James L. Pipkin

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.

Role of Glucocorticoids and “Caloric Stress” in Modulating the Effects of Caloric Restriction in Rodentsa

Caloric restriction (i.e., the balanced reduction of the protein, carbohydrate and fat content of the diet without reduction of its micronutrient content) has been shown, in rodents, to be an extremely powerful modulator of a broad spectrum of age-associated degenerative diseases as well as life span.’-5 Caloric restriction has been found to delay the occurrence of many age-associated neoplastic diseases or to slow their progression, often to such an extent that the clinical expression of the diseases is eliminated.* Since reducing individual dietary components such as protein or fat without reducing the overall caloric intake is less effective in increasing longevity or suppressing neoplasia than caloric restriction by itself, it is reasonable to assume that the observed effects of caloric restriction are primarily dependent upon a specific reduction in calories and not specific dietary compon e n t ~ . ~ ~ ’ The multiple actions of caloric restriction suggest that it may influence primary aging processes themselves; however, recent examination of pathological data suggests that caloric restriction delays different age-dependent pathological conditions at different rates.8 This observation would argue against the modulation by caloric restriction of a simple fundamental process of cellular aging, but rather, it implies that caloric restriction modifies the organism’s homeostatic set-point to one that is less susceptible to some but not all pathologies. Unfortunately, such theories on the nature of the caloric restriction effect will remain merely speculatory until the precise molecular and biochemical mechanisms by which caloric restriction influences the processes of aging and disease are fully elucidated.

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.

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.

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.

The turnover of radiolabeled nuclear proteins in rats exposed to environmental and chemical stress

Exposure to a 12 h light/12 h dark (L/D) cycle for 1 month, followed by reversal to a 12 h D/12 h L (D/L) cycle stimulated within 18 h the incorporation of [3H]leucine and [32P]orthophosphoric acid into new proteins (130-25 kDa) in the G0 phase of the cell cycle of the non-regenerating and regenerating rat liver as observed in two-dimensional gel autoradiograms. Six additional proteins from the rat submaxillary gland (130-20 kDa) revealed labeling with 32P within 3 h following combined administration of isoproterenol and sodium arsenite. Labeling disappeared within 7 days for all stressed proteins.

p53 synthesis and phosphorylation in the aging diet-restricted rat following retinoic acid administration

Multiple doses of retinoic acid (RA) were administered intraperitoneally to three groups of male Fischer 344 rats over a 36 h period. The p53 isoforms from bone marrow nuclei in these three groups of rats were analyzed over time by two-dimensional polyacrylamide gel electrophoresis (PAGE) and fluorography for the incorporation of [35S]methionine (p53-synthesis) and [32P]phosphate (p53-phosphorylation). Two groups of rats, young (3.5 months) ad libitum (Y/AL) and old (28 months) ad libitum (O/AL), had free access to Purina rat chow; a third group of old (28 months) diet-restricted rats (O/DR) were maintained on a restricted caloric intake (60% of the AL diet) from 3 months of age. After 36 h of RA dosing, the PAGE patterns of p53 synthesis and phosphorylation in Y/AL and O/DR rats were very similar. In both groups, an increase in complexity was observed with labeling of additional isotypes possessing more acidic isoelectric values. In contrast, the O/AL animals showed a pattern of p53 isoform synthesis and phosphorylation that was considerably less complex and lacked the pronounced shift to more acidic forms following RA dosing. The p53 isoforms of O/AL rats as recognized by wild type (wt) Pab 246 antibody, were also much less dramatic in their increase to more acidic forms. Two-dimensional phospho-tryptic maps of Y/AL and O/DR rats were also very similar, both exhibiting two additional minor 32P-labeled fragments after RA dosing. The maps of O/AL rats did not show the two additional fragments following RA administration. After RA dosing, cyclin protein inhibitors (p16, p21, p27) revealed robust labeling with their respective antibodies in Y/AL and O/DR rats as analyzed by Western blotting. The O/AL animals showed marginally detectable antibody recognition of the cyclin inhibitors after RA dosing. Taken together, these data suggest that the biosynthesis and phosphorylation of p53 isoforms and the expression of cyclin dependent kinase inhibitor proteins is not significantly different between Y/AL and O/DR rats. Further, these results confirm and extend our previous observations that chronic diet-restriction attenuates the age related decline in the metabolic activity of nuclear protein products.

Age and temperature related changes in behavioral and physiological performance in the Peromyscus leucopus mouse

Age-related and ambient temperature-related changes in motor activity, body temperature, body weight (b.w.), and food consumption were studied in the long-lived Peromyscus leucopus mouse at environmental temperatures of 29 and 21 degrees C. Major changes in physiological performance were observed between the young (6 months) and old (60-72 month) age groups. The number of daily activity episodes, and total activity output was significantly lower in old mice. Maximum, average and minimum daily body temperature was lower in the old mice and a significant ambient temperature-by-age interaction was found. Maximum, minimum, and average daily b.w. was higher in old mice. Motor activity was evenly distributed over the active (night) phase in young mice but in old mice activity was significantly greater in the late night partition of the active cycle than in the early night partition. Both groups were significantly more active at night than during the day. Most of the food consumption in both groups occurred at night, but young mice consumed significantly more during the late night partition than the early night partition, and the consumption rates for old mice were not significantly different between early and late night partitions. The percentage of activity episodes involved with food consumption in both groups was significantly higher during the night partition, but the percentage during the early night partition was significantly higher in old mice than in young mice. Significant episodes of circadian torpor occurred in a high percentage of old mice at 06:00, on consecutive days, at both environmental temperatures, but young mice expressed no evidence of torpor.