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Patatin-like phospholipase domain-containing lipase 8 (PNPLA8), one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. Biallelic variants in PNPLA8 have been associated with a range of paediatric neurodegenerative disorders. However, the phenotypic spectrum, genotype-phenotype correlations and the underlying mechanisms are poorly understood. Here, we newly identified 14 individuals from 12 unrelated families with biallelic ultra-rare variants in PNPLA8 presenting with a wide phenotypic spectrum of clinical features. Analysis of the clinical features of current and previously reported individuals (25 affected individuals across 20 families) showed that PNPLA8-related neurological diseases manifest as a continuum ranging from variable developmental and/or degenerative epileptic-dyskinetic encephalopathy to childhood-onset neurodegeneration. We found that complete loss of PNPLA8 was associated with the more profound end of the spectrum, with congenital microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 led to developmental defects by reducing the number of basal radial glial cells and upper-layer neurons. Spatial transcriptomics revealed that loss of PNPLA8 altered the fate specification of apical radial glial cells, as reflected by the enrichment of gene sets related to the cell cycle, basal radial glial cells and neural differentiation. Neural progenitor cells lacking PNPLA8 showed a reduced amount of lysophosphatidic acid, lysophosphatidylethanolamine and phosphatidic acid. The reduced number of basal radial glial cells in patient-derived cerebral organoids was rescued, in part, by the addition of lysophosphatidic acid. Our data suggest that PNPLA8 is crucial to meet phospholipid synthetic needs and to produce abundant basal radial glial cells in human brain development., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Synthesis of the A/D/E-ring core compounds of maoecrystal V was achieved. The key Diels-Alder reactions between tricyclic α-methylene lactones and Kitahara-Danishefsky dienes afforded the spirocyclic core compounds in a regioselective and stereoselective manner., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Objectives: To analyze the proportion of successful biological disease-modifying antirheumatic drugs (bDMARDs) discontinuation and related factors in patients with rheumatoid arthritis (RA) in clinical settings., Methods: Among 1775 RA patients who started bDMARDs between 2003 and 2012, 43 patients with DAS28-ESR <3.2 at the time of bDMARD discontinuation were extracted. Patients were divided into two groups (bio-free success: BS and bio-free failure: BF groups) based on bDMARD usage and disease activity 1 year after discontinuation. We evaluated the proportion of bio-free success and assessed factors related to bio-free success., Results: Twenty-five patients (58.1%: BS group) maintained discontinuation of bDMARDs and DAS28-ESR <3.2 at 1 year after discontinuation. The median DAS28-ESR at bDMARD initiation was lower in the BS group than in the BF group (3.95 vs 5.04; p  = .04). The BS group experienced a larger decrease in average glucocorticoid (GC) dose during bDMARD use than the BF group (-3.0 mg/day vs 0 mg/day; p  = .01)., Conclusion: bDMARDs were discontinued without flare up of RA in 58.1% of patients with RA in clinical settings. A lower DAS28-ESR at initiation and reduction of GC dose before discontinuation of bDMARD were important factors associated with bio-free success.

A 52-year-old woman with a strong family history of breast cancer was diagnosed as having triple-negative breast cancer (TNBC) in her right breast. Neoadjuvant chemotherapy (NAC; four cycles of epirubicin/cyclophosphamide/5-fluorouracil) was performed, followed by breast-conserving surgery and axillary lymph node dissection. Histopathological analysis of the surgical specimens demonstrated a few focal tumor cells remaining in the stroma, but not a pathological complete response (pCR). Weekly paclitaxel was subsequently added to the treatment regimen. A total of 17 months after the adjuvant treatments, TNBC recurred in her left breast with massive lymph node metastasis. Because of the early recurrence after standard treatment, NAC was administered together with carboplatin and paclitaxel. Histopathological analysis of the partially resected breast and axillary lymph nodes demonstrated a pCR. No recurrent disease was found 2 years after the second TNBC treatment. This case underlines the importance of platinum-based chemotherapy and prophylactic mastectomy for patients with BRCA dysfunction., (Published by Oxford University Press and JSCR Publishing Ltd. All rights reserved. © The Author(s) 2021.)

Background: Inflammation, intrarenal renin-angiotensin system (RAS) activation, oxidative stress, and carbonyl stress have been postulated to play a fundamental role in controlling blood pressure. However, little is known about the association among renal RAS activation, carbonyl stress, and blood pressure elevation., Methods: We evaluated the relationship between blood pressure elevation and either renal RAS activity or carbonyl stress in the general population (N = 355) in Japan. To minimize the effect of antihypertensive drug therapy, we divided participants into 3 groups (normotensive, hypertensive-with-non-medication, and hypertensive-with-medication). Intrarenal RAS activity and carbonyl stress were indicated by the urinary angiotensinogen (AGT) and carbonyl compound excretion levels, respectively., Results: The urinary AGT and carbonyl compound excretion levels were significantly associated with blood pressure. Using a stepwise multiple regression analysis, we found that the urinary AGT excretion levels were strongly associated with blood pressure elevation, compared with inflammation, oxidative stress, and carbonyl stress markers, in all groups. Urinary carbonyl compound excretion was significantly associated with blood pressure in only the hypertensive-without-medication group. Furthermore, blood pressure was significantly increased in these participants, and both the urinary AGT and carbonyl compound levels were high. The urinary AGT excretion levels were strongly associated with elevated blood pressure in normotensive people, and inappropriate renal RAS activity and carbonyl stress independently contributed to the development of hypertension., Conclusions: These findings suggest that RAS activation, particularly renal RAS activation exert a fundamental role in the pathogenesis of hypertension in the general population.

In the current article, we provide in vitro efficacy evaluation of a unique accelerator-based neutron source, constructed at the Budker Institute of Nuclear Physics (Novosibirsk, Russian Federation), for boron neutron capture therapy (BNCT), which is particularly effective in the case of invasive cancers. U251MG, CHO-K1 and V79 cells were incubated and irradiated in various concentrations of boric acid with epithermal neutrons for 2-3 h in a plexiglass phantom, using 2.0 MeV proton energy and 1.5-3.0 mA proton current, resulting in a neutron fluence of 2.16 × 1012 cm-2. The survival curves of cells loaded with boron were normalized to those irradiated without boron (to exclude the influence of the fast neutron and gamma dose components) and fit to the linear-quadratic (LQ) model. Colony formation assays showed the following cell survival rates (means ± SDs): CHO-K1: 0.348 ± 0.069 (10 ppm), 0.058 ± 0.017 (20 ppm), 0.018 ± 0.005 (40 ppm); V79: 0.476 ± 0.160 (10 ppm), 0.346 ± 0.053 (20 ppm), 0.078 ± 0.015 (40 ppm); and U251MG: 0.311 ± 0.061 (10 ppm), 0.131 ± 0.022 (20 ppm), 0.020 ± 0.010 (40 ppm). The difference between treated cells and controls was significant in all cases (P < 0.01) and confirmed that the neutron source and irradiation regimen were sufficient for control over cell colony formation. We believe our study will serve as a model for ongoing in vitro experiments on neutron capture therapy to advance in this area for further development of accelerator-based BNCT into the clinical phase.

Secretogranin III (SgIII), a member of the granin family, binds both to another granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knockdown of SgIII in adrenocorticotropic hormone (ACTH)-producing AtT-20 cells largely impairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretion of active insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors., (Copyright © 2018 Endocrine Society.)

