Effect of amino acids on choline uptake and phosphatidylcholine biosynthesis in the isolated hamster heart

Choline uptake by the hamster heart has been shown to be enhanced by exogenous glycine. In this study, the effect of neutral, basic, and acidic amino acids on choline uptake was assessed. Hamster hearts were perfused with labelled choline, and in the presence of L-alanine, L-serine, or L-phenylalanine (greater than or equal to 0.1 mM), choline uptake was enhanced 20-38%. L-Arginine, L-lysine, L-aspartate, and L-glutamate did not influence choline uptake. The rate of phosphatidylcholine biosynthesis was unaffected by all amino acids tested. Enhancement of choline uptake by neutral amino acids was not additive or dose dependent but required a concentration threshold. The enhancement of choline uptake by neutral amino acids was not influenced by preperfusion with the same amino acid. Exogenous choline had no effect on the uptake of amino acids. We postulate that choline and the neutral amino acids are not cotransported and modulation of choline uptake is facilitated by direct interaction of the neutral amino acids with the choline transport system.     

大杯香菇辐射新株系各类氨基酸的遗传主成分分析

对大杯香菇辐射选育新株系的各类氨基酸总量进行了遗传主成分分析。结果表明,变异系数最大的为硫氨基酸总量,达到13．39％;其次为儿童氨基酸总量,变异系数为7．00％;其它变异系数较小分别为甜味氨基酸总量、芳香族氨基酸总量、必需氨基酸总量、支链氨基酸总量和鲜味氨基酸总量为5．69％、5．64％、5．19％、5．24％和5．06％;在相关性上,鲜味氨基酸总量与甜味氨基酸总量、支链氨基酸总量、芳香族氨基酸总量、儿童氨基酸总量和必需氨基酸总量是呈极显著和显著的正相关,甜味氨基酸总量与支链氨基酸总量、儿童氨基酸总量和芳香族氨基酸总量呈极显著和显著的正相关,硫氨基酸总量与儿童氨基酸总量呈极显著负相关,与必需氨基酸总量是呈极显著正相关;支链氨基酸总量与芳香族氨基酸总量、必需氨基酸总量是呈极显著,与儿童氨基酸总量呈显著正相关;芳香族氨基酸总量与儿童氨基酸总量和必需氨基酸总量是呈极显著和显著的正相关;主成分分析结果表明,前4个特征根在7个特征根中累计贡献率达97．14％,也就是前4个主成分对变异的贡献率达96．07％。在各类氨基酸总量指标选择上,首先对变异大的各类氨基酸总量进行选择是及其重要的。在辐射选育新株系选择时,应注意选择大杯香菇中硫氨基酸总量高的新株系。     

Effect of some essential amino acid deficiency in the medium on the action of insulin on primary cultured hepatocytes of rats. Hepatocytes do not respond to insulin in some essential amino acid-deficient medium

1. The effects of insulin, glucagon and dexamethasone on the amino acid consumption by primary cultures of rat hepatocytes were studied in a medium containing all essential amino acids or in those deficient in some essential or nonessential amino acids. 2. The cells which were cultured in a medium containing all the essential amino acids responded to insulin by enhancing the consumption of amino acids and augmenting protein synthesis. 3. However, the cells did not respond to insulin significantly when they were cultured in a medium deficient in lysine or some other essential amino acids. 4. The results suggest that some essential amino acid deficiency impairs the transmission of the signal of insulin to the site of the metabolic changes induced by the hormone.     

Construction by site-directed mutagenesis of a 39-kilodalton mosquitocidal protein similar to the larva-processed toxin of Bacillus sphaericus 2362.

