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Mammalian Urea Cycle Enzymes
... The kinetics of GOT1 protein levels in virus-specific CD8 + T cells was similar to that of Got1 mRNA (Fig. 1c). Similar to the in vivo observations, TCR-transgenic P14 CD8 + T cells showed significantly elevated GOT1 protein expression 3 d after cognate peptide GP [33][34][35][36][37][38][39][40][41] stimulation in vitro (Fig. 1d). GOT1 protein levels decreased when the GP [33][34][35][36][37][38][39][40][41] peptide was washed out and replaced with interleukin (IL)-15, thus suggesting that antigen persistence was required for maintaining GOT1 expression. ...
... Similar to the in vivo observations, TCR-transgenic P14 CD8 + T cells showed significantly elevated GOT1 protein expression 3 d after cognate peptide GP [33][34][35][36][37][38][39][40][41] stimulation in vitro (Fig. 1d). GOT1 protein levels decreased when the GP [33][34][35][36][37][38][39][40][41] peptide was washed out and replaced with interleukin (IL)-15, thus suggesting that antigen persistence was required for maintaining GOT1 expression. ...
... To further examine the potential role of TCR stimulation in driving Got1 expression, we infected C57BL/6 mice with LCMV clone 13 or a mutated strain LCMV clone 13 V35A 10 . P14 CD8 + T cells recognize the GP 33-41 epitope of LCMV clone 13, but not the mutated GP [33][34][35][36][37][38][39][40][41] V35A epitope of LCMV clone 13 V35A. P14 CD8 + T cells in mice infected with LCMV clone 13 expressed higher levels of Got1 than those in mice infected with LCMV clone 13 V35A, suggesting that TCR stimulation drives Got1 expression (Extended Data Fig. 1a,b). ...
The malate shuttle is traditionally understood to maintain NAD⁺/NADH balance between the cytosol and mitochondria. Whether the malate shuttle has additional functions is unclear. Here we show that chronic viral infections induce CD8⁺ T cell expression of GOT1, a central enzyme in the malate shuttle. Got1 deficiency decreased the NAD⁺/NADH ratio and limited antiviral CD8⁺ T cell responses to chronic infection; however, increasing the NAD⁺/NADH ratio did not restore T cell responses. Got1 deficiency reduced the production of the ammonia scavenger 2-ketoglutarate (2-KG) from glutaminolysis and led to a toxic accumulation of ammonia in CD8⁺ T cells. Supplementation with 2-KG assimilated and detoxified ammonia in Got1-deficient T cells and restored antiviral responses. These data indicate that the major function of the malate shuttle in CD8⁺ T cells is not to maintain the NAD⁺/NADH balance but rather to detoxify ammonia and enable sustainable ammonia-neutral glutamine catabolism in CD8⁺ T cells during chronic infection.
... Argininosuccinate synthetase (ArgG; EC 6.3.4.5) catalyzes the condensation of citrulline and aspartate into argininosuccinate, the immediate precursor of arginine (Haines et al. 2011). ArgG is considered a rate-limiting step in the urea cycle and nitric oxide (NO) production in mammals (Haines et al. 2011;Jackson et al. 1986;Meijer et al. 1990). The crystal structures of ArgG have been revealed in E. coli, Thermus thermophilus, and human (Lemke and Howell 2001;Goto et al. 2002Goto et al. , 2003Karlberg et al. 2008). ...
Cyanobacteria are oxygen-evolving photosynthetic prokaryotes that affect the global carbon and nitrogen turnover. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a model cyanobacterium that has been widely studied and can utilize and uptake various nitrogen sources and amino acids from the outer environment and media. l-arginine is a nitrogen-rich amino acid used as a nitrogen reservoir in Synechocystis 6803, and its biosynthesis is strictly regulated by feedback inhibition. Argininosuccinate synthetase (ArgG; EC 6.3.4.5) is the rate-limiting enzyme in arginine biosynthesis and catalyzes the condensation of citrulline and aspartate using ATP to produce argininosuccinate, which is converted to l-arginine and fumarate through argininosuccinate lyase (ArgH). We performed a biochemical analysis of Synechocystis 6803 ArgG (SyArgG) and obtained a Synechocystis 6803 mutant overexpressing SyArgG and ArgH of Synechocystis 6803 (SyArgH). The specific activity of SyArgG was lower than that of other arginine biosynthesis enzymes and SyArgG was inhibited by arginine, especially among amino acids and organic acids. Both arginine biosynthesis enzyme-overexpressing strains grew faster than the wild-type Synechocystis 6803. Based on previous reports and our results, we suggest that SyArgG is the rate-limiting enzyme in the arginine biosynthesis pathway in cyanobacteria and that arginine biosynthesis enzymes are similarly regulated by arginine in this cyanobacterium. Our results contribute to elucidating the regulation of arginine biosynthesis during nitrogen metabolism.
... Since the urea cycle is restricted to ureotelic organisms, mapping of genes to the urea cycle is simply due to bacteria containing distant homologous enzymes with important roles in mammalian pathways [27]. Nevertheless, the distinction of the two metabolic pathways can shed light on the flux of ammonia nitrogen, in which amino groups donated by ammonia are incorporated into L-arginine and L-ornithine in the urea cycle [51], while ammonia is completely liberated through allantoin degradation by bacteria under anaerobic conditions [52]. Metagenomic data also confirmed that more genes are involved in the amino acid biosynthesis pathway in H-NFC compared to L-NFC, including pathways of L-phenylalanine, L-tyrosine, L-ornithine, arginine and polyamine biosynthesis. ...
