Molecular changes associated with adaptation of equine influenza H3N8 virus in embryonated chicken eggs

Embryonated chicken eggs (ECEs) are routinely used to isolate equine influenza virus. Propagation of the virus in ECEs results in selection of variants. In the present study, we determined nucleotide sequences of entire coding regions of parent A/equine/Tottori/1/07 (H3N8) and its derivatives that have different passage histories in ECE. After 12 passages, nucleotide sequence analysis predicted 3 amino acid substitutions in hemagglutinin (HA; 2 in HA1 and 1 in HA2). The two amino acid substitutions in HA1 were located in the vicinity of the cell receptor-binding site. Three other amino acid substitutions were predicted in internal proteins, 1 in the M1, 1 in the NP and 1 in the PA. This is the first report showing mutations in the internal protein genes of equine influenza virus associated with adaptation to ECE.     

Epigenetic assessment of environmental chemicals detected in maternal peripheral and cord blood samples

Epigenetic alteration is an emerging paradigm underlying the long-term effects of chemicals on gene functions. Various chemicals, including organophosphate insecticides and heavy metals, have been detected in the human fetal environment. Epigenetics by DNA methylation and histone modifications, through dynamic chromatin remodeling, is a mechanism for genome stability and gene functions. To investigate whether such environmental chemicals may cause epigenetic alterations, we studied the effects of selected chemicals on morphological changes in heterochromatin and DNA methylation status in mouse ES cells (ESCs). Twenty-five chemicals, including organophosphate insecticides, heavy metals and their metabolites, were assessed for their effect on the epigenetic status of mouse ESCs by monitoring heterochromatin stained with 4￠,6-diamino-2-phenylindole (DAPI). The cells were surveyed after 48 or 96 h of exposure to the chemicals at the serum concentrations of cord blood. The candidates for epigenetic mutagens were examined for the effect on DNA methylation at genic regions. Of the 25 chemicals, five chemicals (diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se) and octachlorodipropyl ether (S-421)) caused alterations in nuclear staining, suggesting that they affected heterochromatin conditions. Hg and Se caused aberrant DNA methylation at gene loci. Furthermore, DEP at 0.1 ppb caused irreversible heterochromatin changes in ESCs, and DEP-, Hg- and S-421-exposed cells also exhibited impaired formation of the embryoid body (EB), which is an in vitro model for early embryos. We established a system for assessment of epigenetic mutagens. We identified environmental chemicals that could have effects on the human fetus epigenetic status.     

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.     

Effects of hydroxy-aluminum on flocculation and permeability of silt and clay fractions separated from paddy soils

Flocculation tests and permeability measurements were carried out for silt and clay fractions separated from surface and subsurface horizons of four paddy soils. There was not much difference in the flocculating power between A1(OH)_2.2C1_0.8 and CaSO₄. Their flocculation value, 0.3 to 3 mM, was attained by adding the reagents at the rate of 0.24 to 2.4 mmol/100 g of the silt and clay. The flocculation with A1(OH)_2.2C1_0.8 or CaSO₄ did not affect the sediment volume of the silt and clay. This sediment volume was smaller for the surface than for the subsurface horizons of each soil, suggesting that soil organic matter and repetition of wetting and drying contribute to the stable aggregation of the silt and clay in the surface horizon. The permeability was higher for the silt and clay fractions separated from the surface horizon than that from the subsurface horizon, and the addition of 2.5 to 4.2 mmol of A1(OH)_2.2C1_0.8 or CaSO₄/100g of the silt and clay increased its permeability by 1.5 to 4 times. The effect of A1(OH)_2.2C1_0.8 was more lasting than that of CaSO₄ and was recognized even after 200 ml of water passed through 1 g of the silt and clay. The effect of the flocculating reagent on the permeability was more or less enhanced by air-drying the treated silt and clay.     

