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Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages to anchoring enzymes to a stable substrate, including stability and the possibility for substrate channeling. To avoid the necessity of protein purification and chemical immobilization, there has been growing interest in bio-assembly of protein-containing nanoparticles, exploiting the self-assembly of viral capsid proteins or other proteins that form polyhedral structures. But these nanoparticle are limited in size which constrains the packaging and the accessibility of the proteins. The axoneme, the insoluble protein core of the eukaryotic flagellum or cilium, is a highly ordered protein structure that can be several microns in length, orders of magnitude larger than other types of nanoparticles. We show that when proteins of interest are fused to specific axonemal proteins and expressed in living cells, they become incorporated into linear arrays which have the advantages of high protein loading capacity, high stability, and single-step purification with retention of biomass. The arrays can be isolated as membrane enclosed vesicle or as exposed protein arrays. The approach is demonstrated for both fluorescent proteins and enzymes, and in the latter case it is found that incorporation into axoneme arrays provides increased stability for the enzyme.

Some studies have demonstrated that excessive fructose consumption negatively impact brain function. Recently, the Developmental Origins of Health and Disease hypothesis - which suggests that maternal nutritional status during gestation and lactation can alter offspring phenotype - has received much attention. In a previous study, we demonstrated that maternal fructose consumption increases levels of lipid peroxides in hippocampi of offspring. The hypothesis in the present study was that maternal fructose intake would affect hippocampal antioxidant enzyme via epigenetic regulation. Upon confirmation of gestation, female rats were assigned to receive either water (control group) or a 20% fructose solution (fructose-fed group). Water or fructose solution were administered to dams from day 1 of gestation to postnatal day 21. Immediately after weaning, hippocampi of offspring were removed for analysis of antioxidant enzyme (Sod1, Sod2, Gpx1, Gpx4, and Cat) messenger RNA transcript levels. Levels of the Cat transcript were significantly lower in the fructose-fed relative to the control group. The Cat protein level was also significantly lower in the fructose-fed relative to the control group as with the messenger RNA transcript levels. Moreover, Cat promoter DNA methylation levels were higher in the fructose-fed group. The present study indicates that maternal fructose consumption may decrease offspring hippocampal Cat transcript levels via altered DNA methylation, which may result in higher levels of oxidative stress due to a decreased ability to neutralize lipid peroxides.

We examined whether orally administered octacosanol, a long-chain aliphatic saturated alcohol, improves the features of high fructose-induced metabolic syndrome in rats. Five-week-old rats were fed a high fructose diet containing 60% fructose for 3 weeks. Then, the high fructose fed rats received a daily single oral administration of octacosanol (10 or 100 mg/kg body weight) with high fructose feeding for one week. Three- or four-week high fructose feeding increased insulin resistance, serum insulin, triglyceride, total cholesterol, free fatty acids, uric acid, and lipid peroxide concentrations, and hepatic triglyceride and cholesterol contents significantly and decreased serum high-density lipoprotein cholesterol and adiponectin concentrations significantly but did not affect blood pressure and hepatic lipid peroxide and reduced glutathione contents. Four-week high fructose feeding decreased hepatic ascorbic acid content significantly. Oral administration of octacosanol (10 or 50 mg/kg body weight) to high fructose-fed rats for the last 1-week fructose diet feeding attenuated these changes except serum insulin level and insulin resistance significantly and increased hepatic reduced glutathione content significantly. The higher dose of Oct decreased hepatic lipid peroxide content significantly. These results indicate that orally administered octacosanol improves dyslipidemia, hyperuricemia, hypoadiponectinemia, and oxidative stress associated with the features of high fructose-induced metabolic syndrome rats.

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism, Chlamydomonas reinhardtii have focused on the length-dependence of the intraflagellar transport (IFT) system which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella, and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not previously been determined. We found that SHF1 encodes a Chlamydomonas ortholog of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as wild-type cells, but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intra-flagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

Given that fructose consumption has increased by more than 10-fold in recent decades, it is possible that excess maternal fructose consumption causes harmful effects in the next generation. This study attempted to elucidate the mechanism of the harmful effects of excessive maternal fructose intake from the perspective of offspring liver function. Female rats during gestation and lactation were fed water containing fructose, and their offspring were fed normal water. We attempted to elucidate the mechanism of fructose-induced transgenerational toxicity by conducting a longitudinal study focusing on hepatic programming prior to disease onset. Impaired Insulin resistance and decreased high-density lipoprotein-cholesterol levels were observed at 160 days of age. However, metabolic disorders were not observed in 60-day-old offspring. Microarray analysis of 60-day-old offspring livers showed the reduction of hepatic insulin-like growth factor-1 (Igf1) mRNA expression. This reduction continued until the rats were aged 160 days and attenuated Igf1 signaling. Hepatic microRNA-29 (miR-29a) and miR-130a, which target Igf1 mRNA, were also found to be upregulated. Interestingly, these miRNAs were upregulated in the absence of metabolic disorder. In this study, we found that maternal fructose intake resulted in dysregulated expression of Igf1 and its target miRNAs in the offspring liver, and that these offspring were more likely to develop metabolic disorders. Abnormal hepatic programming induced by an imbalanced maternal nutritional environment is maintained throughout life, implying that it may contribute to metabolic disorders.

Background Although previous studies have demonstrated that n-3 polyunsaturated fatty acids (PUFA) are inversely associated with a risk of CVD through gene expression, recent evidence suggests that it may arise from changes in DNA methylation. Therefore, we investigated whether dietary FA intake and serum FA levels are associated with ATP-binding cassette transporter A1 (ABCA1), a gene associated with HDL-cholesterol metabolism. Methods A total of 298 subjects (137 men, mean age of 63.2) without clinical history and medication participated in this cross-sectional study. We used the pyrosequencing method to measure DNA methylation levels at 8 CpG sites within ABCA1 gene and calculated mean DNA methylation level. Dietary FA intake were assessed with the food frequency questionnaire. Results We found that ABCA1 DNA methylation levels were significantly lower with higher dietary intake of n-3PUFA (β = –5.75, p = 0.001), which is observed both in men (β = –4.85, p = 0.001) and women (β = –7.24, p = 0.007). In addition, ABCA1 DNA methylation levels were significantly lower with higher serum levels of n-3PUFA in men (β = –2.78, p = 0.05), but not in women (β = 1.61, p = 0.31). Conclusions The results suggest that higher dietary n-3 PUFA intake and serum levels of n-3 PUFA are associated with lower ABCA1 DNA methylation in a Japanese population. Key messages Higher dietary n-3 PUFA intake can be a prevention from dyslipidemia and CVD via low level of ABCA1 DNA methylation

