Effect of altered sterol levels on the transport of amino acids and membrane structure ofMicrosporum gypseum

Ergosterol and cholesterol supplementation resulted in a significant increase (1·5-fold) in the sterol content while phospholipid remained unaffected inMicrosporum gypseum. The levels of phosphatidylethanolamine and phosphatidylcholine increased in ergosterol supplemented cells. However, a decrease in phosphatidylcholine and an increase in phosphatidylethanolamine was observed in cholesterol grown cells. The ratio of unsaturated to saturated fatty acids decreased on ergosterol/cholesterol supplementation. The uptake of amino acids (lysine, glycine and aspartic acid) decreased in sterol supplemented cells. Studies with fluorescent probe l-anilinonaphthalene-8-sulfonate showed structural changes in membrane organisation as evident by increased number of binding sites in such cells.     

Role of inositol trisphosphate as a second messenger in signal transduction processes: An essay

This essay attempts to summarize some of the best evidence for the role of inositol trisphosphate as a second messenger in signal transduction processes. The following aspects are addressed in the essay: (a) The synthesis of inositol trisphosphate and other inositol lipids, (b) Receptor-phosphatidylinositol bisphosphate phospholipase C coupling and the N-ras protooncogene, (c) Inositol trisphosphate and intracellular calcium, (d) Cell growth and oncogenes, (e) Receptors linked to the phosphatidylinositol cycle, (f) Phototransduction and (g) Interactions between inositol trisphosphate and other second messengers.Abbreviations Cyclic AMP Adenosine 3,5-cyclic monophosphate - Cyclic GMP Guanosine 3,5-cyclic monophosphate - DG sn, 1,2-Diacylglycerol - EGF Epidermal growth factor - GDP Guanosine diphosphate - GTP Guanosine triphosphate - IP Inositol 1-monophosphate - IP₂ Inositol 1,4-diphosphate - IP₃ Inositol 1,4,5-trisphosphate - PA Phosphatidic acid - PDGF Platelet-derived growth factor - PI Phosphatidylinositol - PIP Phosphatidylinositol 4-monophosphate - PIP₂ Phosphatidylinositol 4,5-bisphosphate - PIP₃ Phosphatidylinositol 3,4,5-trisphosphate - PLC Phospholipase C     

Pharmacological activities of branched-chain amino acids: specificity of tissue and signal transduction

Branched-chain amino acid (BCAA: Leu, Ile, and Val) mixture has been used for treatment of hypoalbuminemia in patients with decompensated liver cirrhosis in Japan. It has been known that BCAA, especially leucine, activates mTOR signals and inhibition of protein degradation results in promoting protein synthesis in vitro. Furthermore, leucine activates glycogen synthase via mTOR signals in L6 cell, but not hepatocyte, and it has been shown that leucine improved glucose metabolism in normal and cirrhosis model rats. In this review, it will be proposed about the pharmacological activity of branched-chain amino acids, mainly leucine, on tissue specificity of cirrhotic disease.     

Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria

Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients. These compounds have to pass the membrane. However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems. A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane. This improved model system has been used to analyze the properties of amino acid transport systems in lactococci. Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment. The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed.Abbreviations pmf protonmotive force - transmembrane electrical potential - pH transmembrane pH gradient - PE phosphatidylethanolamine - PC phosphatidylcholine Paper adapted from a treatise Secondary Transport of Amino Acids by Membrane Vesicles Derived from Lactic Acid Bacteria and awarded the Kluyver Prize 1988 by the Netherlands Society of Microbiology.     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Comparative use of amino acids by three auxotypes of Neisseria gonorrhoeae

Auxotypes of Neisseria gonorrhoeae are usually distinguishable by their particular requirements for growth; these requirements often include amino acids. It is possible that strains needing particular substrates to grow can be distinguished not merely by their growth requirements but also by their metabolism of these particular substrates. In this work amino acid utilization and oxidation studies were performed enabling prototype, pro- and thia-strains to be distinguished. The metabolism study also underlined the importance of proline as an energy source and pointed to the probability of distinct relationships with the metabolism of the key amino acids, glutamic and aspartic acids, for the three auxotypes.It is proposed that the specific amino acid required by the naturally occurring auxotype serves as an energy source at the site of infection and has important implications with respect to particular auxotypes at various sites.     

