Individual amino acid balances in young lean and obese Zucker rats fed a cafeteria diet

The amino acid composition of the diet ingested by reference and cafeteria diet-fed lean and obese Zucker rats has been analyzed from day 30 to 60 after birth. Their body protein amino acid composition was measured, as well as the urinary and faecal losses incurred during the period studied. The protein actually selected by the rats fed the cafeteria diet had essentially the same amino acid composition as the reference diet. The mean protein amino acid composition of the rat showed only small changes with breed, age or diet.Cafeteria-fed rats had a higher dietary protein digestion/absorption efficiency than reference diet-fed rats. Obese rats wasted a high proportion of dietary amino acids when given the reference diet, but not on the cafeteria diet. In all cases, the amino acids lost as such in the urine were a minimal portion of available amino acids.In addition to breed, the rates of protein accretion are deeply influenced by diet, but even more by the age — or size — of the animals: cafeteria-fed rats grew faster, to higher body protein settings, but later protein accrual decreased considerably; this is probably due to a limitation in the blueprint for growth which restricts net protein deposition when a certain body size is attained. Obese rats, however, kept accuring protein with high rates throughout.Diet composition — and not protein availability or quality-induced deep changes in amino acid metabolism. Since the differences in the absolute levels of dietary protein or carbohydrate energy ingested by rats fed the reference or cafeteria diets were small, it can be assumed that high (lipid) energy elicits the changes observed in amino acid metabolism by the cafeteria diet. The effects induced in the fate of the nitrogen ingested were more related to the fractional protein energy proportion than to its absolute values. Cafeteria-fed rats tended to absorb more amino acids and preserve them more efficiently; these effects were shown even under conditions of genetic obesity.There were deep differences in handling of dietary amino acids by dietary or genetically obese rats. The former manage to extract and accrue larger proportions of their dietary amino acids than the latter. The effects of both models of amino acid management were largely additive, suggesting that the mechanisms underlying the development of obesity did not run in parallel to those affecting the control of amino acid utilization. Obesity may be developed in both cases despite a completely different strategy of amino acid assimilation, accrual and utilization. (Mol Cell Biochem121: 45–58, 1993)     

The effect of caffeine on some mouse brain free amino acid levels

Changes in free amino acids were examined in the central nervous system of mice treated with caffeine for three weeks. Caffeine was administered in the drinking water, and at the end of three weeks the level of caffeine in the cerebral cortex was 113±19 g/g. When amino acid levels in cerebral hemispheres, midbrain, pons and medulla, and cerebellum were measured a significant increase in glutamine levels was found in all four regions. Glycine, alanine, serine, threonine, and GABA were significantly reduced in some regions. Caffeine appears to alter some of the metabolic or transport processes regulating amino acid pools in the brain. The decrease of GABA found in pons and medulla may contribute to the observed increase in reflex excitability after caffeine.Special issue dedicated to Dr. Elling Kvamme     

Hemidecortication selectively alters release of glutamate in perfusates collected from cerebral cortex of unrestrained rats

The effect of hemidecortication on the in vivo release of amino acids was examined in different areas of the cerebral cortex of the freely-moving rat. After one side of the cortex was lesioned by aspiration, four guide tubes for push-pull perfusion were implanted chronically on the contralateral side so as to rest above the frontal, parietal, temporal and occipital areas of the cortex. After 10–14 days elapsed, each of these regions was perfused with an artificial cerebrospinal fluid (CSF) at a rate of 25.0 l/min. Two types of assays were undertaken to determine the release of either newly synthesized amino acids from [¹⁴C]glucose precursor or the actual endogenous content in samples of perfusate. The separation of the [¹⁴C]amino acids was performed by thin layer chromatography, whereas endogenous amino acids were separated by HPLC with electrochemical detection and quantitated in the range of 1.0–10.0 picomoles. When compared to the control group, samples collected in the hemidecorticate rat showed no significant differences in the new synthesis of glutamate, aspartate, glutamine, glycine, and GABA from the precursor. On the other hand, the analysis of the endogenous amino acid neurotransmitters revealed that the levels of glutamic acid and glutamine declined in samples obtained from the parietal and frontal cortex, respectively. These results implicate further the potential role of glutamic acid as a neurotransmitter of interhemispheric connections in the cerebral cortex.     

