy ratio was lower in the UC patients than in the controls, no significant difference was observed in their fatty acid intakes. Significantly higher linoleic acid levels were observed in the erythrocyte, mononuclear cell and neutrophil phospholipids of the UC patients compared with the controls. Concerning neutrophil phospholipids, two times higher levels of linoleic acid and arachidonic acid and lower levels of eicosapentaenoic acid (EPA) were observed in the UC patients compared with those in the controls. The serum TNF-α concentration was correlated positively with molar percentage of linoleic acid (r = 0.675, p < 0.02) and negatively with the n-3 polyunsaturated fatty acid (PUFA) (r = -0.584, p < 0.05) in the plasma phospholipids of UC patients. Dietary EPA intake was correlated negatively with the n-6 PUFA molar percentage in the phospholipids of the plasma, mononuclear cells and neutrophils. From these findings, we suggest that a diet high in n-3 PUFA and low in linoleic acid should be recommended for UC patients.

Antizyme-1 (AZ1) is a protein that negatively regulates polyamine synthesis by inhibiting the key synthetic enzyme ornithine decarboxylase and targeting it for degradation by the 26 S proteasome. Recent work shows that antizyme protein translocates to the nucleus during mouse development (Gritli-Linde, A., Nilssom, J., Bohlooly, Y. M., Heby, O., and Linde, A. (2001) Dev. Dyn. 220, 259-275). However, the significance and mechanism of this phenomenon remain unclear. In this study, we expressed AZ1 fused with enhanced green fluorescent protein (EGFP) to study its localization in a living cell. We found that EGFP-AZ1 was predominantly localized in the cytoplasm and that treatment with leptomycin B, a specific inhibitor of chromosomal region maintenance 1 (CRM1) induced nuclear accumulation of EGFP-AZ1 in Chinese hamster ovary and NIH3T3 cells. Two independent nuclear export signal (NES) sequences, each containing essential hydrophobic residues, were identified in the 50 N-terminal amino acid residues and in the central part of AZ1. The activity of the second NES was inhibited by an N-terminal adjacent region and was only revealed in N-terminal truncated constructs. Both NESs were active when fused to an artificial nuclear protein SV40-NLS-EGFP-EGFP. The ability of AZ1 to shuttle between the nucleus and the cytoplasm suggests that it has a novel function in the nucleus.

Background Epithelial ovarian cancer (EOC) is the most common cause of gynecological malignancy-related mortality. Ovarian clear cell carcinoma (CCC) has unique clinical characteristics and behaviors that differ from other histological types of EOC, including a frequent association with endometriosis and a highly chemoresistant nature, resulting in poor prognosis. However, factors underlying its malignant behavior are still poorly understood. Aberrant expression of microRNAs has been shown to be involved in oncogenesis, and microRNA-21 (miR-21) is frequently overexpressed in many types of cancers. The aim of this study was to investigate the role of miR-21 in 17q23-25 amplification associated with CCC oncogenesis. Methods We identified 17q23-25 copy number aberrations among 28 primary CCC tumors by using a comparative genomic hybridization method. Next, we measured expression levels of the candidate target genes, miR-21 and PPM1D, for 17q23-25 amplification by real-time RT-PCR analysis and compared those data with copy number status and clinicopathological features. In addition, immunohistochemical analysis of PTEN (a potential target of miR-21) was performed using the same primary CCC cases. We investigated the biological significance of miR-21 overexpression in CCC using a loss-of-function antisense approach. Results 17q23-25 amplification with both miR-21 overexpression and PTEN protein loss was detected in 4/28 CCC cases (14.2%). The patients with 17q23-25 amplification had significantly shorter progression-free and overall survival than those without 17q23-25 amplification (log-rank test: p = 0.0496; p = 0.0469, respectively). A significant correlation was observed between miR-21 overexpression and endometriosis. Both PTEN mRNA and PTEN protein expression were increased by miR-21 knockdown in CCC cells. We also confirmed that miR-21 directly bound to the 3′-untranslated region of PTEN mRNA using a dual-luciferase reporter assay. Conclusions MiR-21 is a possible driver gene other than PPM1D for 17q23-25 amplification in CCC. Aberrant expression of miR-21 by chromosomal amplification might play an important role in CCC carcinogenesis through the regulation of the PTEN tumor suppressor gene. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-799) contains supplementary material, which is available to authorized users.

