Effect of rapid addition and dilution of dimethyl sulfoxide on the viability of frozen-thawed rabbit morulae

The aim of the present study was to examine effects of altering thawing conditions and procedure of addition and dilution of Me2SO on the viability of frozen-thawed rabbit morulae. Five hundred and sixty two rabbit morulae were cooled from room temperature to -80 degrees C at 1 degree C/min in the presence of 1.5 M dimethyl sulfoxide (Me2SO) using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, cooled rapidly, and stored in liquid nitrogen. When Me2SO was added in a single step, the frozen embryos were thawed in ambient air at 40 degrees C/min and Me2SO was diluted in a single step, 99 of 107 (93%) embryos cultured for 48 hr and 12 of 32 (38%) embryos transferred to 6 recipients developed to expanding blastocysts and viable fetuses, respectively. When Me2SO was added in a single step and the frozen embryos were thawed at the same rate and transferred directly without removal of Me2SO to culture media or oviducts of 8 recipients, 67 of 75 (89%) embryos cultured and 12 of 40 (30%) embryos transferred developed to expanding blastocysts and viable fetuses, respectively. There were no significant differences between these survival rates and survival rates obtained by conventional method, i.e., frozen embryos were thawed at 4 degrees C/min by interrupted slow method and Me2SO was added and diluted in a stepwise manner.     

Studies on the production of indole-3-acetic acid with auxin-heterotrophic mutants derived from cultured crown gall cells

Mutants in the indole-3-acetic acid metabolism derived fromcultured crown gall cells were tested to see whether they couldutilize any one of eight indolic compounds in place of indole-3-aceticacid. Two auxin-heterotrophic mutant cell lines could not utilizeindolepyruvic acid, but growth recovered when there was a supplementof indole-3-acetic acid. Indoleacetonitril and indoleacetaldoximeinhibited the growth of mutant cell lines and their parentalcrown gall cells. Cultured crown gall cells may have synthesizedindole-3-acetic acid from tryptophan via indolepyruvic acidand indole-acetaldehyde, and also may be able to produce indole-3-aceticacid from tryptophan via tryptamine (Received May 6, 1980; )     

The Activin A-Dependent Proliferation of PCC3/A/1 Embryonal Carcinoma Cells in Serum-Free Medium

Examination of the growth requirements of murine embryonal carcinoma cells (EC cells) or embryonic stem cells (ES cells) in serum-free medium revealed that PCC3 EC cells required activin A to grow and/or survive in such medium. In the absence of activin A, PCC3 cells began to disintegrate within 3 days under any serum-free conditions examined. P19 and AT805 EC cells grew even in serum-free medium without activin A but their growth rates were slightly facilitated by its addition. F9 EC cells also grew in the medium without activin A and its addition somewhat inhibited their growth rate. Three independently isolated ES cell lines and feeder-dependent PSA-1 EC cells also grew in serum-free medium without activin A if leukemia inhibitory factor (LIF) was supplemented. The addition of activin A had little effect on their growth rates. These findings suggest that PCC3 EC cells are a sort of nutritional mutant requiring activin A, thus making them useful in stidies on the growth regulatory mechanisms of EC/ES cells and/or the action of activin on EC/ES cells.     

Activation of the systemic production of tumor necrosis factor after exposure to acute stress.

Tumor necrosis factor (TNF), which was originally identified as a tumoricidal factor, is now regarded as one of the main regulators of inflammation and various immune systems. Thus it has been considered to be mobilized in case of emergency. However, we assume that TNF and the cytokine network driven by the monokine also function under normal condition for homeostasis of the animal body which is exposed to various kinds of physiological stress. To test this possibility, we exposed C3H/He mice for up to 3 days to five types of acute stress: food deprivation, drinking water deprivation, sleep deprivation, swimming, and physical restraint. After release from the stress, the level of priming for systemic production of TNF was examined using OK-432 (a streptococcal preparation) as a trigger. Priming of TNF production was not observed immediately after 2-day exposure to most of the stressors. Sleep deprivation alone tended to induce a primed state especially when the stress period was lengthened to 3 days. On the other hand, by keeping mice in a normal condition for a 2-day restorative interval after 2-day exposure to the stressors, systemic production of TNF was consistently primed for all the stress examined. The time course of the priming effect was examined in detail for water-deprivation stress. The effect was detected as early as 3 hours after release from stress, was sustained for 2 days, and returned to the basal level by 4 days after the release. Based on these results, we discussed the role of the TNF-driven cytokine circuit in adaptation to stress.     

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging.The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6°C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to sialic acid-associated cell surface charges. The pattern of change in relation to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maximum occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.     

Effect of particle size and temperature on the conformation and physiological behavior of apolipoprotein E bound to model lipoprotein particles

The effect of particle size and structural order/disorder of the lipid domain on the conformation and physiological behavior of lipid-associated apolipoprotein E (apoE) was evaluated. Circular dichroic (CD) spectra of apoE bound to large (LME) and small (SME) microemulsion particles, composed of dimyristoylphosphatidylcholine (DMPC) and cholesteryl oleate (CO), and to DMPC disks revealed that at 4 degrees C, where all of the lipid constituents were in an ordered state, apoE bound to LME displayed approximately 60% alpha-helicity, while apoE bound to SME and DMPC disks displayed 73% and 95% helicity, respectively. Over the temperature range 4-50 degrees C, encompassing the lipid thermal transitions, only apoE bound to LME demonstrated an abrupt change in its CD spectrum (decrease in alpha-helicity) in response to temperature. To determine the source of the abrupt CD change, the constants for dissociation (Kd) of apoE from the surface of the large and small microemulsion particles were determined at 4, 25, and 37 degrees C. These results demonstrated that at 4 degrees C, the KdS for binding of apoE to the LME and SME were approximately equal; however, between 4 and 25 degrees C, there was a 5-fold increase in the Kd for binding of apoE to the LME, whereas the Kd for binding to the SME remained constant. The physiological effects of these differences in apoE secondary structure and equilibrium binding were examined by measuring the capacity of each apoE-containing particle to complete with LDL for binding to human fibroblasts, and by measuring the capacity of the apoE-microemulsion particles to suppress HMG-CoA reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of high activity of ornithine decarboxylase and occurrence of unusual chromophobic cells in anterior pituitary gland of a novel growth-retarded strain of mice, grm/grm

Extremely high activity of ornithine decarboxylase (ODC) was detected in the pituitary gland of growth- retarded mice, grm/grm at 2 months after birth. The elevated enzyme activity gradually decreased to the control level in 14 months after birth. In the pituitary gland of the growth-retarded mice, unusual chromophobic cells were also present from the early stages after birth. The chromophobic cells showed conspicuous proliferations and resulted in a distinct hyperplasia of the tissue after 4 months after birth. These findings suggest that OCD is correlated to the progressive transformation of pituitary cells into the chromophobic cells.