The main objective of our current study was evaluating the effects of NFC supplementation and forage type on rumen microbiota and metabolism, by comparing microbial structures and composition among samples collected from cows fed AH (alfalfa-based diet), H-NFC (CS-based diet with high NFC) and L-NFC (CS-based diet with low NFC) diets. Our results show that microbial communities were structurally different but functionally similar among groups. When compared with L-HFC, NFC increased the population of Treponema, Ruminobacter, Selenomonas and Succinimonas that were negatively correlated with ruminal NH3-N, and urea nitrogen in blood, milk and urine, as well as significantly increasing the number of genes involved in amino acid biosynthesis. However, when compared to the AH group, H-NFC showed a higher abundance of bacteria relating to starch degradation and lactate production, but a lower abundance of bacteria utilizing pectin and other soluble fibers. This may lead to a slower proliferation of lignocellulose bacteria, such as Ruminococcus, Marvinbryantia and Syntrophococcus. Lower fibrolytic capacity in the rumen may reduce rumen rotation rate and may limit dry matter intake and milk yield in cows fed H-NFC. The enzyme activity assays further confirmed that cellulase and xylanase activity in AH were significantly higher than H-NFC. In addition, the lower cobalt content in Gramineae plants compared to legumes, might have led to the significantly down-regulated microbial genes involved in vitamin B12 biosynthesis in H-NFC compared to AH. A lower dietary supply with vitamin B12 may restrict the synthesis of milk lactose, one of the key factors influencing milk yield. In conclusion, supplementation of a CS-based diet with additional NFC was beneficial for nitrogen conversion by increasing the activity of amino acid biosynthesis in rumen microbiota in dairy cattle. However, lower levels of fibrolytic capacity may limit dry matter intake of cows fed H-NFC and may prevent increased milk yield.
... At physiological levels, ammonia helps to stabilize pH and serves as a nitrogen source for glutamine synthesis. 24,25 Excess ammonia is removed by periportal hepatocytes through a detoxification process called the urea cycle, 26,27 where urea is created and released into bloodstream and then converted into urine by the kidneys. 28,29 3. Cytochrome P450 family and other enzymes involved in detoxification ...
Hepatocytes are parenchymal cells of the liver responsible for drug detoxification, urea and bile production, serum protein synthesis, and glucose homeostasis. Hepatocytes are widely used for drug toxicity studies in bioartificial liver devices and for cell-based liver therapies. Because hepatocytes are highly differentiated cells residing in a complex microenvironment in vivo, they tend to lose hepatic phenotype and function in vitro. This paper first reviews traditional culture approaches used to rescue hepatic function in vitro and then discusses the benefits of emerging microfluidic-based culture approaches. We conclude by reviewing integration of hepatocyte cultures with bioanalytical or sensing approaches.
... Urea (nontoxic) is then transported and excreted in the urine by kidney function. Ornithine is recycled to continue the cycle for further rounds of urea production (Jackson et al. 1986). The reduced activity of arginase 1 characterizes ARG1D or hyperargininemia, a rare autosomal inborn error of metabolism, with an estimated incidence of 1/950,000 (Summar et al. 2013). ...
Ammonia is a neurotoxic compound which is detoxified through liver enzymes from urea cycle. Several inherited or acquired conditions can elevate ammonia concentrations in blood, causing severe damage to the central nervous system due to the toxic effects exerted by ammonia on the astrocytes. Therefore, hyperammonemic patients present potentially life-threatening neuropsychiatric symptoms, whose severity is related with the hyperammonemia magnitude and duration, as well as the brain maturation stage. Inherited metabolic diseases caused by enzymatic defects that compromise directly or indirectly the urea cycle activity are the main cause of hyperammonemia in the neonatal period. These diseases are mainly represented by the congenital defects of urea cycle, classical organic acidurias, and the defects of mitochondrial fatty acids oxidation, with hyperammonemia being more severe and frequent in the first two groups mentioned. An effective and rapid treatment of hyperammonemia is crucial to prevent irreversible neurological damage and it depends on the understanding of the pathophysiology of the diseases, as well as of the available therapeutic approaches. In this review, the mechanisms underlying the hyperammonemia and neurological dysfunction in urea cycle disorders, organic acidurias, and fatty acids oxidation defects, as well as the therapeutic strategies for the ammonia control will be discussed.
... Arginine metabolism is highly localized in the hepatic tissue and has also been reported as a "clock-regulated" metabolic pathway. 35,37 It should be mentioned that b-citryl-glutamate and its isomer A were neither detected in plasma nor in the liver tissue. Mass spectrometric intensities of significantly modified metabolites in liver tissue in sleep and wake state (N ¼ 6); (e) Significantly altered metabolites of the arginine metabolic pathway; Error bars: standard deviation (SD), two-tailed unpaired t-test: *P < 0.05, **P < 0.01, ***P < 0.001. ...
Sleep has evolved as a universal core function to allow for restorative biological processes. Detailed knowledge of metabolic changes necessary for the sleep state in the brain is missing. Herein, we have performed an in-depth metabolic analysis of four mouse brain regions and uncovered region-specific circadian variations. Metabolites linked to oxidative stress were altered during sleep including acylcarnitines, hydroxylated fatty acids, phenolic compounds, and thiol-containing metabolites. These findings provide molecular evidence of a significant metabolic shift of the brain energy metabolism. Specific alterations were observed for brain metabolites that have previously not been associated with a circadian function including the microbiome-derived metabolite ergothioneine that suggests a regulatory function. The pseudopeptide β-citryl-glutamate has been linked to brain development and we have now discovered a previously unknown regioisomer. These metabolites altered by the circadian rhythm represent the foundation for hypothesis-driven studies of the underlying metabolic processes and their function.