The mechanism of the regulatory degradation of ornithine decarboxylase (ODC) by polyamines was studied in fission yeast, Schizosaccharomyces pombe. To regulate cellular spermidine experimentally, we cloned and disrupted S-adenosylmethionine decarboxylase gene (spe2) in S. pombe. The null mutant of spe2 was devoid of spermidine and spermine, accumulated putrescine, and contained a high level of ODC. Addition of spermidine to the culture medium resulted in rapid decrease in the ODC activity caused by the acceleration of ODC degradation, which was dependent on de novo protein synthesis. A fraction of ODC forming an inactive complex concomitantly increased. The accelerated ODC degradation was prevented either by knockout of antizyme gene or by selective inhibitors of proteasome. Thus, unlike budding yeast, mammalian type antizyme-mediated ODC degradation by proteasome is operating in S. pombe.

Eukaryotic cells contain various types of proteasomes. Core 20 S proteasomes (abbreviated 20 S below) have two binding sites for the regulatory particles, PA700 and PA28. PA700–20 S-PA700 complexes are known as 26 S proteasomes and are ATP-dependent machines that degrade cell proteins. PA28 is found both in previously described complexes of the type PA28–20 S-PA28 and in complexes that also contain PA700, as PA700–20 S-PA28. We refer to the latter as “hybrid proteasomes.” The relative amounts of the various types of proteasomes in HeLa extracts were determined by a combination of immunoprecipitation and immunoblotting. Hybrid proteasomes accounted for about a fourth of all proteasomes in the extracts. Association of PA28 and proteasomes proved to be ATP-dependent. Hybrid proteasomes catalyzed ATP-dependent degradation of ornithine decarboxylase (ODC) without ubiquitinylation, as do 26 S proteasomes. In contrast, the homo-PA28 complex (PA28–20 S-PA28) was incapable of degrading ODC. Intriguingly, a major immunomodulatory cytokine, interferon-γ, appreciably enhanced the ODC degradation in HeLa and SW620 cells through induction of the hybrid proteasome, which may also be responsible for the immunological processing of intracellular antigens. Taken together, we report here for the first time the existence of two types of ATP-dependent proteases, the 26 S proteasome and the hybrid proteasome, which appear to share the ATP-dependent proteolytic pathway in mammalian cells.

Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals.

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Turnover of ODC is extremely rapid and highly regulated, and is accelerated when polyamine levels increase. Polyamine-stimulated ODC degradation is mediated by association with antizyme (AZ), an ODC inhibitory protein induced by polyamines. ODC, in association with AZ, is degraded by the 26S proteasome in an ATP-dependent, but ubiquitin-independent, manner. The 26S proteasome irreversibly inactivates ODC prior to its degradation. The inactivation, possibly due to unfolding, is coupled to sequestration of ODC within the 26S proteasome. This process requires AZ and ATP, but not proteolytic activity of the 26S proteasome. The carboxyl-terminal region of ODC presumably exposed by interaction with AZ plays a critical role for being trapped by the 26S proteasome. Thus, the degradation pathway of ODC proceeds as a sequence of multiple distinct processes, including recognition, sequestration, unfolding, translocation, and ultimate degradation mediated by the 26S proteasome.

Cellular polyamines are regulated by a unique feedback mechanism involving ornithine decarboxylase (ODC) antizyme. The synthesis of mammalian antizyme requires a programmed translational frameshift event induced by polyamines. Antizyme represses ODC, a key enzyme for polyamine synthesis, through accelerating enzyme degradation by the 26 S proteasome. Antizyme also inhibits the cellular uptake of polyamines. In the present study we isolated two distinct zebrafish (Danio rerio) antizyme cDNA clones (AZS and AZL) from an embryonic library. Their sequences revealed that both clones required translational frameshifting for expression. Taking account of +1 frameshifting, AZS and AZL products were 214 and 218 residues long respectively and shared 51.8% amino acid identity. In rabbit reticulocyte lysates, both mRNA species were translated through spermidine-induced frameshifting. The presence of the two antizyme mRNA species in embryos, adult fish and a cultured cell line was confirmed by Northern blot analysis. The ratio of AZS mRNA to AZL mRNA in the adult fish was 1.8-fold higher than in the embryos. Whole-mount hybridization in situ demonstrated that both mRNA species are expressed in every tissue in embryo, but predominantly in the central nervous system and the eyes. Bacterial expression products of both cDNA species inhibited ODC activity, but only the AZS product accelerated ODC degradation in vitro. These results show that both zebrafish antizymes are induced by polyamines but their mRNA species are expressed differently during development. The difference in activities on ODC degradation suggests their functional divergence.

