The in vivo effects of culture medium. I. Radioprotective effects of vitamins, amino acids and inorganic salts of culture medium in mice.

It has been shown that pretreatment of mice with a five-fold condensed culture medium before irradiation increased the number of multipotential haemopoietic stem cells in the spleen. The degree of cyto- and radioprotection is dependent on both the time of administration and the dose of the culture medium. The administration of culture medium in twofold doses in a volume of 1 ml intraperitoneally 18 h and 8 h before irradiation with a dose of 9 Gy protected 95% of C57Bl/6 mice. On the other hand, no therapeutic effect of different doses of the culture medium was found. Our observation suggests that the culture medium can mediate a radioprotective effect. The possible mechanisms participating in this effect are discussed.     

The effect of low dose rate on recovery of hemopoietic and stromal progenitor cells in gamma-irradiated mouse bone marrow

Long-term recovery of mouse hemopoietic stem cells (CFU-S and CFU-S per colony), granulocyte-macrophage precursor cells (GM-CFC), and stromal colony-forming units (CFU-F) after doses up to 12.5 Gy was almost complete by 1 year when the dose rate was reduced to 0.0005 Gy/min compared to incomplete recovery after doses up to only 6.5 Gy given at greater than 0.7 Gy/min. This sparing effect of dose rate on long-term hemopoietic recovery is in contrast to the generally reported lack of dependence on dose rate for acute survival of hemopoietic progenitors after doses up to 5 Gy. The present results are compatible with the hypothesis that good recovery of the stroma should be reflected in the long-term recovery of hemopoiesis.     

Influence of serum supplements in culture medium on gonadotropin-releasing hormone effects on colony formation

A number of studies have demonstrated that GnRH has anti-proliferative effects on various carcinomas of breast, ovary, endometrium, prostate, pancreas, and liver origin. In contrast, GnRH increases the proliferative activity of lymphoid tissues and cells, which suggests that GnRH is also an important immunomodulator. In a previous study, we demonstrated that the colony-forming efficiencies of HHUA (derived from human endometrial carcinoma) and Jurkat (derived from human mature leukemia) cells are affected by the GnRH agonist Buserelin, and that the conditioned media of HHUA and Jurkat cells severely affect the Buserelin activity. The latter finding suggests that substances in the culture medium have some relation to the GnRH activity. Therefore, in the present study, to evaluate the effect of serum supplements on the colony-forming efficiency assay, the assay was performed using 3 lots of fetal bovine serum (FBS) and 2 lots of Nu-Serum I, a semi-synthetic serum supplement. The results showed that the colony-forming efficiencies of HHUA and Jurkat cells fluctuated greatly depending on the lot of FBS. In contrast, Buserelin significantly affected the colony-forming efficiency to similar extents in the media containing both the lots of Nu-Serum I. These results strongly suggest that the constituents of the serum supplements also influence the effect of GnRH on cell proliferation. For further studies about the relationship between substances in the culture medium and the GnRH effects on cell proliferation, it will be necessary to use a completely defined medium, and that a semi-synthetic serum supplement such as Nu-Serum I will also be useful.     

The effects of dexamethasone on the growth of gamma-irradiated tadpoles.

A plausible hypothesis as to the mode of action of dexamethasone (DXM) has been proposed. Growth of irradiated tadpoles of Bufo melanostictus, treated daily with the corticoid, is more suppressed. Compared with its antagonism with ACTH, its coordinate promotion of TSH production accelerates metamorphosis. This modulated feedback is consistent with the constant course of the hypothalamic nuclei influence upon the adenohypophyseal hormones.     

The adaptation of BHK cells to a non-ammoniagenic glutamate-based culture medium.

Although glutamine is a major carbon source for mammalian cells in culture, its chemical decomposition or cellular metabolism leads to an undesirable excess of ammonia. This limits the shelf-life of glutamine-supplemented media and may reduce the cell yield under certain conditions. We have attempted to develop a less ammoniagenic medium for the growth of BHK-21 cells by a mole-to-mole substitution of glutamine by glutamate. This results in a medium that is thermally stable but unable to support an equivalent growth yield. However, supplementation of the glutamate-based medium with asparagine (3 mM) and a minimal level of glutamine (0.5 mM) restored the original growth capacity of the cultures. Substitution of the low level of glutamine with the glutamine dipeptides, ala-gln (1 mM), or gly-gln (3 mM) resulted in an equivalent cell yield and in a thermally stable medium. The ammonia accumulation in cultures with glutamate-based medium was reduced significantly (>60%). Factors mediating growth and adaptation in medium substituted with glutamate were also investigated. The maximum growth capacity of the BHK-21 cells in glutamate-based medium (without glutamine) was achieved after a period of adaptation of 5 culture passages from growth in glutamine-based cultures. Adaptation was not influenced by increases in glutamate uptake which was constitutively high in BHK cells. Adaptation was associated with changes in the activities of enzymes involved in glutamate or glutamine metabolism. The activities of glutamine synthetase (GS) and alanine aminotransferase (ALT) increased significantly and the activity of phosphate-activated glutaminase (PAG) decreased significantly. The activity of glutamate dehydrogenase (GDH) showed no significant change after adaptation to glutamate. These changes resulted in an altered metabolic profile which included a reduced ammonia production but an increased alanine production. Alanine production is suspected of being an alternative route for removal of excess nitrogen.     