BackgroundThe adverse health effects of sugar-sweetened beverage consumption have been studied worldwide. There are several reports on actual sugar contents in sugar-sweetened beverages. However, there is no recent report on actual sugar contents in Japanese sugar-sweetened beverages. Therefore, we attempted to analyze glucose, fructose, and sucrose contents in Japanese common beverages.MethodsGlucose, fructose, and sucrose contents in 49 beverages including 8 energy drinks, 11 sodas, 4 fruit juices, 7 probiotic drinks, 4 sports drinks, 5 coffee drinks, 6 green tea drinks, and 4 tea drinks were determined using the enzymatic methods.ResultsTow zero calorie drinks, 2 sugarless coffee drinks, and 6 green tea drinks contained no sugar. Three coffee drinks contained only sucrose. The orders of median glucose, fructose, and sucrose contents in categorized beverages containing sugars were as follows: for glucose, fruit juice > energy drink ≥ soda >> probiotic drink > black tea drink > sports drink; for fructose, probiotic drink ≥ energy drink > fruit juice > soda >> sports drink > black tea drink; and for sucrose, black tea drink > energy drink ≥ probiotic drink > fruit juice > soda > coffee drink >> sports drink. The rate of total fructose content in total sugar content in 38 sugar-containing beverages was approximately 40-60%. The total sugar content analyzed was not always equivalent to carbohydrate content indicated on the nutrition label.ConclusionsThese results indicate that actual sugar content in Japanese common beverages is necessary for the exact assessment of beverage-derived sugar intake.

Fructose consumption is rising globally, but maternal high fructose intake might adversely affect offspring. Our previous report demonstrated that excess maternal fructose intake impairs hippocampa...

Summary Any proposed mechanism for organelle size control should be able to account not only for average size but also for the variation in size. We analyzed cell-to-cell variation and within-cell variation of length for the two flagella in Chlamydomonas, finding that cell-to-cell variation is dominated by cell size, whereas within-cell variation results from dynamic fluctuations. Fluctuation analysis suggests tubulin assembly is not directly coupled with intraflagellar transport (IFT) and that the observed length fluctuations reflect tubulin assembly and disassembly events involving large numbers of tubulin dimers. Length variation is increased in long-flagella mutants, an effect consistent with theoretical models for flagellar length regulation. Cells with unequal flagellar lengths show impaired swimming but improved gliding, raising the possibility that cells have evolved mechanisms to tune biological noise in flagellar length. Analysis of noise at the level of organelle size provides a way to probe the mechanisms determining cell geometry., Graphical abstract, Highlights • Chlamydomonas flagella show dynamic length fluctuations • Length variation involves processes inside the cell body and in the flagella • Long-flagella mutants have increased length variability • Flagellar length variation leads to variation in cell motility, Biological Sciences; Molecular Biology; Cell Biology; Biophysics; Mathematical Biosciences

For human risk assessment of toxic chemicals, especially volatile organic compounds (VOCs), the Ministry of the Environment, Government of Japan, has called for the interconversion of inhalation-dose and oral-dose data, two common exposure routes. To address this issue, the present study investigated the time-course changes of ethylbenzene (EB) concentrations in the blood of rats during and after 6-hr inhalation exposure to EB (25, 50, 100, and 200 ppm) and after oral administration of EB by a single oral gavage (25, 50, 100, and 200 mg/kg) of EB. The Area Under the blood concentration-time Curve (AUC) at each blood collection time point (0, 30, 60, 120, 180, 360, 420, 540, and 1440 min, after starting exposure) was determined. The inhalation dose of 25 ppm corresponded closely to the oral administration of 25 mg/kg・bw (r value of 0.859), and the inhalation dose of 200 ppm correlated with the oral administration of 100 mg/kg・bw (r value of 0.948). These results suggest that this comparison using the AUC data at each blood collection time point is valuable for understanding the route- and dose-effects of EB. This study will improve risk assessment of human exposure to EB and other VOCs.

Analysis of fluctuation in organelle size provides a new way to probe the mechanisms of organelle size control systems. By analyzing cell-to-cell variation and within-cell fluctuations of flagellar length inChlamydomonas, we show that the flagellar length control system exhibits both types of variation. Cell to cell variation is dominated by cell size, while within-cell variation results from dynamic fluctuations that are subject to a constraint, providing evidence for a homeostatic size control system. We analyzed a series of candidate genes affecting flagella and found that flagellar length variation is increased in mutations which increase the average flagellar length, an effect that we show is consistent with a theoretical model for flagellar length regulation based on length-dependent intraflagellar transport balanced by length-independent disassembly. Comparing the magnitude and time-scale of length fluctuations with simple models suggests that tubulin assembly is not directly coupled with IFT-mediated arrival and that observed fluctuations involve tubulin assembly and disassembly events involving large numbers of tubulin dimers. Cells with greater differences in their flagellar lengths show impaired swimming but improved gliding motility, raising the possibility that cells have evolved mechanisms to tune intrinsic noise in length. Taken together our results show that biological noise exists at the level of subcellular structures, with a corresponding effect on cell function, and can provide new insights into the mechanisms of organelle size control.

Objectives A diet rich in fish and ω-3 polyunsaturated fatty acids (PUFAs) has been thought to reduce the risk for cardiovascular disease (CVD). The beneficial effects of fish oil and ω-3 PUFA on CVD can be mediated by epigenetic status of the genes associated with lipid metabolism and inflammation. The aim of this study was to investigate whether dietary fish and fatty acid (FA) intakes are associated with leukocyte ATP-binding cassette transporter A1 (ABCA1) DNA methylation levels in a Japanese population. Methods This cross-sectional study included 298 adults (137 men and 161 women) without clinical history of CVD or cancer. The pyrosequencing method was used to measure leukocyte ABCA1 DNA methylation levels. Dietary fish and FA intakes were assessed based on the validated food frequency questionnaire. Results Mean ABCA1 DNA methylation levels were significantly lower in the highest fish intake groups (≥5–6/wk) compared with the lowest intake group (≤1–2/wk; P = 0.004). In multivariable linear regression analyses, higher dietary intake of ω-3 PUFAs and ω-3 highly unsaturated fatty acids was significantly associated with decreased levels of ABCA1 DNA methylation (P = 0.001 and 0.005); whereas no significant associations were seen between intake of dietary saturated fatty acid, monounsaturated fatty acid, and ω-6 PUFAs and ABCA1 DNA methylation. Conclusion Higher dietary fish and ω-3 PUFA intake were associated with lower ABCA1 DNA levels in a Japanese population. The present results may bring potential insights on biological mechanisms underlying the protective effects of dietary fish and ω-3 PUFA intakes on CVD.

