Incomplete Elongation of Ultra-long-chain Polyunsaturated Acyl-CoAs by the Fatty Acid Elongase ELOVL4 in Spinocerebellar Ataxia Type 34.

Spinocerebellar ataxias (SCAs) are autosomal dominant diseases characterized by cerebellar atrophy and ataxia. The SCA subtype SCA34 is caused by specific mutations in the gene ELOVL4, which encodes a fatty acid (FA) elongase that synthesizes ultra-long-chain (ULC; ≥C26) FAs. However, the pathogenesis and molecular mechanism that confers dominant inheritance remains unknown. Here, a cell-based assay demonstrated that each of the five known SCA34 mutants produced shorter ULC polyunsaturated FA-containing phosphatidylcholines (ULC-PCs) than wild-type protein, in the following order of severity: Q180P and T233M > W246G > I171T and L168F. Next, we generated knock-in mouse embryonic stem cells that contained heterozygous Q180P, heterozygous W246G, or homozygous W246G mutations. Neuronal differentiation-dependent production of ULC-PCs was reduced in heterozygous Q180P and homozygous W246G cells relative to control cells, and we observed shortening of the FA moiety in all mutant cells. This FA shortening was consistent with our prediction that amino acid residues substituted by SCA34 mutations are located in the transmembrane helices that interact with the ω-end region of the FA moiety of the substrate acyl-CoA. Hence, reduced levels and shortening of ULC-PCs in neurons may cause SCA34, and incomplete elongation of ULC polyunsaturated acyl-CoAs by mutated ELOVL4 may induce dominant inheritance.

Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes.

The reactivation of X-linked genes is observed in some primary breast tumors. Two active X chromosomes are also observed in female embryonic stem cells (ESCs), but whether double doses of X-linked genes affect DNA repair efficiency remains unclear. Here, we establish isogenic female/male ESCs and show that the female ESCs are more sensitive to camptothecin and have lower gene targeting efficiency than male ESCs, suggesting that homologous recombination (HR) efficiency is reduced in female ESCs. We also generate Xist-inducible female ESCs and show that the lower HR efficiency is restored when X chromosome inactivation is induced. Finally, we assess the X-linked genes with a role in DNA repair and find that Brcc3 is one of the genes involved in a network promoting proper HR. Our findings link the double doses of X-linked genes with lower DNA repair activity, and this may have relevance for common diseases in female patients, such as breast cancer.

Dynamics of transcription-mediated conversion from euchromatin to facultative heterochromatin at the Xist promoter by Tsix.

The fine-scale dynamics from euchromatin (EC) to facultative heterochromatin (fHC) has remained largely unclear. Here, we focus on Xist and its silencing initiator Tsix as a paradigm of transcription-mediated conversion from EC to fHC. In mouse epiblast stem cells, induction of Tsix recapitulates the conversion at the Xist promoter. Investigating the dynamics reveals that the conversion proceeds in a stepwise manner. Initially, a transient opened chromatin structure is observed. In the second step, gene silencing is initiated and dependent on Tsix, which is reversible and accompanied by simultaneous changes in multiple histone modifications. At the last step, maintenance of silencing becomes independent of Tsix and irreversible, which correlates with occupation of the -1 position of the transcription start site by a nucleosome and initiation of DNA methylation introduction. This study highlights the hierarchy of multiple chromatin events upon stepwise gene silencing establishment.

Good prognosis for follicular lymphoma with estrogen receptor α-positive follicular dendritic cells.

Follicular lymphoma (FL) has a meshwork of follicular dendritic cells (FDCs). We previously demonstrated the presence of estrogen receptor alpha (ERα)+ CD23+ FDCs in grades 1-2 FL. The significance of FDCs as a prognostic factor in FL remains unknown. The current study aimed to compare clinicopathological features, including prognosis, between FL with and without ERα+ FDCs. This study evaluated the clinicopathological significance of ERα expression in 70 FL patients by immunostaining. The presence of ERα mRNA on FDCs from 5 FL patients was confirmed by CD21/ERα double staining (immunohistochemistry and in situ hybridization). We defined patients with frequent ERα expression as the ERαhigh group and those with infrequent ERα expression as the ERαlow group. Thirty-two patients were assigned to the ERαhigh group (45.7%), and 38 patients were assigned to the ERαlow group (54.3%). Both overall survival (OS) and progression-free survival (PFS) were significantly better in the ERαhigh group than in the ERαlow group (OS, log-rank, P = .0465; PFS, log-rank, P = .0336). Moreover, high ERα expression on FDCs was an independent prognostic factor for OS in both the univariate ([hazard ratio] HR, 0.163; P = .0260) and multivariate (HR, 0.050; P = .0188) analyses and for PFS in both the univariate (HR, 0.232; P = .0213) and multivariate (HR, 0.084; P = .0243) analyses. ERα mRNA expression was detected in CD21+ FDCs within the neoplastic follicles of FL patients. In conclusion, a neoplastic follicular microenvironment with ERα-positive FDCs might affect the grade and presence of the follicular pattern of FL and improve patient prognosis.