After ingestion of the parasporal crystals of Bacillus sphaericus, mosquito larvae process the 42-kilodalton (kDa) toxin to a protein of 39 kDa, which has an increased toxicity (A. H. Broadwell and P. Baumann, Appl. Environ. Microbiol. 53:1333-1337, 1987). A similar activation is performed by trypsin and chymotrypsin. Using site-directed mutagenesis, we have constructed derivatives of the 42-kDa toxin with a deletion of 10 amino acids at the N terminus and deletions of 7, 17, or 20 amino acids at the C terminus. Toxicity for mosquito larvae was retained upon deletion of 7 or 17 amino acids but was lost upon deletion of 20 amino acids. Evidence is presented indicating that the protein containing deletions of 10 amino acids at the N terminus and 17 amino acids at the C terminus (corresponding to potential chymotrypsin cleavage sites) is similar to the 39-kDa protein produced in mosquito larvae or by digestion with chymotrypsin. Digestion with trypsin appears to generate a protein lacking 16 or 19 amino acids from the N terminus and 7 amino acids from the C terminus. As is the case with the recombinant-made 42-kDa protein, toxicity of its derivatives is dependent on the presence of a 51-kDa protein which is a component of the parasporal crystal of B. sphaericus 2362.     

The mitochondrial pool of free amino acids reflects the composition of mitochondrial DNA-encoded proteins: indication of a post- translational quality control for protein synthesis

Mitochondria can synthesize a limited number of proteins encoded by mtDNA (mitochondrial DNA) by using their own biosynthetic machinery, whereas most of the proteins in mitochondria are imported from the cytosol. It could be hypothesized that the mitochondrial pool of amino acids follows the frequency of amino acids in mtDNA-encoded proteins or, alternatively, that the profile is the result of the participation of amino acids in pathways other than protein synthesis (e.g. haem biosynthesis and aminotransferase reactions). These hypotheses were tested by evaluating the pool of free amino acids and derivatives in highly-coupled purified liver mitochondria obtained from rats fed on a nutritionally adequate diet for growth. Our results indicated that the pool mainly reflects the amino acid composition of mtDNA-encoded proteins, suggesting that there is a post-translational control of protein synthesis. This conclusion was supported by the following findings: (i) correlation between the concentration of free amino acids in the matrix and the frequency of abundance of amino acids in mtDNA-encoded proteins; (ii) the similar ratios of essential-to-non-essential amino acids in mtDNA-encoded proteins and the mitochondrial pool of amino acids; and (iii), lack of a correlation between codon usage or tRNA levels and amino-acid concentrations. Quantitative information on the mammalian mitochondrial content of amino acids, such as that presented in the present study, along with functional studies, will help us to better understand the pathogenesis of mitochondrial diseases or the biochemical implications in mitochondrial metabolism.     

Amino acid transport by prosthecae of Asticcacaulis biprosthecum: evidence for a broad-range transport system

Prosthecae purified from cells of Asticcaulis biprosthecum possess active transport systems that transport all 20 amino acids tested. Using ascorbate-reduced phenazine methosulphate in the presence of oxygen, all 20 amino acids are accumulated against a concentration gradient by isolated prosthecae. Results of experiments testing the inhibition of transport of one amino acid by another, and of experiments testing the exchange of exogenous amino acids with those preloaded in prosthecae, along with characteristics of mutants defective in amino acid transport, suggest the presence in prosthecae of three amino acid transport systems. One, the general or G system, transports at least 18 of the 20 amino acids tested. Another system, referred to as the proline or P system, transports seven amino acids (including proline) that are also transported by the G system. The third system transports only glutamate and aspartate, and is referred to as the acidic amino acid transport system or A system.     

Synthesis of N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units for the assembly of backbone cyclic peptides.

To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH4. N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing efficient purification by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or fluorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both the N-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.     

