Effects of stage of pregnancy on variance components, daily milk yields and 305-day milk yield in Holstein cows, as estimated by using a test-day model.

Pregnancy and calving are elements indispensable for dairy production, but the daily milk yield of cows decline as pregnancy progresses, especially during the late stages. Therefore, the effect of stage of pregnancy on daily milk yield must be clarified to accurately estimate the breeding values and lifetime productivity of cows. To improve the genetic evaluation model for daily milk yield and determine the effect of the timing of pregnancy on productivity, we used a test-day model to assess the effects of stage of pregnancy on variance component estimates, daily milk yields and 305-day milk yield during the first three lactations of Holstein cows. Data were 10 646 333 test-day records for the first lactation; 8 222 661 records for the second; and 5 513 039 records for the third. The data were analyzed within each lactation by using three single-trait random regression animal models: one model that did not account for the stage of pregnancy effect and two models that did. The effect of stage of pregnancy on test-day milk yield was included in the model by applying a regression on days pregnant or fitting a separate lactation curve for each days open (days from calving to pregnancy) class (eight levels). Stage of pregnancy did not affect the heritability estimates of daily milk yield, although the additive genetic and permanent environmental variances in late lactation were decreased by accounting for the stage of pregnancy effect. The effects of days pregnant on daily milk yield during late lactation were larger in the second and third lactations than in the first lactation. The rates of reduction of the 305-day milk yield of cows that conceived fewer than 90 days after the second or third calving were significantly (P<0.05) greater than that after the first calving. Therefore, we conclude that differences between the negative effects of early pregnancy in the first, compared with later, lactations should be included when determining the optimal number of days open to maximize lifetime productivity in dairy cows.

Genetic correlations between production and disease traits during first lactation in Holstein cows.

The aim of this study was to estimate genetic correlations between milk yield, somatic cell score (SCS), mastitis, and claw and leg disorders (CLDs) during first lactation in Holstein cows by using a threshold-linear random regression test-day model. We used daily records of milk, fat and protein yields; somatic cell count (SCC); and mastitis and CLD incidences from 46 771 first-lactation Holstein cows in Hokkaido, Japan, that calved between 2000 and 2009. A threshold animal model for binary records (mastitis and CLDs) and linear animal model for yield traits were applied in our multiple trait analysis. For both liabilities and yield traits, additive genetic effects were used as random regression on cubic Legendre polynomials of days on milk. The highest positive genetic correlations between yields and disease incidences (0.36 for milk and mastitis, 0.56 for fat and mastitis, 0.24 for protein and mastitis, 0.32 for milk and CLD, 0.44 for fat and CLD and 0.31 for protein and CLD) were estimated at about the time of peak milk yield (36 to 65 days in milk). Selection focused on early lactation yield may therefore increase the risk of mastitis and CLDs. The positive genetic correlations of SCS with mastitis or CLD incidence imply that selection to reduce SCS in the early stages of lactation would decrease the incidence of both mastitis and CLD.

Relationships between conception rate in Holstein heifers and cows and milk yield at various stages of lactation.

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.

MO-F-213AB-04: Biological Effect of Dose Shadowing by Fiducial Markers in Spot Scanning Proton Therapy with a Limited Number of Fields.

