Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Primary cultures of fetal hepatocytes from the genetically obese Zucker rat: Protein synthesis

Summary Primary fetal hepatocytes derived from Zucker rats with expectedfa gene frequencies of 0.0 and 0.75 have been established and can be used to detect early effects of thefa gene on hepatocellular metabolism. Paired incubation experiments demonstrate that protein synthesis in 0.75fa gene cultures is significantly less than in 0.0fa gene cultures under basal conditions. Insulin stimulates protein synthesis in 0.0fa gene cultures but has no effect on 0.75fa gene cultures. Cycloheximide inhibits protein synthesis in both types of culture. NH₄Cl inhibits protein synthesis in 0.0 but not in 0.75fa gene cultures. These data suggest that fetal hepatocytes bearing thefa gene have in vitro a generally sluggish anabolic capacity and a blunted capacity to respond to insulin compared to fetal hepatocytes without thefa gene. These diminished capacities may be expressions of a genetic error in lysosomal function. A portion of this work was presented in preliminary form at the 1980 meeting of the Tissue Culture Association. This work was supported in part by National Institutes of Health Grants AM19382 and AM06197.     

Immunologic unresponsiveness of mouse spleen sensitized to allogenic tumors.

CS7BL/6 mice were sensitized with an ip injection of allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the tumor allosensitized mice were cultured and tested for their responsiveness to mitogens and alloantigens, and for their ability to generate cytotoxic cells in vitro. The results indicate that 15 day tumor-sensitized spleen cells are hypo-responsive in mixed lymphocyte culture (MLC) with DBA/2 or AKR as stimulating spleen cells. The cells which are hypo-responsive in MLC can proliferate in response to mitogens and they also can generate cytotoxic cells in vitro. MLC reactivity recovers in about 2–3 months which is $\text{1}\text{1}\text{2}\text{–2}\text{1}\text{2}$ months after the mice have rejected their tumors. The mechanism of MLC hypo-responsiveness was investigated. The results suggest the presence of a suppressor cell which does not appear to be a macrophage or a B-cell. The suppressor cell can be separated from the cytotoxic cell and therefore appears to be a noncytotoxic T-cell.     

Molecular cloning of bovine leukemia virus DNA integrated into the bovine tumor cell genome

The bovine leukemia virus (BLV) DNA harbored in the bovine tumor cell genome was cloned in lambda Charon 4A phage. Using either representative or 3' half-enriched BLV cDNA as a blot hybridization probe, clone lambda BLV-1 was shown to carry 9 kb of the BLV genome, flanked by cellular sequences at both ends. Restriction mapping with twelve endonucleases and hybridization of the DNA fragments to BLV cDNA representing a 3'-end portion of the viral genome revealed the presence and precise location of two long terminal repeats (LTRs) and virus-cell junctions. Thus, lambda BLV-1 appears to contain the complete BLV genome and flanking tumor cellular sequences. The restriction map of the cloned BLV proviral DNA closely resembles that previously reported for unintegrated linear proviral DNA, but differs significantly from that of the integrated provirus of another BLV isolate, the difference occurring preferentially in the putative gag and pol genes.     

Potentiation by calcium ions of the antithrombin III inhibition of thrombin

Calcium ions potentiated heparin-modulated antithrombin III inhibition of amidolysis catalysed by thrombin. Potentiation by calcium ions of heparin-independent antithrombin III inhibition of thrombin activity appeared to contribute to this effect. These results suggest a complex modulatory role for calcium ions in proteinase-catalysed reactions influenced by anti-proteinases and glycosaminoglycans.     

Amino group modification inhibits ristocetin cofactor activity of human von Willebrand factor

Purified von Willebrand factor rapidly loses activity when treated under mild conditions with the highly specific amino group reagent trinitrobenzenesulfonic acid. Greater than 90 percent inhibition of activity is achieved by modification of only 7 percent of the amino groups. Other modifications such as acetylation and succinylation also abolish activity. It is unlikely that the essential rapidly reacting amino groups function simply in an electrostatic manner since modifications such as amidination and methylation which produce derivatives which retain positive charge are also inactive or nearly so.     

Fertilizing competency of multiple ovulated eggs in the domestic fowl (Gallus domesticus)

Fertilizing competency of multiple ovulated eggs in the domestic fowl was examined by fertilization in vitro and early development in culture. Normal laying hens (White Leghorn) were treated with 75 IU of PMSG for 7 days followed by injection of anterior pituitary extracts from chickens (CAPE). Ovulation began to occur 7.5 h after injection of CAPE. These hens ovulated 1-7 ova but some premature ovulation of GV stage ova were observed. In vitro fertilization of the multiple ovulated ova was examined by inseminating 10(6)-10(7) sperm onto the germinal disks in m-Ringer's solution. The gamete or zygote nuclei were detected by DNA specific fluorescence using DAPI (4',6'-diamidino-2-phenylindole) in the histological section prepared from the germinal disk. Process of fertilization was examined in the eggs incubated for 4 h after insemination in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Fertilization rate of the total multiple ovulated eggs was 55% (11/20), in which 90% (9/10) and 10% (1/10) in the eggs recovered 7.5-8.5 h and 9.0-9.5 h after CAPE injection were obtained, respectively. Normal pronuclei were formed in five eggs of those recovered 7.5-8.5 h after CAPE injection. Early development after fertilization in vitro was also examined by incubation for 12 h in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Although development in vitro was delayed compared to that in utero condition, normal development was observed in naturally and multiple ovulated eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture

The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.     

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes

Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system for the study of human cardiac muscle cell biology.