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Abstract: The advantages of supercritical water (SCW) as a reaction medium for upgrading oil sand bitumen were investigated through a comprehensive analysis of the output product, which includes gaseous products, middle distillate, distillation residue, and coke. Canadian oil sand bitumen mined by the steam assisted gravity drainage method was treated in an autoclave at 420–450°C and 20–30MPa for up to 120min with three kinds of reaction media: SCW, high-pressure nitrogen, and supercritical toluene. The yields of gaseous products indicated that a very small amount of water was involved in the upgrading reaction. The analytical results of the middle distillate fractions were almost the same using water and nitrogen at 450°C. The distillation residues produced in SCW had lower molecular weight distributions, lower H/C atomic ratios, higher aromaticities, and consequently more condensed structures compared to those produced in nitrogen. The coke produced using SCW also had lower H/C values and higher aromaticities. Judging from all the analytical results, the upgrading of bitumen by SCW reaction was primarily considered to be physical in nature. As a result, it is possible to highly disperse the heavy fractions by SCW. This dispersion effect of SCW led to intramolecular dehydrogenation of the heavier component and prevention of recombination reactions, and consequently gave the highest conversion. [ABSTRACT FROM AUTHOR]

The article presents an obituary for Takashi Tsuruo, the founding associate editor of "The Journal of Experimental Therapeutics and Oncology," who died on December 16, 2008.

Six to seven narrow fractions from each of six light gas oils obtained from various upgrading processes of Athabasca oilsands bitumen were evaluated as diesel fuels. The relationships among the boiling range, cetane number (CN), and aromatics content were investigated. The correlation between CN and aromatic content of narrow-cut fractions showed three trends; i.e. (1) CN increases as aromatics increase; (2) CN decreases as aromatics increase; and (3) a mixed trend of both. In order to investigate such trends, the narrow-cut samples were separated into saturate and aromatic fractions using HPLC, and analyses of type distribution and average molecular structures were carried out. CN of narrow cuts was estimated from the type distribution and CN data for pure compounds.From the H/C atomic ratio, each narrow-cut sample was represented by six types of compounds, i.e. alkanes, mono- and dicycloalkanes, alkyl and naphthenic benzenes, and diaromatics. The average molecular structures for those types had 4–5 branches. The CN trend of the narrow-cuts were reproducible at a certain assumption from the CN of the average molecule structure by type estimated from the CNs of pure compounds. It was found that for the prediction of CN, not only aromatic content but also hydrocarbon types of saturates are important. [Copyright &y& Elsevier]

Six coals with different ranks and different ash contents have been used to study the effect of demineralization on N2 formation during coal pyrolysis. Chars obtained after pyrolysis have been also gasified with carbon dioxide at 1000 °C to investigate the influence of the demineralization on char gasification reactivity. The pyrolysis results show that the demineralization by acid washing drastically changes N2 formation profiles and decreases nitrogen conversion to N2 for low rank coals; on the other hand, the demineralization has little effect on N2 formation for high rank coals. Addition of 0.5 wt% Fe promotes N2 formation from the demineralized coals, but the catalytic effect depends on the coal type. It is found that the Fe remarkably promotes N2 formation from the demineralized low rank coals, but the effect is much smaller for high rank demineralized coals. These observations suggest that the existing state of Fe-containing minerals and added Fe catalyst is important for catalytic N2 formation during coal pyrolysis. Gasification results show that the demineralization lowers char gasification reactivity not only for low rank coals but also for high rank coals. [Copyright &y& Elsevier]

To characterize the bovine chromosome 6q21 for bovine chondrodysplastic dwarfism (BCD), we developed 48 new microsatellite markers from yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones using a modified magnetic bead capture method. These new markers were used to construct a high-resolution physical map of the region with a total of 85 loci. The physical map will be a powerful tool for successful positional cloning experiments. [ABSTRACT FROM AUTHOR]

Erythropoietin is a glycoprotein factor which specifically regulates the proliferation and differentiation of erythroid progenitor cells. We have investigated here the biochemical mechanisms of erythroid differentiation on mouse erythroleukemia SKT6 cells which can be induced to differentiate either with erythropoietin or dimethyl sulfoxide (Me2SO). cAMP-elevating agents, such as forskolin and 3-isobutyl-l-methyl-xanthine, caused spontaneous erythroid differentiation, and these agents showed the stimulatory effects on erythropoietin- or Me2SO-induced differentiation. An adenylate cyclase inhibitor, 2',5'-dideoxyadenosine, blocked erythropoietin-induced differentiation. The intracellular cAMP level was rapidly increased by addition of erythropoietin but not by Me2SO. These observations suggest that erythroid differentiation induced by erythropoietin is mediated, at least in part, through the cAMP-dependent pathway. When the effect of erythropoietin and Me2SO on the intracellular Ca2+ level was examined using fura 2, no acute change was observed. Measurements of the levels of inositol 1,4,5-trisphosphate and diacylglycerol following stimulation with erythropoietin or Me2SO showed that phosphatidylinositol turnover did not change significantly after erythropoietin stimulation but decreased gradually after Me2SO induction. Taken together, these results indicate that a complex signaling network including the cAMP-dependent pathway is involved in the erythroid differentiation process. [ABSTRACT FROM AUTHOR]

Two-gene vectors with positive or positive-negative drug-selectable markers enable the expansion or elimination of gentetically modified cells in vivo. We have established a bicistronic retroviral vector system which utilizes an internal ribosome entry site (IRES) to co-express two independent genes with high efficiency. As a positive-negative (suicide) marker, Herpes simplex virus thymidine kinase was coexpressed with the human multidrug resistance gene, MDR1. Using this vector, almost all the MDR1-transduced cells showed hypersensitivity to a nucleoside analog, gancielovir. As a dominant selectable marker, the MDR1 gene was co-expressed with a-galactosidase A for the model of gene therapy of Fabry disease. Vineristine selection efficiently enhanced the population of transduced cells expressing the second non-selectable genes. These drug-selectable retroviral vectors could be applicable to the therapy of many diseases. [ABSTRACT FROM AUTHOR]

Abstract The integrated stress response (ISR) is a cellular process that is characterized by activation of eukaryotic initiation factor (eIF)2α kinases and subsequent induction of activating transcription factor (ATF)4. The ISR plays an important role in protecting cells from tumor-related metabolic stresses, such as nutrient deprivation and perturbed proteostasis. Here, we showed that disruption of the ISR, together with increased cellular stress vulnerability, was produced by pharmacological inhibition of BCR-ABL, the oncogenic driver in chronic myeloid leukemia (CML). Treatment of CML-derived K562 cells with BCR-ABL tyrosine kinase inhibitors, including imatinib, dasatinib, nilotinib and ponatinib, prevented activation of eIF2α kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control nonderepressible 2, and downstream ATF4 induction during metabolic stress. Prevention of ATF4 induction likely occurred as a result of the combinatorial suppression of the eIF2α kinase and phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathways. In addition, we found that pharmacological inhibition of PERK mimicked BCR-ABL inhibition to enhance apoptosis induction under stress conditions. These findings indicate that the ISR is under the control of BCR-ABL and may foster adaptation to tumorigenic stresses in CML cells. Highlights •BCR-ABL inhibitors prevent activation of PERK and GCN2. •BCR-ABL inhibitors suppress ATF4 induction, possibly through multiple mechanisms. •Metabolic stress conditions enhance apoptosis induction by BCR-ABL inhibitors. [ABSTRACT FROM AUTHOR]