The enhancement of haemopoietic stem cell recovery in irradiated mice by prior treatment with cyclophosphamide.

Studies are reported of the enhancement of stem cell recovery following whole body irradiation as a result of prior administration of cyclophosphamide. It is shown that the much larger enhancement of regeneration observed for the hosts own surviving stem cells, compared to the regeneration of injected bone marrow stem cells, is due to the different numbers of stem cells initiating the regeneration in conjunction with the time course of stem cell regeneration. The results show that the environmental changes produced by cyclophosphmide greatly enhance haemopoietic recovery even though at the dose used this agent is relatively toxic to stem cells. Furthermore it has been shown that the level of stem cell regeneration is nearly independent of the gamma-ray dose in the range 3-8 gray (300-800 rad). If human bone marrow should respond similarly it follows that regeneration produced by cytotoxic drugs administered prior to radiation embodies a considerable safety factor as far as recovery of the haemopoietic system is concerned.     

Effect of conditioned medium on recovery of circulating blood cells in irradiated mice.

The effect of supernatant of thymus-cell conditioned medium (TCCM) on blood element recovery in peripheral blood was followed in mice exposed to a single whole body dose of 5.8 Gy of gamma radiation. As follows from our results. TCCM administered 18 h before irradiation accelerated the recovery of the reticulocyte and, in part, thrombocyte and granulocyte counts. However, no effect on the rate of lymphocyte recovery was found.     

Cytotoxic effects of antimony trichloride on mice in vivo.

Clastogenic effects of antimony trichloride, used in small industries, were monitored in laboratory bred white Swiss mice in vivo, following oral administration by gavaging, after 6, 12, 18 and 24 h. Chromosomal aberrations were principally breaks and damaged cells observed from bone marrow preparations. The frequencies of chromosomal aberrations were directly related to the dose used and were significantly higher than the control. The chemical did not alter the frequency of dividing cells to any significant level.     

Radiation-induced human chromosome aberrations. II. Human in vitro irradiation compared to in vitro and in vivo irradiation of marmoset leukocytes

Whole blood cultures from humans and from the New World primate, Saguinus fuscicollis, were irradiated with various doses of 250 kV X-rays. The resulting centric ring plus dicentric aberration yields were fitted to the three models, Y = a+bD, Y = a+bD+cD², and Y = a+cD², by least squares regression. In both instances the best fit was to the model Y = a+bD+cD², with coefficients of the one- and two-track components for human and marmoset being: b = (0.78 ± 0.09)·10⁻³, c = (5.92 ± 0.31)·10⁻⁶, and b = (1.11 ± 0.36)·⁻³, c = (7.7 ± 1.7)·10⁻⁶, respectively.     

Nutritional requirements of Plasmodium falciparum in culture. II. Effects of antimetabolites in a semi-defined medium

A semi-defined minimal medium, in which pantothenic acid is the only vitamin, was used to culture Plasmodium falciparum for the analysis of antimetabolite drugs. Analogs of riboflavin, nicotinamide, pyridoxine, and thiamin inhibited the growth of this parasite; for each drug, effects were much more pronounced after 96 h of exposure compared to 48 h. The most potent drug examined was 8-methylamino-8-desmethyl riboflavin (IC50 value approximately 1.0 X 10(-10) M at 96 h). Avidin, a protein which complexes and thus inactivates biotin, did not affect parasite viability. Other antimalarial drugs, including chloroquine and quinine derivatives and antibiotics, were equipotent in the minimal medium and in RPMI 1640. Four strains of P. falciparum showed only minor differences in sensitivity to these antimetabolites. The use of prolonged drug exposure times and a vitamin-depleted medium allowed the preliminary characterization of antimalarial antimetabolites in vitro.     

Influence of prior exposure to low-dose adapting radiation on radiation-induced teratogenic effects in fetal mice with varying Trp53 function

Teratogenesis in tails and limb digits of fetal mice with varying Trp53 status was examined after exposure of pregnant females to 4 Gy gamma radiation with and without a prior 30-cGy exposure. Prior low-dose exposure modified the teratogenic effects of radiation in a manner dependent upon Trp53 status and gestation time. A 4-Gy exposure on gestation day 11 resulted in tail shortening and digit abnormalities. A 30-cGy exposure 24 h prior to a 4-Gy radiation exposure on day 11 reduced the extent of both digit abnormalities and the tail-shortening effects in Trp53(+/+) fetuses and also reduced tail shortening in Trp53(+/-) fetuses, but to a lesser extent. However, the pre-exposure enhanced the tail-shortening effects of 4 Gy in Trp53(-/-) fetuses. In contrast, a 30-cGy exposure given 24 h prior to a 4-Gy exposure on gestation day 12 had no effect on the reduced tail length resulting from the 4-Gy exposure of Trp53(+/+) or Trp53(+/-) fetuses, but it partly protected Trp53(-/-) fetuses against reduced tail length. A 4-Gy exposure alone on day 12 did not result in any increase in the frequency of digit abnormalities in Trp53(-/-) fetuses so any protective effect of the preirradiation could not be detected. However, the preirradiation did result in protection against in digit abnormalities in Trp53(+/-) fetuses. We conclude that radiation-induced teratogenesis reflects both Trp53-dependent and independent processes that lead to apoptosis, and these respond differently to prior adapting doses.     