PURPOSE: In spot scanning proton therapy, accurate patient positioning before and during treatment is essential. A small gold ball marker is suitable as a fiducial for prostate treatment. However, it has been pointed out that the marker causes dose shadowing because the protons are scattered with their energy quickly diminished. In this research we explore the possibility that the biological effect of dose shadowing can be mitigated with a limited number of fields. METHODS: The proton dose distribution in prostate was simulated using Geant4. The simulations include the Hokkaido University spot scanning nozzle and a water phantom positioned isocentrically. The PTV was delineated at the center of the phantom and a gold ball of 2 mm in diameter was placed at the middle of the PTV. The plan was created by single-field optimization and each of the following beam arrangements was investigated; (1) single lateral field (2) two lateral fields (3) two lateral + one anterior fields (4) four-field box. The dose prescription was D95 = 74 GyE (37 fr). The minimum dose and tumor control probability (TCP) were compared for the four beam arrangements. RESULTS: For (1)-(4), the minimum dose values were 55%, 77%, 78%, and 84% of the prescribed dose, respectively. The reduction of the TCP values from those in the absence of the gold marker were 50%, 2%, 1.1%, and 0.7%, using the TCP model by Wang et al. (Int.J.Radiat.Oncol.Biol.Phys. 55, 2003) and 2%, 0.7%, 0.5%, and 0.4%, using the biological parameters in Levegrûn et al. (Int.J.RadiatOncol.Biol.Phys. 51, 2001), respectively. CONCLUSIONS: Although dose shadowing by the gold marker is locally non-negligible, the size of the affected domain is tiny. It was found that with a minimum number of fields, the TCP nearly recovers to the value without the gold marker.

Lactation persistency as a component trait of the selection index and increase in reliability by using single nucleotide polymorphism in net merit defined as the first five lactation milk yields and herd life.

We first sought to clarify the effects of discounted rate, survival rate, and lactation persistency as a component trait of the selection index on net merit, defined as the first five lactation milks and herd life (HL) weighted by 1 and 0.389 (currently used in Japan), respectively, in units of genetic standard deviation. Survival rate increased the relative economic importance of later lactation traits and the first five lactation milk yields during the first 120 months from the start of the breeding scheme. In contrast, reliabilities of the estimated breeding value (EBV) in later lactation traits are lower than those of earlier lactation traits. We then sought to clarify the effects of applying single nucleotide polymorphism (SNP) on net merit to improve the reliability of EBV of later lactation traits to maximize their increased economic importance due to increase in survival rate. Net merit, selection accuracy, and HL increased by adding lactation persistency to the selection index whose component traits were only milk yields. Lactation persistency of the second and (especially) third parities contributed to increasing HL while maintaining the first five lactation milk yields compared with the selection index whose only component traits were milk yields. A selection index comprising the first three lactation milk yields and persistency accounted for 99.4% of net merit derived from a selection index whose components were identical to those for net merit. We consider that the selection index comprising the first three lactation milk yields and persistency is a practical method for increasing lifetime milk yield in the absence of data regarding HL. Applying SNP to the second- and third-lactation traits and HL increased net merit and HL by maximizing the increased economic importance of later lactation traits, reducing the effect of first-lactation milk yield on HL (genetic correlation (rG) = -0.006), and by augmenting the effects of the second- and third-lactation milk yields on HL (rG = 0.118 and 0.257, respectively).

Formation of a three-dimensional network of V trimers in A2V13O22 (A=Ba, Sr).

We found that in A2V13O22 (A=Ba, Sr), which contains a trilayer slab of VO in the sodium-chloride structure with periodically missing ions, the trimerization of V ions occurs at 290 K (A=Ba) and 380 K (A=Sr). V trimers form a three-dimensional network, but some V ions remain untrimerized in these compounds. The suppression of magnetic susceptibility with trimerization and the existence of a Curie tail at low temperatures, together with the result of NMR measurement, indicate that the V trimers are spin singlet, whereas the untrimerized V ions have a magnetic moment; i.e., there is a spontaneous separation between nonmagnetic and magnetic ions in the crystal.

Quantitative trait loci analysis on Sus scrofa chromosome 7 for meat production, meat quality, and carcass traits within a Duroc purebred population.

