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Cow genotypes are expected to improve the accuracy of genomic estimated breeding values (GEBV) for young bulls in relatively small populations such as Thai Holstein-Friesian crossbred dairy cattle in Thailand. The objective of this study was to investigate the effect of cow genotypes on the predictive ability and individual accuracies of GEBV for young dairy bulls in Thailand. Test-day data included milk yield (n = 170,666), milk component traits (fat yield, protein yield, total solids yield, fat percentage, protein percentage, and total solids percentage; n = 160,526), and somatic cell score (n = 82,378) from 23,201, 82,378, and 13,737 (for milk yield, milk component traits, and SCS, respectively) cows calving between 1993 and 2017, respectively. Pedigree information included 51,128; 48,834; and 32,743 animals for milk yield, milk component traits, and somatic cell score, respectively. Additionally, 876, 868, and 632 pedigreed animals (for milk yield, milk component traits, and SCS, respectively) were genotyped (152 bulls and 724 cows), respectively, using Illumina Bovine SNP50 BeadChip. We cut off the data in the last 6 yr, and the validation animals were defined as genotyped bulls with no daughters in the truncated set. We calculated GEBV using a single-step random regression test-day model (SS-RR-TDM), in comparison with estimated breed value (EBV) based on the pedigree-based model used as the official method in Thailand (RR-TDM). Individual accuracies of GEBV were obtained by inverting the coefficient matrix of the mixed model equations, whereas validation accuracies were measured by the Pearson correlation between deregressed EBV from the full data set and (G)EBV predicted with the reduced data set. When only bull genotypes were used, on average, SS-RR-TDM increased individual accuracies by 0.22 and validation accuracies by 0.07, compared with RR-TDM. With cow genotypes, the additional increase was 0.02 for individual accuracies and 0.06 for validation accuracies. The inflation of GEBV tended to be reduced using cow genotypes. Genomic evaluation by SS-RR-TDM is feasible to select young bulls for the longitudinal traits in Thai dairy cattle, and the accuracy of selection is expected to be increased with more genotypes. Genomic selection using the SS-RR-TDM should be implemented in the routine genetic evaluation of the Thai dairy cattle population. The genetic evaluation should consider including genotypes of both sires and cows.

International audience; The objective of this study was to determine whether the linear regression (LR) method could be used to validate genomic threshold models. Statistics for the LR method were computed from estimated breeding values (EBVs) using the whole and truncated data sets with variances from the reference and validation populations. The method was tested using simulated and real chicken data sets. The simulated data set included 10 generations of 4,500 birds each; genotypes were available for the last three generations. Each animal was assigned a continuous trait, which was converted to a binary score assuming an incidence of failure of 7%. The real data set included the survival status of 186,596 broilers (mortality rate equal to 7.2%) and genotypes of 18,047 birds. Both data sets were analysed using best linear unbiased predictor (BLUP) or single-step GBLUP (ssGBLUP). The whole data set included all phenotypes available, whereas in the partial data set, phenotypes of the most recent generation were removed. In the simulated data set, the accuracies based on the LR formulas were 0.45 for BLUP and 0.76 for ssGBLUP, whereas the correlations between true breeding values and EBVs (i.e. true accuracies) were 0.37 and 0.65, respectively. The gain in accuracy by adding genomic information was overestimated by 0.09 when using the LR method compared to the true increase in accuracy. However, when the estimated ratio between the additive variance computed based on pedigree only and on pedigree and genomic information was considered, the difference between true and estimated gain was

Estimating single nucleotide polymorphism (SNP) effects over time is essential to identify and validate candidate genes (or quantitative trait loci) associated with time-dependent variation of economically important traits and to better understand the underlying mechanisms of lactation biology. Therefore, in this study, we aimed to estimate time-dependent effects of SNP and identifying candidate genes associated with milk (MY), fat (FY), and protein (PY) yields, and somatic cell score (SCS) in the first 3 lactations of Canadian Ayrshire, Holstein, and Jersey breeds, as well as suggest their potential pattern of phenotypic effect over time. Random regression coefficients for the additive direct genetic effect were estimated for each animal using single-step genomic BLUP, based on 2 random regression models: one considering MY, FY, and PY in the first 3 lactations and the other considering SCS in the first 3 lactations. Thereafter, SNP solutions were obtained for random regression coefficients, which were used to estimate the SNP effects over time (from 5 to 305 d in lactation). The top 1% of SNP that showed a high magnitude of SNP effect in at least 1 d in lactation were selected as relevant SNP for further analyses of candidate genes, and clustered according to the trajectory of their SNP effects over time. The majority of SNP selected for MY, FY, and PY increased the magnitude of their effects over time, for all breeds. In contrast, for SCS, most selected SNP decreased the magnitude of their effects over time, especially for the Holstein and Jersey breeds. In general, we identified a different set of candidate genes for each breed, and similar genes were found across different lactations for the same trait in the same breed. For some of the candidate genes, the suggested pattern of phenotypic effect changed among lactations. Among the lactations, candidate genes (and their suggested phenotypic effect over time) identified for the second and third lactations were more similar to each other than for the first lactation. Well-known candidate genes with major effects on milk production traits presented different suggested patterns of phenotypic effect across breeds, traits, and lactations in which they were identified. The candidate genes identified in this study can be used as target genes in studies of gene expression.

The objectives of this study were to estimate the heritability of mastitis incidence and genetic correlations between the mastitis and the somatic cell score (SCS) statistics, and to compare the practicability between different models. We used test-day records with the mastitis incidence and SCS collected from Holstein cows calving from 1988 to 2015 in Hokkaido, Japan. As indicators of mastitis, the average SCS (avSCS), the standard deviation of SCS (sdSCS), and the maximum SCS (maxSCS) were calculated using test-day records up to the first 305 days in milk within a lactation. We compared a four-trait repeatability animal model (MTRP) with a four-trait multiple-lactation animal model (MTML). The heritability for mastitis was equal to or lower than 0.05 in all the models. Genetic correlations between lactations with MTML within the same trait were positive and close to 1. With MTRP, the estimated genetic correlations of the mastitis incidence with avSCS, sdSCS, and maxSCS were 0.66, 0.79, and 0.82, respectively. A joint evaluation with SCS statistics is expected to give an extra reliability for mastitis because of high and positive genetic correlations among the traits.

