Temperament Trait Changes in Japanese Black Cows Under Grazing and Confined Conditions.

The objective of the present study was to reveal the effects of grazing on the temperament traits of cows. Nine Japanese Black cows [344 ± 32 kg body weight (BW), 7.7 ± 3.0 year of age], which had various experiences, such as tethering, handling, and grazing, were used in this experiment. Five of the nine cows were grazed for 3 months on a 1.8-ha field composed of a sown pasture with forestland. The remaining cows were fed in confinement. On days 38, 52, 72, and 86 after the start of grazing, the temperament traits observed in various situations, such as moving to the body weight scale, weighing, handling, moving to the stock for blood sampling, holding in the stock, and obtaining a blood sample, were assessed with a visual analog scale (VAS: 1-10) or score (1-5). During weighing and handling, the intensity of resistance exhibited by the grazing cows, as evaluated by head movement, walking/stepping, tail flicking, rope tension, and overall movement, was lower than that exhibited by confined cows (P < 0.05). The resistance score exhibited by the grazing cows during blood sampling was also lower than that exhibited by confined cows (P < 0.01). These results suggest that grazing enhances docility in cows with various experiences in different situations encountered in daily management.

Effects of Grazing in a Sown Pasture with Forestland on the Health of Japanese Black Cows as Evaluated by Multiple Indicators.

The objective of the present study was to evaluate the effects of extensive grazingin a sown pasture with forestland on the health of beef cows by measuring multiple indicators. Ten Japanese Black beef cows were used in this experiment. Five of the ten cows were grazed for two months on a 1.8 ha field. The remaining cows were fed under confinement conditions. Behavioral assessments showed that grazing increased sternum lying with rumination of the cows. The grazing cows did not show any abnormal behaviors. There was a tendency for the numbers of red blood cells and lymphocytes to be lower in grazing cows than in confined cows, whereas the number of neutrophils in grazing cows was significantly higher than that in confined cows. In addition, grazing cows had a higher total antioxidant capacity and glutathione peroxidase activity than confined cows. These results suggest that extensive grazing in a sown pasture with forestland increases natural behaviors, decreases circulating red blood cells and lymphocytes and enhances neutrophil circulation, antioxidant enzyme activity, and antioxidant capacity.

Effects of direct exposure to cold weather under grazing in winter on the physiological, immunological, and behavioral conditions of Japanese Black beef cattle in central Japan.

This study aimed to reveal the effects of direct exposure to cold weather under grazing in winter (GW) on the health of Japanese Black beef cattle, as assessed using physiological, immunological, and behavioral parameters. Ten Japanese Black beef cattle (328 ± 45 kg, 7.6 ± 3.4 years of age) were used in this experiment. In winter, five of the 10 cattle grazed for 2 months in a 1.8 ha pasture (GW), and the remaining cattle were fed under confined conditions with tethering (control [CT]). The two groups were fed similar feed during the experiment, except for the grazing forage. Blood samples were collected approximately every 2 weeks. The numbers of neutrophils and monocytes and antioxidant enzyme activity were higher in the GW group than in the CT group (p < 0.05). The proportions of CD4-single-positive cells were lower in GW cattle than in CT cattle (p = 0.06). This study showed that direct exposure of beef cattle to cold weather under GW enhanced the levels of circulating neutrophils and monocytes and contributed to the kinetic homeostasis of lymphocytes but also activated antioxidant enzymes due to an increase in oxidative stress.

Effects of aging on stress-related responses of serotonergic neurons in the dorsal raphe nucleus of male rats.

Responses to various stressors in the brain change with age. However, little is known about the neural mechanisms underlying age-dependent changes in stress responses. It is known that serotonin, a stress-related transmitter, is closely related with the regulation of stress responses in the brain and that serotonergic function is modulated by various factors, including estrogen, in both sexes. In the present study, to elucidate the effects of aging on stress responses in serotonergic neurons, we examined the expression levels of tryptophan hydroxylase (TPH; a marker of serotonergic neurons) in the dorsal, ventral and lateral parts of the dorsal raphe nucleus (DRN) in young and old intact male rats. In young males, repeated restraint stress significantly increased the number of TPH-positive cells in all subdivisions of the DRN. In contrast, the stress-induced increase in TPH expression was only observed in the ventral part of the DRN in old males. Pretreatment with an estrogen receptor ß antagonist had no effect on the number of TPH-positive cells in the dorsal and lateral DRN in young stressed males, whereas the antagonist decreased the number of TPH-positive cells in all DRN subdivisions in old stressed males. Our results suggest that the effects of repeated stress exposure on the expression of TPH in serotonergic neurons in the DRN change with age and that estrogenic effects via estrogen receptor ß on TPH expression in stressed old males differ from those in young males.