Background Cancer is a complex disease with multiple genetic and epigenetic alterations. Since decades, the hallmark of cancer therapy is chemotherapy. Cytotoxic drugs erase rapidly dividing cells without sufficient differentiation between normal and cancerous cells resulting in severe side effects in normal tissues. Recently, strategies for cancer treatment focused on targeting specific proteins involved in tumor growth and progression. The present study was designed to investigate the cytotoxicity of 65 crude extracts from 35 Sudanese medicinal plants towards various cancer cell lines expressing molecular mechanisms of resistance towards classical chemotherapeutics (two ATP-binding cassette transporters, ABCB1 (P-glycoprotein) and ABCB5, tumor suppressor p53, epidermal growth factors receptor (EGFR). And the aim was to identify plant extracts and isolated compounds thereof with activity towards otherwise drug-resistant tumor cells. Methods Cold maceration was performed to obtain crude extracts from the plants. The resazurin assay was used to determine cytotoxicity of the plant extracts. Microarray-based mRNA expression profiling, COMPARE, and hierarchical cluster analyses were applied to identify, which genes correlate with sensitivity or resistance to ambrosin, the main constituent of the most active extract Ambrosia maritima . Results The results of the resazurin assay on different tumors showed that Lawsonia inermis, Trigonella foenum-graecum and Ambrosia maritma were the most active crude extracts. Ambrosin was selected as one active principle of A. maritima for microarray-based expression profiling. Genes from various functional groups (transcriptional regulators, signal transduction, membrane transporters, cytoskeleton organization, chaperones, immune system development and DNA repair) were significantly correlated with response of tumor cell lines to ambrosin. Conclusion The results revealed cytotoxicity and pharmacogenomics studies of Sudanese medicinal plants provide an attractive strategy for the development of novel cancer therapeutics with activity towards cell lines that resistance to established anticancer agents. [ABSTRACT FROM AUTHOR]

Dystocia is a major problem for the dairy cattle industry, and the observed high rates of this condition stem from genetic selection to increase subsequent milk production of the calving female. Because smaller birth size does not adversely affect subsequent milk production, selecting for cows with a smaller birth size would reduce dystocia rates and be beneficial for both the cattle and the farmers. To identify genes that regulate birth weight, we conducted a genome-wide association study using 1151 microsatellite markers and identified a single nucleotide polymorphism (SNP) associated with birth weight: A-326G in the 5' untranslated region (UTR) of solute carrier family 44, member 5 (SLC44A5). Cows with higher birth weights carried the A polymorphism in the SLC44A5 59 UTR, and the presence of the A polymorphism correlated with a high rate of dystocia. Luciferase assays and quantitative polymerase chain reaction (QPCR) assays revealed that SLC44A5 transcripts with the A polymorphism are expressed at lower levels than those carrying the G polymorphism. SLC44A5 encodes a choline transporter-like protein, and choline is a component of the major phospholipids of cell membranes. Uptake studies in HeLa cells demonstrated that SLC44A5 knockdown reduces choline efflux, whereas SLC44A5 overexpression resulted in the opposite effect. Furthermore, cell viability assays indicated that SLC44A5 knockdown increased cell proliferation, whereas SLC44A5 overexpression repressed proliferation. Taken together, our results suggest that calves with reduced SLC44A5 expression are larger due to enhanced cell proliferation. This study provides novel insights into the molecular mechanisms that control birth weight in Holsteins and suggests that SLC44A5 may serve as a potential target for preventing dystocia. [ABSTRACT FROM AUTHOR]

Abstract: Tankyrase-1 poly(ADP-ribosyl)ates the telomere-binding protein TRF1. This post-translational modification dissociates TRF1 from telomeres, enhancing telomerase-mediated telomere elongation. Tankyrase-1 multimerizes via its sterile alpha motif domain, but its functional implication remains elusive. Here, we found that excessive amounts of tankyrase-1 form punctate nuclear foci. This focus formation depends on both homophilic multimerization and heterophilic protein–protein interaction. These foci are functionally dormant because they do not efficiently release TRF1 from telomeres. Consistently, hyper-overexpression of tankyrase-1 attenuates its ability to elongate telomeres. These observations suggest that tankyrase-1 assembly to large protein complexes masks its telomeric function. Structured summary: MINT-7987689, MINT-7987677: Tankyrase-1 (uniprotkb:O95271) and TRF1 (uniprotkb:P54274) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7987977: Tankyrase-1 (uniprotkb:O95271) physically interacts (MI:0915) with TRF1 (uniprotkb:P54274) by anti tag coimmunoprecipitation (MI:0007) MINT-7987998: Tankyrase-1 (uniprotkb:O95271) physically interacts (MI:0915) with Tankyrase-1 (uniprotkb:O95271) by anti tag coimmunoprecipitation (MI:0007). [ABSTRACT FROM AUTHOR]

Abstract: Eight stilbenoids, 1-(p-hydroxybenzyl)-4,8-dimethoxyphenanthrene-2,7-diol (1), 2,7-dihydroxy-1,3-bis(p-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene (2), 4,7-dihydroxy-1-(p-hydroxybenzyl)-2-methoxy-9,10-dihydrophenanthrene (3), 3,3′-dihydroxy-2′,6′-bis(p-hydroxybenzyl)-5-methoxybibenzyl (4), 3′,5-dihydroxy-2-(p-hydroxybenzyl)-3-methoxybibenzyl (5), blestriarenes B (6) and C (7), and blestrianol A (8) have been isolated by the guidance of inhibitory effect of tubulin polymerization from the tubers of Bletilla striata (Orchidaceae). Among them, both of bisbenzyls 4 and 5 inhibited the polymerization of tubulin at IC50 10μM, respectively. Furthermore bisbenzyl 4 potentiated the cytotoxicity of SN-38 in BCRP-transduced K562 (K562/BCRP) cells. [Copyright &y& Elsevier]

Investigates whether human wild type ABCG2, also known as placenta-specific ABC transporter/breast cancer resistance protein/mitoxantrone resistance-associated protein, transports estrone 3-sulfate using membrane vesicles from cDNA-transfected mouse lymphoma cell line. Expression of ABCG2; Determination of the substrate specificity of ABCG2; Uptake of [sup3H]E[sub1]S into membrane vesicles.

Presents a study that showed that the sumoylation enhances the cleavable complex formation and apoptosis induced by camptothecin (CPT). Reduction of the CPT-induced cleavable complexes without influencing its in vitro catalytic activity; Manner of increase of CPT-induced cleavable complexes of wild-type topoisomerase I (Topo1); Independent occurrence of Topo1 sumoylation.