Many QTL analyses related to meat production and meat quality traits have been carried out using an F(2) resource population produced by crossing 2 genetically different breeds. This experiment was intended to investigate whether these QTL were segregating in a purebred Duroc population that had been selected for meat production and meat quality traits during 7 generations. Sus scrofa chromosome 7, for which significant QTL of intramuscular fat and many other traits have already been reported, was studied. The polymorphism of 10 microsatellite markers that were arranged at about 20-cM intervals was investigated on 1,004 pigs. In the selected population, 954 progeny were produced from mating of 99 sires and 286 dams. The QTL analysis for a full-sib family population was examined with the multigeneration pedigree structure of the population. Variance component analysis was used to detect QTL in this population and was examined for the multigeneration pedigree population. In this study, multigenerational pedigree estimated identical by descent coefficients among sibs were produced using Markov chain Monte Carlo methods. The maximum likelihood of odds score was found at the 70-cM position for the LM area, at the 0-cM position for the pork color standard, and at the 120-cM position for the number of thoracic vertebra, but no significant QTL for intramuscular fat were detected on SSC 7. These results indicate that QTL analysis via a variance component method within a purebred population was effective to determine that QTL were segregating in a population of purebred Durocs.

Non-transcallosal ipsilateral area 3b responses to median nerve stimulus.

We report two patients with left hemisphere lesions who had no normal left hemispheric responses to right median nerve stimulus on magnetoencephalography but displayed right area 3b responses. One patient had suffered a severe left hemispheric contusion and the other left hemispheric infarction. Equivalent current dipoles of these ipsilateral responses were detected on the central sulcus adjacent to the location of the N20m response to left median nerve stimulus. The somatosensory afferent pathway from the hand may extend directly to the ipsilateral area 3b without following the transcallosal pathway in at least part of the population.

Genetic mapping of quantitative trait loci affecting body weight, egg character and egg production in F2 intercross chickens.

Phenotypic measurements of chicken egg character and production traits are restricted to mature females only. Marker assisted selection of immature chickens using quantitative trait loci (QTL) has the potential to accelerate the genetic improvement of these traits in the chicken population. The QTL for 12 traits (i.e. body weight (BW), six for egg character, three for egg shell colour and two for egg production) of chickens were identified. An F2 population comprising 265 female chickens obtained by crossing White Leghorn and Rhode Island Red breeds and genotyped for 123 microsatellite markers was used for detecting QTL. Ninety-six markers were mapped on 25 autosomal linkage groups, and 13 markers were mapped on one Z chromosomal linkage group. Eight previous unmapped markers were assigned to their respective chromosomes in this study. Significant QTL were detected for BW on chromosomes 4 and 27, egg weight on chromosome 4, the short length of egg on chromosome 4, and redness of egg shell colour (using the L*a*b* colour system) on chromosome 11. A significant QTL on the Z chromosome was linked with age at first egg. Significant QTL could account for 6-19% of the phenotypic variance in the F2 population.

Simple deterministic identity-by-descent coefficients and estimation of QTL allelic effects in full and half sibs.

Accurate and rapid methods for the detection of quantitative trait loci (QTLs) and evaluation of consequent allelic effects are required to implement marker-assisted selection in outbred populations. In this study, we present a simple deterministic method for estimating identity-by-descent (IBD) coefficients in full- and half-sib families that can be used for the detection of QTLs via a variance-component approach. In a simulated dataset, IBD coefficients among sibs estimated by the simple deterministic and Markov chain Monte Carlo (MCMC) methods with three or four alleles at each marker locus exhibited a correlation of greater than 0.99. This high correlation was also found in QTL analyses of data from an outbred pig population. Variance component analysis used both the simple deterministic and MCMC methods to estimate IBD coefficients. Both procedures detected a QTL at the same position and gave similar test statistics and heritabilities. The MCMC method, however, required much longer computation than the simple method. The conversion of estimated QTL genotypic effects into allelic effects for use in marker-assisted selection is also demonstrated.

E2F1-mediated transcriptional inhibition of the plasminogen activator inhibitor type 1 gene.