Second messengers involved in the muscarinic control of the heart: the role of the phosphoinositide response

Two heart muscarinic responses are compared, the reduction of cyclic AMP concentration and the hydrolysis of phosphoinositides. It is suggested that the former is more important physiologically, since the latter is a slow response requiring much higher agonist concentrations. Inositol trisphosphate released from phosphoinositides is unlikely to produce a positive inotropic effect by releasing calcium from the sarcoplasmic reticulum. However, long term muscarinic effects may involve activation of protein kinase C by diacylglycerol released from phosphoinositides.     

Changes in fatty acids,amino acids and carbon/nitrogen biomass during nitrogen starvation of ammonium- and nitrate-grownIsochrysis galbana

Growth of cells ofIsochrysis galbana with either nitrate or ammonium as the N-source, and the effects of subsequent N-starvation of these cells, were compared. During exponential N-sufficient growth nitrate-grown cells had double the fatty acid content of the ammonium-grown cells but lower concentrations of a few amino acids. Following resuspension in N-free medium the fatty acid content of the ammonium-grown cells increased to that of the nitrate-grown cells, but there was no further increase in fatty acid content on a C-biomass or cellular basis during the following 4 days for either culture. Fatty acid synthesis was continuous during N-starvation, while it occurred during the light-phase only in exponential growth. The proportion of 18:1n9 fatty acid increased from 10 to 25% total fatty acids during N-starvation. Intracellular free amino acid content decreased in a similar manner in both cultures on N-starvation, the ratio of intracellular free amino-N/cell-C falling more rapidly than overall cellular N/C. It was concluded that optimal amino acid and fatty acid content would be attained by growth in the presence of excess nitrate. Measurements of chlorophyll and carotenoid content and ofin vivo fluorescence indicated that these parameters had potential for monitoring the C and N biomass in cultures grown under relatively constant (not necessarily continuous) illumination.     

Transport of branched-chain amino acids in Corynebacterium glutamicum

The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (K _m[Ile]=5.4 M, K _m[Leu]=9.0 M, K _m[Val]=9.5 M). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na⁺ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na⁺-coupled symport mechanism.Abbreviations CCCP Carboxyl cyanide m-chlorophenylhydrazone - dw dry weight - MES 2[N-morpholino]ethanesulfonic acid - mon monensin - nig nigericin - TPP tetraphenylphosphonium bromide - Tris tris[hydroxymethyl]aminomethane - val valinomycin     

Uptake of free amino acids by the diatom,Melosira mediocris

Individual ¹⁴C-labelled amino acids are rapidly removed from dilute solution in artificial sea water (0.2 mol 1^–1) by suspensions of Meliosira medocris. The rate of disappearance of radioactivity corresponds closely to removal of primary amines as determined by measurement of the rate of decrease of fluorescamine-positive material. Net removal of naturally occurring free amino acids from the sea water habitat from which the alga was isolated is demonstrated using high performance liquid chromatography. Removal of amino acids from natural sources makes a significant contribution to the carbon requirements of the alga as well as supplying significant amounts of amino nitrogen.     

The role of free sugars and amino acids in the regulation of biomass partitioning and plant growth

Theoretical plant growth models postulate an important role for growth substrates such as sugars and amino acids. To test this experimentally, spinach plants were grown under controlled conditions and with nitrogen added daily, following different exponential addition schemes. Plants were harvested during exponential growth. Free amino acid levels or free sugar levels were only weakly correlated with growth and biomass partitioning. Factor analysis showed however that the product of free sugar concentration and amino acid concentration yielded a parameter adequately reflecting the plant's nutritional state.It is concluded that growth and biomass partitioning under limiting N conditions cannot be modelled solely based on N substrate levels.     

