Studies on the mechanism of lymphocyte-mediated cytolysis. VI. A reappraisal of the requirement for protein synthesis during T cell-mediated lysis.

The role of protein synthesis in the mechanism of T cell-mediated cytolysis has been re-investigated. Cytolytically active (C57BL/L anti-DBA/2) spleen cells treated with pactamycin (10-7 M to 10-6 M) exhibited suppressed protein synthesis (100 +/- 5%), but unimpeded lytic activity. Drug-treated effector cells, incubated for prolonged (up to 24 hr) periods of time in the presence and absence of antigen, showed no siginificant diminution of lytic activity although the incorporation of 3H-leucine into protein was totally ablated. Studies with emetine, another irreversible inhibitor of protein synthesis, gave identical results. These findings are difficult to reconcile with the hypothesis that effector T cells lysis via a soluble protein mediator.     

Effect of 6-mercaptopurine on 65Zn distribution in the pregnant rat

The effect of 6-mercaptopurine (6-MP) on the distribution of gavaged 65Zn in maternal and embryonic tissues of Sprague-Dawley rats was examined 24 hr after injection of the drug on day 13 of pregnancy. 6-MP injection resulted in a significantly higher retention of counts of 65Zn in maternal liver and lower counts in maternal plasma, uterus, placenta, and embryo than in controls. Compared to controls, gel chromatography of maternal liver from 6-MP injected dams showed higher counts associated with a protein peak of molecular weight 6,000-8,000, the approximate molecular weight of the zinc-binding protein metallothionein. These results support the idea that the zinc deficiency, which is observed in day 21 fetuses from dams injected with 6-MP during midgestation, may be the result of a drug-induced sequestering of zinc into maternal liver followed by a decrease in maternal plasma zinc and subsequent reduction in fetal zinc uptake. We suggest that this 6-MP-associated redistribution of zinc into maternal liver may be due to induction of maternal metallothionein synthesis by the drug.     

Effect of maternal hypercholesterolemia on fetal sterol metabolism in the Golden Syrian hamster.

The fetus obtains a significant amount of cholesterol from de novo synthesis. Studies have suggested that maternal cholesterol may also contribute to the cholesterol accrued in the fetus. Thus, the present studies were completed to determine whether diet-induced maternal hypercholesterolemia would affect fetal sterol metabolism. To accomplish this, maternal plasma cholesterol concentrations were increased sequentially by feeding hamsters 0.0%, 0.12%, 0.5%, and 2.0% cholesterol. At 11 days into a gestational period of 15.5 days, cholesterol concentrations and sterol synthesis rates were measured in the three fetal tissues: the placenta, yolk sac, and fetus. In the placenta and yolk sac, the cholesterol concentration increased significantly when dams were fed as little as 0.12% cholesterol (P < 0.0167), and sterol synthesis rates decreased in dams fed at least 0.5% or 2% cholesterol, respectively (P < 0.0167). In the fetus, changes in fetal cholesterol concentration and sterol synthesis rates occurred only when dams were fed at least 0.5% cholesterol, which corresponded to a greater than 2-fold increase in maternal plasma cholesterol concentrations. When the cholesterol concentration in the fetal tissues in each animal was plotted as a function of maternal plasma cholesterol concentration, a linear relationship was found (P < 0.001).These studies demonstrate that sterol homeostasis in fetal tissues, including the fetus, is affected by maternal plasma cholesterol concentration in a gradient fashion and that sterol metabolism in the fetus is dependent on sterol homeostasis in the yolk sac and/or placenta.     