... A. woodii cells are at least to some extent able to use arginine as nitrogen source, since a native arginine deiminase could cleave off ammonium and produce citrulline. Small amounts of fumarate detected in the supernatants of A. woodii [pMTL83151] hint to a native utilization of an urea cycle (Jackson et al. 1986), executed by the action of arginine deiminase, argininosuccinate synthetase (AS), and argininosuccinate lyase (AL), whose genes are all present in the genome of A. woodii and are encoded by argA (Awo_c08610 ), argG (Awo_c135 40), and argH (Awo_c13530), respectively (Poehlein et al. 2012). The activity of AS and AL are responsible for the conversion of citrulline into argininosuccinate (Ratner 1954) and the subsequent cleavage to arginine and fumarate (Davison and Elliott 1952). ...
The advantage of using acetogens such as Acetobacterium woodii as biocatalysts converting the cheap substrate and greenhouse gas carbon dioxide (CO2) into value-added chemicals comes together with the disadvantage of a low overall ATP gain due to the bioenergetics associated with the Wood-Ljungdahl pathway. Expanding the product spectrum of recombinant A. woodii strains to compounds with high ATP-demanding biosynthesis is therefore challenging. As a least invasive strategy for improved ATP generation, the exploitation of the arginine deiminase pathway (ADI) was examined under native conditions and via using heterologously expressed genes in A. woodii. Several promoters were analyzed for application of different gene expression levels in A. woodii using β-glucuronidase assays. Heterologous expression of the ADI pathway genes from Clostridium autoethanogenum was controlled using either the constitutive pta-ack promoter from Clostridium ljungdahlii or a tightly regulated tetracycline-inducible promoter Ptet. Unlike constitutive expression, only induced expression of the ADI pathway genes led to a 36% higher maximal OD600 when using arginine (OD600 3.4) as nitrogen source and a 52% lower acetate yield per biomass compared to cells growing with yeast extract as nitrogen source (OD600 2.5). In direct comparison, a 69% higher maximal OD600 and about 60% lower acetate yield per biomass in induced to non-induced recombinant A. woodii cells was noticed when using arginine. Our data suggests the application of the ADI pathway in A. woodii for expanding the product spectrum to compounds with high ATP-demanding biosynthesis.
... In addition, we demonstrated that cyanate is generated by the reaction. Cyanate is known to be mainly generated in vivo by the decomposition of carbamoyl phosphate [31], which is generally used for the biosynthesis of L-arginine (or urea) or pyrimidine nucleotide, in which the carbamoyl group is transferred to L-ornithine or L-aspartate, respectively, by the enzymatic activity of the respective carbamoyltransferase [32][33][34]. However, in the DcsG reaction to synthesize D-CS from D-OUS, the carbamoyl phosphate may not be generated. ...
In the biosynthetic pathway of an antitubercular antibiotic d‐cycloserine (d‐CS), O‐ureido‐d‐serine (d‐OUS) is converted to d‐CS. We have previously demonstrated that DcsG, classified into the ATP‐grasp superfamily enzyme, catalyzes the ring formation to generate d‐CS, which is accompanied by the cleavage of a bond in the urea moiety of d‐OUS to remove a carbamoyl group. Although the general ATP‐grasp enzymes catalyze an ATP‐dependent ligation reaction between two substrates, DcsG catalyzes specifically the generation of an intramolecular covalent bond. In the present study, cyanate was found in the reaction mixture, suggesting that carbamoyl group is eliminated as an isocyanic acid during the reaction. By the crystallographic and mutational investigations of DcsG, we anticipate the residues necessary for the binding of d‐OUS. An acylphosphate intermediate must be bound at the narrow pocket of DcsG in a folded conformation, inducing the bond cleavage and the new bond formation to generate cyanate and d‐CS, respectively.
Database
Structural data are available in Protein Data Bank database under the accession number 6JIL.
... In addition to urea synthesis in the liver of ureotelic species, arginase is also involved in biosynthesis of polyamines and proline [5], conversion of arginine into a-ketoglutarate for oxidation in the Krebs cycle [6], adaptive responses to anoxia in some invertebrates [7] and production of urea for osmoregulatory purposes [8]. It should be noted that arginase is found in various tissues of non-ureotelic organisms, including liver, but is not part of a functional urea cycle [9]. It had previously been thought that there were significant kinetic and structural differences between ureotelic and non-ureotelic arginases [10][11], but many studies suggested that the characteristics of arginases are not consistent with a particular mode of nitrotelism [12][13]. ...
