Correlation between redistribution of a 26 kDa protein and development of chronic thermotolerance in various mammalian cell lines

Previous studies suggested that a 26 kDa protein might play an important role in protein synthesis-independent thermotolerance development in CHO cells. To determine if this phenomenon was universal, four mammalian cell lines, viz., CHO, HA-1, murine Swiss 3T3, and human HeLa, were studied. Cells were heated at 42 degrees C, and the level of 26 kDa protein in the nucleus was measured, together with clonogenic survival and protein synthesis. The results demonstrated that 1) the 26-kDa protein was present in the four different cell lines, and 2) the level of the 26 kDa protein in their nuclei was decreased by 30-70% after heating at 42 degrees C for 1 hr. However, restoration of this protein occurred along with development of chronic thermotolerance. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) neither inhibited the development of chronic thermotolerance nor affected the restoration of the 26 kDa protein in the nucleus. In fact, this drug protected cells from hyperthermic killing and heat-induced reduction of 26 kDa protein in the nucleus. Heat sensitizers, quercetin (0.1 mM), 3,3'-dipentyloxacarbocyanine iodide (DiOC5[3]: 5 micrograms/ml), and stepdown heating (45 degrees C-10 min----42 degrees C), potentiated hyperthermic killing and inhibited or delayed the restoration of the 26 kDa protein to the nucleus. These results support a correlated, perhaps causal relationship between the restoration of the 26 kDa protein and chronic thermotolerance development in four different mammalian cell lines.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Development of acute thermotolerance in L929 cells: lack of HSP28 synthesis and phosphorylation.

We investigated the correlation between the development of acute thermotolerance and the phosphorylation, synthesis, and expression of the HSP28 family in murine L929 cells. Following heating at 43 degrees C for 30 min, thermotolerance developed rapidly in exponential-phase cells and reached its maximum 4-9 h after heat shock. Maximal thermal resistance was maintained for 24 h and then gradually decayed. However, heat-induced phosphorylation of HSP28 was not detected. Furthermore, HSP28 synthesis during incubation at 37 degrees C for 12 h following heat shock was not detected by [3H]-leucine labeling followed by two-dimensional polyacrylamide gel electrophoresis. In addition, Northern blots failed to demonstrate expression of the HSP28 gene. Unlike HSP28, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was observed during incubation at 37 degrees C after heat shock. These results demonstrate that HSP28 synthesis and its phosphorylation are not required to develop acute thermotolerance in L929 cells.     

Reduction of levels of nuclear-associated protein in heated cells by cycloheximide, D2O, and thermotolerance.

Hyperthermia increases levels of nuclear-associated proteins in a manner that correlates with cell killing. If the increase in nuclear-associated proteins represents a lethal lesion then treatments that protect against killing by heat should reduce and/or facilitate the recovery of levels of the proteins in heated cells. This hypothesis was tested using three heat protection treatments: cycloheximide, D2O, and thermotolerance. All three treatments reduced levels of the proteins measured immediately following hyperthermia at 43.0 or 45.5 degrees C, with the greatest reduction occurring at 43.0 degrees C. In addition to reducing the proteins, thermotolerance facilitated the recovery of the proteins to control levels following hyperthermia. Thus thermotolerance may protect cells by both reducing the initial heat damage and facilitating recovery from that damage. Cycloheximide and D2O did not facilitate recovery of nuclear-associated proteins, suggesting that their protection against cytotoxicity related to the proteins resulted solely from their reduction of increases in levels of the proteins. All three treatments have been shown to stabilize cellular proteins against thermal denaturation. The results of this study suggest that the increase in nuclear-associated proteins may result from thermally denatured proteins adhering to the nucleus and that it is the ability of cycloheximide, D2O, and thermotolerance to thermostabilize proteins that reduces the increase in levels of the proteins within heated cells.     

Cycloheximide increases the thermostability of proteins in Chinese hamster ovary cells

Protein denaturation resulting from temperatures between 42.0 degrees C and 50 degrees C has been observed and implicated as the lethal lesion for hyperthermic cell killing. A logical corollary is that protection against hyperthermic killing requires stabilization of cellular proteins against thermal denaturation. To test this, Chinese hamster ovary cells were treated with the heat protector cycloheximide and then subjected to differential scanning calorimetry to measure protein denaturation. Cycloheximide stabilized proteins that denatured between 42 degrees C and 52 degrees C in control cells by increasing their transition (denaturation) temperature by an average of 1.3 degrees C. In addition, cycloheximide reduced the cytotoxicity of actinomycin D and adriamycin, suggesting that protein stabilization protects cells against stresses other than hyperthermia.     

Long-term optimized embryogenic cultures in durum wheat (Triticum durum Desf.)

Five varieties of durum wheat: Appulo, Ofanto, Latino, Creso, and Castello (Triticum durum Desf.) adapted to the semi-arid mediterranean environment have been tested for their in vitro response. Compact, embryogenic, highly regenerable calli originated from primary callus derived through proliferation of the scutellum of immature embryos explanted in the presence of 2,4 dichlorophenoxyacetic acid. Selective subculture of the white, compact, embryogenic sectors led to the establishment of long-term cultures. Regeneration occurred on hormone-free medium either via germination of somatic embryos, or via multiple-shoot formation probably due to precocious germination of somatic embryos. The three varieties, Ofanto, Creso and Appulo, were the best responding genotypes. Callus fragmentation and two subsequent transfers onto fresh medium at 7-day intervals yielded a frequency of plant regeneration of some 25–40 plantlets per gram fresh weight callus in 21 days on Murashige and Skoog's hormone-free medium. Plantlets could be efficiently established in soil, thus confirming the possibility of biotechnological approaches with varieties of this crop species.Abbreviations E embryogenic - NE non embryogenic - MS Murashige and Skoog's (1962) medium - 2,4-D 2,4 dichlorophenoxyacetic acid - DAA days after anthesis - FWT fresh weight tissue