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Biosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis . Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity., (© The Author(s) 2024. Published by Oxford University Press.)

Ribosomal DNA (rDNA) repeat units are organized into tandem clusters in eukaryotic cells. In mice, these clusters are located on at least eight chromosomes and show extensive variation in the number of repeats between mouse genomes. To analyze intra- and inter-genomic variation of mouse rDNA repeats, we selectively isolated 25 individual rDNA units using Transformation-Associated Recombination (TAR) cloning. Long-read sequencing and subsequent comparative sequence analysis revealed that each full-length unit comprises an intergenic spacer (IGS) and a ∼13.4 kb long transcribed region encoding the three rRNAs, but with substantial variability in rDNA unit size, ranging from ∼35 to ∼46 kb. Within the transcribed regions of rDNA units, we found 209 variants, 70 of which are in external transcribed spacers (ETSs); but the rDNA size differences are driven primarily by IGS size heterogeneity, due to indels containing repetitive elements and some functional signals such as enhancers. Further evolutionary analysis categorized rDNA units into distinct clusters with characteristic IGS lengths; numbers of enhancers; and presence/absence of two common SNPs in promoter regions, one of which is located within promoter (p)RNA and may influence pRNA folding stability. These characteristic features of IGSs also correlated significantly with 5'ETS variant patterns described previously and associated with differential expression of rDNA units. Our results suggest that variant rDNA units are differentially regulated and open a route to investigate the role of rDNA variation on nucleolar formation and possible associations with pathology., (Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics 2024.)

We focused on Piper longum L., a herbal drug produced in Myanmar, which has a renoprotective effect. Thus, we attempted to isolate and identify compounds that enhance the expression of the ABCG2 gene from the aerial parts of the plant except for the fruit. Among the various P. longum extracts, we isolated and identified the components. Using Caco-2 cells, the hABCG2 mRNA expression-enhancing effects of the isolated compounds were compared with the positive reference compound (3-methylcholanthrene [3MC]) using real-time polymerase chain reaction. Six compounds were isolated and identified from the methanol extract of P. longum. Among the isolated compounds, licarin A and neopomatene had lower toxicity and higher hABCG2 mRNA expression-enhancing effects in Caco-2 cells. Suppression of hAhR expression by siRNA reduced the activity of licarin A and neopomatene, as well as the hAhR agonist 3MC, suggesting that these 2 compounds may act as hAhR agonists to promote hABCG2 expression., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Introduction: Metastases from renal cell carcinoma develop in various organs. However, the breadth of discrepancy in response to immune checkpoint inhibitors across tumor sites within the same individual remains unclear., Patients and Methods: We reviewed 50 patients with metastatic renal cell carcinoma who had target lesions at multiple sites and received nivolumab monotherapy (n = 36) or nivolumab plus ipilimumab (n = 14). When the best overall response in tumor burden increased at one site but decreased at other sites, the response was defined as a dissociated response. The response was evaluated according to the Response Evaluation Criteria in Solid Tumors 1.1, and patients who met the definition of dissociated response were categorized as dissociated response. The rate of dissociated response and prognosis were evaluated., Results: Eight of 36 (22%) and 4 of 14 (29%) patients treated with nivolumab and nivolumab plus ipilimumab were categorized as having dissociated response, respectively. The median overall survival of the patients treated with nivolumab was 20.2 months for those with a partial response, 6.8 months for those with stable disease, and 13.2 months for those with progressive disease, while dissociated response was not reached. There was no significant difference in the median overall survival between patients categorized as having progressive disease and those with dissociates response (P = 0.224)., Conclusion: A certain proportion of patients with metastatic renal cell carcinoma show dissociated response when treated with immune checkpoint inhibitors. The prognosis of patients with dissociated response and progressive disease was not shown to be significantly different., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Several studies have demonstrated that low back pain (LBP) is related to disease activity in patients with rheumatoid arthritis (RA). However, there is no longitudinal research. This study aimed to determine the impacts and risk factors for LBP increase in RA in a longitudinal cohort study., Methods: The study evaluated 113 patients with RA who completed the secondary survey. LBP increase was defined as ≥1 standard deviation of mean change in visual analogue scale (VAS) between the baseline and secondary surveys. The impacts of LBP increase on quality of life (QOL) and psychological status were evaluated. Risk factors were assessed among patient demographic characteristics and radiological changes., Results: Mean change in VAS for LBP was -0.8 ± 30.4 mm during a mean 7-year follow-up. LBP increase was defined as ≥30-mm increase in VAS for LBP. Patients with LBP increase had significantly lower QOL and worse mental status than patients without it. Poor control of RA was identified as an independent risk factor for LBP increase (odds ratio, 9.82, p = .001)., Conclusion: Patients with poor control of RA were likely to experience LBP increase in the long term. Control of RA disease activity is important for control of LBP, QOL, and mental status., (© Japan College of Rheumatology 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Total arch replacement (TAR) using an endovascular approach has been initially introduced as the frozen elephant trunk technique (FET). In our institute, TAR using the FET with Frozenix has been used as the first-line treatment for distal aortic arch aneurysms since 2014. This study aimed to evaluate the early and long-term outcomes and demonstrate the efficacy of this procedure., Methods: Between 2014 and 2021, 121 consecutive patients were treated with TAR using the FET with Frozenix for distal aortic arch aneurysms. Early and long-term outcomes were retrospectively analysed., Results: The 30-day mortality rate was 2.5% (3/121). Of postoperative complications, paraplegia due to spinal cord injury occurred in 2 (1.7%) patients, stroke in 12 (9.9%) and acute renal failure in 10 (8.3%). At follow-up, 23 secondary aortic interventions were required and 8 (6.6%) patients underwent intended secondary thoracic endovascular aortic repair for residual descending aortic aneurysm. Late and aortic-related deaths occurred in 16 (13.2%) and 4 (3.3%) patients, respectively. The overall long-term survival rates at 1, 3 and 5 years were 87.6%, 83.1% and 65.4%, respectively, while the rates of freedom from aortic-related death at 1, 3 and 5 years were 95.7%, 95.7% and 84.8%, respectively., Conclusions: TAR using the FET with Frozenix for distal aortic arch aneurysms has acceptable early mortality and morbidity. Spinal cord injury and paraplegia occur less frequently than previously reported. The technique has satisfactory long-term survival and freedom from aortic-related death., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery.)

