Search

Regorafenib is a standard salvage line therapy used for advanced colorectal cancer (CRC). Recently, trifluridine/tipiracil (TFTD) plus bevacizumab also showed promising efficacy as a salvage line therapy for advanced CRC. However, the efficacy and safety of regorafenib for patients with advanced CRC who have previously received TFTD plus bevacizumab is unclear. We retrospectively collected clinicopathologic data from patients with advanced CRC who received regorafenib after TFTD plus bevacizumab in multiple institutions between April 2017 and June 2020.Thirty-four advanced CRC patients who received regorafenib were analyzed. The median age was 66.5 (range 43-81 years), 11 patients were male, and all had an ECOG performance status(PS) of 0 or 1. Twenty-two patients had left-sided tumors, 18 patients had RAS mutants, and 1 patient had a BRAF V600E mutation. The response rate was 0%, and the disease control rate was 31%. The median progression-free survival was 70 days (95% CI: 56-91), and the overall survival was 233 days (95% CI: 188-324). Treatment was discontinued in 32 patients, and 28 (82%) discontinued treatment due to progressive disease. The major grade 3 and4 toxicities were proteinurea (29%), hypertension (26%), hand-foot syndrome(15%), and platelet decrease (6%). Regorafenib after TFTD plus bevacizumab showed efficacy similar to that of the previous study, and no new adverse events were observed., (© 2023. The Author(s).)

Introduction: Among the fatty acid ethanolamides (FAEs), oleoylethanolamide (OEA), linoleoylethanolamide (LEA), and palmitoylethanolamide (PEA) are reported to be involved in feeding regulation. In particular, OEA is well characterized as a satiety signal. Following food consumption, OEA is synthesized from oleic acid (OA) via an N -acyl phosphatidylethanolamine-specific phospholipase D-dependent pathway in the gastroenterocytes, and OEA induces satiety by recruiting sensory fibers. Thus, we hypothesized that dietary OA is an important satiety-inducing molecule. However, there has been no direct demonstration of the effect of dietary OA on satiety induction without the influence of the endogenous biosynthesis of OA from stearic acid (SA) or other FAEs., Methods: In this study, we used two experimental diets to test our hypothesis: (i) an OA diet (OAD; 38.4 mg of OA/g and 7.2 mg of SA/g) and (ii) a low OA diet (LOAD; 3.1 mg of OA/g and 42.4 mg of SA/g)., Results: Relative to mice fed the OAD, mice fed the LOAD for two weeks exhibited reduced levels of jejunal OEA but not jejunal LEA and PEA. The LOAD-fed mice showed an increase in food intake and body weight gain. Moreover, LOAD-induced increase in food intake was immediately observed after the switch from the OAD, whereas these effects were diminished by the switch back to the OAD. Furthermore, treatment with OA and OEA diminished the effects of LOAD on food intake., Conclusion: Collectively, these results show that dietary OA is a key factor in the reduction of food intake and increase in satiety mediated by OEA signaling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Igarashi, Iwasa, Hayakawa, Tsuduki, Kimura, Maruyama and Yoshikawa.)

Background: Paclitaxel or nanoparticle albumin-bound paclitaxel combined with ramucirumab (PTX/nab-PTX + RAM) is widely used as second-line chemotherapy for advanced gastric cancer (AGC), but severe neutropenia often develops with this regimen. Although previous studies have reported that severe neutropenia is a favorable prognostic factor in cancer chemotherapy, it is unclear in AGC patients receiving PTX/nab-PTX + RAM. In addition, the risk factors for early-onset of severe neutropenia (EOSN) still remain unknown., Methods: Among patients with AGC treated with PTX/nab-PTX (on day 1, 8, and 15) + RAM (on day 1 and 15) every 4 weeks as second-line therapy from January 2017 to June 2020, those with grade 0 or 1 neutropenia before the treatment were retrospectively studied. Blood tests were performed on the day of treatment each time, and disease progression was primarily determined by computed tomography every 8±2 weeks. EOSN was defined as grade 4 neutropenia that occurred during the first 28 days. The risk factors for EOSN were investigated using multivariate logistic regression analysis. Progression-free survival (PFS) and overall survival (OS) in patients with and without EOSN were investigated using multivariate analysis with a Cox proportional hazards model., Results: The clinical data of 244 patients were analyzed. EOSN was observed in 51 (20.9%) patients. Multivariate analysis identified the following five risk factors for EOSN: age ≥65 years [odds ratio (OR), 2.75], presence of primary tumor (OR, 2.82), presence of peritoneal metastasis (OR, 2.52), grade 1 neutropenia (OR, 3.32), and high serum level of alkaline phosphatase (OR, 2.34). The PFS was significantly longer in patients with EOSN than in those without EOSN [adjusted hazard ratio (HR), 0.61; 95% CI, 0.41-0.92] and the OS tended to be longer in patients with EOSN than in those without EOSN (adjusted HR, 0.73; 95% CI, 0.47-1.12). HR was adjusted with patient background factors and blood test data considered important as predictive or prognostic factors., Conclusions: EOSN may be associated with favorable outcomes in patients with AGC treated with PTX/nab-PTX + RAM. We should carefully try to treat them keeping the risk factors in mind., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-499/coif). TM received honoraria for lectures from Chugai, Takeda, Yakult, Taiho, Ono, Daiichi-Sankyo, Bristol Myers Squibb, Lilly, and Merck Serano. YY received honoraria for lectures from Sanofi, Nihon Kayaku, Eisai, Bayer, Lilly, Taiho, Daiichi Sankyo, Yakult, Nihon Servier, Asahi Kasei, and Ono. TK received honoraria for lectures from Taiho, Chugai, Ono, and Lilly. TN received honoraria for lectures from Chugai, Takeda, Yakult-Honsha, Taiho, Ono, Daiichi-Sankyo, Bristol Myers Squibb, Lilly, and Merck Serano, Pharmaceutical and a Data Safety Monitoring Board member from Janssen. TM received honoraria for speakers bureaus from Lilly, Taiho, and Bristol-Myers Squibb, and a grant from Taiho outside the submitted work. IH received honoraria for lectures from Chugai, Takeda, and Yakult, and payment as an Advisory Board member from Asahi-Kasei and a Data Safety Monitoring Board member from Chugai, Taiho, Ono, Daiichi-Sankyo, and Merck Serano. The other authors have no conflicts of interest to declare., (2022 Journal of Gastrointestinal Oncology. All rights reserved.)