Arginase (EC 3.5.3.1) catalyzes the hydrolysis of arginine to ornithine and urea. Arginase is a metalloenzyme in which manganese acts as a cofactor as well as an activator in almost all reported arginases. A variety of function has been proposed for arginase in different environments and organisms. Examples include its involvement in bulb growth and sprouting in plants, normal mammary gland development in rat, ammonia detoxification, hormone secretion, immune modulation and cellular replication in humans. In vertebrates, as well as in insects, uric acid is produced by degradation of purines. The action of arginase in insects is limited to arginine from dietary sources or from endogenous protein turnover. This is because insects lack one or more genes encoding enzymes required for the urea cycle. Arginase was partially purified and characterized from Ametermes eveuncifer by dialysis, ammonium sulphate precipitation using 85% saturation. Affinity chromatography was performed on treated resin (Agarose). The column was washed and equilibrated with 0.005 M of Tris-HCl buffer, pH 7.5, pKa 8.1. The specific activity of the enzyme was 1.217 μmol per min per mg of protein and a yield of 0.16% was obtained. The Michaelis-Menten constant (Km) of the enzyme was 142.85 mM with arginine as substrate. The optimum pH was 4.0 and the optimum temperature was 40 °C for A. eveuncifer arginase. The enzyme's activity was strongly enhanced in the presence of Ni 2+ and Mn 2+. K + , Ba 2+ , Zn 2+ and Na + slightly inhibited A. eveuncifer arginase. Chelating agents (Urea, EDTA and 2-Mercaptoethanol) with various concentrations (0.001, 0.01 and 0.1 M) showed different effects on the activity of arginase in A. eveuncifer. Urea enhanced the activity at all concentrations while Mercaptoethanol (ME) enhanced the activity of the enzyme only at 0.001 M and showed moderate inhibition at 0.01 M and 0.1 M. Ethylenediaminetetraacetate (EDTA) showed moderate inhibition at all concentrations. The amino acids Proline, Serine and Valine showed moderate inhibition with percentage residual activity of 94.05, 89.29 and 97.62 respectively while Aspartic acid and Glutamic acid (>100% respectively) stimulated arginase activity. The biochemical properties of Ametermes eveuncifer will be useful in assigning physiological role to the enzyme; especially its involvement in the growth of the exoskeleton, excretion of nitrogen waste efficiently and for survival
... Urea is formed in the urea cycle, a series of enzymatic steps to neutralize ammonia, which is released with degradation of amino acids [54]. It is a small nitrogen containing compound, with a molecular weight of 60 Da. ...
Although glomerular filtration rate (GFR) in children can be measured using a gold-standard technique following injection of an exogenous marker, this invasive and cumbersome technique is not widely available and GFR is commonly estimated using serum levels of endogenous markers. Creatinine, urea, cystatin C, beta-trace protein, and beta-2 microglobulin are well-established endogenous markers of kidney function. These markers differ in site of production and effects of diet and medication, as well as renal-tubular handling and extra-renal elimination. For each marker, different methods are available for measurement. Importantly, the measurements of creatinine and cystatin C have recently been standardized with the introduction of international reference standards. In order to allow estimation of GFR from serum marker concentrations, different equations for estimated GFR (eGFR) have been developed in children, using simple or more complex regression strategies with gold standard GFR measurements as a dependent variable. As a rule, estimation strategies relying on more than one marker – either by calculating the average of single parameter equations or by using more complex equations incorporating several parameters – outperform eGFR estimations using only a single marker. This in-depth review will discuss the physiology, measurement and clinical use of creatinine, urea, cystatin C, beta-trace protein, and beta-2 microglobulin in children. It will also address the generation of eGFR equations in children and provide an overview of currently available eGFR equations for the pediatric age group.
... The urea cycle, often referred to as "urea synthesis" or "ureagenesis" was discovered by Krebs and Henseleit in 1932 [1]. It occurs predominantly in the liver [2][3][4][5]. Its overall equation is: ...
This article is a guided pedagogical approach, devoted to postgraduate students specializing in biochemistry, aimed at presenting all single reactions and overall equations leading to the metabolic interaction between ureagenesis and citric acid cycle to be incorporated into a two-three lecture series about the interaction of urea cycle with other metabolic pathways. We emphasize that citrate synthetase, aconitase, and isocitrate dehydrogenase, three enzymes of the citric acid cycle are not involved, thus creating a shunt in citric acid cycle. In contrast, the glutamic-oxaloacetate transaminase, which does not belong to citric acid cycle, has a paramount importance in the metabolic interaction of the two cycles, because it generates aspartate, one of the two fuel molecules of urea cycle, and a-ketoglutarate, an intermediate of the citric acid cycle. Finally, students should appreciate that balancing equations for all atoms and charges is not only a stoichiometric task, but strongly facilitates the discussion of the physiological roles of metabolic pathways. Indeed, this exercise has been used in the classroom, to encourage a deeper level of understanding of an important biochemical issue. © 2017 by The International Union of Biochemistry and Molecular Biology, 2017.
... THE SYNTHESIS OF UREA, often referred to as "ureagenesis" or "urea cycle" was discovered by Krebs and Henseleit in 1932 (12). It occurs predominantly in the liver (3,6,11,14,15,28). Its physiological importance in handling the toxic ammonium ions discharged during catabolism of amino acids is satisfactorily discussed in many texts. ...
It is well known that a strong metabolic interrelationship exists between ureagenesis and gluconeogenesis. In this paper, we present a detailed, overall equation, describing a possible metabolic link between ureagenesis and gluconeogenesis. We adopted a guided approach in which we strongly suggest that students, when faced with the problem of obtaining the overall equation of a metabolic pathway, carefully account for all atoms and charges of the single reactions, as well as the cellular localizations of the substrates, and the related transport systems. If this suggestion is always taken into account, a balanced, overall equation of a metabolic pathway will be obtained, which strongly facilitates the discussion of its physiological role. Unfortunately, textbooks often report unbalanced overall equations of metabolic pathways, including ureagenesis and gluconeogenesis. Most likely the reason is that metabolism and enzymology have been neglected for about three decades, owing to the remarkable advances of molecular biology and molecular genetics. In this paper, we strongly suggest that students, when faced with the problem of obtaining the overall reaction of a metabolic pathway, carefully control if the single reactions are properly balanced for atoms and charges. Following this suggestion, we were able to obtain an overall equation describing the metabolic interrelationship between ureagenesis and gluconeogenesis, in which urea and glucose are the final products. The aim is to better rationalize this topic and to convince students and teachers that metabolism is an important and rewarding chapter of human physiology.