Frontoparietal networks contribute to complex cognitive functions in humans and macaques, such as working memory, attention, task-switching, response suppression, grasping, reaching, and eye movement control. However, there has been no comprehensive examination of the functional organization of frontoparietal networks using functional magnetic resonance imaging in the New World common marmoset monkey (Callithrix jacchus), which is now widely recognized as a powerful nonhuman primate experimental animal. In this study, we employed hierarchical clustering of interareal blood oxygen level-dependent signals to investigate the hypothesis that the organization of the frontoparietal cortex in the marmoset follows the organizational principles of the macaque frontoparietal system. We found that the posterior part of the lateral frontal cortex (premotor regions) was functionally connected to the anterior parietal areas, while more anterior frontal regions (frontal eye field [FEF]) were connected to more posterior parietal areas (the region around the lateral intraparietal area [LIP]). These overarching patterns of interareal organization are consistent with a recent macaque study. These findings demonstrate parallel frontoparietal processing streams in marmosets and support the functional similarities of FEF-LIP and premotor-anterior parietal pathways between marmoset and macaque., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Amyloid formation and the deposition of the amyloid-β peptide are hallmarks of Alzheimer's disease pathogenesis. Immunotherapies using anti-amyloid-β antibodies have been highlighted as a promising approach for the prevention and treatment of Alzheimer's disease by enhancing microglial clearance of amyloid-β peptide. However, the efficiency of antibody delivery into the brain is limited, and therefore an alternative strategy to facilitate the clearance of brain amyloid is needed. We previously developed an artificial photo-oxygenation system using a low molecular weight catalytic compound. The photocatalyst specifically attached oxygen atoms to amyloids upon irradiation with light, and successfully reduced the neurotoxicity of aggregated amyloid-β via inhibition of amyloid formation. However, the therapeutic effect and mode of actions of the photo-oxygenation system in vivo remained unclear. In this study, we demonstrate that photo-oxygenation facilitates the clearance of aggregated amyloid-β from the brains of living Alzheimer's disease model mice, and enhances the microglial degradation of amyloid-β peptide. These results suggest that photo-oxygenation may represent a novel anti-amyloid-β strategy in Alzheimer's disease, which is compatible with immunotherapy., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: We evaluated the operative and long-term outcomes of the frozen elephant trunk (FET) technique for acute type A aortic dissection., Methods: This study evaluated 426 consecutive patients who underwent aortic repair for acute type A aortic dissection from June 2007 to December 2018 at our centre. Of these, 139 patients underwent total arch replacement with FET (FET group), and 287 underwent other procedures (no FET group). Ninety-two patients in the FET group were matched to 92 patients in the no FET group by using propensity score matching analysis., Results: Thirty-day mortality and neurological dysfunction were not significantly different between the FET and no FET groups (1.4% vs 2.4%, P = 0.50 and 5.0% vs 6.3%, P = 0.61, respectively). Long-term survival was better in the FET group than in the no FET group (P = 0.008). Freedom from distal thoracic reintervention was similar in the FET and no FET groups (P = 0.74). In the propensity-matched patients, freedom from aortic-related death was better in the FET group than in the no FET group (P = 0.044)., Conclusions: Operative outcomes showed no significant difference between the 2 groups. FET contributes to better long-term survival in patients with acute type A aortic dissection., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

The common marmoset (Callithrix jacchus) is a New World primate that is becoming increasingly popular as a preclinical model. To assess functional connectivity (FC) across the marmoset brain, resting-state functional MRI (RS-fMRI) is often performed under isoflurane anesthesia to avoid the effects of motion, physiological stress, and training requirements. In marmosets, however, it remains unclear how isoflurane anesthesia affects patterns of FC. Here, we investigated the effects of isoflurane on FC when delivered with either medical air or 100% pure oxygen, two canonical methods of inhalant isoflurane anesthesia delivery. The results demonstrated that when delivered with either medical air or 100% oxygen, isoflurane globally decreased FC across resting-state networks that were identified in awake marmosets. Generally, although isoflurane globally decreased FC in resting-state networks, the spatial structure of the networks was preserved. Outside of the context of RS networks, we indexed pair-wise functional connectivity between regions across the brain and found that isoflurane substantially altered interhemispheric and thalamic FC. Taken together, these findings indicate that RS-fMRI under isoflurane anesthesia is useful to evaluate the global structure of functional networks, but may obfuscate important nodes of some network components when compared to data acquired in fully awake marmosets., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

We report a case of pulmonary artery aneurysm and pulmonary stenosis with a quadricuspid pulmonary valve in a 64-year-old woman. While pulmonary artery disease had been found as a child, she needed no treatment because of the absence of symptoms and her normal physical development. She began experiencing dyspnoea on exertion 3 years ago. Echocardiography showed pulmonary artery aneurysm and severe pulmonary stenosis with a quadricuspid valve. Computed tomography showed a pulmonary artery aneurysm extending from its main trunk to the bilateral branches. The pulmonary valve was replaced, and the pulmonary trunk and its bilateral branch were reconstructed. The patient's postoperative course was uneventful., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada-/- and Sadb-/- double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada-/- mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada-/- mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada-/- mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Labelling technologies developed over the past few years have changed the way of looking at biomolecules and have made a considerable contribution to our understanding of the functions and regulation of dynamic biological processes. One of the robust technologies employed to image proteins in a cellular environment is based on the use of chemical tags and their fluorescent probes, which provides flexibility in developing probes with a wide range of synthetic fluorophores. A variety of chemical tags, ranging from short amino acid sequences to small proteins, have been employed to generate protein-labelling systems. One such chemical tag is the photoactive yellow protein (PYP)-tag, which is a small bacterial protein, developed for the selective labelling and imaging of proteins. Herein, we briefly discuss the protein-labelling system developed based on PYP-tag technology, with a focus on the design strategy for PYP-tag labelling probes and their applications in protein imaging., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Objectives: This study aimed to assess differences in midterm outcomes between total arch replacement (TAR) and debranching thoracic endovascular aortic repair (d-TEVAR) and to evaluate the validity of d-TEVAR as the preferred treatment choice for aortic arch aneurysm in the elderly., Methods: We reviewed the case histories of 86 patients who had undergone TAR (64 men; mean age 78 ± 2.9 years) and 121 patients who had undergone d-TEVAR (90 men; mean age 82 ± 4.5 years) between 2007 and 2017; of these patients, 50 from each group were matched based on propensity scores to adjust for differences in patient characteristics., Results: Rates of freedom from all-cause mortality at 2 and 4 years were similar between the 2 groups (88% and 77% in the TAR group vs 82% and 64% in the d-TEVAR group, P = 0.11), but rates of freedom from reintervention at 2 and 4 years were significantly higher in the TAR group (100% and 96%) than in the d-TEVAR group (97% and 88%) (P = 0.004). Propensity score matching yielded similar survival rates of 88% and 85% for TAR vs 86% and 71% for d-TEVAR (P = 0.53) and comparable freedom from reintervention rates (100% and 97% in TAR, 98% and 90% in d-TEVAR, P = 0.16) at 2 and 4 years. Cox regression analysis identified previous cerebral infarction [hazard ratio (HR) 3.9; P = 0.005 in TAR/HR 3.1; P = 0.002 in d-TEVAR] as an independent positive predictor of overall mortality in both groups., Conclusions: Midterm outcomes after TAR and d-TEVAR were satisfactory and propensity score matching-based evaluation revealed no significant differences in outcomes, implying that d-TEVAR is an acceptable first-choice procedure for aortic arch aneurysm in patients older than 75 years., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

