Extracellular heme enhances the antimalarial activity of artemisinin.

Artemisinin exerts the antimalarial activity through activation by heme. The hemolysis in malaria results in the elevated levels of plasma heme which may affect the activity of artemisinin. We hypothesized that the extracellular heme would potentiate the antimalarial activity of artemisinin. Hemin (ferric heme) at the pathologic concentrations enhanced the activity of artemisinin against Plasmodium falciparum in vitro and increased the levels of the lipid peroxidation products in the presence of artemisinin. The antimalarial activity of artemisinin and potentiation by hemin was decreased by vitamin E. Hemin had no effect on the activity of quinoline drugs (chloroquine, quinine and mefloquine). Furthermore, the oxidative effect of hemin in the presence of artemisinin or quinoline drugs was studied using low-density lipoprotein (LDL) oxidation as a model. Artemisinin enhanced the effects of hemin on lipid peroxidation and a decrease of tryptophan fluorescence in LDL whereas the quinoline drugs inhibited the oxidation by hemin. In conclusion, the extracellular hemin enhances the antimalarial activity of artemisinin as a result of the increasing oxidative effect of hemin.

The effect of diet composition on tryptophan requirement of young piglets.

The aim of the study was to evaluate the requirement for Trp in relation to diet composition in piglets in the period after weaning (BW range of 9 to 24 kg). Two Trp-deficient [relative to the Dutch (CVB, 1996) and NRC (NRC, 1998) requirement values for piglets of 10 to 20 kg of BW] basal diets were formulated: one based on corn and soybean meal and a second one based on wheat, barley, soybean meal, peas, and whey powder [10.0 g/kg of apparent ileal digestible (AID) Lys; 1.4 g/kg of AID Trp; 1.5 g/kg of standardized ileal digestible (SID) Trp]. Both basal diets were supplemented with 0.3, 0.6, and 0.9 g of l-Trp per kg of diet to obtain diets with 1.7, 2.0, and 2.3 g of AID Trp per kg (1.8, 2.1, and 2.4 g of SID Trp per kg), respectively. Each of the 8 treatments was evaluated in 8 replicates (pens with 8 male or female piglets). Average daily feed intake, ADG, and G:F were measured as response criteria. Over the 28-d experimental period, ADG and G:F were greater for the treatments on the wheat/barley diet compared with those on the corn/soybean meal and were increased by the level of Trp in the diet (P < 0.05). Average daily feed intake was only increased by the level of Trp supplementation (P < 0.05). Increasing the Trp level increased ADFI for the corn/soybean meal diet up to 2.3 g of AID Trp per kg (2.4 g of SID Trp per kg) and up to 2.0 g of AID Trp per kg (2.1 g of SID Trp per kg) in the wheat/barley diet (P < 0.05). For both diet types supplementation of free l-Trp increased the G:F up to 1.7 g of AID Trp per kg (1.8 g of SID Trp per kg). Nonlinear regression analysis of the response curves for ADFI using an exponential model for estimating a requirement value for Trp (defined as the Trp level resulting in 95% of the maximum response) revealed a requirement estimate of 2.3 g of AID Trp per kg for the corn/soybean meal-based diet and 2.1 g of AID Trp per kg for the wheat/barley-based diet, equivalent to 2.4 and 2.2 g of SID Trp per kg of diet, respectively. For ADG, a requirement estimate of 2.1 g of AID Trp per kg for both types of diets was derived, equivalent to 2.2 g of SID Trp per kg of diet. The Trp requirement for young piglets seems to be greater than indicated by some commonly used recommendations and does not seem largely dependent on diet ingredient composition.

Effects of experimental acute tryptophan depletion on acoustic startle response in females.