Objectives: To evaluate the cost-effectiveness of biological disease modifying antirheumatic drugs (bDMARDs) in patients with rheumatoid arthritis (RA) in a real-world setting in Japan., Methods: We used a state-transition model and parameters were determined from RA patients registered in the Institute of Rheumatology, Rheumatoid Arthritis (IORRA) cohort study on 421 patients who had failed at least one DMARD and started either 1 of 4 bDMARDs (bDMARD group; adalimumab, etanercept, infliximab, and tocilizumab) or methotrexate (control group). bDMARD group was evaluated as two groups: sequence of any 1 of 4 bDMARDs with and without tocilizumab. The incremental cost-effectiveness ratios (ICERs) for bDMARD group were estimated using base-case analysis, probabilistic sensitivity analysis (PSA) and scenario sensitivity analyses., Results: ICERs of bDMARD group with or without tocilizumab were $38,179 and $48,855, respectively. By PSA, these sequences had respective probabilities of 86.8% and 75.1% of falling below the assumed cost-effectiveness threshold of $50,000 in Japan. Scenario sensitivity analyses showed that the best population for initiating bDMARD was RA patients less than 50 years old with Japanese version of HAQ between 1.1 and 1.6 and using tocilizumab as the bDMARD., Conclusion: bDMARDs were cost-effective for RA patients based on a real-world setting in Japan.

A 37-year-old woman with rheumatoid arthritis and interstitial lung disease (ILD) developed clinically amyopathic dermatomyositis (CADM) after achieving pregnancy through in vitro fertilization. She was given oral prednisolone, which improved her respiratory status, and delivered a healthy baby at 35 weeks' gestation. There are few reports of successful outcomes for CADM during pregnancy; to the best of our knowledge, this is the first report of successful delivery in a patient with both CADM and ILD.

The diagnosis of macrophage activation syndrome (MAS) in patients with systemic lupus erythematosus (SLE) may be challenging as it can mimic the clinical features of the underlying disease or be confused with an infectious complication. In this report, a Japanese woman in her forties had diverse clinical features of MAS at initial presentation of SLE, where erythematosus plaques with histiocytic infiltrates focally surrounding degenerated collagen might be the earliest indicator of MAS.

Objective: Along with the advances of newly developed medical therapies in rheumatoid arthritis (RA), the number of pharmacoeconomical issues has been paid attention rapidly. For cost-utility analysis and determination of quality-adjusted life years, measurement of the EuroQol 5-dimensional descriptive system (EQ-5D) is essential, and has been used in several clinical studies. However, EQ-5D utility measure in Japanese patients with RA, especially in daily practice has not been fully documented. We analyzed the distribution of EQ5D utility scores and investigated the relationship between other clinical measures based on our Institute of Rheumatology, Rheumatoid Arthritis (IORRA) database., Method: Among 5,284 outpatients who participated in the IORRA cohort study on October 2007, data from 5,043 patients who completed the EQ-5D questionnaire were cross-sectionally analyzed. EQ-5D scores in each subgroup for baseline feature such as gender, age, disease activity score 28 (DAS28), and Japanese version of health assessment questionnaire (J-HAQ) were evaluated. For the evaluation of variables that influenced EQ-5D score, the contribution of each variable was evaluated by ANOVA., Results: Average EQ-5D score was 0.76 in 5,284 patients (84% females, average age: 59.0 years, average disease duration: 12.4 years) whose average DAS28 was 3.3 and average J-HAQ was 0.74. EQ-5D scores were highly correlated with J-HAQ and DAS28, and were significantly lower in females and rheumatoid factor-positive patients. Older age, longer disease duration, higher DAS28, and higher J-HAQ were also significantly associated with lower EQ-5D scores. In multivariate analysis, the factor that most strongly influenced EQ-5D was J-HAQ (57.6%), followed by pain visual analog score (VAS; 12.5%)., Conclusion: This study clearly demonstrated the distribution of EQ-5D score in the daily practice of RA patients, and provides important information for the pharmacoeconomical studies in rheumatology.

Unlabelled: : Chronic rotator cuff (RC) tears are a common and debilitating injury, characterized by dramatic expansion of adipose tissue, muscle atrophy, and limited functional recovery. The role of adipose expansion in RC pathology is unknown; however, given the identified paracrine/endocrine regulation by other adipose depots, it likely affects tissue function outside its boundaries. Therefore, we characterized the epimuscular (EM) fat depot of the human rotator cuff, defined its response to RC tears, and evaluated its influence on myogenesis in vitro. EM fat biopsies exhibited morphological and functional features of human beige fat compared with patient-matched s.c. biopsies, which appeared whiter. The transcriptional profile of EM fat and isolated EM adipose-derived stem cells (ASCs) shifted as a function of the tear state; EM fat from intact cuffs had significantly elevated expression of the genes associated with uncoupled respiration, and the EM fat from torn cuffs had increased expression of beige-selective genes. EM ASC cocultures with human- and mouse-derived myogenic cells exhibited increased levels of myogenesis compared with s.c. cultures. Increased fusion and decreased proliferation of myogenic cells, rather than changes to the ASCs, were found to underlie this effect. Taken together, these data suggest that EM fat in the human rotator cuff is a novel beige adipose depot influenced by cuff state with therapeutic potential for promoting myogenesis in neighboring musculature., Significance: Rotator cuff tears affect millions of people in the U.S.; however, current interventions are hindered by persistent muscle degeneration. This study identifies the therapeutic potential for muscle recovery in the epimuscular fat in the rotator cuff, previously considered a negative feature of the pathology, and finds that this fat is beige, rather than white. This is important for two reasons. First, the stem cells that were isolated from this beige fat are more myogenic than those from white fat, which have been the focus of stem cell-based therapies to date, suggesting epimuscular fat could be a better stem cell source to augment rotator cuff repair. Second, these beige stem cells promote myogenesis in neighboring cells in culture, suggesting the potential for this fat to be manipulated therapeutically to promote muscle recovery through secreted signals., (©AlphaMed Press.)

Objectives: To assess the effectiveness of the golimumab (GLM) 50-mg and 100-mg regimens in patients with rheumatoid arthritis (RA) in daily practice., Methods: We retrospectively analyzed RA patients who started GLM between September 2011 and July 2012. Patients were divided into three groups: a 50-mg group; a 50/100-mg group (had a dose increase to 100 mg); and a 100-mg group (started GLM at 100 mg). We assessed Disease Activity Score 28 (DAS28) and treatment continuation rate. Risk factors associated with time to discontinuation of the 50-mg regimen were determined with proportional hazards analysis., Results: We analyzed 74 patients: 43 in the 50-mg group, 23 in the 50/100-mg group, and 8 in the 100-mg group. DAS28 improved from 4.0 ± 1.0, 4.8 ± 1.0, and 4.7 ± 1.9, respectively, at baseline to 2.4 ± 1.2, 3.3 ± 1.5, and 2.5 ± 0.7, respectively, at week 52. Treatment continuation rates at week 52 were 73.7%, 60.9%, and 87.5%, respectively. In the 50/100-mg group, the mean DAS28 improved significantly from 4.4 ± 1.2 before to 3.6 ± 1.3 12 weeks after the dose increase. Oral corticosteroid therapy ≥ 5 mg/day, previous use of two biologic agents, and DAS28 > 5.1 at initiation of GLM were significantly associated with discontinuation of the 50-mg regimen., Conclusions: Both GLM 50-mg and 100-mg regimens are effective in patients with RA in daily practice.

