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Phorbol ester treatment of K562 cells regulates the transcriptional activity of AML1c through phosphorylation.

We find that phorbol ester (PE) treatment of K562 cells greatly stimulates promoters (T cell receptor beta, myeloperoxidase, macrophage colony-stimulating factor receptor, and granulocyte macrophage colony-stimulating factor receptor) containing AML1 transcription factor binding sites. This stimulation of AML1c transcriptional activity is mediated by direct phosphorylation of the AML1c molecule on multiple phosphorylation sites. Eleven AML1c (S/T)P sites in the transcriptional activating domain are phosphorylated at a basal level in untreated K562 cells; treatment of the K562 cells with PE results in increased phosphorylation at five of these sites (serines 276, 293, 303, 462, and threonine 300). Mutation of these five sites to alanine inhibits PE-induced transcriptional activity; mutation of the sites to an acidic amino acid, aspartic acid, stimulates constitutive activity. Single mutations in four amino acids or double mutations (serines 276 and 293 or threonine 300 and serine 303) have little effect on AML1c transcriptional activity. Inhibitor assays suggest that the ERK family of protein kinases is activated by PEs to phosphorylate the (S/T)P sites within the AML1c molecule and markedly enhance the transcriptional activity of AML1c.

Aspartic Acid↗

The evaluation of the potential of botulinum C3 enzyme as an exogenous differentiation inducing factor to neurons.

Botulinum C3 enzyme produced by Clostridium botulinum type C and D strains modifies Rho proteins. In a previous study, we observed that the LDH isozyme pattern of neurons treated with C3 enzyme was different from that induced with endogenous growth factor of neurons such as NGF [21]. This type of change is considered to have an advantage in the medical use of C3 enzyme for neural disorder. To determine the functional similarity of C3-treated neurons to control and NGF-treated neurons, we examined the responses of C3-treated neurons to various drugs, including some neurotransmitters, by measuring the rise of intracellular Ca ions into the neurons. The time course of the rise of intracellular Ca ions induced by high concentration of potassium in the C3-treated neurons was similar to that in the NGF-treated neurons. The C3-treated neurons responded to glutamic acid, aspartic acid, kainic acid, gamma-aminobutylic acid, muscarine and ACh with similar time courses and magnitudes as the control neurons. These results suggest that the C3 enzyme induces the functional differentiation of neurons, and that C3 enzyme has the potential for the medical use as an exogenous differentiation-inducing factor of neurons.

ADP Ribose Transferases↗

Free-standing nanogold membranes as supports for the growth of calcium phosphate crystals.

Current strategies for bone tissue regeneration focus on the development of implantable matrices that mimic biological tissues. Inorganic composites are of special interest for bone substitute applications. It is necessary to create an artificial three-dimensional scaffold-like porous material with certain geometrical structure to induce bone growth. We report here the growth of calcium phosphate crystals on free-standing carboxylic acid functionalized gold nanoparticle membranes. The gold nanoparticle membrane is synthesized by the spontaneous reduction of aqueous chloroaurate ions by a diamine molecule at a liquid-liquid interface. This membrane is robust and malleable, and most importantly, the gold nanoparticles in the membrane may be functionalized with suitable ligands. In this study, the amino acids aspartic acid and cysteine together with an aromatic bifunctional molecule, anthranilic acid, were used to modify the surface of the gold nanoparticles in the membrane. The free carboxylic acid groups on the gold nanoparticles further to functionalization with these molecules were then used to bind Ca(2+) ions and reacted with phosphate ions to yield calcium phosphate. The nature of the nanogold surface modifier directed the formation of either crystalline hydroxyapatite or amorphous calcium phosphate. The nanogold membrane thus suggests potential biomedical application as biocompatible implants and grafts.

Aspartic Acid↗

Aspartame. Review of safety issues. Council on Scientific Affairs.

This report examines the safety issues related to the nutritive sweetener aspartame, including possible toxic effects of aspartame's component amino acids, aspartic acid and phenylalanine, and its major decomposition products, methanol and diketopiperazine, and the potential synergistic effect of aspartame and dietary carbohydrate on brain neurochemicals. Available evidence suggests that consumption of aspartame by normal humans is safe and is not associated with serious adverse health effects. Individuals who need to control their phenylalanine intake should handle aspartame like any other source of phenylalanine.