The Inequality of Patient Profile Information in Japanese Hospitals.

A model dataset of patient profile information was created based on the items used at five Japanese university hospitals, the patient information data elements in Health Level 7 (HL7) v2.5, and the standard datasets for medical information exchange used in Japan. In order to check the validity of the model dataset, a cross-sectional survey was performed. A preliminary analysis of 20 Japanese hospitals found that most items were implemented at some hospitals, but the number of items implemented at many hospitals was rather small. This result strongly shows the necessity for a standardized dataset of patient profile information.

A human thyroid cancer cell line, DH-14-3, newly established from poorly differentiated thyroid carcinoma.

Poorly differentiated thyroid carcinoma (PDTC) is a newly recognized histological type of malignant thyroid tumor, accounting for about 2 - 13% of all thyroid carcinomas. PDTC is considered as a morphologically and biologically intermediate stage between well-differentiated thyroid carcinoma and anaplastic thyroid carcinoma. PDTC preferentially manifests bone metastases. We here established a cell line from a resected tumor specimen from a 70-year-old male patient with PDTC who presented with multiple bone metastases. This new thyroid tumor cell line was designated as DH-14-3 and was subsequently grown in culture for several years. DH-14-3 cells express thyroglobulin in the cytoplasm and thyroid transcription factor-1 in the nuclei, both proteins of which are specific markers for the thyroid gland. Importantly, triiodothyronine (T3) was detected in the cultured medium of DH-14-3 cells, in which, however, thyroxine (T4) was undetectable. Moreover, DH-14-3 cells secreted interleukin-8, transforming growth factor-ß1, vascular endothelial growth factor, matrix metalloproteinase-1 and parathyroid hormone-related protein, all of which may be responsible for the aggressiveness or bone metastasis of PDTC. Thus, the production of these proteins may reflect the metastatic potential of this cell line. DH-14-3 cells also express CXC chemokine receptor-4 and epidermal growth factor receptor, and carry a missense mutation in the p53 tumor suppressor gene. In fact, transplantation of DH-14-3 cells into the back of nude mice resulted in the formation of tumors, thereby confirming the capability of tumorigenesis. DH-14-3 cells may be useful for investigating the biological features of PDTC and will contribute to the therapeutic study of thyroid cancer.

T-reflex studies in human upper limb muscles during voluntary contraction: normative data and diagnostic value for cervical radiculopathy.

OBJECTIVE: To evaluate the diagnostic utility of the T reflexes elicited from the upper limb muscles during standardized volitional contraction monitored by a real-time integrating electromyographic analyzer. DESIGN: Prospective descriptive study. SETTING: Department of orthopedic surgery at a university hospital. PARTICIPANTS: Healthy subjects (n=80) evenly distributed across decades of age from 21 to 79 years, and 12 consecutive patients with a single cervical root lesion based on clinical and magnetic resonance imaging studies and diagnostic block. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Using a special hammer, which externally triggers the sweep on skin contact, we evoked T reflexes in the biceps (C5), brachioradialis (C6), triceps (C7), and the first dorsal interosseous muscles (C8). RESULTS: Simultaneous regression analyses yielded clinically useful upper limits of normative values for latencies, side-to-side differences, and amplitude ratios adjusted to age and arm span. Comparison of the T reflexes between the 2 sides localized the solitary root lesions with a high sensitivity (92%), specificity (81%), and accuracy (83%). T-reflex studies proved helpful to localize the lesion even in patients who solely complained of upper limb pain. CONCLUSIONS: The T reflexes with a standardized facilitation of the upper limb muscles provide a clinically useful, noninvasive measure to localize the C5 to C8 radiculopathies. This study contributes in reassessing the currently underused T reflex as an electrodiagnostic technique.