Diagnostic utility of CD205 in breast cancer: Simultaneous detection of myoepithelial cells and dendritic cells in breast tissue by CD205.

BACKGROUND: CD205 can be used to detect myoepithelial cells (MECs) and dendritic cells (DCs) in breast tissue. However, the usefulness of CD205 immunostaining in the pathological diagnosis of breast tumors is not fully understood. The objective of this study was to re-evaluate CD205 co-expression with other MEC markers, such as p63 and CD10, in nonneoplastic and neoplastic breast tissue and to evaluate its pathological diagnostic utility in these types of breast cancer. MATERIAL AND METHODS: Nonneoplastic breast tissue samples with a terminal duct lobular unit and duct were obtained from fibroadenoma and mastopathy patients. Neoplastic breast tissue samples included ductal carcinoma in situ (DCIS) (n=43) and invasive ductal carcinoma (IDC) (n=60), including the tubule-forming type (n=20). These specimens were investigated by CD205, p63, and CD10 immunostaining. RESULTS: In addition to p63 and CD10, CD205 was expressed on MECs in nonneoplastic breast and DCIS tissue samples; CD205 was simultaneously detected on DCs that had infiltrated DCIS and IDC tumor nests. CD205 was expressed on cancer cells themselves in only 7.3% of the breast cancer samples. The number of intratumoral CD205⁺ DCs in tubular IDC was significantly higher than that in DCIS (P<0.01). CONCLUSION: Because CD205 was simultaneously detected on MECs and DCs in the same breast tissue sections, it may be useful for distinguishing tubular IDC from DCIS.

Changes in Bile Acid Concentrations after Administration of Ketoconazole or Rifampicin to Chimeric Mice with Humanized Liver.

Drug-induced liver injury (DILI) is a common side effect of several medications and is considered a major factor responsible for the discontinuation of drugs during their development. Cholestasis is a DILI that results from impairment of bile acid transporters, such as the bile salt export pump (BSEP), leading to accumulation of bile acids. Both in vitro and in vivo studies are required to predict the risk of drug-induced cholestasis. In the present study, we used chimeric mice with humanized liver as a model to study drug-induced cholestasis. Administration of a single dose of ketoconazole or rifampicin, known to potentially cause cholestasis by inhibiting BSEP, did not result in elevated levels of alkaline phosphatase (ALP), which are known hepatic biomarkers. The concentration of taurodeoxycholic acid increased in the liver after ketoconazole administration, whereas rifampicin resulted in increased tauromuricholic acid and taurocholic acid (TCA) levels in the liver and plasma. Furthermore, rifampicin resulted in an increase in the uniform distribution of a compound with m/z 514.3, presumed as TCA through imaging mass spectrometry. The mRNA levels of bile acid-related genes were also altered after treatment with ketoconazole or rifampicin. We believe these observations to be a part of a feedback mechanism to decrease bile acid concentrations. The changes in bile acid concentrations results may reflect the initial responses of the human body to cholestasis. Furthermore, these findings may contribute to the screening of drug candidates, thereby avoiding drug-induced cholestasis during clinical trials and drug development.

Differential expression of estrogen receptor-α on follicular dendritic cells from patients with grade 1-2 and grade 3 follicular lymphoma.

Hormone therapy has been used for patients with estrogen receptor alpha (ERα)-positive breast cancers. Recently, some studies reported the expression of ERα on neoplastic cells from B-cell lymphomas. However, there has been only one report of ERα expression on the follicular dendritic cells (FDCs) that structurally and functionally support the microenvironment of follicular lymphomas (FLs). The objective of this study was to investigate the frequency of ERα expression on FDCs in nonneoplastic reactive lymphoid tissues and to compare the frequency of ERα expression on FDCs in the axillary lymph nodes between patients with and without antiestrogen therapy and among patients with grades 1-3 of FL. Reverse transcription-polymerase chain reaction was performed to detect ERα mRNA in FL. In nonneoplastic germinal centers (GCs) from patients with tonsillitis or reactive lymphadenitis, ERα was expressed in the light zone. ERα-positive cells strongly correlated with the width of GCs (rs  = 0.81, P < 0.01) and the CD21-positive (rs  = 0.69, P < 0.01) and CD23-positive (rs  = 0.83, P < 0.01) FDC meshwork. The axillary lymph nodes had fewer ERα-positive cells, smaller GCs, and a looser CD21- and CD23-positive FDC meshwork with hormone therapy than without hormone therapy (P < 0.01). Neoplastic follicles of G1-2 FL had more ERα-positive cells and a larger CD23+ FDC meshwork than those of G3 FL (P < 0.01). ERα mRNA was detected in both G1-2 FL and G3 FL by reverse transcription-polymerase chain reaction. In conclusion, these results suggested that antiestrogen hormone therapy may decrease the number of ERα-positive FDCs and that the responses mediated by the estrogen-ERα interaction on FDCs may differ between G1-2 FL and G3 FL.

