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Biochemical analysis of proteins extracted from mammalian cell nuclei which were sorted according to DNA content was greatly simplified using a special collection device to recover sorted nuclei. The device was developed for use with a sorting flow cytometer to provide sufficient numbers of particles to achieve adequate sensitivity and resolution while minimizing sample degradation and loss. The design, evaluation and application of the collection device were described with emphasis on sample preparation. Nuclear populations G0 + G1, S, and G2 + M, were sorted from regenerating rat liver tissue preparations and collected separately in the special chambers. Nuclear proteins were extracted and analyzed by polyacrylamide gel isoelectric focusing or capillary isotachophoresis.

Carcinogenicity and aging are characterized by a set of complex endpoints, which appear as a series of molecular events. Many of these events can be modified by caloric intake. Since most of these processes determine an organism*s ability to cope with various environmental stressors, it is not surprising that a relationship (in the presence of a constant nutrient density) exists between caloric intake and time-to-tumor and/or life span. Our studies have clearly shown that generally, the greater the caloric intake, the greater the body weight, the higher the incidence of spontaneous tumor occurrence, the greater the susceptibility to chemical carcinogens, and the shorter the life span. It is also recognized that variables other than body weight influence the life span and carcinogenesis. We have focused our attention on the questions of how and to what extent caloric intake modifies those homeostatic processes believed to be critical in determining the ability of an organism to cope with endogenous and exogenous stresses such as chemical, physical, and biological carcinogens. The response of an organism to its environment can be divided into four categories—physiological, metabolic, molecular, and cellular. We have found that, from a physiological perspective, decreasing caloric intake causes body temperature in rodents to be decreased by 0.5 to 1.8°C and water consumption to be increased by 80%, as is running activity. However, metabolic output per gram of lean body mass is not altered. Reproductive capacity declines, whereas the ECG waveform is preserved as caloric intake decreases. Alterations in these and other physiological functions suggests that energy intake serves as a signal to up-regulate or down-regulate functions related to the flight-or-fight response observed in placental mammals. A number of key metabolic pathways are altered as a function of lowered caloric intake, even though the rate of food consumption per gram of lean body mass remains steady during body weight decreases caused by decreasing caloric intake. Pharmacological compartmentalization, however, is altered. As caloric intake declines, changes occur in the expression of a number of drug-metabolizing enzymes, with the most striking effect seen in sex-specific growth hormones and liver-dependent phase I and phase II enzymes. Additionally, oxidative stress (free-radical and mediated damage to macromolecules) appears to decrease as a function of reduced caloric intake. A number of molecular processes also change with changes in energy consumption. Our studies have shown that, regardless of the source and nature of DNA damage, DNA repair is better preserved and/or enhanced when caloric consumption decreases. In addition, the fidelity of DNA replication increases and oncogene expression is stabilized, P53 gene expression is increased, and apoptosis is elevated by up to 500% with decreased caloric intake. At the cellular level, cell proliferation is decreased in direct proportion to lower energy intake in some but not all tissues. Studies have also shown an enhancement in immune capacity, changes in IGF1, and accelerated rates of wound healing proportionate to declines in energy consumption. Our most recent findings, however, have shown that the benefits associated with decreases in caloric intake only occur in the presence of sufficient nutrient quality and density. In the absence of proper nutrition, however, sensitivity to carcinogens and toxic substances appears to be enhanced. These findings are supported by independent studies. These observations have led us to conclude that, in certain organisms, when caloric intake is decreased, there is an up-regulation of those processes that modulate the responses to a wide range of environmental stressors. This response allows for a better survival rate and a down-regulation of reproductive activity. It is our belief that, during periods of environmental stress, these systems may be essential to perpetuation of the species and the gene pool of the individual.

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.

Administration of phytohemagglutinin initiated cycling of rat lymphocytes in vitro, and following treatment with this drug and other drugs in combination, lymphocytes were pulse labeled with [3H] leucine of [32P] phosphate. The nuclei were isolated from lymphocytes and collected from partitions of the cell cycle, and the proteins analyzed from fluorographs following gel electrophoresis for protein biomarkers after drug exposure. Stress proteins (sps) were dependent on a specific drug or drugs in combination (i.e., interleukin-2, bleomycin) for their synthesis that occurred only during the G1-phase of the cell cycle. An "actin-like" protein (A4) with electrophoretic mobilities similar to the actin complex, was synthesized in S and G2 phases and phosphorylated in all phases of the cell cycle only following the administration of drugs in combination. A4 exhibited a binding affinity for sp 24 that was cell cycle regulated (i.e., A4 from S phase did not bind with sp 24, but A4 from G2 phase did bind with the sp. Protein A4 appeared similar in some structural aspects to the nonmuscular actin isoform family but differed in epitope, suggesting a unique relationship and represented a stable protein, perhaps a product from the mutation of an actin gene. The dependence of certain sps and protein A4 for their induction by drugs in combination may serve as biomarkers of chemical interaction and toxicity.