Background: A trial with trifluridine/tipiracil (FTD/TPI) versus placebo in patients with heavily pretreated metastatic gastric cancer showed that FTD/TPI is effective with manageable toxicity in these patients. However, real-world data on the effects of FTD/TPI in patients with advanced gastric cancer (AGC) are limited., Methods: We retrospectively collected and analyzed the clinicopathological data of patients with AGC who received FTD/TPI monotherapy at our institutions (Kobe City Medical Center General Hospital, Osaka Red Cross Hospital, Himeji Red Cross Hospital, and Kansai Medical University Hospital) between September 2019 and July 2021. Tumor responses were evaluated based on the Response Evaluation Criteria in Solid Tumors, version 1.1. Overall survival (OS) and progression-free survival were estimated using the Kaplan-Meier method., Results: A total of 53 patients were included in the study. The median age was 70 (range, 37-85) years; 39 patients (74%) were men; the numbers of patients with Eastern Cooperative Oncology Group performance status scores of 0, 1, and 2 were 10 (19%), 39 (74%), and 4 (8%), respectively; and 27 patients (51%) had diffuse-type histology. A total of 29 patients (56%) had ascites. Prior nivolumab therapy was administered to 49 patients (92%). The response rate and disease control rate (DCR) were 2% and 35%, respectively. The median progression-free survival was 2.4 months, and OS was 5.8 months. Patients with ascites exhibited significantly shorter OS (8.6 vs 4.7 months, P = .0291) than those without ascites, and DCR (54% vs 18%, P = .0055) was significantly worse in patients with ascites. There was no significant difference in the frequency of adverse events of grade 3 or higher between patients with and without ascites., Conclusion: In a real-world setting, FTD/TPI has similar effectiveness as late-line chemotherapy for patients with AGC, including those who previously had received nivolumab., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Toshihiko Matsumoro received research funding from Ono Pharmaceutical Co, Ltd and Sanofi Co, Ltd; honoraria from Bayer Co, Ltd, Bristol-Myers Squibb Co, Ltd, Chugai Pharmaceutical Co, Ltd, Daiichi Sankyo Co, Ltd, Eli Lilly Japan Co, Ltd, Merck Bio Pharma Co, Ltd, MSD Co, Ltd, Ono Pharmaceutical Co, Ltd, Sanofi Co, Ltd, Taiho Pharmaceutical Co, Ltd, Takeda Co, Ltd, Teijin Pharmaceutical Co, Ltd and Yakult Honsha Co, Ltd. Hironaga Satake received research funding from Ono Pharmaceutical Co, Ltd, Daiichi Sankyo Co, Ltd, Taiho Pharmaceutical Co, Ltd, Takeda Pharmaceutical Co, Ltd, and Sanofi Co, Ltd; honoraria from Bayer Co, Ltd, Bristol-Myers Squibb Co, Ltd, Chugai Pharmaceutical Co, Ltd, Daiichi Sankyo Co, Ltd, Eli Lilly Japan Co, Ltd, Merck Bio Pharma Co, Ltd, MSD Co, Ltd, Ono Pharmaceutical Co, Ltd, Sanofi Co, Ltd, Taiho Pharmaceutical Co, Ltd, Takeda Co, Ltd, and Yakult Honsha Co, Ltd. Hisateru Yasui received honoralia from Taiho Pharmaceutical Co, Ltd. All the remaining authors declare that they have no competing interests., (© The Author(s) 2022.)

Trastuzumab deruxtecan (T-DXd) has shown promising efficacy against HER2- positive advanced gastric cancer (AGC). However, data on its real-world efficacy in AGC patients are insufficient, and the predictive marker of T-DXd is unclear. In this multi-center retrospective study, we collected clinical information of 18 patients with HER2 -positive AGC who received T-DXd after intolerant or refractory responses to at least two prior regimens and analyzed predictive factors. The median age was 71 years (range: 51-85), 13 men were included, and ECOG performance status (PS): 0/1/2/3 was 9/6/2/1. A total of 11 patients (61%) received prior immune checkpoint inhibitors (ICIs), 14 patients were HER2 3+, and 4 patients were HER2 2+/FISH positive. The median trastuzumab (Tmab)-free interval was 7.7 months (range: 2.8-28.6). The overall response rate was 41%, and the disease control rate was 76%. Median progression-free survival (PFS) was 3.9 months (95% CI: 2.6-6.5), and median overall survival (OS) was 6.1 months (95% CI: 3.7-9.4). PFS (6.5 vs. 2.9 months, p = 0.0292) and OS (9.2 vs. 3.7 months, p = 0.0819) were longer in patients who received prior ICIs than in those who had not. PFS (6.5 vs. 3.4 months, p = 0.0249) and OS (9.4 vs. 5.7 months, p = 0.0426) were longer in patients with an 8 month or longer Tmab-free interval. In patients with ascites, PFS (6.5 vs. 2.75 months, p = 0.0139) and OS (9.4 vs. 3.9 months, p = 0.0460) were shorter. T-DXd showed promising efficacy in HER2 -positive AGC patients in a real-world setting. Pre-administration of ICIs and a sufficient Tmab-free interval may be predictive factors of T-DXd efficacy.

