Pharmacokinetics and pharmacodynamics of single and multiple doses of the glucagon receptor antagonist LGD-6972 in healthy subjects and subjects with type 2 diabetes mellitus.

AIM: To evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of single and multiple doses of a novel, oral glucagon receptor antagonist, LGD-6972, in healthy subjects and subjects with type 2 diabetes (T2DM). METHODS: In the single ascending dose study, LGD-6972 (2-480 mg) was administered to healthy subjects (n = 48) and T2DM subjects (n = 8). In the multiple ascending dose study, healthy subjects (n = 12) received a dose of 15 mg LGD-6972 and T2DM subjects (n = 36) received doses of 5, 10 or 15 mg of LGD-6972 daily for 14 days. RESULTS: LGD-6972 had linear plasma pharmacokinetics consistent with once-daily dosing that was comparable in healthy and T2DM subjects. Dose-dependent decreases in fasting plasma glucose were observed in all groups with a maximum of 3.15 mmol/L (56.8 mg/dL) on day 14 in T2DM subjects. LGD-6972 also reduced plasma glucose in the postprandial state. Dose-dependent increases in fasting plasma glucagon were observed, but glucagon levels decreased and insulin levels increased after an oral glucose load in T2DM subjects. LGD-6972 was well tolerated at the doses tested without dose-related or clinically meaningful changes in clinical laboratory parameters. No subject experienced hypoglycaemia. CONCLUSION: Inhibition of glucagon action by LGD-6972 was associated with decreases in glucose in both healthy and T2DM subjects, the magnitude of which was sufficient to predict improvement in glycaemic control with longer treatment duration in T2DM patients. The safety and pharmacological profile of LGD-6972 after 14 days of dosing supports continued clinical development.

Phase IIa cross-over study of propylene glycol-free melphalan (LGD-353) and alkeran in multiple myeloma autologous transplantation.

Propylene Glycol-Free melphalan HCL for Injection (PGF-Mel) is a new formulation that incorporates Captisol, a specially modified cyclodextrin, to improve melphalan stability. In this phase IIa, open-label, randomized, cross-over design bioequivalence study, the pharmacokinetics of PGF-Mel were compared with the marketed formulation of melphalan, or Alkeran. Patients received half of the total dose of melphalan in the form of Alkeran and the other half in the form of PGF-Mel in an alternating manner. The pharmacokinetic measures were determined using WinNonlin 6.2 and bioequivalence was assessed using log-transformed systemic exposure parameters. Twenty-four patients, 11 females and 13 males, were enrolled between 4 February 2010 and 16 May 2011 at The University of Kansas Medical Center and The University of Kansas Cancer Center. The median age of enrolled subjects was 58 years (range: 48-65). All patients achieved myeloablation 3 days post autologous graft followed by successful neutrophil engraftment with a median of 11 days after transplant. Pharmacokinetic analysis showed that PGF-Mel was bioequivalent with Alkeran and also revealed that maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC) were higher (~10%) after PGF-Mel administration. In conclusion, PGF-Mel is considered bioequivalent to Alkeran while also demonstrating a marginally higher systemic drug exposure.

The effects of substituted cyclodextrins on the colloidal and conformational stability of selected proteins.

The effects of various types of substituted and nonsubstituted cyclodextrins (CDs) on the physical and colloidal stability of three different proteins were studied to further ascertain the mechanism by which cyclodextrins stabilize proteins. The three proteins examined in this study are the Clostridium difficile Toxoid A, Yersinia pestis low-calcium-response V or V antigen (LcrV), and fibroblast growth factor 10 (FGF-10). These three pharmaceutically relevant proteins differ in molecular weight, pI, as well as in their secondary and tertiary structure. The effects of three parent cyclodextrins (alpha, beta, and gamma), as well as several hydroxypropyl (HP-CDs) and sulfobutylether (SBE-CDs) cyclodextrins of varying degrees of substitution, on the three proteins were examined as a function of pH and temperature. Structural changes and aggregation behavior were monitored in the presence and absence of the 17 cyclodextrins using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering. Overall, the major effect of the cyclodextrins on the proteins was the ability of a majority of them to inhibit thermally induced aggregation. This study suggests that the stabilization of proteins by cyclodextrins is dictated by their type and degree of substitution, as well as the physical and chemical properties of the protein being examined.

