Biochemistry, Genetics and Molecular Biology › Biochemistry

Amino Acid Enzymes and Metabolism

Works Count: 75660

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This cluster of papers explores the transport, metabolism, and physiological roles of amino acids, with a focus on D-serine, NMDA receptor function, and their implications in neurological disorders such as schizophrenia. It also delves into the role of amino acid transporters in cancer, metabolic disorders, and neuronal function.

Keywords

Amino Acid Transporters; D-Serine; NMDA Receptor; Schizophrenia; Hartnup Disorder; Glutamine Transport; Neurotransmitters; Cancer; Metabolic Disorders; Neuronal Function

[6] Determination of cystine as cysteic acid

1967-01-01

C.H.W. Hirs

Methoden der enzymatischen Analyse

1962-01-01

Hans Ulrich Bergmeyer

Glucose Transport in Isolated Brush Border Membrane from Rat Small Intestine

1973-01-01

Ulrich Hopfer , Kristine Nelson , Joseph L. Perrotto +1 more

Abstract Uptake of labeled d- and l-glucose has been shown to occur with highly purified brush border membranes from the epithelial cells of rat small intestine using a Millipore filtration … Abstract Uptake of labeled d- and l-glucose has been shown to occur with highly purified brush border membranes from the epithelial cells of rat small intestine using a Millipore filtration technique. An intact glucose carrier system in the isolated membranes was demonstrated as evidenced by the following. (a) d-Glucose was taken up and released faster than l-glucose. (b) Sodium ions increased initial rate and extent of d-glucose uptake 3- to 5-fold; no other cation showed this effect. (c) d-Glucose uptake and release was inhibited by phlorizin. (d) Countertransport of d-glucose was demonstrated. (e) d- and l-glucose reached the same level of uptake after prolonged incubation. (f) Uptake of labeled d-glucose was inhibited by higher concentrations of unlabeled d-galactose and vice versa. Glucose uptake by membrane vesicles represented entry into an intravesicular aqueous space rather than binding to the membrane. Exposure of the membrane to increasing cellobiose concentrations led to osmotic shrinkage of the intravesicular space and decreased glucose uptake. Infinite medium osmolarity and therefore zero intravesicular space resulted in no glucose uptake. Sodium in the medium (but not in the intravesicular spaces) stimulated d-glucose transport. It is concluded that isolated brush border membranes of intestinal epithelial cells retain the glucose carrier system. The reported findings are consistent with the concept that (a) glucose transport across the brush border membrane represents facilitated diffusion; (b) the glucose carrier is dependent on sodium ions; and (c) high extracellular, but not intracellular sodium concentrations lead to increased glucose transport.

THE FREE AMINO ACIDS OF HUMAN BLOOD PLASMA

1954-12-01

William H. Stein , Stanford Moore

Complete amino acid analysis of proteins from a single hydrolysate.

1976-04-01

Robert J. Simpson , M R Neuberger , T Y Liu

An analytical procedure which affords the precise amino acid composition of a protein or a peptide from a single hydrolysate is described. This method utilizes 4 N methanesulfonic acid containing … An analytical procedure which affords the precise amino acid composition of a protein or a peptide from a single hydrolysate is described. This method utilizes 4 N methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole, rather then 6N HCl as a catalyst for hydrolysis. The hydrolysis is carried out in vacuo (20 mu) at 115 degrees for 22 to 72 hours. Half-cystine is determined as S-sulfocysteine by treating the hydrolysate with dithiothreitol followed by an excess of tetrathionate. The values of all amino acids, including tryptophan and half-cystine, were close to the expected theoretical values for the proteins examined. The method has the advantage that the neutralized hydrolysate can be applied directly to an ion exchange column. Further, the method is capable of distinguishing between free sulfhydryl groups as S-carbosymethylcysteine and disulfides as S-sulfocysteine. A limitation of the procedure is that tryptophan remains sensitive to the presence of carbohydrate in the sample.

Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography.

1976-09-01

Y. Yasukochi , Bettie Sue Siler Masters

NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an … NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.

Excitatory amino acid receptors in the vertebrate central nervous system.

1989-06-01

Graham L. Collingridge , R.A.J. Lester

Ifenprodil discriminates subtypes of the N-methyl-D-aspartate receptor: selectivity and mechanisms at recombinant heteromeric receptors.

1993-10-01

Keith Williams

The effects of the atypical N-methyl-D-aspartate (NMDA) receptor antagonist ifenprodil were investigated by voltage-clamp recording of Xenopus oocytes expressing heteromeric NMDA receptors from cloned NR1 and NR2 subunit RNAs. In … The effects of the atypical N-methyl-D-aspartate (NMDA) receptor antagonist ifenprodil were investigated by voltage-clamp recording of Xenopus oocytes expressing heteromeric NMDA receptors from cloned NR1 and NR2 subunit RNAs. In oocytes voltage-clamped at -70 mV, ifenprodil inhibited NMDA-induced currents at NR1A/NR2B receptors with high affinity (IC50 = 0.34 microM). The affinity of NR1A/NR2A receptors for ifenprodil (IC50 = 146 microM) was 400-fold lower than that of NR1A/NR2B receptors. The rate of onset of inhibition by low concentrations of ifenprodil acting at NR1A/NR2B receptors was considerably slower than the onset of inhibition seen with high concentrations of ifenprodil acting at NR1A/NR2A receptors. The onset and recovery of blockade by ifenprodil at NR1A/NR2B receptors were not activity dependent. The inhibitory effects of low concentrations of ifenprodil at NR1A/NR2B receptors were not voltage dependent. In contrast, the inhibitory effects of high concentrations of ifenprodil at NR1A/NR2A receptors were partially voltage dependent, and a greater inhibition of NMDA-induced currents was seen at hyperpolarized membrane potentials than at depolarized membrane potentials. The reversal potential of NMDA currents was not altered in the presence of ifenprodil. Ifenprodil may act as a weak open-channel blocker of NR1A/NR2A receptors. The degree of inhibition seen with 100 microM ifenprodil at NR1A/NR2A receptors was not altered by changes in the concentration of extracellular glycine. However, the inhibitory effect of 1 microM ifenprodil at NR1A/NR2B receptors was reduced by increasing the concentration of glycine. Thus, part of the mechanism of action of ifenprodil at NR1A/NR2B receptors may involve noncompetitive antagonism of the effects of glycine. These results indicate that the mechanism of action of ifenprodil, as well as the potency of this antagonist, is different at NR1A/NR2B and NR1A/NR2A receptors expressed in Xenopus oocytes.

A METHOD FOR THE FLUOROMETRIC ASSAY OF HISTAMINE IN TISSUES

1959-11-01

Parkhurst A. Shore , Alan Burkhalter , Victor Cohn

A sensitive and specific fluorometric method for the estimation of histamine in tissues is described. The method involves extraction of histamine into butanol from alkalinized perchloric acid tissue extracts, return … A sensitive and specific fluorometric method for the estimation of histamine in tissues is described. The method involves extraction of histamine into butanol from alkalinized perchloric acid tissue extracts, return to an aqueous phase, and condensation with o -phthalaldehyde to form a highly fluorescent product which is estimated in a spectrofluorometer. Concentrations of histamine as low as 0.005 µg/ml can be assayed. Low levels of histamine (0.2-0.4 µg/g) occur in brain. Gross dissection of brain showed no marked localization of brain histamine.

A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids

1967-08-01

MK Gaitonde

Research Article| August 01 1967 A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids MK Gaitonde MK Gaitonde Search for other … Research Article| August 01 1967 A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids MK Gaitonde MK Gaitonde Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd © 1967 The Biochemical Society1967 Biochem J (1967) 104 (2): 627–633. https://doi.org/10.1042/bj1040627 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation MK Gaitonde; A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J 1 August 1967; 104 (2): 627–633. doi: https://doi.org/10.1042/bj1040627 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1967 The Biochemical Society1967 Article PDF first page preview Close Modal You do not currently have access to this content.

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Role of amino acid transport and countertransport in nutrition and metabolism.