Amino acid regulation of insulin action in isolated adipocytes. Selective ability of amino acids to enhance both insulin sensitivity and maximal insulin responsiveness of the protein synthesis system

Using the number and concentration of amino acids in Dulbecco's modified Eagle's medium as reference (DMEM = 100%), we found that a maximally effective concentration of insulin (10 ng/ml) stimulated protein synthesis by 125% over basal rate in the presence of 50% amino acids (EC50 = 19%), but by only 48% in amino acid-free buffer. Moreover, time course experiments revealed that amino acid regulation of insulin action was very rapid (t1/2 of 9.5 min) and readily reversible (less than 30 min). This effect was specific in that basal rates of protein synthesis were unaltered by amino acids. A second effect of amino acids was to markedly enhance insulin sensitivity of the protein synthesis system in a dose-dependent manner. Thus, the half-maximally effective concentrations of insulin required to stimulate protein synthesis fell from 0.43 to 0.25 to 0.15 ng/ml in the presence of 0, 50, and 150% amino acids. Neither insulin sensitivity nor maximal insulin responsiveness of the glucose transport system was altered by amino acids, nor did amino acids affect the insulin binding capacity of cells. When we divided the 14 amino acids found in DMEM into two groups, we found that one group of 7 amino acids had little or no effect on insulin sensitivity or responsiveness, whereas the other group was fully active (a 157% increase in insulin responsiveness, ED50 of 0.21 ng/ml versus a 68% increase, ED50 of 0.51 ng/ml, with no amino acids). Isoleucine and serine together increased both insulin sensitivity and responsiveness to 60-70% of that seen with the full complement of amino acids. In conclusion: 1) amino acids modulate insulin action by enhancing maximal insulin responsiveness and insulin sensitivity of the protein synthesis system, and the regulatory site of amino acid action appears to be distal to the common signal pathway, within the insulin action-protein synthesis cascade, and 2) the effects of amino acids are specific, in that basal rates of protein synthesis are unaffected, only certain amino acids influence insulin action, and amino acids fail to alter insulin binding or the insulin-responsive glucose transport system. These studies, together with those in the companion paper, demonstrate that the pleiotropic actions of insulin on enhancing glucose uptake and protein synthesis are mediated through divergent pathways that can be independently regulated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrical evidence for different mechanisms of uptake for basic, neutral, and acidic amino acids in oat coleoptiles

The application of neutral or acidic amino acids to oat coleptiles induced transient depolarizations of the membrane potentials. The depolarizations are considered to reflect H⁺ -amino acid co-transport, and the spontaneous repolarizations are believed to be caused by subsequent electrogenic H⁺ extrusion. The basic amino acids depolarized the cell membrane strongly, but the repolarizations were weak or absent. The depolarizations induced by the basic amino acids were weakly sensitive to manipulations of the extracellular and intracellular pH. The depolarizations induced by the other amino acids, in contrast, were more strongly affected by the pH changes. Several amino acids induced distinct but diminished depolarizations in the presence of 2,4-dinitrophenol or cyanide, but the repolarizations were generally eliminated. These experiments support the co-transport theory but suggest somewhat different mechanisms for the transport of the neutral, acidic, and basic amino acids. We suggest that the neutral amino acids are co-transported with a single H⁺ and that accumulation depends upon both the ΔpH and the membrane potential components of the proton motive force. The acidic amino acids appear to be accumulated by a similar mechanism except that the transport of each molecule may be associated with a cation in addition to a single proton. The permanently protonated basic amino acids appear not to be co-transported with an additional proton. Accumulation would depend only on the membrane potential component of the proton motive force.     

Promoting effect of amino acids added to a chemically defined medium on blastocyst formation and blastomere proliferation of bovine embryos cultured in vitro