Seasonality of factors controlling N mineralization rates among slope positions and aspects in cool-temperate deciduous natural forests and larch plantations

This study aimed to evaluate the spatial patterns of soil nitrogen (N) transformations in relation to slope aspect and position, and to investigate the main factors controlling N transformation patterns during both the growing and dormant seasons in cool-temperate deciduous natural forests and larch plantations in eastern Hokkaido, northern Japan. Net rates of N mineralization (NR_minN) and of nitrification (NR_nit) in surface soils on north-facing and lower slopes were higher than those on south-facing and upper slopes, whereas the net rate of ammonium-N production (NR_amm) on south-facing and upper slopes was higher than that on north-facing slopes in both the natural forests and larch plantations. Both NR_minN and NR_nit were higher in the growing than in the dormant season, whereas NR_amm was higher in the dormant season. The soil C/N ratio, water content, soil pH and frequency of freeze–thaw cycles were important variables affecting N transformation patterns in any season. In relation to seasonality, the solar radiation index, daily temperature range and earthworm biomass were important controlling factors only during the growing season, and watershed area and soil N concentration only during the dormant season, suggesting that biological control accompanied with wet–dry events were important factors affecting N transformations during the growing season, but that run-off water and chemical controls were important determinants of spatial variation in N transformations during the dormant season.     

Effects of topography and planted trees on the distribution of naturally regenerated broad-leaved trees in a 140-year-old <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> plantation in northern Kyushu,Japan

We investigated factors affecting the distribution of naturally regenerated broad-leaved trees in a 140-year-old Cryptomeria japonica plantation. We used path analysis to examine the relationship among microtopography, the biomass of planted trees, and the biomasses of canopy and subcanopy trees of broad-leaved species. The study plot was divided into three topographic types (ridge, slope, and valley), and we discuss how the different topographic types are affected. For all topographic types, the biomass of canopy trees of broad-leaved species decreased with convexity. For slope and valley topographies, the biomass of subcanopy trees of broad-leaved species also decreased with convexity. For ridge topography, the biomass of subcanopy trees of broad-leaved species increased with the biomass of planted trees, and decreased with the biomass of canopy trees of broad-leaved species. These results suggest the effects of microtopography on the biomass of subcanopy trees were much larger than the effects of canopy trees for slope and valley topographies, while the effects of microtopography were smaller for ridge topography.     

Effect of increased feeding of dietary α‐linolenic acid by grazing on formation of the cis9,trans11–18:2 isoform of conjugated linoleic acid in bovine milk

Feeding systems such as grazing affect the fatty acid profile of bovine milk fat. In addition, milk fat is formed as the product of fatty acid metabolism in cow bodies before being secreted into milk. However, how grazing influences milk fatty acid profile through the metabolism has not been completely characterized. When fatty acid concentrations in Holstein milk were compared between grazing and non‐grazing periods, α‐linolenic acid was significantly higher in the grazing period than in the non‐grazing period. This could be explained with an increase in α‐linolenic acid feeding with grazing. α‐linolenic acid had a linear positive correlation with conjugated linoleic acid (9c,11t‐18:2) (CLA) and vaccenic acid (VA) during the grazing period, whereas CLA had higher correlation with linoleic acid rather than with α‐linolenic acid during the non‐grazing period. These data indicate that the high content of dietary α‐linolenic acid affects CLA and VA formation in milk of grazing periods via α‐linolenic acid metabolism into VA.     