Gene expression of the plasminogen activation system is cell-cycle dependent. Previously, we showed that ectopic expression of E2F1 repressed the plasminogen activator inhibitor type 1 (PAI-1) promoter in a manner dependent on the presence of DNA-binding and transactivation domains of E2F1 but independent of binding to pocket-binding proteins, suggesting a novel mechanism for E2F-mediated negative gene regulation [Koziczak, M., Krek, W. & Nagamine, Y. (2000) Mol. Cell. Biol. 20, 2014-2022]. However, it remains to be seen whether endogenous E2F can exert a similar effect. We report here that down-regulation of PAI-1 gene expression correlates with an increase in endogenous E2F activity. When cells were treated with a cdk2/4-specific inhibitor, which maintains E2F in an inactive state, the decline of serum-induced PAI-1 mRNA levels was suppressed. In mutant U2OS cells expressing a temperature-sensitive retinoblastoma protein (pRB), a shift to a permissive temperature induced PAI-1 mRNA expression. In U2OS cells stably expressing an E2F1-estrogen receptor chimeric protein that could be activated by tamoxifen, PAI-1 gene transcription was markedly reduced by tamoxifen even in the presence of cycloheximide. These results all indicate that endogenous E2F can directly repress the PAI-1 gene. DNase I hypersensitive-site analysis of the PAI-1 promoter suggested the involvement of conformation changes in chromatin structure of the PAI-1 promoter. 5' deletion analysis of the PAI-1 promoter showed that multiple sites were responsible for the E2F negative regulation, some of which were promoter dependent. Interestingly, one of these sites is a p53-binding element.

p53 Phosphorylation at serine 15 is required for transcriptional induction of the plasminogen activator inhibitor-1 (PAI-1) gene by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.

The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a widely spread environmental carcinogen that causes DNA lesions leading to cell killing. MNNG can also induce a cell-protective response by inducing the expression of DNA repair/transcription-related genes. We recently demonstrated that urokinase-type plasminogen activator, an extracellular protease to which no DNA repair functions have been assigned, was induced by MNNG. Here, we show that the physiological inhibitor of urokinase-type plasminogen activator, PAI-1, is also induced by MNNG in a p53-dependent fashion, because MNNG induced PAI-1 in p53-expressing cells but not in p53-/- cells. MNNG induced p53 phosphorylation at serine 15, resulting in stabilization of the p53 protein, and this phosphorylation event was central for p53-dependent PAI-1 transcription. Finally, we showed that PAI-1 transcriptional induction by MNNG required a p53-responsive element located at -136 base pairs in the PAI-1 promoter, because specific mutation of this site abrogated the induction. Because PAI-1 is a prognostic factor in many metastatic cancers, being involved in the control of tumor invasiveness, our finding that a genotoxic agent induces the PAI-1 gene via p53 adds a new feature to the role of the tumor-suppressor p53 protein. Our results also suggest the possibility that genotoxic agents contribute to tumor metastasis by inducing PAI-1 without involving genetic modification.

Chicken microsatellite primers are not efficient markers for Japanese quail.

Domestic fowl or chicken (Gallus gallus) and Japanese quail (Coturnix japonica) belong to the family Phasianidae. The exchange of marker information between chicken and quail is an important step towards the construction of a high-resolution comparative genetic map in Phasianidae, which includes several poultry species of agricultural importance. We tested chicken microsatellite markers to see if they would be suitable as genetic linkage markers in Japanese quail. Twenty-six per cent (31/120) of chicken primers amplified individual loci in Japanese quail and 65% (20/31) of the amplified loci were found to be polymorphic. Eleven of the polymorphic loci were excluded as uninformative because of the lack of amplification in some individuals or high frequency of nonspecific amplification. The sequence information of the remaining nine loci revealed six of them to contain microsatellites that were nearly identical with those of the orthologous regions in chicken. For these six loci, allele frequencies were estimated in 50 unrelated quails. Although the very few chicken markers that do work well in quail could be used as anchor points for a comparative mapping, most chicken markers are not useful for studies in quail. Therefore, more effort should be committed to developing quail-specific markers rather than attempting to adapt chicken markers for work in quail.

Using the mixed model for interval mapping of quantitative trait loci in outbred line crosses.