Physiological and behavioral performance was monitored in old (24 month) female B6C3F1 hybrid mice that were fed either a caloric restricted (CR) diet (60% of ad libitum) or fed ad libitum (AL). The CR group was fed (CR1) and then fasted (CR2) on alternate days starting at 12–14 weeks of age. The main objective of the study was to determine if the effects of intermittent feeding were different from those for daily CR feeding. The average daily body weight, body temperature, oxygen consumption per gram of lean body mass (LBM), and respiratory quotient were higher on the feed day (CR1) than on the fast day (CR2), whereas the average daily body weight, temperature, activity, and oxygen metabolism per gram of LBM were lower on CR1 than in AL. The daily range in temperature, oxygen metabolism per gram of LBM, and RQ was greater on CR1 and CR2 than in AL; the exception was RQ on CR1. No food was eaten on CR2, and water consumption was reduced by 60% compared to CR1. Average food consumption per meal, and average time eating and drinking per meal was greater in CR mice than in AL mice. The results of this study indicated that mice responded to alternate day CR by changing average daily metabolic output per gram of LBM in direct response to food consumption, whereas when CR mice were fed daily, no change was seen in average metabolism per gram of LBM. The fact that a significant reduction in energy output and temperature occurred in CR mice when metabolic pathways shifted from carbohydrate to fat metabolism may indicate that decreased glucose utilization may have triggered these CR-induced effects.

A novel protein (p34) was observed in polyacrylamide gel fluorographs of gestation day 13 embryonic mouse brain following retinoic acid dosing of dams. Another p34 polypeptide with identical gel migratory characteristics was seen in the hypothalamus of old caloric restricted rats after "food deprivation stress". Western blotting, employing an ultramicro trans-blot cell developed in our laboratory, detected identical immunochemical determinants between these proteins, verifying their homology. Peptide mapping and Western blotting further validated the uniqueness of p34 compared with other stress proteins including heme oxygenase.

Retinoic acid (RA) is teratogenic in rodents and also induces the synthesis of stress proteins in fetal mouse limb buds. To determine if the RA induction of stress proteins is target tissue specific, pregnant CD-1 mice were gavaged with 100 mg/kg RA on day 11 of gestation, and nuclei isolated from tissues susceptible to RA-induced malformations (target tissues) as well as nuclei isolated from nontarget tissues were examined for stress protein synthesis and malformations. Forelimb and hindlimb (target tissues), as well as heart and tail (nontarget tissues), were removed from embryos 2.5 hours after RA treatment (1.5 hr after [3H]leucine labeling). Cell nuclei were isolated, stained with a DNA specific fluorochrome, propidium iodide, and sorted from the G0 + G1 and G2 + M phases of the cell cycle. Forelimb and hindlimb target tissues showed the synthesis in these embryonic nuclear proteins of an 84,000 relative molecular mass (Mr) protein and a 90,000 Mr protein following RA treatment. Two 20,000-25,000 Mr stress proteins were also labeled both in forelimb and hindlimb. Forelimb and hindlimb from untreated dams showed no stress protein labeling. Neither heart nor tail, nontarget tissues, showed any stress protein labeling following RA treatment. Classical teratological evaluation of embryos treated on GD 11 and sacrificed on GD 17 showed that 100% of the fetuses had forelimb and/or hindlimb malformations, while no malformations were observed in either the heart or tail. Based on the correlation of teratological anomalies with the identification of stress proteins in target tissue only, we postulate that stress proteins may be involved in the teratogenic process. Further work is necessary to establish whether a causal relationship exists.