... thereby increasing the dietary requirement (8). Synthesis of L-arginine from citrulline also occurs at a low level in many other cells (46,47), and cellular capacity for arginine synthesis can be markedly increased under circumstances that also induce iNOS (48). Thus, citrulline, a coproduct of the NOScatalyzed reaction, can be recycled to L-arginine in a pathway known as the citrulline-NO or arginine-citrulline pathway (32). ...
In mammals, L-arginine is classified as a semiessential or conditionally essential amino acid, depending on the developmental stage and health status of the individual. It can be derived from proline or glutamate, with the ultimate synthetic step catalyzed by argininosuccinate lyase. L-arginine is catabolized by arginases, nitric oxide synthases, arginine:glycine amidinotransferase, and possibly also by arginine decarboxylase, resulting ultimately in the production of urea, proline, glutamate, polyamines, nitric oxide, creatine, or agmatine. There is considerable diversity in tissue-specific and stimulus-dependent regulation of expression within this group of enzymes, and the expression of several of them can be regulated at transcriptional and translational levels by changes in the concentration Of L-arginine itself. Consequently, the interplay among these enzymes in the regulation of specific aspects of arginine metabolism can be quite complex. For example, nitric oxide production can be affected by the interplay between nitric oxide synthases, arginases, and argininosuccinate synthetase. This metabolic complexity can pose challenges for analyses of arginine metabolism not only because L-arginine is a substrate for several different enzymes but also because ornithine and citrulline, key products of arginine metabolism, can each be produced by multiple enzymes. This overview highlights key features of the arginine metabolic enzymes and their interactions.
... Deficiency in any of the six principal enzymes associated with the urea cycle results in perturbation of ureagenesis, leading to incomplete removal of ammonia and eventual hyperammonemia of varying degrees. The liver is the main site of urea cycle activity where the proximal three enzymes are in the mitochondria [N-acetyl-glutamate synthase (NAGS), carbamoyl phosphate synthetase 1 (CPS1), and ornithine transcarbamylase (OTC)], while the distal three are c yt os ol i c [a rg i n i n o s u cc i n at e s y nt he t a s e ( A S S ) , argininosuccinate lyase (ASL), and arginase] [1]. ...
Arginase-1 (ARG1) deficiency is a rare autosomal recessive disorder that affects the liver-based urea cycle, leading to impaired ureagenesis. This genetic disorder is caused by 40+ mutations found fairly uniformly spread throughout the ARG1 gene, resulting in partial or complete loss of enzyme function, which catalyzes the hydrolysis of arginine to ornithine and urea. ARG1-deficient patients exhibit hyperargininemia with spastic paraparesis, progressive neurological and intellectual impairment, persistent growth retardation, and infrequent episodes of hyperammonemia, a clinical pattern that differs strikingly from other urea cycle disorders. This review briefly highlights the current understanding of the etiology and pathophysiology of ARG1 deficiency derived from clinical case reports and therapeutic strategies stretching over several decades and reports on several exciting new developments regarding the pathophysiology of the disorder using ARG1 global and inducible knockout mouse models. Gene transfer studies in these mice are revealing potential therapeutic options that can be exploited in the future. However, caution is advised in extrapolating results since the lethal disease phenotype in mice is much more severe than in humans indicating that the mouse models may not precisely recapitulate human disease etiology. Finally, some of the functions and implications of ARG1 in non-urea cycle activities are considered. Lingering questions and future areas to be addressed relating to the clinical manifestations of ARG1 deficiency in liver and brain are also presented. Hopefully, this review will spark invigorated research efforts that lead to treatments with better clinical outcomes.
... [22] The peptide is derived from the acetylation site K527 of carbamoyl phosphate synthetase 1( CPS1), whichi nitiates the urea cycle by catalyzing carbamoyl phosphate formation. [23] Schwer et al. showedt hat K527 is hyperacetylated during calorie restriction, thus implicating it in CPS1 regulation. [10b] Other studies revealed succinylation of K527; [24] this could be removed by SIRT5,c onsistent with ar ole of SIRT5 in urea cycle regulation. ...
Mitochondrial enzymes implicated in the pathophysiology of diabetes, cancer, and metabolic syndrome are highly regulated by acetylation. However, mitochondrial acetyltransferases have not been identified. Here, we show that acetylation and also other acylations are spontaneous processes that depend on pH value, acyl-CoA concentration and the chemical nature of the acyl residue. In the case of a peptide derived from carbamoyl phosphate synthetase 1, the rates of succinylation and glutarylation were up to 150 times than for acetylation. These results were confirmed by using the protein substrate cyclophilin A (CypA). Deacylation experiments revealed that SIRT3 exhibits deacetylase activity but is not able to remove any of the succinyl groups from CypA, whereas SIRT5 is an effective protein desuccinylase. Thus, the acylation landscape on lysine residues might largely depend on the enzymatic activity of specific sirtuins, and the availability and reactivity of acyl-CoA compounds.
... The urea cycle shown in Figure 6A consists of five reactions of which two are mitochondrial and three cytosolic. 34 We found altered regulation of arginine and carbamoyl phosphate metabolite upon quercetin treatment, in both HCFs and HKCs. Carbamoyl phosphate reacts with ornithine to give citrulline as shown in Figure 6A. ...