Adolescent↗

Isolation and characterization of a hydroxyproline-containing protein from soluble extracts of the leaves of sandal (Santalum album L.).

1. A hydroxyproline-containing protein was isolated from the soluble fraction of sandal leaves (Santalum album L.) and the purified protein was homogeneous by disc electrophoresis. 2. It is a glycoprotein containing 16% carbohydrate, the components of which were mainly arabinose, with only small amounts (about 5%) of galactose. The principal amino acids were glutamic acid, aspartic acid, glycine, alanine, arginine, lysine, proline and hydroxyproline, which together comprised 60% of the total. The number of acidic amino acids exceeds the number of basic amino acids. By Sephadex gel filtration, the approximate molecular weight was found to be about 63000. The ratio of residues of hydroxyproline to those of arabinose was 1:2. 3. The native protein is resistant to the action of several proteolytic enzymes. After partial hydrolysis with 0.1m-HCl, the protein became susceptible to attack by Pronase but remained resistant to collagenase.

Alanine↗

The turnover rate of rabbit urinary Tamm-Horsfall glycoprotein.

1. The turnover rate of urinary Tamm-Horsfall glycoprotein in rabbits was determined by two different methods. The first involved measurement of the pool size of the glycoprotein in rabbit kidney and the daily urinary excretion rate by a radioimmunoassay from which the turnover rate was calculated. 2. The second method made use of the incorporation in vivo of Na(2) (14)CO(3) and sodium [(14)C]acetate. After a single intramuscular injection of one of these compounds, urine collections were made every 24h and the glycoprotein was isolated and its specific radioactivity was determined. 3. Incorporation of the label into urinary HCO(3) (-), urea and plasma fibrinogen was also examined. The specific radio-activities of the O-acetyl, sialic acid, aspartic acid and glutamic acid residues isolated from the Tamm-Horsfall glycoprotein were compared and their half-lives were compared with that of the intact glycoprotein. The two methods gave results in quite close agreement and indicated a half-life for the glycoprotein of approx. 9h. 4. An attempt was made to localize the glycoprotein within the kidney and within the cell. It is present throughout the kidney, but was not detected in the brush-border fraction isolated from the proximal tubules. From differential cell-centrifugation studies, the glycoprotein seemed to be predominantly present in the soluble fraction (100000g supernatant). This suggests that it is either largely a soluble cytoplasmic component or is very loosely bound to a membrane, being readily released under the gentlest homogenization procedure. 5. The half-life of Tamm-Horsfall glycoprotein in human kidney was found by the radioimmunoassay method to be approx. 16h. The similarity between the composition of Tamm-Horsfall glycoprotein and human erythropoietin is discussed.

Acetates↗

Selective inhibition of synaptosomal proline uptake by leucine and methionine enkephalins.

The high affinity, sodium-dependent uptake of proline by rat brain synaptosomes was inhibited by the opioid pentapeptides, Leu-enkephalin and Met-enkephalin. The synaptosomal uptake of other putative neurotransmitter amino acids including glutamic acid, aspartic acid, gamma-aminobutyric acid, and taurine was not altered in the presence of enkephalins. The uptake of a neuroinactive amino acid, leucine, was also unaffected by enkephalins. The extent of proline uptake was half-maximal at a Leu-enkephalin concentration of 1 microM. Both the initial rate of transport and the overall capacity for proline accumulation were reduced. The effect of the enkephalins was vectorial since carrier-mediated efflux of proline was not altered in the presence of enkephalins. Morphine and the opioid peptides, dynorphin and beta-endorphin, were without effect on proline uptake. The inhibition of proline uptake by enkephalins was not diminished by prior incubation of the synaptosomal preparation with naloxone; however, the inhibition was attenuated by 1-butanol. The des-tyrosyl fragments of the enkephalins were as inhibitory as the intact pentapeptides. A modified enkephalin ([D-Ser2]Leu-enkephalin-Thr) with selective affinity for the delta subclass of enkephalin receptor was effective in inhibiting proline uptake. On the basis of the selectivity of these effects, we propose that there is a specific population of nerve endings in the cerebral cortex that contains both a proline-transport system and binding sites for Leu- and Met-enkephalin and furthermore, that these binding sites may be related to the putative delta receptor.