1990-01-01

H N Christensen

Primary structure and functional characterization of a high-affinity glutamate transporter

1992-12-01

Yoshikatsu Kanai , Matthias A. Hediger

Diphosphofructose-Aldolase, Phosphoglyceraldehyd-Dehydrogenase, Milchsäure-Dehydrogenase, Glycerophosphat-Dehydrogenase und Pyruvat-Kinase aus Kaninchenmuskulatur in einem Arbeitsgang

1953-10-01

G Beisenherz , H.-J. Boltze , Th. Bücher +4 more

Extrakt von Kaninchenmuskulatur wird durch steigende Sättigung mit Ammonsulfat in amorphe Fraktionen aufgeteilt, aus denen sich ohne weitere Reinigungsschritte die obengenannten Fermente kristallisieren lassen. Die Reinigung der Rohkristallisate erfolgt durch … Extrakt von Kaninchenmuskulatur wird durch steigende Sättigung mit Ammonsulfat in amorphe Fraktionen aufgeteilt, aus denen sich ohne weitere Reinigungsschritte die obengenannten Fermente kristallisieren lassen. Die Reinigung der Rohkristallisate erfolgt durch Waschungen mit Ammonsulfatlösungen und Umkristallisationen. Jedes Ferment wird an Hand seines Absorptionsspektrums, der Umsatzzahl, Kristallform und spezifischen Eigenschaft beschrieben und den entsprechenden reinsten Präparaten anderer Autoren gegenübergestellt. Der methodische Teil enthält neben technischen Einzelheiten für die Aufarbeitung im größeren Maßstab eine neue Ableitung zur Errechnung der Salzzugabe für bestimmte Ammonsulfatkonzentrationen und Vorschriften für die Ammonsulfat- und Eiweißbestimmung im Routinebetrieb.

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<scp>d</scp> -Amino Acids Trigger Biofilm Disassembly

2010-04-29

Ilana Kolodkin–Gal , Diego Romero , Shugeng Cao +3 more

Bacteria form communities known as biofilms, which disassemble over time. In our studies outlined here, we found that, before biofilm disassembly, Bacillus subtilis produced a factor that prevented biofilm formation … Bacteria form communities known as biofilms, which disassemble over time. In our studies outlined here, we found that, before biofilm disassembly, Bacillus subtilis produced a factor that prevented biofilm formation and could break down existing biofilms. The factor was shown to be a mixture of D-leucine, D-methionine, D-tyrosine, and D-tryptophan that could act at nanomolar concentrations. D-amino acid treatment caused the release of amyloid fibers that linked cells in the biofilm together. Mutants able to form biofilms in the presence of D-amino acids contained alterations in a protein (YqxM) required for the formation and anchoring of the fibers to the cell. D-amino acids also prevented biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa. D-amino acids are produced by many bacteria and, thus, may be a widespread signal for biofilm disassembly.

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Cloning and Expression of a Plasma Membrane Cystine/Glutamate Exchange Transporter Composed of Two Distinct Proteins

1999-04-01

Hideyo Sato , Michiko Tamba , Tetsuro Ishii +1 more

Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain.

1992-11-15

Thorsten Storck , S. Schulte , Kay Hofmann +1 more

Transport systems specific for L-glutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa … Transport systems specific for L-glutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/L-aspartate transporter (GLAST). A GLAST-specific cDNA clone was isolated from a rat brain cDNA library. The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da). The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters. The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system. GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxy-aspartate. In situ hybridization reveals a strikingly high density of GLAST mRNA in the Purkinje cell layer of cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum. These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.

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Living with Water Stress: Evolution of Osmolyte Systems

1982-09-24

Paul H. Yancey , Mary E. Clark , Steven C. Hand +2 more

Striking convergent evolution is found in the properties of the organic osmotic solute (osmolyte) systems observed in bacteria, plants, and animals. Polyhydric alcohols, free amino acids and their derivatives, and … Striking convergent evolution is found in the properties of the organic osmotic solute (osmolyte) systems observed in bacteria, plants, and animals. Polyhydric alcohols, free amino acids and their derivatives, and combinations of urea and methylamines are the three types of osmolyte systems found in all water-stressed organisms except the halobacteria. The selective advantages of the organic osmolyte systems are, first, a compatibility with macromolecular structure and function at high or variable (or both) osmolyte concentrations, and, second, greatly reduced needs for modifying proteins to function in concentrated intracellular solutions. Osmolyte compatibility is proposed to result from the absence of osmolyte interactions with substrates and cofactors, and the nonperturbing or favorable effects of osmolytes on macromolecular-solvent interactions.

Glutamine Synthetase: Glial Localization in Brain

1977-03-25

Antonio Martinez-Hernandez , Katherine P. Bell , Michael D. Norenberg

Light microscopy immunohistochemical techniques were used to examine the distribution of glutamine synthetase in rat brain. Glutamine synthetase was found to be localized in the glial cells. Neuronal cell bodies, … Light microscopy immunohistochemical techniques were used to examine the distribution of glutamine synthetase in rat brain. Glutamine synthetase was found to be localized in the glial cells. Neuronal cell bodies, endothelial cells, and choroid epithelium contained no enzyme. The findings indicate that glia have a crucial role in glutamic acid, γ-aminobutyric acid, and ammonia metabolism in brain.

D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release.

1995-04-25

Michael J. Schell , M.E. Molliver , S H Snyder

Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the … Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the cerebral cortex, hippocampus, anterior olfactory nucleus, olfactory tubercle, and amygdala. Localizations of D-serine immunoreactivity correlate closely with those of D-serine binding to the glycine modulatory site of the N-methyl-D-aspartate (NMDA) receptor as visualized by autoradiography and are inversely correlated to the presence of D-amino acid oxidase. D-Serine is enriched in process-bearing glial cells in neuropil with the morphology of protoplasmic astrocytes. In glial cultures of rat cerebral cortex, D-serine is enriched in type 2 astrocytes. The release of D-serine from these cultures is stimulated by agonists of non-NMDA glutamate receptors, suggesting a mechanism by which astrocyte-derived D-serine could modulate neurotransmission. D-Serine appears to be the endogenous ligand for the glycine site of NMDA receptors.

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Expression Cloning and Characterization of a Transporter for Large Neutral Amino Acids Activated by the Heavy Chain of 4F2 Antigen (CD98)

1998-09-01

Yoshikatsu Kanai , Hiroko Segawa , Ken–ichi Miyamoto +3 more

A cDNA was isolated from rat C6 glioma cells by expression cloning which encodes a novel Na+-independent neutral amino acid transporter designated LAT1. For functional expression in Xenopus oocytes, LAT1 … A cDNA was isolated from rat C6 glioma cells by expression cloning which encodes a novel Na+-independent neutral amino acid transporter designated LAT1. For functional expression in Xenopus oocytes, LAT1 required the heavy chain of 4F2 cell surface antigen (CD98), a type II membrane glycoprotein. When co-expressed with 4F2 heavy chain, LAT1 transported neutral amino acids with branched or aromatic side chains and did not accept basic amino acids or acidic amino acids. The transport via LAT1 was Na+-independent and sensitive to a system L-specific inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. These functional properties correspond to those of the classically characterized amino acid transport system L, a major nutrient transporter. In in vitro translation, LAT1 was shown to be a nonglycosylated membrane protein consistent with the property of 4F2 light chain, suggesting LAT1 is at least one of the proteins formerly referred to as 4F2 light chain. LAT1 exhibits relatively low but significant amino acid sequence similarity to mammalian cationic amino acid transporters and amino acid permeases of bacteria and yeasts, indicating LAT1 is a new member of the APC superfamily. Because of highly regulated nature and high level of expression in tumor cell lines, LAT1 is thought to be up-regulated to support the high protein synthesis for cell growth and cell activation. The cloning of LAT1 is expected to facilitate the research on the protein-protein interaction in the transporter field and to provide a clue to the search for still unidentified transporters.

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Control of amino-acid transport by antigen receptors coordinates the metabolic reprogramming essential for T cell differentiation

2013-03-22

Linda V. Sinclair , Julia Rolf , Elizabeth Emslie +3 more

The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation

1999-10-08

Andrew P. Halestrap , Nigel T. Price

Monocarboxylates such as lactate and pyruvate play a central role in cellular metabolism and metabolic communication between tissues. Essential to these roles is their rapid transport across the plasma membrane, … Monocarboxylates such as lactate and pyruvate play a central role in cellular metabolism and metabolic communication between tissues. Essential to these roles is their rapid transport across the plasma membrane, which is catalysed by a recently identified family of proton-linked monocarboxylate transporters (MCTs). Nine MCT-related sequences have so far been identified in mammals, each having a different tissue distribution, whereas six related proteins can be recognized in Caenorhabditis elegans and 4 in Saccharomyces cerevisiae. Direct demonstration of proton-linked lactate and pyruvate transport has been demonstrated for mammalian MCT1-MCT4, but only for MCT1 and MCT2 have detailed analyses of substrate and inhibitor kinetics been described following heterologous expression in Xenopus oocytes. MCT1 is ubiquitously expressed, but is especially prominent in heart and red muscle, where it is up-regulated in response to increased work, suggesting a special role in lactic acid oxidation. By contrast, MCT4 is most evident in white muscle and other cells with a high glycolytic rate, such as tumour cells and white blood cells, suggesting it is expressed where lactic acid efflux predominates. MCT2 has a ten-fold higher affinity for substrates than MCT1 and MCT4 and is found in cells where rapid uptake at low substrate concentrations may be required, including the proximal kidney tubules, neurons and sperm tails. MCT3 is uniquely expressed in the retinal pigment epithelium. The mechanisms involved in regulating the expression of different MCT isoforms remain to be established. However, there is evidence for alternative splicing of the 5'- and 3'-untranslated regions and the use of alternative promoters for some isoforms. In addition, MCT1 and MCT4 have been shown to interact specifically with OX-47 (CD147), a member of the immunoglobulin superfamily with a single transmembrane helix. This interaction appears to assist MCT expression at the cell surface. There is still much work to be done to characterize the properties of the different isoforms and their regulation, which may have wide-ranging implications for health and disease. In the future it will be interesting to explore the linkage of genetic diseases to particular MCTs through their chromosomal location.