The innate electric potentials in biased p- and n-type GaAs compound semiconductors and the built-in potential were successfully measured with high accuracy and precision by applying in situ phase-shifting electron holography to a wedge-shaped GaAs specimen. A cryo-focused-ion-beam system was used to prepare the 35°-wedge-shaped specimen with smooth surfaces for a precise measurement. The specimen was biased in a transmission electron microscope, and holograms with high-contrast interference fringes were recorded for the phase-shifting method. A clear phase image around the p-n junction was reconstructed even in a thick region (thickness of ~700 nm) at a spatial resolution of 1 nm and precision of 0.01 rad. The innate electric potentials of the unbiased p- and n-type layers were measured to be 12.96 ± 0.17 V and 14.43 ± 0.19 V, respectively. The built-in potential was determined to be 1.48 ± 0.02 V. In addition, the in situ biasing measurement revealed that the measured electric-potential difference between the p and n regions changed by an amount equal to the voltage applied to the specimen, which indicates that all of the external voltage was applied to the p-n junction and that no voltage loss occurred at the other regions., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To assess the characteristics of biochemical recurrence in the late period (>5 years after radical prostatectomy) and the differences in the predictors of biochemical recurrence in different periods, we conducted a multicenter retrospective study., Methods: We reviewed 478 men who underwent radical prostatectomy for clinically localized prostate cancer. All of the patients were followed up for at least 5 years. The cohort was then divided into three groups; no recurrence group, recurrence <5 years after surgery group and recurrence ≥5 years after surgery group. The background characteristics of each group were compared using the χ2 test. A Cox multivariate regression analysis was performed to determine the predictors of biochemical recurrence in each period., Results: Biochemical recurrence occurred in 135 men. In 113 (84%) of the patients, biochemical recurrence occurred at <5 years after surgery; in 22 (16%), it occurred at ≥5 years after surgery. The proportion of men with a low preoperative prostate-specific antigen level was significantly larger in the latter group (P = 0.0023). A preoperative prostate-specific antigen level and a positive surgical margin were significant predictors of biochemical recurrence at <5 years after surgery (hazard ratio: 1.03 and 3.20). A positive surgical margin was also a significant predictor of biochemical recurrence at ≥5 years after surgery (hazard ratio: 3.03); however, a high preoperative prostate-specific antigen level was not., Conclusions: Biochemical recurrence occurred at ≥5 years after surgery in 16% of the patients. A positive surgical margin predicted biochemical recurrence in both the early and late periods., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Objective: To evaluate long-term clinical outcomes in cT1c-T3a prostate cancer patients following delayed-combined androgen blockade therapy., Methods: From January 2001 to December 2004, 92 cT1c-T3a prostate cancer cases were enrolled. Medical castration and anti-androgen treatment were used sequentially as delayed-combined androgen blockade therapy. Time to prostate-specific antigen biochemical failure was estimated, and risk factors for prostate-specific antigen biochemical failure were evaluated., Results: The average patient age was 76.4 years (range, 59-91 years), the median observation period was 52.8 months (range, 26-106.6 months) and the median pre-treatment prostate-specific antigen level was 14 ng/ml (range, 3.68-492 ng/ml). The TNM classification distribution was as follows: T1c, n= 27; T2a, n = 39; T2b, n = 20; and T3a, n = 6. In the multivariate analysis, Gleason's score ≥8 (P < 0.05; hazard ratio, 3.02), prostate-specific antigen nadir >1.4 ng/ml (P = 0.001; hazard ratio, 8.76) and a half-life of the prostate-specific antigen level >1.2 months (P < 0.005; hazard ratio, 6.3) during the initial 6 months of luteinizing hormone-releasing hormone agonist monotherapy were significant independent risk factors for prostate-specific antigen biochemical failure with luteinizing hormone-releasing hormone agonist monotherapy. The high-risk group, which had at least one of these three risk factors, had a shorter time to prostate-specific antigen biochemical failure than the low-risk group, during luteinizing hormone-releasing hormone agonist monotherapy (P < 0.0001). For the total delayed-combined androgen blockade therapy observation period, the free-prostate-specific antigen biochemical failure rate was 88.3% at 5 years. Only a maintenance period following luteinizing hormone-releasing hormone agonist monotherapy (P < 0.005; hazard ratio, 16.8) was revealed to be a significant independent risk factor for prostate-specific antigen biochemical failure with total delayed-combined androgen blockade., Conclusions: The free-prostate-specific antigen biochemical failure rate of delayed-combined androgen blockade therapy in our study was as valuable as those in other androgen deprivation therapy of previous reports.

No conservative treatments for chylothorax have yet been established, and surgical ligation of the thoracic duct is required in many cases. In the present study, we investigated the management of body fluid in a canine chylothorax model. Twelve beagle dogs were divided evenly into three groups: A, B, and C. Under general anesthesia, the thoracic duct was cut and opened, and the amount of lymph fluid leakage was measured. Intravenous extracellular fluid infusion was started at 5mL/kg/h for the first 2h, and then between 2 and 4h, the infusion rate was increased to 10 mL/kg/h in group A and to 20mL/kg/h in group B. During the first 2h after cutting the thoracic duct, the mean lymph fluid leakage rates in groups A, B, and C were 0.466, 0.635, and 0.575 mL/kg/h, respectively. The rates of leakage did not differ significantly among the groups. Between 2 and 4h, the mean rates of leakage were 0.750, 1.43, and 0.544mL/kg/h, respectively, being significantly higher in groups A and B than in group C. The amount of lymph fluid ascending the thoracic duct correlates with the amount of intravenous fluid infusion. For the management of chylothorax, it is important to avoid fluid overload., (© 2011 Copyright the Authors. Journal compilation © 2011, Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.)