Decreased in vitro interaction between p53 and nuclear stress proteins in the p53-deficient mouse.

In a previous study, the strength of the interaction between the nuclear stress proteins (sps) 25a, 70i, 72c, and 90 and the tumor suppressor protein p53 was determined by an in vitro fluorescence binding assay. The relative binding of the individual sps with p53, derived from the bone marrow of transgenic mice heterozygous at the p53 locus (p53+/-), was reduced compared to the interaction of sps and p53 derived from wild-type (p53+/+) mice. In order to determine if the genotype of the p53 donor or the genotype of the sp donor determined the binding efficiency, p53 expression was induced by retinoic acid and sp synthesis by bleomycin. P53 derived from either wild-type or heterozygous animals was cross-reacted with nuclear sps obtained from either wild-type or heterozygous animals. Each of the sps, 25a, 70i, 72c, and 90, bound to wild-type p53 with a similar efficiency, irrespective of the genotype of the sp donor mouse (p53+/+ or p53+/-). In contrast, when the sp interaction with p53 obtained from the heterozygous mouse was measured, the relative value of the fluorescence complex was significantly reduced. The data suggest that the strength of the interaction between p53 and nuclear sps is related to the genotype of the p53 donor, and not to the genotype of the animals from which the sps are derived.

Dose-response of retinoic acid induced stress protein synthesis and teratogenesis in mice.

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.

Induction of stress proteins by electromagnetic fields in cultured HL-60 cells.

HL-60 cells in culture were exposed for 2 h to a sinusoidal 0.1 or 1 mT (1 or 10 Gauss) magnetic field at 60 Hz and pulse labeled after exposure with radioactive isotopes by incubation by using either [(35)S]methionine, [(3)H]leucine, or [(33)P]phosphate. The radioactive labels were incorporated into cellular proteins through synthesis or phosphorylation. Proteins were extracted from electrostatically sorted nuclei, and the heat shock/stress proteins (sp) were analyzed for synthesis and phosphorylation by two-dimensional polyacrylamide gel electrophoresis. In the control cultures (no exposure to the magnetic field), sp 72c (cognate form) was faintly observed. A 0.1 mT exposure did not show sp metabolism to be different from that of the controls; however, after a 1 mT exposure of the HL-60 cells, sp 70i (inducible form) was synthesized ([(35)S]methionine incorporation). Sp 90 was not synthesized at either field level, but was phosphorylated ([(33)P]phosphate incorporation) in the 1 mT exposure. Sp 27 (isoforms a and b) was induced after a 1 mT exposure as reflected by labeling with [(3)H]leucine. These sps were not detected after a 0.1 mT exposure. After a 1 mT exposure and labeling with [(33)P], sp 27 isoforms b and c were phosphorylated whereas isoform 'a' was not observed. Sps 70i, 72c, and 90 were identified by commercial sp antibodies. Likewise, polypeptides a, b, and c were verified as sp 27 isoforms by Western blotting. Statistical evaluation of sp areas and densities, determined from fluorographs by Western-blot analysis, revealed a significant increase in sps 90 and 27a after a 1 mT magnetic field exposure. The 1 mT magnetic field interacts at the cellular level to induce a variety of sp species. Bioelectromagnetics 20:347-357, 1999. Published 1999 Wiley-Liss, Inc.

The relationship of p53 and stress proteins in response to bleomycin and retinoic acid in the p53 heterozygous mouse.

A single, i.p. dose of bleomycin was administered simultaneously with [35S]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 22000 to 100000 Mr 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 [35S]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 G1 than in S+G2 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.

Adaptive role of caloric intake on the degenerative disease processes.

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 degrees 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 perpetu

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.

The physiologic, neurologic, and behavioral effects of caloric restriction related to aging, disease, and environmental factors.

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 B6C3F1 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.

A circadian study of liver antioxidant enzyme systems of female Fischer-344 rats subjected to dietary restriction for six weeks.

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.