Thyrotropin-releasing hormone and phorbol esters stimulate sphingomyelin synthesis in GH3 pituitary cells. Evidence for involvement of protein kinase C

Previous studies demonstrated that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 14525-14530). Since phosphatidylcholine may serve as the precursor for sphingomyelin synthesis, studies were performed to assess the effect of protein kinase C on sphingomyelin synthesis. The potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulated time- and concentration-dependent incorporation of 32Pi into the head group of sphingomyelin in cells short term labeled with 32Pi and resuspended in medium without radiolabel. TPA (10(-7) M) increased incorporation at a rate 1.4-fold of control after 2 h; EC50 congruent to 2 x 10(-9) M TPA. This correlated closely to TPA-induced phosphatidylcholine synthesis; EC50 congruent to 9 x 10(-10) M TPA. TRH (10(-7) M), which activates protein kinase C via a receptor-mediated mechanism, similarly stimulated 32Pi incorporation into sphingomyelin at a rate 1.5-fold of control; EC50 congruent to 5 x 10(-10) M TRH. This correlated closely with TRH-induced phosphatidylcholine and phosphatidylinositol synthesis; EC50 congruent to 2 x 10(-10) and 1.5 x 10(-10) M TRH, respectively. In cells short term labeled with [3H]palmitate, TRH induced a time- and concentration-dependent reduction in the level of [3H]ceramide and a quantitative increase in the level of [3H]sphingomyelin. Compositional analysis of the incorporated [3H]palmitate revealed that TRH increased radiolabel into both the sphingoid base and the fatty acid moieties of sphingomyelin. Similarly, TRH increased incorporation of [3H] serine into sphingomyelin to 145 +/- 8% of control after 3 h. TPA also stimulated these events. Like the effect of TRH on phosphatidylcholine synthesis, TRH-induced sphingomyelin synthesis was abolished in cells "down-modulated" for protein kinase C. In contrast, TRH-induced phosphatidylinositol synthesis still occurred in these cells. These studies suggest that protein kinase C stimulates coordinate synthesis of phosphatidylcholine and sphingomyelin. This is the first report of stimulation of sphingomyelin synthesis via a cell surface receptor.     

Role of the renin-angiotensin-aldosterone system in the enhancement of salt sensitivity caused by prenatal protein restriction in stroke-prone spontaneously hypertensive rats

We previously demonstrated that maternal protein restriction during pregnancy enhanced salt sensitivity and shortened life span in stroke-prone spontaneously hypertensive rats (SHRSP). The present study was conducted to investigate the participation of the renin-angiotensin-aldosterone system in the development of salt sensitivity in the offspring of dams fed a low-protein diet during pregnancy. We used SHRSP offspring from dams fed a 20% casein diet (CN) or a 9% casein diet (LP) during pregnancy. The CN and LP SHRSP offspring were further subdivided into tap-water-drinking and 1%-saline-drinking groups from the postnatal 10th week. A remarkable elevation in blood pressure in response to salt loading was observed in the LP SHRSP offspring. The protein levels of CYP11B2, an enzyme for aldosterone synthesis, were markedly elevated in response to salt loading in the kidneys of LP offspring. Treatment of the LP offspring with an aldosterone receptor antagonist prevented the blood pressure from elevating and lengthened the average life span in LP offspring in response to the drinking of 1% saline. No difference in the activity of angiotensin-converting enzyme or in the protein level of the angiotensin type 1 receptor was found between the CN and LP offspring in either the tap-water-drinking or saline-drinking conditions. In conclusion, the increment of aldosterone production in response to high-salt loading may contribute to the elevated salt sensitivity of the offspring of protein-restricted dams.     

The administration of oleoyl-estrone to lactating dams induces selective changes in the normal growth pattern of their pups.

To determine the effects of oleoyl-estrone treatment on the lactating dams and on the growth pattern of developing rats, female Wistar rats were randomly divided into two groups after delivery. One group received a daily gavage of 0.2 mL sunflower oil containing 10 micromol oleoyl-estrone/kg (treated group) and the other received a daily intragastric gavage of 0.2 mL sunflower oil (control group). Treatment was carried out during the first 15 days of lactation. Dams were killed on days 1, 15 or 20 after delivery and pups were sacrificed on days 1, 15, 20 or 30. Treated dams showed a transient decrease in food intake, significant lower lipid content than control dams, with a parallel maintenance of protein content and no appreciative changes in plasmatic parameters. However, a significant increase in brown adipose tissue mass was detected in treated group. Pups from treated dams showed a decrease in their growth rate that was reflected in the lower adipose tissue mass in different locations, except in the case of brown adipose tissue and, that continued after cessation of treatment. Thus, treatment affects dams in a selective way that does not coincide with a simple food restriction model.     