Previous studies suggest an important role for serotonergic (5-HT) modulation of the acoustic startle reflex (ASR) and prepulse inhibition (PPI). Acute challenge of brain serotonin by means of tryptophan depletion test (TDT) represents an established human challenge tool for temporary reduction of tryptophan (-TRP) levels and central nervous serotonin. Under these experimental conditions, PPI was found attenuated in males, but greater biochemical effects of TDT in the central nervous system of females are known. Therefore, in order to explore influence of 5-HT on various standard startle parameters in females, 16 young healthy females participated in a double-blind, cross-over TDT study. Acoustic stimuli were presented in 15 pulse-alone trials (100 dB, 40 ms) randomly followed by 25 pulse-alone or prepulse (70 dB, 30 ms; 120 ms interval) trials alongside electromyographic eyeblink recordings and mood state assessments. During 81% depletion of free plasma TRP, mean ASR magnitudes were significantly reduced compared to control (+TRP) condition while there were no differences in habituation or PPI nor did startle parameters correlate with mood states. Changes of plasma TRP and mood states correlated in tendency negatively in (-TRP) for depression and positively in (+TRP) for fatigue. In conclusion, this first study of startle parameters after TDT in a homogenous female population demonstrates that depletion of brain 5-HT in women only influences ASR.

Patients with a major depressive episode responding to treatment with repetitive transcranial magnetic stimulation (rTMS) are resistant to the effects of rapid tryptophan depletion.

Repetitive transcranial magnetic stimulation (rTMS) appears to be efficacious in the treatment of major depression based on the results of controlled studies, but little is known about its antidepressant mechanism of action. Mood sensitivity following rapid tryptophan depletion (RTD) has been demonstrated in depressed patients responding to SSRI antidepressants and phototherapy, but not in responders to electroconvulsive therapy (ECT). We sought to study the effects of RTD in patients with major depression responding to a course of treatment with rTMS. Twelve subjects treated successfully with rTMS monotherapy underwent both RTD and sham depletion in a double-blind crossover design. Depressive symptoms were assessed using both a modified Hamilton Depression Rating Scale (HDRS) and Beck Depression Inventory (BDI). The differential change in depression scores across the procedures was compared. No significant difference in mood symptoms was noted between RTD and the sham-depletion procedure on either continuous measures of depression, or in the proportions of subjects that met predefined criteria for a significant degree of mood worsening. Responders to rTMS are resistant to the mood perturbing effects of RTD. This suggests that rTMS does not depend on the central availability of serotonin to exert antidepressant effects in major depression.

Studies on the tryptophan requirement of lactating sows. Part 2: Estimation of the tryptophan requirement by physiological criteria.

Mature sows were fed for a total of 72 lactations with diets which provided an adequate supply of energy and nutrients except for tryptophan (Trp). By supplementing a basal diet [native 1.2 g Trp/kg, equivalent to 0.8 g apparent ileal digestible (AID) Trp or 0.9 g true ileal digestible (TID) Trp] with L-Trp, five further diets (2-6) containing 1.5-4.2 g Trp/kg were formulated. The dietary Trp content had no effect on amino acid contents in milk on days 20 and 21 of lactation, but Trp in blood plasma on day 28 of lactation reflected the alimentary Trp supply with an increase from 2.74 +/- 1.14 mg/l (diet 1) to 23.91 +/- 7.53 mg/l (diet 6; p < 0.001). There were no directional differences between the diets with regard to the other amino acids. Concentrations of urea in milk and blood were higher with diet 1 (211 and 272 mg/l, respectively) than with diets 3-6 (183 and 227 mg/l, respectively). Serotonin levels in the blood serum were lower with diet 1 (304 ng/ml) than the average of diets 4-6 (540 ng/ml). This study confirms previously given recommendations for the Trp content in the diet of lactating sows, estimated by means of performance, of 1.9 g AID Trp (equivalent to 2.0 g TID Trp; approximately 2.6 g gross Trp) per kg diet.

The tryptophan requirement of growing and finishing barrows.