Corneal scarring is the result of a disease, infection or injury. The resulting scars cause significant loss of vision or even blindness. To-date, the most successful treatment is corneal transplantation, but it does not come without side effects. One of the corneal dystrophies that are correlated with corneal scarring is keratoconus (KC). The onset of the disease is still unknown; however, altered cellular metabolism has been linked to promoting the fibrotic phenotype and therefore scarring. We have previously shown that human keratoconus cells (HKCs) have altered metabolic activity when compared to normal human corneal fibroblasts (HCFs). In our current study, we present evidence that quercetin, a natural flavonoid, is a strong candidate for regulating metabolic activity of both HCFs and HKCs in vitro and therefore a potential therapeutic to target the altered cellular metabolism characteristic of HKCs. Targeted mass spectrometry-based metabolomics was performed on HCFs and HKCs with and without quercetin treatment in order to identify variations in metabolite flux. Overall, our study reveals a novel therapeutic target OF Quercetin on corneal stromal cell metabolism in both healthy and diseased states. Clearly, further studies are necessary in order to dissect the mechanism of action of quercetin. Copyright © 2015 John Wiley & Sons, Ltd.
Copyright © 2015 John Wiley & Sons, Ltd.
... In addition to urea synthesis in the liver of ureotelic species, arginase is also involved in biosynthesis of polyamines and proline [5], conversion of arginine into α-ketoglutarate for oxidation in the Krebs cycle [6], adaptive responses to anoxia in some invertebrates [7] and production of urea for osmoregulatory purposes [8]. It should be noted that arginase is found in various tissues of nonureotelic organisms, including liver, but is not part of a functional urea cycle [9]. It had previously been thought that there were significant kinetic and structural differences between ureotelic and non-ureotelic arginases [10], but many studies suggested that the characteristics of arginases are not consistent with a particular mode of nitrotelism [11,12]. ...
We describe the hepatopancreas arginase activity of freshwater prawn (Macrobrachium rosenbergii). The enzyme was isolated using reactive blue 2- agarose affinity chromatography and gel filtration on Sephadex G-150. The enzyme had a specific activity of 5.70 μmol/min/mg of protein. The enzyme exhibited a maximal activity at pH 8.5 and Km of 12.5 mM. The enzyme was capable of hydrolysing L-arginine and to a lesser extent, L-arginine monohydrochlorate and L-arginine monohydrate. The optimum temperature of the enzyme was 35 0C. The molecular weight as determined by gel filtration was approximately 160,000 dalton and SDS-PAGE, was 22,000 dalton. The different amino acids (L-lysine, L-cysteine, Lvaline, L-proline, L-aspartic acid, L-glutamic acid and L-serine) and metal ions (Ni²⁺, Co²⁺, Zn²⁺, Mn²⁺ and Mg²⁺) did not show any inhibition on the enzyme activity. The enzyme was activated with Mn²⁺ and different concentration of Mn²⁺ had no effect on the enzyme activity. EDTA, citrate and urea showed considerable inhibition on the enzyme activity.
Key words: Freshwater prawn; arginase; uricotelism; invertebrates; hepatopancreas
... Although toadfish hepatocytes might be a suitable model to study the hormonal regulation of gluconeogenesis and glycogenolysis by glucagon, GLP or epinephrine, a scan of some 15 metabolic hormones failed to identify any short-term regulation of the ornithine-urea cycle. In the case of glucagon, which clearly exerts rapid effects on urea cycle activity in the rat (Jackson et al. 1986), this result must be related to the lack of hormonal regulation of ureogenesis by this hormone in the toadfish liver rather than to an artifact induced by our experimental procedure. Since cells respond sensitively to glucagon, epinephrine and GLP, cell surface receptors were not compromised by collagenase digestion of the liver and mechanisms of intracellular message transduction were undisturbed. ...
Short-term exposure of isolated toadfish hepatocytes to high concentrations (100 nM) of glucagon, glucagon-like peptide (GLP) or epinephrine significantly increases the rate of lactate gluconeogenesis (1.3-fold) and glycogenolysis (5- to 7-fold). Half-maximal responsiveness to GLP is reached at about 2 nM for gluconeogenesis and 6 nM for glycogenolysis, while the value for glycogenolysis activated by catfish glucagon is 28 nM. Cells do not to respond to 5 nM epinephrine. Norepinephrine, urotensin II and leucine-enkephalin, each applied at 100 nM, increase the rate of glycogenolysis by 1.3 to 1.5-fold. All other hormones tested (vasotocin, isotocin, VIP, methionine-enkephalin, ovine prolactin, β-endorphin, APY, salmon insulin) failed to affect metabolic flux through glycogenolysis or gluconeogenesis. None of the hormones altered the rate of urea synthesis or the rate of lactate oxidation by hepatocytes. Although toadfish hepatocytes are responsive to hormonal stimuli, they do not appear to be a useful model to study evolutionary trends in short-term hormonal regulation of urea synthesis. However, the obvious differences in mechanisms of control of urea synthesis in this species compared with ureogenic amphibians and mammals open an intriguing avenue for research.
... Sources of arginine might include re-lease of arginine from the intracellular stores of cells dying in culture, contaminating arginine during cell dispersion, or biosynthesis of arginine from media aspartate. Although arginine is generally considered to be an essential amino acid, some cells in culture are able to synthesize arginine [40]. ...
Recent studies suggest that endogenously generated nitric oxide (NO) may mediate the effects of cytokines in a variety of tissues. In an effort to determine whether NO generation mediates any of the intraovarian actions of interleukin-1 beta (IL-1 beta), we have looked for and characterized the accumulation of nitrite by IL-1 beta-treated, cultured whole ovarian dispersates. Application of IL-1 beta significantly enhanced basal nitrite release in a dose-, cell density- and time-dependent manner, the latter characterized by a lag time of about 20 h, suggestive of induction of NO synthase (NOS). Cellular NOS activity was also elevated by IL-1 beta. Sustained nitrite accumulation required continuous application of IL-1 beta. The maximally stimulating dose of IL-1 beta (50 ng/ml) produced a 10-fold increase in nitrite accumulation by 96 h of culture, an effect reduced 23% when cells were cultured in substrate (i.e., arginine)-free media. IL-1 beta-stimulated nitrite accumulation was reduced to control levels by the simultaneous application of an IL-1 beta receptor antagonist, thereby suggesting a specific receptor-mediated effect. Both the control and IL-1 beta-stimulated levels of nitrite accumulation were attenuated in a dose-dependent manner by inhibitors that favor the inducible form of NOS. In contrast, selective inhibitors of the constitutive form of NOS were significantly less potent. No inhibition was noted after application of an inactive stereoisomeric analogue. IL-1 beta-induced nitrite accumulation was shown to require cell-cell interaction between granulosa and theca-interstitial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
... Activated CPSI converts ammonia and bicarbonate to carbamyl phosphate (CP) with the expenditure of 2 ATP molecules ( Figure 1.2) [10][11]. CPSI exhibits high homology between mammalian species. ...
... Since build up of ammonia is toxic, lungfishes (and indeed most aquatic species) excrete ammonia passively by diffusion over branchial and cutaneous surfaces (Anderson, 2001;Graham, 1997;Korsgaard et al., 1995;Wood et al., 2005), a process known as ammonioteley. In terrestrial vertebrates passive loss of ammonia is not possible therefore in these animals urea is the typical nitrogenous waste and it is produced via the ornithine-urea cycle (O-UC) requiring the equivalent of four moles of ATP per mole of urea produced (Jackson et al., 1986;Meijer, 1995;Mommsen and Walsh, 1991). In ureotelic fish (fish that produce urea as the primary nitrogenous waste) ammonia is converted to urea because it is less toxic (Ip et al., 2001;Walsh, 1998) at a metabolic cost of five moles of ATP per mole of urea produced (Mommsen and Walsh, 1991;Mommsen and Walsh, 1992;Walsh, 1998). ...
Molecular aspects of nitrogen metabolism in vertebrates is an interesting area of physiology and evolution to explore due to the different ways in which animals excrete nitrogenous waste as they transition from an aquatic to a terrestrial lifestyle. Two main products of nitrogen metabolism in fishes are ammonia and urea. Ammonia is produced during protein catabolism and build up of ammonia is toxic. Some aquatic vertebrates convert ammonia into a less toxic compound urea via de novo synthesis through the ornithine-urea cycle (O-UC). Five enzymes are involved in the O-UC: carbamoyl phosphate synthetase (CPS), ornithine carbamoyl transferase (OCT), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (ARG). An accessory enzyme, glutamine synthetase (GS) also participates in the "fish-type" O-UC. Teleosts excrete ammonia passively over their gills into the aquatic environment. The teleost, Opsanus beta, has been shown to increase urea production after 48 hours of crowding. This thesis explored how crowding stress affected nitrogen metabolite levels of ammonia and urea and O-UC gene expression and enzyme activity in O. beta. Lungfishes while in an aquatic environment avoid ammonia toxicity by releasing excess ammonia across their gills, but when stranded on land they produce urea through the O-UC. Urea production via the O-UC has a metabolic cost of at least four ATP molecules. This thesis explored the response of a lungfish, Protopterus annectens, to six days of aerial exposure and re-immersion conditions by measuring concentrations of O-UC mRNA expression and enzyme activity and nitrogen metabolites ammonia and urea. CPS acts as the entry point to the O-UC and based on enzymatic studies, most aquatic vertebrates utilize one isoform of this enzyme (CPSIII) while terrestrial vertebrates utilize a different isoform of this enzyme (CPSI). Lungfishes are a particularly interesting group of air-breathing fishes, not only because of their link to the origins of tetrapods, but also because CPS I may have originated within this group. Both CPS III and CPS I have been enzymatically described within this group. This thesis uses phylogenetics to investigate how CPS nucleotide sequences in lungfishes evolved compared to other vertebrates.
... Hence, mitochondria are commonly characterized as the powerhouses of the cell (McBride et al. 2006). However, mitochondria also play a central role in a number of critical cellular and metabolic processes, including cellular proliferation; apoptosis or programmed cell death (cellular suicide), a process aimed at destroying a physiologically unwanted cell (Desagher & Martinou 2000); the regulation and homeostasis of intracellular calcium, which acts as an intracellular signal involved in numerous cellular processes including cellular expression and metabolism; fertilization and embryonic development (Cao & Chen 2009); DNA repair (DNA is constantly exposed to endogenous and exogenous agents that generate DNA lesions and induce DNA instability; see Gredilla et al. 2010); aging (Salvioli et al. 2001); the regulation of steroid hormone synthesis in the adrenal cortex, including estrogen, testosterone, and cortisol (Almahbobi et al. 1992); synthesis of heme, one of the components of hemoglobin (Atamna 2004); and detoxification of ammonia from the liver (Jackson et al. 1986). Mitochondria also play a critically important role in the brain and are involved in the regulation of brain function, including synaptic plasticity and brain size (Chada & Hollenbeck 2004). ...
My response is divided into four sections: (1) is devoted to a potpourri of commentaries that are essentially in agreement with the substance of my target article (with one exception); in (2) I address, in response to one of the commentaries, several issues relating to the use of candidate gene association studies in behavior genetics (in particular those proposing a specific G × E interaction); in (3) I provide a detailed response to several defenses of the twin study methodology; and in (4) I conclude with several reflections on that methodology and the conception of human nature it has fostered.
