Thermal tolerance during S phase for cell killing and chromosomal aberrations

Synchronous Chinese hamster ovary cells in early S phase were obtained by selecting mitotic cells, accumulating them at the G1/S border by incubating them in aphidicolin for 12 h, and then incubating them for 2 h after releasing them from the aphidicolin block. To determine if thermotolerance could be induced, the cells were heated at 43 degrees C for 20 min in early S phase, incubated for 160 min, and then heated a second time at 43 degrees C for different durations (30-100 min). For the control, nontolerant population, the cells in early S phase were incubated for 50 min and then heated once at 43 degrees C for different durations (20-60 min). Flow cytometric analysis indicated that the population receiving the second heat dose was in the same part of S phase as the population receiving the single heat dose. A comparison of the heat response for the two populations indicated that heating during early S phase induced thermotolerance for both cell killing and chromosomal aberrations; i.e., for 10% survival, which corresponded to 10% of the cells being cytologically normal, the thermal dose was twofold greater in the thermotolerant cells than in the control, nontolerant cells. Furthermore, this thermotolerance developed during S phase. These observations support the hypothesis that heating during S phase kills cells primarily by inducing chromosomal aberrations.     

Cholinergic agents: antinociception without morphine type dependence in rats

We have confirmed the work of others showing that loss in body weight is a predictable and consistent sign of opiate withdrawal in rats. Rats that were treated chronically with either oxotremorine or physostigmine displayed no weight loss or other signs of opiate-like withdrawal when the drugs were withdrawn. Furthermore, there was no difference in weight loss between morphine dependent rats substituted with saline and those substituted with either cholinergic drug. However, we did observe an increased mortality among rats substituted with a cholinergic agent compared with saline. Rats infused with a mixture of morphine plus oxotremorine or morphine plus physostigmine showed less weight loss, but not fewer behavioral signs, after the end of the infusion than rats treated only with morphine. It is concluded that the cholinergic agents did not cause a morphine-like physical dependence themselves, but appeared to antagonize to some extent the development or manifestation of opiate dependence.     

Comparative study of the effects of hyperthermia and BCNU on BCNU-sensitive and BCNU-resistant 9L rat brain tumor cells

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU)-sensitive and BCNU-resistant 9L rat brain tumor cells were treated with BCNU at graded temperatures between 37 and 44 degrees C. The cytotoxic effects of hyperthermia alone on both cell lines were the same. Treating both cell lines with BCNU at temperatures above 37 degrees C caused a progressive increase in cell kill. All survival curves for drug-sensitive cells had shoulders followed by a region of exponential cell kill; dose enhancement ratios calculated at the 10% survival level ranged from 1.7 to 3.0. Survival curves for drug-resistant cells were exponential without a shoulder; dose enhancement ratios ranged from 3.3 to 8.4. For each cell line, a similar amount of the increased cell kill could be explained on the basis of the increases concentration of reactive species produced by hydrolysis of BCNU at elevated temperatures. The amount of cell kill that cannot be explained on this basis, however, suggests that factors other than an increase in the concentration of reactive species at higher temperatures are involved in the enhanced cell killing. Possible mechanisms include a heat-induced change in the structure of DNA chromatin and the effect of isocyanate deactivation of repair enzymes, both of which could lead to an increase in the number of crosslinks formed and therefore to an increase in cytotoxicity.     

Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects.

Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.     

Genetic control of whisker antigen of bacteriophage T4D

An antigenic component of T4 whiskers (short fibrils located in the region of the head—tail junction) has been reported to be under the control of gene 49 (Yanagida & Ahmad-Zadeh, 1970; Yanagida, 1972). This was based on immunological evidence using antiserum to particles of T4D adsorbed with gene 49-defective extract made with the mutant amE727. The latter phage, however, is shown here to be a double mutant bearing amber mutations in gene 49 and another gene, herein referred to as wac (whisker antigen control gene). Gene wac maps in the general region of gene 16. Evidence is presented indicating that the whisker antigen is under the control of wac and not gene 49. In wac-defective infections phage are produced that lack a protein. This protein appears by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gels to be the major component of the antigen.The tail fibers of wac-defective bacteriophage are in an open configuration under conditions in which those of wild-type phage are folded alongside the tail. Thus, the wac gene may have a role in the regulation of tail-fiber configuration.     

The Structure of Mytilus Smooth Muscle and the Electrical Constants of the Resting Muscle

The individual muscle fibers of the anterior byssus retractor muscle (ABRM) of Mytilus edulis L. are uninucleate, 1.2–1.8 mm in length, 5 µm in diameter, and organized into bundles 100–200 µm in diameter, surrounded by connective tissue. Some bundles run the length of the whole muscle. Adjacent muscle cell membranes are interconnected by nexuses at frequent intervals. Specialized attachments exist between muscle fibers and connective tissue. Electrical constants of the resting muscle membrane were measured with intracellular recording electrodes and both extracellular and intracellular current-passing electrodes. With an intracellular current-passing electrode, the time constant τ, was 4.3 ± 1.5 ms. With current delivered via an extracellular electrode τ was 68.3 ± 15 ms. The space constant, λ, was 1.8 mm ± 0.4. The membrane input resistance, R_eff, ranged from 23 to 51 MΩ. The observations that values of τ depend on the method of passing current, and that the value of λ is large relative to fiber length and diameter are considered evidence that the individual muscle fibers are electrically interconnected within bundles in a three-dimensional network. Estimations are made of the membrane resistance, R_m, to compare the values to fast and slow striated muscle fibers and mammalian smooth muscles. The implications of this study in reinterpreting previous mechanical and electrical studies are discussed.     

Effects of cold treatment on synchronous Chinese hamster cells treated in mitosis

Chinese hamster mitotic cells (95% in mitosis with 85% in metaphase and 10% in anaphase) were shaken loose from monolayer asynchronous cultures and stored at 1°C for up to 28 hours. During this period, the mitotic index did not decrease and the cells remained cytologically normal. However, over a four-hour period, metaphase cells located within 1.8 minutes of anaphase, 10% of the metaphase population, were able to move into anaphase; this point of 1.8 minutes corresponds in time to that reported for the spindle activation marker. When the cells were warmed to 37°C, they were delayed in entering G₁ and S (35 and 70 minutes, respectively, after a 4-hour treatment). This delay of 70 minutes was maintained for three cell cycles, during which a high degree of synchrony was maintained. Cold treatment for 12 hours produced delays into G₁ and S of 50 and 110 minutes, respectively. A fraction of the metaphase cells (12 or 50% after treatments of 4 or 12 hours, respectively) evidenced chromosomal aggregation, were unable to complete cytokinesis, and appeared in the next division as tetraploid cells. These tetraploid cells were unable to survive and produce macroscopic colonies. It is concluded that this decrease in viability is caused by irreversible effects on the spindle and/or centriolar components in metaphase cells located prior to the spindle activation marker.     

ULTRASTRUCTURE OF THE NUCLEOLUS DURING THE CHINESE HAMSTER CELL CYCLE

Changes in the structure of the nucleolus during the cell cycle of the Chinese hamster cell in vitro were studied. Quantitative electron microscopic techniques were used to establish the size and volume changes in nucleolar structures. In mitosis, nucleolar remnants, "persistent nucleoli," consisting predominantly of ribosome-like granular material, and a granular coating on the chromosomes were observed. Persistent nucleoli were also observed in some daughter nuclei as they were leaving telophase and entering G₁. During very early G₁, a dense, fibrous material characteristic of interphase nucleoli was noted in the nucleoplasm of the cells. As the cells progressed through G₁, a granular component appeared which was intimately associated with the fibrous material. By the middle of G₁, complete, mature nucleoli were present. The nucleolar volume enlarged by a factor of two from the beginning of G₁ to the middle of S primarily due to the accumulation of the granular component. During the G₂ period, there was a dissolution or breakdown of the nucleolus prior to the entry of the cells into mitosis. Correlations between the quantitative aspects of this study and biochemical and cytochemical data available in the literature suggest the following: nucleolar reformation following division results from the activation of the nucleolar organizer regions which transcribe for RNA first appearing in association with protein as a fibrous component (45S RNA) and then later as a granular component (28S and 32S RNA).