Thioredoxin-interacting protein (TXNIP) inhibits the activity of thioredoxin (TXN), leading to increased oxidative stress. Expression of the TXNIP gene is regulated by DNA methylation. However, no study has reported the influence of lifestyle factors on TXNIP DNA methylation. Our goal was to determine the association between smoking habits and TXNIP DNA methylation levels in a Japanese population. We conducted a cross-sectional study of 417 subjects (180 males and 237 females) participating in a health examination. We used a pyrosequencing assay to determine TXNIP DNA methylation levels in leukocytes. The mean TXNIP DNA methylation level in current smokers (75.3%) was significantly lower than that in never and ex-smokers (never: 78.1%, p < 0.001; ex: 76.9%, p = 0.013). Multivariable logistic regression analyses showed that the OR for TXNIP DNA hypomethylation was significantly higher in current smokers than that in never smokers, and significantly higher in current smokers with years of smoking ≥ 35 and Brinkman Index ≥ 600 compared to that in non-smokers. In conclusion, we found that current smokers had TXNIP DNA hypomethylation compared to never and ex-smokers. Moreover, long-term smoking and high smoking exposure also were associated with TXNIP DNA hypomethylation.

Aim: Aberrant global DNA methylation is involved in the development of several diseases, including cardiovascular disease (CVD). We investigated whether the methylation of long interspersed nuclear element-1 (LINE-1) in leukocytes is associated with dyslipidemia, a major risk factor for CVD, in the Japanese general population. Methods: We conducted a cross-sectional study consisting of 420 Japanese subjects (187 men and 233 women) without a clinical history of cancer, stroke, or ischemic heart disease. LINE-1 DNA methylation levels in leukocytes were measured using a pyrosequencing method. Results: Significantly higher odds ratios (ORs) for hypermethylation were observed in the high LDL cholesterol and high LDL/HDL ratio groups than the corresponding normal group (high LDLC group: OR, 1.88; 95% confidence interval [CI], 1.20–2.96, high LDL/HDL ratio group: OR, 1.90; 95% CI, 1.20–3.01). Subjects with 2 or more lipid abnormalities had significantly higher ORs for hypermethylation than those with no lipid abnormality (OR, 2.31; 95% CI, 1.11–4.82). Conclusion: LINE-1 DNA hypermethylation in leukocytes was associated with CVD risk profiles: high LDLC, high LDL/HDL ratio, and the degree of abnormal lipid metabolism. Abbreviations:ANOVA, analysis of variance; BMI, body mass index; CI, confidence interval; CRP, C-reactive protein; CVD, cardiovascular disease; DNMT, DNA methyltransferase; EDTA, ethylenediaminetetraacetic acid; HDLC, high-density lipoprotein cholesterol; IL-6, interleukin-6; KLF2, Kruppel-like factor 2; LDLC, low-density lipoprotein cholesterol; LINE-1, long interspersed nuclear element-1; OR, odds ratio; PCR, polymerase chain reaction; SD, standard deviation; TC, total cholesterol; TG, triglyceride

Aims Recent increases in fructose consumption have raised concerns regarding the potential adverse intergenerational effects, as maternal fructose intake may induce physiological dysfunction in offspring. However, no reports are available regarding the effect of excess maternal fructose on reproductive tissues such as the ovary. Notably, the maternal intrauterine environment has been demonstrated to affect ovarian development in the subsequent generation. Given the fructose is transferred to the fetus, excess fructose consumption may affect offspring ovarian development. As ovarian development and its function is maintained by 17β-estradiol, we therefore investigated whether excess maternal fructose intake influences offspring ovarian estradiol synthesis. Rats received a 20% fructose solution during gestation and lactation. After weaning, offspring ovaries were isolated. Key findings Offspring from fructose-fed dams showed reduced StAR and P450(17α) mRNA levels, along with decreased protein expression levels. Conversely, attenuated P450arom protein level was found in the absence of mRNA expression alteration. Consistent with these phenomena, decreased circulating levels of estradiol were observed. Furthermore, estrogen receptor α (ERα) protein levels were also down-regulated. In accordance, the mRNA for progesterone receptor, a transcriptional target of ERα, was decreased. These results suggest that maternal fructose might alter ovarian physiology in the subsequent generation.

Recent increases in fructose consumption have raised concerns about the potential adverse intergenerational effects of excess fructose intake. In the present study, we investigated whether excess maternal fructose intake affects hippocampal function in offspring. Female Sprague-Dawley rats were divided into 3 experimental groups: one group received distilled water, one group received 20% fructose water, and one group received 20% glucose water in addition to standard chow during gestation and lactation. Hippocampal function of offspring was evaluated by using novel object recognition and fear conditioning tests. Impaired cognitive performance was observed in the offspring of fructose-fed dams at postnatal d 60, potentially a result of decreased hippocampal neurogenesis. Real-time PCR analysis demonstrated that offspring from fructose-fed dams exhibited decreased brain-derived neurotrophic factor ( BDNF) gene expression, whereas pyrosequencing assays revealed increased DNA methylation at the BDNF promoter. The potential association between BDNF transcription and levels of DNA methylation was confirmed on the basis of luciferase activity. Furthermore, longitudinal analysis revealed that increased methylation of the BDNF promoter region was maintained at least until rats reached maturity. These results indicate that epigenetic changes associated with BDNF may underlie hippocampal dysfunction that is induced by early-life exposure to excess maternal fructose consumption.-Yamazaki, M., Yamada, H., Munetsuna, E., Ishikawa, H., Mizuno, G., Mukuda, T., Mouri, A., Nabeshima, T., Saito, K., Suzuki, K., Hashimoto, S., Ohashi, K. Excess maternal fructose consumption impairs hippocampal function in offspring via epigenetic modification of BDNF promoter.