Five experiments were conducted to determine the true ileal digestible Trp (tidTrp) requirement of growing and finishing pigs fed diets (as-fed basis) containing 0.87% (Exp. 3), 0.70% (Exp. 4), 0.61% (Exp. 5), and 0.52% (Exp. 1 and 2) tidLys during the early-grower, late-grower, early-finisher, and late-finisher periods, respectively. Treatments were replicated with three or four replications, with three or four pigs per replicate pen. Treatment differences were considered significant at P = 0.10. Experiment 1 was conducted with 27 pigs (initial and final BW of 78.3 +/- 0.5 and 109.8 +/- 1.9 kg) to validate whether a corn-feather meal (FM) tidTrp-deficient (0.07%) diet, when supplemented with 0.07% crystalline l-Trp, would result in growth performance and carcass traits similar to a conventional corn-soybean meal (C-SBM) diet. Pigs fed the corn-FM diet without Trp supplementation had decreased growth performance and carcass traits, and increased plasma urea N (PUN) concentration. Supplementing the corn-FM diet with Trp resulted in greater ADG and G:F than pigs fed the positive control C-SBM diet. Pigs fed the corn-FM diet had similar carcass traits as pigs fed the C-SBM diet, but loin muscle area was decreased and fat thickness was increased. In Exp. 2, 60 pigs (initial and final BW of 74.6 +/- 0.50 and 104.5 +/- 1.64 kg) were used to estimate the tidTrp requirement of finishing pigs. The levels of tidTrp used in Exp. 2 were 0.06, 0.08, 0.10, 0.12, or 0.14% (as-fed basis). Response variables were growth performance, PUN concentrations, and carcass traits and quality. For Exp. 2, the average of the estimates calculated by broken-line regression was 0.104% tidTrp. In Exp. 3, 4, and 5, barrows (n = 60, 60, or 80, respectively) were allotted to five dietary treatments supplemented with crystalline l-Trp at increments of 0.02%. The basal diets contained 0.13, 0.09, and 0.07% tidTrp (as-fed basis) in Exp. 3, 4, and 5, and initial BW of the pigs in these experiments were 30.9 +/- 0.7, 51.3 +/- 1.1, and 69.4 +/- 3.0 kg, respectively. The response variable was PUN, and the basal diet used in Exp. 3 and 4 contained corn, SBM, and Canadian field peas. The tidTrp requirements were estimated to be 0.167% for pigs weighing 30.9 kg, 0.134% for pigs weighing 51.3 kg, and 0.096% for pigs weighing 69.4 kg. Based on our data and a summary of the cited literature, we suggest the following total Trp and tidTrp requirement estimates (as-fed basis): 30-kg pigs, 0.21 and 0.18%; 50-kg pigs, 0.17 and 0.14%; 70-kg pigs, 0.13 and 0.11%; and in 90-kg pigs, 0.13 and 0.11%.

Serotonergic modulation of prefrontal cortex during negative feedback in probabilistic reversal learning.

This study used functional magnetic resonance imaging to examine the effects of acute tryptophan (TRP) depletion (ATD), a well-recognized method for inducing transient cerebral serotonin depletion, on brain activity during probabilistic reversal learning. Twelve healthy male volunteers received a TRP-depleting drink or a balanced amino-acid drink (placebo) in a double-blind crossover design. At 5 h after drink ingestion, subjects were scanned while performing a probabilistic reversal learning task and while viewing a flashing checkerboard. The probabilistic reversal learning task enabled the separate examination of the effects of ATD on behavioral reversal following negative feedback and negative feedback per se that was not followed by behavioral adaptation. Consistent with previous findings, behavioral reversal was accompanied by significant signal change in the right ventrolateral prefrontal cortex (PFC) and the dorsomedial prefrontal cortex. ATD enhanced reversal-related signal change in the dorsomedial PFC, but did not modulate the ventrolateral PFC response. The ATD-induced signal change in the dorsomedial PFC during behavioral reversal learning extended to trials where subjects received negative feedback but did not change their behavior. These data suggest that ATD affects reversal learning and the processing of aversive signals by modulation of the dorsomedial PFC.

Nutrition, brain function and cognitive performance.

Military interest in the effects of nutritional factors on cognitive function has stimulated considerable research on a variety of food constituents. This paper will review the research on the amino acids tryptophan and tyrosine, caffeine and carbohydrate. It will focus on research that addresses the potential utility of these compounds in military applications, particularly the acute, as opposed to chronic, effects of these substances on cognitive functions such as alertness, vigilance and resistance to stress. Caffeine, the most intensively studied food constituent, has unequivocal beneficial effects on vigilance, and in sleep deprived individuals it enhances other cognitive functions as well. Tryptophan, although it clearly has sedative-like properties, has not been extensively studied by military laboratories for use as a hypnotic, due to safety concerns. Tyrosine has been examined in animal models and human studies, and appears to prevent the substantial decline in various aspects of cognitive performance and mood associated with many kinds of acute stress. Carbohydrate supplementation appears to enhance cognitive performance in soldiers engaged in sustained, intense physical activities that expend high levels of energy.