Ferroptosis is a form of regulated cell necrosis, as a consequence of Fe(II)-dependent lipid peroxidation. Although ferroptosis has been linked to cancer cell death, neurodegeneration and reperfusion injury, physiological roles of ferroptosis have not been elucidated to date mostly due to the lack of appropriate methodologies. Here, we show that 4-hydroxy-2-nonenal (HNE)-modified proteins detected by a HNEJ-1 mouse monoclonal antibody is a robust immunohistochemical technology to locate ferroptosis in tissues in combination with morphological nuclear information, based on various models of ferroptosis, including erastin-induced cysteine-deprivation, conditional Gpx4 knockout and Fe(II)-dependent renal tubular injury, as well as other types of regulated cell death. Specificity of HNEJ-1 with ferroptosis was endorsed by non-selective identification of HNE-modified proteins in an Fe(II)-dependent renal tubular injury model. We further comprehensively searched for signs of ferroptosis in different developmental stages of Fischer-344 rats from E9.5-2.5 years of age. We observed that there was a significant age-dependent increase in ferroptosis in the kidney, spleen, liver, ovary, uterus, cerebellum and bone marrow, which was accompanied by iron accumulation. Not only phagocytic cells but also parenchymal cells were affected. Epidermal ferroptosis in ageing SAMP8 mice was significantly promoted by high-fat or carbohydrate-restricted diets. During embryogenesis of Fischer-344 rats, we found ferroptosis in nucleated erythrocytes at E13.5, which disappeared in enucleated erythrocytes at E18.5. Administration of a ferroptosis inhibitor, liproxstatin-1, significantly delayed erythrocyte enucleation. Therefore, our results demonstrate for the first time the involvement of ferroptosis in physiological processes, such as embryonic erythropoiesis and aging, suggesting the evolutionally acquired mechanism and the inevitable side effects, respectively., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Nivolumab has changed the treatment of advanced gastric cancer (AGC). Nivolumab shows better outcomes compared to best supportive care among AGC patients who received at least two prior regimens. However, there are no reliable data regarding AGC patients with poor performance status (PS) who received nivolumab. We investigated the efficacy and safety of nivolumab among AGC patients with poor PS., Methods: We retrospectively collected clinicopathologic data from patients with AGC who underwent nivolumab monotherapy at our institution from October 2017 to June 2019., Results: Forty-nine AGC patients who received nivolumab were assessed. Twenty-seven patients had PS 0 or 1 (Good group) and 22 had PS 2 or 3 (Poor group). The median progression-free survival and overall survival durations were 2.0 and 6.0 months in the Good group, respectively, and 1.2 and 2.8 months in the Poor group, respectively. The overall survival was significantly shorter in the Poor group (6.0 vs 2.8 months, p = 0.0255). The disease control rates were 23 and 9% in the Good and Poor groups, respectively. Thirty-three percent of patients experienced immune-related adverse events in the Good group, and 18% in the Poor group., Conclusion: Nivolumab is feasible but insufficient as third- or later-line treatment for AGC patients with poor PS.

Mushrooms are familiar ingredients in Japanese cuisine and large numbers are consumed in Japan. Recently, we reported that the consumption of Japanese mushrooms suppressed the accumulation of visceral fat. The purpose of this study was to examine the alteration of lipid metabolism by Japanese mushrooms consumption in high-fat diet (HFD) mice. Multivariate analysis of serum, liver, adipose tissue, cecal contents, large intestinal and fecal lipids showed differing compositions in the mice that had consumed HFD or HFD supplemented with 3% freeze-dried mushroom mixture (HFMD). There were higher concentrations of diacylglycerol in the adipose tissue, non-esterified fatty acids in the serum, and triacylglycerol in the feces of the HFMD group. These results suggest that mushroom consumption promotes the degradation of lipids in visceral fat and limits the absorption of food lipids. Moreover, the HFMD group demonstrated higher concentrations of phospholipids, some of which contained odd-chain fatty acids. Thus, we speculated that the alteration of lipid metabolism in mice such that mushroom consumption prevent obesity progression, as demonstrated by metabolomic analysis., (© 2020 The Authors.)

Dysregulation of the gut microbiome is associated with dementia. However, the relationship between microbiome-associated metabolites and dementia has yet to be identified. Outpatients visiting a memory clinic in Japan enrolled in this cross-sectional study; 107 subjects were eligible for the study, 25 of which had dementia. We collected demographics, activities of daily living, risk factors, cognitive function, and brain imaging data. The gut microbiome was assessed using terminal restriction fragment length polymorphism analysis. Concentrations of faecal metabolite were measured. We used multivariable logistic regression analyses to identify whether metabolites were independently related to dementia. The concentrations of metabolites were significantly different between subjects with and those without dementia. Every 1 standard deviation increment in faecal ammonia concentration was associated with around a 1.6-fold risk for the presence of dementia. A higher faecal lactic acid concentration was related to a lower risk of dementia, by around 60%. A combination of higher faecal ammonia and lactic acid concentrations was indicative of the presence of dementia, and had a similar predictive value as traditional biomarkers of dementia. Thus, faecal ammonia and lactic acid are related to dementia, independently of the other risk factors for dementia and dysregulation of the gut microbiome.

Recent studies have revealed an association between the dysregulation of the gut microbiome and dementia. However, whether this dysregulation is associated with mild cognitive impairment (MCI), an early stage of cognitive decline, in patients without dementia remains unclear. We performed a cross-sectional analysis to determine the association between the gut microbiome and MCI. Data, including patient demographics, risk factors, cognitive function, and brain imaging, were collected. The gut microbiome was assessed through terminal restriction fragment length polymorphism analysis. Multivariable logistic regression models were used to identify factors independently associated with MCI. Graphical modelling was used to illustrate mutual associations between MCI and identified factors. We analysed 82 patients, 61 of whom exhibited MCI. Patients with MCI had a higher prevalence of Bacteroides. Furthermore, patients with more Bacteroides were more likely to present with white matter hyperintensity and high voxel-based specific regional analysis system for Alzheimer's Disease (VSRAD) scores, indicating cortical and hippocampal atrophy. A multivariable logistic regression analysis revealed that a greater prevalence of Bacteroides was independently associated with MCI. Graphical modelling also showed a close association between Bacteroides and MCI. In conclusion, an increased prevalence of Bacteroides is independently associated with the presence of MCI in patients without dementia.