Background A recent study has reported that incidence of chronic kidney disease (CKD) is higher in evacuees, but the molecular mechanism still remains unclear. One plausible hypothesis is a change in vascular function following to psychological distress. In order to assess molecular mechanisms underlying this association, we examined whether cardiovascular disease (CVD)-associated miRNAs (miR-126, miR-197, and miR-223) were associated with CKD among Japanese elderly survivors after an earthquake. Methods We analyzed 1385 individuals (670 men and 715 women) who participated in a post-disaster health check-up after the Great East Japan Earthquake, which occurred in 2011. The check-up involved collection of information about lifestyle, clinical history, the degree of housing damage, and baseline measurement of the estimated glomerular filtration rate. Expression levels of miRNAs were determined using real-time polymerase chain reaction. Estimated glomerular filtration rate (eGFR) was calculated using sex, age, and serum creatinine. CKD was defined as eGFR 2. The multivariable regression analyses were performed to examine the associations between CVD-associated miRNAs and CKD after adjusting potential confounders. Results Mean age (standard deviation) of participants with normal kidney function and CKD was 62.7 (10.6) and 71.9 (8.1) years, respectively. Expression levels of these miRNAs in participants with CKD were significantly lower than normal kidney function (all p p = 0.04). Conclusions This cross-sectional study suggested that CVD-associated miRNAs were an important factor of CKD in an elderly Japanese population after earthquake. Future studies need to examine this association in longitudinal dataset.

Background Higher intake of fruits and vegetables is associated with reduced risk of specific types of cancer and of cardiovascular disease (CVD), but the protective role of the vitamins contained in fruits and vegetables on CVD is controversial. This discrepancy can raise the question of the effects of antioxidants in vitamins on CVD. Recently, we reported that higher vegetable intake was significantly associated with the decreased DNA methylation level of ATP-binding cassette transporter A1 (ABCA1), a gene associated with HDL-cholesterol metabolism. Objective We investigated whether ABCA1 DNA methylation mediates an effect of dietary vitamin intake on lipid profiles, an important risk factor for CVD, in a Japanese population. Methods A total of 225 individuals (108 men and 117 women) with no clinical history and no drug use for dyslipidemia participated in this cross-sectional study. We used the pyrosequencing method to measure the ABCA1 DNA methylation levels at 8 CpG sites, and we used mean DNA methylation level in statistical analysis. Dietary vitamin intake was assessed with the FFQ and adjusted for the residual method. Results In women, higher dietary vitamin intake [vitamin A, β-carotene, folic acid, vitamin C (VC), vitamin D, and vitamin E] was significantly associated with lower mean ABCA1 DNA methylation levels (P = 0.004, 0.03, 0.005, 0.001, 0.03, and 0.04, respectively). In addition, in women, we found a significant inverse association between mean ABCA1 DNA methylation and HDL cholesterol (P = 0.04) but not for other lipid indexes. Mediation analysis showed a significant indirect effect of VC intake on HDL cholesterol through ABCA1 DNA methylation level in women (P = 0.04). Conclusions Although this study does not prove causality, the results suggest that ABCA1 DNA methylation mediates the protective effect of VC on HDL cholesterol in women, which could offer a novel biological mechanism in CVD prevention.

We describe an approach for using the flagella axoneme as the basis for biological self-assembling protein nanoarrays. The axoneme is the insoluble protein core of the eukaryotic flagellum or cilium. By attaching a protein of interest to particular axonemal proteins, it is possible to exploit the intraflagellar transport system to incorporate those proteins into the axoneme as it assembles. Using the axoneme as a protein array confers several advantages, such as high protein loading capacity compared to other bioparticle systems; genetically programmed self-assembly without the need for any linking steps; single-step purification of particles without the need for cell lysis, allowing retention and re-use of biomass; and choice of isolating the particle as a membrane enclosed vesicle or as an exposed protein array. Here we test several potential axonemal proteins as adaptor proteins, using green fluorescent protein as a test case. We find that FAP20 is an ideal scaffold protein for this purpose in that it shows high incorporation and uniform localization. We verify that FAP20-GFP constructs are stably associated with the axoneme during purification and storage, that the GFP moiety can be released by protease cleavage, and that the flagellar array can be easily encapsulated in aqueous-oil emulsion droplets for use in microfluidic assays.

Aims Fructose may play a crucial role in the pathogenesis of metabolic syndrome (MetS). However, the pathogenic mechanism of the fructose-induced MetS has not yet been investigated fully. Recently, several reports have investigated the association between mitochondrial DNA (mtDNA) and MetS. We examined the effect of fructose-rich diets on mtDNA content, transcription, and epigenetic changes. Main methods Four-week-old male Sprague-Dawley rats were offered a 20% fructose solution for 14 weeks. We quantified mRNAs for hepatic mitochondrial genes and analyzed the mtDNA methylation (5-mC and 5-hmC) levels using ELISA kits. Key findings Histological analysis revealed non-alcoholic fatty liver disease (NAFLD) in fructose-fed rats. Hepatic mtDNA content and transcription were higher in fructose-fed rats than in the control group. Global hypomethylation of mtDNA was also observed in fructose-fed rats. Significance We showed that fructose consumption stimulates hepatic mtDNA-encoded gene expression. This phenomenon might be due to epigenetic changes in mtDNA. Fructose-induced mitochondrial epigenetic changes appear to be a novel mechanism underlying the pathology of MetS and NAFLD.

Maternal fructose consumption affects the metabolic functions of offspring later in life. However, the molecular mechanism remains poorly understood. Differences of microRNA expression profile and DNA methylation status are a candidate mechanism to explain the developmental programming that contributes to the development of a metabolic disorder. This study examined the transgenerational effect of maternal fructose consumption from the perspective of epigenetic modification. To do this, we collected serum and liver tissues from male offspring rats that were exposed to maternal distilled water or 20% fructose water during gestation and lactation. A decreased serum high-density lipoprotein cholesterol (HDL-C) level was observed in the offspring of fructose-fed dams at postnatal day (PD) 160. Given research indicating a role of liver X receptor alpha (LXRA) in cholesterol metabolism, we analyzed Lxra expression. Real-time polymerase chain reaction analysis demonstrated that offspring that were delivered from fructose-fed dams exhibited decreased Lxra gene expression in their liver tissue. There is a well-established association between Lxra expression and the level of DNA methylation and miR-206 expression. Pyrosequencing assays revealed no differences in the level of DNA methylation in the Lxra promoter region, whereas miR-206 expression was increased in the liver at PD 60 and 160. Our data indicate that early-life exposure to maternal fructose results in changing of miR-206 expression level in the liver that suppresses the expression of Lxra. This phenomenon may be associated with the decreased serum HDL-C level in offspring.