Photoautotrophic growth of soybean cells in suspension culture. II. Optimization of culture medium and conditions

We have attempted to optimize the conditions under which a photoautotrophic soybean suspension culture line (SB-P; Horn et al. 1983) is grown. Magnesium, phosphate, and calcium concentrations were varied individually from one-tenth to five times the normal level found in the Murashige and Skoog (1962) recipe. After two subcultures, only phosphate at one-tenth the normal level caused the cells to show a substantial reduction in fresh and dry weight increase and chlorophyll level. Nitrate and ammonium levels were inversely varied in 20 millimolar increments of potassium nitrate and ammonium chloride. Neither N-source alone could support growth through two subcultures. A ratio of 40 millimolar potassium nitrate to 20 millimolar ammonium gave significantly better fresh and dry weight increases than did a ratio of 20:40 or 30:30 but the chlorophyll level was unchanged. The minor salts as a group resulted in a small improvement in growth when provided at twice the normal level.Indole-3-acetic acid at five milligrams per liter resulted in significantly better fresh and dry weight increases than did -naphthaleneacetic acid at any level but the final chlorophyll level was not changed. There was no correlation between growth and kinetin level and this resulted in the discovery that SB-P cells are cytokinin-autotrophic, as are heterotrophic SB cells, with regard to both growth and greening ability. Growing SB-P cells under a 14 h:10 h day:night photoperiod resulted in a slow but inevitable death. Increasing the carbon dioxide level to 10% for four weeks gave no increase in SB-P cell growth or chlorophyll level, but SB-P cells would not grow with carbon dioxide levels below 0.4%. The results clearly show that SB-P cells, despite their tenuous existence, are capable of adapting to a wide range of culture conditions. A simplified and improved culture medium for photoautotrophic cultures is given.Abbreviations SB-P photoautotrophic soybean cells - SB-M photomixotrophic soybean cells - SB-H heterotrophic soybean cells     

Antimalarial effects in mice of orally administered peptidyl cysteine protease inhibitors.

The Plasmodium falciparum cysteine protease falcipain is required for the degradation of hemoglobin by erythrocytic malaria parasites. In prior studies, peptidyl inhibitors of falcipain blocked hemoglobin degradation and development by cultured parasites and one of these compounds, when administered parenterally, cured Plasmodium vinckei-infected mice. We now report an evaluation of orally administered peptidyl inhibitors of falcipain in a mouse malaria model. In studies with a fluoromethyl ketone, orally administered morpholine urea-phenylalanine-homophenylalanine-fluoromethyl ketone delayed the progression of murine malaria. In studies of a new series of vinyl sulfones, a set of related compounds demonstrated marked inhibition of falcipain and of parasite biological activities in vitro. One of these compounds, N-methyl piperazine urea-leucine-homophenylalanine-2-naphthalene vinyl sulfone, cured about 40% of mice when administered orally twice-a-day for four days. Our results suggest that peptidyl inhibitors of falcipain have promise as antimalarial chemotherapeutic agents.     

Cryopreservation of mouse pancreatic islets: effects of different glucose concentrations in the post-thaw culture medium on islet recovery

It was the aim of this study to investigate the influence of the glucose concentration of the post-thaw culture medium on islet B-cell survival after cryopreservation by the combined assessments of islet recovery, islet DNA and insulin contents, and insulin release. Collagenase isolated mouse islets were kept in culture for 3 days in the presence of 11.1 mM glucose and then transferred to freezing ampoules containing Hanks' solution supplemented with 10% calf serum and 2 M dimethyl sulfoxide. After a 20-min incubation at 0 degrees C the islets were cooled at a rate of 25 degrees C/min to -70 degrees C and subsequently plunged into liquid nitrogen. After 2 hr the frozen islets were rapidly thawed at 37 degrees C, transferred to culture dishes, and cultured for another 3 days in the presence of 2.8, 5.6, 11.1, 16.7, or 28 mM glucose. Nonfrozen control islets were treated identically after a preceding 3-day culture at 11.1 mM glucose. The percentage recovery of cryopreserved islets was decreased compared to that of nonfrozen islets, but was increased when higher glucose concentrations were used in the post-thaw culture medium. Since the DNA content of the cryopreserved islets was slightly decreased, the overall survival rate of the cryopreserved B-cells, when cultured at the higher glucose concentrations after thawing, was found to be about 75%. The insulin content of the cryopreserved islets was decreased but the glucose-stimulated insulin release was essentially the same as that of the nonfrozen islets.(ABSTRACT TRUNCATED AT 250 WORDS)