Background: Epidemiologic observations have raised expectations that the Japanese dietary pattern could promote longer disability-free survival (DFS) times among the Japanese population; however, no previous study has examined this issue., Objective: The aim of this study was to investigate the association between the Japanese dietary pattern and DFS time in the elderly Japanese population., Methods: We analyzed follow-up data covering a 10-y period for 9456 elderly Japanese individuals (aged ≥65 y) participating in a community-based prospective cohort study. Dietary habits were assessed using a food-frequency questionnaire. Based on previous studies, we used 9 food items to calculate the Japanese Diet Index (JDI) score: rice, miso soup, fish and shellfish, green and yellow vegetables, seaweed, pickled vegetables, green tea (1 point for each item if the consumption value was more than or equal to the median, and 0 points otherwise), beef and pork, and coffee (0 points for each item if the consumption value was more than or equal to the median, and 1 point otherwise). Differences in median age at incident disability or death [50th percentile differences (PDs)] according to quartiles (Q1-Q4) of the JDI score were estimated using Laplace regression., Results: During the follow-up period, 4233 (44.8%) incident disability or death events occurred. In addition, a higher JDI score was significantly associated with longer DFS time: compared with the lowest quartile of JDI scores (Q1), the multivariate-adjusted 50th PD (95% CI) was 7.1 (1.8, 12.4) mo longer for Q4. Each 1-SD increase of the JDI score was associated with 3.7 (1.7, 5.7) additional months of life without disability (P-trend < 0.01). No differences were seen in sex or chronic condition (no or ≥1 chronic condition) at baseline. A post hoc analysis showed a larger effect on DFS time when using a modified JDI score without coffee., Conclusion: These results suggest that the Japanese dietary pattern is associated with improved DFS time in the general elderly population., (Copyright © American Society for Nutrition 2019.)

Objective: This study aimed to investigate whether the intake of the 1975 Japanese diet (JD) could reduce the amount of abdominal fat in people with overweight., Methods: Using a single-blind randomized controlled trial, the modern diet (MD) was compared with the 1975-type JD, which is based on the MD but includes five characteristics of the 1975 JD in an enhanced form. Overweight people were randomly assigned to an MD group (n = 30) and a JD group (n = 30). The participants consumed test diets that were provided three times a day for 28 days. Body composition measurements and blood biochemical examinations were performed before and after the test diet intake, and the proportions of change were compared., Results: Those in the JD group had significantly decreased BMI, fat mass, and levels of low-density lipoprotein cholesterol, glycated hemoglobin, and C-reactive protein (P = 0.002, 0.015, 0.014, 0.012, and 0.039, respectively) and significantly increased high-density lipoprotein cholesterol levels compared with those in the MD group (P = 0.020)., Conclusions: The intake of a diet with the characteristics of the 1975 JD may have beneficial effects on lipid metabolism in people with overweight and reduce the onset risk of metabolism-related disorders, such as obesity and diabetes., (© 2019 The Obesity Society.)

We investigated whether the difference in miso consumption between the Japanese diets of 1975 and 2010 has influenced the observed increase in diet-induced obesity. To recreate the 2010 and 1975 Japanese high-fat diets with the corresponding proportions of miso, freeze-dried miso was added to high-fat mouse feed at 1.6% and 2.6%, respectively. When 5-week-old male Institute of Cancer Research (ICR) mice were provided each of these diets ad libitum for 8 weeks, it was found that the white adipose tissue weight and adipocyte area were lower in mice receiving the 1975 diet than in those receiving the 2010 diet. Therefore, high miso consumption is one reason why the 1975 Japanese diet tended to not lead to obesity. Next, the combined effects of treadmill exercise and miso consumption were investigated. The mice were divided into three groups, which were provided either a high-fat diet (group C), a high-fat diet with exercise (group C + E), or a miso-supplemented high-fat diet with exercise (group M + E) for 8 weeks. In this experiment, the white adipose tissue weight and adipocyte area in group M + E were lower than in group C. When the mRNA expression of lipid metabolism-associated genes in adipose tissue was measured, we found that expression of Hsl (lipase, hormone sensitive), which is involved in lipolysis, and Pparγ (peroxisome proliferator activated receptor gamma), which regulates adipocyte differentiation upstream of Hsl , was increased in group M + E. These results clearly demonstrated that lipid accumulation in the adipose tissues is suppressed by miso consumption in combination with exercise.

Various functions of dietary sphingolipids have been reported; however, little is known about marine sphingolipids. Ceramide 2-aminoethylphosphonate (CAEP), an abundant sphingolipid in marine mollusks, frequently has a unique triene type of sphingoid base [2-amino-9-methyl-4,8,10-octadecatriene-1,3-diol (d19:3)]. We previously reported that dietary CAEP prepared from the skin of squid was digested in the intestinal mucosa of mice via ceramides to yield free sphingoid bases. How dietary CAEP is then used in the body remains unclear. Here, we investigated the absorption of dietary CAEP using a lipid absorption assay on the lymph collected from rats with thoracic duct cannulation. Our results reveal that sphingoid bases derived from CAEP, including d16:1, d18:1, and d19:3, were detected in the lymph after administration of CAEP. Lymphatic recovery of d19:3 was lower than that of other sphingoid bases. A large fraction of the absorbed sphingoid bases was present as complex sphingolipids, whereas a smaller portion was present in the free form. Fatty acids in ceramide moieties found in the lymph were partially different from dietary CAEP, which indicates that sphingoid bases derived from CAEP could be, at least in part, resynthesized into complex sphingolipids. Future studies should elucidate the metabolism of sphingoid bases derived from CAEP., (Copyright © 2019 Tomonaga et al.)

Dysregulation of the gut microbiome is associated with several life-threatening conditions and thus might represent a useful target for the prevention of dementia. However, the relationship between the gut microbial population and dementia has not yet been fully clarified. We recruited outpatients visiting our memory clinic to participate in this study. Information on patient demographics, risk factors, and activities of daily living was collected, and cognitive function was assessed using neuropsychological tests and brain magnetic resonance imaging scans. Faecal samples were obtained, and the gut microbiome was assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis, one of the most well-established and reliable 16S ribosomal RNA-based methods for classifying gut microbiota. Patients were divided into two groups, demented and non-demented. Multivariable logistic regression models were used to identify the variables independently associated with dementia. The T-RFLP analysis revealed differences in the composition of the gut microbiome: the number of Bacteroides (enterotype I) was lower and the number of 'other' bacteria (enterotype III) was higher in demented than non-demented patients. Multivariable analyses showed that the populations of enterotype I and enterotype III bacteria were strongly associated with dementia, independent of the traditional dementia biomarkers. Further studies of the metabolites of gut microbes are needed to determine the mechanism underlying this association.