The effect of some non-protein amino acids on pollen germination and pollen-tube growth in five species of the Vicieae

Summary The effects of canavanine, ,-diaminobutyric acid, homoarginine and lathyrine on the germination of pollen and on in-vitro growth of pollen tubes were studied in the following species: Lathyrus niger, L. silvestris, Vicia unijuga, Pisum sativum and Cicer arietinum.The effects of these non-protein amino acids depended on their quantity and on the plant species. Every amino acid had a promoting effect on germination and growth at some concentration in some species. Inhibition or promotion of pollen germination and pollen-tube growth were usually parallel. The stronger influence of some amino acid on growth than on germination may be due to slow penetration of the acid into the cell.Homoarginine and lathyrine had a promoting effect at all concentrations in L. niger, a species in which these amino acids occur naturally. In most other species they had, if anything, a very slight inhibitory effect, ,-Diaminobutyric acid and canavanine had the strongest inhibitory effects on the species studied. It seems possible that these amino acids are antimetabolites of common amino acids.It is obvious that non-protein amino acids can form effective hybridization barries, although the conditions in nature are more complex than in vitro. The ability to synthesize a new amino acid may therefore be of evolutionary significance in the isolation of new species and genera.     

Use of Chou-Fasman amino acid conformational parameters to analyze the organization of the genetic code and to construct protein genealogies

Summary Chou-Fasman parameters, measuring preferences of each amino acid for different conformational regions in proteins, were used to obtain an amino acid difference index of conformational parameter distance (CPD) values. CPD values were found to be significantly lower for amino acid exchanges representing in the genetic code transitions of purines, GA than for exchanges representing either transitions of pyrimidines, CU, or transversions of purines and pyrimidines. Inasmuch as the distribution of CPD values in these non GA exchanges resembles that obtained for amino acid pairs with double or triple base differences in their underlying codons, we conclude that the genetic code was not particularly designed to minimize effects of mutation on protein conformation. That natural selection minimizes these changes, however, was shown by tabulating results obtained by the maximum parsimony method for eight protein genealogies with a total occurrence of 4574 base substitutions. At the beginning position of the codons GA transitions were in very great excess over other base substitutions, and, conversely, CU transitions were deficient. At the middle position of the codons only fast evolving proteins showed an excess of GA transitions, as though selection mainly preserved conformation in these proteins while weeding out mutations affecting chemical properties of functional sites in slow evolving proteins. In both fast and slow evolving proteins the net direction of transitions and transversions was found to be from G beginning codons to non-G beginning codons resulting in more commonly occurring amino acids, especially alanine with its generalized conformational properties, being replaced at suitable sites by amino acids with more specialized conformational and chemical properties. Historical circumstances pertaining to the origin of the genetic code and the nature of primordial proteins could account for such directional changes leading to increases in the functional density of proteins.In order to further explore the course of protein evolution, a modified parsimony algorithm was developed for constructing protein genealogies on the basis of minimum CPD length. The algorithm's ability to judge with finer discrimination that in protein evolution certain pathways of amino acid substitution should occur more readily than others was considered a potential advantage over strict maximum parsimony. In developing this CPD algorithm, the path of minimum CPD length through intermediate amino acids allowed by the genetic code for each pair of amino acids was determined. It was found that amino acid exchanges representing two base changes have a considerably lower average CPD value per base substitution than the amino acid exchanges representing single base changes. Amino acid exchanges representing three base changes have yet a further marked reduction in CPD per base change. This shows how extreme constraining effects of stabilizing selection can be circumvented, for by way of intermediate amino acids almost any amino acid can ultimately be substituted for another without damage to an evolving protein's conformation during the process.     

Changes in the Sensitivity of the Brain Cortex to the Action of Convulsants within the Remote Period after Electrostimulation-Induced Kindling in Rats

In acute experiments on rats, we measured the intensity of epileptiform events after applications of agonists of the receptors to excitatory amino acids, NMDA (10.0 mg/ml) and kainic acid (2.0 mg/ml), to the cortex. The rats were preliminarily subjected to a kindling procedure using electrical stimulations (ES) of the ventrobasal amygdala. Within the remote post-kindling period (three weeks after the last ES session), the sensitivity to the above drugs increased: the latency of the first EEG spike potential decreased, the amplitude of epileptic discharges within the period of the highest activity increased, and the epileptic nidi existed for a longer period. Applications of NMDA and kainic acid after preliminary intraventricular injections of an agonist of the -opiate receptors, DAMGO (0.5 g), on the one hand, evoked more intensive epileptogenic effects in kindling rats, and, on the other hand, did not change the above-mentioned indices of epileptogenesis in the control animals. The effects of applications of other convulsants (30 mg/ml strychnine or 10,000 IU/ml benzylpenicillin) showed no changes under the influence of DAMGO.     

Formation of amino acids and related oligomers from formaldehyde and hydroxylamine in a solution of transition metal ions

Summary In a solution of transition metal ions with or without kaolin not only amino acids but also their oligomers were formed.The maximum ratio of amino acids after to before acid hydrolysis was 5 – 6.The term transition element is used here in its broader sense, and includes zinc and similar elements     

Cloning and nucleotide sequence of β-mannanase and cellulase genes from Bacillus sp. 5H

The two genes for -mannanase and cellulase of Bacillus sp. 5H have been cloned in Escherichia coli JM 109 by a shotgun method, though the cellulase gene was not expressed in Bacillus sp. 5H. The nucleotide sequences of the -mannanase gene and the cellulase gene revealed open reading frames of 1,086 and 1,503 base pairs, respectively, coding for a proteins of M_r 40,803 Da (-mannanase) and 55,420 Da (cellulase). The deduced primary structure of -mannanase comprised 362 amino acids which had a mature protein of 336 amino acids and a signal peptide of 26 amino acids and that of cellulase comprised 501 amino acid residues.     

Epidermal growth factor (EGF) increases the renal amino acid transport capacity in amino acid loaded rats

Summary In anaesthetized adult female rats, the influence of epidermal growth factor (EGF) on renal amino acid handling was investigated in glutamine, arginine (both 50 mg/100 g b. wt. per hour), or alanine (90 mg/ 100 g b. wt. per hour) loaded animals. Continuous infusions of the three amino acids were followed by an increase in the fractional excretion (FE) of the administered amino acids as well as of the other endogenous amino acids. Under load conditions (alanine, arginine or glutamine), EGF pretreatment (8g/100g b. wt. subcutaneously for 8 days, twice daily 8 a.m. and 4 p.m.) was followed by a stimulation of renal amino acid reabsorption. The increase in the fractional excretion of the administered amino acids was significantly lower than in non-EGF-treated rats. These changes in amino acid transport were connected with a significant reduction of GFR after EGF pretreatment (0.96 ± 0.10 vs. 0.62 ± 0.07 ml/min X 100 g b. wt.) and a distinct increase in sodium excretion (2.98 ± 0.55 vs. 4.97 ± 0.71val/100 g b. wt. X 20 min). After loading with p-aminohippurate (PAH; 200mg/100g b. wt.), PAH excretion in EGF rats was increased by about 20%, whereas urinary protein excretion was lower in EGF pretreated rats (control: 0.45 ± 0.04 vs. EGF: 0.18 ± 0.03 mg/ 100 g b. wt. X 20 min). The PAH load reduced amino acid reabsorption as a sign of overloading of renal tubular transport capacity, but in EGF pretreated animals the amino acid excretion was only slightly increased under these conditions. Furthermore, EGF pretreatment depressed normal kidney weight gain significantly (874 ± 18 vs. 775 ± 32mg/100g b. wt.). EGF can improve the renal tubular transport capacity, but, compared to well-known stimulators of renal transport like dexamethasone or tri-iodothyronine, its effect is only of a moderate degree.     

