Knockout mouse production assisted by Blm knockdown

Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3–4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.     

CO2 efflux from leaf litter focused on spatial and temporal heterogeneity of moisture

Leaf litter respiration (R _LL) was directly measured in situ to evaluate relationships with the water content in leaf litter (WC), which is distributed heterogeneously under natural conditions. To do so, we developed a small, closed static chamber system using an infrared gas analyzer, which can measure instantaneous R _LL. This study focuses on the measurement of CO₂ effluxes from leaf litter using the chamber system in the field and examines the relationship between R _LL and WC among seven broadleaf species in a temperate forest. The measurements focused on the position of leaves within the litter layer, finding that both R _LL and WC were significantly higher in the lower layer. The value of R _LL increased with increasing WC, and the response of R _LL to WC was similar among all seven species. Moreover, the temporal variation in WC differed among three species and was associated with leaf litter thickness. The observed heterogeneity in WC induced by the physical environment (e.g., position and thickness of leaf litter) affects the variation in WC and, therefore, both R _LL and the decomposition rates of organic matter in the litter layer.     

Differences in Organochlorine Accumulation in Masu Salmon Oncorhynchus masou masou between Fluvial and Sea-run Forms

Masu salmon Oncorhynchus masou masou divides into two life histories after a year of life in the river, the sea-run form and the fluvial form. Since salmons are anadromous, the sea-run form salmons are known to pollute the river sediments during spawning migration. In this study, we have studied the accumulation of organochlorines in the fluvial form as well as the sea-run form, and discussed their differences. In order to elucidate the accumulation patterns of organochlorines in both the fluvial and sea-run form masu salmons, the concentrations of organochlorines were determined in the muscle of both life histories. The organochlorines in the sea-run form were 7 to 21 times higher than those of fluvial form. Since salmons are semelparous, the carcasses of polluted sea-run form pollute the river sediments. The trans-nonachlor/trans-chlordane (N/C) ratio in the fluvial form (1.93) was significantly lower than that of sea-run form (23.8). This indicates that the fluvial form is polluted by comparatively newly input chemicals. These results suggest that sea-run form masu salmons have the potential to pollute the river sediments secondarily as vectors, but from the difference in N/C ratio between the two forms, the fluvial fish does not seem to be polluted by those organochlorine compounds of sea-run fish origin yet.     

Plasma cortisol and prolactin secretion rhythms in cattle under varying external environments and management techniques

The secretion rhythms of plasma cortisol (CORT) and prolactin (PRL), hormones related to stress responsiveness and biological rhythm and controlled by light and temperature, were investigated under varying external environments and different management techniques. Serial blood samples were collected from female cattle reared in free‐stall and freely fed (FF) conditions (n = 4) or in tie‐stall and restricted feeding (RF) conditions (hay and concentrate twice daily, n = 4). Plasma CORT and PRL concentrations, eating behavior, and environmental parameters were analyzed. Cyclic patterns for each parameter were examined using spectral analysis, and correlations between CORT, PRL and other parameters were investigated using cross‐spectral analysis. Under FF conditions, CORT secretion was not related to the lighting intensity and eating behavior. However, under RF conditions, the CORT secretion rhythm showed a distinct correlation with lighting intensity and eating behavior. Under FF conditions, the PRL secretion rhythm was similar in all seasons. However, under RF conditions, the PRL rhythm oscillated with high frequency in summer and low frequency in winter, indicating a seasonal change in rhythm. The present study demonstrates that hormone secretion rhythms change under different environments and management techniques.     