We determined whether the anti-obesity effect provided by the consumption of Euglena gracilis (Euglena), which is rich in insoluble dietary fiber, could be enhanced by the co-consumption of vegetables with an abundance of soluble dietary fiber. Nine-week-old male C57BL/6J mice were divided into five groups as follows: group 1 received a normal diet, group 2 received a high-fat diet, and groups 3, 4, and 5 received high fat diets containing 0.3% paramylon, 1.0% Euglena, or 1.0% Euglena plus 0.3% vegetables (barley leaf, kale, and ashitaba), respectively. Mice were fed ad libitum until 18 weeks of age. Euglena intake significantly reduced visceral fat accumulation in obese mice, and co-consumption of vegetables enhanced this effect. Consumption of Euglena with vegetables reduced adipocyte area, suppressed the expression of genes related to fatty acid synthesis, upregulated genes related to adipocyte lipolysis, and suppressed serum markers of inflammation. Notably, we also observed an increase in the fraction of short-chain fatty acid-producing beneficial bacteria, a reduction in harmful bacteria that cause inflammation, and an increase in short-chain fatty acid production. Therefore, the co-consumption of vegetables enhanced the anti-obesity and anti-inflammatory effects of Euglena, likely by modulating the gut microbiota composition.

Low-carbohydrate, high-protein diets, known as carbohydrate-restricted diets, are in contrast to the carbohydrate-centric meals typical of the Japanese diet. Carbohydrate-restricted diets were reported to reduce visceral fat, owing to which they have attracted attention and been widely implemented. If, as proposed, carbohydrate-restricted diets are effective in delaying senescence, then Japanese diets have a hidden potential to evolve further. However, long-term carbohydrate restriction in mice was reported to have a negative effect on the cardiovascular system, with shortening of lifespan due to activation of mechanistic target of rapamycin (mTOR). As a result, the safety of long-term adherence to carbohydrate-restricted diets remains doubtful. Recently, we conducted a study using senescence-accelerated mouse-prone 8 (SAMP8) mice to examine the effects of a carbohydrate-restricted diet on aging and skin senescence, and to determine the effect of long-term carbohydrate restriction on the aging process in mice. Here, we discuss the safety of long-term carbohydrate restriction based on the findings obtained from animal studies.

We determined whether the benefits provided by the consumption of Euglena gracilis (Euglena), which is a unicellular photosynthesizing green alga and rich in insoluble dietary fiber paramylon, can be enhanced by the co-consumption of vegetables that are rich in soluble dietary fiber. Nine-week-old male C57BL/6J mice were divided into four groups: group 1 received normal diet, whereas groups 2, 3 and 4 received normal diet containing 0.3% paramylon, 1.0% Euglena, or 1.0% Euglena plus 0.3% vegetables (barley leaf, kale and ashitaba), respectively. Mice were fed ad libitum until 18 weeks of age. Euglena intake significantly decreased serum markers of inflammation and co-consumption of vegetables enhanced this reduction. Notably, we observed an increase in the fraction of beneficial bacteria producing short-chain fatty acids, a reduction in harmful bacteria that cause inflammation and an increase in short-chain fatty acid production. Visceral fat accumulation was also reduced. Subsequent analyses showed that co-consumption of Euglena with vegetables reduced adipocyte area, suppressed the expression of genes related to fatty acid synthesis and increased the expression of genes related to adipocyte growth and lipolysis. Therefore, co-consumption of Euglena with vegetables enhanced its anti-inflammatory effect and the inhibitory effect on visceral fat accumulation likely by modulating the composition of gut microbiota.

A lot of Japanese people are generally known for having a healthy diet, and consume a variety of mushrooms daily. Many studies have reported anti-obesity effects of mushrooms, but few have investigated the effects of consuming a variety of edible mushroom types together in realistic quantities. In this study, we investigated whether supplementation with a variety of mushroom types affects visceral fat accumulation and gut microbiota in mice. The most popular mushroom varieties in Japan were lyophilized and mixed according to their local production ratios. C57BL/6J mice were fed a normal diet, high-fat (HF) diet, HF with 0.5% mushroom mixture (equivalent to 100 g mushrooms/day in humans) or HF with 3% mushroom mixture (equivalent to 600 g mushrooms/day in humans) for 4 weeks. The mice were then sacrificed, and blood samples, tissue samples and feces were collected. Our results show that mushroom intake suppressed visceral fat accumulation and increased the relative abundance of some short chain fatty acid- and lactic acid-producing gut bacteria. These findings suggest that mushroom intake is an effective strategy for obesity prevention., Competing Interests: Three of the authors (T.S., K.M. and K.O.) are salaried employees of the Hokuto Corporation, which cultivated some of the mushrooms used in this study. The remaining authors (M.K. and T.T.) have no conflicts of interest to disclose. All research funding for this study was provided by the Hokuto Corporation.

The aim of this study was to develop a purified diet that mimics the characteristics of the Japanese diet using readily available materials with a simpler composition and a focus on quality, with the goal of facilitating performance of studies on the Japanese diet worldwide. The utility of the new diet was examined as a mimic of the standard Japanese diet for use in animal experiments. We examined whether a key characteristic of the Japanese diet of being less likely to cause obesity could be reproduced. The mimic diet had a balance of protein, fat and carbohydrate based on the 1975 Japanese diet, which is the least likely to cause obesity, and materials chosen with reference to the National Health and Nutrition Survey (NHNS). To examine similarities of the mimic diet with the model 1975 Japanese diet, we created a menu of the 1975 diet based on the NHNS and prepared the freeze-dried and powdered diet. The mimic diet, the 1975 Japanese diet, a control AIN-93G diet and a Western diet were fed to mice for 4 weeks. As a result, the mimic diet and the 1975 diet resulted in less accumulation of visceral fat and liver fat. Mice given these two diets showed similar effects. This indicates that the mimic diet used in this study has characteristics of the 1975 Japanese diet and could be used as a standard Japanese diet in animal experiments.

The effect of 1-deoxynojirimycin, a caloric restriction mimetic, was examined in ICR mice with azoxymethane dextran sodium sulfate-induced colorectal cancer. Azoxymethane is a carcinogen (10 mg/kg body weight), and 2% dextran sodium sulfate (w/v) used as a colitis-inducing agent. Mice were separated into 5 groups: a group without colorectal cancer fed a normal diet (CO- group), and groups with colorectal cancer fed a normal diet (CO+ group), a calorie-restricted diet (caloric restriction group), and diets including 0.02% and 0.1% 1-deoxynojirimycin (l-1-deoxynojirimycin and H-1-deoxynojirimycin groups). The tumor incidence and number were reduced significantly in the caloric restriction group compared to the CO+ group, and were also suppressed in a dose-dependent manner by 1-deoxynojirimycin. mRNA for anti-apoptotic Bcl-2 was decreased and that for pro-apoptotic Bax was increased in the carcinoma tissue of CR, l-1-deoxynojirimycin and H-1-deoxynojirimycin groups. These results suggest that caloric restriction and 1-deoxynojirimycin inhibit growth of colorectal cancer by inducing apoptosis in an induced cancer model in mice., Competing Interests: No potential conflicts of interest were disclosed.