Objectives: We evaluated the durability of aortic valve preservation with root reconstruction for acute type A aortic dissection (AAAD)., Methods: From January 2002 to March 2011, 140 patients [70 males, 68 ± 12 (SD) years] underwent emergency operation for AAAD. The aortic valve was preserved and one or more Valsalva sinuses were reconstructed. Techniques used for reconstruction were valve resuspension and additional reinforcement of the aortic root with Teflon felt patches, and gelatin-resorcinol-formaldehyde-glue (GRF-glue) was used for mending the dissection. The mean follow-up period was 44.0 ± 26.2 months. We classified the degree of aortic regurgitation (AR) into four grades (0, 1+, 2+ and 3+) using echocardiography. Based on a retrospective analysis of pre-operative echocardiographic findings, the 127 survivors were divided into two groups: group 1 (G1) included 98 patients with 0 or 1+ AR, and group 2 (G2) 29 patients with 2+ or 3+ AR. In addition, we measured the post-operative native aortic root dimension of AAAD patients with use of echocardiography or CT scan., Results: The operative mortality rate was 9.3% (13/140). Freedom from aortic root re-operation was 100%. Aortic root pseudoaneurysm formation and severe AR requiring aortic valve replacement did not occur. Pre-operative AR of 0.2 ± 0.4 in G1 did not deteriorate (0.5 ± 0.5 at discharge, 0.4 ± 0.4 at follow-up). Meanwhile, pre-operative AR of 2.4 ± 0.5 in G2 improved to 0.6 ± 0.5 (P < 0.05) at discharge and 1.0 ± 0.6 (P < 0.05) at follow-up. The native aortic root dimension in G2 at follow-up was significantly larger than G1 (36.0 ± 4.7 vs. 33.9 ± 5.0 mm)., Conclusions: Aortic valve preservation and root reconstruction appear to be an appropriate surgical approach to AAAD.

Background: Particle radiotherapy is a novel treatment for malignant tumours. The present study aimed to evaluate risk factors for overall survival and local control after particle radiotherapy of single small hepatocellular carcinoma (HCC), and to identify suitable candidates for this treatment., Methods: All patients with a single HCC smaller than 5 cm in diameter treated by particle radiotherapy between 2001 and 2008 were identified retrospectively from a prospectively collected database. Clinical outcomes and prognostic factors were analysed., Results: A total of 150 patients were included. Five-year overall survival and local control rates were 50.9 and 92.3 per cent respectively. Multivariable analysis revealed that several factors, including age and Child-Pugh classification, significantly influenced overall survival. Proximity to the digestive tract and Child-Pugh classification were independent risk factors for local recurrence. Other tumour factors including size, gross classification, previous treatment, macroscopic vascular invasion, and tumour location in relation to the diaphragm and large vessels did not influence local control rate., Conclusion: Particle radiotherapy seems safe and effective, and may be a novel treatment for small HCC. Recurrences are more frequent when the tumour is located close to the gut., (Copyright © 2011 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

A 43-year-old woman with systemic sclerosis (SSc) developed IgA deficiency (IgAD) after cholecystitis. The severe decrease of IgA (<10 mg/dl) partially recovered after 5 years. She had repeated episodes of infection during IgAD. Anti-IgA antibody was not detected. Flow cytometric analysis showed that peripheral CD19(+)IgA(+) and CD38(+)IgA(+) cells were normally present. Although the mechanism of secondary IgAD is still vague, its association with autoimmune diseases including SSc and also with bacterial infection is discussed.

Aims: Long-standing right ventricular apical pacing (RVAP) may result in impaired left ventricular (LV) function and systolic heart failure (HF) in selected patients. However, which patients are susceptible to those harmful effects is unknown., Methods and Results: In 367 consecutive patients undergoing pacemaker implantations (PMIs) and RVAP, the clinical, laboratory, and echocardiographic data before the PMIs, electrocardiographic parameters [baseline and paced QRS duration (QRSd)], and echocardiography were analysed. The cumulative per cent of those ventricularly paced (Cum%VP) was >90% in all subjects. During a mean follow-up period of 113±69 months, the occurrence of HF requiring hospitalization for the intravenous administration of HF medications was found in 60 patients (16%; HF group), but not in the remaining 307 (84%; no-HF group). The prevalence of structural heart disease (SHD; P<0.0001), cardiothoracic ratio (P<0.0001), baseline left atrial size (P=0.0001), LV end-diastolic volume (P<0.005) and end-systolic volume (P<0.0005), LV mass index (P<0.001), and baseline and paced QRSd (both for P<0.001) were greater in the HF group than in the no-HF group. Inversely, the LV ejection fraction (LVEF) in the HF group was smaller than that in the no-HF group (P<0.001). The multivariate Cox regression analysis revealed that the presence of SHD [hazard ratio (HR)=3.12; 95% confidence interval (CI), 1.7-5.7; P<0.001] and the LVEF (<40%; HR=2.57; 95% CI, 1.09-6.07; P<0.05) were associated with hospitalizations due to HF after RVAP., Conclusion: The presence of SHD and an impaired LV systolic function before the PMI may predict hospitalizations due to HF after RVAP.

In the past, pulmonary arterial (PA) structure has been extensively investigated with the aim of providing an insight into operative indication for patients with congenital heart disease (CHD). Although PA histological analysis is applied less frequently in the current era, demographic changes of CHD patients require a refocussing of attention. With an exponential increase in the number of adult CHD patients, it is important to realise how structural changes evolve long after previous procedures as a certain proportion of such cases necessitate surgical or interventional manipulation on their PAs. Herein we present our findings on main PA tissues obtained from a 35-year-old woman who had been palliated with a classic Fontan operation 23 years earlier. Immunohistological analysis showed severe alterations, especially in the medial layer; not only attenuation of muscular component but also disarray and fragmentation of elastic fibres were remarkable, which should represent the adaptive response to longstanding diminished lung perfusion. To our knowledge, these observations have not been well described in the literature, presumably because previous studies were conducted primarily with respect to 'increased' pulmonary flow, and hence little is known regarding structural alterations in response to 'decreased' perfusion. Our findings are provided with a review of the literature.