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The Cystine/Glutamate Antiporter System xc−in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities

2012-06-05

Jan Lewerenz , Sandra J. Hewett , Ying Huang +7 more

The antiporter system x(c)(-) imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, … The antiporter system x(c)(-) imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system x(c)(-) is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x(c)(-), including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x(c)(-). Moreover, the roles of system x(c)(-) in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system x(c)(-) inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System x(c)(-) is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x(c)(-) in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS.

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Requirement for Glycine in Activation of NMDA-Receptors Expressed in Xenopus Oocytes

1988-08-12

Nancy W. Kleckner , Raymond Dingledine

Receptors for N -methyl-D-aspartate (NMDA) are involved in many plastic and pathological processes in the brain. Glycine has been reported to potentiate NMDA responses in neurons and in Xenopus oocytes … Receptors for N -methyl-D-aspartate (NMDA) are involved in many plastic and pathological processes in the brain. Glycine has been reported to potentiate NMDA responses in neurons and in Xenopus oocytes injected with rat brain messenger RNA. Glycine is now shown to be absolutely required for activation of NMDA receptors in oocytes. In voltage-clamped oocytes, neither perfusion nor rapid pressure application of NMDA onto messenger RNA-injected oocytes caused a distinct ionic current without added glycine. When glycine was added, however, NMDA evoked large inward currents. The concentration of glycine required to produce a half-maximal response was 670 nanomolar, and the glycine dose-response curve extrapolated to zero in the absence of glycine. Several analogs of glycine could substitute for glycine, among which D-serine and D-alanine were the most effective. The observation that D-amino acids are effective will be important in developing drugs targeted at the glycine site.

CD44 Variant Regulates Redox Status in Cancer Cells by Stabilizing the xCT Subunit of System xc− and Thereby Promotes Tumor Growth

2011-03-01

Takatsugu Ishimoto , Osamu Nagano , Toshifumi Yae +7 more

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Serine racemase: A glial enzyme synthesizing <scp>d</scp> -serine to regulate glutamate- N -methyl- <scp>d</scp> -aspartate neurotransmission

1999-11-09

Herman Wolosker , Seth Blackshaw , Solomon H. Snyder

Although d amino acids are prominent in bacteria, they generally are thought not to occur in mammals. Recently, high levels of d -serine have been found in mammalian brain where … Although d amino acids are prominent in bacteria, they generally are thought not to occur in mammals. Recently, high levels of d -serine have been found in mammalian brain where it activates glutamate/ N -methyl- d -aspartate receptors by interacting with the “glycine site” of the receptor. Because amino acid racemases are thought to be restricted to bacteria and insects, the origin of d -serine in mammals has been puzzling. We now report cloning and expression of serine racemase, an enzyme catalyzing the formation of d -serine from l -serine. Serine racemase is a protein representing an additional family of pyridoxal-5′ phosphate-dependent enzymes in eukaryotes. The enzyme is enriched in rat brain where it occurs in glial cells that possess high levels of d -serine in vivo . Occurrence of serine racemase in the brain demonstrates the conservation of d -amino acid metabolism in mammals with implications for the regulation of N -methyl- d -aspartate neurotransmission through glia-neuronal interactions.

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Amino Acid Transport Across Mammalian Intestinal and Renal Epithelia

2008-01-01

Stefan Bröer

The transport of amino acids in kidney and intestine is critical for the supply of amino acids to all tissues and the homeostasis of plasma amino acid levels. This is … The transport of amino acids in kidney and intestine is critical for the supply of amino acids to all tissues and the homeostasis of plasma amino acid levels. This is illustrated by a number of inherited disorders affecting amino acid transport in epithelial cells, such as cystinuria, lysinuric protein intolerance, Hartnup disorder, iminoglycinuria, dicarboxylic aminoaciduria, and some other less well-described disturbances of amino acid transport. The identification of most epithelial amino acid transporters over the past 15 years allows the definition of these disorders at the molecular level and provides a clear picture of the functional cooperation between transporters in the apical and basolateral membranes of mammalian epithelial cells. Transport of amino acids across the apical membrane not only makes use of sodium-dependent symporters, but also uses the proton-motive force and the gradient of other amino acids to efficiently absorb amino acids from the lumen. In the basolateral membrane, antiporters cooperate with facilitators to release amino acids without depleting cells of valuable nutrients. With very few exceptions, individual amino acids are transported by more than one transporter, providing backup capacity for absorption in the case of mutational inactivation of a transport system.

Synthesis of a Fluorescent Derivatizing Reagent, 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate, and Its Application for the Analysis of Hydrolysate Amino Acids via High-Performance Liquid Chromatography

1993-06-01

Steven A. Cohen , Dennis P. Michaud

The SLC16 gene family?from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond

2004-02-01

Andrew P. Halestrap , David Meredith

THE DOMINANT ROLE OF THE LIVER IN PLASMA PROTEIN SYNTHESIS

1951-11-01

L. L. Miller , C. G. Bly , Michael L. Watson +1 more

A direct study of the isolated rat liver perfused with oxygenated blood containing amino acids and lysine-ϵ-C14 has yielded facts indicating that the liver synthesizes practically all the plasma fibrinogen, … A direct study of the isolated rat liver perfused with oxygenated blood containing amino acids and lysine-ϵ-C14 has yielded facts indicating that the liver synthesizes practically all the plasma fibrinogen, the albumin fraction, and probably more than 80 per cent of the plasma globulin fraction. The response of the isolated perfused liver in protein synthesis is qualitatively and quantitatively analogous to that of the intact animal, notably in (a) the ability to discriminate between natural L-lysine and D-lysine, (b) the per cent of isotopic amino acid converted to CO2, (c) the per cent utilized in liver and plasma protein synthesis. The results obtained with the perfused liver are compared and contrasted with those reported for tissue homogenates, minces, and slices.

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Expression cloning and cDNA sequencing of the Na+/glucose co-transporter

1987-11-01

Matthias A. Hediger , Michael J. Coady , Tyson S. Ikeda +1 more

HISTOCHEMICAL AND ULTRASTRUCTURAL DEMONSTRATION OF γ-GLUTAMYL TRANSPEPTIDASE ACTIVITY

1969-08-01

Alexander M. Rutenburg , Hwakyu Kim , J Fischbein +3 more

A simultaneous coupling azo dye method for the histochemical demonstration of γ-glutamyl transpeptidase activity using the new substrate γ-glutamyl-4-methoxy-2-naphthylamide has been described. The method appears superior to previously reported methods … A simultaneous coupling azo dye method for the histochemical demonstration of γ-glutamyl transpeptidase activity using the new substrate γ-glutamyl-4-methoxy-2-naphthylamide has been described. The method appears superior to previously reported methods for γ-glutamyl transpeptidase activity and can easily be modified for the electron microscopic localization of the enzyme by bridging osmium to the copper chelate of the azo dye via thiocarbohydrazide. The optimum conditions for the histochemical reaction were developed and the distribution of enzymatic activity in the tissues of the rat is described for light microscopy and with rat pancreas for electron microscopy. The electron-opaque deposits were seen in the endoplasmic reticulum in the vicinity of the zymogen granules in the apical portion of the acinar cell.

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Sequence and Structure of a Human Glucose Transporter

1985-09-06

Mike Mueckler , Carla Caruso , Stephen A. Baldwin +6 more

The amino acid sequence of the glucose transport protein from human HepG2 hepatoma cells was deduced from analysis of a complementary DNA clone. Structural analysis of the purified human erythrocyte … The amino acid sequence of the glucose transport protein from human HepG2 hepatoma cells was deduced from analysis of a complementary DNA clone. Structural analysis of the purified human erythrocyte glucose transporter by fast atom bombardment mapping and gas phase Edman degradation confirmed the identity of the clone and demonstrated that the HepG2 and erythrocyte transporters are highly homologous and may be identical. The protein lacks a cleavable amino-terminal signal sequence. Analysis of the primary structure suggests the presence of 12 membrane-spanning domains. Several of these may form amphipathic α helices and contain abundant hydroxyl and amide side chains that could participate in glucose binding or line a transmembrane pore through which the sugar moves. The amino terminus, carboxyl terminus, and a highly hydrophilic domain in the center of the protein are all predicted to lie on the cytoplasmic face. Messenger RNA species homologous to HepG2 glucose transporter messenger RNA were detected in K562 leukemic cells, HT29 colon adenocarcinoma cells, and human kidney tissue.