Myostatin down‐regulates the IGF‐2 expression via ALK‐Smad signaling during myogenesis in cattle

Myostatin (MSTN) is a negative regulator during muscle differentiation, whereas insulin‐like growth factors (IGFs) are essential for muscle development. MSTN and IGFs act oppositely during myogenesis, but there is little information on the mutual relationship of MSTN and IGFs. The present study was conducted to examine whether MSTN affects IGF expression during early myogenesis in cattle. IGF‐1 mRNA was similarly expressed in M. longissimus thoracis of double‐muscled (DM) and normal (NM) Japanese shorthorn cattle. IGF‐2 mRNA expression was consistently higher in the normal and regenerating muscle of DM cattle than those of NM cattle. When myoblasts were isolated from regenerating M. longissimus thoracis, IGF‐2 mRNA expression showed a significant increase in differentiating DM derived myoblasts (DM‐myoblasts) as compared with differentiating NM derived myoblasts (NM‐myoblasts). An addition of recombinant mouse myostatin (rMSTN) to myoblast cultures attenuated IGF‐2 mRNA expression and decreased myotube formation, but did not effect IGF‐1 mRNA expression. An activin‐like kinase (ALK) inhibitor, SB431542, mediates MSTN action, suppressed the translocation of Smad2/3 into the nucleus in DM‐myoblasts, and restored the attenuated IGF‐2 mRNA expression and the decreased myotube formation induced by rMSTN in myoblast cultures. The findings indicate that MSTN may negatively regulate myoblast differentiation by suppressing IGF‐2 expression via ALK‐Smad signaling.     

Feeding Solanum glaucophyllum to preparturient multiparous cows prevents postparturient hypocalcemia

Solanum glaucophyllum (SG) contains 1,25‐dihydroxyvitamin D₃ (1,25‐(OH)₂D₃) glycosides. We investigated the effect of SG on hypocalcemia in cows. Serum levels of 1,25‐(OH)₂D₃, total calcium and phosphorus dose‐relatedly increased after feeding with SG, while serum magnesium and chloride levels fell (P < 0.05). We also performed an ethylenediaminetetraacetic acid (EDTA) infusion to induce artificial hypocalcemia. Cows that had been fed 4.0 mg/kg body weight of SG daily for 2 weeks had a higher serum concentration of total calcium at the end of EDTA infusion than those not fed SG (P < 0.05). In a field trial, multiparous cows were assigned to one of four groups: (1) no SG, (2) 1.3 g or (3) 2.6 g of SG daily from 14 days before the estimated calving day until 3 days after calving, or (4) a single feed of 35.75 g SG at 3 days before the estimated calving day. The concentrations of serum total calcium after the calving in each treatment group were (1) 7.4, (2) 7.9, (3) 8.0 and (4) 8.9 mg/dL and higher for (4) than for (1) (P < 0.05). The data suggests that feeding a high dose of SG before the calving may maintain higher concentrations of serum calcium after the calving.     

Differential expression of myostatin and its receptor in the porcine anterior pituitary gland

Myostatin (MSTN), known as growth and differentiation factor 8 (GDF-8), is a member of the transforming growth factor β (TGF-β) superfamily that negatively regulates skeletal muscle mass. Myostatin binds with high affinity to the receptor serine threonine kinase activin receptor type IIB (ActRIIB). Activins that also belong to the TGF-β superfamily, stimulate follicle-stimulating hormone production in gonadotrophs and suppress growth hormone and adrenocorticotropic hormone production in somatotrophs and corticotrophs, respectively. The aim of the present paper was therefore to clarify the endocrine action of MSTN in adenohypophysis. The present study details the expression and cellular localization of MSTN and ActRIIB in porcine anterior pituitary gland. The mRNA of MSTN and ActRIIB was consistently expressed in RT-PCR. Immunohistochemistry of MSTN and specific hormones showed that MSTN localized in thyrotrophs and gonadotrophs, in which most of the MSTN immunoreactive cells were identified as thyrotrophs. The immunostaining of ActRIIB was restricted to corticotrophs. These results indicate that MSTN was mainly produced in thyrotrophs and its receptor, ActRIIB, was restrictively contained in corticotrophs. Interestingly, thyrotrophs immunoreactive for MSTN were frequently close to corticotrophs immunoreactive for ActRIIB. The present study suggests that MSTN from thyrotrophs may regulate corticotroph function as a paracrine mediator among the porcine anterior pituitary cells.