ADAMTS13 is a metalloproteinase that specifically cleaves unusually large von Willbrand factor multimers under high-shear stress. Deficiency of ADAMTS13 activity induces a life-threatening generalized disease, thrombotic thrombocytopenic purpura. We established a simple and efficient method to purify plasma ADAMTS13 (pADAMTS13) from cryosupernatant using an anti-ADAMTS13 monoclonal antibody (A10) that recognizes a conformational epitope within the disintegrin-like domain. Using the purified pADAMTS13, the amino acid residues involved in cleavage by thrombin, plasmin and leucocyte elastase were determined, and the carbohydrate moieties of this enzyme was analysed by lectin blots. Purified pADAMTS13 had a specific activity of 300 U/mg (25,057-fold purification) and the pI was 5.1-5.5. Cleavage sites of the purified pADAMTS13 by three proteases were identified; thrombin cleaved the four peptidyl bonds between Arg257-Ala258, Arg459-Ser460, Arg888-Thr889 and Arg1176-Arg1177, plasmin cleaved the three peptidyl bonds between Arg257-Ala258, Arg888-Thr889 and Arg1176-Arg1177, and elastase cleaved the two peptidyl bonds between Ile380-Ala381 and Thr874-Ser875. Lectin blot analysis indicated the presence of non-reducing terminal α2-6 and α2-3-linked sialic acid residues with penultimate β-galactose residues on the N- and O-linked sugar chains of pADAMTS13, suggesting that pADAMTS13 is cleared from the circulation via the hepatic asialoglycoprotein receptor like other plasma glycoproteins.

Success in islet transplantation-based therapies for type 1 diabetes mellitus and an extreme shortage of pancreatic islets have motivated recent efforts to develop renewable sources of islet-replacement tissue. Although pancreatic progenitor cells hold a promising potential, only a few attempts have been made at the prospective isolation of pancreatic stem/progenitor cells, because of the lack of specific markers and the development of effective cell culture methods. We found that prominin1 (also known as CD133) recognized the undifferentiated epithelial cells, whereas platelet-derived growth factor receptor beta (PDGFRbeta) was expressed on the mesenchymal cells in the mouse embryonic pancreas. We then developed an isolation method for putative stem/progenitor cells by flow cytometric cell sorting and characterized their potential for differentiation to pancreatic tissue using both in vitro and in vivo protocols. Flow cytometry and the subsequent reverse transcription-polymerase chain reaction and microarray analysis revealed pancreatic epithelial progenitor cells to be highly enriched in the prominin1(high)PDGFRbeta(-) cell population. During in vivo differentiation, these cell populations were able to differentiate into endocrine, exocrine, and ductal tissues, including the formation of an insulin-producing cell cluster. We established the prospective isolation of putative pancreatic epithelial progenitor cells by sorting for prominin1 and PDGFRbeta. Since this strategy is based on the cell surface markers common to human and rodents, these findings may lead to the development of new strategies to derive transplantable islet-replacement tissues from human pancreatic stem/progenitor cells. Disclosure of potential conflicts of interest is found at the end of this article.

Objective: To investigate the relationship between active ghrelin and oxidative stress in obese subjects., Design: We measured the plasma levels of free 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha), a reliable and systemic marker of oxidative stress) and the active form of ghrelin in 17 obese and 17 normal subjects. The biologically active forms of ghrelin were measured using a commercially available radio-immunoassay kit and free 8-epi-PGF(2alpha) was measured using an enzyme immunoassay kit., Results: The circulating level of active ghrelin was significantly decreased (20.4 +/- 2.6 vs 40.9 +/- 3.9 fmol/ml, P < 0.01) while that of 8-epi-PGF(2alpha) was significantly increased (61.5 +/- 9.6 vs 17.3 +/- 3.4 pg/ml, P < 0.01) in obese subjects compared with normal subjects. The plasma levels of active ghrelin and 8-epi-PGF(2alpha) were significantly correlated in obese (r = -0.507, P < 0.05) and in all (r = -0.577, P < 0.01) subjects. Multivariate analysis showed that the plasma levels of active ghrelin and 8-epi-PGF(2alpha) were significantly and independently correlated in all subjects (F = 7.888, P < 0.01)., Conclusions: There is an inverse correlation between circulating levels of active ghrelin and oxidative stress in obesity. Low circulating levels of active ghrelin may enhance oxidative stress and the process of atherosclerosis in obese subjects.

We discovered FR207944 produced by Chaetomium sp. No. 217 in the course of screening for antifungal antibiotics from natural products. FR207944 is identical with fuscoatroside, described in the preceding paper as an anti-Aspergillus flavus agent. Determination of the relative stereochemistry of fuscoatroside was made formally by comparison with WF11605 (16-Oxo-FR207944). We confirmed the stereochemistry on the basis of single crystal X-ray analysis.

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.

To investigate the direct relationship of oxidative stress with obesity and insulin resistance in men, we measured the plasma levels of 8-epi-prostaglandin F2alpha (PGF2alpha) in 14 obese and 17 nonobese men and evaluated their relationship with body mass index; body fat weight; visceral, sc, and total fat areas, measured by computed tomography; and glucose infusion rate during a euglycemic hyperinsulinemic clamp study. Obese men had significantly higher plasma concentrations of 8-epi-PGF2alpha than nonobese men (P < 0.05). The plasma levels of 8-epi-PGF2alpha were significantly correlated with body mass index (r = 0.408; P < 0.05), body fat weight (r = 0.467; P < 0.05), visceral (r = 0.387; P < 0.05) and total fat area (r = 0.359; P < 0.05) in all (obese and nonobese) men. There was also a significant correlation between the plasma levels of 8-epi-PGF2alpha and glucose infusion rate in obese men (r = -0.552; P < 0.05) and all men (r = -0.668; P < 0.01). In all subjects, the plasma levels of 8-epi-PGF2alpha were significantly correlated with fasting serum levels of insulin (r = 0.487; P < 0.01). In brief, these findings showed that the circulating levels of 8-epi-PGF2alpha are related to adiposity and insulin resistance in men. Although correlation does not prove causation, the results of this study suggest that obesity is an important factor for enhanced oxidative stress and that this oxidative stress triggers the development of insulin resistance in men.