Adaptive threonine increase in transmembrane regions of mitochondrial proteins in higher primates.

BACKGROUND: The mitochondrial (mt) gene tree of placental mammals reveals a very strong acceleration of the amino acid (AA) replacement rate and a change in AA compositional bias in the lineage leading to the higher primates (simians), in contrast to the nuclear gene tree. Whether this acceleration and compositional bias were caused by adaptive evolution at the AA level or directional mutation pressure at the DNA level has been vigorously debated. METHODOLOGY/PRINCIPAL FINDINGS: Our phylogenetic analysis indicates that the rate acceleration in the simian lineage is accompanied by a marked increase in threonine (Thr) residues in the transmembrane helix regions of mt DNA-encoded proteins. This Thr increase involved the replacement of hydrophobic AAs in the membrane interior. Even after accounting for lack of independence due to phylogeny, a regression analysis reveals a statistical significant positive correlation between Thr composition and longevity in primates. CONCLUSION/SIGNIFICANCE: Because crucial roles of Thr and Ser in membrane proteins have been proposed to be the formation of hydrogen bonds enhancing helix-helix interactions, the Thr increase detected in the higher primates might be adaptive by serving to reinforce stability of mt proteins in the inner membrane. The correlation between Thr composition in the membrane interior and the longevity of animals is striking, especially because some mt functions are thought to be involved in aging.

Treatment with proteasome inhibitor MG132 during cloning improves survival and pronuclear number of reconstructed rat embryos.

In several mammalian species including rats, successfully cloned animals have been generated using somatic cell nuclear transfer (SCNT). However, in the case of rats, additional treatment with MG132, a proteasome inhibitor, before enucleation of oocytes seems to be required for successful cloning because ovulated rat oocytes are spontaneously activated, and hence, their suppression is the key to successful cloning. A previous study on rats demonstrated that matured oocytes potentially possess lower cytostatic factor (CSF) activity compared to mouse oocytes, resulting in a low incidence of premature chromosome condensation in the reconstructed embryos after SCNT. It is known that mice having more than two pronuclei are generally observed in nuclear-transferred oocytes after induction of premature chromosome condensation, which implies successful reprogramming. This leads us to the hypothesis that MG132 treatment affects not only the inhibition of spontaneous activation but also the reprogramming and developmental ability of reconstructed rat embryos. If so, prolonged MG132 treatment during and/or after SCNT may further improve the survivability. However, the effect of MG132 treatment on reconstructed embryos after SCNT has been very limited in rats and other species. We show here that prolonged MG132 treatment during and after SCNT improves survival and the number of pronuclei in reconstructed rat embryos after activation. These reconstructed embryos treated before, during, and after SCNT showed significantly higher p34(cdc2) kinase activity involving CSF activity compared to that of the control embryos. On the other hand, p34(cdc2) kinase activity was not recovered in nuclear-transferred oocytes without MG132, which suggested that the enucleation had detrimental effects on the development of reconstructed oocytes. Taken together, MG132 treatment during SCNT increases survival and pronuclear numbers in reconstructed rat embryos via maintenance of high CSF activity. The data suggest that MG132 treatment is indispensable for at least rat SCNT.

Robust time estimation reconciles views of the antiquity of placental mammals.

BACKGROUND: Molecular studies have reported divergence times of modern placental orders long before the Cretaceous-Tertiary boundary and far older than paleontological data. However, this discrepancy may not be real, but rather appear because of the violation of implicit assumptions in the estimation procedures, such as non-gradual change of evolutionary rate and failure to correct for convergent evolution. METHODOLOGY/PRINCIPAL FINDINGS: New procedures for divergence-time estimation robust to abrupt changes in the rate of molecular evolution are described. We used a variant of the multidimensional vector space (MVS) procedure to take account of possible convergent evolution. Numerical simulations of abrupt rate change and convergent evolution showed good performance of the new procedures in contrast to current methods. Application to complete mitochondrial genomes identified marked rate accelerations and decelerations, which are not obtained with current methods. The root of placental mammals is estimated to be approximately 18 million years more recent than when assuming a log Brownian motion model. Correcting the pairwise distances for convergent evolution using MVS lowers the age of the root about another 20 million years compared to using standard maximum likelihood tree branch lengths. These two procedures combined revise the root time of placental mammals from around 122 million years ago to close to 84 million years ago. As a result, the estimated distribution of molecular divergence times is broadly consistent with quantitative analysis of the North American fossil record and traditional morphological views. CONCLUSIONS/SIGNIFICANCE: By including the dual effects of abrupt rate change and directly accounting for convergent evolution at the molecular level, these estimates provide congruence between the molecular results, paleontological analyses and morphological expectations. The programs developed here are provided along with sample data that reproduce the results of this study and are especially applicable studies using genome-scale sequence lengths.

Core set approach to reduce uncertainty of gene trees.

BACKGROUND: A genealogy based on gene sequences within a species plays an essential role in the estimation of the character, structure, and evolutionary history of that species. Because intraspecific sequences are more closely related than interspecific ones, detailed information on the evolutionary process may be available by determining all the node sequences of trees and provide insight into functional constraints and adaptations. However, strong evolutionary correlations on a few lineages make this determination difficult as a whole, and the maximum parsimony (MP) method frequently allows a number of topologies with a same total branching length. RESULTS: Kitazoe et al. developed multidimensional vector-space representation of phylogeny. It converts additivity of evolutionary distances to orthogonality among the vectors expressing branches, and provides a unified index to measure deviations from the orthogoality. In this paper, this index is used to detect and exclude sequences with large deviations from orthogonality, and then selects a maximum subset ("core set") of sequences for which MP generates a single solution. Once the core set tree is formed whose all the node sequences are given, the excluded sequences are found to have basically two phylogenetic positions on this tree, respectively. Fortunately, since multiple substitutions are rare in intra-species sequences, the variance of nucleotide transitions is confined to a small range. By applying the core set approach to 38 partial env sequences of HIV-1 in a single patient and also 198 mitochondrial COI and COII DNA sequences of Anopheles dirus, we demonstrate how consistently this approach constructs the tree. CONCLUSION: In the HIV dataset, we confirmed that the obtained core set tree is the unique maximum set for which MP proposes a single tree. In the mosquito data set, the fluctuation of nucleotide transitions caused by the sequences excluded from the core set was very small. We reproduced this core-set tree by simulation based on random process, and applied our approach to many sets of the obtained endpoint sequences. Consequently, the ninety percent of the endpoint sequences was identified as the core sets and the obtained node sequences were perfectly identical to the true ones.

Multidimensional vector space representation for convergent evolution and molecular phylogeny.

With growing amounts of genome data and constant improvement of models of molecular evolution, phylogenetic reconstruction became more reliable. However, our knowledge of the real process of molecular evolution is still limited. When enough large-sized data sets are analyzed, any subtle biases in statistical models can support incorrect topologies significantly because of the high signal-to-noise ratio. We propose a procedure to locate sequences in a multidimensional vector space (MVS), in which the geometry of the space is uniquely determined in such a way that the vectors of sequence evolution are orthogonal among different branches. In this paper, the MVS approach is developed to detect and remove biases in models of molecular evolution caused by unrecognized convergent evolution among lineages or unexpected patterns of substitutions. Biases in the estimated pairwise distances are identified as deviations (outliers) of sequence spatial vectors from the expected orthogonality. Modifications to the estimated distances are made by minimizing an index to quantify the deviations. In this way, it becomes possible to reconstruct the phylogenetic tree, taking account of possible biases in the model of molecular evolution. The efficacy of the modification procedure was verified by simulating evolution on various topologies with rate heterogeneity and convergent change. The phylogeny of placental mammals in previous analyses of large data sets has varied according to the genes being analyzed. Systematic deviations caused by convergent evolution were detected by our procedure in all representative data sets and were found to strongly affect the tree structure. However, the bias correction yielded a consistent topology among data sets. The existence of strong biases was validated by examining the sites of convergent evolution between the hedgehog and other species in mitochondrial data set. This convergent evolution explains why it has been difficult to determine the phylogenetic placement of the hedgehog in previous studies.