Background: Calf mortality generally occurs in calves prior to weaning, which is a serious problem in cattle breeding. Several causative variants of monogenic Mendelian disorders in calf mortality have been identified, whereas genetic factors affecting the susceptibility of calves to death are not well known. To identify variants associated with calf mortality in Japanese Black cattle, we evaluated calf mortality as a categorical trait with a threshold model and performed a genome-wide copy number variation (CNV) association study on calf mortality. Results: We identified a 44-kb deleted-type CNV ranging from 103,317,687 to 103,361,802 bp on chromosome 5, which was associated with the mortality of 1–180-day-old calves. The CNV harbored C1RL, a pseudogene, and an IncRNA localized in the C1R and C1S gene cluster, which is a component of the classical complement activation pathway for immune complexes for infectious pathogens. The average complement activity in CNVR_221 homozygotes at postnatal day 7 was significantly lower than that of wild-type animals and heterozygotes. The frequency of the risk allele in dead calves suffering from diarrhea and pneumonia and in healthy cows was 0.35 and 0.28, respectively (odds ratio = 2.2, P = 0.016), suggesting that CNVR_221 was associated with the mortality of Japanese Black calves suffering from an infectious disease. Conclusions: This study identified a deleted-type CNV associated with the mortality of 1–180-day-old calves. The complement activity in CNVR_221 homozygotes was significantly lower than that in heterozygotes and wild type animals. The frequency of the risk allele was higher in dead calves suffering from an infectious disease than in healthy cows. These results suggest that the existence of CNVR_221 in calves could be attributed to a reduction in complement activity, which in turn leads to susceptibility to infections. Thus, the risk allele could serve as a useful marker to reduce the mortality of infected Japanese Black calves. [ABSTRACT FROM AUTHOR]

JQ1 disrupts the binding of bromodomain and extra-terminal (BET) family of proteins to acetylated histones, modulates the expression of various genes, and inhibits the proliferation of cancer cells. We established two JQ1-resistant sublines from human colorectal cancer HCT116 cells. These resistant cells showed an 8- to 9-fold higher resistance to JQ1, and a 2- to 4-fold higher resistance to various anti-cancer agents, such as doxorubicin, etoposide, mitoxantrone, SN-38, cisplatin, and methotrexate than the parental HCT116 cells. The JQ1-resistant cells expressed higher levels of TRAF2 and NCK-interacting protein kinase (TNIK), cyclin D1 (CCND1), cyclin E1 (CCNE1), and their corresponding mRNAs than the parental cells. TNIK is a regulator of Wnt/β-catenin signaling and is known to transactivate CCND1. Transient transfection of HCT116 cells with a TNIK expression plasmid resulted in the upregulation of cyclin D1, cyclin E1, and their corresponding mRNAs, as well as an increase in CCNE1 promoter activity. Furthermore, luciferase assay revealed that the JQ1-resistant cells showed high CCNE1 promoter activity. These results suggest that TNIK also transactivates CCNE1. Three stable TNIK transfectant clones of HEK293 cells expressed 1.5- to 2-fold higher levels of TNIK, cyclin D1, and cyclin E1 than the parental cells. The 293/TNIK-6 cells, which expressed the highest level of TNIK among the transfectants, showed a 2.3-fold higher resistance to JQ1 than the parental cells. These results suggest the possible involvement of TNIK in cellular resistance to JQ1. • Cross-resistance of JQ1-resistant cells to DNA-damaging agents. • Upregulation of TNIK, cyclin D1, and cyclin E1 in JQ1-resistant cells. • Upregulation of cyclin D1 and cyclin E1 in TNIK -transfected cells. • Increased CCNE1 promoter activity in JQ1-resistant cells and TNIK -transfected cells. • JQ1 resistance of stable TNIK -transfected cells. [ABSTRACT FROM AUTHOR]

We previously reported the upregulation of cellular Glu and glutathione levels in human ABCB5 - and murine Abcb5 -transfected cells. Here, we demonstrate the upregulation of STAT1 and glutaminase (GLS) in ABCB5 / Abcb5 -transfected cells. Among a total of four ABCB5/Abcb5 high-expressing clones with docetaxel resistance, three of the clones expressed STAT1 and GLS highly and showed resistance to docetaxel and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. Neither STAT1 nor GLS upregulation was observed in the remaining ABCB5 high-expressing clone, as well as in another two ABCB5 low-expressing clones; these three clones did not show BSO resistance. The ABCB5/STAT1 high-expressing clones showed higher cellular levels of Ala, Glu, and Asp and lower cellular levels of Phe, Trp, Leu, Ile, Gly, Met, Tyr, Val, and His compared to the ABCB5/STAT1 low-expressing clones. The former clones also showed a higher resistance to Glu. The STAT1 -transfected clones expressed high levels of GLS and the corresponding mRNA, suggesting the transactivation of GLS by STAT1. These clones showed resistance to Glu and BSO, similar to the ABCB5/STAT1 high-expressing clones. The cellular glutathione levels of the STAT1 -transfected clones were significantly higher than that of the control. The STAT1 -transfected clones also showed greater resistance to the effect of BSO on the cellular glutathione depletion compared to the control. These results demonstrate that STAT1 upregulates GLS and modulates amino acids and glutathione metabolism. Although we were unable to directly prove STAT1 upregulation by ABCB5, our results suggest that ABCB5 expression, directly or indirectly, leads to the overexpression of STAT1. • STAT1 and GLS upregulation and resistance to docetaxel and BSO in 3/4 ABCB5/Abcb5 high-expressing clones. • Docetaxel resistance in ABCB5 high-expressing clone lacking STAT1 upregulation, without GLS upregulation or BSO resistance. • Higher Ala, Glu, and Asp levels in ABCB5/STAT1 high-versus low-expressing clones. • Upregulation of GLS and resistance to BSO in STAT1 -transfected clones. • High levels of glutathione and resistance to BSO in STAT1 -transfected clones upon glutathione depletion. [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) is associated with cancer malignancies such as invasion, metastasis, and drug resistance. In this study, HCT116 human colorectal cancer cells were transduced with SLUG or SNAIL retroviruses, and EMT cells with mesenchymal morphology were established. The EMT cells showed a high invasive activity and resistance to several anticancer agents such as methotrexate, SN-38, and cisplatin. Furthermore, they contained about 1–10% side population (SP) cells that were not stained by Hoechst 33342. This SP phenotype was not stable; the isolated SP cells generated both SP and non-SP cells, suggesting a potential for differentiation. Gene expression analysis of SP cells suggested the alteration of genes that are involved in epigenetic changes. Therefore, we examined the effect of 74 epigenetic inhibitors, and found that two inhibitors, namely I-BET151 and bromosporine, targeting the bromodomain and extra-terminal motif (BET) proteins, decreased the ratio of SP cells to <50% compared with the control, without affecting the immediate efflux of Hoechst 33342 by transporters. In addition, compared with the parental cells, the EMT cells showed a higher sensitivity to I-BET151 and bromosporine. This study suggests that EMT development and SP phenotype can be independent events but both are regulated by BET inhibitors in SLUG - or SNAIL -transducted HCT116 cells. • SNAIL and SLUG induce EMT and drug resistance in human colorectal cancer cells. • The EMT cells contained 1–10% side population cells. • BET inhibitors decreased the ratio of side population cells. • The EMT cells showed higher sensitivity to BET inhibitors. [ABSTRACT FROM AUTHOR]

Abstract Poly (ADP-ribose) polymerases (PARPs) are involved in various cellular events, including DNA repair. PARP inhibitors including olaparib and rucaparib, have been specially developed against breast and ovarian cancers deficient in DNA repair systems. In this study, we found that PARP1-defective olaparib-resistant A2780 cells (ola-R cells) cells were still sensitive to two PARP inhibitors, rucaparib and veliparib. Metabolomic analysis revealed that rucaparib suppressed the lactate dehydrogenase (LDH)-mediated conversion of pyruvic acid to lactic acid in A2780 cells, although olaparib did not. The inhibition of LDH by siRNA-mediated knockdown or by LDH inhibitors suppressed the growth of ovarian cancer cells. Our results suggested that the suppression of the LDH-associated pathway contributed to the pharmacological effects of rucaparib. Highlights • Several PARP inhibitors have been developed for clinical use in cancer chemotherapy. • PARP1-deficient A2780 cells are resistant to a PARP inhibitor olaparib, but not to another PARP inhibitor rucaparib. • Rucaparib suppresses lactate dehydrogenase (LDH)-mediated pyruvate to lactate metabolism in ovarian cancer cells. • Suppression of LDH-pathway shows antiproliferative effect on ovarian cancer cells. [ABSTRACT FROM AUTHOR]

FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) is a constitutively active mutant of FLT3 and causes 20%–30% of acute myeloid leukemia (AML) cases. FLT3-ITD upregulates the proviral integration site for Moloney murine leukemia virus 1 (PIM-1) expression and promotes the proliferation of AML cells. In this study, we investigated the role of protein kinase C (PKC)-mediated phosphorylation on the expression and function of PIM-1L. Drug screening in leukemia cell lines revealed that sotrastaurin (a PKC inhibitor) suppressed the proliferation of the FLT3-ITD-positive AML cell line MV4-11 but not of K562, HL60, or KG-1a cells, similar to SGI-1776 (a PIM-1/FLT3 inhibitor) and quizartinib (an FLT3 inhibitor). Sotrastaurin decreased the expression of pro-survival protein myeloid cell leukemia (MCL-1) and the phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), both of which are downstream effectors of PIM-1. PKCα directly phosphorylated Ser65 of PIM-1L, which is a long isoform of PIM-1. The PKCα-mediated phosphorylation stabilized PIM-1L. The phosphorylation-mimicked mutant, PIM-1L-S65D, was more stable and showed higher kinase activity than PIM-1L-S65A. Expression of PIM-1L-wildtype or -S65D reduced sotrastaurin-mediated apoptosis and growth inhibition in MV4-11 transfectants. These results suggest that PKCα directly upregulates PIM-1L, resulting in promotion of the survival and proliferation of AML cells. [ABSTRACT FROM AUTHOR]

Weak calf syndrome (WCS) is a major cause of calf death in Japanese Black cattle. Among IARS disorders, theisoleucyl-tRNA synthetasec.235G>C mutation has been identified as one of the causes of WCS. However, calf deaths differing from those attributed to IARS disorder has been occurring. To identify other genes potentially responsible for these calf deaths, we constructed three populations of three bulls (Bull-1, -2 and -3) that did not carry theIARSmutation, and dead calves (18, 28, and 31 calves) and healthy cattle (18, 15, and 10 cattle) sired by these bulls. The populations were genotyped using the BovineSNP50 BeadChip, but homozygosity mapping did not detect any associated genomic regions with calf death. Linkage analysis performed using each population as a paternal half-sib family of Bull-1, Bull-2, and Bull-3 revealed that, in the Bull-1 population, calf death was mapped to the 8.94 Mb–14.53 Mb and 29.82 Mb–33.77 Mb regions of BTA29. The findings suggested that the incidence of calf death in calves sired by Bull-1 was a hereditary disease exhibiting a dominant, not recessive, inheritance pattern. [ABSTRACT FROM PUBLISHER]

Hydrallantois is the excessive accumulation of fluid in the allantoic cavity in a pregnant animal and is associated with fetal death. We recently identified a recessive missense mutation in the solute carrier family 12, member 1 ( SLC12A1) gene (g.62382825G>A, p.Pro372Leu) that is associated with hydrallantois in Japanese Black cattle. Unexpectedly, we found a case of the homozygous risk-allele for SLC12A1 in a calf, using a PCR-based direct DNA sequencing test. The homozygote was outwardly healthy up to 3 months of age and the mother did not exhibit any clinical symptoms of hydrallantois. In order to validate these observations, we performed confirmation tests for the genotype and a diuretic loading test using furosemide, which inhibits the transporter activity of the SLC12A1 protein. The results showed that the calf was really homozygous for the risk-allele. In the homozygous calf, administration of furosemide did not alter urinary Na+ or Cl− levels, in contrast to the heterozygote and wild-type calves in which these were significantly increased. These results demonstrate that the SLC12A1 (g.62382825G>A, p.Pro372Leu) is a hypomorphic or loss-of-function mutation and the hydrallantois with this mutation shows incomplete penetrance in Japanese Black cattle. [ABSTRACT FROM AUTHOR]

The influence of genotype imputation using low-density single nucleotide polymorphism (SNP) marker subsets on the genomic relationship matrix (G matrix), genetic variance explained, and genomic prediction (GP) was investigated for carcass weight and marbling score in Japanese Black fattened steers, using genotype data of approximately 40,000 SNPs. Genotypes were imputed using equally spaced SNP subsets of different densities. Two different linear models were used. The first (model 1) incorporated one G matrix, while the second (model 2) used two different G matrices constructed using the selected and remaining SNPs. When using model 1, the estimated additive genetic variance was always larger when using all SNPs obtained via genotype imputation than when using only equally spaced SNP subsets. The correlations between the genomic estimated breeding values obtained using genotype imputation with at least 3,000 SNPs and those using all available SNPs without imputation were higher than 0.99 for both traits. While additive genetic variance was likely to be partitioned with model 2, it did not enhance the accuracy of GP compared with model 1. These results indicate that genotype imputation using an equally spaced low-density panel of an appropriate size can be used to produce a cost-effective, valid GP. [ABSTRACT FROM AUTHOR]

Isoleucyl-tRNA synthetase ( IARS) c.235G > C (p.V79L) is a causative mutation for a recessive disease called IARS disorder in Japanese black cattle. The disease is involved in weak calf syndrome and is characterized by low birth weight, weakness and poor suckling. The gestation period is often slightly extended, implying that intrauterine growth is retarded. In a previous analysis of 2597 artificial insemination (AI) procedures, we suggested that the IARS mutation might contribute toward an increase in the incidence of prenatal death. In this study, we extended this analysis to better clarify the association between the IARS mutation and prenatal death. The IARS genotypes of 92 animals resulting from crosses between carrier (G/C) × G/C were 27 normal (G/G), 55 G/C and 10 affected animals (C/C) (expected numbers: 23, 46 and 23, respectively). Compared to the expected numbers, there were significantly fewer affected animals in this population ( P < 0.05), suggesting that more than half of the affected embryos died prenatally. When the number of AI procedures examined was increased to 11 580, the frequency of re-insemination after G/C × G/C insemination was significantly higher at 61-140 days ( P < 0.001). The findings suggested that the homozygous IARS mutation not only causes calf death, but also embryonic or fetal death. [ABSTRACT FROM AUTHOR]

Days open ( DO), which is the interval from calving to conception, is an important trait related to reproductive performance in cattle. To identify quantitative trait loci for DO in Japanese Black cattle, we conducted a genome-wide association study with 33 303 single nucleotide polymorphisms ( SNPs) using 459 animals with extreme DO values selected from a larger group of 15 488 animals. We identified a SNP on bovine chromosome 2 ( BTA2) that was associated with DO. After imputation using phased haplotype data inferred from 586 812 SNPs of 1041 Japanese Black cattle, six SNPs associated with DO were located in an 8.5-kb region of high linkage disequilibrium on BTA2. These SNPs were located on the telomeric side at a distance of 177 kb from the parathyroid hormone 2 receptor ( PTH2R) gene. The association was replicated in a sample of 1778 animals. In the replicated population, the frequency of the reduced- DO allele ( Q) was 0.63, and it accounted for 1.72% of the total genetic variance. The effect of a Q-to- q allele substitution on DO was a decrease of 3.74 days. The results suggest that the Q allele could serve as a marker in Japanese Black cattle to select animals with superior DO performance. [ABSTRACT FROM AUTHOR]