Stress proteins are synthesized in response to a variety of stressors, including several teratogenic agents. However, their role, if any, in the teratogenic process is unknown. We have previously demonstrated that all-trans-retinoic acid administered to pregnant CD-1 mice on gestational day 11 or 13 produced limb defects and cleft palate near term in a dose-responsive manner. This chemical also induced the synthesis of several nuclear stress proteins in embryonic tissues within several hours of dosing. The stress proteins were only observed in tissues that eventually became malformed and not in tissues that appeared normal at term. In the current work, we examined the stress response in embryonic target tissues after several different doses of retinoic acid. The nuclear stress proteins were synthesized in a dose-related manner and at a lower retinoic acid dose than doses producing malformations in the corresponding tissue at birth. Each individual stress protein and the total stress protein response were highly correlated, across dose, with the respective malformations observed at term.

A single, i.p. dose of bleomycin was administered simultaneously with [ 35 S]methionine to 4-month-old p53 wild type (+/+) and p53 heterozygous (+/−) C57BL/6 mice. Following a period of 3.5 h from dosing, the bone marrow nuclei were examined by two-dimensional PAGE and fluorography for induction of stress proteins (sps). Eight sps ranging from 22 000 to 100 000 M r were synthesized in p53 +/− and p53 +/+ mice following elicitation by bleomycin. No quantitative or qualitative differences were observed in sp expression in these two groups of animals. In a second experiment, three doses of retinoic acid were given i.p. to p53 +/− and p53 +/+ mice over a 36 h period. The p53 isoforms in bone marrow nuclei from these mice were analyzed by PAGE for incorporation of [ 35 S]methionine following retinoic acid injections. Quantitative and qualitative alterations in p53 isotypes were substantially increased in p53 +/+ as compared with p53 +/− mice. The increased complexity in the synthesis patterns in both groups of dosed mice consisted of additional isoforms possessing more acidic isoelectric values. In an in vitro binding assay, individual p53 isoforms demonstrated varying degrees of association with sps 25a, 70i, 72c and 90 which was consistently greater in p53 +/+ mice. Both the synthesis and binding of isoforms were greater in G 1 than in S+G 2 phase, in both groups of animals, reflecting a cell cycle regulated mechanism for these events. Collectively, these data implied that the synthesis and the binding characteristics of p53 isoforms with sps were enhanced in the p53 +/+ mice relative to the p53 +/− mouse; however, sp labeling was not affected by p53 genotype.

We examined the influences of dietary restriction (DR) on the circadian profile of liver catalase (CAT), glutathione peroxidase (GPx), and interacting systems required for removal of H2O2 (support systems), in 18-week old female Fischer 344 rats fed 60% of their ad libitum (AL) diet for six weeks. Food was presented to the DR animals during the early light-span. Regardless of diet, enzyme levels were generally consistent with circadian patterns. In CR animals, maximum activities often occurred at the time of food presentation. CAT and GPx activities generally were significantly higher in DR animals than in AL animals at the time of feeding. When assessing glucose-6-phosphate dehydrogenase (G6PDH) activity using saturating substrate (NADP(+)) concentrations, higher activities were seen at all times of day in the AL animals; however, when activity was measured in the presence of lower (i.e., physiologic) NADP(+) concentrations, the reverse was true. In contrast, glutathione reductase (GR) activity was not influenced by DR. Cytosolic levels of NADPH peaked and were higher in DR than in AL rodents prior to feeding. NADH levels were not influenced by diet, but did manifest a significant circadian pattern with a maximum occurring toward the middle of the dark span. These data suggest that even at a young age and following only a relatively brief duration of DR, there exists an enhanced enzymatic capability in rats subjected to DR to remove free radicals generated as a consequence of normal oxidative metabolism. Further, these data support emerging trends suggesting metabolic regulation of antioxidant defense systems in response to free radical generation.

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.

Little is known about the mechanisms by which acute and chronic caloric restriction (CR) modulate disease, longevity, and toxicity. To study these endpoints, behavioral parameters such as food and water consumption and physiologic parameters such as motor activity, body temperature, metabolic output (oxygen use), and respiratory quotient (RQ) were continuously monitored in 26-month-old male B 6 C 3 F 1 mice and Fischer 344 rats fed either ad libitum (AL) or a CR diet (60% of AL). Different dietary regimens were used: rodents were (1) chronically food-restricted using daily feeding starting at 14 months of age, (2) chronically food-restricted using alternate day feeding, or (3) abruptly switched from CR to AL (acute CR). The physiologic and behavioral changes seen with chronic and acute CR were consistent across strains and species. Average body temperature, the number of meals, and the ratio of food/water consumption were significantly lower in CR rodents than in AL rodents. Also, the daily range of body temperature, oxygen metabolism, RQ, average water consumption, and motor activity was significantly higher in CR rodents. CR caused the onset of altered neurobehavioral functions such as abnormal water consumption; increases in motor activity, serum corticosterone, and stress proteins (HSP); and decreases in the basal setpoint for body temperature and brain metabolism. These changes strongly suggest that many beneficial effects of CR are controlled by the hypothalamic–pituitary–adrenal axis via hormonal regulation. This study supports the assertion that nutritional status may be a primary factor of consideration in development of safety standards and assessment of risk.