The Production of Malignant Primary Hepatic Tumours in the Rat by Feeding Dimethylnitrosamine

1956-03-01

P N Magee , J. M. Barnes

Structure of a glutamate transporter homologue from Pyrococcus horikoshii

2004-10-01

Dinesh Yernool , Olga Boudker , Yan Jin +1 more

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N<scp>OVEL</scp>N<scp>EURAL</scp>M<scp>ODULATORS</scp>

2003-03-01

Darren Boehning , Solomon H. Snyder

▪ Abstract The discovery that nitric oxide (NO) is produced by neurons and regulates synaptic activity has challenged the definition of a neurotransmitter. NO is not stored in synaptic vesicles … ▪ Abstract The discovery that nitric oxide (NO) is produced by neurons and regulates synaptic activity has challenged the definition of a neurotransmitter. NO is not stored in synaptic vesicles and does not act at conventional receptors on the surface of adjacent neurons. The toxic gases carbon monoxide (CO) and hydrogen sulfide (H 2 S) are also produced by neurons and modulate synaptic activity. D-serine synthesis and release by astrocytes as an endogenous ligand for the “glycine” site of N-methyl D-aspartate (NMDA) receptors defy the concept that a neurotransmitter must be synthesized by neurons. We review the properties of these “atypical” neural modulators.

Glia-Derived d-Serine Controls NMDA Receptor Activity and Synaptic Memory

2006-05-01

Aude Panatier , Dionysia T. Theodosis , Jean‐Pierre Mothet +4 more

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Long-term potentiation depends on release of d-serine from astrocytes

2010-01-12

Christian Henneberger , Thomas Papouin , Stéphane H. R. Oliet +1 more

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Pyridoxal Phosphate Enzymes: Mechanistic, Structural, and Evolutionary Considerations

2004-06-01

Andrew C. Eliot , Jack F. Kirsch

Pyridoxal phosphate (PLP)-dependent enzymes are unrivaled in the diversity of reactions that they catalyze. New structural data have paved the way for targeted mutagenesis and mechanistic studies and have provided … Pyridoxal phosphate (PLP)-dependent enzymes are unrivaled in the diversity of reactions that they catalyze. New structural data have paved the way for targeted mutagenesis and mechanistic studies and have provided a framework for interpretation of those results. Together, these complementary approaches yield new insight into function, particularly in understanding the origins of substrate and reaction type specificity. The combination of new sequences and structures enables better reconstruction of their evolutionary heritage and illuminates unrecognized similarities within this diverse group of enzymes. The important metabolic roles of many PLP-dependent enzymes drive efforts to design specific inhibitors, which are now guided by the availability of comprehensive structural and functional databases. Better understanding of the function of this important group of enzymes is crucial not only for inhibitor design, but also for the design of improved protein-based catalysts.

<scp>d</scp> -Serine is an endogenous ligand for the glycine site of the N -methyl- <scp>d</scp> -aspartate receptor

2000-04-25

Jean‐Pierre Mothet , Angèle Parent , Herman Wolosker +5 more

Functional activity of N -methyl- d -aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although d -serine … Functional activity of N -methyl- d -aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although d -serine is a more potent agonist. Localizations of d -serine and it biosynthetic enzyme serine racemase approximate the distribution of NMDA receptors more closely than glycine. We now show that selective degradation of d -serine with d -amino acid oxidase greatly attenuates NMDA receptor-mediated neurotransmission as assessed by using whole-cell patch–clamp recordings or indirectly by using biochemical assays of the sequelae of NMDA receptor-mediated calcium flux. The inhibitory effects of the enzyme are fully reversed by exogenously applied d -serine, which by itself did not potentiate NMDA receptor-mediated synaptic responses. Thus, d -serine is an endogenous modulator of the glycine site of NMDA receptors and fully occupies this site at some functional synapses.

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Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria

1993-09-01

P.W. Postma , J W Lengeler , Gary R. Jacobson

Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject … Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the regulation of gene expression. By sensing the presence of PTS carbohydrates in the medium and adjusting the phosphorylation state of IIAGlc, cells can adapt quickly to changing conditions in the environment. In gram-positive bacteria, it has been demonstrated that HPr can be phosphorylated by ATP on a serine residue and this modification may perform a regulatory function.

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A Continuum of Anionic Charge: Structures and Functions of<scp>d</scp>-Alanyl-Teichoic Acids in Gram-Positive Bacteria

2003-12-01

Francis C. Neuhaus , J. Baddiley

Teichoic acids (TAs) are major wall and membrane components of most gram-positive bacteria. With few exceptions, they are polymers of glycerol-phosphate or ribitol-phosphate to which are attached glycosyl and D-alanyl … Teichoic acids (TAs) are major wall and membrane components of most gram-positive bacteria. With few exceptions, they are polymers of glycerol-phosphate or ribitol-phosphate to which are attached glycosyl and D-alanyl ester residues. Wall TA is attached to peptidoglycan via a linkage unit, whereas lipoteichoic acid is attached to glycolipid intercalated in the membrane. Together with peptidoglycan, these polymers make up a polyanionic matrix that functions in (i) cation homeostasis; (ii) trafficking of ions, nutrients, proteins, and antibiotics; (iii) regulation of autolysins; and (iv) presentation of envelope proteins. The esterification of TAs with D-alanyl esters provides a means of modulating the net anionic charge, determining the cationic binding capacity, and displaying cations in the wall. This review addresses the structures and functions of D-alanyl-TAs, the D-alanylation system encoded by the dlt operon, and the roles of TAs in cell growth. The importance of dlt in the physiology of many organisms is illustrated by the variety of mutant phenotypes. In addition, advances in our understanding of D-alanyl ester function in virulence and host-mediated responses have been made possible through targeted mutagenesis of dlt. Studies of the mechanism of D-alanylation have identified two potential targets of antibacterial action and provided possible screening reactions for designing novel agents targeted to D-alanyl-TA synthesis.

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Molecular Biology of Mammalian Plasma Membrane Amino Acid Transporters

1998-10-01

Manuel Palacı́n , Raúl Estévez , Joan Bertran +1 more

Palacı́n, Manuel, Raúl Estévez, Joan Bertran, and Antonio Zorzano. Molecular Biology of Mammalian Plasma Membrane Amino Acid Transporters. Physiol. Rev. 78: 969–1054, 1998. — Molecular biology entered the field of … Palacı́n, Manuel, Raúl Estévez, Joan Bertran, and Antonio Zorzano. Molecular Biology of Mammalian Plasma Membrane Amino Acid Transporters. Physiol. Rev. 78: 969–1054, 1998. — Molecular biology entered the field of mammalian amino acid transporters in 1990–1991 with the cloning of the first GABA and cationic amino acid transporters. Since then, cDNA have been isolated for more than 20 mammalian amino acid transporters. All of them belong to four protein families. Here we describe the tissue expression, transport characteristics, structure-function relationship, and the putative physiological roles of these transporters. Wherever possible, the ascription of these transporters to known amino acid transport systems is suggested. Significant contributions have been made to the molecular biology of amino acid transport in mammals in the last 3 years, such as the construction of knockouts for the CAT-1 cationic amino acid transporter and the EAAT2 and EAAT3 glutamate transporters, as well as a growing number of studies aimed to elucidate the structure-function relationship of the amino acid transporter. In addition, the first gene ( rBAT) responsible for an inherited disease of amino acid transport (cystinuria) has been identified. Identifying the molecular structure of amino acid transport systems of high physiological relevance (e.g., system A, L, N, and x − c ) and of the genes responsible for other aminoacidurias as well as revealing the key molecular mechanisms of the amino acid transporters are the main challenges of the future in this field.

Bidirectional Transport of Amino Acids Regulates mTOR and Autophagy

2009-02-01

Paul Nicklin , Philip J. Bergman , Bailin Zhang +7 more

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Genetic and physiological data implicating the new human gene G72 and the gene for <scp>d</scp> -amino acid oxidase in schizophrenia

2002-10-03

Ilya Chumakov , Marta Blumenfeld , Oxana Guerassimenko +7 more

A map of 191 single-nucleotide polymorphism (SNPs) was built across a 5-Mb segment from chromosome 13q34 that has been genetically linked to schizophrenia. DNA from 213 schizophrenic patients and 241 … A map of 191 single-nucleotide polymorphism (SNPs) was built across a 5-Mb segment from chromosome 13q34 that has been genetically linked to schizophrenia. DNA from 213 schizophrenic patients and 241 normal individuals from Canada were genotyped with this marker set. Two 1,400- and 65-kb regions contained markers associated with the disease. Two markers from the 65-kb region were also found to be associated to schizophrenia in a Russian sample. Two overlapping genes G72 and G30 transcribed in brain were experimentally annotated in this 65-kb region. Transfection experiments point to the existence of a 153-aa protein coded by the G72 gene. This protein is rapidly evolving in primates, is localized to endoplasmic reticulum/Golgi in transfected cells, is able to form multimers and specifically binds to carbohydrates. Yeast two-hybrid experiments with the G72 protein identified the enzyme d-amino acid oxidase (DAAO) as an interacting partner. DAAO is expressed in human brain where it oxidizes d-serine, a potent activator of N-methyl-D-aspartate type glutamate receptor. The interaction between G72 and DAAO was confirmed in vitro and resulted in activation of DAAO. Four SNP markers from DAAO were found to be associated with schizophrenia in the Canadian samples. Logistic regression revealed genetic interaction between associated SNPs in vicinity of two genes. The association of both DAAO and a new gene G72 from 13q34 with schizophrenia together with activation of DAAO activity by a G72 protein product points to the involvement of this N-methyl-d-aspartate receptor regulation pathway in schizophrenia.