Detection of Minimal Bone Marrow involvement of Blastic Plasmacytoid Dendritic Cell Neoplastic Cells - CD303 immunostaining as a diagnostic tool.

Blastic plasmacytoid dendritic cell (pDC) neoplasm (BPDCN) is a relatively rare hematological malignancy with significantly complex clinicopathological features that are still unclear. This study aimed to analyze the clinicopathological data of BPDCN and evaluate immunohistochemical detection of minimal bone marrow (BM) involvement. In this study, we examined skin and BM lesions from 6 patients with BPDCN. Neoplastic cells tested positive for CD303 (polyclonal, 100%; monoclonal, 40%) in the skin lesions and for CD303 (polyclonal, 100%; monoclonal, 67%) in the BM clots. Although immunostaining of CD4, CD56, CD123, CD303, and TCLl detected minimal BM involvement in 3 patients, morphological identification was challenging in the BM clots stained with hematoxylin-eosin. In conclusion, our results demonstrate the significance of observing BM smears to detect neoplastic cells and that immunohistochemical examination, including CD303 antibodies, is useful to detect minimal BM involvement. This study is the first to report the expression of thymic stromal lymphopoietin (TSLP) and its receptor in BPDCN cells. Therefore, the TSLP/TSLP receptor axis may be associated with the proliferation of BPDCN, and consequently, the survival of patients.

Assessment of amiodarone-induced phospholipidosis in chimeric mice with a humanized liver.

It is important to consider susceptibility to drug-induced toxicity between animals and humans. Chimeric mice with a humanized liver are expected to predict hepatotoxicity in humans. Drug-induced phospholipidosis (DIPL), in which phospholipids accumulate, is a known entity. In this study, we examined whether chimeric mice can reveal species differences in DIPL. Changes in various phosphatidylcholine (PhC) molecules were investigated in the liver of chimeric mice after administering amiodarone, which induces phospholipidosis. Liquid chromatography-tandem mass spectrometry revealed that levels of PhCs tended to increase in the liver after administration of amiodarone. The liver of chimeric mice consists of human hepatocytes and residual mouse hepatocytes. We used imaging mass spectrometry (IMS) to evaluate the increase of PhCs in human and mouse hepatocytes after administration of amiodarone. IMS visualizes localization of endogenous and exogenous molecules in tissues. The IMS analysis suggested that the localized levels of several PhCs tended to be higher in the human hepatocytes than those in mouse hepatocytes, and PhC levels changed in response to amiodarone. Chimeric mice with a humanized liver will be useful to evaluate species differences in DIPL between mice and humans.

Antimutagenic Effects of Polymethoxy Flavonoids of Citrus unshiu.

Citrus fiits have been used as edible fruit and traditional medicine for various diseases such as cancer. In the courses of our study to find antimutagens, we have found that.the ethanolic extract of the peel of Citrus unshiu Marc showed antimutagenic effects against several mutagens in the Ames test using Salmonella typhimurium TA98 strain. Three polymethoxy flavonoids, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin, were identified in the extract as major constituents. These three polymethoxy flavonoids showed antimutagenic effects in the Ames test in vitro and in the micronucleus test in vivo.

Selective transport of microplastics and mesoplastics by drifting in coastal waters.

The quantity and size distributions of small plastic fragments in the Seto Inland Sea, Japan were investigated using field surveys and a numerical particle-tracking model. The model was used to interpret the distributions of small plastic fragments and the possible transport processes in coastal waters. Of note, the size and quantity of mesoplastics (approximately >5mm) gradually increased close to the coast irrespective of the existence of river mouths, which probably act as a major source of anthropogenic marine debris. Additionally, microplastics were more dominant as we moved further offshore. The numerical model reproduced the near-shore trapping of mesoplastics, suggesting that mesoplastics are selectively conveyed onshore by a combination of Stokes drift and terminal velocity, dependent on fragment sizes. It is suggested that mesoplastics washed ashore on beaches degrade into microplastics, and that the microplastics, which are free from near-shore trapping, are thereafter spread offshore in coastal waters.

A cross-sectional survey of factors influencing bone mass in junior high school students.