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.

1. 1. Cultured mouse spleen cells were exposed to the mitogen Concanavalin A followed by isoproterenol, and nuclei were electronically sorted from seven partitions of the cell cycle. 2. 2. Several nuclear proteins, including stress proteins, which were cell-cycle-stage specific, were elicited by isoproterenol as determined by micro-electrophoresis and fluorography. 3. 3. Two novel S-phase proteins (X 0 and X′) demonstrated differing synthesis and phosphorylation patterns during the cell-cycle phases. 4. 4. X′ showed DNA binding characteristics and proteolytic properties (hydrolyzing X 0 or β-galactosidase); both proteins were cell-cycle regulated.

The responses to stress in living cells are well known. Thermal stress causes decreased protein synthesis as well as rapid induction of heat shock proteins (hsps), or alternately termed stress proteins (sps). The exposure of cultured promyelocytic leukemia cells (HL-60) to a 45 degrees C lethal heat shock for 1 h elicited synthesis and phosphorylation of a polypeptide M(r) 48,000 and pI 7.5 (p 48) as visualized by two-dimensional polyacrylamide gel ultra-microelectrophoresis. p 48, which was not observed at sublethal temperatures (39 and 41 degrees C), was synthesized during all phases of the cell cycle but was phosphorylated only in G0 + G1 and S-phases. The appearance of p 48 was marked by a concomitant and reciprocal reduction in hsps or sps 70 and 90. Distinct protease V8 fragment maps of p 48, hsps 70 and 90 in conjunction with immunochemical determination indicated vast differences in their primary structures. These facts suggest that p 48 was not formed from coalesced breakdown products of hsps 70 or 90. Western blotting showed that p 48 possessed the same immunochemical determinants as two other proteins with the same molecular mass but different isoelectric points. In an association assay, p 48 was shown to bind with actins and hsp 90 from HL-60 nuclei.

We have modified an existing technique in order to perform DNA analysi by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statsitically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken (1) from three individuals who had epithelial dysplasia and (2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amont of DNA or had higher than normal percentages of cells in the S and G2 + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G2 + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.

The effect of interleukin-2 (IL-2) on cell proliferation, sister-chromatid exchange (SCE) frequency, and the phosphorylation of nuclear stress proteins was evaluated in phytohemagglutinin (PHA)-stimulated spleen lymphocytes isolated from Fischer 344 rats. In addition, the ability of 2-mercaptoethanol (2-ME) to modulate the induction of these biological responses was characterized. Cell proliferation, as measured by the mitotic index, increased significantly (P less than .003) from a range of 3-4% in PHA-stimulated cultures to a range of 8-11% in PHA-stimulated cultures exposed to IL-2. The average generation time (AGT) did not respond to IL-2 in a concentration-dependent manner and decreased significantly (P less than .05) when 20 microM 2-ME was included with IL-2 in the culture medium. The number of SCE increased significantly (P less than .004) from control frequencies, which ranged from 13.1 to 15.6 SCE per cell, to frequencies of 18.5 to 21.5 SCE per cell as the concentration of IL-2 in the culture medium increased to 50 half-maximal units per ml. A reduction in SCE frequency was observed when cells were cultured with 20 microM 2-ME and IL-2 compared to IL-2 alone. Three nuclear proteins, with relative molecular masses of approximately 13,000-18,000, 20,000, and 80,000, were phosphorylated in IL-2-exposed G1-phase nuclei. Elicitation of these nuclear proteins in IL-2-exposed cells was not affected by exposure to 2-ME.

The patterns of [3H]-leucine and [32P]-phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the G0 + G1 and G2 + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two-dimensional polyacrylamide gel ultra-microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of [3H]-leucine- and [32P]-phosphate-labeled protein regions from the autoradiographs occurring primarily in the G2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the G2 + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of G2 + M and early mitosis were composed, in part, of nucleolar proteins.

Cultured mouse lymphoma cells incorporated [ 3 H]leucine and [ 32 P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45°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 [ 3 H]leucine incorporation into stress proteins occurred during the G 0 + G 1 phase with minimal labeling in the G 2 phase. On the other hand, 32 P labeling of stress proteins occurred in both the G 0 + G 1 and G 2 phases after exposure to sodium arsenite, while incorporation of 32 P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80–84 kDa) was detected with [ 3 H]leucine only in G 0 + G 1 phase, but with [ 32 P]phosphate these stress proteins were labeled in both G 0 + G 1 and G 2 . There was differential [ 32 P]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 G 2 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 G 2 4 h after exposure to sodium arsenite and very modest labeling with 32 P of stress proteins was observed at this time.

Rat hepatocyte nuclei were electrostatically sorted and collected from specific regions of the cell cycle. Proteins were extracted from homogeneous populations of nuclei with cetyltrimethylammonium bromide and chromatographed by one- and two-dimensional acid-urea polyacrylamide gel electrophoresis. Small quantitative differences were observed in amido black stained two-dimensional patterns of the nuclear proteins of the G0 + G1 and G2 + M populations. However, comparison of the two-dimensional autoradiographs of 3H-leucine labeled nuclear proteins in these populations revealed dramatic differences. After isoproterenol administration, animals showed enhanced labeling of certain nuclear proteins in G2 + M populations, but no noticeable effect in protein labeling was observed in the G0 + G1 populations. Four days after the administration of isoproterenol, fluorescent histograms exhibited increased amounts of DNA in the G2 + M populations. Comparison of the total amino acid composition of two proteins from control animals revealed the distinct chemical contrast of these proteins. Both proteins possessed higher ratios of acidic to basic residues than anticipated for basic proteins. These procedures and findings should prove useful for further understanding of mechanisms of toxicant effects upon nuclear protein metabolism.

The incorporation of leucine and phosphate in proteins extracted with two different solvents from nuclei sorted physically from the G0+G1 phase of the rat submaxillary gland cell cycle was observed by two-dimensional microscale polyacrylamide gel electrophoresis. Sodium phenobarbital did not initiate additional isotope ([3H]leucine and [32P]phosphate) incorporation on its own. After dosing with DL-isoproterenol, several proteins incorporated leucine and phosphate during the G0+G1 phase. The inclusion of phenobarbital in the dosing regime enhanced labeling of several proteins already present in the G0+G1 phase. Treatment of animals with sodium arsenite prompted labeling of phosphate of two “stress” proteins in the non-dividing G0 phase of the submaxillary gland. Enhanced incorporation of proteins was exhibited after combined dosing of isoproterenol and sodium arsenite, but no new incorporated proteins were seen in the G0+G1 phase. Likewise, no new proteins were seen to incorporate in the G2+M phase, but there was some increased incorporation in a few already present proteins under these conditions. The similar mobilities of several stress proteins, which were determined by coelectrophoresis of stress marker proteins, suggested that several of these proteins, even though they were extracted with different solvents, were identical.

In vivo injections of plant growth-promoting hormones increase the growth of animals as well as plants. Plant growth-promoting hormones and positive plant growth regulators are known to increase RNA and protein synthesis. Since cyclic GMP also increases RNA and protein synthesis, the object of the present investigation was to determine whether physiological levels of plant growth-promoting hormones and positive plant growth regulators have part of their mechanism(s) of action through stimulation of the guanylate cyclase (EC 4.6.1.2)-cyclic GMP system. Representatives of the three classes of growth-promoting hormones were investigated. Thus, auxins (indole-3-acetic acid, indole-3-butyric acid, beta-naphthoxyacetic acid, and 2,4,5-trichlorophenoxy acetic acid), gibberellins (gibberellic acid), and cytokinins [N6-benzyl adenine, kinetin (6-furfuryl aminopurine), and beta-(2-furyl) acrylic acid] all increased rat lung, small intestine, liver, and renal cortex guanylate cyclase activity 2- to 4-fold at the 1 microM concentration. Dose response curves revealed that maximal stimulation of guanylate cyclase by these plant growth regulators was at 1 microM; there was no augmented cyclase activity at 1 nM. The guanylate cyclase cationic cofactor manganese was not essential for augmentation of guanylate cyclase by these plant growth-promoting regulators. The antioxidant butylated hydroxytoluene did not block the enhancement of guanylate cyclase by these plant growth-promoting factors. These data suggest that guanylate cyclase may play a role in the mechanism of action of plant growth-promoting hormones and even of positive plant regulators at the cellular level.