Background: Copy number variation (CNV) is an important source of genetic variability associated with phenotypic variation and disease susceptibility. Comprehensive genome-wide CNV maps provide valuable information for genetic and functional studies. To identify CNV in Japanese Black cattle, we performed a genome-wide autosomal screen using genomic data from 1,481 animals analyzed with the Illumina Bovine High-Density (HD) BeadChip Array (735,293 single-nucleotide polymorphisms (SNPs) with an average marker interval of 3.4 kb on the autosomes). Results: We identified a total of 861 CNV regions (CNVRs) across all autosomes, which covered 43.65 Mb of the UMD3.1 genome assembly and corresponded to 1.74 % of the 29 bovine autosomes. Overall, 35 % of the CNVRs were present at a frequency of > 1 % in 1,481 animals. The estimated lengths of CNVRs ranged from 1.1 kb to 1.4 Mb, with an average of 50.7 kb. The average number of CNVR events per animal was 35. Comparisons with previously reported cattle CNV showed that 72 % of the CNVR calls detected in this study were within or overlapped with known CNVRs. Experimentally, three CNVRs were validated using quantitative PCR, and one CNVR was validated using PCR with flanking primers for the deleted region. Out of the 861 CNVRs, 390 contained 717 Ensembl-annotated genes significantly enriched for stimulus response, cellular defense response, and immune response in the Gene Ontology (GO) database. To associate genes contained in CNVRs with phenotypes, we converted 560 bovine Ensembl gene IDs to their 438 orthologous associated mouse gene IDs, and 195 of these mouse orthologous genes were categorized into 1,627 phenotypes in the Mouse Genome Informatics (MGI) database. Conclusions: We identified 861 CNVRs in 1,481 Japanese Black cattle using the Illumina BovineHD BeadChip Array. The genes contained in CNVRs were characterized using GO analysis and the mouse orthologous genes were characterized using the MGI database. The comprehensive genome-wide CNVRs map will facilitate identification of genetic variation and disease-susceptibility alleles in Japanese Black cattle. [ABSTRACT FROM AUTHOR]

Using target and reference fattened steer populations, the performance of genotype imputation using lower-density marker panels in Japanese Black cattle was evaluated. Population imputation was performed using BEAGLE software. Genotype information for approximately 40 000 single nucleotide polymorphism ( SNP) markers by Illumina BovineSNP50 BeadChip was available, and imputation accuracy was assessed based on the average concordance rates of the genotypes, varying equally spaced SNP densities, and the number of individuals in the reference population. Two additional statistics were also calculated as indicators of imputation performance. The concordance rates tended to be lower for SNPs with greater minor allele frequencies, or those located near the ends of the chromosomes. Longer autosomes yielded greater imputation accuracies than shorter ones. When SNPs were selected based on linkage disequilibrium information, relative imputation accuracy was slightly improved. When 3000 and 10 000 equally spaced SNPs were used, the imputation accuracies were greater than 90% and approximately 97%, respectively. These results indicate that combining genotyping using a lower-density SNP chip with genotype imputation based on a population of individuals genotyped using a higher-density SNP chip is a cost-effective and valid approach for genomic prediction. [ABSTRACT FROM AUTHOR]

Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia. [ABSTRACT FROM AUTHOR]

Conception rates among dairy cows in Japan have declined in recent decades. To enhance our understanding of the genes involved in conception rates, we conducted a genome-wide association study (GWAS) using 822 Holsteins and identified a single-nucleotide polymorphism (SNP) associated with conception rate: A+169G in the 3’ untranslated region (UTR) of unc-5 homolog C (UNC5C). Cows with higher conception rates carried the A polymorphism in the UNC5C 3’UTR. Luciferase assays and quantitative analysis of allele ratios revealed that UNC5C transcripts with the A polymorphism were expressed at higher levels than those carrying the G polymorphism. UNC5C transmits either pro- or anti-apoptotic signals depending on the availability of its ligand, Netrin-1. UNC5C expression is negatively regulated by reproductive homeobox X-linked 5 (Rhox5), and the Rhox5 locus is methylated by G9a methyltransferase. G9a-knockout mice have previously been demonstrated to be subfertile, and we found that UNC5C, G9a, and Netrin-1 expression levels increased from the 4-cell stage to the blastocyst stage in fertilized murine embryos, whereas Rhox5 expression decreased. Repression of UNC5C, G9a, or Netrin-1 or forced expression of Rhox5 in the anterior nucleus stage inhibited development to the blastocyst stage, suggesting that cows carrying the G polymorphism in UNC5C might have lower conception rates because of the poor development of preimplantation embryos. This study provides novel insights into the role of UNC5C during embryonic development. [ABSTRACT FROM AUTHOR]

Apigenin is a common dietary flavonoid with considerable cytotoxic activity in vitro and in vivo . Despite many mechanistic studies, less is known about resistance factors hampering apigenin's activity. We investigated the ATP-binding cassette (ABC) transporters BCRP/ ABCG2 , P-glycoprotein/ ABCB1 and its close relative ABCB5 . Multidrug-resistant cells overexpressing these ABC transporters were not cross-resistant toward apigenin. Moreover, apigenin inhibited not only P-glycoprotein but also BCRP by increasing cellular uptake of doxorubicin and synergistic inhibition of cell viability in combination with doxorubicin or docetaxel in multidrug-resistant cells. To perform in silico molecular docking studies, we first generated homology models for human P-glycoprotein and ABCB5 based on the crystal structure of murine P-glycoprotein. Their nucleotide binding domains (NDBs) revealed the highest degrees of sequence homologies (89%–100%), indicating that ATP binding and cleavage is of crucial importance for ABC transporters. Molecular docking of apigenin bound to the NDBs of P-glycoprotein and ABCB5 in molecular docking studies. Hence, apigenin may compete with ATP for NDB-binding leading to energy depletion to fuel the transport of ABC transporter substrates. Furthermore, we performed COMPARE and hierarchical cluster analyses of transcriptome-wide mRNA expression profiles of the National Cancer Institute tumor cell line panel. Microarray-based mRNA expressions of genes of diverse biological functions (signal transduction, transcriptional regulation, ubiquitination, autophagy, metabolic activity, xenobiotic detoxification and microtubule formation) significantly predicted responsiveness of tumor cells to apigenin. In conclusion, apigenin's activity is not hampered by classical mechanisms of multidrug resistance and the inhibition of ABC transporters by apigenin indicates that apigenin may overcome multidrug resistance in otherwise refractory tumors. [ABSTRACT FROM AUTHOR]

Abstract: P-glycoprotein (P-gp)/ABCB1 is a key molecule of multidrug resistance in cancer. Protein phosphatase (PP) 2A, regulatory subunit B, gamma (PPP2R3C), which is a regulatory subunit of PP2A and PP5, was identified as a binding candidate to P-gp. Immunoprecipitation–western blotting revealed that PP5 and PPP2R3C were coprecipitated with P-gp, while PP2A was not. PP5/PPP2R3C dephosphorylated protein kinase A/protein kinase C-phosphorylation of P-gp. Knockdown of PP5 and/or PPP2R3C increased P-gp expression and lowered the sensitivity to vincristine and doxorubicin. Consequently, our results indicate that PP5/PPP2R3C negatively regulates P-gp expression and function. [Copyright &y& Elsevier]