Interval mapping by simple regression is a powerful method for the detection of quantitative trait loci (QTLs) in line crosses such as F2 populations. Due to the ease of computation of the regression approach, relatively complex models with multiple fixed effects, interactions between QTLs or between QTLs and fixed effects can easily be accommodated. However, polygenic effects, which are not targeted in QTL analysis, cannot be treated as random effects in a least squares analysis. In a cross between true inbred lines this is of no consequence, as the polygenic effect contributes just to the residual variance. In a cross between outbred lines, however, if a trait has high polygenic heritability, the additive polygenic effect has a large influence on variation in the population. Here we extend the fixed model for the regression interval mapping method to a mixed model using an animal model. This makes it possible to use not only the observations from progeny (e.g. F2), but also those from the parents (F1) to evaluate QTLs and polygenic effects. We show how the animal model using parental observations can be applied to an outbred cross and so increase the power and accuracy of QTL analysis. Three estimation methods, i.e. regression and an animal model either with or without parental observations, are applied to simulated data. The animal model using parental observations is shown to have advantages in estimating QTL position and additive genotypic value, especially when the polygenic heritability is large and the number of progeny per parent is small.

Somatosensory evoked fields in comatose survivors after severe traumatic brain injury.

OBJECTIVE: To evaluate the cortical function quantitatively in patients in the chronic phase of severe traumatic brain injury. METHODS: Thirteen patients with severe traumatic brain injury due to traffic accident followed by persistent consciousness disturbance and disability were studied. Somatosensory evoked magnetic fields (SEFs) for unilateral median nerve stimulation were measured using a whole-head magnetoencephalography system. The latency and electrical current dipole (ECD) moment for the N20m, P30m, N45m and P60m components were calculated and compared with those of 14 age-matched healthy adults. RESULTS: The peak latency of N20m was longer (P<0.05) and those of P30m and N45m were shorter (P<0.01) in the patients than in normal adults. The ECD moment of N20m and P30m was smaller and that of N45m and P60m was larger in the patients than in normal adults (P<0.01). CONCLUSIONS: These results can be explained by the hypothesis that diffuse brain injury induces decreased and delayed input of the somatosensory afferent and compensational amplification of the response in the primary somatosensory cortex. Middle-latency SEFs may be applicable as a cortical functional measure for patients with severe traumatic brain injury.

Pocket protein-independent repression of urokinase-type plasminogen activator and plasminogen activator inhibitor 1 gene expression by E2F1.

Expression of genes of the plasminogen activator (PA) system declines at the G(0)/G(1)-S-phase boundary of the cell cycle. We found that overexpression of E2F1-3, which acts mainly in late G(1), inhibits promoter activity and endogenous expression of the urokinase-type PA (uPA) and PA inhibitor 1 (PAI-1) genes. This effect is dose dependent and conserved in evolution. Mutation analysis indicated that both the DNA-binding and transactivation domains of E2F1 are necessary for this regulation. Interestingly, an E2F1 mutant lacking the pRB-binding region strongly repressed the uPA and PAI-1 promoters. An E2F-mediated negative effect was also observed in pRB and p107/p130 knockout cell lines. This is the first report that E2F can act as a repressor independently of pocket proteins. Mutation of AP-1 elements in the uPA promoter abrogated E2F-mediated transcriptional inhibition, suggesting the involvement of AP-1 in this regulation. Results shown here identify E2F as an important component of transcriptional control of the PA system and thus provide new insights into mechanisms of cellular proliferation.

Mode of action of anhydrofulvic acid against Candida utilis ATCC 42402 under acidic condition.

The mode of action of anhydrofulvic acid against Candida utilis ATCC 42402 was investigated under acidic conditions. Anhydrofulvic acid inhibited the incorporation of radioactive precursors into DNA, RNA, protein and lipid fractions. Although it did not induce leakage of intracellular materials from the treated cells, it had inhibitory effects on both endogenous and exogenous cellular respiration. Moreover, it inhibited mitochondrial respiration of Candida utilis ATCC 42402 using both succinate and cytochrome c as respiratory substrates, but not using NADH. Unexpectedly, the inhibition against isolated mitochondria was observed at pH 7.0. These results suggested that the action site against the respiratory inhibition of anhydrofulvic acid might be involved in succinate dehydrogenase, complex II in the mitochondrial electron transport chain of the yeast cells. Judging from the inhibitory effect of anhydrofulvic acid on mitochondria detected at pH 7.0, it was postulated that the antifungal activity at a low pH level might depend on the elevation of drug permeability to the cell membrane under acidic conditions.