In male mouse kidney, ornithine decarboxylase (ODC) is induced after feeding, and the induction depends on dietary protein content. 24 h after feeding with 50% casein-containing meal, ODC activity and amount of immunoreactive ODC protein increased more than 10-fold, ODC mRNA level increased 2-fold, and the ODC half-life extended 7-fold. The renal ODC induction after feeding is, therefore, due mainly to stabilization of ODC protein. Urinary excretion of putrescine increased in response to the ODC induction, but the renal polyamine contents scarcely changed. Consistently, the level of antizyme, a polyamine-inducible protein, determined as the ODC–antizyme complex level, scarcely changed after feeding, and the antizyme/ODC ratio in the kidney largely decreased, resulting in the stabilization of ODC protein. The present results suggest that the strong excretion system of the kidney for newly synthesized polyamines enables renal ODC escape from antizyme-mediated feedback regulation.

The 26S proteasome was purified from cultured rice cells to near homogeneity by ultracentrifugation fo r5ha t85,000g, chromatography on Biogel A-1.5m, and glycerol density-gradient centrifugation analysis. The purified enzyme had two distinct forms, termed 26Sa- and 26Sb-type proteasomes, with different electrophoretic mobilities by nondenaturing polyacrylamide gel electrophoresis. It consisted of multiple polypeptides with molecular masses of 25‐35 and 42‐120 kDa, which presumably corresponded to those of the 20S proteasome and an associated PA700 regulatory complex, respectively. The rice 26S proteasome resembled, but was not identical to, one from other sources in their subunit composition and immunochemical reactivity. Intriguingly, both rice and spinach 26S proteasomes could not degrade rat ornithine decarboxylase in the presence of antizyme and ATP, unlike the rat 26S proteasome, implying the existence of functional differences between mammalian and plant 26S proteasomes. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.

The 26S proteasome is a eukaryotic ATP-dependent protease, but the molecular basis of its energy requirement is largely unknown. Ornithine decarboxylase (ODC) is the only known enzyme to be degraded by the 26S proteasome without ubiquitinylation. We report here that the 26S proteasome is responsible for the irreversible inactivation coupled to sequestration of ODC, a process requiring ATP and antizyme (AZ) but not proteolytic activity. Neither the 20S proteasome (catalytic core) nor PA700 (the regulatory complex) by itself contributed to this ODC inactivation. Analysis with a C-terminal mutant ODC revealed that the 26S proteasome recognizes the C-terminal degradation signal of ODC exposed by attachment of AZ, and subsequent ATP-dependent sequestration of ODC in the 26S proteasome causes irreversible inactivation, possibly unfolding, of ODC and dissociation of AZ. These processes may be linked to the translocation of ODC into the 20S proteasomal inner cavity, centralized within the 26S proteasome, for degradation.

Addition of spermidine to culture medium of insect cells, Trichoplusia ni 5, at a low cellular density suppressed ornithine decarboxylase (ODC; EC 4.1.1.17) activity and induced ODC inhibitory activity. The inhibitory factor was non-dialyzable, temperature-sensitive, and could reversibly form an inactive complex with ODC. It showed a time-independent and non-stoichiometric pattern of inhibition. Upon addition of spermidine to cultured cells with preinduced ODC, the enzyme decayed more rapidly than after addition of cycloheximide. These data strongly suggested that ODC of Tn5 cells is under negative feedback control by polyamines, in which an antizyme-like regulatory protein plays an essential role.

Alcoholic liver disease (ALD), such as fatty liver, hepatitis, or fibrosis, is frequently observed in patients with a long history of excessive alcohol intake. These types of ALD are considered alcohol-associated lifestyle diseases and involve both genetic and environmental factors [1]. Interactions between alcohol and nutritional status, which are one of the secondary risk factors, may also be important. Indeed, the presence and extent of protein-calorie malnutrition have important roles in determining the outcome of patients with ALD. Micronutrient abnormalities, such as hepatic vitamin A depletion or depressed vitamin E levels, may also potentially aggravate liver disease [2]. Additionally, obesity and excess body weight have been associated with an increased risk of ALD [3, 4]. Alcoholic and nonalcoholic fatty liver each begin with the accumulation of lipids in the liver, which, although a reversible condition, is understood to play an important role in the development of advanced liver disease. With continued alcohol intake, the development of steatosis may progress to hepatitis and fibrosis and might lead to liver cirrhosis. Excessive alcohol intake adversely influences the liver through the production of toxic products such as acetaldehyde and potentially highly reactive oxygen molecules, which are generated by alcohol dehydrogenase and the microsomal ethanol oxidizing system. These products can directly and indirectly interfere with the normal metabolism of other nutrients, particularly lipids, contributing to liver cell damage [5].

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. It is a short-lived protein and negatively regulated by its products, polyamines. Its degradation is accelerated by the binding of antizyme, an ODC-inhibitory protein induced by polyamines. To evaluate the physiological importance of antizyme we examined the effect of forced expression of antizyme on cellular ODC and polyamine levels and cell growth. Antizyme almost completely abolished the induction of ODC by growth stimuli. This may have been caused by antizyme-induced rapid degradation of newly synthesized ODC, since the half-life of ODC complexes with antizyme was less than 5 min. Forced expression of antizyme caused reductions of cellular putrescine and spermidine levels, and inhibited cell growth, which was partially restored by the addition of putrescine. These observations suggested a critically important role of antizyme in polyamine metabolism.

Antizyme (AZ) is a key molecule in feedback regulation of cellular polyamines. It is induced by polyamines through stimulation of ribosomal frameshifting during its translation. In mammals, AZ is diverged into three paralogs, AZ1-3. Tissue and subcellular distribution are different among the paralogs, as determined by immunochemical methods or expression of fluorescent-tagged proteins. Only AZ2 is known to be phosphorylated. AZ regulates cellular polyamine levels through multiple mechanisms. It binds to ornithine decarboxylase (ODC) to form an inactive complex and to trigger degradation of ODC by 26S proteasomes. The AZ activity to promote ODC degradation can be measured both in vitro and in cells. AZ also inhibits cellular uptake of polyamines. This chapter comprises seven subchapters describing methods for studying expression and function of AZ.

Ornithine decarboxylase (ODC) degradation in a freshly prepared reticulocyte lysate was examined. Immunodepletion of proteasomes from the reticulocyte lysate resulted in almost complete loss of ODC degradation. In contrast with the previously reported degradation in extracts of hepatoma tissue-culture (HTC) and Chinese-hamster ovary (CHO) cells or that by the purified 26 S proteasome, efficient degradation of ODC was observed in the lysate without exogenous antizyme, an ODC protein inhibitor induced by polyamines, owing to the presence of a significant amount of antizyme in the lysate. The degradation of ODC in the lysate was strongly suppressed on inactivation of antizyme in the lysate with antizyme inhibitor, a protein which binds to the antizyme and releases ODC from the ODC-antizyme complex. Thus the main pathway for ODC degradation in a reticulocyte lysate was essentially the same as that characterized previously in extracts of HTC and CHO cells, namely an ATP- and antizyme-dependent 26 S proteasome-catalysed pathway that is presumed to be responsible for ODC degradation in whole cells.

Human pancreatic tumor cell lines – AsPC-1, PANC-1, MIA paca2, KP-1 and KP-59 cells – can be induced to differentiate into pancreatic hormone-producing cells by brief trypsin treatment and subsequent culture in a serum-free, chemically defined medium. During culture, AsPC-1 cells formed cell clusters resembling the pancreatic islets, expressed genes associated with the pancreatic development and produced glucagon but not insulin. When PANC-1, MIA paca2, KP-1 and KP-59 cells were treated and cultured the same way, they underwent similar morphological changes and produced insulin and glucagon. We used these systems to identify intracellular regulatory molecules involved in the conversion of pancreatic tumor cells into glucagon-producing cells. We found that the expression of antizyme 1 (AZ1), a negative regulator of ornithine decarboxylase, was increased and its localization was altered from the nucleus to the cytoplasm during AsPC-1 cell differentiation. Transient transfection of AsPC-1 cells with AZ1 siRNA resulted in inhibition of the morphological and functional cell differentiation as well as the specific suppression of AZ1 expression. By contrast, constitutive overexpression of AZ1 in AsPC-1 cells led to the enhancement of glucagon production. We also found that PANC-1 cells reduced the expression of glucagon mRNA when treated with AZ1 siRNA. These results suggested that AZ1 was necessary for the conversion of pancreatic tumor cells into glucagon-producing cells. Glucagon production in AsPC-1 cells was not affected by addition of putrescine, suggesting that the polyamines were not directly involved in the AZ1-mediated conversion of pancreatic tumor cells to differentiated state.