Unusual regulation of the uptake system for branched-chain amino acids in Corynebacterium glutamicum

The uptake of branched-chain amino acids in threonine-dehydratase deficient mutants of Corynebacterium glutamicum is dependent on the presence of relatively high (>1 mM) intracellular concentrations of isoleucine, valine or leucine. This indicates that the respective uptake-system is induced by its substrate, i.e. branched-chain amino acids, at the internal side. This unusual regulation presumably is the reason for the failure to obtain mutants deficient in isoleucine uptake by use of a selection scheme which starts from isoleucine auxotroph mutants. The physiological meaning of this regulation is discussed with respect to isoleucine efflux and the cyclic retention hypothesis.Abbreviations amp ampicillin - dw dry weight - Km kanamycin - kb kilobase(s) - NMG N-methyl-N-nitro-N-nitrosoguanidine - ®, resistant resistance     

Local Differences in GABA Release Induced by Excitatory Amino Acids During Retina Development: Selective Activation of NMDA Receptors by Aspartate in the Inner Retina

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.     

Enzyme-catalysed deprotection of N-acetyl and N-formyl amino acids

To investigate the full potential of hydrolases for the removal of two amine-protecting groups, 15 different, commercially available lipases, acylases, proteases and esterases were studied for the hydrolyses of N-acetyl and N-formyl protecting groups. In addition to the well-known acylases from porcine kidney and Aspergillus melleus, this screening revealed that porcine liver esterase and the lipases from Rhizomucor miehei and Pseudomonas stutzeri are also catalysts for the hydrolysis of N-acetylalanine. The activity of lipases in this reaction was unexpected, since lipases are commonly believed not to hydrolyse amides. In addition, from these 15 enzymes, three were found to be active in the hydrolysis of N-formylalanine, i.e. porcine liver esterase and the two acylases. This is the first example where esterase is employed to deprotect N-formyl amides.     

The uptake of copper ions by cell suspension cultures of Agave amaniensis,and its effect on the growth,amino acids and hecogenin content

Cell suspension cultures of Agave amaniensiswere able to grow in media containing 10 – 240 M copper ions, and could remove more than 67% copper ions from the media. The cells accumulated up to 106 mg g^–1 copper ions in the biomass. Copper ions at 240 M caused a decrease in growth index and packed cell volume of the cultures of 61.5 and 53.3%, respectively. The presence of copper ions caused the cell walls to thicken and to be more wrinkled. Certain amino acids were released in high concentration into the media. The hecogenin content in the biomass increased up to 157.9% at 20 M copper ions.     

The involvement of amino acids in latex lipid synthesis in Euphorbia lathyris seedlings

The breakdown of triglycerides and proteins in the endosperm of Euphorbia lathyris was assayed in a 14 day germination period. Six days after germination, the average daily production was 2.7 μmol of amino acids. Arginine, glutamine, asparagine and glutamic acid accounted for 53% of the total amino acids. Excised cotyledons with 1 cm hypocotyls were used for amino acid uptake and their involvement in terpenoid synthesis was studied. Glutamine and aspartate were hardly involved in apolar lipid synthesis. Leucine, isoleucine, valine and threonine were mainly incorporated into the triterpenes in the laticifers. Alanine and serine were also involved in phytosterol synthesis in the adjacent tissue. In the 14 day germination period, ca3% of the daily yield of latex triterpenes may be synthesized from a variety of amino acids.     