Protein synthesis in Volvox carteri f. nagariensis

Incorporation of radioactive amino acids was used to examine protein synthesis during the asexual reproductive cycle of the HK10 (female) strain of Volvox carteri f. nagariensis. It was observed that arginine is incorporated into TCA-precipitable material 500 to 1000 times as rapidly as other amino acids. Arginine is taken up by the organisms by a saturable transport system and concentrated against a steep concentration gradient. Intracellular arginine is incorporated with high efficiency: after 1 hr, more than 80% of the intracellular radioactivity is in a TCA-precipitable form. N-terminal analysis ruled out the possibility that this tracer arginine was being incorporated via a ribosome-independent arginyl tRNA-protein transferase and supported the alternative explanation that it was being incorporated via conventional protein synthesis. Proteins labeled with arginine were found to decay with the same time course as those labeled with another amino acid.A method for isolation of somatic and reproductive cells free of cross-contamination is reported. Using this method to isolate cells of prelabeled Volvox, it was observed that arginine is preferentially incorporated into reproductive cells. Reproductive cells exhibited four times as high an arginine-to-lysine incorporation ratio as somatic cells. Over half the total incorporated arginine that was recovered was in reproductive cells. Thus arginine incorporation should serve well as a probe to investigate developmentally significant protein synthesis in the reproductive cells of this species.     

Studies on the possible role of vitreous humor on protein synthesis and morphology of organ cultured adult rabbit lens. II. Epithelial cells.

The protein synthetic activities of epithelial cells of lenses organ-cultured without adhered vitreous humor manifest significant increase compared to the epithelial cells of lenses incubated with attached vitreous humor. This effect is not due to trauma of vitreous removal, as the addition of freeze-dried vitreous humor to the culture medium of lenses without attached vitreous humor could inhibit protein synthesis. However, the protein synthesis inhibitor in the vitreous humor has no visible effect on the lens morphology. It was also found that the factor from vitreous humor has no effect on mRNA production or cell-free protein synthesis. Thus, it seems that the effect on protein synthesis must be mediated via some other pathway.     

Endothelial nitric oxide synthase is myristylated.

The enzyme responsible for the synthesis of endothelium-derived relaxing factor and/or nitric oxide in the endothelium has been described as a particulate enzyme, whereas other isoforms of nitric oxide synthase are soluble enzymes. Here we are reporting that endothelial cells metabolically incorporate myristate (C14), but not palmitate (C16), into nitric oxide synthase. We are postulating that the endothelial-derived nitric oxide synthase is a particulate enzyme because of the fatty acid acylation of the protein which 'anchors' the enzyme into the membrane either directly or via another membrane-bound protein.     

SYNTHESIS OF PARAMECIUM SURFACE PROTEINS : I. Puromycin Insensitive Amino Acid Incorporation

The synthesis of a surface protein has been studied in Paramecium through double-labeling experiments using [¹⁴C]- and [³H]leucine-labeled bacteria as the source of radioactive amino acid. Over a 4–5 h incubation period, the turnover rate was found to be higher than that of overall cell protein. In addition, the initial label is apparently utilized during the chase period, being incorporated into protein via a puromycin insensitive pathway.     

A rapid posttranslational myristylation of a 68-kD protein in D. discoideum

Cells incubated with [3H]myristate were shown to rapidly and specifically acylate a 68-kD protein, p68, in a developmentally-regulated manner. The fatty acid incorporated into p68 was identified as myristate, and is linked to the protein via an amide bond, apparently to an NH2-terminal glycine. The acylation of p68 in D. discoideum displays some unusual properties. Unexpectedly, myristylation of p68 is a posttranslational event and occurs in the presence of inhibitors of protein synthesis. Another unusual finding was that although p68 is a stable protein, the acyl moiety is removed with a half time of approximately 15 min.     

Protein-energy malnutrition during pregnancy alters caffeine's effect on brain tissue of neonate rats

We studied whether protein-energy malnutrition changed brain susceptibility to a small dose of caffeine in newborn rats. Since we had demonstrated previously that caffeine intake during lactation increased the brain neuropeptide on newborns, we investigated further the effects of the prenatal administration of caffeine on TRH and cyclo (His-Pro). From day 13 of gestation to delivery day, pregnant rats in one group were fed either a 20% or a 6% protein diet ad libitum, and those in the other group were pair-fed with each protein diet supplemented with caffeine at an effective dose of 2 mg/100 g body weight. Upon delivery, brain weight, brain protein, RNA, DNA and the neuropeptides thyrotropin-releasing hormone (TRH) and cyclo (His-Pro) were measured in the newborn rats. A 6% protein without caffeine diet caused reductions in brain weights and brain protein, RNA and DNA contents, but did not alter brain TRH and cyclo (His-Pro) concentrations in the newborn animals. In the offspring from dams fed a 6% protein diet, caffeine administration significantly elevated brain weights and brain contents of protein, RNA and DNA. In contrast, these values were similar between noncaffeine and caffeine-supplemented animals in a 20% protein diet group. Brain TRH and cyclo (His-Pro) concentrations were not changed by caffeine administration. These data suggest that caffeine augments protein synthesis in the newborn rat brain when malnourished, but that the same dose of caffeine did not affect protein synthesis in brains of newborn rats from normally nourished dams. Therefore, the present findings indicate that the nutritional status of mothers during pregnancy has important implication in the impact of caffeine on their offspring's brains.     

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.     

1,2-Diacylglycerols overcome cyclic AMP-mediated inhibition of phosphatidylcholine synthesis in GH3 pituitary cells.

Previous studies showed that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells [Kolesnick (1987) J. Biol. Chem. 262, 14525-14530]. In contrast, 1,2-diacylglycerol-stimulated phosphatidylcholine synthesis appeared independent of protein kinase C. The present studies compare phosphatidylcholine synthesis stimulated by these agents with inhibition via the cyclic AMP system. The potent phorbol ester phorbol 12-myristate 13-acetate (PMA, 10 nM) increased [32P]Pi incorporation into phosphatidylcholine at 30 min to 159 +/- 6% of control. The adenylate cyclase activator cholera toxin (CT; 10 nM) and the cyclic AMP analogue dibutyryl cyclic AMP (1 mM) abolished this effect. CT similarly abolished TRH-induced phosphatidylcholine, but not phosphatidylinositol, synthesis. This is the first report of inhibiton of receptor-mediated phosphatidylcholine synthesis by the cyclic AMP system. The 1,2-diacylglycerol 1,2-dioctanoylglycerol (diC8) also stimulated concentration-dependent phosphatidylcholine synthesis. DiC8 (3 micrograms/ml) induced an effect quantitatively similar to that of maximal concentrations of PMA and TRH, whereas a maximal diC8 concentration (30 micrograms/ml) stimulated an effect 3-4-fold greater than these other agents. CT decreased the effect of diC8 (3 micrograms/ml) by 80%. Higher diC8 concentrations overcame the CT inhibition. Similar results were obtained with dibutyryl cyclic AMP. Additional differences were found between low and high concentrations of diC8. Low concentrations of diC8 failed to induce additive phosphatidylcholine synthesis with maximal concentrations of PMA, whereas high concentrations were additive. Hence, low concentrations of 1,2-diacylglycerols appear to be regulated similarly to phorbol esters, and higher concentrations appear to act via a pathway unavailable to phorbol esters.     