Genomic selection using high-density single nucleotide polymorphism ( SNP) genotype data may accelerate genetic improvements in livestock animals. In this study, we attempted to estimate the variance components of six carcass traits in fattened Japanese Black steers using SNP genotype data. Six hundred and seventy-three steers were genotyped using an Illumina Bovine SNP50 Bead Chip and phenotyped for cold carcass weight, ribeye area, rib thickness, subcutaneous fat thickness, estimated yield percent and marbling score. Additive polygenic variance and the variance attributable to a set of SNPs that had statistically significant effects on the trait were estimated via Gibbs sampling with two models: (i) a model with the chosen SNPs and the additive polygenic effects; and (ii) a model with the polygenic effects alone. The proportion of the estimated variance attributable to the SNPs became higher as the number of SNP effects that fit increased. High correlations between breeding values estimated with the model containing the polygenic effect alone and those estimated by chosen SNPs were obtained. No fraction of the total genetic variance was explained by SNPs associated with the trait at P ≥ 0.1. Our results suggest that for the carcass traits of Japanese Black cattle, a maximum of half of the total additive genetic variance may be explained by SNPs between 100 several tens to several 100s. [ABSTRACT FROM AUTHOR]

MCC1381 may be a novel pan-inhibitor of HDACs, evidenced by binding to HDAC6, inducing cell apoptosis, arresting cell cycle, inducing excessive ROS, MMP collapse and in vivo anti-cancer effects. [Display omitted] New and potent agents that evade multidrug resistance (MDR) and inhibit epigenetic modifications are of great interest in cancer drug development. Here, we describe that a moniliformin derivative (IUPAC name: 3‐(naphthalen‐2‐ylsulfanyl)‐4‐{[(2Z)‐1,3,3‐trimethyl‐2,3‐dihydro‐1H‐indol‐2‐ylidene]methyl}cyclobut‐3‐ene‐1,2‐dione; code: MCC1381) bypasses P-gp-mediated MDR. Using transcriptomics, we identified a large number of genes significantly regulated in response to MCC1381, which affected the cell cycle and disturbed cellular death and survival. The potential targets of MCC1381 might be histone deacetylases (HDACs) as predicted by SwissTargetPrediction. In silico studies confirmed that MCC1381 presented comparable affinity with HDAC1, 2, 3, 6, 8 and 11. Besides, the inhibition activity of HDACs was dose-dependently inhibited by MCC1381. Particularly, a strong binding affinity was observed between MCC1381 and HDAC6 by microscale thermophoresis analysis. MCC1381 decreased the expression of HDAC6, inversely correlated with the increase of acetylated HDAC6 substrates, acetylation p53 and α-tubulin. Furthermore, MCC1381 arrested the cell cycle at the G 2 /M phase, induced the generation of reactive oxygen species and collapse of the mitochondrial membrane potential. MCC1381 exhibited in vivo anti-cancer activity in xenografted zebrafish. Collectively, MCC1381 extended cytotoxicity towards P-gp-resistant leukemia cancer cells and may act as a pan-HDACs inhibitor, indicating that MCC1381 is a novel candidate for cancer therapy. [ABSTRACT FROM AUTHOR]

MCC1734 can bypass P-gp-mediated resistance. The anti-cancer mechanisms of MCC1734 include activation of PERK/eIF2α/ATF4 pathway, cell cycle arrest, excessive ROS induction, and MMP collapse. [Display omitted] After decades of research, multidrug resistance (MDR) remains a huge challenge in cancer treatment. In this study, the cytotoxic of 4-hydroxy-N-(naphthalen-1-yl)-2-oxo-2H-chromene-3-carboxamide (MCC1734) has been investigated towards multidrug-resistant cancer cell lines. MCC1734 exerted cytotoxicity on cell lines expressing different mechanisms of drug resistance (P-glycoprotein, BCRP, ABCB5, EGFR, p53 knockout) to a different extent. Interestingly, sensitive CCRF-CEM cells and multidrug-resistant P-gp-overexpressing CEM/ADR5000 cells represented similar sensitivity towards MCC1734, indicating MCC1734 can bypass P-gp-mediated resistance. Microarray-based mRNA expression revealed that MCC1734 affected cells by multiple pathways, including cell cycle regulation, mitochondrial dysfunction, apoptosis signaling, and EIF2 signaling. MCC1734 stimulated the generation of excessive reactive oxygen species and the collapse of mitochondria membrane potential in CCRF-CEM cells, companied by the arrest of the cell cycle in the G 2 M phase and apoptosis induction as determined by flow cytometry. In addition, our immunoblotting analysis highlighted that MCC1734 triggered endoplasmic reticulum (ER) stress, evidenced by the activation of p-PERK, p-eIF2α, ATF4 and CHOP. The anti-cancer effects of MCC1734 were further observed in vivo using human xenograft tumors transplanted to zebrafish, providing further support for MCC1734 as a promising new candidate for cancer drug development. [ABSTRACT FROM AUTHOR]

The c. 1326T>G single nucleotide polymorphism ( SNP) in the NCAPG gene, which leads to an amino acid change of Ile442 to Met442, was previously identified as a candidate causative variation for a bovine carcass weight quantitative trait loci ( QTL) on chromosome 6, which was associated with linear skeletal measurement gains and daily body weight gain at puberty. Recently, we identified the stature quantitative trait nucleotides ( QTNs) in the PLAG1-CHCHD7 intergenic region as the causative variations for another carcass weight QTL on chromosome 14. This study aimed to compare the effects of the two QTL on growth and carcass traits using 768 Japanese Black steers from a progeny testing program and to determine whether a genetic interaction was present between them. The FJX_250879 SNP representing the stature QTL was associated with linear skeletal measurements and average daily body weight gain at early and late periods during adolescence. A genetic interaction between FJX_250879 and NCAPG c. 1326T>G was detected only for body and rump lengths. Both were associated with increased carcass weight and Longissimus muscle area, and NCAPG c. 1326T>G was also associated with reduced subcutaneous fat thickness and increased carcass yield estimate. These results will provide useful information to improve carcass weight in Japanese Black cattle. [ABSTRACT FROM AUTHOR]

We identified an IARS (isoleucyl-tRNA synthetase) c.235G>C (p.Val79Leu) substitution as the causative mutation for neonatal weakness with intrauterine growth retardation (perinatal weak calf syndrome). In Japanese Black cattle, the syndrome was frequently found in calves sired by Bull A. Hence, we employed homozygosity mapping and linkage analysis. In order to identify the perinatal weak calf syndrome locus in a 4.04-Mb region of BTA 8, we analysed a paternal half-sibling family with a BovineSNP50 BeadChip and microsatellites. In this critical region, we performed exome sequencing to identify a causative mutation. Three variants were detected as possible candidates for causative mutations that were predicted to disrupt the protein function, including a G>C (p.Val79Leu) mutation in IARS c.235. The IARS c.235G>C mutation was not a homozygous risk allele in the 36 healthy offspring of Bull A. Moreover, the IARS Val79 residue and its flanking regions were evolutionarily and highly conserved. The IARS mutant (Leu79) had decreased aminoacylation activity. Additionally, the homozygous mutation was not found in any of 1526 healthy cattle. Therefore, we concluded that the IARS c.235G>C mutation was the cause of hereditary perinatal weak calf syndrome. [ABSTRACT FROM AUTHOR]

ABSTRACT Two taurine breeds, Japanese Black and Holstein, established from geographically distant origins and selected for different uses, beef and dairy, were extensively genotyped using a genome-wide single nucleotide polymorphism (SNP) chip with more than 1000 animals of each breed. The genetic structure was examined by principal component analysis, in which the first principal component clearly separated the two breeds and explained more than 15% of the variance. Highly differentiated SNPs were detected throughout the genome, some of which were clustered within small regions on BTA4 (79.2-79.7 Mb, Btau4.0) and BTA26 (22.2-23.6 Mb). A breed assignment test was developed using 18 highly differentiated SNPs to distinguish Japanese Black from F1 (Japanese Black × Holstein) and Holstein. The error rate that an F1 or Holstein animal is misjudged as Japanese Black was expected to be < 0.8%, while the error rate that a Japanese Black animal is misjudged as F1 or Holstein was expected to be < 0.001%. This test provides a reliable and powerful method to detect breed label falsification in retail beef. [ABSTRACT FROM AUTHOR]