Background: The aim of this study is to evaluate the magnitude of the aggregation of a stomach cancer history in parents and their offspring in comparison with that of a history at other sites., Methods: We used the baseline data from the Japan Collaborative Cohort Study for Evaluation of Cancer Risk (JACC Study), which was initiated during 1988-1990 in Japan. Association of the cancer history of the subjects' parents with that of the subjects themselves and any of the subjects' siblings was evaluated with odds ratios (OR) by the crude and generalized estimating equations (GEE) technique for four sites: stomach, colorectum, liver, and lung/bronchus., Results: The aggregation of a history of stomach cancer between parents and their offspring was evident with significant OR >2.5. The magnitude of the parent-offspring association of a disease history of the colorectum and liver was found to be greater than that for stomach cancer. Conversely, lung and bronchus cancer failed to demonstrate a significant aggregation., Conclusions: The hereditary and environmental influences shared by parents and offspring are likely to play a strong aetiological role in colorectal or liver cancer versus a weaker but still significant role in stomach cancer. In contrast, the aetiological role of familial predisposition to lung cancer was indeterminate, which suggests a predominant role of non-familial factors in the development of lung cancer.

Objective: It is well known that nitric oxide synthase (NOS) is expressed and that it modulates glucose transport in skeletal muscles. Recent studies have shown that adipose tIssues also express inducible and endothelial nitric oxide synthase (eNOS). In the present study, we investigated whether nitric oxide (NO) induces glucose uptake in adipocytes, and the signaling pathway involved in the NO-stimulated glucose uptake in 3T3-L1 adipocytes., Methods: First, we determined the expression of eNOS in 3T3-L1 adipocytes, and then these cells were treated with the NO donor sodium nitroprusside (SNP) and/or insulin, and glucose uptake and phosphorylation of insulin receptor substrate (IRS)-1 and Akt were evaluated. Moreover, we examined the effects of a NO scavenger, a guanylate cyclase inhibitor or dexamethasone on SNP-stimulated glucose uptake and GLUT4 translocation., Results: SNP at a concentration of 50 mmol/l increased 2-deoxyglucose uptake (1.8-fold) without phosphorylation of IRS-1 and Akt. Treatment with the NO scavenger or guanylate cyclase inhibitor decreased SNP-stimulated glucose uptake to the basal level. Dexamethasone reduced both insulin- and SNP-stimulated glucose uptake with impairment of GLUT4 translocation., Conclusion: NO is capable of stimulating glucose transport through GLUT4 translocation in 3T3-L1 adipocytes, via a mechanism different from the insulin signaling pathway.

Bacterial ribonuclease P RNA ribozyme can do the hyperprocessing reaction, the internal cleavage reaction of some floppy eukaryotic tRNAs. The hyperprocessing reaction can be used as a detection tool to examine the stability of the cloverleaf shape of tRNA. Until now, the hyperprocessing reaction has been observed in the heterologous combination of eukaryotic tRNAs and bacterial RNase P enzymes. In this paper, we examined the hyperprocessing reaction of Escherichia coli tRNAs by homologous E. coli RNase P, to find that these homologous tRNAs were resistant to the toxic hyperprocessing reaction. Our results display the evidence for molecular co-evolution between homologous tRNAs and RNase P in the bacterium E. coli.

In Japan, poisonings by the glyphosate (GLYP)-containing herbicide Roundup and the gluphosinate (GLUF)-based herbicide BASTA have been increasing since about 1987. We applied the gas chromatography-mass spectrometry (GC-MS) method of analysis, on which we have already reported in regard to the determination of the blood serum level of GLUF and its metabolite, for the determination of serum and urinary levels of GLYP and its metabolite aminomethyl phosphonic acid (AMPA). Derivatization using N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide was completed at a temperature of 80 degrees C after 30 min, and the detection limit of GLYP was 10 pg using m/z 454 and that of AMPA was 1 pg using m/z 396. The full mass spectra of 100 pg GLYP and of 10 pg AMPA were obtained easily. In extractions for which the Isolute HAX cartridge was employed, the mean recovery rate of GLYP and AMPA added to serum to yield concentrations of 10-0.1 microg/mL (n = 5) was 91.6 +/- 10.6% (or better), whereas that of GLYP and AMPA added to urine to yield concentrations of 100-1.0 microg/mL (n = 10) was 93.3 +/- 6.6% (or better), both of which were good rates. Also, using this method of analysis, the presence of GLYP was identified in the full mass spectra obtained from the serum of a patient who may or may not have ingested Roundup.

Hypofibrinolysis is a common finding in patients with diabetes mellitus and a risk factor for diabetic nephropathy. Recently, a new potent inhibitor of fibrinolysis, the thrombin-activatable fibrinolysis inhibitor (TAFI), has been isolated from human plasma. The possibility that TAFI also participates in the mechanism of hypofibrinolysis has not been appraised in diabetic patients with microalbuminuria. In the present study, we investigated the plasma levels of TAFI and its relation to urinary albumin excretion in normotensive diabetic patients with normo- and microalbuminuria. Thirty-nine normotensive nonobese type 2 diabetic patients (27 with normoalbuminuria, 12 with microalbuminuria) and 20 age-matched normal subjects were enrolled in this study. The plasma level of thrombin-antithrombin complex was significantly increased (22.1 +/- 2.6 vs. 8.3 +/- 1.0 nmol/liter; P < 0.05), whereas the D-dimer/thrombin-antithrombin complex ratio was significantly decreased (15.7 +/- 1.4 vs. 26.5 +/- 2.2; P < 0.05), showing the occurrence of hypercoagulability and hypofibrinolysis in diabetic patients. The plasma level of TAFI in diabetic patients was significantly elevated, compared with normal subjects (147.4 +/- 11.6 vs. 99.5 +/- 4.9%; P < 0.05). The plasma level of TAFI in diabetic patients with microalbuminuria was significantly higher than the level in diabetic patients with normoalbuminuria (194.1 +/- 24.5 vs. 128.8 +/- 12.3%; P < 0.02) or normal subjects (194.1 +/- 24.5 vs. 99.5 +/- 4.9%; P < 0.005). Univariate analysis showed that the plasma TAFI levels are significantly and proportionally correlated with urinary albumin excretion rate (r = 0.58; P < 0.005) and with plasma soluble thrombomodulin level, a marker of endothelial cell damage, in all diabetic patients (r = 0.42; P < 0.01). These data suggest that increased plasma level of TAFI may be involved in the mechanism of vascular endothelial damage in patients with type 2 diabetes mellitus.

