Exercise-induced stress enhances mammary tumor growth in rats: beneficial effect of the hormone melatonin

We hypothesized that intense exercise training (forced swimming for 30 min, 5 days/week) may enhance the progression of mammary carcinogenesis through the involvement of stress hormones, such as catecholamines and prolactin, which can promote breast cancer. After the appearance of the DMBA-induced tumors in Sprague-Dawley rats, the effect was evaluated of exercise-induced stress (with or without administration of the hormone melatonin) on the survival time, tumor multiplicity, and tumor growth until the death of the animals. In a second set of experiments, after one month of exercise, the NK cells count in blood, and the plasma concentrations of catecholamines and prolactin were determined. Although no significant change was found in either the survival time of the rats or the tumor multiplicity, exercise significantly increased the tumor growth rate. Stress was confirmed by the enhanced adrenaline and prolactin concentrations in the blood of the exercised rats. Exercise-induced stress did not change the percentage of NK cells in the tumor-bearing rats. Melatonin counteracted the increased tumor growth, returning the prolactin and adrenaline concentrations to their optimal physiological levels in the exercised tumor-bearing rats, thus confirming an “anti-stress” role of this hormone. In conclusion, intense exercise-induced stress enhances mammary carcinogenesis through the involvement of adrenaline and prolactin. The results also confirmed a role of melatonin as a therapeutic aid against breast cancer in general, and in particular during situations of stress.     

Bisexual behavior in the male rat: Influence of masculine sexual activity on the display of lordosis behavior

Sexually inexperienced male Wistar rats (strain WI in our colony) known to very infrequently display spontaneous lordosis behavior (Schaeffer et al., 1990b) were used. A first group was tested four times at 5-day intervals for lordosis with vigorous stimulus males (heterotypic sexual behavior), immediately following testing for masculine sexual activity with highly receptive females (homotypic sexual behavior). A small number of animals displayed lordosis during the first test, but more and more animals displayed this behavior from the first to the fourth test. There was no relationship between the degree of masculine sexual activity--intromission without ejaculation or ejaculation--and the occurrence of lordosis behavior. A second group was tested only once for both masculine sexual activity and lordosis behavior as above and afterwards three times at 5-day intervals for lordosis behavior in the absence of any previous testing for masculine sexual activity. A few animals displayed lordosis during their first test. As compared to the first group, the animals which had not displayed lordosis in the first test never showed lordosis responses in the following tests. It is concluded that both homotypic and heterotypic sexual interactions are required for the display of lordosis behavior in the strain of Wistar rats used in this study.     

Skeletal muscle lipid peroxidation in exercised and food-restricted rats during aging

The effects of chronic endurance exercise and food restriction on nonenzymatic lipid peroxidation (LP) of gastrocnemius muscle during aging were studied in male, Fischer 344 rats. One set of rats aged 6 and 18 mo were assigned to an exercise group (treadmill running) or an age-matched sedentary control group. After 6 mo (at the ages of 12 and 24 mo), LP and levels of alpha-tocopherol and its oxidized form, alpha-tocopheryl quinone, were measured. The extent of LP was determined in homogenates by measuring the content of thiobarbituric acid-reactive substances. After homogenization, the muscles were immediately evaluated for basal LP and also incubated in the presence of oxidant stressors for 2 h to assess antioxidant capacity (AOC) and for 24 h to estimate total peroxidizable lipid (TPL). Basal LP was not affected by age or exercise. AOC was not affected by exercise at either age. However aging significantly decreased AOC and increased alpha-tocopheryl quinone in both sedentary and exercised groups. TPL was not affected by age, but was increased by exercise training (P less than 0.05). Another set of rats was divided into the following three groups at 3 mo of age: sedentary, fed ad libitum (S); sedentary, caloric restricted by alternate day feeding (R); and exercised by forced treadmill running (E). Two years later, when the rats were 27 mo of age, the extent of LP was assessed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exercise-induced HSP-72 elevation and cardioprotection against infarct and apoptosis.

