An improved dry ash procedure for the detection of titanium dioxide in cattle feces.

We improved the dry ash procedure for detecting titanium dioxide (TiO2 ) in cattle feces containing chromium oxide [corrected] (Cr(2)O(3) ). First, the effect of amount of sodium sulfate (Na2 SO4 ) on the recovery of TiO2 from cattle feces that contained Cr2 O3 was evaluated. Average recovery of TiO2 at the 2.5 g Na2 SO4 level was significantly higher (P < 0.0001) than that at 0.75 g Na2 SO4 . Second, the effect of Cr2 O3 concentration on the recovery of TiO2 of cattle feces by using two levels of Na2 SO4 addition was examined. The recovery of TiO2 decreased with the increase in the amount of Cr2 O3 at the 0.75 g Na2 SO4 level but was consistently high at 2.5 g Na2 SO4 . Third, the recovery of Cr2 O3 from cattle feces was checked. The recoveries of TiO2 and Cr2 O3 were high enough at the 2.5 g Na2 SO4 level. Fourth, the improved dry ash procedure (5 mL of concentrated H2 SO4 and 2.5 g of Na2 SO4 were used for sample digestion) was compared to the wet ash procedure. Average recovery of TiO2 by the improved dry ash procedure was significantly higher (P = 0.0077) than that by the wet ash procedure. Thus, the improved dry ash procedure can be used for TiO2 analysis in cattle feces containing Cr2 O3 .

Cloned porcine embryos can maintain developmental ability after cryopreservation at the morula stage.

The aim of the present study was to clarify the overall efficiency of porcine somatic cell nuclear transfer (SCNT) by incorporating cryopreservation of the cloned embryos before transfer. The SCNT embryos reconstructed with preadipocytes and in vitro-matured (IVM) oocytes were cultured to harvest morula stage embryos; they were then subjected to delipation (removal of cytoplasmic lipid droplets) and vitrification. After warming and culture, the embryos developing to blastocysts were transferred to recipients to obtain cloned piglets. From 372 reconstructed embryos, 188 (50.5%) reached the morula stage and 117 (31.5%) developed to blastocysts after vitrification. Transfer of 98 (26.3%) morphologically normal blastocysts gave rise to 6 (1.6%) piglets, including 1 stillborn. The efficiency of the cloned piglet production was comparable with that obtained using SCNT embryos without cryopreservation (2.7%, 17/635). Here, we demonstrate that porcine somatic cell cloning can be performed without a significant reduction in efficiency even when the SCNT embryos are cryopreserved before transfer.

Large-scale identification by shotgun proteomics of proteins expressed in porcine liver and salivary gland.

Protein catalogs containing a large number of proteins expressed in a variety of organs can be powerful tools for stem-cell research, because this requires accurate knowledge about how cells differentiate. Salivary gland progenitor (SGP) cells are somatic stem cells isolated from the salivary gland that can differentiate into hepatic or pancreatic cell lineages. Their differentiation state has been assessed by the expression of major protein markers, but to use these cells in regenerative medicine, it will be necessary to establish additional means of quality assessment. We examined the use of shotgun proteomics for porcine salivary gland (a source of SGP cells) and liver (a destination of differentiated SGP cells) for determining the state of SGP cell differentiation. Protein complexes from each organ were digested into peptides and separated by two-dimensional liquid chromatography involving strong cation-exchange chromatography followed by reversed-phase liquid chromatography. The separated peptides were analyzed by on-line electrospray ionization tandem mass spectrometry using a quadrupole-time of flight mass spectrometer (ESI Q-TOF MS/MS), and the spectra obtained were processed to search peptides against a mammalian database for protein identification. Using this method, we identified 117 proteins in porcine salivary gland and 154 proteins in porcine liver. Of these, 72 and 109 were specific to salivary gland and liver, respectively, and some of these were previously shown to be organ specific. The current study can be utilized in the future as a basis to study the pattern of differentiation in protein expression by stem cells.

Production efficiency and telomere length of the cloned pigs following serial somatic cell nuclear transfer.

