Hybrid sterility between two isozymic types of the fernCeratopteris thalictroides in Japan

JapaneseCeratopteris thalictroides are composed of two allopatric entities called the south type and the north type which are isozymically distinct. Both types are tetraploid at 2n=156. To examine the crossability of these isozymic types, artificial hybridization was conducted among ten representative populations. Allozymes of PGI were used as markers to identify the hybrids among populations. Hybrids within the north type produced normal-shaped spores whose germination rate reached 60.7 to 96.0%. In contrast, hybrids between the north type and the south type formed irregular-shaped spores which were aborted with a low germination rate of 0.0 to 7.0%. It is thus evident that hybrid sterility is present between these types. Morphological analyses revealed that the two types were part of a continuous cline of variability and hence morphologically inseparable. The two types can thus be regarded so far as cryptic species that are morphologically indistinguishable but reproductively isolated.     

The 1975 Japanese diet has a stress reduction effect in mice: search for physiological effects using metabolome analysis

Abstract

We aimed to find new physiological effects of the Japanese diet. First, to determine the key components in serum from mice fed the 1975 diet, serum from mice fed the 1960, 1975, 1990 or 2005 Japanese diet was analyzed using CE-TOFMS and LC-TOFMS. Based on these results, the key components were determined by principal component analysis. Among the identified compounds, GABA was included. Therefore, a stress reduction effect was inferred as a novel physiological effect of this diet. Next, we tested whether the 1975 diet had an actual stress reduction effect in mice. Mice were given the 1975 diet or a control diet for 4 weeks, after which they were divided into restraint stress and non-stress groups. Mice fed the 1975 diet had significantly decreased stress parameters compared with those fed the control diet. These results provide the first evidence that the 1975 Japanese diet has a stress reduction effect.     

A Measure of Biochemical Individual Variability

Once a questionable substance is found in a polluted area, we determine its MLD, LD₅₀ etc. by animal experiments and then taking the species difference into consideration, we provide its MTD, LD₅₀ etc. for the man. However, there exists clearly a wide difference in its tolerance among persona even with the same sex and age belonging to the same race. Our main themes are (i) what is a rational measure of the biochemical individual variability, (ii) what kind of empirical law holds with respect to this measure, and (iii) what is the reason why it holds. This is a summary of my research sponsored by our Agency of Science and Technology [1979a] with some additional results.     

Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.     

CD4+ T cell responses to HLA-DP5-restricted wild-type sequence p53 peptides in patients with head and neck cancer

Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines. Evidence has been accumulating which indicates that CD4+ Th cells have an important role in generating and maintaining antitumor immune responses. To elucidate the nature of CD4+ Th responses to wt p53 epitopes in patients with squamous cell carcinoma of the head and neck (SCCHN), peripheral blood mononuclear cells (PBMCs) from HLA-DP5+ patients were stimulated with HLA-DP5-restricted wt p53 peptides, p53_108–122 or p53_153–166, and tested for the release of IFN-γ and IL-5 in ELISPOT assays. Immunohistochemistry for p53 accumulation in tumors, and ELISA for serum antibodies to p53 were also performed. Eleven (57.9%) of 19 HLA-DP5+ patients but none of 5 healthy donors had detectable Th1 and/or Th2 responses to wt p53 peptides by ELISPOT assay. Among these 11 responding patients, 9 (81.8%) and all 11 (100%) patients had a tumor burden and p53 accumulation, respectively. On the other hand, two responding patients were in post-operative condition. Interestingly, among nine patients with a tumor burden, four patients with early disease showed either Th1-polarized or mixed Th1/Th2 responses, while five patients with advanced disease showed either Th2-polarized or mixed Th1/Th2 responses. Our results suggest that wt p53_108–122 and p53_153–166 peptides stimulate both Th1- and Th2-type CD4+ T cell responses in patients with SCCHN, and anti-p53 Th responses may persist even after surgical resection of the tumor; however, the presence of a tumor and its progression may affect the nature of immune responses to wt p53 peptides.     

Overexpression of taurine transporter in Chinese hamster ovary cells can enhance cell viability and product yield,while promoting glutamine consumption

Transporters mediate the uptake of nutrients such as amino acids and the excretion of metabolites. The fact that transporters play crucial roles in regulating cell metabolism suggests that they might be useful targets for cell engineering to enhance the yield and/or quality of monoclonal antibody (MAb) produced by CHO cells. The taurine transporter (TAUT) is stably expressed in CHO‐DXB11 cells and is upregulated late in the culture period. We found that forcing the overexpression of TAUT delayed apoptotic cell death, extending the culture period. Thus, under fed‐batch small‐culture conditions, CHO cells that expressed pHyg‐TAUT plasmid (TAUT/CHO cells), but not those that contained the null plasmid pHyg (HYG/CHO cells), produced more MAb (P < 0.01) and less lactate (P < 0.05). In a 1‐L bioreactor, a representative high‐yield TAUT/CHO cell line (T10) showed >80% viability for more than 1 month and a 47% increase in medium MAb concentration. In T10 cells, the upregulation of TNF‐α mRNA (an apoptosis marker) and the accumulation of ammonia late in the culture period were suppressed. Moreover, if an excess of taurine was added, T10 cells efficiently consumed glutamine but not other amino acids, so T10 cells may have gained a glutamine transporter‐like function. Because a considerable amount of metabolic energy is derived from glutamine, this active glutamine consumption in T10 cells might be a reason for the improved cell viability and MAb concentration. These results demonstrate that forcing the overexpression of TAUT in CHO cells can enhance cell culture performance and increase MAb titer. Biotechnol. Bioeng. 2010;107: 998–1003. © 2010 Wiley Periodicals, Inc.     

Body mass and composition responses to short-term low energy intake are seasonally dependent in Steller sea lions (Eumetopias jubatus)

Steller sea lions (Eumetopias jubatus) were fed restricted iso-caloric amounts of Pacific herring (Clupea pallasi) or walleye pollock (Theragra chalcogramma) for 8–9 days, four times over the course of a year to investigate effects of season and prey composition on sea lion physiology. At these levels, the sea lions lost body mass at a significantly higher rate during winter (1.6 ± 0.14 kg day⁻¹), and at a lower rate during summer (1.2 ± 0.32 kg day⁻¹). Decreases in body fat mass and standard metabolic rates during the trials were similar throughout the seasons and for both diet types. The majority of the body mass that was lost when eating pollock derived from decreases in lipid mass, while a greater proportion of the mass lost when eating herring derived from decreases in lean tissue, except in the summer when the pattern was reversed. Metabolic depression was not observed during all trials despite the constant loss of body mass. Our study supports the hypothesis that restricted energy intake may be more critical to Steller sea lions in the winter months, and that the type of prey consumed (e.g., herring or pollock) may have seasonally specific effects on body mass and composition.