Various physiological functions of dietary sphingolipids, such as preventing inflammation and improving the skin barrier function, have been recently demonstrated. The sphingolipid most commonly used as a foodstuff is glucosylceramide from plant sources, which is composed of sphingoid bases that are distinctive from those found in mammals. Although the structure of sphingoid bases in higher plants is more complicated than the structure of those in mammals, the fate of dietary sphingolipids of plant origin is still not understood. In the present study, we investigated the absorption of 4,8-sphingadienine that originated from maize glucosylceramide in the rat intestine by using a lipid absorption assay of lymph collected from the thoracic duct. The cumulative recovery of 4,8-sphingadienine in the lymph was lower than that of sphingosine. Verapamil, a P-glycoprotein inhibitor, significantly increased the absorption of 4,8-sphingadienine but did not affect the absorption of sphingosine. Plant-derived sphingoid bases were detected in the ceramide fraction of lymph fluid by using liquid chromatography-mass spectrometry analysis. These results indicate that 4,8-sphingadienine that originates from the glucosylceramide of higher plants is poorly absorbed in the intestine because of efflux by P-glycoprotein and can be incorporated into a ceramide moiety, at least in part, in intestinal endothelial cells.

We examined the effect of a high-fat diet from senescence as a means of preventing malnutrition among the elderly. The senescence-accelerated mouse P8 was used and divided into three groups. The 6C group was given a normal diet until 6 months old. The 12N group was given a normal diet until 12 months old. The 12F group was given a normal diet until 6 months old and then a high-fat diet until 12 months old. In the oral fat tolerance test, there was a decrease in area under the curve for serum triacylglycerol level in the 12N group and a significant increase in the 12F group, suggesting that the attenuation of lipid absorption ability with aging was delayed by a high-fat diet from senescence. To examine this mechanism, histological analysis in the small intestine was performed. As a result, the degeneration of villi with aging was inhibited by the high-fat diet. There was also a significant decrease in length of villus in the small intestine in the 12N group and a significant increase in the 12F group. The high-fat diet from senescence inhibited the degeneration of villi with aging in the small intestine, and inhibited the attenuation of lipid absorption ability.

This study examined how a maternal high-fat diet (HD) during lactation and exposure of offspring to isolation stress influence the susceptibility of offspring to the development of obesity. C57BL/6J mice were fed a commercial diet (CD) during pregnancy and a CD or HD during lactation. Male offspring were weaned at three weeks of age, fed a CD until seven weeks of age, and fed a CD or HD until 11 weeks of age. Offspring were housed alone (isolation stress) or at six per cage (ordinary circumstances). Thus, offspring were assigned to one of eight groups: dams fed a CD or HD during lactation and offspring fed a CD or HD and housed under ordinary circumstances or isolation stress. Serum corticosterone level was significantly elevated by isolation stress. High-fat feeding of offspring reduced their serum corticosterone level, which was significantly elevated by a maternal HD. A maternal HD and isolation stress had combined effects in elevating the serum corticosterone level. These findings suggest that a maternal HD during lactation enhances the stress sensitivity of offspring. White adipose tissue weights were significantly increased by a maternal HD and isolation stress and by their combination. In addition, significant adipocyte hypertrophy was induced by a maternal HD and isolation stress and exacerbated by their combination. Thus, a maternal HD and isolation stress promote visceral fat accumulation and adipocyte hypertrophy, accelerating the progression of obesity through their combined effects. The mechanism may involve enhanced fatty acid synthesis and lipid influx from blood into adipose tissue. These findings demonstrate that a maternal HD during lactation may increase the susceptibility of offspring to the development of stress-induced obesity.

In this study, to study the effect of aging and Apolipoprotein E (ApoE) deficiency on antioxidant ability in mice, we examined whether lipid peroxidation is promoted by aging in ApoE deficient (ApoE(-/-)) mice, which have a shorter lifespan than normal mice. The levels of thiobarbituric acid-reactive substances (TBARS), a biomarker of lipid peroxidation, were measured in plasma and liver in ApoE(-/-) mice aged 12 weeks (young) and 52 weeks (early stage of senescence). TBARS in plasma and liver were significantly increased by aging. Next, we examined the reasons why lipid peroxidation was promoted by aging, based on measurement of protein and mRNA levels for antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in liver in ApoE(-/-) mice aged 12 and 52 weeks. The levels of superoxide dismutase 1 and 2 in liver were significantly decreased by aging. The mRNA level of catalase was also significantly decreased and the mRNA levels of superoxide dismutase 1, superoxide dismutase 2 and glutathione peroxidase 1 all showed a tendency to decrease with age. These results suggest that lipid peroxidation is caused by reduction of antioxidant activity with aging and that this promotes senescence and shortens lifespan in ApoE(-/-) mice.

Recently, we administered fish oil containing eicosapentaenoic acid and docosahexaenoic acid (DHA) to senescence-accelerated mice P8 (SAMP8), in order to investigate the effects on lifespan. Surprisingly, the lifespan of SAMP8 that were fed fish oil was shortened significantly, through a mechanism that likely involved lipid peroxidation. In this study, we investigated this phenomenon in further detail. To examine whether this phenomenon occurs only in SAMP8, we investigated the effect of fish oil on the lifespan of another organism species, Caenorhabditis elegans (C. elegans). C. elegans fed fish oil were cultured and the lifespan monitored. As a consequence of the provision of large amounts of fish oil the lifespan of C. elegans was shortened significantly, whereas an appropriate amount of fish oil extended their lifespan significantly. Lipid peroxide levels in C. elegans that were fed fish oil increased significantly in a dose-dependent manner. However, lipid peroxide levels in C. elegans were inhibited by the addition of fish oil and an antioxidant, α-tocopherol, and completely abrogated the changes in the lifespan. To further confirm whether the oxidation of n-3 polyunsaturated fatty acid in fish oil would change the lifespan of C. elegans, the effect of oxidized DHA was examined. Large amounts of oxidized DHA were found to shorten their lifespan significantly. Thus, fish oil changes the lifespan of C. elegans through lipid peroxidation.

