William G. Hinson

Chronic caloric restriction induces stress proteins in the hypothalamus of rats

The induction of stress proteins (sps) in the hypothalamus of female Fischer 344 rats in response to caloric restriction (CR) and to heat stress was investigated. Caloric restriction was found to elicit sps 27, 34, 70, and 90 in the hypothalamus of both young and old rats while none was found in the hypothalamus of ad libitum (AL) fed controls. Heat stress initiated heat shock proteins (hsps/sps) 27, 70, and 90 in the hypothalamus of the young (AL) fed animals, the same proteins evoked by feeding stress. The same sps were induced in the old (AL) rats although the expression showed substantial decline with age. This reduction was less marked, however, with the old CR rats. Stress protein 34, an infrequently reported protein, was related to feeding and was not induced by heat shock. Recent reports point to the important role sps play in the cellular reaction to stress, as well as their involvement in the higher functions. The findings reported here suggest that sps are involved in the regulatory mechanisms allowing CR animals to tolerate stress related to metabolic substrate deprivation.

Retinoic acid induction of stress proteins in fetal mouse limb buds

Retinoic acid (RA) is teratogenic in rodent embryos. Several teratogens have been shown to induce the synthesis of a subset of heat shock proteins (stress proteins) in Drosophila. To determine if RA induces the synthesis of these proteins in rodent embryos, pregnant ICR mice were dosed with 100 mg/kg RA on Day 11 of gestation. Forelimb buds were removed from embryos 2.5 hr post-RA-treatment and nuclei were isolated, stained, and sorted from stages of the cell cycle. Nuclear proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. Nuclear proteins with molecular weights of approximately 84 and 25 kDa were synthesized in embryos in the G0 + G1 phase after pregnant dams were treated with RA. Isoelectric points, molecular weights, immunochemical blotting, and polypeptide mapping demonstrated that these proteins are indistinguishable from stress proteins isolated under a variety of conditions from rat submaxillary glands and mouse lymphoma cells. These results suggest that treatment with RA induces the synthesis of a subset of stress proteins; the role of these proteins in the teratogenic effects of RA is not known.

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.

Retinoic acid-induced stress protein synthesis in the mouse

We have previously demonstrated that stress proteins (SPs) are synthesized in tissues in which malformations are later observed following treatment with the developmental toxicant, retinoic acid (RA), on day 11 of gestation (GD 11). These proteins were not synthesized in tissues which did not present with malformations near partuition. The purpose of the present investigation was to determine if this correlation between early SP synthesis and later malformation was present at other times during gestation. CD-1 strain mice were dosed orally with corn oil or 100 mg/kg body weight RA on GD 10 or 13. Some of the mice in each group were given an intraperitoneal injection of 3H-leucine to label embryonic protein synthesis one hour after dosing with RA. These animals were sacrificed 1.5 hour later, and embryonic protein synthesis was determined by two-dimensional gel electrophoresis followed by autoradiography. Other animals in each group were sacrificed on day 17 of gestation, and fetuses were examined for the presence of malformations. Following treatment with RA on day 10 of gestation, malformations were observed in the forelimbs, the hindlimbs and the tail; heart defects were not observed. SPs of 20-25,000 and 90,000 relative molecular mass (Mr) were synthesized in the forelimb bud and tail; in addition, a second low molecular weight (20-25,000) and a 84,000 Mr SPs were synthesized in forelimb buds. No SPs were synthesized in the hindlimb bud or the heart. Following RA treatment on GD 13, cleft palate was observed in 58% of fetuses; no other malformations were found. Proteins of 34,000, 84,000 and 90,000 Mr were synthesized in craniofacial tissue; SPs were not observed in forelimb bud, hindlimb bud, heart or tail tissues at this time. Therefore, it appears that there may be a correlation between tissue-specific SP synthesis early in organogenesis and the presence of a malformation later in gestation.