Objectives: To evaluate the cost-effectiveness of tocilizumab in patients with rheumatoid arthritis (RA) in a real-world setting in Japan., Methods: The cost-effectiveness was determined using a Markov model-based probabilistic simulation. Data from RA patients registered in the Institute of Rheumatology, Rheumatoid Arthritis (IORRA) cohort study between April 2007 and April 2011 were extracted using a pair-matching method: tocilizumab group (n = 104), patients who used at least 1 disease-modifying anti- rheumatic drug and in whom tocilizumab treatment was initiated; methotrexate group (n = 104), patients in whom methotrexate treatment was initiated for the first time or after an interruption of 6 or more months. Assuming a 6-month cycle length, health benefits and costs were measured over a lifetime and discounted at an annual rate of 3%., Results: Compared with methotrexate treatment, lifetime costs and quality-adjusted life years (QALYs) for tocilizumab treatment were approximately 1.5- and 1.3-times higher, respectively. Incremental cost per QALY gained with tocilizumab was $49,359, which was below the assumed cost-effectiveness threshold of $50,000 per QALY. The probability of tocilizumab being cost- effective was 62.2%., Conclusion: The simulation model using real-world data from Japan showed that tocilizumab (at a certain price) may improve treatment cost-effectiveness in patients with moderate-to-severe RA by enhancing quality-adjusted life expectancy.

Background/purpose: The use of biologic disease-modifying anti-rheumatic drugs (DMARDs) for rheumatoid arthritis (RA) has been increasing since 2003. In this study, we evaluated changes in the characteristics of patients receiving biologic DMARDs daily, in Japan., Methods: The characteristics of all RA patients who received any biologic DMARD at the Institute of Rheumatology, Tokyo Women's Medical University, within 1 year after its approval in Japan, were retrospectively evaluated. The periods of patient enrollment for each biologic agent were: infliximab (IFX), 2003-2004; etanercept (ETN), 2005-2006; tocilizumab (TCZ), 2008-2009; adalimumab (ADA), 2008-2009; abatacept (ABT), 2010-2011; and golimumab (GLM), 2011-2012. We retrospectively collected individual patient characteristics, concomitant medication usage, and disease activity assessed by disease activity score 28 (DAS28) at the time of administration, from the medical records. The retention rate for each agent at 6 months after treatment initiation was also assessed., Results: The numbers of patients who received each biologic DMARD at our institute within 1 year after its approval were: IFX, 49; ETN, 50; TCZ, 62; ADA, 52; ABT, 40; and GLM, 77. From 2003 to 2012, the proportion of patients with prior use of any biologic DMARD increased, as did concomitant use and dose of methotrexate (MTX); however, corticosteroid use and doses decreased. DAS28, at the time of treatment initiation, gradually decreased. At the time of IFX administration, 75% and 25% of patients had high and moderate disease activity respectively, compared to 25% and 58% respectively, of patients who received GLM. No significant difference was observed in the retention rate of biologic DMARDs at 6 months (range, 75.0% to 89.6%)., Conclusion: Baseline disease activity of RA patients who received biologic DMARDs between 2003 and 2012 has changed from high to moderate in daily practice in Japan.

Neurotensin (NT) has multiple functions, ranging from acting as a neurotransmitter to regulating intestinal movement. However, its function in reproductive physiology is unknown. Here, we confirmed the expression and localization of NT receptors (NTR1) in mouse epididymal spermatozoa and investigated the effect of NT on sperm function. Sperm protein tyrosine phosphorylation, one of the indices of sperm capacitation, was facilitated dose-dependently by NT administration. In addition, the acrosome reaction was promoted in capacitated spermatozoa, and addition of a selective antagonist of NTR1 and NTR2 blocked the induction. Furthermore, intracellular calcium mobilization by NT addition was observed. This showed that NT was an accelerator of sperm function via its functional receptors. The presence of NT was confirmed by immunohistochemistry and its localization was observed in epithelia of the uterus and oviduct isthmus and ampulla, which correspond to the fertilization route of spermatozoa. The NT mRNA level in ovulated cumulus cell was remarkably increased by treatment with human chorionic gonadotropin (hCG). Using an in vitro maturation model, we analyzed the effects of FSH, epidermal growth factor (EGF), estradiol, and progesterone in NT production in cumulus cells. We found that FSH and EGF upregulated NT release and mRNA expression. Both FSH- and EGF-induced upregulation were inhibited by U0126, an MAPK kinase inhibitor, indicating that FSH and EGF regulate NT expression via a MAPK-dependent pathway. This evidence suggests that NT can act as a promoter of sperm capacitation and the acrosome reaction in the female reproductive tract., (© 2014 by the Society for the Study of Reproduction, Inc.)

Objectives: To examine the effectiveness of tocilizumab (TCZ) in preventing joint destruction in patients with inadequate response to tumor necrosis factor inhibitors (TNF-IR) by assessing X-rays., Methods: RA patients were extracted from the Retrospective actemra investigation for optimal needs of RA patients (REACTION) study. Parameters and components of disease activity were evaluated during anti-TNF treatment and during TCZ treatment. X-ray images of hands and feet at the beginning of this study during anti-TNF treatment (Pre), at the start point of TCZ treatment (Baseline) and after TCZ treatment (Post) were collected for assessing joint destruction., Results: Forty-five patients from the REACTION study fulfilled the criteria of clinical TNF-IR. During anti-TNF treatment, mean DAS28-ESR rose from 5.35 to 5.87 (mean observation duration, 16 months) but improved significantly to 2.94 (P < 0.0001) at 52 weeks after switching to TCZ. Mean change in van der Heijde-modified Sharp score (TSS) during anti-TNF treatment was 3.17 in this TNF-IR population. After switching to TCZ, mean change in TSS was 1.20 (P < 0.05). Rate of radiographic non-progression improved to 66.7% during TCZ treatment from 40.0% during anti-TNF treatment. The predictive factor for no radiographic progression after switching to TCZ was a HAQ disability index (HAQ-DI) score of ≤ 1.88 at switching to TCZ., Conclusion: TCZ was a good treatment option for improving signs and symptoms and inhibiting progression of joint damage in patients with clinical and structural TNF-IR.

Migration and invasion of malignant glioma play a major role in tumor progression and can be increased by low doses of gamma or X-ray irradiation, especially when the migrated tumor cells are located at a distance from the main tumor mass or postoperative cavity and are irradiated in fractions. We studied the influence of proton beam irradiation on migration and invasion of human U87 malignant glioma (U87MG) cells. Irradiation at 4 and 8 Gy increased cell migration by 9.8% (±4, P = 0.032) and 11.6% (±6.6, P = 0.031) and invasion by 45.1% (±16.5, P = 0.04) and 40.5% (±12.7, P = 0.041), respectively. After irradiation at 2 and 16 Gy, cell motility did not differ from that at 0 Gy. We determined that an increase in proton beam irradiation dose to over 16 Gy might provide tumor growth control, although additional specific treatment might be necessary to prevent the potentially increased motility of glioma cells during proton beam therapy.

Objective: To analyze the relationship between the progression of disability and disease activity in patients with rheumatoid arthritis (RA) in daily practice., Methods: Patients from an observational cohort, IORRA, who completed surveys during 2009-2011 were eligible. Linear regression of disease activity score 28 (DAS28), Japanese version of Health Assessment Questionnaire (J-HAQ), and EQ-5D from baseline were calculated, and the angles of the regression lines were designated DAS28 slope, J-HAQ slope, and EQ-5D slope, respectively, in each patient; averages were compared between treatment groups., Results: A total of 5,038 patients [84.0% female, mean age 59.4 (SD 13.1) years, disease duration 13.2 (9.6) years, DAS28 3.29 (1.14), and J-HAQ 0.715 (0.760)] were analyzed. The average DAS28 slope indicated improvement in all groups, whereas J-HAQ slopes were negative in patients on methotrexate (MTX), biologics, combination biologics/disease-modifying antirheumatic drugs (DMARDs), and combination biologics/MTX at baseline, but positive in patients on prednisolone >5 mg/day [0.010 (0.153)] and not on MTX at baseline [0.007 (0.122)], representing a worsening of disability., Conclusion: There is some disparity between improvement of disease activity and progression of disability, suggesting that quality of remission must be considered.