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A fluorometric method for the estimation of tyrosine in plasma and tissues.

1957-11-01

T. Phillip Waalkes , Sidney Udenfriend

10 Methoden der enzymatischen Analyse

2019-01-01

Jürgen Hallbach

Intracavitary bacillus Calmette-Grerin in the treatement of superficial bladder tumors

1976-01-01

A Morales

The Human ATP-Binding Cassette (ABC) Transporter Superfamily

2001-01-18

Michael Dean

Samuel Deutsch1,2, Christian Iseli3,4, Philipp Bucher4,5, Stylianos E. Antonarakis1,7, and Hamish S. Scott1,6 Division of Medical Genetics, University of Geneva Medical School, Geneva, Switzerland; 2Graduate Program in Molecular and Cellular … Samuel Deutsch1,2, Christian Iseli3,4, Philipp Bucher4,5, Stylianos E. Antonarakis1,7, and Hamish S. Scott1,6 Division of Medical Genetics, University of Geneva Medical School, Geneva, Switzerland; 2Graduate Program in Molecular and Cellular Biology, University of Geneva Medical School, Geneva, Switzerland; 3Ludwig Institute for Cancer Research, Epalinges, Switzerland; 4Swiss Bioinformatics Institute, Epalinges, Switzerland; 5Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland

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The biochemistry of methylotrophs

1983-01-01

C. Anthony

Bictegravir decreases expression of system L-amino acid transporters and inhibits leucine uptake activity in a human placental cell line

2025-06-25

Ratul Sabrina Rasna , Caroline Dunk , Md. Tozammel Hoque +2 more | Biomedicine & Pharmacotherapy

d-Amino acid analysis in solution using the photochemical properties of protonated adenosine

2025-06-23

Riyo Nakanishi , Akimasa Fujihara | Photochemical & Photobiological Sciences

The Charge of the Substrate Determines Sodium-Coupling Stoichiometry of the Amino Acid Transporter SLC6A14

2025-06-20

Yueyue Shi , Dong Yang , Noah B. Herrington +4 more | Biochemistry

SLC6A14 is a member of the SLC6 family of amino acid transporters and is known for its wide selectivity in transporting various amino acids across the cell membrane. A recent … SLC6A14 is a member of the SLC6 family of amino acid transporters and is known for its wide selectivity in transporting various amino acids across the cell membrane. A recent report detailed the Na+ coupling stoichiometry of SLC6A14 as 3 Na+: 1 amino acid substrate, focusing on the transport of the neutral amino acid glycine as the transported substrate. However, it is still unknown how SLC6A14 can also accommodate the transport of amino acids with positively charged side chains. Here, we employed structural modeling and multiple electrophysiological methods to investigate the unique Na+/Cl- coupling mode of SLC6A14. Our results revealed distinct, variable Na+ coupling modes when the transporter was subjected to either cationic or neutral amino acids (+, 0). In addition, our data provide further insight into the kinetic mechanism of SLC6A14, demonstrating that positively charged amino acids are transported with a 1.4- to 4-fold slower turnover rate compared to neutral amino acid substrates. We propose a binding mode in which the positively charged amino acid allows binding of and coupling of transport to only two Na+ ions, with no effect on Cl- coupling. Results from molecular dynamics (MD) simulations are consistent with this proposal. These findings have significant implications for our understanding of the substrate selectivity of the transport process as well as the development of new pharmacological compounds targeting this transporter.

Biological Functions and Therapeutic Potential of SLC7A11 Across Diseases

2025-06-20

Zhongping Ma , Qishan Ran | International Journal of Biology and Life Sciences

SLC7A11, a key component of the cystine/glutamate antiporter System Xc⁻, regulates redox balance and ferroptosis. Its dysregulation is linked to cancer, metabolic, neurodegenerative, immune, and kidney diseases. In tumors, SLC7A11 … SLC7A11, a key component of the cystine/glutamate antiporter System Xc⁻, regulates redox balance and ferroptosis. Its dysregulation is linked to cancer, metabolic, neurodegenerative, immune, and kidney diseases. In tumors, SLC7A11 promotes growth and therapy resistance, while in non-cancerous tissues, it offers antioxidant protection. This review outlines the biological functions of SLC7A11, its dual disease roles, and therapeutic strategies including small molecule inhibitors, exosome delivery, and metabolic interventions. Though promising, clinical translation faces challenges in tissue specificity and metabolic compensation. Advances in precision targeting may help realize its full therapeutic potential.

Exploring the pathophysiology underlying clozapine‐induced enhancement of glutamatergic transmission through L‐glutamate and D‐serine release associated with pannexin1 hemichannels

2025-06-18

Motohiro Okada , Ruri Okubo , Eishi Motomura | British Journal of Pharmacology

Abstract Background and purpose Clozapine, an approved antipsychotic for treatment‐resistant schizophrenia (TRS), enhances glutamatergic transmission by increasing exocytosis and non‐exocytosis glutamate release; however, its full action remained to be clarified. … Abstract Background and purpose Clozapine, an approved antipsychotic for treatment‐resistant schizophrenia (TRS), enhances glutamatergic transmission by increasing exocytosis and non‐exocytosis glutamate release; however, its full action remained to be clarified. Experimental approach This study determined the effects of chronic administration of clozapine on tripartite‐synaptic glutamatergic transmission associated with N‐methyl‐D‐aspartate (NMDA)/glutamate receptors (NMDARs) by using microdialysis, cultured astrocytes and capillary immunoblotting. Key results Chronic clozapine administration dose‐ and time‐dependently increased the basal release of L‐glutamate and D‐serine from astrocytes in the prefrontal cortex. A therapeutically relevant concentration of clozapine increased the expression of phosphorylated‐Src, but not pannexin1, whereas a supratherapeutic concentration of clozapine increased the expression of both pannexin1 and phosphorylated‐Src. Clozapine‐induced increase of L‐glutamate and D‐serine release was inhibited by inhibitors of Src kinase and pannexin1 hemichannels, but not by inhibitors of NMDAR and connexin43‐hemichannels. Clozapine enhanced synthesis of L‐β‐aminoisobutyrate (L‐BAIBA) also increased astroglial release of L‐glutamate and D‐serine through pannexin1 hemichannels and increased expression of pannexin1 and phosphorylated Src. Activation of pannexin1‐hemichannels by chronic administration of clozapine and L‐BAIBA was regulated by Src kinase, which was inhibited by an inhibitor of group‐III metabotropic glutamate receptors (III‐mGluR). L‐BAIBA enhances III‐mGluR, although clozapine did not directly affect III‐mGluR activity. Conclusions and implications Enhanced glutamatergic transmission by chronic administration of therapeutically relevant concentrations of clozapine involves increased L‐BAIBA signalling, which activates Src signalling via III‐mGluR agonistic action, without affecting pannexin1 expression. Activation of pannexin1‐hemichannel activity induced by Src signalling and III‐mGluR may play an important role in the effectiveness of clozapine in TRS.

L-3-[18F]-Fluoro-α-Methyl Tyrosine as a PET Tracer for Tumor Diagnosis: A Systematic Review from Mechanisms to Clinical Applications

2025-06-18

Mei Bao , Xiang Gu , Kai Tong +6 more | International Journal of Molecular Sciences

L-3-[18F]-fluoro-α-methyl tyrosine ([18F]FAMT) is an amino acid positron emission tomography (PET) tracer with high specificity for malignant tumors through its selective transport via L-type amino acid transporter (LAT) 1. Although … L-3-[18F]-fluoro-α-methyl tyrosine ([18F]FAMT) is an amino acid positron emission tomography (PET) tracer with high specificity for malignant tumors through its selective transport via L-type amino acid transporter (LAT) 1. Although extensively studied for its diagnostic performance, a comprehensive review of its molecular and clinical characteristics remains lacking. A systematic literature review (1997-2025) was conducted using PubMed and Web of Science, with keywords including "L-3-[18F]-fluoro-α-methyl tyrosine", "[18F]FAMT", "amino acid PET", and "tumor imaging". The review covered aspects of synthesis, structural properties, pharmacokinetics, and clinical applications. Notably, while research on [18F]FAMT has declined significantly in recent years, [18F]FAMT PET demonstrates superior specificity to [18F]FDG PET in distinguishing malignancies from inflammatory lesions and offers distinct advantages in lung, esophageal, and oral cancers, though with slightly lower sensitivity. Its key features include tumor-specific uptake patterns, rapid blood clearance, and a significant correlation between its uptake levels and both LAT1 expression and tumor proliferation. In conclusion, [18F]FAMT is a promising PET tracer with notable advantages in tumor imaging, particularly due to its LAT1 selectivity and favorable pharmacokinetics. Despite challenges in production, these characteristics underscore its clinical value in cancers requiring precise imaging. Future research should focus on optimizing synthesis, expanding clinical validation, and exploring theranostic applications.