Successive bouts of endurance exercise are associated with both increased cardiac levels of heat shock protein-72 (HSP-72) and improved cardioprotection against ischemia-reperfusion (I/R)-induced cardiac cell death. Although overexpression of HSP-72 has been shown to be cardioprotective in transgenic animals, it is unclear whether increased levels of HSP-72 are essential for exercise-induced cardioprotection against I/R-mediated cell death. We tested the hypothesis that exercise-induced increases in myocardial levels of HSP-72 are required to achieve exercise-mediated protection against I/R-induced cardiac cell death. To test this postulate, we investigated the effect of preventing the exercise-induced increase in cardiac HSP-72 on myocardial infarction and apoptosis after 50 min of in vivo ischemia and 120 min of reperfusion. Adult male rats remained sedentary or performed successive bouts of endurance exercise in cold (8 degrees C) or warm (22 degrees C) environments. We found that, compared with sedentary control animals, exercise in a warm environment significantly increased myocardial HSP-72 content. In contrast, exercise in the cold environment prevented the exercise-induced increase in myocardial HSP-72 levels. After in vivo myocardial I/R, infarct size was reduced in both exercised groups compared with sedentary animals. Furthermore, compared with sedentary rats, I/R-induced myocardial apoptosis (as indicated by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling-positive nuclei and caspase-3 activity) was attenuated in both groups of exercised animals. Therefore, although HSP-72 has cardioprotective properties, our results reveal that increased myocardial levels of HSP-72 (above control) are not essential for exercise-induced protection against I/R-induced myocardial infarction and apoptosis.     

Variation in the responsiveness of female rats to ovarian hormones as a function of preceding hormonal deprivation

Ovariectomized female rats were tested for the display of lordosis behavior 30 days after gonadectomy. They were then tested 7, 14, 21 and 81 days later following estrogen and progesterone treatment. Finally, on Day 88 of the experiment the animals were tested after either estrogen and progesterone treatment or after progesterone alone. The response to estrogen and progesterone treatment was found to be limited on the first test and on the fifth test which occurred after 2 mo without hormone treatment. When hormone treatment was repeated at seven day intervals (Tests 2–4) the tendency to show lordosis increased markedly. On the final test the animals given both hormones showed lordosis, while those which received only progesterone did not. The data suggest that the response to estrogen decreases after estrogen deprivation.     

Ozone inhalation effects consequent to continuous exercise in females: comparison to males

Exposure to ozone (O3) at ambient photochemical smog alert levels has been shown to cause alteration in pulmonary function and exercise response in humans, but there is a paucity of data on females. The initial purpose of the present investigation was to study the effects of O3 inhalation on pulmonary function and selected exercise respiratory metabolism and breathing pattern responses in young adult females. Six female subjects exercised continuously on a bicycle ergometer for 1 h on 10 occasions at one of three intensities, while exposed to 0.0, 0.20, 0.30, or 0.40 ppm O3. Forced expiratory volume and flow rates and residual volume (RV) were measured before and immediately following each protocol. During exercise, expired minute ventilation (VE), respiratory frequency (fR), tidal volume, O2 uptake (VO2), and heart rate (HR) were measured every 10 min. O3 dose-dependent decrements were observed for forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1.0), and forced expiratory flow rate during the middle half of FVC, coupled with an increase in RV and altered exercise ventilatory pattern. There was also an increased VE but no significant O3 effect on VO2 or HR. Comparison of the females' responses to those of a group of young adult males (previously studied) at the same total O3 effective dose (i.e., expressed as the simple product of O3 concentration, VE, and exposure time) revealed significantly greater effects on FVC, FEV1.0, and fR for the females. With VE reduced for females as a function of exercise intensity at the same percent of maximum VO2, these differences were considerably attenuated, although not negated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of proceptive and receptive behavior by ovarian hormones in the Syrian hamster (Mesocricetus auratus)

Two studies examined the roles of estrogen with progesterone and of estrogen alone on the proceptive and receptive behavior of female hamsters. Proceptivity was measured in terms of proximity (approaching, leaving, and following by the female) and in time spent sniffing the male partner. During the 4-day natural estrous cycle, these measures change systematically although lordosis is seen only on Day 1 (estrus). With a constant dose of progesterone, both proceptive and receptive behavior were found to be estrogen dose dependent in ovariectomized females. At estrogen levels too low to induce lordosis, changes in proceptive behavior were seen; at the two highest levels of estrogen, lordosis was maximal but proceptive behavior continued to increase. With estrogen alone, levels of proceptive behavior were attained characteristic both of estrus and of the higher estrogen and progesterone dosage but were not accompanied by spontaneous lordosis. Factors indicating that proceptivity and receptivity may be under separate hormonal and neural control are discussed.     