Animals↗

Dependence of apparent viscosity on mycelial morphology of Streptomyces fradiae culture in various nitrogen sources.

To examine what causes increased viscosity in culture broth in Streptomyces fradiae culture, various natural nitrogen sources were investigated. Extracellular protease activity increased with culture time and decomposed the natural nitrogen source into amino acids. In the case of gluten meal, after a culture time of 5 d, concentrations of glutamic acid and aspartic acid had increased to 600 and 200 mg/L, respectively, which were about 3- and 2-fold as high as levels in cultures under similar conditions using Pharmamedia. For various amino acids tested, the addition of glutamic acid or aspartic acid mixture to the culture medium raised the apparent viscosity to its highest demonstrated value, 260 mPa.s after 5 d of culture, which was 3-fold higher than without amino acids. Consumption of the decomposed glutamic acid and aspartic acid was dependent on the activities of glutamate dehydrogenase and aspartate aminotransferase, respectively. When ammonium ion was used as the nitrogen source, cell concentration reached 1.75 g/L measured as an intracellular nucleic acid concentration, which was about 2.3-fold higher than that with any other natural nitrogen source. However, apparent viscosity was only 75 mPa.s, a value one-third that of the amino acid mixture, and 70% of the pellets were bigger than 1.2 x 10(4) microm(2). In the case of gluten meal or the amino acid mixture, pellets bigger than 1.2 x 10(4) microm(2) comprised only 8%. This demonstrates that consumption of some amino acids affected the formation of filamentous morphology, which caused an increase in the apparent viscosity of the culture broth, and the apparent viscosity was not caused by the mycelial concentration but the mycelial morphology.

Amino Acid Oxidoreductases↗

The relative potency of pentobarbital in suppressing the kainic acid- or the N-methyl-D-aspartic acid-induced enhancement of cGMP in cerebellar cells.

Primary cultures of rat cerebellar cells were pretreated with various dosages of pentobarbital before the addition of kainic acid or N-methyl-D-aspartic acid in order to assess effects of this drug on the enhancement of cyclic guanosine-3',5'-phosphate (cyclic GMP) mediated by these excitatory agonists. Pentobarbital significantly suppressed kainic acid-induced increases in this cyclic nucleotide at concentrations as low as 5 microM but was only effective in suppressing the N-methyl-D-aspartic acid enhancement at dosages of 100 microM or greater. These data suggest that this barbiturate is a more effective depressant of the stimulatory effects of kainic acid as compared to N-methyl-D-aspartic acid.

Animals↗

Carbon dioxide fixation by cells of Streptococcus faecalis var. liquefaciens.

Fixation of NaH(14)CO(3) by a heavy cell suspension of Streptococcus faecalis var. liquefaciens was studied. Several nutrients, pyridoxal, riboflavine, adenine, uracil, and O(2) stimulated (14)CO(2) incorporation into cells only under conditions that were adequate for synthesis of cell macromolecules. Biotin increased CO(2) incorporation in the absence of extensive synthesis of macromolecules, whereas O(2) inhibited incorporation under these conditions. When (14)CO(2) fixation was occurring during synthesis of macromolecules, 71% of the (14)C was incorporated into cells and 29% occurred extracellularly. Ninety-three per cent of the cellular (14)C was in protein and 5.5% was in nucleic acid. Aspartic acid was the only amino acid in the protein fraction that was radioactive. Eighty-three per cent of the extracellular (14)C was resistant to precipitation by trichloroacetic acid. When (14)CO(2) fixation was occurring in cells that were not carrying on extensive synthesis of macromolecules, 38% of the (14)C was incorporated into cells and 59% occurred in the supernatant fluid. Sixty-nine per cent of the cellular (14)C was in protein, 21% was in low-molecular-weight compounds, and 9% was in nucleic acid. Addition of unlabeled aspartate to the medium inhibited incorporation of (14)CO(2). Based on studies of the rate of (14)CO(2) fixation, the cells fix CO(2) into a pool of intermediates which are either used for synthesis, primarily protein, or are excreted into the medium.