Regulation of Gene Expression of Mouse D‐Amino Acid Oxidase

2025-06-18

Huong Thi Thanh Trinh , Yuji Shishido , Nam Hoang Tran +4 more | ChemBioChem

D‐amino acid oxidase (DAO) catalyzes oxidative deamination of D‐amino acids, producing 2‐oxo acids, hydrogen peroxide and ammonia. In mammals, DAO is essential for metabolizing both endogenous and exogenous D‐amino acids. … D‐amino acid oxidase (DAO) catalyzes oxidative deamination of D‐amino acids, producing 2‐oxo acids, hydrogen peroxide and ammonia. In mammals, DAO is essential for metabolizing both endogenous and exogenous D‐amino acids. Our previous studies on human DAO identified two promoter regions (P1 and P2), a negative regulatory element in intron 1, and several transcription factor binding sites. In this study, we investigated the regulatory mechanism of mouse DAO gene expression to compare with the human system. Poly (I:C) or Interleukin‐1βtreatment induced DAO expression by 1.8‐2.0 folds in the mouse brain, cerebellum and cerebral cortex. To determine the promoter activity in the upstream region of initiation site, we constructed plasmids containing mouse DAO gene fragments inserted into pGL4 [luc2P/Hygro] vector and assessed luciferase activity in LLC‐PK1 cells. We analyzed a series of deletion constructs, revealing promoter activity in all tested fragments. The highest promoter activity was detected in the ‐333/‐87 subregion, with residual activities in the ‐87/+111 region. Bioinformatics analysis identified transcription factors, including NEUR, EGRF, ZF07, ZF11, KLFS, SP1F and ZF02, which bind to both the human and mouse DAO genes at conserved positions, suggesting their critical role in regulating DAO promoter activity.

Rhodamine-Based Molecules

2025-06-16

Esther Rani Motamarri , Abha Sharma | CRC Press eBooks

N-Nitroso-ethylisopropylamine mutagenicity in rat liver using the cII transgenic mutation assay and Duplex Sequencing analysis of genomic DNA

2025-06-16

Li Xia , Jian Yan , Ying Chen +5 more | Chemico-Biological Interactions

Mapping genetic modifiers of epimutation rates reveals a punctuated-equilibrium model of CG methylome evolution

2025-06-16

Zhilin Zhang , Wilma Wanney , Yangyang Xu +4 more | bioRxiv (Cold Spring Harbor Laboratory)

Spontaneous epimutations—stochastic changes in cytosine methylation—can persist across generations in plants and are thought to contribute to phenotypic variation. Although epimutations are increasingly studied for their potential long-term effects, it … Spontaneous epimutations—stochastic changes in cytosine methylation—can persist across generations in plants and are thought to contribute to phenotypic variation. Although epimutations are increasingly studied for their potential long-term effects, it remains unclear why their accumulation varies across genotypes. Here, we tracked DNA methylation across ten generations in ~400 mutation accumulation lineages derived from ~70 Arabidopsis Ler × Cvi recombinant inbred lines. Treating epimutation rates as quantitative molecular traits, we mapped a major QTL to a Cvi-derived deletion near VIM2 and VIM4 , two genes involved in CG methylation (mCG) maintenance. We show that this deletion rapidly reduces genome-wide methylation to a lower steady-state and compromises mCG maintenance fidelity across generations, resulting in a ~1.5-fold increase in epimutation rates. Genotypes with elevated rates exhibited accelerated epigenetic drift and phenotypic divergence. Our findings support a punctuated-equilibrium model of mCG evolution, in which sudden disruptions to methylation homeostasis can destabilize epigenetic inheritance over longer time-scales.

Glycine as a conditionally essential amino acid and its relationship to l-serine

2025-06-15

Milan Holeček | Metabolism

A New Ammonia Determination Assay Based on the Linear Activation of Horseradish Peroxidase for the Determination of Protein-Glutamine Glutaminase Activities

2025-06-14

Katrin Reichenberger , Gudrun Horstmann , Sabine Lutz‐Wahl +1 more | Food Analytical Methods

Abstract A new horseradish peroxidase (HRP)-based assay for the determination of aqueous ammonia was developed. This new assay utilizes the concentration-dependent, linearly increasing activation effect of aqueous ammonia on the … Abstract A new horseradish peroxidase (HRP)-based assay for the determination of aqueous ammonia was developed. This new assay utilizes the concentration-dependent, linearly increasing activation effect of aqueous ammonia on the enzyme HRP at alkaline pH values. The special feature of this assay is that the analyte, ammonia, is not directly involved in the reaction catalyzed by the enzyme HRP. Instead, the concentration of the analyte is determined by measuring the increase of the activity of the HRP in the absence and presence of the sample. Accordingly, all relevant assay parameters and concentrations of the components were first carefully evaluated to enable the determination of low aqueous ammonia concentrations while simultaneously achieving a broad linear range. The optimized conditions in the newly developed method for the determination of ammonia were pH 10.0, 4.8 nkat mL −1 HRP, 2.3 mmol L −1 o -dianisidine, and 400 µM hydrogen peroxide. The limit of detection and the limit of quantification of the new method were 0.24 mmol L −1 and 0.36 mmol L −1 , respectively. One possible application for the newly developed ammonia determination assay was the determination of protein-glutamine glutaminase (PG) activities. This assay was employed as a two-step assay, starting with the PG reaction conducted at the optimal pH value for the PG used. After this step, the pH was increased to 10, and the ammonia released was measured in a second reaction. The results obtained with this method showed less than 10% variation compared to two established methods.

An Overview of Glutaminyl Cyclase as a Promising Drug Target for Alzheimer’s Disease

2025-06-13

Rasajna Madhusudhana , Emily Boyle , Yana Cen | Biomedicines

Alzheimer's disease (AD) has become an increasingly pressing concern for the aging population. Current AD treatments mainly focus on cognitive and neuropsychiatric symptoms-with few FDA-approved treatments targeting disease progression itself. … Alzheimer's disease (AD) has become an increasingly pressing concern for the aging population. Current AD treatments mainly focus on cognitive and neuropsychiatric symptoms-with few FDA-approved treatments targeting disease progression itself. The amyloid cascade hypothesis describes the formation and accumulation of β-amyloid (Aβ) oligomers and plaques as a primary event in AD pathogenesis. This hypothesis has served as the foundation of disease-modifying treatment development over the last decade. Recently, glutaminyl cyclase (QC) has been identified as a potential drug target in the amyloid cascade. QC catalyzes the cyclization of Aβ to form pyroglutamated Aβ (pEAβ). pEAβ acts as the seed for the formation of Aβ plaques, thus preventing the formation of pEAβ via QC inhibition, and offers a promising therapeutic strategy against AD. Here, we offer an overview of the pathway QCI research has followed-from the initial testing of imidazole-based inhibitor scaffolds to QCI structural optimization via pharmacophore identification, Varoglutamstat entering clinical trials, and further avenues of bettering specificity and potency for future QCI development.