D-penicillamine affects lipid peroxidation and iron content in the rat brain cortex

d-Penicillamine, a trifunctional aminoacid known for its ability to form metal complexes and for being a radical scavenger, has been investigated in vitro and in vivo in the rat brain cortex. At 50 M the drug facilitate lipid hydroperoxides and TBARS formation in brain cortex homogenates, while at higher concentrations a clear inhibition of the lipid peroxidative process was observed. The activity of thed-penicillamine (25 and 50 mg/Kg i.p) was evaluated in vivo after a 7-day treatment in rats in whose brain cortex a slow process of lipid peroxidation was induced by iron-saccharate injection. Lipid hydroperoxides, lipid soluble fluorescent compounds and the iron content of both iron-injected and contralateral hemicortices showed a significant decrease in comparison to rats untreated withd-penicillamine. The higher dose also induced in normal rats a significant decrease in basal TBARS and iron content of the brain cortex. In the iron-injected cortex the observed Fe²⁺/Fe³⁺ ratio was significantly different from that of normal rats. On the contrary ratios obtained formd-penicillamine treated animals were higher in comparison to both normal and iron-injected animals. These results suggest thatd-penicillamine, acting as a reducing agent, inhibits the iron redox system and, as a chelating agents, can remove metal from action sites where lipid peroxidation may occur.     

Excitatory and inhibitory amino acid changes in ischemic brain regions in spontaneously hypertensive rats

Excitatory (glutamate, aspartate) or inhibitory amino acids (-aminobutyric acid: GABA, taurine) and glutamine contents were examined in acutely induced cerebral ischemia in spontaneously hypertensive rats. At 20 min ischemia most of these amino acids remained unchanged, but glutamine significantly decreased by 14% in the CA3 hippocampal subfield. At 60 min ischemia glutamate significantly decreased by 14% in the CA3, aspartate by 17–26% in the CA3, cingulate cortex, septum and striatum. In contrast, GABA significantly increased by 48–106% in the cortices (frontal, parietal and cingulate), striatum and nucleus accumbens, but insignificantly in hippocampal subrïelds. Likewise, taurine increased in the parietal cortex and nucleus accumbens. Glutamine showed heterogeneous changes (increase in the nucleus accumbens and decrease in the CA3). Amino acid levels change during ischemia, but their changes are varied in each area, implying that different reaction of amino acids may explain the selective vulnerability to cerebral ischemia.     

Alterations in synaptosomal neurotransmitter amino acids in “petit-mal” rats at a daytime and a nighttime

The synaptosoma fractions of 6 brain areas-olfactory tubercles (OT), frontal cortex (FC), striatum (Sr), amygdala (A), thalamus (Th), hypothalamus (Hy)-have been analyzed for their neurotransmitter amino acids (AA) content in Wistar rats exhibiting petit-mal epilepsy (PM-E) and in controls (C). The analysis was carried out at 11 p.m. (nighttime corresponding to the acrophase for the hourly number of spike-wave complexes) and at 11 a.m. (daytime). A day versus night rhythmicity is recorded for synaptosomal inhibitory AA in control and in PM-E rats. However, day versus night variations are more frequent and more prominent in C rats than in PM-E rats. Two day versus night variations exist only in PM-E rats: increases of GABA level in Sr and of Asp in Hy. Differences between PME-and C in synaptosomal AA content are more likely to be present during the nighttime. During this period lower AA values for PM-E rats are found for one or several inhibitory AA in OT, Th, and FC. It seems that the differences between PM-E and C concerning the inhibitory AA correlate with the number of spike-wave discharges. Only in one brain area is there a similar difference for PM-E and C during daytime and nighttime: a decreased GABA content for PM-E rats in OT. The decrease is larger in nighttime than in daytime. This difference may serve as a marker for this epileptic disorder. Moreover, it is in OT that the greatest number of PM-E versus C differences in synaptosomal neurotransmitter AA are observed. In view of these and former data, the existence of different alterations in synaptosomal neurotransmitter AA for different types of epilepsy is suggested.Abbreviations used GABA 4-aminobutyrate - Tau taurine - Gly glycine - Asp aspartate - Glu glutamate - Gln glutamine - OT offactory tubereles - FC fronto-parietal cortex - Sr striatum - A amygdala - Th lateral thalamus - Hy lateral hypothalamus - AA neurotransmitter amino acids - I inhibitory - E excitatory - C control rats - PM-E petit-mal rats     