Two puromycin-based techniques, in vitro virus (IVV) and C-terminal labelling of proteins, were developed based on the observation that puromycin binds the C-terminus of a protein. Puromycin technology is a useful tool for the detection of proteins and analysis of protein-protein interactions (PPIs); however, problems arise due to the existence of stop codons in the native mRNAs. Release factors (RFs) that enter the A-site of the ribosome at stop codons compete with puromycin. To overcome this issue, we have used a highly controllable reconstituted cell-free system for puromycin-based techniques, and observed efficient IVV formation and C-terminal labelling using templates possessing a stop codon. The optimal conditions of IVV formation using templates possessing a stop codon was RF (-), while that of C-terminal labelling was RF (-) and the ribosome recycling factor (RRF) (+). Thus, we have overcome the experimental limitations of conventional IVV. In addition, we discovered that RRF significantly increases the efficiency of C-terminal protein labelling, but not IVV formation.

Objectives: Our goal was to determine the annual direct medical and nonmedical costs for the care of patients with rheumatoid arthritis (RA) using data from a large cohort database in Japan., Methods: Direct medical costs [out of pocket to hospitals and pharmacies and for complementary and alternative medicine (CAM)] and nonmedical costs (caregiving, transportation, self-help devices, house modifications) were determined for RA patients who were participants in the Institute of Rheumatology, Rheumatoid Arthritis (IORRA) studies conducted in October 2007 and April 2008. Correlations between these costs and RA disease activity, disability level, and quality of life (QOL) were assessed., Results: Data were analyzed from 5,204 and 5,265 RA patients in October 2007 and April 2008, respectively. The annual direct medical costs were JPY132,000 [out of pocket to hospital (US$1 = JPY90 in 2007)], JPY84,000 (out of pocket to pharmacy), and JPY146,000 (CAM). Annual direct nonmedical costs were JPY105,000 (caregiving), JPY22,000 (transportation), JPY30,000 (self-help devices), and JPY188,000 (house modifications). Based on the utilization rate for each cost component, the annual medical and nonmedical costs for each RA patient were JPY262,136 and JPY61,441, respectively. Costs increased with increasing RA disease activity and disability level or worsening quality of life (QOL)., Conclusions: Based on the IORRA database, patients with RA bear heavy economic burdens that increase as the disease is exacerbated. The results also suggest that the increase in medical and nonmedical costs may be ameliorated by the proactive control of disease activity.

Objective: To investigate the point prevalence of major depressive disorder (MDD) as diagnosed by the Mini-International Neuropsychiatric Interview (M.I.N.I) in patients with rheumatoid arthritis (RA) and to determine whether MDD is related to features of RA disease, such as disease activity or physical dysfunction., Methods: Of the patients with RA who participated in the IORRA survey conducted in October 2005, 162 were evaluated using the M.I.N.I., the Center for Epidemiologic Studies-Depression (CES-D) scale, and the two-question depression screen for MDD. RA clinical features were obtained from the concomitant IORRA cohort database. Relationships between MDD and RA disease features were analyzed by the Wilcoxon rank sum test and Pearson's chi-square test., Results: The point prevalence of MDD as diagnosed by the M.I.N.I. was 6.8 % in our Japanese patients with RA. The percentage of depressive patients was determined to be 23.5, 17.3, or 7.4 % according to the CES-D scale with cut-off points of 16, 19, or 27, respectively, and 14.2 % according to the two-question depression screen. The best cut-off point for CES-D for risk of MDD diagnosed by M.I.N.I. in this study was determined to be 23, with 11.7 % depressive patients having the highest sum of sensitivity and specificity. No relationship between MDD and RA disease activity was detected., Conclusion: By using the well-established structural interview instrument M.I.N.I., we determined the point prevalence of MDD in the RA patients enrolled in this study to be 6.8 %, leading to the conclusion that concomitant MDD does not seem to influence disease activity in RA patients.

Background: In previous studies, we made the unexpected finding that in mice, ultraviolet (UV)B irradiation of the eye increased the concentration of α-melanocyte-stimulating hormone (α-MSH) in plasma, and systemically stimulated epidermal melanocytes., Aims: To compare the extent of the pigmentation induced by social and restraint stress (which activate the hippocampus-pituitary system) with that induced by UVB irradiation., Methods: DBA/2 and sham-operated or hypophysectomized DBA/2 mice were subjected to local UVB exposure using a sunlamp directed at the eye, and two types of stress (social and restraint) were imposed., Results: UVB irradiation of the eye or exposure to stress loading both increased the number of Dopa-positive melanocytes in the epidermis, and hypophysectomy strongly inhibited the UVB-induced and stress-induced stimulation of melanocytes. Irradiation of the eye caused a much greater increase in dopamine than did the stress load. Both UVB eye irradiation and stress increased the blood levels of α-MSH and adrenocorticotropic hormone (ACTH). In addition, the increase in plasma α-MSH was greater in animals subjected to UVB eye irradiation than in those subjected to stress loading, whereas the reverse occurred for plasma ACTH. UVB irradiation to the eye and stress loading increased the expression of prohormone convertase (PC)1/3 and PC2 in the pituitary gland. The increase in expression of pituitary PC2 was greater in animals subjected to UVB eye irradiation than to stress, whereas no difference was seen between the two groups for the increase in PC1/3., Conclusions: UVB eye irradiation exerts a stronger effect on pigmentation than stress loading, and is related to increased levels of α-MSH and PC2., (© The Author(s). CED © 2012 British Association of Dermatologists.)

Unlabelled: Protein-protein interactions (PPIs) are mediated through specific regions on proteins. Some proteins have two or more protein interacting regions (IRs) and some IRs are competitively used for interactions with different proteins. IRView currently contains data for 3417 IRs in human and mouse proteins. The data were obtained from different sources and combined with annotated region data from InterPro. Information on non-synonymous single nucleotide polymorphism sites and variable regions owing to alternative mRNA splicing is also included. The IRView web interface displays all IR data, including user-uploaded data, on reference sequences so that the positional relationship between IRs can be easily understood. IRView should be useful for analyzing underlying relationships between the proteins behind the PPI networks., Availability: IRView is publicly available on the web at http://ir.hgc.jp/

Although animal cloning is becoming more practicable, there are many abnormalities in cloned embryos, and the success rate of producing live animals by cloning has been low. Here, we focused on the procedure for preventing pseudo-second polar body extrusion from somatic cell nuclear transfer (SCNT)-derived oocytes. Typically, reconstructed oocytes are treated with cytochalasin B (CB), but here latrunculin A (LatA) was used instead of CB to prevent pseudo-second polar body extrusion by inhibiting actin polymerization. CB caps F-actin, LatA binds G-actin, and both drugs prevent their polymerization. When the localization of F-actin was examined using phalloidin staining, it was abnormally scattered in the cytoplasm of CB-treated 1-cell embryos, but this was not detected in LatA-treated or in vitro fertilization-derived control embryos. The spindle was larger in CB-treated oocytes than in LatA-treated or untreated control oocytes. LatA treatment also doubled the rate of full-term development after embryo transfer. These results suggest that cloning efficiency in mice can be improved by optimizing each step of the SCNT procedure. Moreover, by using LatA, we could simplify the procedure with a higher birth rate of cloned mice compared with our original method.

Neuroendocrine regulatory peptide (NERP)-3, derived from the neurosecretory protein VGF (non-aconymic), is a new biologically active peptide identified through peptidomic analysis of the peptides secreted by an endocrine cell line. Using a specific antibody recognizing the C-terminal region of NERP-3, immunoreactive (ir)-NERP-3 was identified in acid extracts of rat brain and gut as a 30-residue NERP-3 with N-terminal pyroglutamylation. Assessed by radioimmunoassay, ir-NERP-3 was more abundant in the brain, including the posterior pituitary (PP), than in the gut. Immunohistochemistry demonstrated that ir-NERP-3 was significantly increased in the suprachiasmatic nucleus, the magnocellular division of the paraventricular nucleus, and the external layer of the median eminence, but not in the supraoptic nucleus, after dehydration. The immunoreactivity was, however, markedly decreased in all of these locations after chronic salt loading. Intracerebroventricular administration of NERP-3 in conscious rats induced Fos expression in a subset of arginine vasopressin (AVP)-containing neurons in the supraoptic nucleus and the magnocellular division of the paraventricular nucleus. On in vitro isolated rat PP preparations, NERP-3 caused a significant AVP release in a dose-related manner, suggesting that NERP-3 in the PP could be an autocrine activator of AVP release. Taken together, the present results suggest that NERP-3 in the hypothalamo-neurohypophyseal system may be involved in the regulation of body fluid balance.

We aimed to demonstrate the incidence of serious respiratory infections in patients with rheumatoid arthritis (RA) treated with tocilizumab (TCZ) monotherapy. We analyzed the incidence of serious respiratory infections in 601 RA patients enrolled in TCZ clinical trials and their extension studies (TCZ cohort) and in 601 age- and sex-standardized RA patients treated in daily clinical practice at Tokyo Women's Medical University (IORRA subsample cohort). The rates of serious respiratory infections were 1.77 per 100 patient-years from 1999 to 2008 in the TCZ cohort and 0.53 per 100 patient-years from 2000 to 2009 in the IORRA subsample cohort. With the IORRA subsample cohort regarded as a standard population, the standardized incidence ratio (SIR) of serious respiratory infection in the TCZ cohort was 3.64 [95% confidence interval (CI) 2.56-5.01], standardized for age and sex; 2.35 (95% CI 1.66-3.24), standardized for age sex, and corticosteroid use; 1.85 (95% CI 1.30-2.55), standardized for age sex, and pre-existing pulmonary involvement; and 2.41 (95% CI 1.68-3.34) standardized for age sex, and disease activity. The risk of serious respiratory infection in the TCZ cohort was approximately double that in the IORRA subsample cohort after standardizing for corticosteroid use, pre-existing pulmonary involvement, or disease activity. This is comparable to the risk reported when tumor necrosis factor (TNF) inhibitors are used.

Irradiation by ultraviolet (UV)B is known to increase the number of dopamine (Dopa)-positive melanocytes in the skin. In this study, a 2.5-kJ/m(2) dose of UVB radiation was delivered by a sunlamp to the ear or the eye of wild-type C57BL/6j mice and of gp91 phox(-/-) C57BL/6j mice that had a knockout mutation of the gp91 phox subunit of reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH). The degree of change in the Dopa-positive melanocyte expression in was reduced in gp91 phox(-/-) mice given UVB irradiation to the eye, but not in those given irradiation to the ear. The plasma level of α-melanocyte-stimulating hormone (α-MSH) in the blood increased in the C57BL/6j mice after irradiation to either the eye or the ear, but it did not increase in the gp91 phox(-/-) mice given UVB irradiation to the eye. Both gp91 phox and α-MSH in the central nervous system seem to contribute to pigmentation after UVB irradiation of the eye in mice., (© The Author(s). CED © 2011 British Association of Dermatologists.)

The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.

We aimed to clarify the degree of improvement in disease control following early treatment of rheumatoid arthritis (RA) in daily clinical practice in 2006 compared to that in 2001. Using a large observational Japanese RA cohort (IORRA), we analyzed changes in clinical parameters, including disease activity assessed by the disease activity score 28 (DAS28) and physical disability assessed by the Japanese version of the Health Assessment Questionnaire (J-HAQ), which occurred within 2 years of cohort inception. All patients had enrolled in the IORRA cohort within 1 year of RA onset, in either 2001 (2001-cohort) or 2006 (2006-cohort). For both cohorts, changes in clinical features over 2 years were compared by Fisher's exact test or the Wilcoxon test. The 2001-cohort included 71 patients and the 2006-cohort included 56 patients. Over the 2-year period for each cohort, DAS28 significantly decreased from 3.9 to 3.5 in the 2001-cohort (p < 0.001) and from 4.1 to 3.1 in the 2006-cohort (p < 0.0001), and J-HAQ significantly decreased from 0.62 to 0.49 (p < 0.02) in the 2001-cohort and from 0.71 to 0.41 (p < 0.001) in the 2006-cohort. Greater improvement in disease activity over 2 years occurred in the 2006-cohort than in the 2001-cohort (p < 0.05). Better disease control was obtained following changes in RA treatment strategy that occurred in Japan between 2001 and 2006.

Aim: The arrhythmogenic relationship between the presence of J-waves during sinus rhythm and idiopathic ventricular tachycardia (VT) or pre-mature ventricular contractions (PVCs) originating from the right ventricular outflow tract (RVOT) has not been reported. The aim of this study was to investigate the prevalence and characteristics of idiopathic RVOT-VT/PVCs associated with J-waves., Methods and Results: The study enrolled 138 consecutive idiopathic RVOT-VT/PVC patients undergoing radiofrequency catheter ablation (RFCA) and 276 age- and gender-matched control subjects. The prevalence of J-waves was assessed in each cohort, and the clinical and electrophysiological data were compared between the RVOT-VT/PVC patients with J-waves (J-RVOT-VT/PVC group) and those without (non-J-RVOT-VT/PVC group). J-waves were more common among patients with idiopathic RVOT-VT/PVCs than among the matched control subjects (40 vs. 16% P < 0.001). The J-RVOT-VT/PVC group had a higher incidence of sustained VT (25 vs. 5%, P < 0.01), shorter VT cycle length (302 ± 57 vs. 351 ± 58 ms, P < 0.001), and more episodes of syncope (25 vs. 2%, P < 0.001) than did the non-J-RVOT-VT/PVC group. However, no patients demonstrated any ventricular fibrillation (VF) or cardiac sudden death in either group., Conclusions: There was a high prevalence of J-waves in the idiopathic RVOT-VT/PVC patients referred for RFCA. Although patients with idiopathic RVOT arrhythmias associated with J-waves might have a more enhanced arrhythmogenicity than those without J-waves, the significance of those J-waves was limited in terms of the prognosis and VF.

Objectives: To evaluate the effectiveness and safety of tocilizumab in RA patients in clinical practice., Methods: We observed 232 consecutive RA patients who began tocilizumab in three rheumatology centres in Japan for 52 weeks. Clinical, radiographic and functional status and safety were evaluated., Results: Mean age of the 232 patients was 59.1 years, mean duration of disease was 12.4 years and average DAS using the 28-joint count (DAS-28) was 5.6. Although 62.8% of the patients had been treated previously with anti-TNF biologics, clinical remission at Week 52 was achieved in 43.7%, radiographic non-progression in 62.8% and functional remission in 26.4%. Retention rate at Week 52 was 71.1%, and the same for those with or without previous anti-TNF treatment. Adverse drug reactions leading to tocilizumab discontinuation were observed in 15.5% of patients, the most frequent adverse drug reaction being pneumonia in eight cases. On multivariate logistic regression analysis, DAS-28, HAQ-disability index (HAQ-DI), concomitant MTX and concomitant glucocorticoids (GCs) were predictive variables for clinical remission at Week 52 of tocilizumab treatment. In particular, HAQ-DI was found to be a predictive variable for remission of all three types-clinical, radiographic and functional-at Week 52 of tocilizumab treatment., Conclusions: In daily clinical practice, tocilizumab exhibited excellent effectiveness in established RA patients, some of whom had failed to respond to previous anti-TNF treatment. Although further detailed safety findings are required, this study provides valuable real-world findings on the management of RA with tocilizumab.

The nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the xanthine oxidase (XOD) systems generate reactive oxygen species (ROS). In the present study, to characterize the difference between the two systems, the kinetics of ROS generated by both the NADH oxidase and XOD systems were analysed by an electron spin resonance (ESR) spin trapping method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide (DEPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). As a result, two major differences in ROS kinetics were found between the two systems: (i) the kinetics of (•)OH and (ii) the kinetics of hydrogen peroxide. In the NADH oxidase system, the interaction of hydrogen peroxide with each component of the enzyme system (NADPH, NADH oxidase and FAD) was found to generate (•)OH. In contrast, (•)OH generation was found to be independent of hydrogen peroxide in the XOD system. In addition, the hydrogen peroxide level in the NADPH-NADH oxidase system was much lower than measured in the XOD system. This lower level of free hydrogen peroxide is most likely due to the interaction between hydrogen peroxide and NADPH, because the hydrogen peroxide level was reduced by ~90% in the presence of NADPH.

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) has been cloned from mouse adrenocortical cells and is known to be closely associated with zonal differentiation of adrenocortical cells. In cell culture systems, TINAGL1 is a matricellular protein that interacts with both structural matrix proteins and cell surface receptors. However, the physiological roles of TINAGL1 and regulation of its expression are still not clearly understood. In the present study, the expression and localization of TINAGL1 in peri-implantation mouse embryos was examined. During preimplantation, the expression of both Tinagl1 mRNA and TINAGL1 protein was increased just prior to implantation. In blastocysts, TINAGL1 expression was localized to the trophectoderm. Using a progesterone-treated, delayed-implantation model, TINAGL1 was found to be upregulated in implantation-competent blastocysts after estrogen treatment. During postimplantation, TINAGL1 expression was restricted to extraembryonic regions. Marked expression was detected in the Reichert membrane on Embryonic Days 6.5 (E6.5) and E7.5. Colocalization of laminin 1 and TINAGL1 was also examined. Using an anti-LAMA1 antibody, colocalization of LAMA1 and TINAGL1 was observed in postimplantation embryos. Colocalization was also detected in the Reichert membrane. Immunoprecipitation analysis determined that LAMA1 and TINAGL1 interact in embryos on E7.5. These results demonstrate that after implantation, TINAGL1 is an extraembryonic tissue-specific protein. In particular, TINAGL1 is a novel component of the Reichert membrane that interacts with laminin 1. These results suggest that TINAGL1 most likely plays a physical and physiological role in embryo development at postimplantation.

In vitro antibody-display technologies are powerful approaches for isolating monoclonal antibodies from recombinant antibody libraries. However, these display techniques require several rounds of affinity selection which is time-consuming. Here, we combined mRNA display with a microfluidic system for in vitro selection and evolution of antibodies and achieved ultrahigh enrichment efficiency of 10(6)- to 10(8)-fold per round. After only one or two rounds of selection, antibodies with high affinity and specificity were obtained from naive and randomized single-chain Fv libraries of approximately 10(12) molecules. Furthermore, we confirmed that not only protein-protein (antigen-antibody) interactions, but also protein-DNA and protein-drug interactions were selected with ultrahigh efficiencies. This method will facilitate high-throughput preparation of antibodies and identification of protein interactions in proteomic and therapeutic fields.

CD44 on macrophages is recognized as a phagocytic receptor involved in the phagocytosis of apoptotic cells. Recently, we detected CD44 on macrophages in atretic follicles during atresia. In this study, we evaluated the distribution of the principal CD44 ligand hyaluronan (HA) and the expressions of HA synthases (HAS: HAS1, HAS2, and HAS3) during atresia in pig ovaries. We determined the 2139-bp sequence of Sus scrofa HAS1 and raised an anti-HAS1 polyclonal antibody. The S. scrofa HAS1 sequence contained six putative HA-binding motifs and conserved amino acid residues crucial for GlcNac transferase activity. HAS1 mRNA expression was upregulated during atresia; however, HAS2 and HAS3 mRNA expression levels were low and very low to undetectable, respectively. Western blotting showed that HAS1 was markedly upregulated during atresia. Immunohistochemical analyses revealed HAS1 distribution in theca cells of healthy and early atretic (stages I and II) follicles and in progressing atretic (stage III) follicles. Hyaluronan was visualized with the HA-binding protein; it accumulated in the theca layer during all stages and in stage III follicles. Hyaluronan assay showed a significantly increased HA concentration in follicular fluid at stage III. Flow cytometry showed HAS1 expression in 55.7% of SIRPA-positive macrophages in stage III follicles. Our results suggest that the HA concentration in follicular fluids increased during atresia and that HAS1 may be the dominant HAS protein in theca cells to produce HA in pig ovaries.

Prodigiosins are natural red pigments that have multi-biological activities. Recently, we discovered a marine bacterial strain, which produces a red pigment. Extensive two-dimensional nuclear magnetic resonance and mass spectrometry analysis showed that the pigment is a prodigiosin analogue (PG-L-1). Here, we investigated the effect of PG-L-1 on NADPH oxidase activity in macrophage cells. PG-L-1 significantly inhibited superoxide anion (O(2)(-)) production by phorbol 12-myristate 13-acetate (PMA)-stimulated RAW 264.7 cells, a mouse macrophage cell line. The ED(50) value was estimated to be approximately 0.3 microM. PG-L-1 had no direct scavenging effect on O(2)(-) generated by hypoxanthine/xanthine oxidase system in electron spin resonance-spin trapping determinations, suggesting that this compound directly acts on the O(2)(-) production system, NADPH oxidase, in macrophage cells. We further investigated the effect of PG-L-1 on the behaviour of the cytosolic components of the NADPH oxidase, p67(phox), p47(phox), p40(phox), Rac and protein kinase C (PKC), in PMA-stimulated RAW 264.7 cells. Although PG-L-1 showed no effect on the activation of PKC, the immunoblotting analysis using specific antibodies showed that PG-L-1 strongly inhibits the association of p47(phox) and Rac in the plasma membrane of PMA-stimulated RAW 264.7 cells. These results suggest that PG-L-1 inactivates NADPH oxidase through the inhibition of the binding of p47(phox) and Rac to the membrane components of NADPH oxidase.

Biologics targeting TNF have brought about a paradigm shift in the treatment of rheumatoid arthritis (RA) and infliximab, anti-TNF-alpha chimeric monoclonal antibody, was marketed in 2003 in Japan. We previously reported on the RECONFIRM study, a retrospective clinical study on the efficacy of infliximab therapy in a RA management group in Japan, where we evaluated the clinical response after 22 weeks of the therapy in 258 patients. The study reported here was aimed at reconfirming the clinical efficacy of the infliximab therapy and demographic factors related to the efficacy over a 54-week study period in 410 RA patients in the same study group. Infliximab was infused according to the domestically approved method, and the clinical response was evaluated following 54 weeks of infliximab therapy using the European League Against Rheumatism (EULAR) response criteria. Disease activity was assessed by DAS28-CRP (Disease Activity Score including a 28-joint count/C-reactive protein). Infliximab was discontinued in 24.4% of the 410 patients at 54 weeks and 9.3% and 8.1% discontinued the therapy due to adverse events and inefficiency, respectively. Average DAS28-CRP decreased from 5.5 at week 0 to 3.1 at week 54 after the therapy. Patients in remission and those showing low-, moderate-, and high-disease activity changed from 0.0, 1.0, 9.0 and 90.0%, respectively, at the start of the study to 27.6, 11.7, 34.4 and 26.3%, respectively, at week 54. Younger age, RF-negativity and low scores of DAS28-CRP showed significant correlations with remission at week 54. EULAR response criteria -- good, moderate, and no response to infliximab -- were 37.0, 41.7 and 21.2%, respectively. In conclusion, we reconfirmed the clinical efficacy of infliximab and demographic factors related to the efficacy over a 54-week study period in 410 Japanese patients with RA using DAS28-CRP and EULAR response criteria.

The anti-TNF-alpha chimeric monoclonal antibody infliximab is the first biologic to be approved for rheumatoid arthritis (RA) in Japan, and post-marketing surveillance of all of the Japanese cases treated with infliximab has been conducted to explore the safety of infliximab therapy. In addition, a retrospective clinical study on the notable efficacy and related factors of infliximab therapy in an RA management group in Japan (RECONFIRM and RECONFIRM-2) has demonstrated clinical responses. However, information on the effect of infliximab on joint destruction in Japanese RA patients remains insufficient. In this study, we retrospectively analyzed X-ray data from 67 patients in whom both hand and foot X-rays at baseline and at 54 weeks had been available among the 410 cases in the RECONFIRM-2 study. By scoring the X-rays according to the modified van der Heijde (vdH)-Sharp method, we found that the total vdH-Sharp score in the RA patients before infliximab therapy was 104.40+/-87.34 and the yearly progression was 21.33, indicating relatively rapid progression. After infliximab therapy for 54 weeks, the total vdH-Sharp score at 54 weeks was 104.37+/-86.87 and the estimated yearly progression was -0.03, indicating the almost complete inhibition of progression. The RECONFIRM-2J study confirmed the significant ability of infliximab to halt joint destruction in Japanese RA patients, and showed that joint destruction was significantly associated with disease activity and the dose of MTX in the patients with moderate and advanced disease durations, respectively, before infliximab therapy.

Introduction: Patients with systemic lupus erythematosus (SLE) have recognized reduction in endothelium-dependent vasodilation. Evidence demonstrates that statins are able to improve endothelial function independently on their hypolipemic action., Objectives: To evaluate the efficacy of atorvastatin in improving vasodilation in SLE patients with and without conventional risk factors for coronary heart disease (CHD)., Patients and Methods: Sixty-four SLE women, mean age 31 +/- 8 yrs, received atorvastatin 20 mg/day during 8 weeks. Thirty-one patients in this intervention group did not have conventional risk factors for CHD, while 33 others had hypertension, dyslipidaemia and/or obesity. Twenty-four SLE control patients, mean age 34 +/- 7.5 yrs, not receiving atorvastatin were followed during the same time period. High-resolution ultrasound was used to measure brachial artery diameter in resting conditions, during reactive hyperaemia and after sub-lingual glyceryl trinitrate (GTN). Measurements were performed at baseline and at the end of the study (8 weeks)., Results: Atorvastatin was associated with a significant increase in flow-mediated dilation (FMD) [3.8 (2.8-7.9%) vs 6.9 (4.2-10.7%), P < 0.001] while GTN-mediated dilation (GTND) was unaffected [20.9 (16.6-26.1%) vs 20.1(16.6-25.4%), P = 0.514]. FMD increase was observed in patients with conventional risk factors [4.1 (3.1-8.7%) vs 6.5 (4-10%), P = 0.046] and also for those without conventional risk factors for CHD [3.6 (2.6-7.3%) vs 7.1 (4.5-10.9%), P = 0.001]. Resting brachial artery diameter also increased significantly in patients receiving atorvastatin (2.79 +/- 0.30 mm vs 2.92 +/- 0.40 mm, P < 0.001). No significant difference in artery diameter and FMD was seen in control patients at the end of the study. When compared to the control patients, atorvastatin treatment was associated with significant increase in resting diameter (+0.13 +/- 0.1 mm vs -0.02 +/- 0.07 mm, P < 0.001) and FMD (+1.9 +/- 3.9% vs -0.3 +/- 1.8%, P = 0.009)., Conclusion: Our results demonstrate that an 8-week 20 mg/day atorvastatin series improved endothelium-dependent vasodilation in SLE patients independently on the presence of conventional risk factors for atherosclerotic disease.

In a previous study of ours, the superoxide scavenging activity of aqueous extracts from dinophycean red tide flagellates was detected by an electron spin resonance (ESR)-spin trapping method, but not by an L-012 (luminol analog)-dependent chemiluminescence (CL) method. To investigate the discrepancy between the two methods, the effect of ferric-protein complexes on superoxide scavenging activity was examined. The reduced signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OOH due to superoxide dismutase (SOD) did not change with the addition of horseradish peroxidase (HRP), while the reduced CL response due to SOD was restored by the addition of different concentrations of HRP. Since HRP is a ferric-protein complex, the effects of other ferric-protein complexes, catalase and hemoglobin, on the reduced CL response due to SOD were examined, and similar results were obtained. As is the case with SOD, the reduced CL response activity due to an aqueous extract from a raphidophycean red tide flagellate, Chattonella ovata, was also enhanced by HRP, catalase, and hemoglobin. ESR spectra analyzed at 77 K indicated that aqueous extracts of Gymnodinium impudicum and Alexandrium affine, both of which are dinophycean red tide flagellates, contained a ferric-protein complex, and that an extract of C. ovata did not. These results suggest that the presence of such a ferric-protein complex is a causative factor in the discrepancy between the ESR and luminol CL methods when determining superoxide scavenging activity.

The antioxidant properties of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined. An electron spin resonance (ESR)-spin trapping method coupled with steady state kinetic analysis showed that all of the extracts directly scavenge superoxide, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. As for hydroxyl radical scavenging, the Fenton reaction and the method of ultraviolet radiation to hydrogen peroxide were used as hydroxyl radical generation systems. All of extracts reduced the level of hydroxyl radicals in both of the systems, indicating that the extracts also directly scavenge hydroxyl radicals. Since the levels of phenolic compounds did not correlate with the antioxidant activities of the extracts, substances other than phenolic compounds also appeared to be attributable to the activities. It is of our interest that the scavenging activities of extract from G. impudicum against superoxide and hydroxyl radicals were increased by heat exposure at 100 degrees C and 200 degrees C respectively. Although the reason for the increased activities of the aqueous extract from G. impudicum is not clear, the heat-resistance of the extract from G. impudicum might make it a desirable antioxidant.

Background: Reactive oxygen species (ROS) released from inflammatory cells constitute one of the critical causative factors in inflammatory skin diseases such as seborrhoeic dermatitis and atopic dermatitis., Objectives: To investigate inhibitory effects of ketoconazole (KCZ) and ciclopiroxolamine (CPO), both of which have been used for the treatment of seborrhoeic dermatitis, on ROS released from inflammatory cells., Methods: The methyl-Cypridina-luciferin analogue-dependent chemiluminescence method was employed for the detection of ROS production by phorbol 12-myristate 13-acetate (PMA)-stimulated inflammatory cells. Moreover, the radical scavenging activities of both agents were examined by using a hypoxanthine-xanthine oxidase system and the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). NADPH oxidase activity was determined in particulate (membrane) fractions prepared from PMA-stimulated RAW 264 x 7 cells, a macrophage-like cell line., Results: Both of these antifungal agents inhibited PMA-stimulated ROS production. However, only CPO significantly scavenged both ROS generated by the hypoxanthine-xanthine oxidase system and DPPH, and the scavenging activity of CPO seemed to act on ROS other than superoxide anions. Although KCZ inhibited PMA-stimulated ROS production, it did not show radical-scavenging activities. The inhibition of ROS production by KCZ is probably attributable to the inhibition of NADPH oxidase activity., Conclusions: The mechanism of the inhibitory action of KCZ against PMA-stimulated ROS production is distinct from that of CPO. Knowledge of the inhibitory or scavenging effects of both antifungal agents on ROS released from inflammatory cells may be useful in developing a therapeutic strategy for dermatitis.