The objective of this study was to model differences in pedigree accuracy caused by selective genotyping. As genotypes are used to correct pedigree errors, some pedigree relationships are more accurate than others. These accuracy differences can be modeled with uncertain parentage models that distribute the paternal (maternal) contribution across multiple sires (dams). In our case, the parents were the parent on record and an unknown parent group to account for pedigree relationships that were not confirmed through genotypes. Pedigree accuracy was addressed through simulation and through North American Holstein data. Data were simulated to be representative of the dairy industry with heterogeneous pedigree depth, pedigree accuracy, and genotyping. Holstein data were obtained from the official evaluation for milk, fat, and protein. Two models were compared: the traditional approach, assuming accurate pedigrees, and uncertain parentage, assuming variable pedigree accuracy. The uncertain parentage model was used to add pedigree relationships for alternative parents when pedigree relationships were not certain. The uncertain parentage model included 2 possible sires (dams) when the sire (dam) could not be confirmed with genotypes. The 2 sires (dams) were the sire (dam) on record with probability 0.90 (0.95) and the unknown parent group for the birth year of the sire (dam) with probability 0.10 (0.05). An additional set of assumptions was tested in simulation to mimic an extensive dairy production system by using a sire probability of 0.75, a dam probability of 0.85, and the remainder attributed to the unknown parent groups. In the simulation, small bias differences occurred between models based on pedigree accuracy and genotype status. Rank correlations were strong between traditional and uncertain parentage models in simulation (≥0.99) and in Holstein (≥0.99). For Holsteins, the estimated breeding value differences between models were small for most animals. Thus, traditional models can continue to be used for dairy genomic prediction despite using genotypes to improve pedigree accuracy. Those genotypes can also be used to discover maternal parentage, specifically maternal grandsires and great grandsires when the dam is not known. More research is needed to understand how to use discovered maternal pedigrees in genetic prediction.

Test-day traits are important for genetic evaluation in dairy cattle and are better modeled by multiple-trait random regression models (RRM). The reliability and bias of genomic estimated breeding values (GEBV) predicted using multiple-trait RRM via single-step genomic best linear unbiased prediction (ssGBLUP) were investigated in the 3 major dairy cattle breeds in Canada (i.e., Ayrshire, Holstein, and Jersey). Individual additive genomic random regression coefficients for the test-day traits were predicted using 2 multiple-trait RRM: (1) one for milk, fat, and protein yields in the first, second, and third lactations, and (2) one for somatic cell score in the first, second, and third lactations. The predicted coefficients were used to derive GEBV for each lactation day and, subsequently, the daily GEBV were compared with traditional daily parent averages obtained by BLUP. To ensure compatibility between pedigree and genomic information for genotyped animals, different scaling factors for combining the inverse of genomic (G−1) and pedigree (A−122) relationship matrices were tested. In addition, the inclusion of only genotypes from animals with accurate breeding values (defined in preliminary analysis) was compared with the inclusion of all available genotypes in the analyzes. The ssGBLUP model led to considerably larger validation reliabilities than the BLUP model without genomic information. In general, scaling factors used to combine the G−1 and A−122 matrices had small influence on the validation reliabilities. However, a greater effect was observed in the inflation of GEBV. Less inflated GEBV were obtained by the ssGBLUP compared with the parent average from traditional BLUP when using optimal scaling factors to combine the G−1 and A−122 matrices. Similar results were observed when including either all available genotypes or only genotypes from animals with accurate breeding values. These findings indicate that ssGBLUP using multiple-trait RRM increases reliability and reduces bias of breeding values of young animals when compared with parent average from traditional BLUP in the Canadian Ayrshire, Holstein, and Jersey breeds.

In our previous study, we showed that prexasertib, a checkpoint kinase 1 (Chk1) inhibitor, enhances the effects of standard drugs for pancreatic cancer, including gemcitabine (GEM), S-1, and the combination of GEM and S-1 (GS). The combination of prexasertib and GS has a strong antitumor effect and induces apoptosis in pancreatic cancer cells by downregulating anti-apoptotic protein Bcl-2. In the present study, we investigated the combined effect of GEM, S-1, and prexasertib with a selective Bcl-2 inhibitor (venetoclax) and a non-selective Bcl-2 inhibitor (navitoclax) in SUIT-2 pancreatic cancer cells. An MTT assay revealed that the combination of prexasertib with navitoclax showed a synergistic effect but the combination with venetoclax did not. Investigation of the pancreatic cancer cell lines SUIT-2, MIA PaCa-2, and BxPC-3 revealed that BxPC-3 also showed a high synergistic effect when combined with prexasertib and navitoclax but not venetoclax. Mechanistic analysis of the combined effect showed that apoptosis was induced. Bcl-2 knockdown with siRNA and prexasertib treatment did not induce apoptosis, whereas Bcl-xL knockdown with siRNA and prexasertib treatment resulted in strong induction of apoptosis. In addition, among the three cell lines, the combined effect of prexasertib and navitoclax resulted in increased apoptotic cell death because the protein expression levels of Bcl-xL and Chk1 were higher. Our results demonstrate that the combination of prexasertib and navitoclax has a strong antitumor effect and induces apoptosis in pancreatic cancer cells by downregulating Bcl-xL. Simultaneous inhibition of Chk1 and Bcl-xL could be a new strategy for treating pancreatic cancer.