This study was conducted to elucidate the role of amino acids added singly or in groups to a chemically defined culture medium in blastocyst formation and blastomere proliferation of bovine embryos. Embryos were generated by in vitro fertilization, and blastocyst formation and hatching, and blastomere number of blastocysts were subsequently monitored after the culture of embryos in synthetic oviduct fluid medium (SOFM). First, one of four non-essential amino acids (asparagine, aspartate, glutamate or serine) was added to SOFM and, compared with no addition, a significant (P <0.05) increase in blastocyst formation was found after the addition of asparagine, aspartate, or glutamate (35-42% versus 22%). Second, one of four essential amino acids (arginine, cystine, isoleucine or leucine) was added and arginine or isoleucine greatly improved blastocyst formation (30-36% versus 16%). Third, the addition of five stimulatory amino acids (aspartate, asparagine, glutamate, arginine and isoleucine) to SOFM significantly improved blastocyst formation compared with no addition (12% versus 21%) and such value was similar to that obtained after the addition of 19 amino acids consisting of MEM amino acid solutions (21-27%). However, five amino acids yielded fewer hatched blastocysts than 19 amino acids. Finally, although five amino acids yielded more cell number of blastocysts than no addition (93 versus 74 cells per blastocyst), it was lower than that from 19 amino acids (131 cells per blastocyst). In conclusion, either single or combined addition of asparagine, aspartate, glutamate, arginine and isoleucine stimulated blastocyst formation, while other amino acids might be necessary for further stimulating blastomere proliferation and blastocyst hatching.     

Synthesis of proteins and nucleic acids by the pea aphid Acyrthosiphon pisum (aphididae) in the absence of a full complement of dietary amino acids

Protein, nucleic acids, and nucleotide syntheses were studied in pea aphids, Acyrthosiphon pisum (Harris), by feeding them labeled ¹⁴C-amino acids and [5-³H]-orotic acid in sucrose. It was demonstrated that in the absence of dietary essential amino acids, aphids were capable of synthesizing nucleic acids, nucleotides, and proteins when provided with a single dietary amino acid in sucrose. It is suggested that other required amino acids were possibly supplied by the symbionts present in the pea aphid and/or were obtained from the amino acid pool in the hemolymph or glucose, one of the end products of sucrose digestion. Of the various amino acids tested, synthesis of measurable amounts of protein or other compounds occurred when alanine, aspartic acid, glutamic acid, glycine, proline, or serine were provided, but no synthesis occurred with cysteine.     

Hindered migration of amino acids toward their isoelectric positions in gel electrofocusing, and facilitation of the migration at high ionic strength

Amino acids with a largepI -pK_p difference are known to be poor carrier ampholytes in electrofocusing, exhibiting isoelectric zones with poor conductivity across as many as 4 pH units. Accordingly, radioactive amino acids of this type, e.g., glycine, are found to be distributed over the entire pH gradient formed by Ampholine in electrofocusing gels, while radioactive amino acids like histidine or glutamic acid with small pI - pK_p differences form single peaks at or near their pI's. When poor carrier ampholyte amino acids are subjected to gel electrofocusing in 0.1 KCl, their distribution sharpens into single peaks, at or near the pI, indistinguishable from those of the good carrier ampholyte amino acids. At an intermediate stage of peak coalescence of the original broad distributions of poor carrier ampholyte amino acids, in 0.01 KCl, acidic and basic peaks of amino acid can be observed, possibly analogous to acidie and basic distributions previously observed with labeled Ampholine. The rate of peak coalescence of anionic amino acids seems higher than that of the cationic species. The mechanism by which high ionic strength facilitates the condensation of poor carrier ampholyte amino acids at their pI remains unknown. Possibly, the current within zones of poor carrier ampholyte amino acids is insufficient, or poor carrier ampholyte amino acids are not sufficiently charged, to allow for electrophoretic migration of the bulk of loaded amino acid to its isoelectric position, unless the current density is increased by electrofocusing at high ionic strength. Alternatively, 0.1 KCl may interfere with electrovalent interactions between amino acids and isoelectric carrier ampholyte zones, analogous to the action of urea in preventing the interaction between polyanions and carrier ampholytes.     

The atmosphere of the primitive Earth and the prebiotic synthesis of amino acids

The atmosphere of the Earth at the time of its formation is now generally believed to have been reducing, an idea proposed by Oparin and extensively discussed by Urey. This atmosphere would have contained CH₄, N₂ with traces of NH₃, water and hydrogen. Only traces of NH₃ would have been present because of its solubility in water. UV light and electric discharges were the major sources of energy for amino acid synthesis, with electric discharges being the most efficient, although most other sources of energy also give amino acids.The first prebiotic electric discharge synthesis of amino acids showed that surprisingly high yields of amino acids were synthesized. Eleven amino acids were identified, four of which occur in proteins. Hydroxy acids, simple aliphatic acids and urea were also identified. These experiments have been repeated recently, and 33 amino acids were identified, ten of which occur in proteins, including all of the hydrophobic amino acids.Methionine can be synthesized by electric discharges if H₂S or CH₃SH is added to the reduced gases. The prebiotic synthesis of phenylalanine, tyrosine and trytophan involves pyrolysis reactions combined with plausible solution reactions.Eighteen amino acids have been identified in the Murchison meteorite, a type II carbonaceous chondrite, of which six occur in proteins. All of the amino acids found in the Murchison meteorite have been found among the electric discharge products. Furthermore, the ratios of amino acids in the meteorite show a close correspondence to the ratios from the electric discharge synthesis, indicating that the amino acids on the parent body of the carbonaceous chondrites were synthesized by electric discharges or by an analogous process.     

Control by amino acids of the activity of system A-mediated amino acid transport in isolated rat hepatocytes.

The effect of amino acids, in concentrations corresponding to those found in the portal vein of rats given a high-protein diet, was investigated on the activity of system A amino acid transport in hepatocytes from fed rats. Amino acids counteracted the induction of system A by insulin or glucagon. This effect was observed at all concentrations of hormones tested, up to 1 microM. Amino acids did not affect the basal cyclic AMP concentration in hepatocytes, or the large rise in cyclic AMP elicited by glucagon. The reversal of system-A induction was observed at relatively low concentration of amino acids, corresponding to plasma values reported in rats given a basal diet. Amino acids were separately tested: substrates of system A were particularly efficient, but so were glutamine and histidine. Non-metabolizable substrates of system A, such as 2-aminoisobutyrate, were also inhibitory, suggesting that a part of the effect of amino acids is independent of their cellular metabolism. Provision of additional energy substrates such as lactate and oleate did not affect induction of system A or the inhibitory effects of amino acids. Thus amino acids do not act by serving as an energy source and by maintaining the integrity of hepatocytes. Inhibition of mRNA synthesis by actinomycin practically abolished the effect of amino acids on the induction of system A by glucagon. The results suggest that amino acids may promote the synthesis of protein(s) affecting the activity of system A either directly at the carrier unit or at an intermediate stage of its emergence.     

Derepression of amino acid transport by amino acid starvation in rat hepatoma cells.

Amino acid starvation causes an adaptive increase in the initial rate of transport of selected neutral amino acids in an established line of rat hepatoma cells in tissue culture. After a lag of 30 min, the initial rate of transport of alpha-aminoisobutyric acid (AIB) increases to a maximum after 4 to 6 h starvation of 2 to 3 times that seen in control cells. The increased rate of transport is accompanied by an increase in the Vmax and a modest decrease in the Km for this transport system, and is reversed by readdition of amino acids. The enhancement is specific for amino acids transported by the A or alanine-preferring system (AIB, glycine, proline); uptake of amino acids transported by the L or leucine-preferring system (threonine, phenylalanine, tyrosine, leucine) or the Ly+ system for dibasci amino acids (lysine) is decreased under these conditions. Amino acids which compete with AIB for transport also prevent the starvation-induced increase in AIB transport; amino acids which do not compete fail to prevent the enhancement. Paradoxically threonine, phenylalanine, tryptophan, and tyrosine, which do not compete with AIB for transport, block the enhancement of transport upon amino acid starvation. The starvation-induced enhancement of amino acid transport does not appear to be the result of a release from transinhibition. After 30 min of amino acid starvation, AIB transport is either unchanged or slightly decreased even though amino acid pools are already depleted. Furthermore, loading cells with high concentrations of a single amino acid following a period of amino acid starvation fails to prevent the enhancement of AIB transport, whereas incubation of the cells with the single amino acid for the entire duration of amino acid starvation prevents the enhancement; intracellular amino acid pools are similar under both conditions. The enhancement of amino acid transport requires concomitant RNA and protein synthesis, consistent with the view that the adaptive increase reflects an increased amount of a rate-limiting protein involved in the transport process. Dexamethasone, which dramatically inhibits AIB transport in cells incubated in amino acid-containing medium, both blocks the starvation-induced increase in AIB transport, and causes a time-dependent decrease in transport velocity in cells whose transport has previously been enhanced by starvation.     

Influence of amino acid availability on protein turnover in perfused skeletal muscle

The effects of amino acids on protein turnover in skeletal muscle were determined in the perfused rat hemicorpus preparation. Perfusion of preparations from fasted young rats (81±2 g) with medium containing either a complete mixture of amino acids at five times (5×) their normal plasma levels, a mixture of leucine, isoleucine, and valine at 5× or 10× levels, or leucine alone (10×) resulted in a 25–50% increase in muscle protein synthesis and a 30% decrease in protein degradation compared to fasted controls perfused in the absence of exogenously added amino acids. When the branched-chain amino acids were omitted from the complete mixture, the remaining amino acids (5×) had no effect on protein turnover. The complete mixture at 1× levels was also ineffective. Comparison of the effects of amino acids with those of glucose and palmitate indicated that amino acids were not acting by providing substrates for energy metabolism. The stimulatory effect of amino acids on protein synthesis was associated with a facilitated rate of peptide-chain initiation as evidenced by a relative decrease in the level of ribosomal subunits. This response was not as great as that produced by insulin, and the amino acids did not augment the effect of insulin. Although protein synthesis in preparations from fed young rats (130±3 g) was stimulated by the addition of a mixture of the branched-chain amino acids (5×) to about the same extent as that observed in the fasted young rats, protein degradation was not affected. Furthermore, neither synthesis nor degradation were affected in preparations from fasted older rats (203±9 g) suggesting that the age and or nitritional state of the animal may influence the response of skeletal muscle to altered amino acid levels.     

Electro-olfactogram and multiunit olfactory receptor responses to complex mixtures of amino acids in the channel catfish, Ictalurus punctatus

In vivo electrophysiological recordings from populations of olfactory receptor neurons in the channel catfish, Ictalurus punctatus, clearly showed that both electro-olfactogram and integrated neural responses of olfactory receptor cells to complex mixtures consisting of up to 10 different amino acids were predictable with knowledge of (a) the responses to the individual components in the mixture and (b) the relative independence of the respective receptor sites for the component stimuli. All amino acid stimuli used to form the various mixtures were initially adjusted in concentration to provide approximately equal response magnitudes. Olfactory receptor responses to both multimixtures and binary mixtures were recorded. Multimixtures were formed by mixing equal aliquots of 3-10 different amino acids. Binary mixtures were formed by mixing equal aliquots of two equally stimulatory solutions. Solution 1 contained either one to nine different neutral amino acids with long side-chains (LCNs) or one to five different neutral amino acids with short side-chains (SCNs). Solution 2, comprising the binary mixture, consisted of only a single stimulus, either a LCN, SCN, basic, or acidic amino acid. The increasing magnitude of the olfactory receptor responses to mixtures consisting of an increasing number of neutral amino acids indicated that multiple receptor site types with highly overlapping specificities exist to these compounds. For both binary mixtures and multimixtures composed of neutral and basic or neutral and acidic amino acids, the receptor responses were significantly enhanced compared with those mixtures consisting of an equal number of only neutral amino acids. These results demonstrate that receptor sites for the basic and acidic amino acids, respectively, are highly independent of those for the neutral amino acids, and suggest that a mechanism for synergism is the simultaneous activation of relatively independent receptor sites by the components in the mixture. In contrast, there was no evidence for the occurrence of mixture suppression.     

Differential effects of amino acids on the period of the circadian rhythm from the Aplysia eye

The effect of continuous treatments of single L -amino acids (0.1mM) on the free running rhythm from the isolated Aplysia eye was examined. A variation in the change in free running period produced by different amino acids was observed. Two well-known precursors of neurotransmitter (tyrosine, tryptophan) had the largest effects. These amino acids lengthened the period ca. 1.7 h. Another group of amino acids (alanine, threonine, proline) lengthened the period by about 1 h. Smaller effects were produced by aspartic acid and leucine and no effects were caused by lysine, glycine, valine, and serine. Phenylalanine may shorten the period a small amount. Glucose (5mM) lengthens the period a small amount (0.4 h), decreases the effect of tyrosine somewhat, and has no effect on the lengthening of the period produced by tryptophan. Three amino acids not involved in protein synthesis (ornithine, β-alanine, citrulline) had at most small effects on the free running period. Also, D -tryptophan lengthened the period by 0.6 h but the effect of D -tryptophan was considerably smaller than the effect of L -tryptophan. A few of the amino acids had small short-term effects on spike rate and longer-term effects on the amplitudes of the rhythms but these effects did not correlate with the effects of the amino acids on the free running period. Though continuous treatments of certain amino acids lengthened the periods, shorter treatments (tryptophan, 6 h) did not phase-shift the rhythm. Since eyes maintained in a commonly used culture medium have longer periods than eyes in a simple seawater medium, the amino acids of the culture medium must be responsible, at least in part, for the lengthening effect of the culture medium. The mechanism of action of the amino acids is unknown. The magnitude of the effects did not correlate with physical-chemical properties of the amino acids nor with whether the amino acids were “essential” or “nonessential.” The effects of the amino acids may be mediated by their effects on neurotransmitter and/or protein synthesis.     

EVIDENCE FOR SEPARATE SYSTEMS FOR THE TRANSPORT OF NEUTRAL AND BASIC AMINO ACIDS ACROSS THE BLOOD-BRAIN BARRIER

Abstract— The effects of high circulating concentrations of several amino acids on the free amino acids of rat brain were measured, to see whether or not the results followed any consistent pattern. High circulating concentrations of large, neutral amino acids (phenylalanine, valine or isoleucine) caused significantly decreased values only of other large, neutral amino acids in the brains. High circulating concentrations of the basic amino acids lysine or arginine caused significantly decreased values only of each other. The data suggest that there are separate systems for the transport of neutral and basic amino acids across the blood-brain barrier. The effects of valine and lysine on the uptake by brain and the con-vulsant action of allylglycine (a neutral amino acid) were consistent with the concept of separate systems for the transport of amino acids across the blood-brain barrier. Valine inhibited the uptake by brain and the convulsant action of allylglycine in mice, but lysine did not. The data suggest that allylglycine and valine are transported into the brain by a common mechanism that does not transport lysine.     

Substrate recognition by the ClpA chaperone component of ClpAP protease

ClpA, a member of the Clp/Hsp100 ATPase family, is a molecular chaperone and regulatory component of ClpAP protease. We explored the mechanism of protein recognition by ClpA using a high affinity substrate, RepA, which is activated for DNA binding by ClpA and degraded by ClpAP. By characterizing RepA derivatives with N- or C-terminal deletions, we found that the N-terminal portion of RepA is required for recognition. More precisely, RepA derivatives lacking the N-terminal 5 or 10 amino acids are degraded by ClpAP at a rate similar to full-length RepA, whereas RepA derivatives lacking 15 or 20 amino acids are degraded much more slowly. Thus, ClpA recognizes an N-terminal signal in RepA beginning in the vicinity of amino acids 10-15. Moreover, peptides corresponding to RepA amino acids 4-13 and 1-15 inhibit interactions between ClpA and RepA. We constructed fusions of RepA and green fluorescent protein, a protein not recognized by ClpA, and found that the N-terminal 15 amino acids of RepA are sufficient to target the fusion protein for degradation by ClpAP. However, fusion proteins containing 46 or 70 N-terminal amino acids of RepA are degraded more efficiently in vitro and are noticeably stabilized in vivo in clpADelta and clpPDelta strains compared with wild type.