1993-LB: BGM1812, a Novel Amylin Analog, Enhances Receptor Agonism and Induces Superior Weight Loss in Preclinical Models

2025-06-13

Jiandong Yuan , YANGQING HUANG , HAIFENG DING +1 more | Diabetes

Introduction and Objective: Amylin receptor agonists (e.g. petrelintide and cagrilintide) have shown promise for weight management. BGM1812 is a novel amylin analog designed using AI/ML-driven optimization to enhance agonist activities … Introduction and Objective: Amylin receptor agonists (e.g. petrelintide and cagrilintide) have shown promise for weight management. BGM1812 is a novel amylin analog designed using AI/ML-driven optimization to enhance agonist activities and formulation properties. This study evaluates BGM1812’s pharmacological properties, weight loss efficacy, and potential synergy with a GLP-1/GIP dual agonist (BGM0504) in diet-induced obese (DIO) rats. Methods: Receptor activation: cAMP assays measured EC50 for amylin and calcitonin receptor activation. Pharmacokinetics: PK studies were conducted in rats to assess exposure and clearance profiles after subcutaneous administration. Weight loss efficacy: DIO rats received subcutaneous BGM1812 (0.012-0.12 mg/kg, Q3D) for 28 days, with weight monitored. Combination therapy: BGM1812 (0.4 mg/kg) and BGM0504 (0.15 mg/kg) were tested alone and in combination, with weight loss effects compared to monotherapies in DIO rats. Formulation study: Solubility and stability of BGM1812 formulations were evaluated at physiological pH (6.5-7.5). Results: Stronger receptor activation: BGM1812 showed 1.8x and 2.2x increased agonist activity (EC50) at the amylin and calcitonin receptors, respectively, versus petrelintide. Enhanced weight loss: BGM1812 demonstrated dose-dependent weight loss in 0.012 - 0.12 mg/kg dose range. At 0.04 mg/kg, BGM1812 induced greater weight reduction than petrelintide. Combination therapy: BGM1812 + BGM0504 led to deeper and more sustained weight loss (-28%) compared to BGM0504 (-15%) or BGM1812 (-13%) alone, suggesting a synergistic effect. PK properties: BGM1812 displayed similar PK profile to petrelintide. Formulation properties: BGM1812 exhibits high solubility and maintains stability without fibril formation. Conclusion: BGM1812 demonstrates superior receptor activation, robust weight loss effects, and synergistic potential with GLP-1/GIP dual agonism, supporting its development as a next-generation amylin analog for obesity treatment. Disclosure J. Yuan: Employee; BrightGene Bio-Medical Technology Co., Ltd. Y. Huang: Employee; BrightGene Bio-Medical Technology Co., Ltd. H. Ding: Employee; BrightGene Bio-Medical Technology Co., Ltd. X. Jiang: Consultant; BrightGene Bio-Medical Technology Co., Ltd.

Abstract P1-05-02: Medium chain fatty acids shift metabolism towards the de novo serine pathway fostering epigenetic plasticity and oxidative DNA damage

2025-06-13

Mariana Bustamante Eduardo , Curtis W. McCloskey , Gannon Cottone +7 more | Clinical Cancer Research

Abstract Introduction: We have investigated the breast microenvironment to identify factors that promote Estrogen Receptor Negative Breast Cancer (ERneg BC) and that may be disrupted for prevention. To that end, … Abstract Introduction: We have investigated the breast microenvironment to identify factors that promote Estrogen Receptor Negative Breast Cancer (ERneg BC) and that may be disrupted for prevention. To that end, we have identified a lipid metabolism gene signature associated with the risk of ERneg BC. To better understand lipid metabolism in the breast, we studied the effect of fatty acids (FA) on non-transformed breast epithelial cells and tissues. FA exposure alters histone methylation, affecting gene expression and increase flux through serine, one-carbon, glycine (SOG) and methionine pathways. The association of the serine pathway and ERneg BC was first observed over a decade ago. A SOG pathway gene signature is significantly correlated with ERneg status. We hypothesized that the metabolism of FA results in a metabolic shift toward the de novo serine synthesis pathway (SSP), which ultimately increases S-adenosylmethionine (SAM), altering histone methylation, profoundly changing gene expression and fostering ERneg oncogenesis. Methods: Non-transformed MCF-10A cells were used for in vitro metabolic and epigenomic analyses. Cells exposed to the medium-chain FA octanoic acid (OA) were utilized for proteomics and U13C-glucose tracing. SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG) concentrations were measured following treatment with OA ± blockade of the serine pathway. Reactive Oxygen Species (ROS)-induced redox changes were monitored live cells. Comet assay was performed to detect DNA damage. CUT&RUN was performed for H3K4me3. Human breast tissue derived microstructures were utilized for genomic analysis. Single-cell RNA-seq (scRNAseq) was performed in microstructures exposed to ± OA. Metabolic flux analyses was performed using Compass. Results: 13C flux analysis revealed that OA led to increased flux to methylation. OA significantly increased the main methyl donor SAM, the antioxidant GSH via the transsulfuration pathway and the oncometabolite 2-HG after 15 min exposure. Blocking the first and rate limiting enzyme in the SSP, PHGDH, prevented these increases. Proteomics revealed the overexpression of PHGDH following OA exposure. Upon exposure to OA, scRNAseq analysis revealed increased expression of the SSP transcription factor (TF) ATF3 and the SSP genes PHGDH and PSAT1 in epithelial and non-epithelial clusters. Upon OA the proportion of three subtypes within the epithelial compartment increased: basal BSL1, Hormone sensing HS1 and luminal progenitor LP3. Compass, an algorithm to characterize cellular metabolic states, revealed flux greatly increased through the three enzymes of the SSP: PHGDH, PSAT1 and PSPH secondary to OA exposure in BSL1, LP3 and HS1 cells. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR &lt; 0.05) comparing OA to control. Motif analysis revealed an overrepresentation of binding sites for SSP TFs ATF3/4 (p &lt; 0.05). After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p &lt; 0.01), peaking at 15 min. OA exposure triggered DNA damage likely due to ROS increase in the nucleus. Compass predicted an increase in GSH metabolism and ROS detoxification in BSL1. Conclusions: Protein levels of PHGDH are elevated in 70% of ERneg BCs. This cannot be explained by gene amplification alone as PHGDH gene amplification is observed in only approximately 6% of all breast cancers. This suggests that there are mechanisms other than gene amplification that contribute to PHGDH dysregulation. One of those mechanisms may be the lipid induced metabolic shift toward the SOG and methionine pathways that we have identified. The increased SAM and 2-HG foster epigenetic phenotypic plasticity via altered histone methylation. ROS increase shortly after OA exposure and are controlled by antioxidant defenses (e.g. GSH), which favors the survival of specific cell subtypes with acquired DNA damage which likely facilitates malignant transformation. Citation Format: Mariana Bustamante Eduardo, Curtis W. McCloskey, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B.M.M.K. Islam, Jason Locasale, Marcelo G. Bonini, Maxim V. Frolov, Elizaveta V. Benevolenskaya, Rama Khokha, Navdeep S. Chandel, Seema A. Khan, Susan E. Clare. Medium chain fatty acids shift metabolism towards the de novo serine pathway fostering epigenetic plasticity and oxidative DNA damage [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-05-02.

Abstract P3-04-04: The Butyrate Transporter SLC5A8 Selectively Inhibits Breast Tumor Metastasis

2025-06-13

Sonia Batan , Jabunnesa Khanom , Sabarish Ramachandran +7 more | Clinical Cancer Research