Abstract: Human ABCB5, an ATP-binding cassette (ABC) transporter gene, has two major mRNA species. One transcript encodes an 812 amino acid polypeptide, ABCB5β, with a transmembrane domain and a nucleotide-binding domain. We isolated the cDNA of another ABCB5 mRNA that encodes a 1257 amino acid polypeptide. The translated ABCB5 protein is a full-sized ABC transporter that has an internally duplicated structure with two transmembrane domains and two nucleotide-binding domains. The 5′ and 3′ parts of the ABCB5 mRNA were expressed in the prostate and testis. HEK293 cells transfected with the ABCB5 cDNA expressed a 150–160kDa protein. The ABCB5 transfectants showed approximately 1.5-fold higher resistance to doxorubicin, and 2- to 3-fold higher resistance to paclitaxel and docetaxel. Cellular uptake of radiolabeled paclitaxel and docetaxel in the transfectants was lower than that in the parental HEK293 cells. Treatment of the transfectants with ABCB5-targeted siRNA lowered their resistance to docetaxel. Revertant cells that express a reduced amount of ABCB5 also showed a lowered level of docetaxel resistance. These results indicated that the expression of ABCB5 conferred resistance to taxanes and anthracyclines. Membrane vesicles prepared from ABCB5 baculovirus-infected Sf21 cells showed higher vanadate-sensitive ATPase activity than the Sf21 control vesicles. The k m and V max values of ATPase activity in the ABCB5 vesicles were 1.8mM and 65nmol/min/mg protein, respectively. ABCB5 ATPase activity was 1.25-fold higher in the presence of 100μM docetaxel than it was in the absence of docetaxel. These results indicates that the full-length ABCB5 protein has ATPase activity that is sensitive to docetaxel. [Copyright &y& Elsevier]

Abstract: Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) is associated with malignancy. KSHV-derived vFLIP is structurally related to cellular FLIP and binds to NEMO/IκB kinase (IKKγ) to activate NF-κB signaling. NF-κB activation is postulated to confer chemoresistance to various anticancer drugs. However, here we showed that vFLIP expression uniquely sensitized HEK293 cells to bleomycin and its derivatives. Chemosensitization to bleomycin by vFLIP accompanied accumulation of γ-H2AX and G2/M-arrest of cells, while bleomycin-induced DNA damage checkpoints, such as phosphorylation of Chk2 and foci formation of Rad51, were similarly detected in both parental and vFLIP-expressing cells, suggesting that primary DNA damage was not affected by vFLIP. Paradoxically, while NF-κB activity was little affected by bleomycin treatment, vFLIP-stimulated NF-κB activity was suppressed by it. Additionally, cAMP-response element (CRE)- and p53-dependent transcriptional reporter activity was negatively regulated by vFLIP in the presence of bleomycin. Interestingly, a negative regulatory phosphatase essential for G2 checkpoint recovery and for dephosphorylation of γ-H2AX, Wip1/PPM1D, whose gene promoter is regulated by p53, CRE and NF-κB, was selectively downregulated in vFLIP-expressing cells after bleomycin treatment. These results suggest that vFLIP-mediated transcriptional regulation such as Wip1/PPM1D repression is involved in chemosensitization to bleomycin. [Copyright &y& Elsevier]

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1Asn has a weaker affinity to glutamate than GRIA1Ser, both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1Asn have a slower luteinizing hormone (LH) surge than cows with GRIA1Ser. In addition, cows with GRIA1Asn possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1Ser. Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy. [ABSTRACT FROM AUTHOR]

Abstract: Viral K-cyclin derived from Kaposi’s sarcoma-associated herpesvirus is homologous with mammalian D-type cyclins. Here, we demonstrated the regulatory mechanisms for K-cyclin function and degradation in human embryonic kidney HEK293 and primary effusion lymphoma JSC-1 cell lines. Proteasome inhibitor MG132 treatment induced an accumulation of ubiquitinated K-cyclin in these cells, and co-expression of CDK6 prevented K-cyclin ubiquitination. Also K-cyclin mutants incompetent for CDK6-binding were destabilized by proteasome pathway. Furthermore, silencing of p16INK4a promoted K-cyclin–CDK6 complex formation and hence induced K-cyclin-associated kinase activity in HEK293 cells. These observations indicate that CDK6-bound K-cyclin is functionally stable but monomeric K-cyclin is targeted to ubiquitin-dependent degradation pathway in these cells. Our data suggest that the balance between CDK6 and p16INK4a regulates the availability of functional K-cyclin in human cells. [Copyright &y& Elsevier]

Abstract: The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics. [Copyright &y& Elsevier]

Abstract: The ATP-binding cassette (ABC) transporter protein, BCRP (breast cancer resistance protein)/ABCG2 pumps out some types of photosensitizers used in photodynamic therapy (PDT) and causes resistance to the antitumor effect of PDT. The purpose of this study was to investigate the association between the expression of BCRP and the efficacy of PDT using Photofrin, or the second-generation photosensitizer, NPe6, for centrally located early lung cancers. Using human epidermoid carcinoma cells, A431 cells and the BCRP-overexpressing A431/BCRP cells, we examined the effects of BCRP expression on the effect of PDT by cell viability assay in vitro, and investigated the expression of BCRP by immunohistochemical analysis in 81 tumor samples obtained from patients with centrally located early lung cancers. The A431/BCRP cells were more resistant to Photofrin-PDT than A431 cells in vitro, and Fumitremorgin C, a specific inhibitor of BCRP, reversed the resistance. However, there was no significant difference in the antitumor effect of NPe6-PDT between these cells. All of the 81 centrally located early lung cancer lesions were BCRP-positive (2+, 45 lesions; 1+, 30 lesions) and all the patients were male and heavy smokers (>30 pack-years). The expression of BCRP significantly affected the efficacy of Photofrin-PDT in cancer lesions ≥10mm in diameter (P =0.04). On the other hand, NPe6-PDT exhibited a strong antitumor effect, regardless of the expression status of BCRP. Photofrin may be a substrate of BCRP and be pumped out from the cells, therefore, BCRP may be a molecular determinant of the outcome of Photofrin-PDT. [Copyright &y& Elsevier]

Abstract: Multiple ocular defects (MOD) in cattle is an autosomal recessive hereditary disorder characterized by dysplasia of the lens, retinal detachment, persistence of the hyaloid artery, and microphthalmia. The locus responsible for MOD has been mapped to the proximal region of bovine chromosome 18. In the present study, we refined the localization of the MOD locus to a 1.0-Mb interval by haplotype analysis using a pedigree of affected animals. Comparison of nucleotide sequence of genes in this region revealed a one-nucleotide insertion in the WFDC1 gene, which resulted in a frame shift mutation and premature termination codon at the middle of the protein. WFDC1 is a small secretory protein containing a WAP-type four disulfide core domain. Specific expression of Wfdc1 was observed in the lens, retina, and optic nerves of embryonic and adult mouse eyes by immunohistochemical staining and in situ hybridization. The present finding demonstrated the essential role of WFDC1 in mammalian eye development. [Copyright &y& Elsevier]