The maternal nutritional status during pregnancy and lactation influences the risk of obesity in offspring, but the details of this phenomenon are unclear. In particular, there is little information on the influence on the offspring of the maternal nutritional status during lactation only. Therefore, in this study, we examined the influence of high dietary fat intake in dams during lactation on the risk of obesity in offspring, using C57BL/6J mice. The mice were fed a control diet (CD) during pregnancy. After birth, dams were fed a CD or a high-fat diet (HD) during lactation (3 wk). Fat and energy were significantly increased in milk from dams fed a HD during lactation. Male offspring were weaned at 3 wk old and fed a CD for 4 wk, which resulted in no significant difference in their physique. Four weeks after weaning, the offspring (7 wk old) were fed a CD or HD for 4 wk to induce obesity. High dietary fat intake in dams and offspring promoted lipid accumulation in white adipose tissue and adipocyte hypertrophy in male offspring. The underlying mechanism may involve an increase in expression of Lpl and a decrease in expression of Hsl in white adipose tissue of offspring. In conclusion, our results show that high dietary fat intake during lactation promotes development of diet-induced obesity in male offspring.

Various factors have been reported to influence lipid metabolism and cause metabolic syndrome. However, the influence of allergy on the liver that plays important role of lipid metabolism has not been clarified. The aim of this study was to examine the influence of allergy on lipid metabolism of liver. A model of atopic dermatitis was developed in the NC/Nga mouse using picryl chloride to induce allergy. Lipid metabolism parameters were measured and the mechanism of changes in these parameters was examined using DNA microarray analysis and quantitative reverse transcriptase PCR. Triacylglycerol accumulation was promoted in the liver in the mouse atopic dermatitis model despite reductions in food intake, body weight gain, and serum glucose. As this mechanism, it was thought that atopic dermatitis caused the suppression of fatty acid β-oxidation. These results suggest that atopic dermatitis causes lipid accumulation in the liver.

Unlabelled: Amyloid β-peptide (Aβ) is hypothesized to play a key role by oxidatively impairing the capacity of red blood cells (RBCs) to deliver oxygen to the brain. These processes are implicated in the pathogenesis of Alzheimer's disease (AD). Although plasma Aβ has been investigated thoroughly, the presence and distribution of Aβ in human RBCs are still unclear. In this study, we quantitated Aβ40 and Aβ42 in human RBCs with ELISA assays, and provided evidence that significant amounts of Aβ could be detected in RBCs and that the RBC Aβ levels increased with aging. The RBC Aβ levels increased with aging. On the other hand, providing an antioxidant supplement (astaxanthin, a polar carotenoid) to humans was found to decrease RBC Aβ as well as oxidative stress marker levels. These results suggest that plasma Aβ40 and Aβ42 bind to RBCs (possibly with aging), implying a pathogenic role of RBC Aβ. Moreover, the data indicate that RBC Aβ40 and Aβ42 may constitute biomarkers of AD. As a preventive strategy, therapeutic application of astaxanthin as an Aβ-lowering agent in RBCs could be considered as a possible anti-dementia agent., Trial Registration: Controlled-Trials.com ISRCTN42483402.

Supplementation to an AIN93G-based diet of tocotrienol (T3) for 13 weeks administered to Fischer 344/slc rats showed a safety profile with no side effects. Dose-dependent T3 levels were detected in many tissues. Under the present experimental conditions, a continuous intake of the T3 concentrate would be safe in the rats as long as the T3 content was less than 0.20% of the dietary intake.

The presence of amyloid β-peptide (Aβ) in human blood has recently been established, and it has been hypothesized that Aβ readily contacts red blood cells (RBC) and oxidatively impairs RBC functions. In this study, we conducted in vitro and in vivo studies, which provide evidence that Aβ induces oxidative injury to RBC by binding to them, causing RBC phospholipid peroxidation and diminishing RBC endogenous carotenoids, especially xanthophylls. This type of damage is likely to injure the vasculature, potentially reducing oxygen delivery to the brain and facilitating Alzheimer's disease (AD). As a preventive strategy, because the Aβ-induced RBC damage could be attenuated by treatment of RBC with xanthophylls, we suggest that xanthophylls may contribute to the prevention of AD., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Conjugated linoleic acid (CLA) is a collective term used for fatty acids with a conjugated double bond that are geometrical and positional isomers of linoleic acid. Anti-obesity and anti-cancer properties, an immunopotentiation effect, and promotion of bone formation by CLA have been shown in cell culture and animal studies. A mixture of 9c11t- and 10t12c-CLA is now used as a health food supplement after testing in clinical trials. These trials focused on improvement of lipid metabolism by CLA, whereas few studies have examined absorption and metabolism of CLA in humans. In addition, there is no report concerning absorption and metabolism of CLA in Japanese. This study was designed to examine CLA concentration in blood, the elimination rate of CLA, and metabolic differences between 9c11t-CLA and 10t12c-CLA in blood in Japanese who ingested CLA (about 2 g/d, equal weights of 9c11t-CLA and 10t12c-CLA) for 3 wk. Blood samples were collected 1 wk before the 3-wk period, on the first and last days of the period, and 1 wk after the end of the period, and the CLA concentration and distribution in blood were investigated. The CLA concentration in blood was significantly increased by CLA ingestion and reached 36 µmol/L. The CLA concentration in blood one week after the intake period was significantly lower than that at the end of CLA intake. The 10t12c-CLA level in plasma decreased faster than that of 9c11t-CLA. This suggests faster metabolism (fatty acid β oxidation) of 10t12c-CLA compared with 9c11t-CLA.

We investigated whether replicative senescence of endothelial cells contributed to the pathogenesis of atherosclerosis in human umbilical vein endothelial cells (HUVECs). HUVECs at a population-doubling level of 30 (PDL30) divided much more slowly than those at PDL9. The percentage of SA-β-Gal-positive cells and the mRNA expression levels of PAI-1 and p21 at PDL30 were significantly higher than those at PDL9. The changes induced by aging were evaluated according to the mRNA expression level of genes related to the endothelial cell function. The expression level of many adhesion molecules promoting monocytic adhesion was significantly increased, and monocytic adhesion on HUVECs was found to be significantly promoted by aging. Monocytic adhesion is an essential early event in the development of atherosclerosis, and our results suggest that replicative senescence of the vascular endothelial cells induced increased expression of adhesion molecules. The consequent increase in monocytic adhesion may then promote the pathogenesis of atherosclerosis.

The population of elderly persons has increased worldwide. However, few studies have examined age-dependent changes in lipid and carbohydrate metabolism related to age-related diseases. The number of cases of metabolic syndrome is increasing worldwide and prevention of lifestyle diseases may lead to prolongation of lifespan. In this study, we examined age-dependent changes in lipid and carbohydrate metabolism in the senescence-accelerated (SAM) P10 mouse. Tissue weights and biochemical parameters in plasma and liver were examined in SAMP10 mice aged 3, 6, 9, and 12 mo. White adipose tissue weight and the levels of liver triacylglycerol, plasma free fatty acids, and plasma insulin all showed increases with aging of the mice. To examine this mechanism in detail, aging-related changes in mRNA expression of genes related to lipid and carbohydrate metabolism were examined by DNA microarray analysis. The mRNA level for Hsd11b1 (hydroxysteroid 11-beta dehydrogenase 1), which increases insulin secretion and resistance, was elevated with aging in the liver of SAMP10 mice. These results show that lipid accumulation in liver and white adipose tissue is promoted by aging in SAMP10 mice through an increase in plasma insulin levels.

Sphingolipids are ubiquitous in all eukaryotic organisms. Various physiological functions of dietary sphingolipids, such as preventing colon cancer and improving the skin barrier function, have been recently reported. One of the common sphingolipids used as a foodstuff is glucosylceramide from plant sources, which is composed of sphingoid bases distinct from those of mammals. However, the fate of dietary sphingolipids derived from plants is still not understood. In this study, we investigated the absorption of maize glucosylceramide in the rat intestine using a lipid absorption assay of lymph from the thoracic duct. The free and complex forms of trans-4,cis-8-sphingadienine, the predominant sphingoid base of maize glucosylceramide, were found in the lymph after administration of maize glucosylceramide. This plant type of sphingoid base was detected in the ceramide fraction and N-palmitoyl-4,8-sphingadienine (C16:0-d18:2) and N-tricosanoyl-4,8-sphingadienine (C23:0-d18:2) were identified by LC-MS/MS. The cumulative recovery of 4t,8c-sphingadienine in the lymph was very low. These results indicate that dietary glucosylceramide originating from higher plants is slightly absorbed in the intestine and is incorporated into ceramide structures in the intestinal cells. However, it appears that the intact form of sphingoid bases is not reutilized well in the tissues.

Based on the ratios of (-)-epigallocatechin-3-gallate (EGCG) and caffeine (CAF) levels found in commercial tea drinks, EGCG and CAF were co-administered to human volunteers at various EGCG/CAF ratios, and plasma EGCG was determined by high performance liquid chromatography with chemiluminescence detection. As for the results, in plasma taken after ingestion of a beverage containing 95 mg of EGCG alone, the area under the plasma EGCG concentration-time curve (AUC) was 857 ngxh/ml. A higher AUC (1,370 ngxh/ml) was observed when subjects ingested a beverage containing EGCG (95 mg) and a low amount of CAF (40 mg). In the case of ingestion of a beverage containing EGCG (95 mg) and a high amount of CAF (180 mg), the AUC tended to be somewhat higher (1,165 ngxh/ml), but not significantly so, compared with the beverage with EGCG alone. These findings (modulation of plasma EGCG level by CAF) provide ideas for modulating the bioavailability of tea catechins, which can be applied to tea-related drinks and foods.

Proteins harbor a number of cavities of relatively small volume. Although these packing defects are associated with the thermodynamic instability of the proteins, the cavities also play specific roles in controlling protein functions, e.g., ligand migration and binding. This issue has been extensively studied in a well-known protein, myoglobin (Mb). Mb reversibly binds gas ligands at the heme site buried in the protein matrix and possesses several internal cavities in which ligand molecules can reside. It is still an open question as to how a ligand finds its migration pathways between the internal cavities. Here, we report on the dynamic and sequential structural deformation of internal cavities during the ligand migration process in Mb. Our method, the continuous illumination of native carbonmonoxy Mb crystals with pulsed laser at cryogenic temperatures, has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino acid residues around the cavity, which results in the expansion of the cavity with a breathing motion. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand migration channel arising by induced fit, which is further supported by computational geometry analysis by the Delaunay tessellation method. This result suggests a crucial role of the breathing motion of internal cavities as a general mechanism of ligand migration in a protein matrix.

Tocotrienol (T3), unsaturated vitamin E, has recently gained considerable attention as a potent antiangiogenic agent minimizing tumor growth, the exact intracellular mechanisms of which remain poorly understood. Because hypoxia-inducible factor-1alpha (HIF-1alpha), its downstream target vascular endothelial growth factor (VEGF), and other angiogenic factors such as interleukin-8 (IL-8) and cyclooxygenase 2 (COX-2) play critical roles in neovascularization, we tested the hypothesis that the inhibitory effect of T3 on tumor angiogenesis is via regulation of these angiogenic factors. We used 2 cancer cell lines, human colorectal adenocarcinoma cells (DLD-1) and human hepatoma cells (HepG2). T3 isomers (2 micromol/L) inhibited hypoxia-induced VEGF secretion from DLD-1, with delta-T3 showing potent inhibition. Delta-T3 suppressed hypoxia-induced VEGF and IL-8 expression in DLD-1 at both mRNA and protein levels, and we found the inhibitory mechanism of delta-T3 by reducing HIF-1alpha protein expression or increasing HIF-1alpha degradation. Also, delta-T3 (2 micromol/L) did not affect hypoxia-induced COX-2 mRNA expression; however, delta-T3 tended to suppress (P = 0.044) hypoxia-induced COX-2 protein expression, implying a possible post-transcriptional mechanism by delta-T3. Overall, our results confirmed that T3 has an inhibitory effect on angiogenic factor secretion from cancer cells and revealed the possible mechanisms, providing new information about the antiangiogenic effects of T3.

Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing alpha-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input alpha-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.