Study on teratogenic effect of ultrasonics. I. Effect on pregnant mice]

An experimental study in pregnant mice shows no statistically significative teratogenic influence of ultrasounds.     

Flow cytometric analysis of isolated rat liver nuclei during growth

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.     

Genotoxicity testing of chloramphenicol in rodent and human cells

The results of this work, carried out to extend the limited information at present available on the genotoxic potential of chloramphenicol (CAP), indicate that in millimolar concentrations this antibacterial agent produced a minimal amount of DNA fragmentation in both V79 cells and metabolically competent rat hepatocytes. Moreover, a level of DNA-repair synthesis indicative of a weak but positive response was detected in primary cultures of liver cells obtained from 2 of 3 human donors, and a borderline degree of repair was present in those prepared from rats. The promutagenic character of CAP-induced DNA lesions was confirmed by a low but significant increase in the frequency of 6-thioguanine-resistant clones of V79 cells, which, however, was absent when the exposure was done in the presence of co-cultured rat hepatocytes. Finally, oral administration to rats of 1/2 LD50 CAP did not increase the incidence of either micronucleated polychromatic erythrocytes or micronucleated hepatocytes. Taken as a whole these findings suggest that CAP should be considered a compound intrinsically capable of producing a very weak genotoxic effect, but only at concentrations about 25 times higher than those occurring in patients treated with maximal therapeutic dosages.     

On the association of DNA polymerase alpha activity with the nuclear matrix in HeLa cells

The association of DNA polymerase alpha activity with the nuclear matrix has been reinvestigated in HeLa cells. Isolated nuclei were extracted with 2M NaCl and then digested with Dnase I and the final structures were recovered by centrifugation through a sucrose cushion. Typically over 98% of the total DNA synthesized in the matrix fraction on either endogenous matrix-associated DNA or activated calf thymus DNA was due to DNA polymerase alpha as defined by inhibition to n-ethylmaleimide or aphidicolin. DNA polymerase beta activity was absent or recovered in only trace amounts. Matrix-bound DNA polymerase alpha activity demonstrated a remarkable degree of stability: DNA synthesis was essentially linear up to 3 hours at 37 degrees C. Overall, these results substantiate previous findings from regenerating rat liver, unlike other data obtained from tissue culture cells.     

Critical nuclear DNA size and distribution associated with S phase initiation

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G₁ and middle S phases: Our results show two distinct [³H]-thymidine labeling patterns. The first “peripheral” labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern—homogeneous or inhomogeneous grain distribution throughout the nucleus—are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells—synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography—confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.     

DNA fragmentation by N-nitrosodimethylamine and methyl methanesulfonate in human hepatocyte primary cultures

The ability of N-nitrosodimethylamine (DMN) and methyl methanesulfonate (MMS) to induce DNA damage in primary cultures of human hepatocytes was examined by the alkaline elution technique. Both the agents induced a dose-dependent increase in DNA elution rate, but appreciable differences in the degree of response to the procarcinogen DMN were observed among cultures obtained from the livers of four patients. A comparative analysis of DNA fragmentation indicated a substantial similarity between human and concurrently studied rat hepatocytes in their response to both DMN and MMS.     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Analysis by Confocal Microscopy of the Behavior of Heat Shock Protein 70 within the Nucleus and of a Nuclear Matrix Polypeptide during Prolonged Heat Shock Response in HeLa Cells

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M_r = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42°C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.     

Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor

Pinocytosis was measured in monkey aortic smooth muscle cells (SMC), bovine aortic endothelial cells, and Swiss 3T3 cells in culture as cellular uptake of [U-(14)C]sucrose and horseradish peroxidase (HRP) from the tissue culture medium. Monkey arterial SMC and Swiss 3T3 cells were maintained in a quiescent state of growth at low cells density in medium containing 5 percent monkey plasma-derived serum (PDS). Replacement of PDS with 5 percent monkey whole blood serum (WBS) from the same donor, or addition to PDS of partially purified platelet-derived growth factor(s) (PF), resulted in a marked stimulation of pinocytosis as well as of cellular proliferation. In SMC, enhancement of the rate of pinocytosis occurred 4-6 h after exposure to WBS or PF, and the rate was up to twofold higher than the rate in medium containing PDS. In contrast, [(3)H]thymidine uptake by SMC did not increase until 12-16 h after exposure to PF. In endothelial cells the presence of PF or WBS did not enhance either the rate of pinocytosis or the rate of proliferation over that in PDS. Thus, endothelial cells did not become quiescent at subconfluent densities in PDS but maintained rates of proliferation and pinocytosis that were equivalent to those in WBS. By autoradiography, the fraction of labeled nuclei in SMC cultures 24 h after change of medium increased from 0.061 +/- 0.004 in quiescent cultures to 0.313 +/- 0.028 after exposure to WBS or PF. In contrast, labeling indices of endothelial cells were similar for cultures grown in PDS, WBS, or PF at any single time point after change of medium. These findings suggest that the rate of pinocytosis maybe be coupled in some fashion to growth regulation, which may be mediated in part by specific growth factors, such as that derived from the thrombocyte.