Effects of tryptophan depletion and catecholamine depletion on immune parameters in patients with seasonal affective disorder in remission with light therapy.

BACKGROUND: Altered immunologic parameters are found in symptomatic depressed patients relative to remitted depressed patients and healthy controls. We investigated whether tryptophan depletion and catecholamine depletion induce alterations in immunologic parameters in patients with seasonal affective disorder remitted on light therapy, and whether these changes are associated with changes in mood. METHODS: Remitted patients with seasonal affective disorder underwent tryptophan depletion, catecholamine depletion, and sham depletion in a prospective randomized, double-blind crossover design. Measures of depression, plasma levels of tryptophan and catecholamine metabolites, and plasma levels of cytokines (sIL-4, IL-6, neopterin, sTNF-R1 and sTNF-R2) were obtained at baseline, and 7, 24, and 30 hours after monoamine depletion. RESULTS: Tryptophan depletion decreased plasma total and free tryptophan levels; catecholamine depletion decreased plasma 3-methoxy-4-hydroxyphenylethyleneglycol and homovanillic acid levels. Tryptophan depletion and catecholamine depletion, but not sham depletion, induced a transient exacerbation of depressive symptoms (p <.001); plasma neopterin levels increased during tryptophan depletion and catecholamine depletion (p <.05). Tryptophan depletion and catecholamine depletion induced a transient reduction of plasma sIL-4 levels (p <.05). A significant correlation was found between sIL-4R levels and depression ratings after tryptophan depletion (r = -.61, p <.05). CONCLUSIONS: The monoamine depletion-induced alterations of humoral and cellular immunity suggest a potential role of immunologic parameters in the pathophysiology of seasonal affective disorder; however, the results must be considered preliminary and require further study.

Monoaminergic function in the pathogenesis of seasonal affective disorder.

Seasonal affective disorder/winter type (SAD) is characterized by recurrent depressive episodes during autumn and winter alternating with non-depressive episodes during spring and summer. Light therapy with full-spectrum, bright white light has been shown to be effective for this condition. Several hypotheses have been discussed in the literature about the pathogenesis of SAD. The most prominent includes disturbances in central monoaminergic transmission. Evidence can be inferred from studies showing a seasonal rhythm of central and peripheral serotonergic functioning which may be a predisposing factor for SAD. Some of the symptoms of SAD are believed to represent an attempt to overcome a putative deficit in brain serotonergic transmission. Moreover, 5-HT receptor challenge studies suggest altered activity at or downstream to central 5-HT receptors. Monoamine depletion studies support hypotheses about serotonergic and catecholaminergic dysfunctions in SAD and suggest that light therapy may well compensate for this underlying deficit. Further, albeit indirect, support for the importance of monoaminergic mechanisms in SAD and its involvement in the mechanism of the action of light therapy comes from studies showing antidepressant efficacy of serotonergic and noradrenergic antidepressants in the treatment of SAD. Altogether, disturbances in brain monoaminergic transmission seem to play a key role in the pathogenesis of SAD; monoaminergic systems may also play an important role in the mechanisms of the action of light therapy.

Effects of acute tryptophan depletion on prepulse inhibition of the acoustic startle (eyeblink) response and the N1/P2 auditory evoked response in man.