Dietary intake of vegetables is one of the key lifestyle factors associated with preventing cancer and cardiovascular disease (CVD). Although previous studies have provided evidence that dietary factors can alter global DNA methylation levels in humans, little work has been done on dietary factors influencing methylation levels of specific genes associated with CVD. The aim of this study was to examine whether dietary intake of vegetables was associated with adenosine triphosphate-binding membrane cassette transporter A1 (ABCA1) DNA methylation levels in leukocytes in a Japanese population.This cross-sectional study included 279 Japanese adults (125 men, 154 women) without any clinical history of cancer, stroke, or ischemic heart disease. ABCA1 DNA methylation levels in leukocytes were measured using a pyrosequencing method. Information on dietary vegetable intake was obtained from the validated food frequency questionnaire.Mean ABCA1 DNA methylation levels in men and women were 35.6% ± 6.5% and 36.9% ± 6.7%, respectively. In women, multivariable linear regression analysis showed that the group with the highest dietary vegetable intake (carrot, broccoli, pumpkin, and all vegetables) showed significantly lower levels of ABCA1 DNA methylation than the lowest intake group (P = 0.04,0.001, 0.001, and 0.02, respectively). No significant association was observed between dietary intake of vegetables and DNA methylation levels in men.High dietary intake of vegetables was associated with decreased ABCA1 DNA methylation levels in Japanese women. This may contribute to a better understanding of the protective effects of dietary vegetable intake on CVD.

Global fructose consumption is on the rise; however, maternal high-fructose intake may have adverse effects on offspring. We previously demonstrated that excessive fructose intake by rat dams altered steroidogenic gene transcription in the hippocampus of offspring. Herein, we examined how maternal high-fructose intake influences the regulation of adrenal glucocorticoid levels in offspring. Rat dams received 20% fructose solution during gestation and lactation. After weaning, the offspring were provided normal water. Maternal high-fructose intake did not alter mRNA expression levels of adrenal corticosterone-synthesizing and corticosterone-inactivating proteins or the circulating adrenocorticotropic hormone levels of offspring at postnatal day (PD) 21; however, it increased circulating corticosterone levels and decreased mRNA and protein levels of adrenal 5α-reductase type 1 and 11β-hydroxysteroid dehydrogenase type 2 in offspring at PD160. Furthermore, maternal high-fructose intake enhanced DNA methylation of the adrenal 5α-reductase 1 promoter region in PD160 offspring. Thus, maternal high-fructose intake was found to affect adrenal steroid hormone clearance in adult offspring - at least in part - through epigenetic mechanisms.

Background MicroRNAs are present not only in exosomes but also in high-density lipoprotein (HDL) and have the potential as biomarkers for various diseases. Various purification methods have been developed to quantify HDL-miRNAs; however, they are unsuitable for clinical applications. Therefore, we aimed to establish a simpler analytical method to quantify HDL-miRNAs for clinical applications. Methods We purified HDL fraction from pooled plasma using a three-step protocol consisting of ultracentrifugation, phosphotungstic acid/MgCl2 precipitation and desalting/buffer exchange followed by the quantification of HDL-miRNAs by quantitative real-time PCR. In order to establish a method to quantify HDL-miRNAs by quantitative real-time PCR, we prepared standard curves for miR-223 and miR-92. The HDL-miRNAs of 10 volunteers were assessed. Results Exosomes and LDL were not detected in the purified HDL fraction. Furthermore, we confirmed that only HDL was purified and that the HDL recovery rate of our method was at least approximately 50%. The threshold cycle values of miR-223, miR-92, miR-146a and miR-150 in the same subject were 32.11 ± 0.58, 32.50 ± 0.35, 34.30 ± 0.70 and 34.91 ± 0.77, respectively ( n = 10). The coefficient of variation values for these miRNAs were 1.08–2.21%. In addition, the standard curve for the quantitative analysis of miRNAs showed high linearity (30–30,000 copies/ μL) with a correlation coefficient of >0.99. The concentrations of HDL-miR-223 and HDL-miR-92 in the plasma of 10 subjects were 1.98 ± 0.32 and 0.90 ± 0.14 copies/mL (×104). Conclusions We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.

Background Circulating microRNAs (miRs) may be promising biomarkers for several diseases. We previously found that miR-122 can function as a biomarker for non-alcoholic fatty liver disease (NAFLD). However, little is known regarding the time course of circulating miR-122 levels during the development of NAFLD. Here, we examined circulating miR-122 levels using a rat model of NAFLD. Methods To clarify changes in serum levels of miR-122 during development of NAFLD, experimental rats were fed a high-fat diet (HFD) for 2–10 weeks, while control rats received standard chow. Serum and liver tissue was collected from all animals at 2, 6, and 10 weeks of feeding. Clinical laboratory parameters (cholesterol, TG, AST, ALT, NEFA) were determined by biochemistry analyzer. Hepatic lipid accumulation was estimated by Oil red O staining. Circulating miR-122 levels were then measured by real-time polymerase chain reaction. Results Over the 10 weeks of feeding, body weight, total liver lipids, and liver and serum triacylglycerol were increased in the HFD group compared to the control group. However, no significant changes in serum alanine aminotransferase activity were observed, suggesting that NAFLD status was mild. In contrast, we observed drastic up-regulation of circulating miR-122 levels. Our findings suggest that serum miR-122 level is indeed useful for assessing early NAFLD and might be superior to clinical markers traditionally used to monitor hepatic disease.

Hippocampal functions such as neuronal protection and synapse formation are positively modulated by neurosteroids, which are synthesized de novo within the brain. However, the mechanisms regulating neurosteroidogenesis remain unclear. Fructose, which is used as a sweetener, affects steroid hormone synthesis in peripheral endocrine organs. This monosaccharide can penetrate the blood-brain barrier and impair hippocampal function. Also, fructose is secreted into milk and is thus delivered to the fetus. Based on these observations, we hypothesized that the hippocampal neurosteroidogenesis in the offspring may be affected by maternal fructose consumption. Female rats were fed with normal water or 20% fructose solution during gestation and lactation. Maternal calorie intake did not change significantly, and no significant change in body weight was observed. The levels of messenger RNAs (mRNAs) for steroidogenic enzymes and proteins in the hippocampus of the offspring were analyzed by real-time reverse transcriptase polymerase chain reaction. Maternal fructose consumption during gestation and lactation increased mRNA levels of P450(11β)-2, 11β-HSD-2, and 17β-HSD-1 in the offspring hippocampus, and reduced levels of mRNAs for StAR, PBR, and 17β-HSD-3. Maternal fructose consumption might influence hippocampal neurosteroidogenesis in offspring.

An efficient two-step procedure for the preparation of a new type of 1H-isoindoles, i.e., N-(3-alkyl-1-aryl- or 1,3-diaryl-1H-isoindol-1-yl)-O-methylhydroxylamines 5, from readily available aryl(2-bromophenyl)methanones 1 has been developed. Aryl(2-bromophenyl)methanone O-methyloximes 2, derived from the corresponding ketones, were treated with BuLi in Et2O at 0° to generate novel lithium compounds, aryl(2-lithiophenyl)methanone O-methyloximes 3, which were allowed to react with nitriles to give the desired products 5 in moderate-to-fair yields.