Partially hepatectomized mice were injected intraperitoneally with isoproterenol (IPR), sodium phenobarbital (PB), or a combination of the two, and the regenerating lobe of the liver was removed. Nuclei were isolated, electrostatically sorted from various phases of the cell cycle, and the acid-soluble proteins were extracted. Synthesis of several high-molecular weight non-histone proteins were observed in two-dimensional polyacrylamide gel electrophoretic autoradiographs after independent administration of IPR and PB. The histones revealed alterations in synthesis patterns and the histone variants demonstrated concentration changes in response to these drugs. A protein designated as “a” appeared in G0 + G1 phase nuclei after IPR treatment and showed an increase in concentration after PB was included in the dosing regime. This protein displayed homology with a non-histone protein from calf thymus when its mobility and amino acid content was examined.

A study was performed to determine the role formalin fixation plays in flow cytometric (FCM) analysis of DNA in nucleated blood cells. Blood was frozen and stored at −80°C in a DMSO-citrate-sucrose buffer. When FCM analysis was needed the sample was thawed and added to a large volume of propidium iodide (PI) stain and analyzed after not less than 10 min of staining. No centrifugation steps were necessary. This protocol has been used by us for the discrimination of triploid from diploid Chinese grass carp (Ctenophary ngodon idella). Fixation of PI-staned nucleated RBCs with a final concentration of 1–2% phosphate-buffered formalin significantly lowered the amount of fluorescence recorded from the sample even when the fixed and stained RBCs were washed free of the fixative. Fixation with formalin prior to staining with PI yielded similar results. Our results show that formalin fixation of samples prior to or after staining with PI may lead to problems in data interpretation not previously appreciated. A FCM study was done to select the most appropriate biological DNA reference standard for grass carp RBCs. In this study DNA histograms of red blood cells from chicken, rainbow trout and diamondback watersnake as well as human nucleated white blood cells were compared to grass carp DNA histograms. Trout RBCs were selected as the most appropriate DNA reference standard for grass carp.

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.

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.

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.

1. Incorporation of radioactive phosphate or leucine by stress proteins from rodent lymphoma, submaxillary and liver nuclei were observed in two-dimensional gels following chemical and environmental stress. 2. The stress proteins were isolated from second-dimension gels and their similarities compared. Mol. wt determinations, immunochemical blotting and protease V8 peptide mapping confirmed the identical nature of the stress proteins possessing identical Mr, but from diverse tissue types. 3. These data imply that highly similar stress proteins exist in diverse tissues, are conserved during evolution, and possess some elemental and essential function.

The in vivo administration of hydroxyurea for 12 h counteracts DNA synthesis and cell cycling stimulated by 72 h of isoproterenol treatment in rat salivary gland, as determined by fluorescence-activated flow cytometry. Hydroxyurea has little effect on [3H]leucine incorporation (protein synthesis) of the nuclear proteins soluble in 0.35 M NaCl, when examined by polyacrylamide gel chromatography and autoradiography from electrostatically sorted nuclei of (G0 + G1) and (G2 + M) phases of the in vivo cell cycle. Differential incorporation of [3H]leucine into nuclear proteins was observed during various phases of the cell cycle. Proteins 'X' and 'Z', observed in stained gel chromatographs of the 0.35 M NaCl-soluble nuclear proteins, were identified by biochemical analyses as ubiquitin and protein A24, respectively. Ubiquitin appeared transiently while A24 increased in gel chromatograms concomitant with progressive quiescence of the salivary gland induced by hydroxyurea.

The H1 subtype proteins a, b, c, d, e, and 1 degree and the high mobility group (HMG) proteins 14 and 17 were extracted from submaxillary gland (SMG) nuclei treated in vivo and sorted from specific phases of the cell cycle, and analyzed by gel electrophoresis. Significant differences in these proteins were noted between quiescent (GO) phase nuclei and proliferating nuclei in both stained and autoradiographic gels. Stimulation of the SMG into cell division with DL-isoproterenol-HCl alone or in conjunction with sodium phenobarbital (PB) provided a system for the analysis of drug effects in stained, 3H lysine, and 32P pulse-labeled nuclear proteins obtained from different phases of the cell cycle.