Contraction of the orbicularis oculi muscle in response to a sudden loud sound (acoustic startle response) and the N1/P2 component of the auditory evoked potential are both attenuated when a brief low-intensity stimulus is presented 30-500 ms before the 'startle-eliciting' stimulus (prepulse inhibition). Here, we report the effect of acute tryptophan depletion on prepulse inhibition of these responses. Thirteen males (21-52 years) participated in two sessions separated by 7 days, in which they ingested a drink containing a mixture of amino-acids, which either included (+ TP) or did not include (- TP) tryptophan, according to a balanced double-blind design. Electromyographic (EMG) responses of the orbicularis oculi muscle and N1/P2 auditory evoked potentials were recorded in a 20-min session, 6 h after ingestion of the mixture. Subjects received 40 trials in which 1-kHz sounds were presented: (i) 40 ms, 115 dB ('pulse alone' trials) and (ii) 40 ms, 85 dB, followed after 120 ms by 40 ms, 115 dB ('prepulse/pulse' trials). Mean amplitudes of the EMG response and the N1/P2 potential were derived from the pulse-alone trials and, in each case, percentage prepulse inhibition was calculated. Plasma tryptophan levels were measured from blood samples taken before and 7 h after each treatment. Under the + TP condition, both the EMG response and the N1/P2 complex showed > 60% prepulse inhibition. The - TP condition was associated with (i) significant suppression of prepulse inhibition of the EMG response, with no significant change in response amplitude and (ii) reduction of the amplitude of the N1/P2 potential, with no significant change in prepulse inhibition of this response. Tryptophan levels rose by 90+/-15% under the + TP condition and fell by 81+/-3% under the - TP condition. The suppression of prepulse inhibition of the acoustic startle response under the - TP condition suggests that central 5-hydroxytryptaminergic mechanisms may be involved in regulating prepulse inhibition of this response. The lack of effect of tryptophan depletion on prepulse inhibition of the N1/P2 potential suggests that different mechanisms are involved in prepulse inhibition of the startle response and the N1/P2 complex.

[Pathogenetic and therapeutic aspects of secondary anorexia]. / Aspetti patogenetici e terapeutici dell'anoressia secondaria.

Anorexia is an often underrated symptom in the clinical management of patients suffering from chronic diseases. Moreover, the anorexia accompanying chronic diseases (secondary anorexia) is often confused with anorexia nervosa, a typically neuropsychiatric disorder involving completely different pathogenic mechanisms and therapeutic strategies. Secondary anorexia is one of the main factors responsible for the development of malnutrition, which in turn negatively affects patient morbidity and mortality. Different mechanisms have been proposed to explain the pathogenesis of secondary anorexia. However, consistent experimental and clinical evidence seems to point to hypothalamic serotonergic system hyperactivity as a preeminent cause; this hyperactivity appears to be triggered by enhanced brain availability of tryptophan, the aminoacid precursor of serotonin. The hyperactive hypothalamic serotonergic system might also represent the final effector where different regulatory and modulating pathways, including cytokines, converge. The involvement of tryptophan and the hypothalamic serotonergic system is further supported by the effectiveness of a therapeutic strategy, based on the inhibition of tryptophan entry into the brain, in increasing the food intake of anorectic patients. Although these results represent an encouraging approach to the treatment of secondary anorexia, with possible beneficial effects on the nutritional status of patients, they need to be validated in larger trials.

Can nutrient supplements modify brain function?

Over the past 40 y, several lines of investigation have shown that the chemistry and function of both the developing and the mature brain are influenced by diet. Examples are the effect of folate deficiency on neural tube development during early gestation, the influence of essential fatty acid deficiency during gestation and postnatal life on the development of visual function in infants, and the effects of tryptophan or tyrosine intake (alone or as a constituent of dietary protein) on the production of the brain neurotransmitters derived from them (serotonin and the catecholamines, respectively). Sometimes the functional effects are clear and the underlying biochemical mechanisms are not (as with folate and essential fatty acids); in other cases (such as the amino acids tyrosine and tryptophan), the biochemical effects are well understood, whereas the effect on brain function is not. Despite the incomplete knowledge base on the effects of such nutrients, investigators, physicians, and regulatory bodies have promoted the use of these nutrients in the treatment of disease. Typically, these nutrients have been given in doses above those believed to be required for normal health; after they have been given in pure form, unanticipated adverse effects have occasionally occurred. If this pharmacologic practice is to continue, it is important from a public safety standpoint that each nutrient be examined for potential toxicities so that appropriate purity standards can be developed and the risks weighed against the benefits when considering their use.

The role of tryptophan in fatigue in different conditions of stress.