... Hence, mitochondria are commonly characterized as the powerhouses of the cell (McBride et al. 2006). However, mitochondria also play a central role in a number of critical cellular and metabolic processes, including cellular proliferation; apoptosis or programmed cell death (cellular suicide), a process aimed at destroying a physiologically unwanted cell (Desagher & Martinou 2000); the regulation and homeostasis of intracellular calcium, which acts as an intracellular signal involved in numerous cellular processes including cellular expression and metabolism; fertilization and embryonic development (Cao & Chen 2009); DNA repair (DNA is constantly exposed to endogenous and exogenous agents that generate DNA lesions and induce DNA instability; see Gredilla et al. 2010); aging (Salvioli et al. 2001); the regulation of steroid hormone synthesis in the adrenal cortex, including estrogen, testosterone, and cortisol (Almahbobi et al. 1992); synthesis of heme, one of the components of hemoglobin (Atamna 2004); and detoxification of ammonia from the liver (Jackson et al. 1986). Mitochondria also play a critically important role in the brain and are involved in the regulation of brain function, including synaptic plasticity and brain size (Chada & Hollenbeck 2004). ...
Critically significant parental effects in behavioral genetics may be partly understood as a consequence of maternal brain structure and function of caregiving systems recently studied in humans as well as rodents. Key parental brain areas regulate emotions, motivation/reward, and decision making, as well as more complex social-cognitive circuits. Additional key environmental factors must include socioeconomic status and paternal brain physiology. These have implications for developmental and evolutionary biology as well as public policy.
... Hence, mitochondria are commonly characterized as the powerhouses of the cell (McBride et al. 2006). However, mitochondria also play a central role in a number of critical cellular and metabolic processes, including cellular proliferation; apoptosis or programmed cell death (cellular suicide), a process aimed at destroying a physiologically unwanted cell (Desagher & Martinou 2000); the regulation and homeostasis of intracellular calcium, which acts as an intracellular signal involved in numerous cellular processes including cellular expression and metabolism; fertilization and embryonic development (Cao & Chen 2009); DNA repair (DNA is constantly exposed to endogenous and exogenous agents that generate DNA lesions and induce DNA instability; see Gredilla et al. 2010); aging (Salvioli et al. 2001); the regulation of steroid hormone synthesis in the adrenal cortex, including estrogen, testosterone, and cortisol (Almahbobi et al. 1992); synthesis of heme, one of the components of hemoglobin (Atamna 2004); and detoxification of ammonia from the liver (Jackson et al. 1986). Mitochondria also play a critically important role in the brain and are involved in the regulation of brain function, including synaptic plasticity and brain size (Chada & Hollenbeck 2004). ...
Against the opinion that DNA as program is not sufficiently explanatory, we maintain that the cellular machinery is entirely computational, and we identify the crucial notion of the interpreter that expresses the gene with the minimal gene set. Epigenetics research does not so much need paradigm shifts as the unraveling of an exceedingly complex computational machine.
... Hence, mitochondria are commonly characterized as the powerhouses of the cell (McBride et al. 2006). However, mitochondria also play a central role in a number of critical cellular and metabolic processes, including cellular proliferation; apoptosis or programmed cell death (cellular suicide), a process aimed at destroying a physiologically unwanted cell (Desagher & Martinou 2000); the regulation and homeostasis of intracellular calcium, which acts as an intracellular signal involved in numerous cellular processes including cellular expression and metabolism; fertilization and embryonic development (Cao & Chen 2009); DNA repair (DNA is constantly exposed to endogenous and exogenous agents that generate DNA lesions and induce DNA instability; see Gredilla et al. 2010); aging (Salvioli et al. 2001); the regulation of steroid hormone synthesis in the adrenal cortex, including estrogen, testosterone, and cortisol (Almahbobi et al. 1992); synthesis of heme, one of the components of hemoglobin (Atamna 2004); and detoxification of ammonia from the liver (Jackson et al. 1986). Mitochondria also play a critically important role in the brain and are involved in the regulation of brain function, including synaptic plasticity and brain size (Chada & Hollenbeck 2004). ...
Several new molecular findings and concepts furnish evidence in support of gene-environment interdependence, challenging some of the current tenets and basic statistics of behavioral genetics. I, however, argue that (1) some of the expectations evoked by "neogenomics" are contradicted by findings; and (2) while epigenetic and gene expression effects are complex, they can to some extent be incorporated into "classical" behavioral genetics modeling.
... Hence, mitochondria are commonly characterized as the powerhouses of the cell (McBride et al. 2006). However, mitochondria also play a central role in a number of critical cellular and metabolic processes, including cellular proliferation; apoptosis or programmed cell death (cellular suicide), a process aimed at destroying a physiologically unwanted cell (Desagher & Martinou 2000); the regulation and homeostasis of intracellular calcium, which acts as an intracellular signal involved in numerous cellular processes including cellular expression and metabolism; fertilization and embryonic development (Cao & Chen 2009); DNA repair (DNA is constantly exposed to endogenous and exogenous agents that generate DNA lesions and induce DNA instability; see Gredilla et al. 2010); aging (Salvioli et al. 2001); the regulation of steroid hormone synthesis in the adrenal cortex, including estrogen, testosterone, and cortisol (Almahbobi et al. 1992); synthesis of heme, one of the components of hemoglobin (Atamna 2004); and detoxification of ammonia from the liver (Jackson et al. 1986). Mitochondria also play a critically important role in the brain and are involved in the regulation of brain function, including synaptic plasticity and brain size (Chada & