ABSTRACT We recently isolated a cardiomyogenic (CMG) cell line from murine bone marrow stroma, and in this paper characterize regenerated cardiomyocytes derived from adult mesenchymal stem cells at the molecular level. Stromal cells were immortalized, exposed to 5-azacytidine, and repeatedly screened for spontaneously beating cells. CMG cells began to beat spontaneously after 2 weeks, and beat synchronously after 3 weeks. They exhibited sinus-node-like or ventricular-cell-like action potentials. Analysis of the isoforms of contractile protein genes, such as of myosin and α-actin, indicated that their phenotype was similar to that of fetal ventricular cardiomyocytes. The cells expressed Nkx2.5, GATA4, TEF-1, and MEF2-C mRNA before 5-azacytidine exposure, and MEF2-A and MEF2-D after exposure. CMG cells expressed α1A, α1B, and α1D-adrenergic receptor mRNA prior to differentiation, and β1, β2-adrenergic and M1, M2-muscarinic receptors after acquiring the cardiomyocyte phenotype. Phenylephrine induced phosphorylation of ERK1/ 2, and the phosphorylation was inhibited by prazosin. Isoproterenol increased the cAMP level 38-fold and beating rate, cell motion, % shortening, and contractile velocity by 48%, 38%, 27%, and 51%, respectively, and the increases were blocked by CGP20712A (β1-selective blocker). Car-bachol increased IP3 32-fold, and the increase was inhibited by AFDX116 (M2-selective blocker). These findings demonstrated that the regenerated cardiomyocytes were capable of responding to adrenergic and muscarinic stimulation. This new cell line provides a model for the study of cardiomyocyte transplantation.
ABSTRACT Application of diagnostic X-ray examination to pregnant women is complicated since risks to both mother and embryo/fetus must be considered. Embryos and fetuses are more sensitive to radiation than adults or children. The threshold doses for fetal death, malformations and mental retardation which are deterministic effects, are reported to be 100–200 mGy or higher. The relative risk for childhood cancer due to radiation at an absorbed dose of 10 mGy during embryonic/fetal development has been estimated at 1.4. However, the absorbed dose of the embryo/fetus during X-ray diagnostic examination in which the X-ray beam does not irradiate the embryo/fetus directly such as maternal skull and chest X-ray is extremely low, less than 0.01 mGy. Thus these diagnostic procedures are not a problem from the perspective of radiological protection of the embryo/fetus. However, for pelvic CT scan and barium enema in which the uterus is directly within the X-ray beam, the absorbed doses to the embryo/fetus are about 20–80 mGy and 10–20 mGy, respectively. Therefore, medical staff must pay careful attention to the embryo/fetus in application of these examinations. Pregnant women who were not aware of pregnancy at the time of their diagnostic exposure have great anxiety about radiation from such X-ray examinations. However, fetal doses below 100 mGy should not be considered a reason for terminating a pregnancy.
ABSTRACT We previously showed that PSK, a biological response modifier, suppressed X-ray irradiation induced ocular anomalies in mouse embryos. In the present study, in mouse embryos irradiated at E7.5, PSK, when administered immediately after irradiation, suppressed mitosis and increased apoptosis as compared with embryos not treated with PSK at 12 hrs after irradiation. In the irradiated embryos, p53, which is normally expressed at a high level in early embryos, increased at 6 hrs and decreased at 12 hrs after irradiation. In the irradiated and PSK-treated embryos, the p53 level did not change at 6 hrs, increased at 12 hrs and decreased at 24 hrs after irradiation. This timing of PSK-induced delayed increase of p53 coincided with that of the PSK-induced decrease in mitosis and increase in apoptosis. These results suggested that PSK modified the p53 level and affected cell proliferation and apoptosis, which might contribute to the suppression of teratogenesis.
ABSTRACT Chromosomes of Slc:ICR mouse blastocysts cultured under the diabetic condition were analyzed to clarify the effect of glucose and ketone body (DL-β-hydroxybutyric acid). In the group exposed to glucose plus ketone body or glucose alone, blastocysts showed higher incidences of chromosome abnormalities, especially numerical abnormalities such as aneuploidy and polyploidy, than in the control group (p < 0.01). The association of nucleolus organizing regions was increased in the blastocysts exposed to glucose plus ketone body, which seems to be related to the increase in numerical abnormalities. Structural abnormalities such as break and fragment were also observed, but there was no significant difference between the diabetic and nondiabetic conditions.
These results from chromosome analysis of the cultured blastocysts suggest that the diabetic condition may directly cause chromosome abnormalities in early embryos, especially aneuploidies, and may thus induce duplications or deletions of genes. These chromosomal damages may disrupt the developmental programs for organogenesis and may be involved in diabetes-induced teratogenesis.
ABSTRACT The genetic polydactyly/ariiinencephaly mouse, Pdn/Pdn, exhibits severe polydactyly both in the fore-and hindlimbs, hydrocephalus, and agenesis of the olfactory bulbs, corpus callosum, and anterior commissure. The mechanism of hydrocephalus manifestation in Pdn/Pdn was investigated in the present study. Ink was injected into the left lateral ventricle in the Pdn/Pdn and +/+ newborn mice. After incubation at 32°C for different time intervals, the heads were fixed in Bouin's solution and were subsequently decalcified in 0.5 mol/L of EDTA solution, paraffin sectioned, and stained with hematoxylin and eosin.
Ink spread into the 3rd and right lateral ventricles and flowed to the 4th ventricle and Magendie's foramen rapidly in Pdn/Pdn mice. This rapid spread was due to the dilatation of the interventricular foramen and that the lateral ventricle was directly connected with enlarged 3rd ventricle in Pdn/Pdn. In spite of the rapid spread of ink in the cerebrospinal fluid pathway, ink was not observed in the subarachnoid space around the superior sagittal sinus at 3.5 or 10 hours in Pdn/Pdn mice.
The superior sagittal sinus was narrower in Pdn/Pdn than in +/+, and the arachnoid villi were not observed in Pdn/Pdn. From these observations, we suggested that absorption of cerebrospinal fluid from the arachnoid villi in the superior sagittal sinus stagnated and that stagnation of the fluid in the ventricles was the cause of hydrocephalus in Pdn/Pdn mice.
ABSTRACT In previous report on prenatal developmental effects of pure 2,4-dichlorophenoxyacetic acid (2,4-D) in rat, we found that this chemical was maternally toxic, embryolethal, and that it induced urogenital malformations in the fetuses. In the present report, we investigated the postnatal survival of the offspring prenatally exposed to 2,4-D during organogenesis, to determine the participation of urogenital malformations on postnatal survival. We used doses of 70 mg, 110 mg and 150 mg, which were each found to induce significant urogenital malformations, when administered in different periods of organogenesis: GD 6 to 15, GD 6 to 10, and GD 11 to 15. We found that 2,4-D has a significant influence on progeny viability by increasing the postnatal death. The kidney and urinary tract malformations induced in the fetuses might be the cause of the increased rate of postnatal death. 2,4-D did not impair the postnatal growth of the unaffected offspring.