Tryptophan is the precursor for the neurotransmitter 5-hydroxytryptamine (5-HT), which is involved in fatigue and sleep. It is present in bound and free from in the blood, where the concentration is controlled by albumin binding to tryptophan. An increase in plasma free tryptophan leads to an increased rate of entry of tryptophan into the brain. This should lead to a higher level of 5-HT which may cause central fatigue. Central fatigue is implicated in clinical conditions such as chronic fatigue syndrome and post-operative fatigue. Increased plasma free tryptophan leads to an increase in the plasma concentration ratio of free tryptophan to the branched chain amino acids (BCAA) which compete with tryptophan for entry into the brain across the blood-brain barrier. The plasma concentrations of these amino acids were measured in chronic fatigue syndrome patients (CFS) before and after exercise (Castell et al., 1998), and in patients undergoing major surgery (Yamamoto et al., 1997). In the CFS patients, the pre-exercise concentration of plasma free tryptophan was higher than in controls (p < 0.05) but did not change during or after exercise. This might indicate an abnormally high level of brain 5-HT in CFS patients leading to persistent fatigue. In the control group, plasma free tryptophan was increased after maximal exercise (p < 0.001), returning towards baseline levels 60 min later. The apparent failure of the CFS patients to change the plasma free tryptophan concentration or the free tryptophan/BCAA ratio during exercise may indicate increased sensitivity of brain 5-HT receptors, as has been demonstrated in other studies (Cleare et al., 1995). In post-operative recovery after major surgery plasma free tryptophan concentrations were markedly increased compared with baseline levels; the plasma free tryptophan/BCAA concentration ratio was also increased after surgery. Plasma albumin concentrations were decreased after surgery: this may account for the increase in plasma free tryptophan levels. Provision of BCAA has improved mental performance in athletes after endurance exercise (Blomstrand et al., 1995, 1997). It is suggested that BCAA supplementation may help to counteract the effects of an increase in plasma free tryptophan, and may thus improve the status of patients during or after some clinically stressful conditions.

The significance of tryptophan in infant nutrition.

In the newborn, tryptophan (Trp) and its metabolites are essential to brain maturation and to the development of neurobehavioral regulations of food intake, satiation and sleep-wake-rhythm. Due to the high Trp concentration in human milk in relation to the total of neutral amino acids, the blood-brain transfer of tryptophan as a precursor of its metabolites serotonin and melatonin is optimal. In contrast, commercial infant formulas are lower in Trp and higher in neutral amino acid levels resulting in comparatively lower Trp serum concentrations. alpha-lactalbumin enriched, protein-reduced formulas adapted to 2.2% Trp were shown to be capable of producing Trp serum values that did not differ from those in breast-fed infants.

Lack of behavioral effects of monoamine depletion in healthy subjects.

This study was designed to determine the behavioral effects of a reduction in catecholamine and indoleamine function in healthy subjects. Eight healthy subjects received the tyrosine hydroxylase inhibitor, alpha-methyl-para-tyrosine (AMPT) in combination with a full-strength tryptophan-depleting amino acid drink during one 4-day test session, and AMPT and tryptophan-supplemented amino acid drink (n = 2), or a 25% strength tryptophan-depleting amino acid drink (n = 6) during a second 4-day test session. The combined administration of AMPT and the tryptophan-free amino acid drink did not produce statistically significant or even clinically noticeable changes in mood among the healthy subjects. The implications of these observations for the monoamine hypotheses of depression are discussed.

Trp-16 is essential for the activity of alpha-galactosidase and alpha-N-acetylgalactosaminidase.

By expressing site-directed mutants in the methylotrophic yeast strain Pichia pastoris, the role of a tryptophan residue at position 16 in the activity of alpha-galactosidase and alpha-N-acetylgalactosaminidase, two closely related exoglycosidases, was studied. A substitution of Trp-16 with an arginine residue in alpha-N-acetylgalactosaminidase abolished the enzyme activity, which was confirmed by replacing a 600 bp fragment containing the mutation with the corresponding wild-type sequence. The same tryptophan residue was then substituted with an alanine in both enzymes by site-directed mutagenesis to reveal a possible relationship between their active sites. The purified alpha-N-acetylgalactosaminidase mutant demonstrated a specific activity of 2.8 x 10(-2) U/mg and a Vmax/K(m) of 4.3 x 10(-2), which were both more than a thousandfold lower than corresponding values for the wild-type enzyme. Furthermore, the mutant failed to bind to an affinity resin, suggesting the involvement of Trp-16 in substrate-binding. In addition, the purified alpha-galactosidase mutant resulted in more than a 10(4)-fold decrease in specific activity. Thus our data suggest that Trp-16 in both alpha-galactosidase and alpha-N-acetylgalactosaminidase is critical for enzymatic activity, which in turn supports the hypothesis that these two enzymes may share a catalytic mechanism involving similar residues in their active sites.

Two critical hydrophobic amino acids in the N-terminal domain of the p53 protein are required for the gain of function phenotypes of human p53 mutants.

Some mutant forms of the p53 protein have been shown to gain new functions that are not shared by the wild-type p53 protein; (1) mutant p53 proteins can transcriptionally transactivate the multi-drug resistance gene-1 (MDR-1) and (2) when expressed in non-tumorigenic cells with no endogenous p53 protein, mutant p53 proteins can enhance the tumorigenic potential of these cells (Dittmer et al., 1993). It has recently been shown (Lin et al., 1994b) that the transcriptional activator domain of the p53 protein contains two amino acids, leu-22 and trp-23, which are required by the wild-type p53 protein for transcriptional activity. To determine whether these same amino acid residues are utilized by mutant p53 proteins for their gain of function phenotype, the triple mutant p53 protein (at residues 22 and 23 in the transactivation domain and residue 281 in the DNA binding domain--a gain of function mutant) was made. While the p53-281 mutant transcriptionally activates the MDR-1 gene and enhances the tumorigenic potential of cells it is expressed in, the 22, 23, 281 triple mutant failed to carry out either of these functions.

Dietary carbohydrates: effects on self-selection, plasma glucose and insulin, and brain indoleaminergic systems in rat.

The aim of the study was to investigate the effect of different dietary carbohydrates such as corn starch, sucrose, fructose and glucose on carbohydrate and protein self-selection and on arterial and venous concentrations of glucose and insulin, and brain indoleamines in rats. Fructose and sucrose feeding induced the lowest food intakes which were due respectively to a lower carbohydrate and protein selection. The present data showed that feeding with dietary glucose as the main carbohydrate source gave the highest glycemic response, the lowest one being found with fructose and corn starch, and an intermediate one with sucrose feeding. The insulin response to the dietary carbohydrates followed a somewhat different pattern with the highest insulin secretion observed after fructose feeding whereas highly variable and inconsistent results were obtained following corn starch, sucrose and glucose feeding. Feeding chemically different sugars was also characterized by decreased serotonin synthesis in the raphe nuclei, brainstem and thalamus, and increased 5-HT synthesis in the hypothalamus of rats fed fructose when compared to glucose fed animals. The present results highlight the importance of considering the nature of dietary carbohydrates in the regulation of feeding.

Serotonin release varies with brain tryptophan levels.

This study examines directly the effects on serotonin release of varying brain tryptophan levels within the physiologic range. It also addresses possible interactions between tryptophan availability and frequency of membrane depolarization in controlling serotonin release. We demonstrate that reducing tryptophan levels in rat hypothalamic slices (by superfusing them with medium supplemented with 100 microM leucine) decreases tissue serotonin levels as well as both spontaneous and electrically-evoked serotonin release. Conversely, elevating tissue tryptophan levels (by superfusing slices with medium supplemented with 2 microM tryptophan) increases both tissue serotonin levels and serotonin release. Serotonin release was found to be affected independently by tryptophan availability and frequency of electrical field-stimulation (1-5 Hz), since increasing both variables produced nearly additive increases in release. These observations demonstrate for the first time that both precursor-dependent elevations and reductions in brain serotonin levels produce proportionate changes in serotonin release, and that the magnitude of the tryptophan effect is unrelated to neuronal firing frequency. The data support the hypothesis that serotonin release is proportionate to intracellular serotonin levels.