Investigation of Staphylococcus aureus positive for Staphylococcal enterotoxin S and T genes

Staphylococcus aureus produces staphylococcal enterotoxins (SEs) and causes food poisoning. It is known that almost all SE-encoding genes are present on various types of mobile genetic elements and can mobilize among S. aureus populations. Further, plasmids comprise one of SE gene carriers. Previously, we reported novel SEs, SES and SET, harbored by the plasmid pF5 from Fukuoka5. In the present study, we analyzed the distribution of these SEs in various S. aureus isolates in Japan. We used 526 S. aureus strains and found 311 strains positive for at least one SE/SE-like toxin gene, but only two strains (Fukuoka5 and Hiroshima3) were positive for ses and set among the specimens. We analyzed two plasmids (pF5 and pH3) from these strains and found that they were different. Whereas these plasmids partially shared similar sequences involved in the ser/selj/set/ses gene cluster, other sequences were different. A comparison of these plasmids with those deposited in the NCBI database revealed that only one plasmid had the ser/selj/set/ses cluster with a stop mutation in set similar to that in pH3. In addition, the chromosomes of Fukuoka5 and Hiroshima3, positive for ses and set, were classified into different genotypes. Despite the low rate of gene positivity for these SEs, it is suggested that there is diversity in plasmids and strains carrying these two SEs. Consequently, regarding the entire feature of SE prevalence, we improved the multiplex PCR detection method for the SE superfamily to obtain further insight.     

Differences between coarse woody debris and leaf litter in the response of heterotrophic respiration to rainfall events

Heterotrophic respiration strongly influences carbon cycles at the ecosystem and global scales. We used an automated chamber system to measure the heterotrophic respiration of coarse woody debris (CWD) and leaf litter in a secondary broadleaved forest in southern Kyoto Prefecture. This system, which targeted only organic matter, could detect heterotrophic respiration responses to changes in environmental factors, especially rainfall events. The temporal trends and responses of respiration to environmental factors differed dramatically between CWD and leaf litter. CWD respiration showed a clear diurnal change corresponding to changes in CWD temperature and a clear decrease during rainfall events. Leaf litter respiration did not change clearly but increased at the beginning of rain events and returned to pre-rain rates when soil water content declined. The temporal patterns of the residuals between the observed respiration and baseline respiration, developed from the temperature?Cresponse curves under pre-rain conditions, differed between CWD and leaf litter respiration. The typical trend in CWD respiration response to rainfall events was a clear decrease and then gradual increase in the residuals after the event. The response of leaf litter respiration to wetting was an increase in the residuals during rainfall events and then a gradual decrease during drying. The difference in the responses of these respirations to wetting and drying processes are likely caused by differences in the physical characteristics of the CWD and the leaf litter layer. Measurements targeting only organic matter using an automated chamber system could detect the responses of heterotrophic respiration to environmental factors.     

Stage-specific effect of growth hormone on developmental competence of bovine embryos produced in-vitro

Many efforts have been made to develop effective culture conditions for the production of bovine blastocysts. Growth hormone (GH) and glucose are known to affect in vitro embryo development. To improve in vitro culture conditions, the culture medium containing fetal calf serum (FCS) or bovine serum albumin (BSA) was supplemented with GH at various periods of development, and the effects of GH on the rate of development and the quality of the blastocysts were studied. Then, starting at the morula stage, the effect of glucose and GH on the rate of development was studied. In all experimental periods, FCS was more effective than BSA at improving the development rate and increasing the cell number of blastocysts. Adding GH to the culture medium between 18 and 48 h after fertilization (1-8 cell stage embryo) did not affect either the rate of blastulation or the cell number regardless of the serum protein (FCS or BSA). From 48 to 120 h after fertilization (5-cell to morula stage) GH increased the cell number of the blastocysts in the presence of BSA, but not in the presence of FCS. From 120 to 192 h after fertilization (morula to blastocyst stage), GH improved the developmental rate and cell number in the presence of FCS, although there was no significant difference when BSA was used instead of FCS as the serum protein. When cows were implanted with blastocysts developed in the presence of GH from the morula stage, their pregnancy rate did not differ from that of the control. Increasing the glucose concentration in the medium from 1.5 mM to 3 mM starting at the morula stage (120 h after fertilization) slightly decreased the rate of development, but on the other hand, decreasing the glucose concentration to 0 mM did not affect either the rate of development or the cell number. Also, then GH had no effect on the developmental rate or the cell number in the absence of glucose. In conclusion, when the medium was supplemented with serum, GH improved embryo development from the morula stage, but an increased concentration of glucose decreased embryo development. Furthermore, GH did not improve the pregnancy rate of blastocysts developed in vitro.