Recent studies indicate that some raphidophycean red tide flagellates produce substances able to scavenge superoxide, whereas there have been no reports on superoxide scavenger production by dinophycean red tide flagellates. In this study, we examined the superoxide-scavenging activity of aqueous extracts from dinophycean red tide flagellates, Gymnodinium spp., Scrippsiella trochoidea, and Karenia sp., by a luminol analog L-012-dependent chemiluminescence (CL) method and an electron spin resonance (ESR)-spin trapping method, and compared the activity to that of raphidophycean red tide flagellates, Chattonella spp., Heterosigma akashiwo, and Fibrocapsa japonica. In the experiment applying the L-012-dependent CL method, only the aqueous extracts from raphidophycean red tide flagellates showed superoxide-scavenging activity. On the other hand, applying the ESR-spin trapping method, we found that the aqueous extracts from dinophycean red tide flagellates also showed superoxide-scavenging activity. This is the first report on the production of a superoxide-scavenger by dinophycean red tide flagellates.

Macrophages are essential in cleaning up apoptotic debris during follicular atresia. However, the key factors of this process are still unclear. In the present study, we evaluated CD44 mRNA, CD44 protein, and CD44 antigen glycosylation on macrophages during follicular atresia in the pig. Atresia was classified into five stages: stage I, healthy follicles; stage II, early atretic follicles having apoptotic granulosa cells with an unclear basement membrane; stage III, progressing atretic follicles having apoptotic granulosa cells completely diffused from the basement membrane; stage IV, late atretic follicles with increasing lysosomal activity; and stage V, disintegrated atretic follicles having collapsed theca cells and strong lysosomal activity. Immunohistological analysis showed that macrophages expressing CD44 invaded the inside of stage III follicles, accompanied by a collapse of basement membrane. Semiquantitative RT-PCR showed that only mRNA of the CD44 standard isoform (CD44s) was present in inner cells of follicles, and not any CD44 variant isoform (CD44v) mRNAs. The amount of CD44s mRNA was increased at stage III. Western blot and lectin blot analyses showed that CD44 was markedly expressed at stage III and glycosylated with polylactosamine at the same time. After macrophages invaded atretic follicles at stages III-V, the CD44 expressed on macrophages was glycosylated with polylactosamine. The lysosomal activity began to increase at stage IV, and reached the highest level at stage V. Increased CD44s protein and posttranslational modification of CD44 with polylactosamine on macrophages from stage III could be involved in the cleaning up apoptotic granulosa cells.

Comprehensive analysis of DNA-protein interactions is important for mapping transcriptional regulatory networks on a genome-wide level. Here we present a new application of mRNA display for in vitro selection of DNA-binding protein heterodimeric complexes. Under improved selection conditions using a TPA-responsive element (TRE) as a bait DNA, known interactors c-fos and c-jun were simultaneously enriched about 100-fold from a model library (a 1:1:20 000 mixture of c-fos, c-jun and gst genes) after one round of selection. Furthermore, almost all kinds of the AP-1 family genes including c-jun, c-fos, junD, junB, atf2 and b-atf were successfully selected from an mRNA display library constructed from a mouse brain poly A(+) RNA after six rounds of selection. These results indicate that the mRNA display selection system can identify a variety of DNA-binding protein complexes in a single experiment. Since almost all transcription factors form heterooligomeric complexes to bind with their target DNA, this method should be most useful to search for DNA-binding transcription factor complexes.

We report the first case of pseudogout attack in the distal interphalangeal (DIP) joints during etidronate disodium therapy. A 64-year-old woman had intermittent administration of etidronate disodium (Didronel; Sumitomo, Osaka, Japan) alone because of osteoporosis. Each cycle consisted of a daily dose of 200 mg for 2 weeks, repeating every 12 weeks. Two weeks after completing the third cycle, severe pain and swelling occurred in the DIP joints of the right middle, ring, and left ring finger; and skin ulcer formation was observed on the dorsal side of the DIP joints of the right middle and ring fingers as well as the left ring finger. Because monoclinic calcium pyrophosphate crystals were detected in the synovial fluid from the DIP joints of the right middle finger, we diagnosed these symptoms as induced by pseudogout attack. Oral loxoprofen sodium at a daily dose of 180 mg resulted in rapid symptom resolution. A decrease in function of calcium metabolism in elderly persons has been reported to be a cause of pseudogout attack. On the other hand, distal interphalangeal joint arthritis presenting as Heberden's nodes is a common condition in elderly patients. Therefore, pseudogout attack should be considered as an adverse drug reaction when administering bisphosphonate in elderly patients with Heberden's nodes.

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein-protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein-protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.

Here we describe the application of the in vitro virus mRNA display method, which involves covalent linkage of an in vitro-synthesized antibody (phenotype) to its encoding mRNA (genotype) through puromycin, for in vitro evolution of single-chain Fv (scFv) antibody fragments. To establish the validity of this approach to directed antibody evolution, we used random mutagenesis by error-prone DNA shuffling and off-rate selection to improve the affinity of an anti-fluorescein scFv as a model system. After four rounds of selection of the library of mRNA-displayed scFv mutants, we obtained six different sequences encoding affinity-matured mutants with five consensus mutations. Kinetic analysis of the mutant scFvs revealed that the off-rates have been decreased by more than one order of magnitude and the dissociation constants were improved approximately 30-fold. The antigen-specificity was not improved by affinity maturation, but remained similar to that of the wild type. Although the five consensus mutations of the high-affinity mutants were scattered over the scFv sequence, analysis by site-directed mutagenesis demonstrated that the critical mutations for improving affinity were the two that lay within the complementarity determining regions (CDRs). Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.

To improve the feasibility of nylon-mesh holder for vitrification of bovine cumulus-oocytes complexes (GV-COCs) having germinal vesicle, this study was conducted to demonstrate effects of sugars and protocol of exposure in vitrification on subsequent in vitro maturation, ultrastructural changes, and in vitro development in bovine immature oocytes after cryopreservation using nylon mesh. Before vitrification, GV-COCs were exposed to the cryoprotectant, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40), either by single step or in a stepwise way. The maturation rates in the stepwise exposure with EFS40 or EFT40 were significantly higher (P < 0.05) compared with the corresponding rates in the single step. In the stepwise exposure, few abnormalities were observed compared with the single-step exposure, where most oocytes showed a highly vacuolated cytoplasm with many ruptured mitochondria. Cleavage rates in fertilized oocytes previously exposed stepwise to EFS40 or EFT40 were significantly higher than those exposed by the single-step procedure. The cleaved embryos derived from the stepwise exposure to EFS40 developed to blastocysts. After transfer of blastocysts derived from vitrified GV oocytes, a female calf was born. These results indicate that vitrification of large numbers of bovine GV-COCs using a nylon-mesh holder accompanied with stepwise exposure minimizes structural damage in organelles, resulting in yield of viable blastocysts following in vitro embryo production.