Abstract Introduction: The mammary gland is a dynamic organ that undergoes significant developmental changes during pregnancy, lactation, and involution. The process of involution is a highly orchestrated series of molecular … Abstract Introduction: The mammary gland is a dynamic organ that undergoes significant developmental changes during pregnancy, lactation, and involution. The process of involution is a highly orchestrated series of molecular and physical events that can be divided into two distinct phases. (Lund et al., 1996). Accumulation of milk in the alveolar lumen (milk stasis) is required to initiate the first phase during which the secretory cells begin to enter apoptosis. Here we provide genetic and molecular biological evidence to shows that the short-chain fatty acid Butyrate (BTR), a significant component in milk, contributes to milk stasis-induced apoptosis in the mammary gland, and SLC5A8, a BTR transporter, is obligatory for this effect. Slc5a8 deletion in mice is associated with delayed mammary gland involution, susceptible forto chemical, xenograft, and syngeneic transplant, and predisposes to early onset of mammary tumorigenesis and accelerated lung metastasis driven by genetically engineered mouse models of breast and lung cancers. Objective: To establish the functional link between mammary gland remodeling and its relevance to mammary tumor growth (primary) and distant metastasis. Methods: All animals were housed and handled according to approved protocols established by the Georgia Health SciencesAugusta University (GHSUAU) Animal Care and Use Committee and NIH guidguidelinesance’s. For the measurement of HDAC activity, A commercially available HDAC assay kit (BioVisionBio Vision) was used. Statistical analysis was done using one-way ANOVA followed by Bonferroni multiple comparison test. The software used was Graph Pad Prism, version 5.0. A p-value &lt;0.05 was considered statistically significant. Results: SLC5A8 is a tumor suppressor, and its expression is silenced in many human cancers, including breast cancer (Thangaraju et al., 2006a; Babu et al., 2011). Here we analyzed Slc5a8 expression at various stages of mammary gland development. Slc5a8 expression was evident at mRNA and protein levels in the virgin mammary gland; it was induced marginally during pregnancy and lactation. Slc5a8 was primarily expressed in the lumen-facing apical membrane of the mammary ductal epithelium. deletion of Slc5a8 in mice leads to a delay in mammary gland involution. We also analyzed the expression of involution markers (Stat3, p53, Bax, BMF, survivin, and IGFBP-5) and apoptotic cell death in wild wild-type (Slc5a8-/-) and Slc5a8-knockout (Slc5a8-/-) mice mammary glands at different stages of involution. pStat3 expression was increased dramatically on Inv d1 and d2 in wild- type glands but to a much less extent in Slc5a8 -/- glands. Further, the expression of p53 and Bax (pro-apoptotic genes), BMF (anoikis inducer), and IGFBP-5 (inhibitor of IGF-induced prosurvival signaling) were significantly increased in wild wild-type glands compared to Slc5a8-/- glands. The anti-apoptotic protein survivin was significantly reduced on Inv d1, d2, and d3 in wild wild-type glands, but this reduction was not observed in Slc5a8-/- glands. Slc5a8 transports into cells the HDAC inhibitors butyrate, pyruvate, and propionate into cells the. (Thangaraju et al., 2009). These inhibitors are selective for HDAC1 and HDAC3. Butyrate, a significant component of breast milk, promotes differentiation in normal cells but induces apoptosis in highly proliferative cells and cancer cells. Slc5a8 induces apoptosis in lactating mammary epithelial cells during involution through modulation of HDAC expression and activity. Butyrate, a substrate of Slc5a8, plays a critical role in promoting mammary gland involution through HDAC inhibition and death receptor activation. Administration of exogenous butyrate induces precocious mammary gland involution in mice. Next, we wanted to exploretested whether if the deletion of Slc5a8 plays any a role in mammary tumorigenesis. The biological changes that occur in mammary epithelial cells during pregnancy/lactation and involution are similar in many respects to those associated with tumor development and tumor regression, respectively. Deletion of Slc5a8 in mice is associated with early onset of mammary tumor formation, accelerated lung metastasis, and decreased overall survival. Finally, we wanted to analyze whether if mammary gland-specific overexpression of Slc5a8 SLC5A8 would influence mammary gland involution and mammary tumorigenesis. Mammary gland-specific overexpression of Slc5a8 SLC5A8 induces precocious mammary gland involution and protects from MMTV-Neu-driven mammary tumorigenesis. Conclusion: Slc5a8 is a tumor suppressor in the mammary gland, and butyrate is essential for its tumor-suppressive function. Mammary gland-selective overexpression of Slc5a8 SLC5A8 in mice enhances mammary gland involution and protects against breast cancer. Citation Format: Sonia Batan, Jabunnesa Khanom, Sabarish Ramachandran, Selvakumar Elangovan, Nanditi N. Thangaraju, Subha Sundaram, Snigdha Ganjikunta, Breanna Kennedy, Vadivel Ganapathy, Puttur D. Prasad, Muthusamy Thangaraju. The Butyrate Transporter SLC5A8 Selectively Inhibits Breast Tumor Metastasis [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-04-04.

Major Macronutrients

2025-06-12

James W. Murray | Oxford University Press eBooks

Abstract This chapter summarizes the distributions of and controls on species of the major nutrients nitrogen, phosphorus and silica in seawater. These major macronutrients forge a direct connection between chemical … Abstract This chapter summarizes the distributions of and controls on species of the major nutrients nitrogen, phosphorus and silica in seawater. These major macronutrients forge a direct connection between chemical and biological oceanography. The distributions in representative regions of the surface ocean are covered with emphasis on the controlling processes. The time series at HOT and BATS are highlighted. Ocean sections and vertical distributions are described. Particles of biological origin play a major role and the composition of hard parts and soft parts are discussed. The pros and cons of the Redfield Model which links marine biology with dissolved distributions are analyzed.

D-alanine synthesis and exogenous alanine affect the antimicrobial susceptibility of Staphylococcus aureus

2025-06-12

Y Suzuki , Miki Kawada‐Matsuo , Vy Ton That Thuan +3 more | Antimicrobial Agents and Chemotherapy

ABSTRACT D-alanine is an important amino acid for peptidoglycan biosynthesis in Staphylococcus aureus . In addition, D-alanine is used for the modification of teichoic acids to weaken the net surface … ABSTRACT D-alanine is an important amino acid for peptidoglycan biosynthesis in Staphylococcus aureus . In addition, D-alanine is used for the modification of teichoic acids to weaken the net surface negative charge, leading to decreased susceptibility to cationic antimicrobial agents. D-alanine synthesis is dependent on only two enzymes. One is alanine racemase, encoded by the alr1 gene, which reversibly converts L-alanine and D-alanine. The other is D-amino acid transaminase, encoded by the dat gene, which synthesizes D-amino acids from α-keto acids and other D-amino acids. In addition, the uptake of L- and D-alanine is dependent on the alanine transporter CycA. To reveal the relationship between D-alanine supply and antimicrobial susceptibility, we evaluated antimicrobial susceptibility in alr1, dat, and cycA inactivation mutants. These mutants, especially the Δ alr1 and Δ cycA mutants, presented increased susceptibility to β-lactams, D-cycloserine, bacitracin, lysostaphin, and cationic antimicrobial agents such as aminoglycosides, nisin A, and daptomycin. The net negative charge of the cell surface increased in the Δ alr1 and Δ cycA mutants. The changes in susceptibility to antimicrobial agents and cell surface charge were restored in their gene-complemented mutants. Furthermore, in an alanine-depleted medium, the MIC for oxacillin decreased significantly, and the MIC for gentamicin also decreased slightly. Clinical MRSA strains also showed significantly increased susceptibility to oxacillin in the alanine-depleted medium. These results indicate that D-alanine deficiency leads to impaired peptidoglycan and increased net surface negative charge, resulting in increased antimicrobial susceptibility.

Dehydrogenases in the Flavoprotein Amine Oxidoreductase Superfamily

2025-06-12

Javeria Akram , Tavishi Budagavi , Zhiyao Zhang +4 more | Biochemistry

Enzymes of the ubiquitous flavoprotein amine oxidoreductase (FAO) superfamily catalyze C-N bond oxidation of amine-containing substrates using flavin adenine dinucleotide (FAD) as a prosthetic group. Their reaction proceeds via a … Enzymes of the ubiquitous flavoprotein amine oxidoreductase (FAO) superfamily catalyze C-N bond oxidation of amine-containing substrates using flavin adenine dinucleotide (FAD) as a prosthetic group. Their reaction proceeds via a two-step mechanism involving hydride transfer from the substrate to the bound FAD cofactor, and the reduced flavin is subsequently reoxidized by a physiological electron acceptor. For nearly a century, it has been generally accepted that all enzymes in the FAO superfamily are oxidases, i.e., donating the electrons from substrate oxidation to dioxygen (O2). However, we recently showed that the FAO family enzymes nicotine oxidoreductase and pseudooxynicotine amine oxidase are not oxidases because they do not utilize oxygen efficiently, and instead are cytochrome c utilizing dehydrogenases. Here we characterized other bacterial FAOs and show that many are actually dehydrogenases that react poorly with O2 in favor of using a cytochrome c (CytC) protein closely encoded in the genome, indicating that there are many exceptions to the "oxidase" paradigm that has been traditionally ascribed to the FAO superfamily. These dehydrogenases are highly specific for the CytC from the same organism and are phylogenetically clustered with other FAOs that appear to be oxidases. Our findings further undermine the long-held view that all FAO family enzymes are oxidases and suggest that evolutionary switches between different oxidants are surprisingly frequent in this enzyme family.

An Alternative Mechanism of Glutamate Dehydrogenase Inhibition by EGCG: Promotion of Protein Degradation

2025-06-12

Ziying Zeng , Lin Chen-shui , Chuqiao Pan +2 more | Pharmaceuticals

Backgroud: Glutamate dehydrogenase (GDH) is involved in the metabolism of glutamate and ammonia. It is regulated by multiple ligand variants, and hyper-active GDH mutants have been reported for hyperinsulinism hyperammonemia … Backgroud: Glutamate dehydrogenase (GDH) is involved in the metabolism of glutamate and ammonia. It is regulated by multiple ligand variants, and hyper-active GDH mutants have been reported for hyperinsulinism hyperammonemia syndrome (HHS). Methods: Here, we constructed the wild-type human GDH and three human GDH454 mutants and investigated their degradation activity and performance under different GDH inhibitors. Results: Protein activity test and SDS-PAGE analysis of the purified proteins showed that the GDH454 mutant from HHS has weaker GDH enzymatic activity but greater resistance to trypsin hydrolysis than the wild type. Interestingly, using the biomolecular interactions technique, it showed that the GDH454 mutant has 109 times weaker affinity for trypsin and 10-fold weaker for epigallocatechin gallate (EGCG) than the wild-type GDH. Subsequently, native-PAGE gel analysis demonstrated that EGCG could break down the GDH hexamer into monomers and form a complex with trypsin to enhance the degradation of both types of GDH. Conclusions: EGCG showed good affinity to both the wild-type and the mutant GDH proteins, promoting protein degradation; this provides a new strategy for the treatment of HHS and other hyper-active GDH-related diseases.