Na?ve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.     

Analysis of mitochondrial DNA of an endangered beech species,Fagus hayatae Palibin ex Hayata

Mitochondrial DNA restriction fragment length polymorphisms were used to examine cytoplasmic diversity within a relic-like population of Fagus hayatae, located in northern Taiwan. Fifteen trees were surveyed for three restriction endonucleases (BamHI, EcoRI and HindIII) and five mitochondrial gene probes (atpA, atp6, atp9, coxI and coxII). The analysis failed to reveal any polymorphisms, an observation that suggests cytoplasmic uniformity in the F. hayatae population examined. It is also interesting to note that the chondriome type of our F. hayatae samples is very close to that characteristic of F. crenata populations in the southernmost area of Japan.     

An intact mitochondrial cox1 gene and a pseudogene with different genomic configurations are present in apple cultivars ‘Golden Delicious’ and ‘Delicious’: Evolutionary aspects

We have characterized the mitochondrial cox1 gene copies in two apple cultivars ‘Golden Delicious’ and ‘Delicious’. Both the cultivars contained an intact copy and a truncated copy of cox1. The intact ‘Golden Delicious’ and ‘Delicious’ cox1 genes, designated G-cox1 and D-cox1, respectively, were both found to be actually transcribed to give an RNA of approximately 1.7 kb. The two intact cox1 and two truncated copies (G-φcox1 and D-φcox1) shared a common 1115-bp segment flanked by four combinations of two different 5′- and 3′-sequences. PCR assay demonstrated that the configurations bearing G-cox1 and G-φcox1 existed in substoichiometric amounts within the mitochondrial genome of ‘Delicious’ whereas substoichiometric molecules carrying D-φcox1 were present in the ‘Golden Delicious’ mitochondrial genome. Although ancestor/descendant relationships cannot be inferred between the G-cox1 and D-cox1 arrangements, the results led us to hypothesize that (1) the 1115-bp segment containing part of the progenitor cox1 was duplicated, thereby generating a pseudo-cox1 copy, and (2) this was followed by homologous recombination across a portion of the 1115-bp repeats which gave rise to the descendant cox1 and pseudo-cox1 arrangements.     

Flow cytometric immune profiling of specific-pathogen-free chickens before and after infectious challenges

Broilers and layer chickens have been intensively selected for production parameters. This selection has affected immune capacity. Consequently, the fine-tuning of immune responses is becoming important for maximum productivity. Flow cytometry is a recurrent technology used for the immunophenotyping of birds. Studies, however, have focused on the mechanism of specific diseases or have used animals whose immunological condition could be biased-by vaccination or environmental stressors, for example. The aim of this study was to evaluate the immune status of specific-pathogen-free birds across different age ranges to characterize the natural changes that occur over time. Additionally, specific-pathogen-free chickens were challenged with four infectious agents, allowing identification of the subpopulations of peripheral blood immune cells that are consistently altered under various conditions. Several lymphocyte subsets vary naturally with aging, so the interpretation of results using animals of different age ranges must proceed with care. Parameters such as CD8(+)CD28(-), CD8αα(+), CD4(+)CD8(+), and CD8(+)TCRVβ1(+) have been shown to be valuable in understanding immune changes during disease. The use of these data allows a determination of the consistency of cytometric parameters under various conditions, which should ease the interpretation of immunophenotyping and the future application of cytometric analysis in the poultry industry.     

Origin of plasma insulin-like growth factor-I (IGF-I) during estrus in goats

The objective of this study was to clarify the origin of the increase in plasma insulin-like growth factor-I (IGF-I) during estrus in goats. Focusing on the uterus, the effect of estradiol-17 beta (E2) on the secretion of IGF-I was examined using ovariectomized and hysterectomized animals. A single 5 microg/kg BW of E2 was injected intramuscularly into ovariectomized and hysterectomized goats for 3 consecutive days, and plasma IGF-I concentrations in the two groups were compared. The concentrations of IGF-I rose after the treatments in both groups. The concentrations were significantly higher from 3 to 8 days after the treatment than before the treatment in ovariectomized goats (P<0.05), and from 1 to 3 days after the treatment than before in hysterectomized goats (P<0.05). Thus higher concentrations of plasma IGF-I tended to last longer in ovariectomized than hysterectomized goats. The area under the IGF-I response curve for the 8-day period after the first injection of E2 tended to be greater in ovariectomized than in hysterectomized goats. The results show that E2 increases plasma IGF-I concentrations in goats, and suggest that E2-stimulated IGF-I in plasma may originate mainly from the uterus.     