Regulation by p38 mitogen-activated protein kinase of adenylate- and uridylate-rich element-mediated urokinase-type plasminogen activator (uPA) messenger RNA stability and uPA-dependent in vitro cell invasion.

MDA-MB-231 cells are highly metastatic breast tumor cells. Their high invasiveness is thought to be due to constitutively high levels of urokinase-type plasminogen activator (uPA) and its receptor. Previously (R. Nanbu et al., C. Eur. J. Biochem., 247: 169-174, 1997), we showed that uPA mRNA in these cells is stable and that mRNA degradation mediated by an AU-rich element (ARE) is impaired. Here we report that treatment of MDA-MB-231 cells with SB203580, an inhibitor of the stress-activated p38 mitogen-activated protein (MAP) kinase, strongly destabilized uPA mRNA in an ARE-dependent manner. In contrast, in LLC-PK1 and HeLa cells, uPA mRNA is unstable, and an ARE present in the 3' untranslated region plays a role in its degradation. Enhanced ARE-mediated mRNA destabilization induced by SB203580 was also observed in both LLC-PK1 and HeLa cells with a globin chimeric mRNA harboring two copies of the ARE (globin-2ARE) from uPA mRNA. Overexpression of constitutively active MKK6, a p38 upstream activator kinase, increased the stability of the globin-2ARE message in LLC-PK1 cells, confirming the participation of p38 in the regulation of ARE-mediated mRNA decay. Interestingly, the half-life of the uPA mRNA in the three cell lines studied correlated with the basal levels of active p38. SB203580 treatment of MDA-MB-231 cells decreased cell-associated uPA activity and dramatically reduced in vitro cell invasiveness. These results suggest the participation of p38 in the control of invasiveness through regulation of the stability of uPA and uPA receptor mRNA, which is also destabilized by p38.

Cytoskeletal reorganization leads to induction of the urokinase-type plasminogen activator gene by activating FAK and Src and subsequently the Ras/Erk signaling pathway.

Previously, we showed that cytoskeletal reorganization (CSR) induced by colchicine or cyochalasins leads to activation of the urokinase-type plasminogen activator (uPA) gene in LLC-PK(1) cells via the Ras/Erk signaling pathway [Irigoyen et al. (1997) J. Biol. Chem. 272, 1904]. It remained to be seen how CSR activates Ras/Erk signaling. Changes in cell morphology triggered by extracellular signals are often mediated by integrin-associated proteins, such as focal adhesion kinase (FAK) and Src. We found that CSR induced the activation of FAK and Src and the association of FAK and Shc, a signaling molecule linking growth factor receptor tyrosine kinase and Grb2. Furthermore, expression of either FRNK, a kinase-minus FAK-like molecule acting as a dominant negative FAK, or a dominant negative Src suppressed CSR-induced uPA gene promoter activation. These results suggest that cells respond to a morphology change, using the cytoskeleton as a sensor, by activating FAK and Src and subsequently the Ras/Erk signaling pathway.

Detection of human papilloma virus type 56 in extragenital Bowen's disease.

A case of Bowen's disease arising on the medial part of the first metatarsal bone of an 81-year-old Japanese woman is described. Histopathologically, proliferation of atypical cells was found throughout the epidermis. Electronmicroscopy revealed virus particles 40-50 nm in diameter in the nuclei of tumour cells at the granular cells just on or below the horny layer. Positive bands were obtained by polymerase chain reaction using a consensus primer of human papilloma virus L1 portion. Sequencing analysis of the amplified DNA revealed the same base sequences and homology as human papilloma virus 56. To the best of our knowledge, this case is the first report in which human papilloma virus 56 was found in a case of extragenital Bowen's disease. We consider it important to understand that human papilloma virus 56, often found in cervical lesions, can be detected in extragenital Bowen's diseases.