Supplementary immunocytochemistry of hepatocyte growth factor production in activated macrophages early in muscle regeneration

Regenerative intramuscular motor‐innervation is thought to reside in the spatiotemporal expression of axon‐guidance molecules. Our previous studies showed that resident myogenic stem cells, satellite cells, up‐regulate a secreted neural‐chemorepellent semaphorin 3A (Sema3A) during the early‐differentiation period, in response to hepatocyte growth factor (HGF) elevated in injured muscle. However, a paracrine source of the HGF release is still unknown. Very recently, we proposed a possible contribution of anti‐inflammatory macrophages (CD206‐positive M2) by showing that M2 cells infiltrate predominantly at the early‐differentiation phase (3–5 days post‐injury) and produce/secrete large amounts of HGF. However, in understanding this concept there still remains a critical need to examine if phagocytotic pro‐inflammatory macrophages (CD86‐positive M1), another activated‐phenotype still present at the early‐differentiation phase concerned, produce HGF upon muscle injury. The current immunocytochemical study demonstrated that the HGF expression is negative for M1 prepared from cardiotoxin‐injured Tibialis anterior muscle at day 5, in contrast to the intense fluorescent‐signal of M2 served as a positive control. This supplementary result advances our understanding of a spatiotemporal burst of HGF secretion from M2 populations (not M1) to impact Sema3A expression, which ensures a coordinated delay in attachment of motoneuron terminals onto damaged and generating fibers during the early phase of muscle regeneration.     

Expression profiles of perforin,granzyme B and granulysin genes during the estrous cycle and gestation in the bovine endometrium

The conceptus is susceptible to destruction by maternal cytotoxic lymphocytes, which have cytotoxic potential. Therefore, it is expected that mechanisms for regulating cytotoxic lymphocytes exist, but little is known about the expression of cytotoxic genes in the endometrium. In the present study, we examined the spatial and temporal expression patterns of the cytotoxic genes perforin, granzyme B, and granulysin during the estrous cycle and gestation in the bovine endometrium. Endometrial tissues were collected from cows during the estrous cycle and gestation. The gene expression patterns of the three cytotoxic genes were examined using quantitative polymerase chain reaction and in situ hybridization, and cytotoxic lymphocyte subsets were characterized using immunohistochemistry. During mid‐ to late gestation in the intercaruncular (ICAR), granulysin expression was significantly increased, and a large number of granulysin‐expressing cells were localized in the luminal epithelium. Perforin and granzyme B displayed similar expression profiles and were highly expressed in the peri‐implantation endometrium, but few cells expressing these genes were found in the endometrial stroma. In conclusion, these findings suggest that in the ICAR epithelium granulysin may play important roles in the establishment and maintenance of gestation during normal pregnancy.     

Production of Functional Gametes from Cryopreserved Primordial Germ Cells of the Japanese Quail

The Japanese quail (Coturnix japonica) is a valuable bird as both an experimental animal, for a wide range of scientific disciplines, and an agricultural animal, for the production of eggs and meat. Cryopreservation of PGCs would be a feasible strategy for the conservation of both male and female fertility cells in Japanese quail. However, the effects of freeze-thaw treatment on viability, migration ability and germline transmission ability of quail PGCs still remain unclear. In the present study, male and female PGCs were isolated from the blood of 2-day-old embryos, which were cooled by slow freezing and then cryopreserved at –196 C for 77–185 days, respectively. The average recovery rate of PGCs after freeze-thawing was 47.0%. The viability of PGCs in the frozen group was significantly lower than that of the control group (P<0.05) (85.5% vs. 95.1%). Both fresh and Frozen-thawed PGCs that were intravascularly transplanted into recipient embryos migrated toward and were incorporated into recipient gonads, although the number of PGCs settled in the gonads was 48.5% lower in the frozen group than in the unfrozen control group (P<0.05). Genetic cross analysis revealed that one female and two male recipients produced live progeny derived from the frozen-thawed PGCs. The frequency of donor-derived offspring was slightly lower than that of unfrozen controls, but the difference was not significant (4.0 vs. 14.0%). These results revealed that freeze-thaw treatment causes a decrease in viability, migration ability and germline transmission ability of PGCs in quail.     

Autophagy in health and disease: a double-edged sword

Autophagy, the process by which cells recycle cytoplasm and dispose of excess or defective organelles, has entered the research spotlight largely owing to the discovery of the protein components that drive this process. Identifying the autophagy genes in yeast and finding orthologs in other organisms reveals the conservation of the mechanism of autophagy in eukaryotes and allows the use of molecular genetics and biology in different model systems to study this process. By mostly morphological studies, autophagy has been linked to disease processes. Whether autophagy protects from or causes disease is unclear. Here, we summarize current knowledge about the role of autophagy in disease and health.     

Development and evaluation of event-specific qualitative PCR methods for genetically modified Bt10 maize

In 2005 it was reported that the genetically modified (GM) maize strain or "event" called Bt10 had been distributed inadvertently in the United States over the previous 4 years. In order to ensure that grain for food and feed production did not contain trace amounts of Bt10 maize and complied with the applicable regulation, highly sensitive and specific detection of Bt10 maize was required. Accordingly, we developed a novel qualitative PCR system for specific detection of Bt10 maize. Moreover, we amply evaluated the performance characteristics of two PCR systems, our own and the one provided by the developer of Bt10, Syngenta Co. Ltd. It was confirmed that both of the qualitative PCR systems can specifically detect Bt10 maize, and the results of a single-laboratory examination suggested that the limit of detection was approximately less than 0.05% for both methods. To evaluate the reproducibility of the methods, we organized an interlaboratory study with the participation of 6 laboratories and analysis of 240 blind test samples. In this paper, we report, for the first time, the statistical analysis of the qualitative PCR data obtained from the interlaboratory study. The results of this analysis also revealed that there was no significant difference in the sensitivity between the two aforementioned methods and that the limit of detection of both the methods was less than 0.05%. Thus, we conclude that both of the methods are equally suitable for correct identification and sensitive detection of the unapproved GM maize Bt10 event in test samples.     

Induced maturation and spawning: opportunities and applications for research on oogenesis

Broodstock management requires the ability to detect and regulate oocyte growth, acquisition of maturational competence, maturation of oocytes, and onset of ovarian atresia. Our research on temperate basses (genus Morone) has supported development of these capabilities. These investigations have revealed that accumulation of neutral lipid droplets and deposition of vitellogenin-derived yolk proteins in growing oocytes are independent processes with different sensitivities to changing day length and water temperature. In these fishes, completion of oocyte growth is marked by disappearance of vitellogenin from ovarian biopsy samples. Competence of females for induced spawning is predicted by the ability of biopsied follicles to initiate oocyte meiosis in vitro in response to insulin-like growth factor I. Cytoplasmic maturation of the oocytes is triggered by the maturation-inducing steroid hormone and can be monitored by evaluating degradation of the yolk proteins. Onset of ovarian atresia is indicated by the appearance of edema in the granulosa cell layer of biopsied follicles, and can be delayed for months by holding gravid females at abnormally low temperature (`cold banking'). These novel findings hold strong promise for application to other farmed fishes.     

Relationship Between Physicochemical Properties of Starches and White Salted Noodle Quality in Japanese Wheat Flours

This study describes the effect of starch properties of Japanese wheat flours on the quality of white salted noodles (WSN). Starch was isolated from 24 flours of 17 Japanese cultivars and amylose content was determined along with pasting properties by Rapid Visco Analyser (RVA), thermal properties by differential scanning calorimetry (DSC), and the distribution of amylopectin chain length by high‐performance anion exchange chromatography (HPAEC). Twenty flours were used to prepare WSN. As expected, 5–6% lower amylose content was associated with good WSN quality (higher scores in softness, elasticity, and smoothness). RVA analysis indicated that the pasting temperature had the greatest influence on WSN quality, while breakdown and setback showed slight effects on WSN quality. DSC results showed that lower endothermal enthalpy (ΔH) in the amylose‐lipid complex was associated with good WSN quality. Chainlength distribution of amylopectin by HPAEC was not an important factor in relation to WSN quality.     

Properties of Starches from Several Low‐Amylose Rice Cultivars

The starch properties of five low‐amylose rice cultivars, Yawarakomachi, Soft 158, Hanabusa, Aya, and Snow Pearl, were compared with those of two normal amylose rice cultivars, Nipponbare and Hinohikari. There were no large differences in the distributions of the amylopectin chain length determined by high‐performance anion‐exchange chromatography, and the starch gelatinization properties determined by differential scanning calorimetry, between normal and low‐amylose rice cultivars. Results obtained using rapid viscosity analysis indicated that low‐amylose rice starches had lower peak viscosity, breakdown, and setback values than normal amylose rice starches. Starch granules from low‐amylose rice cultivars had a higher susceptibility to glucoamylase than those from normal amylose rice cultivars. The results of this study showed some differences between normal and low‐amylose rice starches in pasting properties and enzymatic digestibility.