Hysterectomy-induced facilitation of lordosis behavior in the rat

The present study investigated the effect of hysterectomy on hormone-induced lordosis behavior. Lordosis quotients (LQ) were measured in hysterectomized-ovariectomized (HO) and ovariectomized-sham hysterectomized (OSH) rats after several treatments including either estradiol benzoate (EB) alone or EB plus progesterone (P) 44 hr later. Testing consisted of placing the females with sexually active males 48 hr after EB. In Experiment 1, HO animals treated with 5 μg/kg EB and 0.5 mg P had significantly higher LQs than OSH animals; groups treated with 10 μg/kg plus P were not different. Experiment 2 showed that a single injection of 50 μg/kg EB resulted in equally high levels of receptivity in both groups. The LQs of HO animals injected with 3 μg/kg for 4 days did not differ from those of OSH animals; however, the administration of 0.5 mg P 24 hr after the fourth EB injection resulted in significantly higher LQs in the HO group (Experiment 3). In Experiment 4, HO rats injected with 5 μg/kg EB and 0.1 mg P 44 hr later displayed higher levels of lordosis behavior than OSH animals. It was concluded that hysterectomy facilitated the lordosis behavior of ovariectomized rats injected with both EB and P and that the mechanism for this potentiation remains to be determined.     

Lordosis behavior in male rats after lesions in different regions of the corticomedial amygdaloid nucleus

The aim of the experiment was to study the effects of stereotaxic lesions of the anterior and the posterior regions of the corticomedial amygdaloid nucleus (CMN) on the display of lordosis behavior by the male rat. Animals were orchidectomized as adults and given estradiol benzoate and progesterone (P) sequentially. Sexual behavior testing was performed by 9 +/- 1 hr after P injection. Lesions placed into the posterior region of the CMN significantly decreased the proportion of animals showing lordosis behavior as compared to sham-operated and control animals. By contrast lesions in the anterior region of the CMN did not cause any changes in the proportion of animals displaying lordosis but markedly increased the lordosis quotient (LQ) of responding animals. The CMN was then concluded to exert a dual control in the display of lordosis behavior in the male rat with a posterior region regulating the willingness of animals to display lordosis behavior and rostral region subserving inhibitory mechanisms related to the sexual performance (LQ values).     

N-Acetyl-L-cysteine prevents exercise-induced intestinal lymphocyte apoptosis by maintaining intracellular glutathione levels and reducing mitochondrial membrane depolarization

Intense exercise leads to post-exercise lymphocytopenia and immunosuppression, possibly by triggering lymphocyte apoptosis. To test the role of oxidative stress on exercise-induced lymphocyte apoptosis, we administered the antioxidant N-acetyl--cysteine (NAC) and measured apoptosis in intestinal lymphocytes (IL) from exhaustively exercised animals. Eighty-seven female C57BL/6 mice were randomly assigned to receive NAC (1 g/kg) or saline 30 min prior to treadmill exercise for 90 min at 2degrees slope (30 min at 22 m min(-1), 30 min at 25 m min(-1), and 30 min at 28 m min(-1)) and sacrificed immediately (Imm) or 24 hours (24 h) after cessation of exercise. Control mice (nonexercised) were exposed to treadmill noise and vibration without running. Exercise increased IL phosphatidylserine externalization (p<0.001), mitochondrial membrane depolarization (p<0.05), and decreased intracellular glutathione concentrations (p<0.05) immediately following exercise in saline relative to nonexercised mice. At 24 h post-exercise, saline injected mice had fewer total (p<0.001) and CD3+ (p<0.005) IL compared to nonexercised animals. NAC injection in mice maintained intracellular glutathione levels, prevented phosphatidylserine externalization, mitochondrial membrane depolarization, and loss of IL immediately and 24 h after exercise. These data suggest that lymphocyte apoptosis precedes post-exercise lymphocytopenia and may be due to oxidative stress.     