OBJECTIVE: The purpose of this study is to investigate factors influencing the osteo-sono assessment index (OSI) in junior high school students (boys, girls who had reached menarche, and girls who had not). METHODS: A total of 9,743 students (4,974 boys and 4,769 girls) in Ehime Prefecture participated in this study. We measured body mass index (BMI) and calcaneal bone mass using OSI. In parallel, participants answered a questionnaire relating to age, sex, menarche, exercise habits, milk intake, and history of bone fractures during the preceding year. To determine the factors influencing OSI, we calculated an individual standardized partial regression coefficient (ß) using multiple linear regression (MLR) analysis. RESULTS: For boys, MLR showed that BMI (ß = 0.300), age (ß = 0.260), current exercise habits (ß = 0.106), and milk intake per day in primary school (ß = 0.085) statistically significantly influenced OSI. For girls who had reached menarche, BMI (ß = 0.302), current exercise habits (ß = 0.237), age (ß = 0.140), and bone fracture during the preceding year (ß = 0.036) influenced OSI. For girls who had not reached menarche, current exercise habits (ß = 0.242), BMI (ß = 0.135), and age (ß = 0.085) influenced OSI. CONCLUSIONS: There were differences between the factors related to OSI among boys, girls who had reached menarche, and girls who had not. BMI, exercise habits, and age were the common factors related to OSI. Particularly for girls, exercise habits had a great influence on OSI.

Comparison of regional differences in health indicators and standard mortality ratio for stroke in subjects in ehime prefecture.

OBJECTIVE: The purpose of this study was to investigate regional differences in the standard mortality ratio (SMR) and risk factors (including dietary habits) for stroke across the three regions of Ehime Prefecture - Toyo (east), Chuyo (central), and Nanyo (south). PARTICIPANTS AND METHODS: We obtained medical records derived from 956,979 medical examinations carried out at JA Ehime Kouseiren Medical Examination Centers between April 1994 and March 2006. We analyzed data from 132,090 subjects (Toyo - 47,654, Chuyo - 38,435, Nanyo - 46,001) who underwent their first medical examination during this period. To analyze differences between the three regions, we first calculated the SMR for stroke based on data from the Basic Residential Registers and Health Statistics Bureau. Secondly, we calculated significant differences in body mass index, systolic blood pressure (SBP), diastolic blood pressure (DBP), blood glucose (Glu), and total cholesterol (T-CHO). Thirdly, we used the Chi-square test to calculate significant differences in the percentage of subjects who consumed the following foods on a daily basis: rice, bread, eggs, fish, meat, vegetables, dairy products, and fruit juice. RESULTS: Despite the fact that regional differences in the SMR for stroke have been decreasing, in both men and women in Nanyo, the mean values for SBP and DBP were significantly higher and the mean value for T-CHO was significantly lower than in Toyo and Chuyo. In Nanyo, the percentage of subjects who consumed rice and fish (men and women), meat (men), and juice (women) on a daily basis was higher than in Toyo and Chuyo. CONCLUSION: In Nanyo, higher SMR for stroke may be related to high SBP and DBP and low T-CHO. As background to these results, it is also thought that regional differences in dietary habits may have an influence.

Analysis of [(2)H7]methionine, [(2)H4]methionine, methionine, [(2)H4]homocysteine and homocysteine in plasma by gas chromatography-mass spectrometry to follow the fate of administered [(2)H7]methionine.

Homocysteine plays a key role in several pathophysiological conditions. To assess the methionine-homocysteine kinetics by stable isotope methodology, we developed a simultaneous quantification method of [(2)H(7)]methionine, [(2)H(4)]methionine, methionine, [(2)H(4)]homocysteine and homocysteine in rat plasma by gas chromatography-mass spectrometry (GC-MS). [(13)C]Methionine and [(13)C]homocysteine were used as analytical internal standards to account for losses associated with the extraction, derivatization and chromatography. For labeled and non-labeled homocysteine measurements, disulfide bonds between homocysteine and other thiols or proteins were reduced by dithiothreitol. The reduced homocysteine and methionine species were purified by cation-exchange chromatography and derivatized with isobutyl chlorocarbonate in water-ethanol-pyridine. Quantification was carried out by selected ion monitoring of the molecular-related ions of N(O,S)-isobutyloxycarbonyl ethyl ester derivatives on the chemical ionization mode. The intra- and inter-day precision of the assay was less than 6% for all labeled and non-labeled methionine and homocysteine species. The method is sensitive enough to determine pharmacokinetics of labeled methionine and homocysteine.

Volatile organic compounds with characteristic odor in bamboo vinegar.

Bamboo vinegar solutions had pHs of 2.5 to 2.8, and the amounts of organic constituents were estimated to be 2.3 to 4.6% (w/w). Volatile organic compounds (28 components) were detected by GC-MS, and among of these, 11 compounds were common to three samples of bamboo vinegar. Perhaps acetic acid, 3-methyl-1,2-cyclohexadione, guaiacol, p-cresol, and syringol contributed to the characteristic odors (sour, smoky, and medicinal note) in bamboo vinegar.