To clarify whether homeostasis model assessment (HOMA IR) and quantitative insulin sensitivity check index (QUICKI) may be indicators of insulin resistance in elderly patients with type 2 diabetes mellitus, their relationship with the glucose infusion rate during the euglycemic hyperinsulinemic clamp study (clamp IR) was assessed. This study comprised 56 Japanese patients with type 2 diabetes mellitus; of these, 28 were 70 yr of age or older (group 1) and 28 were less than 70 yr of age (group 2). Their blood sugars were in poor control (fasting plasma glucose levels: group 1, 9.0 +/- 2.6 mmol/liter; group 2, 8.9 +/- 2.3 mmol/liter; hemoglobin A1c: group 1, 9.5 +/- 2.0%; group 2, 9.2 +/- 1.7%). Log-transformed HOMA IR was significantly correlated with the clamp IR in group 2 patients (r = -0.51, P < 0.01), but not in group 1 patients (r = -0.28, P = 0.15). There was a significant positive correlation between QUICKI and clamp IR in group 2 patients (r = 0.50, P < 0.01). However, no significant correlation was observed between QUICKI and clamp IR in group 1 patients (r = 0.31, P = 0.12). There was a significant correlation between log-transformed HOMA IR (r = -0.37, P < 0.01) or QUICKI (r = 0.37, P < 0.01) and clamp IR when both groups were combined. In conclusion, neither HOMA IR nor QUICKI should be used as an index of insulin resistance in elderly patients with poorly controlled type 2 diabetes mellitus. The results of this study suggest the need for developing a new noninvasive method for evaluating insulin resistance in those patients.

Human tyrosine tRNA and fly alanine, histidine, and initiator methionine tRNAs are generally cleavable internally by bacterial ribonuclease P ribozyme. The unusual internal cleavage reaction of tRNA, called hyperprocessing, occurs when the cloverleaf structure of the tRNA molecule is denatured to form a double-hair-pin-like structure. The hyperprocessing reaction of these tRNAs requires magnesium ions. We analyzed details of this reaction using human tyrosine tRNA and Escherichia coli RNase P ribozyme. The usual processing reaction occurred efficiently with magnesium at 5 mM, but for the hyperprpocessing reaction, higher concentrations were needed. With such high concentrations, hyperprocessing cleaved both mature tRNA and tRNA precursor as substrates. When mature tRNA was the substrate, the apparent K(M) was almost the same as in the usual reaction, but k(cat) was smaller. These results indicated that the occurrence of hyperprocessing depends on the magnesium ion concentration, and suggested that magnesium ions contribute to the recognition of the shape of the substrate by bacterial RNase P enzymes.

Objective: To investigate the effect of acute hyperinsulinemia on the plasma levels of adrenomedullin (AM) in patients with type 2 diabetes mellitus., Design: We measured the plasma levels of AM in 18 patients with type 2 diabetes mellitus and in 19 normal subjects before and during a euglycemic hyperinsulinemic clamp study (the goal was for blood sugar levels of 5.24 mmol/l and insulin levels of 1200 pmol/l). Both plasma AM and serum insulin were measured by immunoradiometric assays., Results: Before the glucose clamp study there was no significant difference in the plasma levels of AM between patients with type 2 diabetes mellitus and normal subjects. During the glucose clamp study, the serum levels of insulin significantly increased (from 33.0+/-3.6 to 1344.6+/-67.8 pmol/ml, P<0.001), as did the plasma levels of AM (from 12.8+/-0.7 to 14.2+/-0.9 fmol/ml, P<0.03) only in patients with type 2 diabetes mellitus. There was a significant correlation between the change in circulating levels of insulin and AM (r=0.755, P<0.01)., Conclusions: Acute hyperinsulinemia induced a significant increase in the plasma levels of AM in patients with type 2 diabetes mellitus. Increased insulin may regulate circulating levels of AM in patients with type 2 diabetes mellitus.

To investigate whether quantitative insulin sensitivity check index (QUICKI) would be useful as an index of insulin resistance during the clinical course of type 2 diabetes mellitus, correlation between QUICKI and the index of the euglycemic hyperinsulinemic clamp study [clamp insulin resistance (clamp IR)] was evaluated in 60 patients with type 2 diabetes mellitus before and after treatment. The therapy program consisted of diet (1440-1720 kcal/d) and exercise (walking 10,000 steps daily) for 6 wk. QUICKI and clamp IR were significantly correlated before (r = 0.598, P < 0.0001) and after (r = 0.583, P < 0.0001) treatment. Neither the slope nor the intercept of the linear correlation between QUICKI and clamp IR measured before treatment was significantly different from those measured after treatment (slopes; F = 0.002, P = 0.96, intercepts; F = 2.65, P = 0.11). During treatment, the values of both QUICKI (8% change; P < 0.0001) and clamp IR (38% change; P < 0.0001) significantly increased and their changes were significantly correlated (r = 0.415, P < 0.01). In conclusion, QUICKI may become a useful method for the follow-up of insulin resistance during the treatment of patients with type 2 diabetes mellitus.

Recently, we revealed that the cloverleaf structure of some eukaryotic tRNAs is not always stable in vitro, and the denatured structures of these tRNAs are sometimes detected in bacterial RNase P reactions. We have designated the unusual internal cleavage reaction of these tRNAs as hyperprocessing. We have developed this hyperprocessing strategy as a useful tool for examining the stability of the tRNA cloverleaf structure. There are some common features in such unstable, hyperprocessible tRNAs, and the criteria for the hyperprocessing reaction of tRNA are extracted. Metazoan initiator methionine tRNAs and lysine tRNAs commonly fit the criteria, and are predicted to be hyperprocessible. The RNase P reactions of two metazoan lysine tRNAs from Homo sapiens and Caenorhabditis elegans, which fit the criteria, resulted in resistance to the internal cleavage reaction, while one bacterial lysine tRNA from Acholeplasma laidlawii, which also fits the criteria, was internally cleaved by the RNase P. The results showed that the metazoan lysine tRNAs examined are very stable without base modifications even under in vitro conditions. We also examined the 3'-half short construct of the human lysine tRNA, and the results showed that this RNA was internally cleaved by the enzyme. The results indicated that the human lysine tRNA has the ability to be hyperprocessed but is structurally stabilized in spite of lacking base modifications. A comparative study suggested, moreover, that the acceptor-stem bases should take part in the stabilization of metazoan lysine tRNAs. Our data strongly suggest that the cloverleaf shape of other metazoan lysine tRNAs should also be stabilized by means of similar strategies to in the case of human tRNA(Lys3).