Biological Diversity Conservation: A Public Policy Perspective

/ While extinctions of individual species are part of a normal cycle, the current rate of extinctions should be a concern to us all. The maintenance of biological diversity is important for utilitarian reasons, quality of life considerations, and because biodiversity is important to sustainable regional economies. Single-species approaches are too limited to protect biodiversity at the landscape, habitat, and watershed levels. New approaches are necessary to deal with the complexity of biological diversity. The administration is using provisions in the Endangered Species Act to bring about broader multispecies habitat protection. The ecosystem approach provides a framework for ensuring that ecological considerations are taken into account, along with economic and social factors, and that all interested parties are able to participate in the decision-making process.KEY WORDS: Biodiversity; Ecosystem management; Endangered Species Act; Public policy; Stewardship; Regional economies; Habitat protection

P48: a novel nuclear protein possibly associated with aging and mortality.

The synthesis ([35S]-incorporation) of stress proteins (sps, i.e., 24, 25, 70, 90 Mr) and of nuclear protein 48 (p48) was investigated in the heart and bone marrow cells of three groups of male Fischer 344 rats following administration of isoproterenol (IPR). Two groups of rats, young ad libitum (Y/AL-3 1/2 months) and old/AL (O/AL-28 months), had full access to rat chow; a third group of old diet restricted (O/DR-28 months) rats was maintained on a diet restricted intake of 40% of the Y/AL animals. Sp synthesis in the bone marrow (25, 70, 90 Mr) and heart (24, 70, 90 Mr) nuclei of O/AL was significantly reduced, as compared with Y/AL and O/DR rats, following their induction with IPR. A unique sp24 was expressed in heart following IPR dosing. A 1 mg/kg dose of IPR was lethal for O/AL, but not for Y/AL or O/DR animals. This lethal dose induced synthesis of p48 in heart and bone marrow nuclei of O/AL rats only. P48 existed in isoform states in bone marrow, and when a lethal dose of IPR was administered in this tissue, it was expressed in O/AL rats in a cell-cycle regulated pattern. Stress proteins and other non-sps were seen as cell cycle regulated following IPR administration. P48 in bone marrow and heart nuclei from O/AL rats showed an antigenic response identical to that of p48 in HL60 nuclei. The presence of p48 is correlated with mortality and with an ad libitum diet in old rats, since it is absent in old diet restricted animals; therefore, DR may impede the expression of p48 through a mechanism(s) that is undisclosed at this time.

The effect of aging and dietary restriction on the retinoylation of nuclear matrix proteins in rats.

The labeling in vivo of young ad libitum (Y/AL) and old diet restricted (O/DR) rats with [3H]retinoic acid (RA) for 6 hours, and the exposure of electrophoretically separated nuclear matrix proteins from bone marrow tissue on film for 48 days revealed the presence of eleven retinoylated proteins. Dosing with RA (100 mg/kg body weight) for 96 hours and exposure to [3H]RA enhanced the levels of radioactive incorporation of several nuclear matrix proteins, including p51, and p55, similarly in Y/AL and O/DR rats. Dosing of old ad libitum (O/AL) rats with [3H]RA for 6 hours showed the incorporation of six proteins following 48 days of exposure on film. Long-term dosing of RA (96 hours) did not increase [3H]RA incorporation in these proteins, including p51 and p55, in O/AL rats. Increasing the level of RA by two-fold (200 mg/kg body weight) in Y/AL and O/DR rats elicited an increase in the incorporation levels of [3H]RA in five proteins. This dose response following increased levels of RA was not seen in the retinoylated proteins of O/AL animals. Analysis by the Western blotting technique showed p51 and p55 from rat bone marrow cells to have the same immunochemical determinates with proteins of identical molecular masses in HL60 cells. The levels of retinoylation of nuclear matrix proteins in O/DR animals, altered by age- and diet-dependent factors, suggests a condition that is more reminiscent of Y/AL than of O/AL animals.

Comparison of the cell cycle regulated synthesis and phosphorylation of stress proteins, actin isoforms and a novel actin-like protein following drug administration in cultured rat lymphocytes.

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.

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.

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.

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.