Effects of leptin on the release of luteinizing hormone, growth hormone and prolactin from cultured bovine anterior pituitary cells

The effects of leptin on the release of luteinizing hormone (LH), growth hormone (GH) and prolactin (PRL) were studied in cultured bovine anterior pituitary (AP) cells in vitro. The AP cells were obtained from fully‐fed Japanese Black steers and were incubated for 3 h with 10^?13 to 10^?7 mol/L of leptin after incubating in Dulbecco's modified Eagle's Medium for 3 days. Leptin significantly increased the concentration of LH in the culture medium by 45 and 44% at doses of 10^?8 and 10^?7 mol/L, respectively, compared with the controls (P < 0.05). Leptin significantly increased the concentration of GH in the culture medium by 14 and 12% at doses of 10^?8 and 10^?7 mol/L, respectively (P < 0.05). Leptin also significantly increased the concentration of PRL in the culture medium by 26% compared with the controls at a dose of 10^?7 mol/L (P < 0.05). These results show that leptin stimulates the release of LH, GH and PRL by acting directly on bovine AP cells from fully‐fed steers.     

Disulfide Proteome Analysis of Buckwheat Seeds to Screen Putative Allergens

Accumulating evidence suggests a correlation between disulfide bonding and the allergenicity of proteins. Also, a significant characteristic of most food allergens is that they are stable to proteases. We sought to identify putative allergens of buckwheat seed comprehensively by combining a disulfide proteome technique with an in vitro digestibility test. First, a dilute acetic acid fraction of buckwheat seed was found to be rich in disulfide proteins. Internal amino acid sequence analyses of these proteins showed that most of them were known allergens or putative allergens sharing high amino acid sequence similarities to known allergens. Next, the fraction was subjected to in vitro protease digestion, which revealed relatively large fragments that were resistant to prolonged enzymatic digestion. These protease‐resistant fragments contained disulfide bonds that should have protected the potential enzyme cleavage sites by forming compact structures. These results confirm and extend our knowledge of the correlations among the disulfide bonding of proteins, their protease stability, and their allergenicity. Also, these observations suggest a new strategy to identify putative allergens by proteomic approaches as well as to mitigate them.     

Effects of leptin and leptin peptide amide on the release of luteinizing hormone, growth hormone and prolactin from cultured porcine anterior pituitary cells

The present study was carried out to determine whether leptin or leptin (116–130) peptide amide (lep (116–130)), an active fragment of the native protein in rats, is able to stimulate the release of luteinizing hormone (LH), growth hormone (GH) or prolactin (PRL) from cultured porcine anterior pituitary (AP) cells in vitro. The AP cells were obtained from 6 month‐old pigs and were incubated for 3 h with 10^?11?10^?7 mol/L leptin or lep (116–130) after being cultured in Dulbecco's modified Eagle's medium for 3–4 days. Leptin significantly increased the concentration of LH and GH in the culture medium at concentrations of 10^?8 and 10^?7 mol/L, respectively, compared with the controls (P < 0.05). Leptin did not increase the concentration of PRL in the culture medium. In contrast to these results, no effects of lep (116–130) on the release of LH, GH or PRL were seen in the cultured cells. These results suggest that leptin stimulates the release of LH and GH by acting directly on porcine AP cells, and that a fragment of leptin protein comprising amino acids 116–130 is not associated with the secretion of hormones in pigs.     

Effects of intracerebroventricular injections of leptin on the release of luteinizing hormone and growth hormone in castrated calves

The purpose of the present study was to clarify the hypothalamic action of leptin on the secretion of luteinizing hormone (LH) and growth hormone (GH) in cattle. Intracerebroventricular (the third ventricle) injections of leptin were given to fully fed castrated Holstein calves. Blood samples were collected at 10‐min intervals for 60 min after injection and 20‐min intervals for 60 min before injection and for 60–180 min after injection through an indwelling catheter in the external jugular vein. Plasma LH and GH levels were examined by homologous radioimmunoassay. The administration of 10 µg of leptin stimulated a significant (P < 0.05) release of GH but not LH. Average GH levels began to rise after 30 min and were significantly increased at 40, 50 and 60 min after the injection, compared with the respective control values (P < 0.05). The present result suggests that leptin may act partly on the hypothalamus to stimulate the release of GH in castrated calves.