The effect of CMC-544, a calicheamicin-conjugated anti-CD22 monoclonal antibody, was analysed in relation to CD22 and P-glycoprotein (P-gp) in B-cell chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma (NHL) in vitro. The cell lines used were CD22-positive parental Daudi and Raji, and their P-gp positive sublines, Daudi/MDR and Raji/MDR. Cells obtained from 19 patients with B-cell CLL or NHL were also used. The effect of CMC-544 was analysed by viable cell count, morphology, annexin-V staining, and cell cycle distribution. A dose-dependent, selective cytotoxic effect of CMC-544 was observed in cell lines that expressed CD22. CMC-544 was not effective on Daudi/MDR and Raji/MDR cells compared with their parental cells. The MDR modifiers, PSC833 and MS209, restored the cytotoxic effect of CMC-544 in P-gp-expressing sublines. In clinical samples, the cytotoxic effect of CMC-544 was inversely related to the amount of P-gp ( P = 0·003), and to intracellular rhodamine-123 accumulation ( P < 0·001). On the other hand, the effect positively correlated with the amount of CD22 ( P = 0·010). The effect of CMC-544 depends on the levels of CD22 and P-gp. Our findings will help to predict the clinical effectiveness of this drug on these B-cell malignancies, suggesting a beneficial effect with combined use of CMC-544 and MDR modifiers. [ABSTRACT FROM AUTHOR]

Abstract: The breast cancer resistance protein, BCRP/ABCG2, is a half-molecule ATP-binding cassette transporter that facilitates the efflux of various anticancer agents from the cell, including 7-ethyl-10-hydroxycamptothecin, topotecan and mitoxantrone. The expression of BCRP can thus confer a multidrug resistance phenotype in cancer cells, and its transporter activity is involved in the in vivo efficacy of chemotherapeutic agents. Thus, the elucidation of the substrate preferences and structural relationships of BCRP is essential to understanding its in vivo functions during chemotherapeutic treatments. Single nucleotide polymorphisms (SNPs) have also been found to be key factors in determining the efficacy of chemotherapeutics, and those therapeutics that inhibit BCRP activity, such as the SNP that results in a C421A mutant, may result in unexpected side effects of the BCRP- anticancer drugs interaction even at normal dosages. In order to modulate the BCRP activity during chemotherapy, various compounds have been tested as inhibitors of this protein. Estrogenic compounds including estrone, several tamoxifen derivatives in addition to phytoestrogens and flavonoids have been shown to reverse BCRP-mediated drug resistance. Intriguingly, recently developed molecular targeted cancer drugs, such as the tyrosine kinase inhibitors imatinib mesylate, gefitinib and others, can also interact with BCRP. Since both functional SNPs and inhibitory agents of BCRP modulate the in vivo pharmacokinetics and pharmacodynamics of its substrate drugs, BCRP activity is an important consideration in the development of molecular targeted chemotherapeutics. [Copyright &y& Elsevier]

Abstract: In order to study solid base catalyst for biodiesel production with environmental benignity, transesterification of edible soybean oil with refluxing methanol was carried out in the presence of calcium oxide (CaO), -hydroxide (Ca(OH)2), or -carbonate (CaCO3). At 1h of reaction time, yield of FAME was 93% for CaO, 12% for Ca(OH)2, and 0% for CaCO3. Under the same reacting condition, sodium hydroxide with the homogeneous catalysis brought about the complete conversion into FAME. Also, CaO was used for the further tests transesterifying waste cooking oil (WCO) with acid value of 5.1mg-KOH/g. The yield of FAME was above 99% at 2h of reaction time, but a portion of catalyst changed into calcium soap by reacting with free fatty acids included in WCO at initial stage of the transesterification. Owing to the neutralizing reaction of the catalyst, concentration of calcium in FAME increased from 187ppm to 3065ppm. By processing WCO at reflux of methanol in the presence of cation-exchange resin, only the free fatty acids could be converted into FAME. The transesterification of the processed WCO with acid value of 0.3mg-KOH/g resulted in the production of FAME including calcium of 565ppm. [Copyright &y& Elsevier]

Abstract: Although 5-fluorouracil (5-FU) plus leucovorin (LV) is a standard chemotherapy regimen for colorectal cancer, the factors that determine the LV-mediated enhancement of the antitumor activity of 5-FU have remained unknown. We investigated the roles of folylpolyglutamate synthase (FPGS) and γ-glutamyl hydrolase (GGH), which are the main enzymes involved in folate metabolism, in the effect of LV. LV enhanced the anticancer activity of 5-FU and the level of reduced folate in human colon cancer cells. Small-interfering RNA (siRNA) transfected into DLD-1 cells to downregulate FPGS reduced the basal level of reduced folate, the folate level after LV treatment, and the enhancement of 5-fluoro-2′-deoxyuridine (FdUrd)-induced cytotoxicity elicited by LV. By contrast, the downregulation of GGH by siRNA increased cellular sensitivity to FdUrd combined with LV. These results suggest that FPGS and GGH expression levels in tumors are determinants of the efficacy of LV in enhancing the antitumor activity of 5-FU. [Copyright &y& Elsevier]

Forelimb-girdle muscular anomaly is a hereditary disorder of Japanese Black cattle characterized by tremors and astasia caused by hypoplasia of the forelimb-girdle muscles. The locus responsible for this disorder has been mapped on a middle region of bovine chromosome 26. In this study, we applied marker-assisted selection to identify the carriers of this disorder. Four microsatellite markers, DIK4440, BM4505, MOK2602 and IDVGA-59, linked to the disorder locus were genotyped in 37 unaffected offspring of a carrier sire. Transmission of the mutant or wild-type allele of the disorder locus of the sire to the 37 offspring was determined by examining the haplotypes of these markers. The results showed that nine and 18 of the 37 animals possessed the paternally transmitted mutant and wild-type alleles, respectively, and therefore, the nine animals with the mutant allele were identified as carriers. We concluded that the marker-assisted selection using these four markers can be applied for the identification of the carriers of forelimb-girdle muscular anomaly of Japanese Black cattle. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-related protein 1 (MRP1), confer resistance to various anticancer agents. We previously reported that some flavonoids have BCRP-inhibitory activity. Here we show the reversal effects of an extensive panel of flavonoids upon BCRP-, P-gp-, and MRP1-mediated drug resistance. Reversal effects of flavonoids upon BCRP-, P-gp-, or MRP1-mediated drug resistance were examined in the BCRP- or MDR1-transduced human leukemia K562 cells or in the MRP1-transfected human epidermoid carcinoma KB-3-1 cells using cell growth inhibition assays. The IC50 values were determined from the growth inhibition curves. The RI50 values were then determined as the concentration of inhibitor that causes a twofold reduction of the IC50 in each transfectant. The reversal of BCRP activity was tested by measuring the fluorescence of intracellular topotecan. The BCRP-inhibitory activity of 32 compounds was screened, and 20 were found to be active. Among these active compounds, 3′,4′,7-trimethoxyflavone showed the strongest anti-BCRP activity with RI50 values of 0.012 μM for SN-38 and 0.044 μM for mitoxantrone. We next examined the effects of a panel of 11 compounds on P-gp- and MRP1-mediated drug resistance. Two of the flavones, 3′,4′,7-trimethoxyflavone and acacetin, showed only low anti-P-gp activity, with the remainder displaying no suppressive effects against P-gp. None of the flavonoids that we tested inhibited MRP1. Our present results thus indicate that many flavonoids selectively inhibit BCRP only. Moreover, we examined the structure–BCRP inhibitory activity relationship from our current study. [ABSTRACT FROM AUTHOR]