Antizyme inhibitor (AIn), a homolog of ODC, binds to antizyme and inactivates it. We report here that AIn increased at the G1 phase of the cell cycle, preceding the peak of ODC activity in HTC cells in culture. During interphase AIn was present mainly in the cytoplasm and turned over rapidly with the half-life of 10 to 20 min, while antizyme was localized in the nucleus. The level of AIn increased again at the G2/M phase along with ODC, and the rate of turn-over of AIn in mitotic cells decreased with the half-life of approximately 40 min. AIn was colocalized with antizyme at centrosomes during the period from prophase through late anaphase and at the midzone/midbody during telophase. Thereafter, AIn and antizyme were separated and present at different regions on the midbody at late telophase. AIn disappeared at late cytokinesis, whereas antizyme remained at the cytokinesis remnant. Reduction of AIn by RNA interference caused the increase in the number of binucleated cells in HTC cells in culture. These findings suggested that AIn contributed to a rapid increase in ODC at the G1 phase and also played a role in facilitating cells to complete mitosis during the cell cycle.

Ornithine decarboxylase (ODC) antizyme inhibitor (AZI) has been shown to regulate ODC activity in cell cultures. However, its biological functions in an organism remain unknown. An embryonic stem (ES) cell clone was established, in which the Azin1 gene was disrupted by the gene trap technique. To identify the function of Azin1 gene in vivo, a mutant mouse line was generated using these trapped ES cells. Homozygous mutant mice died at P0 with abnormal liver morphology. Further analysis indicated that the deletion of Azin1 in homozygous mice resulted in the degradation of ODC, and reduced the biosynthesis of putrescine and spermidine. Our results thus show that AZI plays an important role in regulating the levels of ODC, putrescine and spermidine in mice, and is essential for the survival of mice.

Ornithine decarboxylase antizyme is a unique inhibitory protein induced by polyamines and involved in the regulation of ornithine decarboxylase. A cDNA was isolated from a rat liver cDNA library by the screening with monoclonal antibodies to rat liver antizyme as probes. The expression products of the cDNA in bacterial systems inhibited rat ornithine decarboxylase activity in a manner characteristic of antizyme and rabbit antisera raised against its direct expression product reacted to rat liver antizyme, confirming the authenticity of the cDNA. On RNA blot analysis with the cDNA probe, an antizyme mRNA band of 1.3 kb was detected in rat tissues. Antizyme mRNA did not increase upon administration of putrescine, an inducer of antizyme, and its half-life after actinomycin D treatment was as long as 12 h in rat liver, suggesting that antizyme mRNA is constitutively expressed and antizyme synthesis is regulated at the translational level. Similar-sized mRNAs hybridizable to the cDNA were also found in various mammalian and non-mammalian vertebrate tissues under physiological conditions. In addition, chicken and frog antizymes showed immunocrossreactivity with rat antizyme. The ubiquitous presence and the evolutionally conserved structure of antizyme in vertebrate tissues suggest that it has an important function.

A sensitive method for the determination of polyamines in mammalian cells was described using electrospray ionization and time-of-flight mass spectrometer. This method was 50-fold more sensitive than the previous method using ionspray ionization and quadrupole mass spectrometer. The method employed the partial purification and derivatization of polyamines, but allowed a measurement of multiple samples which contained picomol amounts of polyamines. Time required for data acquisition of one sample was approximately 2 min. The method was successfully applied for the determination of reduced spermidine and spermine contents in cultured cells under the inhibition of aminopropyltransferases. In addition, a new proper internal standard was proposed for the tracer experiment using (15)N-labeled polyamines.