Food deprivation inhibits estrous behavior in hormone-treated Syrian hamsters despite elevated estradiol levels

Estradiol and progesterone (P) induce female mammalian reproductive behaviors, which are, in turn, sensitive to food availability. When ovariectomized, steroid-primed hamsters are food deprived for 48 h, estrous behavior is suppressed. While this suppression of estrous behavior may be due to alterations in neural steroid receptor levels, it is also possible that decreased levels of circulating estradiol could be involved in mediating this suppression. Ovariectomized Syrian hamsters given varying doses of estradiol benzoate (EB) and P were tested to determine whether increasing doses of sex steroids would overcome the suppressive effects of food deprivation on estrous behavior. As expected, lordosis duration decreased in food-deprived animals. Increasing the levels of EB, but not P, increased lordosis duration in the food-deprived animals so that animals who were given 20 microg of EB had lordosis durations significantly longer than food-deprived hamsters that received 1.5 microg and 2.5 microg EB. Following an injection of 2.5 microg of EB, food-deprived hamsters actually had higher circulating levels of estradiol than ad libitum-fed animals. Therefore, increasing circulating levels of estradiol can increase lordosis durations in fasted animals; however, the suppression of estrous behavior occurs despite increased circulating estradiol levels in ovariectomized, steroid-treated animals. The most parsimonious explanation for this phenomenon is a deprivation-induced reduction in neural responsiveness to estradiol.     

Bimodal respiration and ventilatory behavior in two species of central American turtles: effects of forced submergence

Respiratory gas exchange in both air and water was measured at rest and during recovery from forced submergence in the giant Mexican musk turtle (Staurotypus triporcatus) and the white-lipped mud turtle (Kinosternon leucostomum). Diving and ventilatory behavior were also measured in unrestrained animals of each species. Despite large differences in cutaneous surface area, both species exhibited an aquatic V(O(2)) and V(CO(2)) of approximately 16 and 45%, respectively, with the remainder explained by aerial gas exchange. Aquatic V(O(2)) and V(CO(2)) did not significantly change during forced submergence or during the recovery period. Aerial V(O(2)) and V(CO(2)), however, profoundly increased after forced submergence in both species and were not significantly different from resting values until approximately 60 min following the treatment. At rest, K. leucostomum took significantly more breaths per breathing bout than S. triporcatus. This inherent ventilation pattern in each species remained unaltered following forced submergence. Cutaneous surface area, therefore, remains a minor component for these two species which rely heavily on pulmonary gas exchange to recover from forced submergence.     

Effect of exercise-induced hyperthermia on serum iron concentration

Serum iron levels have been shown to decline both with fever and with strenuous exercise, leading to the supposition that the decrease might be the result of a rise in core body temperature. To evaluate this hypothesis, the serum iron response to an exercise-induced 1.5°C rise in core body temperature was measured. To increase core temperature, five females and two males exercised in an environmental chamber heated to 41°C with a relative humidity of 40%. Blood samples were taken before exercise and immediately after body temperature increased approximately 1.5°C. Blood was also collected 1 h, 6 h, and 24 h postexercise. Results showed that the core body temperature significantly increased (p<0.001) from a mean baseline value of 36.5±0.1°C to 38.1±0.1°C following exercise. A one-way repeated measures analysis of variance was used to examine the effect of increased core body temperature on serum iron levels over the five time periods: preexercise, immediate postexercise, and 1 h, 6 h, and 24 h postexercise. The results indicated that there were no significant differences in serum iron levels among time periods. This suggests that the previously reported depression of serum iron levels that occurs with fever and after prolonged exercise is not the result of hyperthermia. Rather, the change in serum iron occurs in response to biological or physiological stressors, such as bacterial infection, muscle damage, or unusual trauma. Further studies are needed to explicate the mechanisms responsible for these changes.     

Facilitation of female sexual behavior in male rats by septal lesions: an interaction with estrogen.