Possible role of glutamate, aspartate, glutamine, GABA or taurine on cadmium toxicity on the hypothalamic pituitary axis activity in adult male rats

This work was designed to evaluate the possible changes in glutamate, aspartate, glutamine, GABA and taurine within various hypothalamic areas the striatum and prefrontal cortex after oral cadmium exposure in adult male rats, and if these changes are related to pituitary hormone secretion. The contents of glutamine, glutamate, aspartate, GABA and taurine in the median eminence, anterior, mediobasal and posterior hypothalamus, and in prefrontal cortex in adult male rats exposed to 272.7 mol l^–1 of cadmium chloride (CdCl₂) in the drinking water for one month. Cadmium diminished the content of glutamine, glutamate and aspartate in anterior hypothalamus as compared to the values found in the untreated group. Besides, there is a decrease in the content of glutamate, aspartate and taurine in the prefrontal cortex. The amino acids studied did not change in median eminence, mediobasal and posterior hypothalamus or the striatum by cadmium treatment. Plasma prolactin and LH levels decreased in rats exposed to the metal. These results suggest that (1) cadmium differentially affects amino acid content within the brain region studied and (2) the inhibitory effect of cadmium on prolactin and LH secretion may be partially explained by a decrease in the content of both glutamate and aspartate in anterior hypothalamus, but not through changes in GABA and taurine.     

Stimulation of renal amino acid reabsorption after treatment with triiodothyronine or dexamethasone in amino acid loaded rats

Summary In adult female anaestetized rats, the influence of triiodothyronine or dexamethasone on renal amino acid handling was investigated in leucine (20mg/100g b.wt.) or glutamine (45mg/100g b.wt.) loaded animals. Bolus injections of both amino acids were followed by temporary increase in fractional excretion of the administered amino acids as well of the endogenous amino acids which were not administered.Under load conditions (leucine and glutamine), dexamethasone treatment (60 g/100 g b.wt. for 3 days, i.p. once daily) was followed by a stimulation of renal amino acid reabsorption. The increase in fractional amino acid excretion after amino acid load was significantly lower than in untreated rats. The effect of triiodothyronine (20,g/1008 b.wt. for 3 days, i.p. once daily) was different in leucine and glutamine loaded animals: after leucine bolus injection a comparable stimulatory effect as shown for dexamethasone could be demonstrated, but after glutamine administration the stimulatory action of T3 was masked. T3 even increases fractional amino acid excretion in glutamine loaded rats as a sign of enhanced house-keeping in the renal tubular cells. These results confirm previous findings and indicate different effects of both hormones on the renal handling of amino acids.     

Facile preparation of optically pure [α-2H]-α-amino acids

Summary Racemic [-²H]--amino acids were prepared by heating the corresponding amino acids (Phe, nor-Leu and Dopa) with 0.05 equivalents of benzaldehyde in deuterated-acetic acid. Based on¹H-nmr measurement, the isotopic purities of these racemized [-²H]--amino acids were found to be higher than 99.5%. Methylation of these isotope-labelled amino acids was achieved in methanol/thionyl chloride without affecting isotopic purity. Optically pure [-²H]--amino acids were obtained in high yield with high enantiomeric excess via alcalase catalysed resolution.     

Influence of protein structure on selection of nitrous acid induced mutants of TMV

Summary Calculated values for amino acid replacements in nitrous acid induced mutants of TMV are compared with experimentally found data. 60 of 68 analysed mutants (Wittmann, 1962, 1964; Tsugita and Fraenkel-Conrat, 1962) may be explained by transitions CU and A(H)G (Schuster, Schramm, 1958; Mundry, Gierer, 1958). Replacements of non-polar amino acids are less frequent than replacements of polar amino acids. Threonine is the most frequently replaced amino acid. Since substitutions are predominantly by leucine it is assumed that the non-polar character of threonine is predominant. 66% of all replacements are found in the tryptic peptides I, IV, and XI, indicating that these peptides or at least part of them are of less importance for the structure than the others. Based on the results on hemoglobins and myoglobin (Perutz, Kendrew, and H. C. Watson, 1965), and the amino acid replacements of TMV, one may predict helical parts of the TMV protein chain. A correlation of amino acid replacements and symptoms on host plants is evident. It is concluded that special types of replacements were selected preferentially because symptoms were used as the selection system.     

Phylogenetic Analysis of the Aminoacyl-tRNA synthetases

Numerous aminoacyl-tRNA synthetase sequences have been aligned by computer and phylogenetic trees constructed from them for the two classes of these enzymes. Branching orders based on a consensus of these trees have been proposed for the two groups. Although the order of appearance can be rationalized to fit many different scenarios having to do with the genetic code, the invention of a system for translating nucleic acid sequences into polypeptide chains must have predated the existence of these proteins. In the past, a variety of schemes has been proposed for matching amino acids and tRNAs. Most of these have invoked direct recognition of one by the other, whether or not the anticodon was involved. Often ignored is the possibility of a nonprotein (presumably RNA) matchmaker for bringing the two into conjunction. If such had been the case, then the contemporary aminoacyl-tRNA synthetases could have entered the system gradually, each specific type replacing its matchmaking RNA counterpart in turn. A simple displacement scheme of this sort accommodates the existence of two different families of these enzymes, the second being introduced well before the first had undergone sufficient genetic duplications to specify the full gamut of amino acids. Such a scheme is also consistent with similar amino acids often, but not always, being the substrates of enzymes with the most similar amino acid sequences.Based on a presentation made at a workshop—Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: R.F. Doolittle     

Rats that consume caffeine show decreased brain protein synthesis

The effect of caffeine on protein synthesis in brain and liver was studied. When caffeine was added to a post-mitochondrial supernatant from rat brain protein synthesis was inhibited, i.e. 1 mM caffeine about 20%. The effect on protein synthesis of two weeks administration of large doses of caffeine in the drinking fluid of rats was also measured. Caffeine decreased protein synthesis in rat brain by about 32% and 20% compared with ad libitum and pair-fed controls. Protein synthesis was calculated taking into account the levels of free leucine determined by HPLC: 0.10 mol/g brain of ad libitum; 0.11 for pair-fed and 0.07 for caffeine. The pattern of proteins synthesized was not significantly altered by caffeine as shown by gel-electrophoresis and fluorography. There was no effect on protein synthesis of liver. The possible significance of these results is briefly discussed.     

Transformation of some hydroxy amino acids to other amino acids

It has been observed that -hydroxy--amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehyde reactions, glycine also reacts with acetone to give -hydroxyvaline under prebiologically feasible conditions. It is suggested, therefore, that the formation of -hydroxy--amino acids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions.     

Amino acid uptake by Lemna gibba by a mechanism with affinity to neutral L-and D-amino acids

Earlier work suggested that amino acid uptake by Lemna gibba cells is a H⁺-cotransport mechanism driven by a proton-electrochemical gradient at the plasmalemma. The present investigations of the transient membrane depolarizations elicited by amino acids and tracer-uptake experiments show that all neutral -L-amino acids, D-alanine and analogues, like -alanine and p-fluorophenylalanine, are transported by the same system. It remains to be seen if there are separate mechanisms for the uptake of acidic and basic amino acids.