Carbon Dioxide↗

[Amino acid metabolism in neurodegenerative diseases].

Although various neurological diseases occur in patients with inborn error of metabolism of amino acids, amino acids also act as neurotransmitters. Glutamic acid, aspartic acid and glycine play roles as an excitatory neurotransmitter, but exert a neurodegenerative effect in case of the excessive release. Extensive studies have recently been performed on glutamate receptors, especially N-methyl-D-aspartate (NMDA) receptor in the hippocampus. Alzheimer brain shows a decreased number of NMDA receptors in the frontal cortex. The parkinsonian changes caused by MPTP is abolished by the administration of a NMDA antagonist. gamma-Aminobutyric acid (GABA) acts as an inhibitory amino acid. The content of GABA is low in the striatum of patients with Huntington's disease. The number of NMDA receptor is decreased also in Huntington striatum. These observations may give a clue for the prevention of various neurodegenerative diseases.

Alzheimer Disease↗

Aspartame: review of recent experimental and observational data.

In this report the neurotoxicity of aspartame and its constituent amino acids aspartic acid and phenylalanine is reviewed. The adverse reactions ascribed to the consumption of aspartame-containing products, as reported in the U.S.A., are discussed and placed in perspective with the results of recent behavioural studies in humans and animals. The issue of common intake levels associated with proposed uses of aspartame is addressed. In brief, the following conclusions can be drawn: When aspartame is consumed at levels within the ADI-limit of 40 mg/kg body wt, there is no significant risk for an aspartate-induced neurotoxic effect in the brain. When aspartame is consumed at levels within the ADI-limit by normal subjects or persons heterozygous for phenylketonuria (PKU) the resultant plasma phenylalanine concentrations are practically always within the normal postprandial range; elevation to plasma concentrations commonly associated with adverse effects has not been observed. Persons suffering from phenylketonuria (PKU-homozygotes) on a phenylalanine-restricted diet should avoid consumption of aspartame. PKU-homozygotes on the (less strict) phenylalanine-liberalized diet should be made aware of the phenylalanine content of aspartame. In the available behavioural studies in humans with acute dosing, no adverse effects were observed. Long-term studies on behaviour and cognitive function in (sensitive) humans are lacking. Analyses of adverse reaction reports made by consumers in the U.S.A. have not yielded a specific constellation of symptoms clearly related to aspartame that would suggest a widespread public health hazard associated with aspartame use. Focussed clinical studies are now being carried out in the U.S.A.; the results should provide additional evidence concerning the interpretation of the reports on adverse reactions ascribed to aspartame. In the regulation of admitted uses for aspartame the possibility of intake levels exceeding the ADI-limit in some groups of consumers should be a point of attention.

Animals↗

Voltage-induced release of nucleic acids from palaeontological samples.

Most of the protocols for the recovery of ancient DNA from palaeontological specimens are time-consuming and tend to yield inconsistent polymerase chain reaction (PCR) results. "Voltage-induced release" is a novel and rapid approach for the extraction of ancient DNA. Nucleic acids are directly electrophoresed out of powder derived from hard and soft tissues. This technique is much faster than other methods in which pulverized tissue conventionally undergoes time-consuming crude lysis steps. The total preparation time is 5-6 h. The reliability of the voltage-induced release method was validated by (i) measuring the ratio of D-to L-enantiomers of the amino acids aspartic acid, alanine, and leucine, and (ii) by specific PCR amplification of four single-copy markers of human chromosome 17 and 18. We compare voltage-induced release to a frequently used silica-based protocol. DNA extracted employing voltage-induced release was more effective in PCR amplifications, which may be attributed to the effective removal of PCR inhibitors.

DNA↗

Mollusk shell formation: a source of new concepts for understanding biomineralization processes.

The biological approach to forming crystals is proving to be most surprising. Mollusks build their shells by using a hydrophobic silk gel, very acidic aspartic acid rich proteins, and apparently also an amorphous precursor phase from which the crystals form. All this takes place in a highly structured chitinous framework. Here we present ideas on how these disparate components work together to produce the highly structured pearly nacreous layer of the mollusk shell.

Animals↗

Electrophoresis-related protein modification: alkylation of carboxy residues revealed by mass spectrometry.

In recent years, the combination of gel electrophoresis and mass spectrometry has developed into one of the most powerful approaches for the analysis of proteins. However, a number of gel electrophoresis-induced protein modifications have been described. Cysteine is the most endangered amino acid readily reacting with mercaptoethanol or free acrylamide. In the course of studies on glucan phosphorylases (E.C.2.4.1.1) from white potato (Solanum tuberosum L.) and the T cell receptor, we noticed that proteolytic peptides from these proteins can undergo an unexpected modification, giving rise to a mass increment of 14 Da. By post-source decay (PSD) analysis the modification was identified as methylation of the glutamic acid side chain carboxyl group. The methylation takes place during Coomassie blue staining of proteins if both trichloroacetic acid and methanol are present in the staining solution. Replacement of methanol by ethanol under otherwise unchanged conditions results in ethylation of the peptides. The in vitro alkylation was further studied by using synthetic peptides which contain, at different positions: glutamic acid, aspartic acid or the corresponding amides. The kinetic analysis of the observed reactions revealed that glutamic acid is preferentially methylated. The three other amino acid residues can be methylated but with a velocity at least one order of magnitude lower. Although these modifications complicate the interpretation of the spectra, they provide valuable structural information.

Alkylation↗

Biochemical studies of some non conventional sources of protein. Part 4. The proteins of mango waste stone kernels.

The gross chemical composition, protein content and amino acid composition of mango stone kernels of four Egyptian varieties (Ewsi, Hendi, Fonso and Timour) were investigated. Carbohydrates are the main components of the seed. The protein content varies between 5.0 and 7.2% and the ether extract ranged from 10.8% to 13.6%. High glutamic acid, aspartic acid and leucine content and low concentration of sulfur-containing amino acids are characteristic for the proteins of mango kernels. Lysine level is also low, the concentration of other essential amino acids is acceptable. Sulfur-containing amino acids are the first limiting ones followed by lysine and threonine.

Amino Acids↗

A thermodynamic scale for leucine zipper stability and dimerization specificity: e and g interhelical interactions.

The leucine zipper is a dimeric coiled-coil protein structure composed of two amphipathic alpha-helices with the hydrophobic surfaces interacting to create the dimer interface. This structure has been found to mediate the dimerization of two abundant classes of DNA binding proteins: the bZIP and bHLH-Zip proteins. Several workers have reported that amino acids in the e and g positions of the coiled coil can modulate dimerization stability and specificity. Using the bZIP protein VBP as a host molecule, we report a thermodynamic scale (delta delta G) for 27 interhelical interactions in 35 proteins between amino acids in the g and the following e positions (g<==>e') of a leucine zipper coiled coil. We have examined the four commonly occurring amino acids in the e and g positions of bZIP proteins, lysine (K), arginine (R), glutamine (Q), glutamic acid (E), as well as the only other remaining charged amino acid aspartic acid (D), and finally alanine (A) as a reference amino acid. These results indicate that E<==>R is the most stable interhelical pair, being 0.35 kcal/mol more stable than E<==>K. A thermodynamic cycle analysis shows that the E<==>R pair is 1.33 kcal/mol more stable than A<==>A with -1.14 kcal/mol of coupling energy (delta delta Gint) coming from the interaction of E with R. The E<==>K coupling energy is only -0.14 kcal/mol. E interacts with more specificity than Q. The R<==>R pair is less stable than the K<==>K by 0.24 kcal/mol. R interacts with more specificity than K. Q forms more stable pairs with the basic amino acids K and R rather than with E. Changing amino acids in the e position to A creates bZIP proteins that form tetramers.

Amino Acid Sequence↗