Although destruction of the septal region markedly facilitates the lordosis behavior of female rats in response to estrogen priming, comparable lesions were found to be ineffective in facilitating the lordotic behavior of estrogen primed male rats. Neither the age at the time of septal destruction nor castration influenced the lordosis behavior of males. However, if prepubertal castrated males were given subcutaneous ovarian grafts or injected daily with 2 μgm estradiol benzoate (EB) during the 30 day period following septal destruction, a prolonged facilitation of the activational effects of EB on lordosis behavior was observed. Male rats subjected to septal destruction alone, chronic exposure to EB alone, exposure to ovarian grafts for 30 days prior to septal destruction, or chronic treatment with EB started 6 mo after septal lesioning, failed to show an increase in behavioral responsiveness to estrogen. Thus, in order for septal lesions to facilitate lordosis behavior of male rats, exposure to EB or ovarian tissue must occur within an apparent critical period following septal destruction. Adult male rats were found to be more responsive to this interaction of septal lesions and EB exposure than pubertal animals. It is suggested that the prolonged facilitation of lordosis behavior which follows septal destruction and estrogen exposure in the male rat may be due to hormonal modifications of the recovery process following brain damage.     

Effects of nitric oxide synthase inhibitor on decrease in peripheral arterial stiffness with acute low-intensity aerobic exercise

We previously reported that even low-intensity, short-duration acute aerobic exercise decreases arterial stiffness. We aimed to test the hypothesis that the exercise-induced decrease in arterial stiffness is caused by the increased production of NO in vascular endothelium with exercise. Nine healthy men (age: approximately 22-28 yr) performed a 5-min single-leg cycling exercise (30 W) in the supine position under an intravenous infusion of NG-monomethyl-L-arginine (L-NMMA; 3 mg/kg during the initial 5 min and subsequent continuous infusion of 50 mug.kg(-1).min(-1) in saline) or vehicle (saline) in random order on separate days. The pulse wave velocity (PWV) from the femoral to posterior tibial artery was measured on both legs before and after the infusion at rest and 2 min after exercise. Under the control condition, exercised leg PWV significantly decreased after exercise (P <0.05), whereas nonexercised leg PWV did not show a significant change throughout the experiment. Under L-NMMA administration, exercised leg PWV was increased significantly by the infusion (P <0.05) but decreased significantly after the exercise (P <0.05). Nonexercised leg PWV increased with L-NMMA administration and maintained a significantly higher level during the administration compared with baseline (before the infusion, all P <0.05). The NO synthase blockade x time interaction on exercised leg PWV was not significant (P=0.706). These results suggest that increased production of NO is not a major factor in the decrease of regional arterial stiffness with low-intensity, short-duration aerobic exercise.     

Estrogen and progesterone interactions influencing sexual and social behavior in the brown lemming, Lemmus trimucronatus.

The effects of estrogen and progesterone on the social and sexual behavior of brown lemmings, Lemmus trimucronatus, were investigated. The behavior of hormone-treated and untreated ovariectomized females and sexually vigorous males was observed in six consecutive daily 5-min dyadic encounters. Sexual receptivity, as measured by lordosis, and other social behaviors including nasonasal contact, boxing postures, allogrooming, perineal investigation, and male mounting increased following 48 hr of exposure to daily injections of 0.5 μg estradiol benzoate (EB). Lordosis in EB-primed females was not facilitated or inhibited by short-term (4 hr) exposure to 0.5 mg progesterone (P). Long-term (greater than 24 hr) exposure to P apparently inhibited lordosis and other social behaviors in EB-treated females, although males continued to attempt to mount these females. In EB-treated females a dramatic increase in threat-leaps, directed by the female toward the male, was observed within 4 hr of P injection. Threat-leaps declined when P was withdrawn. Threat-leaps were also observed in ovariectomized females after prolonged exposure to P only (0.5 mg/day). Vaginal perforation and cornification were first apparent 48 hr after EB injection. P-alone treated ovariectomized females also showed vaginal perforation but cornified cells were infrequent and these animals did not show lordosis.     

Effects of progesterone implants in the habenula and midbrain on proceptive and receptive behavior in the female rat

Two brain areas behaviorally responsive to progesterone (P) were examined to determine their possible involvement in the control of rat preceptive behavior, i.e., solicitation behavior directed at the male. Progesterone implants were placed in the habenular nuclei and the interpeduncular nucleus-ventral tegmental area of the midbrain reticular formation (MRF). Different testing procedures and levels of priming with estradiol benzoate (EB) were used in order to distinguish the effects of P in either region on proceptive and receptive behavior during exposure to 10 mounts by stimulus males. To test for receptivity, sexually experienced 60-day-old ovariectomized (ovx) rats bearing stereotaxically placed guide cannulas extending to the habenula or MRF were given 10 μg EB subcutaneously. Forty-eight hours later, lordosis quotient (LQ) was determined. Immediately following this test, each animal was implanted with cholesterol (C) or P and was retested 2 hr later. Treatments for the proceptivity test were similar except that the animals received 2.5 μg EB/100 g body wt sc for 7 days before testing on the eighth day; LQ as well as hopping, darting, and ear wiggling were scored. In the receptivity test, P implantation in both the medial portions of the habenula and the MRF significantly increased lordosis above the levels found both in their preimplantation tests and following control implantation of C. Little proceptivity was observed. In the proceptivity test, P implants in both regions also significantly increased proceptive behavior above both types of control tests. All animals were highly receptive, and there was no difference in LQ among the groups. There was no increase of plasma P levels in similarly implanted animals during a 24-hr monitoring period, indicating that systemic leakage of the hormone was not responsible for the observed behavior. The data indicate that both the habenula and MRF are P-sensitive regions. Progesterone's action on the two areas facilitates expression of both proceptive and receptive components of female sexual behavior, indicating that the neural regulation of the two kinds of behavior is integrated at these levels.     

The reversible inhibition of steroid-induced sexual behavior by intracranial cycloheximide

Cycloheximide(Cyclo), an inhibitor of protein synthesis by a direct action on protein synthesis at the ribosomal level, was used to reversibly inhibit estrogen-induced sexual receptivity. Cyclo (100 μg per rat) was infused into the preoptic area(POA) of ovariectomized rats at varying times before, simultaneously with, and after 3 μg of subcutaneous estradiol benzoate (EB). All animals received 0.5 mg progesterone (P) 36 hr after EB, and were tested for sexual receptivity 4–6 hr after P. The females were placed with stud males and a lordosis quotient was computed for each female (lordosis quotient = number of lordosis responses/20 mounts by the male × 100). Females receiving Cyclo 6 hr before, simultaneously with, or 12 hr after EB showed significantly lower levels of sexual receptivity when compared to females receiving Cyclo 36 hr before and 18 and 24 hr after EB. When those animals that showed low levels of sexual behavior after Cyclo infusion were reprimed with EB and P 7 days later and presented with a male they showed high levels of sexual receptivity. Thus, the effect of Cyclo was reversible. Only Cyclo infusions into the POA (bilateral) and third ventricle were effective in suppressing sexual behavior. Caudate nucleus, lateral ventricle, and unilateral POA infusions were without effect.The data presented are in agreement with earlier work that utilized actinomycin D to inhibit steroid-induced sexual behavior. Cyclo was found to be less toxic than actinomycin D. All of the available evidence is consistent with the hypothesis that estrogen stimulates RNA and/or protein synthesis in its facilitation of sexual behavior in the female rat.     

Regulation of lipoprotein lipase in muscle and adipose tissue during exercise.

Lipoprotein lipase (LPL) is regulated in a tissue-specific manner; exercise increases LPL activity in muscle at the same time it is reduced in adipose tissue. The purpose of this study was to determine the relationship between LPL activity and LPL mRNA in muscle and adipose tissue in rats exposed to one bout of exercise. Immediately after a 2-h swim, LPL activity [pmol free fatty acids (FFA).min-1.mg tissue-1] in the exercised animals was reduced 43% in adipose tissue (110 +/- 26 to 63 +/- 17) and increased almost twofold in the soleus muscle (203 +/- 26 to 383 +/- 59) compared with sedentary control animals. At the same time, LPL mRNA was reduced 42% in adipose tissue and increased 50 and 100% in the red vastus and white vastus muscles, respectively. Twenty-four hours after the swim, LPL activity had returned to control levels in adipose tissue and the soleus muscle. At hour 24 of recovery, LPL mRNA was still reduced 23% in the adipose tissue of exercised animals but was not significantly different between exercised and control animals in any of the muscle tissues analyzed. Changes in total RNA concentration could not account for the changes in relative LPL mRNA expression. The relationship between LPL enzyme activity and LPL mRNA in muscle and adipose tissue was +0.86 and +0.93 at 0 and 24 h postexercise, respectively. Thus the tissue-specific changes in enzyme activity induced by exercise could be mediated, in part, through pretranslational control.