資料番号: SA1004962000

The chlorination process utilizing ammonium chloride as chlorination reagent has been employed for the recovery of elements with emphasis on indium from bearing alloy through vaporization of the chlorides. It was found that the chlorination took place by the heating of the model sample in the presence of ammonium chloride. The influence of the process parameters, such as reaction temperature and gas flow rate, has been investigated. The addition of activated carbon resulted in the adsorption of chloride on the carbon surface. The present result indicates the possibility of the recovery process through transport of the target element from the sample to pores of activated carbon. (doi:10.2320/matertrans.M2013206) The development of recovery process of valuable metals from wastes is of great importance in the view of resource securities. Conventional processes are based on the technique of hydrometallurgy, where target elements are dissolved in e.g., acid and enriched by various types of extracting reagent, which have been developed in the past decades. As for the pyrometallurgical processes, the chlorination metallurgy is one of the attractive methods for the extraction of target elements, as it enables the spatial separation of the elements in a single step according to the difference in the vapor pressure of the chlorides. So far, series of basic studies have been reported in literature, 1­8) in which chlorine or hydrogen chloride gases are mostly used as chlorination reagent. In the view of the practical application of the processes, however, one has to pay special attention for the handling of the corrosive gases. Ammonium chloride is an alternative chlorination reagent, which is utilized, for instance, in the laboratory-scale synthesis of rare earth chlorides. 9) The handling is much easier than the gaseous reagents, so that the utilization of ammonium chloride is an attractive way. Recently, Itoh et al. demonstrated that neodymium in Nd­Fe­B magnet scrap could be selectively chlorinated. 10) In our previous studies 11,12) we have reported the results of the pyrometallur- gical chlorination treatment utilizing ammonium chloride for the recovery of indium from wastes. It has been demonstrated that indium can be recovered from dental metal recycling sludge as well as indium tin oxide, transparent conductor, deposited on glass surface. The reaction is apparently described as In2O3ðs Þþ 6NH4ClðsÞ ! 2InCl3ðg Þþ 6NH3ðg Þþ 3H2Oðg Þð 1Þ One of the advantages of the proposed process is that the chemicals used in the reaction can be considerably reduced when the position of the target element is known: the chlorination reagent is directly put onto the sample specimen where the target element exists, e.g., glass surface in the case of ITO-deposited glass. In contrary, the entire sample should be immersed in treatment bath in conventional hydro- metallurgical processes, which may lead to higher cost in case that very small amount of the target element exists in the sample, e.g., in the form of thin film. In the present paper, we address the chlorination treatment of bearing alloy made of lead, indium and tin, which is plated on substrate metal for the improvement of tribological property. The influence of the process parameters has been studied and discussed. 2. Experimental Procedure 2.1 Materials As for the material for the chlorination treatment, model samples were kindly supplied from Daido metal Co., Ltd. A sample specimen has iron substrate (20mm © 50mm © 2mm) above which copper, then nickel intermediate layer and finally Pb­In­Sn alloy (Pb:In:Sn = 8:1:1 in mass%) are plated. The nickel layer is plated in order to avoid the diffusion of the elements of the upper layer into the inner copper layer. The average contents of Pb, Sn and In in a sample specimen were 21, 2.7 and 2.7mg, respectively. Given amount of ammonium chloride (99.5%, Wako pure chem. Co., Ltd.) was put onto the sample surface. Typical composition of NH4Cl, defined as 100 � mNH4Cl=mSample, where mNH4Cl and mSample are the mass of ammonium chloride and sample specimen, respectively, was 6mass%. In order to ensure the sufficient contact between NH4Cl and alloy surface, small amount of ethanol was poured on the surface, which was then dried.

Neurosteroids, steroidal hormones synthesized de novo from cholesterol within the brain, stimulate hippocampal functions such as neuron protection and synapse formation. Previously, we examined the effect of maternal fructose on the transcriptional regulation of neurosteroidogenic enzymes. We found that the mRNA expression level of the steroidogenic acute regulatory protein (StAR), peripheral benzodiazepine receptor (PBR), cytochrome P450(11β), 11β-hydroxysteroid dehydrogenase (HSD), and 17β-HSD was altered. However, we could not determine whether maternal fructose intake played a role in the gestation or lactation period because the dam rats were fed fructose solution during both periods. Thus, in this study, we analyzed the hippocampi of the offspring of dams fed fructose during the gestation or lactation period. Maternal fructose consumption during either the gestation or lactation period did not affect the mRNA levels of StAR, P450(17α), 11β-HSD-2, and 17β-HSD-1. PBR expression was down-regulated, even when rats consumed fructose during the lactation period only, while fructose consumption during gestation tended to activate the expression of P450(11β)-2. We found that maternal fructose intake during gestation and lactation differentially affected the expression of hippocampal neurosteroidogenic enzymes in the offspring.

Background Recently, several studies have shown that microRNAs are present in high-density lipoprotein, and high-density lipoprotein-microRNA may be a promising disease biomarker. We investigated the stability of high-density lipoprotein-microRNAs in different storage conditions as this is an important issue for its application to the field of clinical research. Methods microRNAs were extracted from the high-density lipoprotein fraction that was purified from the serum. miR-135 a and miR-223, which are known to be present in high-density lipoprotein, were quantified by quantitative real-time PCR. The influence of preanalytical parameters on the analysis of high-density lipoprotein-miRNAs was examined by the effect of RNase, storage conditions, and freezing and thawing. Results The concentrations of microRNA in high-density lipoprotein were not altered by RNase A treatment (0–100 U/mL). No significant change in these microRNAs was observed after storing serum at room temperature or 4℃ for 0–24 h, and there was a similar result in the cryopreservation for up to two weeks. Also, high-density lipoprotein-microRNAs were stable for, at least, up to five freeze–thaw cycles. Conclusions These results demonstrated that high-density lipoprotein-microRNAs are relatively resistant to various storage conditions. This study provides new and important information on the stability of high-density lipoprotein-microRNAs.