Toxic Effects of Maternal Zearalenone Exposure on Intestinal Oxidative Stress,Barrier Function,Immunological and Morphological Changes in Rats

The present study was conducted to investigate the effects of maternal zearalenone (ZEN) exposure on the intestine of pregnant Sprague-Dawley (SD) rats and its offspring. Ninety-six pregnant SD rats were randomly divided into four groups and were fed with diets containing ZEN at concentrations of 0.3 mg/kg, 48.5 mg/kg, 97.6 mg/kg or 146.0 mg/kg from gestation days (GD) 1 to 7. All rats were fed with mycotoxin-free diet until their offspring were weaned at three weeks of age. The small intestinal fragments from pregnant rats at GD8, weaned dams and pups were collected and studied for toxic effects of ZEN on antioxidant status, immune response, expression of junction proteins, and morphology. The results showed that ZEN induced oxidative stress, affected the villous structure and reduced the expression of junction proteins claudin-4, occludin and connexin43 (Cx43) in a dose-dependent manner in pregnant rats. Different effects on the expression of cytokines were also observed both in mRNA and protein levels in these pregnant groups. Ingestion of high levels of ZEN caused irreversible damage in weaned dams, such as oxidative stress, decreased villi hight and low expression of junction proteins and cytokines. Decreased expression of jejunal interleukin-8 (IL-8) and increased expression of gastrointestinal glutathione peroxidase (GPx2) mRNA were detected in weaned offspring, indicating long-term damage caused by maternal ZEN. We also found that the Nrf2 expression both in mRNA and protein levels were up-regulated in the ZEN-treated groups of pregnant dams and the high-dose of ZEN group of weaned dams. The data indicate that modulation of Nrf2-mediated pathway is one of mechanism via which ZEN affects gut wall antioxidant and inflammatory responses.     

Prostaglandin E2 synthesis elicited by adrenergic stimuli in guinea pig trachea is mediated primarily via activation of beta 2 adrenergic receptors.

Prostaglandin (PG) E2 synthesis elicited by adrenergic agonists in the guinea pig trachea has been shown to be mediated via activation of beta-adrenergic receptors. The purpose of this study was to examine arachidonic acid (AA) metabolism and to characterize the subtype of beta receptor involved in PG synthesis. [14C]AA was incubated with guinea pig tracheal rings, and the radiolabelled products were extracted from the medium. Thin layer chromatographic analysis and radioimmunoassay of the extract showed that [14C]AA was incorporated into guinea pig tracheal rings and metabolized mainly into radiolabeled and immunoreactive PGE2 (iPGE2) and smaller amounts into PGF2 alpha. Trace amounts of PGD2, TxB2 and 6-keto-PGF1 alpha but not LTB4 or LTC4 were detected by enzyme immunoassay. Incubation of guinea pig tracheal rings for 10 min with isoproterenol or salbutamol resulted in a significant increase in PGE2 synthesis (optimum concentration 0.1 microM for both compounds). In contrast, dobutamine, BRL 37344, BRL 28410, norepinephrine, phenylephrine, and xylazine (up to 1 microM) did not significantly increase PGE2 production. Isoproterenol-induced iPGE2 production was inhibited by the selective beta 2 receptor antagonist butoxamine (0.1-1.0 microM) and somewhat reduced by the beta 1 receptor antagonist practolol (1 microM). The increase in PGE2 synthesis was diminished with increasing concentrations of isoproterenol (0.5-5.0 microM) or salbutamol (0.5-1.0 microM); but it was reversed by pretreatment of tracheal rings with the protein synthesis inhibitors cycloheximide (0.9 microM) and actinomycin D (2 microM) but not by phenylisopropyl adenosine (0.1-1.0 microM), an inhibitor of adenylyl cyclase. These data suggest that isoproterenol-induced iPGE2 synthesis is primarily via activation of a beta 2 adrenergic receptor. Failure to enhance iPGE2 synthesis by a high concentration of isoproterenol is likely to be due to an induction of new inhibitory protein synthesis.     

The Synthesis of Ribosomes in E. coli: IV. The Synthesis of Ribosomal Protein and the Assembly of Ribosomes

The incorporation of C¹⁴ leucine into the protein moiety of ribosomes has been studied as a sequel to the studies of ribosomal RNA synthesis. In contrast to the latter studies, labeled leucine is incorporated directly into 50S and 30S ribosomes without measurable delay by precursor stages. There is, however, evidence of some transfer of radioactivity from the 43S group of particles to the 50S. The inhibition of protein synthesis by chloramphenicol results in the accumulation of material similar to the eosome—the primary precursor in ribosome synthesis. There is also evidence for the synthesis of some neosome. The results of the studies of ribosomal RNA and protein synthesis are combined into a model of ribosome synthesis. Finally, consideration is made of the significance of these studies of ribosome synthesis for general problems of protein synthesis and information transfer.     

Maternal postpartum learned helplessness (LH) affects maternal care by dams and responses to the LH test in adolescent offspring

It is known that the early environment affects the mental development of rodent and human offspring. However, it is not known specifically whether a postpartum depressive state influences the depressive state in offspring. Using learned helplessness (LH) in rats as an animal model of depression, we examined the influence of maternal postpartum LH on responses to the LH test of offspring. Dam rats were judged as LH or non-helpless (nLH) on postnatal days (PN) 2-3, and maternal behavior was recorded during PN2-14. On PN 45-46, offspring were subjected to the LH test. Plasma corticosterone (CORT) levels, hippocampal levels of glucocorticoid receptor (GR) and brain-derived neurotrophic factor (BDNF) mRNA were measured before and after the LH test in offspring. Active nursing in LH dams was significantly lower than that in nLH dams. Susceptibility to LH in the offspring of LH dams was significantly higher than in those of nLH dams, and was negatively correlated with active nursing by LH dams. The GR mRNA levels before and after the LH test were lower in the offspring of LH dams than in those of nLH dams, and the reduced basal GR mRNA and protein might have resulted in the higher CORT response after the LH test. There was no significant difference in BDNF mRNA in the offspring of LH and nLH dams. These findings suggest that early postpartum LH decreased active nursing and increased depression-like behavior in the adolescent offspring via dysfunction of the hypothalamic-pituitary-adrenal axis.     

Deficiency of joining of Okazaki-type fragments in absence of cellular protein synthesis.

The dependence of integration of newly formed DNA chain ( less than 10 S) into larger DNA on concomitant protein synthesis was studied in a special cellular system. Exponentially growing Ehrlich ascites tumor cells in vivo show decreasing rated and finally complete cessation of protein and DNA synthesis upon transfer into an isotonic but non-nutritive environment (Hanks' balanced salt solution). Both protein and DNA synthesis is stimulated in these cells for a period of 30 min when they are placed into fresh Hanks' balanced salt solution; however, stimulation of protein synthesis is completely prevented in Hanks' balanced salt solution containing cycloheximide. This system allowed us to investigate the formation and fate of newly formed DNA chains ( less than 10 S) in dependence of protein synthesis. Analysis of DNA produced in [3H]thymidine pulses showed that DNA chains smaller than 18 S were still formed during the phase of totally delayed protein synthesis and in the presence of cycloheximide, but they were not converted into DNA molecules sedimenting faster than 18 S under these conditions. Stimulation of protein synthesis for a period of 30 min allowed the short DNA pieces to be chased into larger DNA 30 min post stimulation of protein synthesis. The results clearly indicate that DNA chain growth, by sealing of DNA chains smaller than 18 S, is strongly dependent of concomitant protein synthesis. Direct chain elongation by addition of new deoxyribonucleotides is less dependent on concomitant cellular protein synthesis.