Micro-size polyacrylamide gel electrophoresis system

The development and characterization of a micro‐size two‐dimensional polyacrylamide gel electrophoresis system is described. Some of the techniques which have evolved with use of the system are also discussed. This apparatus has unique features which provide advantages over other small scale units. Up to ten first‐ and second‐dimension gels can be processed simultaneously with excellent resolution of protein regions. Consistent reproducibility is possible from protein samples as small as 400 ng and individual protein regions as small as 1 pg can be visualized by silver staining of the two‐dimensional gels. Similar sensitivities are achieved in autoradiographs of 3H‐labeled proteins extracted from the nuclei of cultured cells. The application of this system in conjunction with flow cytometric examination of nuclear DNA and electrostatic cell sorting of specific cell nuclei to provide homogeneous sample populations, allows subtle variations in isotope incorporation in proteins to be detected; whereas many t...

Phenytoin-induced stress protein synthesis in mouse embryonic tissue

Abstract Several proteins haVe been shown to be synthesized in response to various environmental stimuli, including treatment with teratogens. The role of these proteins in the teratogenic process is unknown. Pregnant A/J mice were treated with either a teratogenic or a non-teratogenic dose of the anticonvulsant drug, phenytoin (PHT). Protein synthesis in embryonic craniofacial (target) tissue or forelimb buds (non-target) was determined by incorporation of radiolabeled leucine and analysis by two-dimensional polyacrylamide gel electrophoresis. Synthesis of three proteins in target tissue and one protein in non-target tissue was stimulated by drug treatment. These results suggest that synthesis of specific stress proteins may serve as biomarkers of drug-target tissue interaction.

Temporal and substrate-dependent patterns of stress protein expression in the hypothalamus of caloric restricted rats

Stress proteins (sps) 27, 34, 70 and 90 (Mr x 10(3)) were induced in the hypothalamus of caloric restricted (CR) rats by feeding stress. A definite time pattern for sps synthesis was observed when their induction was examined at several time points after the rats were fed, and the level of sps expression was found to vary significantly at different times of the day. The same group of proteins was induced in ad libitum fed rats when they were subjected to food deprivation for 48 h. Stress protein 34 expression in the hypothalamus of old caloric restricted rats was found to be dependent on blood glucose levels, and was substantially reduced when insulin was added to the glucose infusion. The expression of sps 27, 70 and 90, however, was little changed with glucose and/or insulin infusion.

Enhancement of anchorage-independent growth in JB6 cells exposed to 60 hertz magnetic fields

Abstract Extremely low frequency electromagnetic fields (EMFs) are implicated in the genesis of childhood leukemias and lymphomas after residential exposure, and of tumors in adults after occupational exposure. Experiments in vitro have demonstrated biochemical changes with cultured cells including modulation of signal transduction pathways. We examined the ability of 60 Hz sinusoidal EMFs to alter the growth of a promotion-sensitive mouse epidermal cell line (JB6) under anchorage-independent conditions. Cells (10 4 per dish) were suspended in 0,3% agar in 60 mm dishes and exposed to a 1.1 × 10 −3 T field generated by a Helmholtz coil held in an incubator at 37°C, 5% 002 and 90%–97% relative humidity. Growth of colonies more than 60 nm in diameter was scored after 10–14 days. Exposure to the EMF significantly increased the colony-forming efficiency, by 40%–70% compared with control cells ( > 95% confidence level), suggesting interaction with a growth control/signal transduction pathway.

Cell proliferation by cell cycle analysis in young and old dietary restricted mice

The effect of dietary restriction (DR) on cell proliferation determined by cell cycle analysis in tissues of young and old mice was investigated. Using the percentage of S-phase cells as an index of cell proliferation, we found that DR inhibited cell proliferation in spleen and thymus in young mice. No significant changes were found in bone marrow and kidney in the ad libitum (AL) or DR mice regardless of age. In old mice, the DR effect was observed in spleen only. When age increased, a parallel decline in cell proliferation was evidenced by a reduced % of S-phase cells. DR produces a greater cell cycle effect in the young mice than in the old mice. The present data suggests that inhibition of cell proliferation by DR may be affected by type of tissue, age, length of DR, and capacity or rate of cell proliferation.

The use of stimulated primary spleen cell cultures in evaluating cell cycle response to toxicant insult

Isolated rodent spleen cells, cultured for 48 hours with the mitogen Concanavalin A (Con A), consistently provide a cycling population with which to test the cell cycle effects (both temporal and quantitative) of various toxicants. Any modification in the cell cycle is then analyzed by flow cytometric techniques and computer-aided statistical comparisons of the DNA histogram of the cell populations.