The aim of the present study was to examine the production efficiency of cloned pigs by serial somatic cell nuclear transfer (SCNT) and to ascertain any changes in the telomere lengths of multiple generations of pigs. Using fetal fibroblasts as the starting nuclear donor cells, porcine salivary gland progenitor cells were collected from the resultant first-generation cloned pigs to successively produce second- and third-generation clones, with no significant differences in production efficiency, which ranged from 1.4% (2/140) to 3.3% (13/391) among the 3 generations. The average telomere lengths (terminal restriction fragment values) for the first, second and third generation clones were 16.3, 18.1 and 20.5 kb, respectively, and were comparable to those in age-matched controls. These findings suggest that third-generation cloned pigs can be produced by serial somatic cell cloning without compromising production efficiency and that the telomere lengths of cloned pigs from the first to third generations are normal.

Production of cloned pigs from salivary gland-derived progenitor cells.

To achieve tissue stem cell transplantation in clinical settings, translational studies using large animal models are essential to confirm the efficacy and safety of therapy. Therefore, with the ultimate objective of constructing a porcine model of stem cell transplantation in the present study we attempted to clone pigs using porcine salivary gland-derived progenitor cells (pSGPs) as nuclear donors. Normal chromosomal compositions of pSGPs were maintained after five to eight passages (73%, 41 of 56). Cell cycle was efficiently synchronized in G(0)/G(1) phase after 2 days of serum-starved culture (79%). Characteristics of multipotent pSGPs, that is, CD49f and intracellular laminin staining patterns, were unchanged after serum-starved culture. Developmental rate of blastocysts from embryos reconstructed using pSGPs as nuclear donors was significantly higher when compared to embryos reconstructed using fetal fibroblasts (27.7%, 38 of 137 vs. 12.8%, 17 of 138; p < 0.05). When a total of 615 reconstructed embryos were transplanted into four recipient gilts, all gilts became pregnant and produced 12 piglets. These findings suggest that pSGPs represent appropriate donor cells for porcine somatic cell nuclear transfer.

Isolation of tissue progenitor cells from duct-ligated salivary glands of swine.

Tissue stem cells participate in the repopulation of tissue after injury. Tissue injury stimulates the normally quiescent tissue stem cells to differentiate and proliferate, in the process of replacing and/or repairing the damaged cells, and hence effecting tissue regeneration. The salivary glands retain the ability for frequent regeneration. Previously, we isolated progenitor cells from the injured salivary glands of mice and rats that differentiated into hepatic and pancreatic lineages. The isolated progenitors were CD49f-positive and intracellular laminin-positive, and proliferated on type I collagen while maintaining their multipotency. In this study, we analyzed the tissue stem cells induced by ligating the main excretory duct of the salivary gland in swine. After duct ligation of the gland, acinar cells receded due to apoptosis, and epithelial cells subsequently proliferated. We cultured cells obtained from the duct-ligated salivary gland and purified the cells by limited dilution. The isolated cells were positive for CD29, CD49f, intracellular laminin, AFP, CK19, CK18, and Thy-1(CD90), and weakly positive for c-Kit (CD117). After three-dimensional formation, the cells expressed insulin and albumin. We designated the cells as swine salivary gland-derived progenitor cells. Gene expression of insulin and albumin was significantly increased (five-fold) and that of insulin was also increased (3.8-fold) with differentiation medium with nicotinamide and/or GLP-1 treatment in spherical culture. The expressions of albumin and insulin were 1/10-fold and 1/4-fold compared to porcine hepatocytes and pancreatic endocrine cells. The differentiated SGP cells could release insulin, which were stimulated by glucose and potassium. These results indicate that swine SGP cells could differentiate into hepatocytes and beta-cells, functionally. Swine SGP cells were useful tools for therapy and analyzing endodermal regenerative models in large animals.

Sensory perception of unexpected sudden changes in floor level during human gait.

Possible clues for sensory perception of an unexpected sudden change in floor level during human gait could be discrepancies in the time of heel contact, location of force application point (center of pressure, CoP) in the sole at the heel contact and subsequent stance period from the ones prescribed prior to the gait execution prepared for the normal level gait, if they exist. In order to get insight for this perception mechanism, a psychophysical experiment during gait on a walk way with a small, unexpected and sudden change in the floor level was performed, in which the subject was forced to answer among step-down, flat, and step-up immediately after the walk. During the gait, foot pressure distribution (FPD), electromyogram (EMG) of the ankle muscles, and gait trajectory (GT) were measured. The experiment could quantify accuracy of the perception. The floor level change dependent changes in FPD, CoP trajectory, EMG, GT were examined to suggest that these changes might be the clues for the perception.