This paper proposes a mean time-to-failure (MTTF) aware design methodology for minimizing power dissipation while satisfying target chip lifetime. The key contributions of the proposed design methodology are to explicitly introduce MTTF as a design constraint and optimize the design with activation-aware slack assignment (ASA). Conventionally, the gates included in nonintrinsic critical paths are downscaled or replaced with high- $V$ th gates for power savings, where the nonintrinsic critical paths are timing paths which originally had large timing slacks before the downscaling and replacement. On the other hand, ASA gives timing slacks to nonintrinsic critical paths and reduces the number of active paths whose delays are very close to those of intrinsic critical paths whose timing slacks cannot be increased by resynthesis and sizing. The proposed optimization includes both pre-ASA circuit design and ASA implementation. The former pre-ASA design prepares several design candidates that laid out with different timing constraints and selects the most promising candidate regarding power. For this selection, every candidate is analyzed to estimate minimum supply voltage after ASA that can achieve the target MTTF. Then, the proposed methodology selects a set of flip-flops for ASA using integer linear programming, such that it reduces the sum of gatewise failure probability maximumly, and performs ASA. We evaluate MTTF of circuits with and without ASA and examine how much power saving can be obtained while satisfying the target MTTF, e.g., 10 years. Evaluation results show that the circuits with ASA achieve up to 49.6% power saving.

The objectives of this research were to investigate trends for accuracy of genomic predictions over time in a broiler population accumulating data, and to test if data from distant generations are useful in maintaining the accuracy of genomic predictions in selection candidates. The data contained 820k phenotypes for a growth trait (GROW), 200k for two feed efficiency traits (FE1 and FE2), and 42k for a dissection trait (DT). The pedigree included 1.2M animals across 7 years, over 100k from the last 4 years were genotyped. Accuracy was calculated by the linear regression method. Before genotypes became available for training populations, accuracy was nearly stable despite the accumulation of phenotypes and pedigrees. When the first year of genomic data was included in the training population, accuracy increased 56, 77, 39, and 111% for GROW, FE1, FE2, and DT, respectively. With genomic information, the accuracies increased every year except the last one, when they declined for GROW and FE2. The decay of accuracy over time was evaluated in progeny, grand-progeny, and great-grand-progeny of training populations. Without genotypes, the average decline in accuracy across traits was 41% from progeny to grand-progeny, and 19% from grand-progeny to great-grand-progeny. Whit genotypes, the average decline across traits was 14% from progeny to grand-progeny, and 2% from grand-progeny to great-grand-progeny. The accuracies in the last 3 generations were the same when the training population included 5 or 2 years of data, and a marginal decrease was observed when the training population included only 1 year of data. Training sets including genomic information provided an increased accuracy and persistence of genomic predictions compared to training sets without genomic data. The two most recent years of data were enough to maintain the accuracy of predictions in selection candidates.

The genomic relationship matrix (GRM) can be inverted by the algorithm for proven and young (APY) based on recursion on a random subset of animals. While a regular inverse has a cubic cost, the cost of the APY inverse can be close to linear. Theory for the APY assumes that the optimal size of the subset (maximizing accuracy of genomic predictions) is due to a limited dimensionality of the GRM, which is a function of the effective population size (Ne). The objective of this study was to evaluate these assumptions by simulation. Six populations were simulated with approximate effective population size (Ne) from 20 to 200. Each population consisted of 10 nonoverlapping generations, with 25,000 animals per generation and phenotypes available for generations 1–9. The last 3 generations were fully genotyped assuming genome length L = 30. The GRM was constructed for each population and analyzed for distribution of eigenvalues. Genomic estimated breeding values (GEBV) were computed by single-step GBLUP, using either a direct or an APY inverse of GRM. The sizes of the subset in APY were set to the number of the largest eigenvalues explaining x% of variation (EIGx, x = 90, 95, 98, 99) in GRM. Accuracies of GEBV for the last generation with the APY inverse peaked at EIG98 and were slightly lower with EIG95, EIG99, or the direct inverse. Most information in the GRM is contained in ∼NeL largest eigenvalues, with no information beyond 4NeL. Genomic predictions with the APY inverse of the GRM are more accurate than by the regular inverse.

Background: Nobiletin is contained in Shiikuwasa fruit, a popular citrus fruit from Okinawa Prefecture in Japan. Nobiletin reportedly acts as a strong antioxidant, an anti-inflammatory agent, and an anti-cancer agent, and it suppresses the expression of TF which triggers blood coagulation. However, in vivo verification of in vitro reports is necessary. This study used a rat model of LPS-induced microthrombosis based on the in vivo studies as previously reported. Sustained intravenous injection of LPS changed all blood coagulation indicators in the direction of thrombus formation. The aim of this study was to determine if intake of nobiletin could suppress DIC-like symptoms. Methods: Experimental SD rats were fully anesthetized and fixed to an operating table. Either LPS alone or nobiletin (50 mg/kg) plus LPS was given to rats to investigate the repressive effects of nobiletin on the expression of blood coagulation factors. Results: After 4 h of LPS infusion (12.5 mg/kg/h, i.v.), PLT counts and Fbg levels in rat plasma decreased by 80% and 74%, respectively. PT and APTT were extended by 180% and 256%, respectively. TF activity and PAI-1 antigen levels were remarkably increased (54- and 86-fold, respectively vs. control). Pretreatment on nobiletin (50 mg/kg, p.o.) reduced or suppressed fluctuations in blood coagulation indices caused by LPS. TF activity was repressed almost completely by nobiletin pretreatment. After 4 h, PAI-1 antigen levels in nobiletin-treated animals were repressed 82.6% compared to LPS-treated rats. Nobiletin repressed LPS-induced changes in TF and PAI-1 more effectively than other parameters. Further, nobiletin repressed fibrin thrombi formation in the renal glomeruli induced by LPS treatment. Conclusions: Nobiletin was found to reduce LPS-induced DIC-like symptoms in rats. In the fluctuations of blood indices related to the coagulation cascade, nobiletin suppressed the LPS-induced expression of PAI-1 and TF more effectively than other indices. The binding sites of transcription factors that are activated by LPS-induced signals reside in the promoter areas of TF and PAI-1 gene sequences. Thus, the suppression of TF and PAI-1 expression by nobiletin appears similar to mechanisms previously evaluated during in vitro experiments. Importantly, nobiletin repressed fibrin deposition in the renal glomeruli induced by LPS treatment and improved overall health. Nobiletin may function as an anti-thrombogenic agent when ingested daily. Keywords: nobiletin; LPS; DIC model; blood coagulation; anti-thrombogenic

International audience; Single-step genomic evaluation became a standard procedure in livestock breeding, and the main reason is the ability to combine all pedigree, phenotypes, and genotypes available into one single evaluation, without the need of post-analysis processing. Therefore, the incorporation of data on genotyped and non-genotyped animals in this method is straightforward. Since 2009, two main implementations of single-step were proposed. One is called single-step genomic best linear unbiased prediction (ssGBLUP) and uses single nucleotide polymorphism (SNP) to construct the genomic relationship matrix; the other is the single-step Bayesian regression (ssBR), which is a marker effect model. Under the same assumptions, both models are equivalent. In this review, we focus solely on ssGBLUP. The implementation of ssGBLUP into the BLUPF90 software suite was done in 2009, and since then, several changes were made to make ssGBLUP flexible to any model, number of traits, number of phenotypes, and number of genotyped animals. Single-step GBLUP from the BLUPF90 software suite has been used for genomic evaluations worldwide. In this review, we will show theoretical developments and numerical examples of ssGBLUP using SNP data from regular chips to sequence data.

The objective of this study was to estimate genetic parameters for various fertility traits on Holstein upgraded dairy heifers and cows in a smallholder system under tropical conditions using data sets from the Thailand national recording scheme. The investigated traits were age at first service (AFS), age at first calving (AFC), days from calving to first service (DTFS), days between first and last service (DFLS), days open (DO), calving interval (CI), number of services per conception (NSPC), and conception at first service (FSC). The data consisted of 68,555, 34,401, and 54,004 records on heifers, primiparous, and multiparous cows, respectively, calving between 1996 and 2011. Gibbs sampling was employed to obtain (co)variance components using both univariate and bivariate analyses with linear and threshold animal models. Virgin heifers had better fertility performance than primiparous and multiparous cows. The reproductive performance in primiparous cows was inferior compared with multiparous cows. Cows with higher Holstein-Friesian blood showed lower reproductive efficiency. Estimated heritabilities for most of the fertility traits were 0.04 or less except for AFS (0.26) and AFC (0.25). The estimated genetic correlations among fertility traits within parity indicated that selection for cows with high conception rate could lead to shortened DO and CI, as well as DTFS. The FSC and NSPC could be used as the best indicators for heifer and cow fertility and could be complemented by other traits, which were genetically considered as different traits such as DTFS and DFLS in terms of a fertility index. This would enable efficient selection for better fertility. Genetic correlations for fertility traits in primiparous and multiparous cows were very high (>0.90), but those between heifers and cows were lower (0.03 to 0.83). The latter results indicated that fertility traits of heifers and cows should be considered as different traits.

International audience; The purpose of this study was to evaluate the accuracy of genomic selection in single-step genomic BLUP (ssGBLUP) when the inverse of the genomic relationship matrix (G) is derived by the "algorithm for proven and young animals" (APY). This algorithm implements genomic recursions on a subset of "proven" animals. Only a relationship matrix for animals treated as "proven" needs to be inverted, and the extra costs of adding animals treated as "young" are linear. Analyses involved 10,102,702 final scores on 6,930,618 Holstein cows. Final score, which is a composite of type traits, is popular trait in the United States and was easily available for this study. A total of 100,000 animals with genotypes were used in the analyses and included 23,000 sires (16,000 with >5 progeny), 27,000 cows, and 50,000 young animals. Genomic EBV (GEBV) were calculated with a regular inverse of G, and with the G inverse approximated by APY. Animals in the proven subset included only sires (23,000), sires + cows (50,000), only cows (27,000), or sires with >5 progeny (16,000). The correlations of GEBV with APY and regular GEBV for young genotyped animals were 0.994, 0.995, 0.992, and 0.992, respectively Later, animals in the proven subset were randomly sampled from all genotyped animals in sets of 2,000, 5,000, 10,000, 15,000, and 20,000; each sample was replicated 4 times. Respective correlations were 0.97 (5,000 sample), 0.98 (10,000 sample), and 0.99 (20,000 sample), with minimal difference between samples of the same size. Genomic EBV with APY were accurate when the number of animals used in the subset is between 10,000 and 20,000, with little difference between the ways of creating the subset. Due to the approximately linear cost of APY, ssGBLUP with APY could support any number of genotyped animals without affecting accuracy.

The purpose of this study was to examine accuracy of genomic selection via single-step genomic BLUP (ssGBLUP) when the direct inverse of the genomic relationship matrix (G) is replaced by an approximation of G(-1) based on recursions for young genotyped animals conditioned on a subset of proven animals, termed algorithm for proven and young animals (APY). With the efficient implementation, this algorithm has a cubic cost with proven animals and linear with young animals. Ten duplicate data sets mimicking a dairy cattle population were simulated. In a first scenario, genomic information for 20k genotyped bulls, divided in 7k proven and 13k young bulls, was generated for each replicate. In a second scenario, 5k genotyped cows with phenotypes were included in the analysis as young animals. Accuracies (average for the 10 replicates) in regular EBV were 0.72 and 0.34 for proven and young animals, respectively. When genomic information was included, they increased to 0.75 and 0.50. No differences between genomic EBV (GEBV) obtained with the regular G(-1) and the approximated G(-1) via the recursive method were observed. In the second scenario, accuracies in GEBV (0.76, 0.51 and 0.59 for proven bulls, young males and young females, respectively) were also higher than those in EBV (0.72, 0.35 and 0.49). Again, no differences between GEBV with regular G(-1) and with recursions were observed. With the recursive algorithm, the number of iterations to achieve convergence was reduced from 227 to 206 in the first scenario and from 232 to 209 in the second scenario. Cows can be treated as young animals in APY without reducing the accuracy. The proposed algorithm can be implemented to reduce computing costs and to overcome current limitations on the number of genotyped animals in the ssGBLUP method.

Aims Ephedra preparations are known to cause a false-positive reaction for amphetamine analogs when a Triage® kit is used for urinary drug screening. Maoto, a traditional Japanese herbal medicine that consists of Ephedra herb, cinnamon bark, apricot kernel, and Glycyrrhiza, is used for the treatment of colds and influenza. We compared two urinary drug-screening kits for false-positive reactions for amphetamine analogs after treatment with maoto from five different manufacturers over 12 h. Methods Six healthy adult volunteers received five different preparations of maoto in random order. One packet of maoto was given on each study day. Urine samples were collected before treatment, and at 3, 6, 9, and 12 h after drug treatment. Two urinary drug-screening kits (Triage® and Medical Stat®) were used for testing. Results Twenty-nine of 30 samples produced a false-positive reaction for amphetamine analogs using the Triage® kit at 3 h after treatment. No significant difference was observed among the products of the five pharmaceutical companies (P = 0.5771). All samples produced a negative reaction using the Medical Stat® kit. Conclusions Urine samples that gave false-positive reactions with the Triage® kit gave negative reactions with the Medical Stat® kit. This suggests that comparison of results from the Triage® kit and the Medical Stat® kit may be useful in screening urine samples for amphetamine analogs. The quality of maoto from five pharmaceutical companies appears to be the same with regard to interaction with the drug screening tests, adverse effects, and excretion in the urine.

SummaryThrombin-activatable fibrinolysis inhibitor (TAFI, carboxypeptidase B2) is a 58-kDa plasma glycoprotein secreted by hepatocytes as an inactive form. TAFI is activated by the thrombin-thrombomodulin complex, and activated TAFI (TAFIa) plays an important role in regulating the balance between coagulation and fibrinolysis through inhibition of fibrinolysis. It has been suggested that high levels of TAFI in circulating plasma increase the risks of cardiovascular death and acute phase in ischaemic stroke. However, the mechanisms of regulating TAFI expression have been unclear. The present study investigated the effects of nobiletin (a polymethoxy flavonoid contained in the rind of citrus fruits) on TAFI gene (CPB2) and TAFI antigen expression in cultured human hepatoma HepG2 cells. Nobiletin decreased the release of TAFI antigen from HepG2 cells into conditioned medium in parallel with decreased levels of CPB2 mRNA and antigen. The half-life time of CPB2 mRNA in nobiletin-treated cells was unchanged compared to that of untreated control cells. Using nobiletin-treated cells that were transfected with a luciferase CPB2 promoter reporter plasmid, activity decreased to half of that in untreated control cells. A series of luciferase reporter constructs containing 5´-flanking region deletions of the human CPB2 gene showed that the sequences from –150 bp to –50 bp were essential for transcription of CPB2 and contained an AP-1 binding sequence at ∼ –119 bp to – 99 bp in the CPB2 promoter. The amount of complexed nuclear protein and sequences from ∼ –119 bp to –99 bp was decreased in nobiletin-treated cells. ChIP assays showed that c-Jun bound to the ∼ –119 bp to –99 bp region of the CPB2 promoter and that the amount of the immunocomplex decreased after nobiletin treatment. Therefore, nobiletin-induced repression of CPB2 transcription might involve AP-1 inhibition and/or prevention of AP-1 binding in a specific region on the CPB2 gene in HepG2 cells.

We investigated the relationships between conception rates (CRs) at first service in Japanese Holstein heifers (i.e. animals that had not yet had their first calf) and cows and their test-day (TD) milk yields. Data included records of artificial insemination (AI) for heifers and cows that had calved for the first time between 2000 and 2008 and their TD milk yields at 6 through 305 days in milk (DIM) from first through third lactations. CR was defined as a binary trait for which first AI was a failure or success. A threshold-linear animal model was applied to estimate genetic correlations between CRs of heifers or cows and TD milk yield at various lactation stages. Two-trait genetic analyses were performed for every combination of CR and TD milk yield by using the Bayesian method with Gibbs sampling. The posterior means of the heritabilities of CR were 0.031 for heifers, 0.034 for first-lactation cows and 0.028 for second-lactation cows. Heritabilities for TD milk yield increased from 0.324 to 0.433 with increasing DIM but decreased slightly after 210 DIM during first lactation. These heritabilities from the second and third lactations were higher during late stages of lactation than during early stages. Posterior means of the genetic correlations between heifer CR and all TD yields were positive (range, 0.082 to 0.287), but those between CR of cows and milk yields during first or second lactation were negative (range, −0.121 to −0.250). Therefore, during every stage of lactation, selection in the direction of increasing milk yield may reduce CR in cows. The genetic relationships between CR and lactation curve shape were quite weak, because the genetic correlations between CR and TD milk yield were constant during the lactation period.

In Mongolia, milk production must be improved both quantitatively and qualitatively, and we must find the most desirable method for predicting 305-day milk yields for cows in Mongolia. Therefore, the predicted 305-day milk yield from the test interval method (TIM), multiple-trait prediction (MTP) and the random regression model (RRM) were compared. The daily milk production records during 305 days for cows calving from 1986 to 2007 from the National Livestock Breeding Center in Japan, and the test day records every month for cows calving from 1985 to 2005 from the Hokkaido Dairy Milk Recording and Testing Association were used. Wilmink's model for the average of lactation curve in MTP was adopted. A cubic Legendre polynomials and the parameters of Wilmink's function were used for RRM. The results suggested that RRM was the most desirable method for predicting 305-day milk yields in Mongolia because estimates by RRM were the most accurate when using over four records. Further analyses will be required to examine the properties of RRM when predicting 305-day milk yields using test day records in Mongolia.

Objective: In order to investigate whether repeated immunization induces changes in IgG glycosylation, we analyzed the composition of oligosaccharides on antigen-specific IgGs obtained from mice that received different amounts of immunization boosts. Methods: Three groups of mice were immunized with ovalbumin (OVA) and boosted once, twice, or three times, respectively, with an interval of 1 week. The patterns of oligosaccharides present in anti-OVA specific IgGs were analyzed using lectin-enzyme-linked immunosorbent assay (ELISA) and lectin-blot. Results: The repeated injection of OVA induced both the production of specific IgGs and an increase of fucose content in N -linked oligosaccharides of the IgGs. The lowest IgG fucosylation was observed in mice boosted once, whereas the highest fucosylation rate was observed in mice boosted three times. ELISA assay demonstrated that there was a positive relationship between the fucose content and amount of immunization boosts. Conclusions: IgG fucosylation increases during repeated immunization with ovalbumin. The alteration of IgG fucosylation may have important biological significance.

Tissue engineering may offer patients new options when replacement or repair of an organ is needed. However, most tissues will require a microvascular network to supply oxygen and nutrients. One strategy for creating a microvascular network would be promotion of vasculogenesis in situ by seeding vascular progenitor cells within the biopolymeric construct. To pursue this strategy, we isolated CD34(+)/CD133(+) endothelial progenitor cells (EPC) from human umbilical cord blood and expanded the cells ex vivo as EPC-derived endothelial cells (EC). The EPC lost expression of the stem cell marker CD133 but continued to express the endothelial markers KDR/VEGF-R2, VE-cadherin, CD31, von Willebrand factor, and E-selectin. The cells were also shown to mediate calcium-dependent adhesion of HL-60 cells, a human promyelocytic leukemia cell line, providing evidence for a proinflammatory endothelial phenotype. The EPC-derived EC maintained this endothelial phenotype when expanded in roller bottles and subsequently seeded on polyglycolic acid-poly-l-lactic acid (PGA-PLLA) scaffolds, but microvessel formation was not observed. In contrast, EPC-derived EC seeded with human smooth muscle cells formed capillary-like structures throughout the scaffold (76.5 +/- 35 microvessels/mm(2)). These results indicate that 1) EPC-derived EC can be expanded in vitro and seeded on biodegradable scaffolds with preservation of endothelial phenotype and 2) EPC-derived EC seeded with human smooth muscle cells form microvessels on porous PGA-PLLA scaffolds. These properties indicate that EPC may be well suited for creating microvascular networks within tissue-engineered constructs.

We have previously demonstrated that costunolide, a biologically active compound that was isolated from the stem bark of Magnolia sieboldii, induced apoptosis in human cancer cells. In the present study, we investigated the underlying mechanisms and suggest that costunolide induces apoptosis in human promonocytic leukemia U937 cells by depleting the intracellular thiols. Costunolide treatment rapidly depleted the intracellular reduced glutathione (GSH) and protein thiols, and this preceded the occurrence of apoptosis. Pretreatment with sulfhydryl compounds such as GSH, N-acetyl-L-cysteine, dithiothreitol and 2-mercaptoethanol almost completely blocked the costunolide-induced apoptosis, highlighting the significance of the intracellular thiol level in the process. Furthermore, overexpression of Bcl-2 also significantly attenuated the effects of costunolide. The apoptosis-inducing activity of costunolide is likely to depend on the exomethylene moiety because derivatives in which this group was reduced, such as dihydrocostunolide and saussurea lactone, did not deplete the cellular thiols and showed no apoptotic activity. Taken together, the present study demonstrates that the costunolide-induced apoptosis depends on intracellular thiols contents, which are modulated by Bcl-2.

Beta-hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from Lithospermium radix, most efficiently induced cell-death in two lines of lung cancer cells, namely, NCI-H522 and DMS114, whereas shikonin was effective against a wide variety of tumor cell lines. During our studies of the mechanism of action of beta-HIVS on tumor cells, we found that this compound inhibited protein tyrosine kinase (PTK) activity. The tyrosine kinase activities of a receptor for EGF (EGFR) and v-Src were strongly inhibited and that of KDR/Flk-1 was weakly inhibited by beta-HIVS. The inhibition by beta-HIVS of the activities of EGFR and v-Src was much stronger than that by shikonin. The IC50 values of beta-HIVS for EGFR and v-Src were approximately 0.7 microM and 1 microM, respectively. Moreover, the inhibition of v-Src by beta-HIVS was non-competitive with respect to ATP. These results strongly suggest that the action of beta-HIVS, as well as that of shikonin, involves the inhibition of PTK, and they also suggest the possibility of producing a novel group of PTK inhibitors based on shikonin as the parent compound.

Purpose. For assessment of the antinociceptive potency of antidepressants, we compared the antinociceptive effects of serotonin selective reuptake inhibitors (SSRIs) and classical tricyclic antidepressants (TCAs) in rats. We also attempted to elucidate the monoamine receptor subtypes predominantly involved in the antinociceptive effect of antidepressants. Methods. Male Wistar rats received SSRIs (sertraline, fluvoxamine, and citalopram) or TCAs (imipramine and desipramine) intraperitoneally, and the reaction time until pain response in the hot plate test and licking time in the formalin test were measured 60 min later. We also observed the effects of prazosin (an α1 antagonist), WB-4101 (a selective α1A antagonist), yohimbine (an α2 antagonist), WAY-100635 (a selective 5-HT1A antagonist), and ketanserin (a 5-HT2 antagonist), which were simultaneously administered with imipramine or desipramine, on the antidepressant-induced antinociceptive effect in the formalin test. Results. In the hot plate test, desipramine, 20 mg·kg−1, but not imipramine or sertraline, produced a significant increase in reaction time. In the formalin test, desipramine and imipramine produced significant reductions in the licking time at over 5 mg·kg−1 and at over 10 mg·kg−1, respectively. These reductions were nearly complete at 20 mg·kg−1. On the other hand, both SSRIs induced significant reductions in the licking time only at 20 mg·kg−1. Prazosin, WB-4101, and ketanserin significantly antagonized the antinociceptive effect of 10 mg·kg−1 of imipramine. However, imipramine-induced antinociception was not affected by yohimbine and WAY-100635. Prazosin and ketanserin also significantly suppressed antinociception by 5 mg·kg−1 of desipramine. Conclusion. These findings suggest that classical TCAs are likely to have a therapeutic advantage over SSRIs for pain control. In addition, it is likely that central α1 adrenoceptors and 5-HT2 receptors are predominantly involved in imipramine- and desipramine-induced antinociception.

Bufalin, a component of the Chinese medicine chan'su, induces apoptosis in various lines of human tumor cells, such as leukemia HL60 and U937 cells, by altering the expression of apoptosis-related genes, for example, bcl-2 and c-myc. In this study, we characterized a gene that is involved in bufalin-induced apoptosis by the differential display (DD) technique. The partial nucleotide sequence of one of the differentially expressed clones obtained after treatment with bufalin was identical to that of the human gene for Tiam1. When U937 cells were treated with 10(-7) M bufalin, expression of both Tiam1 mRNA and the protein was induced 1 h after the start of the treatment. The increase of Tiam1 mRNA was transient but the level of Tiam1 protein continued to increase at least for 6 h. In addition, the activities of Rac1 and p21-activated kinase (PAK) were also stimulated by bufalin treatment. To evaluate the role of Tiam1 in the apoptotic process, we examined the effects of the expression of sense and antisense RNA for Tiam1 in U937 cells. Apoptosis was strongly induced by bufalin in cells that expressed sense RNA for Tiam1 as compared to apoptosis in control cells treated with bufalin only. Cells expressing antisense RNA for Tiaml were significantly more resistant than the control bufalin-treated cells to induction of DNA fragmentation in response to bufalin. Moreover, sense transformants had elevated activities of PAK and c-Jun NH2-terminal kinase (JNK). These results suggest that Tiaml might play a critical role in bufalin-induced apoptosis through the activation of Rac1, PAK, and JNK pathway.

Asterriquinone D was easily synthesized in three steps from 2,5-dichloro-1,4-benzoquinone. The reaction of benzoquinone with indole in the presence of Pd(OAc)2, followed by oxidation with cerium (IV) ammonium nitrate (CAN) produced 3,6-dichloro-2,5-bis (3-indolyl)-1,4-benzoquinone. The methoxylation of the dichloride with NaOH in CH3OH afforded asterriquinone D.

We experienced a case of a 38-year-old woman with a persistent cough, 24 years after repair of absent pulmonary valve syndrome with anomalous origin of the left pulmonary artery. The right pulmonary artery was massively dilated, thus it caused the compression of the bronchi, which was thought to result in her respiratory symptom. This dilatation of the right pulmonary artery seemed to have progressed because of the following two reasons. The first is the pulmonary hypertension caused by the late reconstruction of the left pulmonary artery. The second is residual pulmonary stenosis and regurgitation after the initial operation without a pulmonary valve insertion. We performed a reoperation consisting of reconstruction of the right ventricular outflow tract using a valved conduit and plication of the right pulmonary artery. Her postoperative course has been without any complications and satisfactory for the past 2 years.

It has been suggested that plasma sulfoconjugated dopamine (DA) may serve as a source or reservoir for free DA in plasma. Moreover, it has also been reported that the plasma levels of conjugated DA may be used as an index predicting heart failure in patients with heart disease. Therefore, in the present study, we have measured the plasma levels of free and sulfoconjugated DA in patients with congenital heart disease who underwent total corrective operations. The patients were divided into two groups with (6 patients with tetralogy of Fallot, TOF) or without (5 patients with ventricular septal defect without pulmonary hypertension, VSD) cyanosis (mean age of 2.11 years). Blood samples were collected before and after operation from the patients, and plasma free and sulfoconjugated DA levels were measured using high performance liquid chromatography. Preoperative levels of free DA in patients in both groups were higher than the level in age matched control subjects. The plasma level of conjugated DA in TOF was higher than that in the controls and was the highest in VSD before operation. DA infusion early after operation caused a rise in plasma free and conjugated DA, however, the levels of increased free DA were lower in the VSD than in the TOF group. After discontinuing DA infusion, the plasma levels of free DA remained higher, while those of conjugated DA decreased to a level lower than the preoperative values in both groups. As the plasma levels of free and sulfoconjugated DA vary with hemodynamics, it was assumed that the difference in the plasma sulfoconjugated DA level between the groups before operation was due to the influence of pulminary blood flow on catecholamine homeostasis. Since the decrease in conjugated DA has been postulated to be an index of sustained heart failure, it is conceivable that it takes a long time for patients who underwent cardiac operations in infancy to recover from heart failure.

We isolated and characterized almost the entire cDNA encoding BLM hydrolase from rat spleen cDNA libraries. The cDNA encoded a polypeptide composed of 454 amino acids, that had a slightly larger molecular mass than that was previously estimated by SDS-PAGE for purified BLM hydrolase subunit. Amino acid sequence alignments showed that rat BLM hydrolase is homologous to that of rabbit (94% identity of partial amino acid sequence), yeast cysteine protease (39% identity), and the pepC gene products of three bacteria (34-40% identity). In addition, it contained the three regions that are conserved in other cysteine proteases and thought to constitute the catalytic center. These results indicated that rat BLM hydrolase is a member of the papain superfamily of cysteine proteases. Sequencing revealed several putative sites phosphorylated by different types of protein kinases, but no signal sequence, transmembrane domain, N-linked glycosylation site or DNA-binding motif. The yeast homolog is a DNA-binding cysteine protease [Xu et al. (1994) J. Biol. Chem, 269, 21177-21183]. We demonstrated that rat BLM hydrolase also binds the single-stranded form of the Ga14 DNA-binding site oligonucleotide with high affinity compared with that of the double-stranded form. Northern blots revealed that the level of BLM hydrolase mRNA expression was very low in the rat skin, lung, and skeletal muscle. Furthermore, BLM hydrolase mRNA was ubiquitously expressed in the human cell lines, HeLa, SKG-IIIa, FL, KB, HEp-2, U373 GM, P3HR-1, Raji, THP-1, Jurkat, and Molt-4. These results suggested that BLM hydrolase plays important physiological roles, including the metabolism of antibiotics.

Bleomycin (BLM) hydrolase, which hydrolyzes the carboxyamide bond in the beta-amino-alanine moiety, was purified from newborn rat skin. The enzyme was purified 2,500-fold over the crude extract to apparent homogeneity in five steps in the presence of 2-mercaptoethanol: 45-55% ammonium sulfate fractionation, followed by chromatographies on Sephacryl S-200, DEAE-cellulofine, Phe-Superose, and Mono Q ion-exchange. The native enzyme had a molecular mass of 280 kDa according to gel filtration. The subunit molecular mass was estimated as 48 kDa by SDS-PAGE, indicating that the enzyme was comprised of six identical subunits. The amino acid sequence of its NH2-terminus was determined to be acetyl-Met-Asn-Asn-Ala-Gly-Leu-Asn-Ser-Glu-Lys-, which was not found in the amino acid sequence database. The optimum pH of the enzyme was 7.5 with pepleomycin (PLM). The Km and Vmax values were 2.1 mM and 6.8 mu mol center dot mg-1 center dot h-1 for PLM, and 1.8 mM and 7.2 mu mol center dot mg-1 center dot h-1 for BLM-A2, respectively. The enzyme activity was inhibited by iodoacetic acid, N-ethylmaleinimide (NEM), and p-chloromercuribenzoic acid (pCMB) as well as divalent cations such as Cu2+, Cd2+, Hg2+, and Zn2+. It was effectively inhibited by a cysteine protease inhibitor E-64. However, cystatins A and C did not inhibit the activity. BLM hydrolase exhibited broad aminopeptidase substrate specificity towards aminoacyl-beta-naphthylamides such as basic, neutral, and hydrophobic amino acid residues, as well as acidic residues. These results indicated that BLM hydrolase represents a new family of cysteine proteases. Western blotting and immunohistochemical analyses showed that BLM hydrolase is ubiquitous in various rat tissues but at low levels in lung and adult skin tissues, suggesting that this enzyme plays an important role in the metabolism of antibiotics.

The aim of the study was to determine the optimal size and technique for construction of the systemic-to-pulmonary arterial shunt which will provide suitable pulmonary blood flow in first-stage Norwood palliation for hypoplastic left heart syndrome in neonates. Our clinical experience suggested that an arterial oxygen tension of about 30 mm Hg immediately after cardiopulmonary bypass, with the patients being ventilated at the lowest possible mean airway pressure with an FiO2of 1.0, provided a suitable pulmonary-to-systemic flow ratio. We also aimed to clarify the characteristics of pulmonary blood flow in accordance with the size of the shunt and the change in the pulmonary vascular resistance in a simplified rigid model of the Norwood procedure. A hole of2.0 mm diameter proved adequate to provide a suitable pulmonary blood flow of 200−300 mlx002F;min in the presence of a pressure gradient of 20−40 mm Hg between the systemic and pulmonary circulations in neonates weighing 3 kg. A short central shunt with a prosthesis of4 mm in diameter produced an excessive flow of pulmonary blood. Our data suggest that using a smaller shunt than that commonly used is necessary to decrease the early and intermediate postoperative mortality. A prosthesis of 3.0 or 3.5 mm in diameter arising from the brachiocephalic artery would be acceptable and can be recommended for first-stage Norwood palliation in small infants, especially in view of the operative difficulties encountered in taking down the shunt at the time of subsequent operations.

A gene encoding phosphoglycerate kinase (PGK) was isolated from the genomic library of C. maltosa to construct an expression vector for this yeast. The PGK gene had an open reading frame of 1,251 base pairs encoding approximately 47-kDa polypeptide of 417 amino-acid residues. Expression of this gene assayed by Northern-blot analysis was significantly induced in cells grown on glucose but not in cells grown on n-tetradecane, n-tetradecanol, or oleic acid. By using the promoter region of this gene, an expression vector (termed pMEA1) for C. maltosa was constructed and expression of an endogenous gene (P450alk1 encoding one of cytochrome P450s for n-alkane hydroxylation in C. maltosa) and a heterologous gene (LAC4 encoding Kluyveromyces lactis beta-galactosidase) was tested. Expression of P450alk1 gene was confirmed at both mRNA and protein levels. LAC4 gene expression was confirmed by determining beta-galactosidase activity. The activity in cells grown on various carbon sources correlated very well with the expression levels of PGK mRNA in these cells.

We detected a peptide having a behavioral activity on mouse forced swimming from sera of healthy volunteers without affective and psychotic diseases. The amino acid sequence was GVNDNEEGF, which was found in the sequence of human β-fibrinogen. The synthesized peptide also showed the behavioral activity dose-dependently, but human fibrinopeptide B (QGVDNEEGFFSAR) did not. The activity was decreased by dopamine 1 antagonist SCH-23390, but not by dopamine 2 antagonist sulpiride. These findings strongly suggest that metabolism of human β-fibrinogen induces the fragment affecting the mouse behavior via the dopamine1 neuronal activity.