In this paper, we describe the use of ozone microbubbles in photoresist removal from silicon wafers. Ozonized water has attracted much attention as an environmental friendly cleaning method in semiconductor manufacturing. However, it would be desirable to enhance the oxidative ability of ozonized water for practical application. The existence of microbubbles in ozonized water has been shown to significantly enhance the photoresist removal rate due to an elevated dissolved ozone concentration (approximately 2.5 times that of ordinary ozone bubbling) and a direct effect of the microbubbles (removal rate is approximately 1.3 times faster than water with the same concentration of dissolved ozone without microbubbles). Additionally, the ozone microbubble solution was able to effectively remove a high-dose ion-implanted photoresist, which is extremely resistant to removal by ozonized water and other wet chemicals because of its amorphous carbon-like layer, or "crust". Electron spin resonance experiments were also performed without the influence of serious metal contamination and indicated the presence of hydroxyl radicals, which are thought to be formed by interaction of ozone with hydroxide ions adsorbed at the gas-water interface upon collapse of the microbubbles. The hydroxyl radicals play an important role in photoresist removal by the ozone microbubble treatment.

We developed a sample holder for in situ measurement of hydrogen absorbing alloy. In order to prevent the hydrogen absorption by vanadium, copper is coated with 2 μm thickness on inner surface of the vanadium holder. The effect of copper coating and the performance of the holder were evaluated by neutron diffraction and PDF profiles. The lattice parameters a and c of La2Ni7 with Ce2Ni7-type structure were refined as 0.505921(4) and 2.468608(4) nm by Rietveld analysis. The Cu–Cu correlation peak around r = 0.255 nm was not observed in the PDF profile. Thus the holder is useful for in situ measurement of hydrogen absorbing alloy. The diffraction and PDF profiles of La2Ni7Dx (0

Vascular endothelial growth factor (VEGF) is a disulfide-linked dimeric glycoprotein that enhances vascular permeability, induces chemotaxis and activation of monocytes/macrophages, and promotes growth of vascular endothelial cells. Furthermore, VEGF is a multifunctional cytokine, which influences neural cells directly, enhancing neuronal survival, axonal outgrowth, and Schwann cell proliferation. So far studies of the skin of amyotrophic lateral sclerosis (ALS) have shown unique pathological and biochemical abnormalities in collagen, elastic fibers, and the ground substance. However, the expression of VEGF in ALS skin has not previously been studied. We made a quantitative immunohistochemical study of the expression of VEGF in the skin from 15 patients with ALS and 15 control subjects. VEGF immunoreactivity was markedly positive in the epidermis and moderately positive in some dermal blood vessels and glands in ALS patients. These findings became more conspicuous as ALS progressed. The optical densities for VEGF immunoreactivity of the epidermis in ALS patients were significantly higher (p

In order to investigate the effect of mental stress on the tryptophan metabolism, a stress model of visual display terminals work was loaded on healthy students. Before and just after stress, plasma samples were collected and serotonin, tryptophan and kynurenine were assayed. The plasma concentration of serotonin was increased by visual display terminals work. Both Trp and Kyn concentrations were decreased after stress load. These results suggest that serotonin formation from tryptophan may be accelerated, resulting in the suppressing of kynurenine metabolism from tryptophan. Furthermore, when these subjects were divided into two groups, one group of those accustomed to the personal computer and another group of those not accustomed, the changes of above amines and scores of Profile of Mood States in the latter group were larger than that in the former group. These results also suggest that the activation of the person with a strong psychological load is larger than that of a weak person.

We established a highly reproducible, simple and rapid simultaneous measurement method in which blood plasma can be directly injected into high-performance liquid chromatography (HPLC) without conducting conventional extraction of kynurenine (Kyn) and tryptophan (Trp) from blood plasma. In this method, Kyn and Trp were first separated from proteins present in blood plasma using a pre-column. Then, by column-switching, the separated Kyn and Trp were automatically injected into a separation column. The recoveries of Kyn and Trp determined in blood plasma by this method were 100.0 ± 0.9 and 100.0 ± 1.4%, respectively.

DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status.

From a gaseous mixture of Fe(CO)5 and CS2, sedimentary aerosol particles involving organometal compounds were produced under UV light irradiation at 313 nm. Chemical composition of the sedimentary aerosol particles was controlled by a magnetic field and by post-exposure with UV light. By applying a magnetic field up to 5 T, photochemical reactivity of Fe(CO)5 molecules was promoted and the amount of chemical species originating from Fe(CO)5 increased with increasing magnetic field. By irradiating UV light upon the deposited particles, solid-state photochemical reactions took place in the particles, and CO groups bonded to Fe atom were effectively evolved from the sedimentary aerosol particles. These methods can be utilized to modify chemical composition of the metal-containing sedimentary particles.

A systematic approach to understanding the safety fundamentals of Li-ion batteries was undertaken. Firstly, we present thermal characterization experiments of charged prismatic polymer lithium-ion batteries (PLBs). These cells, at different state of charge (SOC), were tested inside an accelerated rate calorimeter (ARC) to determine the onset-of-thermal runaway (OTR) temperatures. In addition, the thermally activated components of these cells were followed by monitoring both the impedance (at 1 kHz) and the open circuit voltage (OCV) as a function of temperature. An increase in the impedance was observed at around 133 °C corresponding to the polyethylene separator shutdown. Secondly, an original in situac impedance measurement was performed over a wide range of frequencies instead of 1 kHz when the battery was heated from ambient to 130 °C. Resulting impedance spectra were modeled using an appropriate equivalent circuit. It is concluded that the high frequency and the low frequency semicircles observed in the impedance spectra are due to processes occurring the anode/electrolyte and cathode/electrolyte interfaces, respectively. The activation energy E a was found in the ranges of 0.4–0.6 and 0.36–0.53 eV, for cathode and anode processes, respectively. In addition, it is assumed that change in the electrolyte composition is the main factor responsible for the rise in the cell impedance at high temperatures.

The title compounds (V) are efficiently obtained in a two-step procedure starting from readily available aryl(2-bromophenyl)methanones (I).

A 85-year-old female was admitted to our hospital because of a fever and unconsciousness. Three days prior to admission, she had been diagnosed to have influenza A, and oseltamivir was therefore prescribed. The symptoms due to the influenza infection, including the fever, thereafter rapidly resolved. She regularly took 10 mg zopiclone for insomnia before sleeping. On the day of admission, she was drowsy with fever. Chest radiography showed bilateral massive infiltration of the lungs. Chest CT images revealed multilobar and nodular infiltration on both lungs. She underwent the partial gastrectomy 10 years ago due to the gastrointestinal bleeding. After that, gastro-esophageal reflux syndrome was occurred in the patient. A bronchoscope was easily inserted into the trachea without anesthesia. Aspirated saliva was found in trachea. Based on her post-gastrectomy state, post-gastrectomy aspiration pneumonia was diagnosed. Sulbactam/ampicillin (SBT/ABPC) (6 g) was administered daily, which led to reduced inflammatory responses and lung infiltration. Although influenza itself is sometimes critical for the elderly, careful attention should be paid to subsequent bacterial infections in patients who are at risk for developing aspiration pneumonia.

The function of ERM (ezrin/radixin/moesin) proteins as general cross-linkers between actin filaments and plasma membranes is regulated downstream of Rho, through the transition between active and inactive forms. To directly examine the conformational change between the active and inactive forms of ERM proteins, we applied low-angle rotary-shadowing electron microscopy to the radixin molecules, wild-type, T564A-non-phosphorylated-type, and T564E-phosphorylated-type, since most of the active forms are reportedly stabilized in cells by the C-terminal threonine phosphorylation. As a result, the T564A- and wild-type radixin molecules yielded the globular closed forms, ∼8-14 nm in diameter, with some striations on their surfaces. In contrast, the T564E-radixin molecules tended to take elongated open forms, in which two globular structures measuring ∼8 nm and ∼5 nm in diameter were associated with both ends of the filamentous structures. The filamentous structure took either a ∼20-25 nm-long straight course or a folded course. Taken together with the biochemical and the crystal structural results obtained to date, the closed and open forms represent the inactive and active forms of radixin as cross-linkers between actin filaments and plasma membranes.

Laminated lithium-ion polymer cells exhibit the attractive characteristics of high energy density, high working voltage, and great flexibility in configuration. Previous work demonstrated the impressive cycle life and endurance performance of this type of cell in a simulated space environment (vacuum, radiation, and vibration). To verify the potential for space application of these laminated lithium-ion polymer cells, this work further investigates the thermal stability of 0.60 Ah-class laminated lithium-ion polymer cells using accelerated-rate-calorimetry (ARC) measurement. Because these cells are expected to operate at a taper voltage of 3.95 V for space applications, two lithium-ion polymer cells were fully charged to 3.8 and 4.0 V, and then applied for ARC measurement. The examined cells exhibited a high onset of thermal runaway temperature (OTRT) of 148 ◦ C. In contrast to common lithium-ion cells with liquid-state electrolytes, OTRT of the examined cells was almost independent of the state of charge in the examined taper-voltage range. Furthermore, cell internal shorting did not occur until at a high temperature of 183 ◦ C, indicating good thermal stability of this type of cell.

Both mizoribine (MZR) and mycophenolate mofetil (MMF) are immunosuppressive agents that inhibit the proliferation of lymphocytes selectively, via inhibition of IMPDH. MZR is a nucleoside of the imidazole class, isolated from the culture medium of the mold Eupenicillium brefeldianum M-2166. Although this compound has been found to have weak antimicrobial activity against Candida albicans, it has proved ineffective against experimental candidiasis. Unlike azathioprine, this compound is not taken up by nucleic acids in the cell. Instead, after phosphorylation MZR-5 -monophosphate inhibits GMP synthesis by the antagonistic blocking of IMPDH (Ki = 10(-8)M) and GMP- synthetase (Ki =10(-5) M). The drug has been found to inhibit both humoral and cellular immunity, and on this basis it was developed in Japan as an immunosuppressant. MZR has been shown in animal experiments to lack oncogenicity, and has been shown clinically to be associated with a low incidence of severe adverse reactions. MZR has been registered in Japan for the prevention of rejection in renal transplantation, and for the treatment of lupus nephritis, rheumatoid arthritis and the nephrotic syndrome. MMF is the morpholinoethyl ester prodrug of mycophenolic acid (MPA), which was first isolated in 1896 from the culture media of several Penicillium species. MPA has been evaluated for its unique properties as an anticancer, antiviral, antifungal and antibacterial agent, as well as for its therapeutic use in psoriasis and rheumatoid arthritis. MMF was designed to enhance the oral bioavailability of the parent compound. After beneficial effects were observed in animals, the clinical efficacy of MMF as an immunosuppressant in renal transplantation was studied in the United States. In 1995 the US Food and Drug Administration (FDA) approved the use of MMF for the prevention of rejection in renal transplantation, the drug also available on a number of European markets.

Background: Serum ornithine carbamoyltransferase is a diagnostic marker of hepatic disorders due to its localization in periportal mitochondria. Methods: We have developed a new method for the determination of serum ornithine carbamoyltransferase. It is based on the reverse reaction of ornithine carbamoyltransferase, using ornithine-ketoacid aminotransferase, ∆1-pyrroline-5-carboxylate dehydrogenase and glutamate dehydrogenase, which together convert citrulline through ornithine to glutamate. The glutamate is then quantitatively measured using glutamate oxidase and Trinder's reagent. Results: The results obtained by this method agreed well with those obtained using the diacetylmonoxime method as a gold standard [correlation coefficient (r) = 0·973 P1-pyrroline-5-carboxylate) were eliminated by the initial futile reaction. The new method appears to be more accurate at low levels of ornithine carbamoyltransferase activity than the diacetylmonoxime method. Conclusions: Here we report a new method for serum ornithine carbamoyl-transferase assay which might be useful for clinical diagnosis of hepatic disorders, including hepatic cancer.

We examined the effect of dietary taurine on the concentrations of serum cholesterol and apolipoprotein in lipoprotein fractions of Six-month-old ovariectomized, which were used as a model of hypercholesterolemia in postmenopausal woman, or sham operated rats. Taurine significantly reduced the serum total and low-density lipoprotein cholesterol concentrations only in the ovariectomized rats. In contrast, taurine significantly lowered the serum apolipoprotein B concentration and serum very low-density lipoprotein-apolipoprotein E concentration only in the sham operated rats. The serum total and high density lipoprotein-apolipoprotein E concentrations were significantly lower in the rats fed taurine than in those fed the control diet regardless of whether they had undergone ovariectomy. The esterified cholesterol level in the liver was significantly lower and the level of hepatic cholesterol 7α-hydroxylase activity was significantly higher in the rats fed taurine than in those fed the control diet. The total bile acids concentration in the feces and intestinal contents of rats fed taurine were significantly higher than those in rats fed the control diet regardless of whether they had undergone ovariectomy. In the sham-rats, taurine accelerated bile acid synthesis and excretion, thereby increasing cholesterol consumption. The increased cholesterol consumption might be compensated by accelerating cholesterol synthesis and/or reducing the synthesis and release of very low-density lipoprotein from the liver. But in the ovariectomized rats, although taurine also accelerated bile acid synthesis and excretion, cholesterol demand might be compensated by excess cholesterol in the blood.