Background: Proliferation of mesangial cells (MCs) is the initial step in glomerulonephritis, and platelet-derived mediators have been shown to play a significant role in this proliferation. Sphingosine 1-phosphate (S1P), one of the sphingolipids, is abundantly stored in platelets and is released upon stimulation. We examined the effects of S1P and related sphingolipids on the cell fate of cultured MCs in order to elucidate potential roles of these lipid mediators in glomerulonephritis., Methods: Cell proliferation was evaluated by bromodeoxy uridine (BrdU) incorporation together with MTS assay. Apoptosis of MCs was evaluated by examining annexin V staining and typical morphological changes in nuclei. We also examined the metabolism of [(3)H]sphingosine in MCs in either the presence or absence of platelet-derived growth factor (PDGF). The expression of endothelial differentiation genes (edg), which are the cell surface receptors for S1P in MCs, was examined by RT-PCR., Results: S1P, but not the other sphingolipids, stimulated MC proliferation. In contrast, dimethylsphingosine (DMS) induced apoptosis in the MCs. The amount of sphingosine (Sph) converted into S1P was small and was not affected by PDGF. This observation suggested that Sph kinase activity producing S1P from Sph was low in the MCs. Furthermore, expression of edg-1, -2 and -5 in MCs was confirmed by RT-PCR., Conclusions: Our observations suggest that S1P stimulates MC proliferation from outside the cells, and not as a second messenger for PDGF. The modulation of MC fate with sphingolipids may provide possible strategies for the treatment of glomerulonephritis.

The 4-O-methylpyridoxine (MPN) present in the seeds of the Ginkgo biloba (maidenhair tree) has anti-vitamin B6 actions, and ginkgo seed poisoning can induce convulsions. We developed a specific quantitative method using gas chromatography-mass spectrometry for the analysis of MPN in human serum. The trifluoroacyl (TFA) derivative of MPN was obtained by treating MPN with trifluoroacetic anhydride at 50 degrees C for 5 min and remained stable for 6 h. The calibration curve of standard MPN obtained in the selective ion mode using the base ion (m/z 343) was linear between 100 pg and 10 ng, and the detection limit was 50 pg. The full mass spectrum of 100 pg of the TFA derivative of MPN was obtained easily. MPN was extracted from the serum with the use of a C18 solid-phase extraction cartridge. The recovery rate of MPN added to the serum at a concentration of 0.1 microg/mL was 90.0%.

Hypofibrinolysis is a common finding in patients with diabetes mellitus (DM) and obesity and a risk factor for the development of cardiovascular disease. Recently, a new potent inhibitor of fibrinolysis, the thrombin-activatable fibrinolysis inhibitor (TAFI) has been isolated and characterized from human plasma. The present study was undertaken to assess the activity and circulating level of TAFI and its relation to fibrinolytic function and obesity in patients with type 2 DM. Fifty-seven patients with type 2 DM (38 men, 19 women) were enrolled in this study. DM patients were categorized in age-matched obese [body mass index (BMI) > or = 25] and nonobese (BMI < 25) groups. The plasma concentration and activity of TAFI were significantly (P < 0.05) higher in DM patients than in healthy controls. The plasma levels and activity of TAFI were significantly (P < 0.05) elevated in obese DM patients compared with nonobese DM and nonobese healthy subjects. RT-PCR demonstrated the expression of TAFI in human adipose tissue and in human endothelial cells. The plasma levels of TAFI were independently and significantly correlated with glucose intolerance (HbA(1c)), with obesity (BMI, visceral fat area), and with an indicator of insulin resistance (glucose infusion rate). This study showed that increased circulating level of TAFI may be an important causative factor of hypofibrinolysis in patients with type 2 diabetes, obesity and insulin resistance.

Porphyrin has been reported to bind to the T psi C stem of tRNA. This site is also recognized by ribonuclease P, which is essential and ubiquitous endoribonuclease responsible for the maturation of 5' ends of tRNA precursors. Thus, we investigated the effects of porphyrins on the in vitro reaction of ribonuclease P from Escherichia coli. The results showed that some of porphyrins inhibited the reaction more strongly than any other inhibitors reported so far. In addition to the benzimidazole inhibition that we have previously reported, these unusual substrate-binding inhibitions may provide new leads for the novel anti-bacterial reagent design.

The RNA subunit of bacterial ribonuclease P (RNase P) is a ribozyme which can cleave a canonical cloverleaf tRNA precursor and a hairpin RNA with a CCA-3' tag sequence as its substrate. With high concentration of Mg ion, the ribozyme as well as holo enzyme internally cleaves certain tRNAs in vitro. We denoted this unusual reaction as hyperprocessing. By controlling magnesium ion concentration for the reaction and also by forcing the RNA shape with external guide DNAs, we could regulate the hyperprocessing reaction by the bacterial RNase P enzymes. These techniques will lead the RNase P ribozyme to more designable and more applicable RNA-cleaving enzyme.

Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA(Ala), tRNA(His), and tRNA(iMet)) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool detect destablized tRNA molecules from any species.

A method for the analysis of glufosinate ammonium (GLUF) and its metabolite 3-methylphosphinicopropionic acid (MPPA) in human serum by gas chromatography-mass spectrometry (GC-MS) was developed. Employing a mixed-mode cartridge with both anion exchange action and weak nonpolar interaction, we extracted GLUF and MPPA from the serum and carried out GC-MS analysis of their tert-butyldimethylsilyl derivatives. The detection limits of GLUF and MPPA were 10 pg and 1 pg, respectively. Full mass spectra of 100 pg GLUF and of 10 pg MPPA were easily obtainable. The recovery rate of 90.0+/-11.9% (or better) when the serum concentrations of GLUF and MPPA were 10-0.1 microg/mL. Results of 23 serum samples, from patients with GLUF poisoning, measured by this method correlate well with those derived from the conventional high-performance liquid chromatography method (r = 0.996). The developed GC-MS method is likely to become a useful analytical technique in clinical settings.