Interactions between the branched-chain amino acids in the growth of Spirodela polyrhiza

The joint action of L-valine and L-isoleucine, L-leucine and L-isoleucine, and L-valine and L-leucine on the growth of Spirodela polyrhiza was established. The effect of one branched-chain amino acid on growth inhibition by another one was compared with the non-specific antagonisms which glycine and L-alanine exert on growth inhibition by singly supplied branched-chain amino acids. In this way specific and non-specific interactions could be distinguished. It appeared that: (1) L-isoleucine was a specific antagonist of L-valine; (2) L-leucine was a specific antagonist of L-isoleucine; (3) L-valine and L-leucine were synergistic growth inhibitors. Further, it was found that: (4) growth inhibition by L-leucine was specifically antagonized by simultaneously supplied L-valine and L-isoleucine; (5) an excess of L-isoleucine strongly inhibited the conversion of exogenous valine into leucine; (6) accumulation of valine was typical of isoleucine-induced growth inhibition. The results are consistent with the view that growth inhibition by L-valine and L-leucine is due to the blocking of acetohydroxy acid synthetase, the first common enzyme in the valine-isoleucine biosynthetic pathway. Growth inhibition by L-isoleucine, however, seems to result from inhibition of leucine synthesis at a step after 2-oxoisovaleric acid. Some aspects of the regulation of branched-chain amino acid biosynthesis in higher plants are discussed.     

Carbachol-induced accumulation of inositol phosphates and its modulation by excitatory amino acids in cortical slices of young and aged rats with down-regulation of muscarinic M-1 receptors

The effects of a subacute intoxication with diisopropyl fluorophosphate (DPF) on total muscarinic acetylcholine receptor sites (mAChRs) and M-1 AChRs were evaluated in the cerebral cortex of young (2–4 months) and aged (22–24 months) Fischer 344 rats. Since M-1 AChRs are coupled to the metabolism of phosphoinositides, carbachol-induced accumulation of inositol phosphates (IP) and its inhibition by glutamate and NMDA was also measured in the cortical slices. DFP treatment caused about 75% inhibition of cholinesterase and 35% down-regulation of mAChRs (measured as [³H]quinuclidinyl benzylate binding) in both young and aged rats. The down-regulation of M-1-ACHRs (measured as [³H]pirenzepine binding) was more pronounced in aged (30%) than in young (17%) DFP-treated rats. There was a significant increase in carbachol-induced IP accumulation in aged, with respect to young, untreated rats. DFP treatment caused a considerable decrease in such IP accumulation in aged but not in young rats. Glutamate and NMDA antagonized carbachol-induced IP accumulation in untreated young and aged rats (and the effects of NMDA were reversed by carboxy-piperazinyl-propyl phosphonic acid). In DFP-treated rats such antagonism was somewhat less pronounced. The data appear of interest in relation to the use of anticholinesterase compounds in the therapy of senile dementia of Alzheimer's type. They suggest that beside their primary action (increasing brain ACh levels) such compounds also act on post-receptor mechanisms and on the interactions between cholinergic and glutamatergic neurotransmitter systems.     

Nitrogen deficiency induced changes of free amino acids and coumarin contents in the leaves of Matricaria chamomilla

Through o-hydroxycinnamic acids, the biosynthesis of coumarins is connected with aromatic amino acid metabolism and nitrogen uptake. Therefore the quantitative changes in levels of some free amino acids and coumarins (herniarin and its glucosidic precursors (Z) - and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids; umbelliferone) in the leaf rosettes of chamomile (Matricaria chamomilla L.) subjected to nitrogen deficiency were studied. Nitrogen content decreased in the leaf rosettes and in the roots of N-deficient plants during the course of the experiment, but these plants produced significantly higher root biomass. Among secondary metabolites, the sum of 2-β-D-glucopyranosyloxy-4-methoxycinnamic acids increased sharply, herniarin increased slowly and the content of umbelliferone was low in N-deficient plants. We have concluded that nitrogen deficiency is not an inducing factor for stress accumulation of herniarin and umbelliferone. A decrease in levels of all detected amino acids, besides histidine, was found. Within aromatic amino acids, tyrosine was the most abundant. The content of free phenylalanine was significantly lower in both, control and N-deficient plants when compared to the content of tyrosine. In this view, the increase of herniarin glucosidic precursors is apparently due to enhancing phenylalanine ammonia-lyase activity under nitrogen deficiency and nitrogen-free carbon skeletons are shunted in to the phenylpropanoid metabolism, including biosynthesis of (Z)-and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids.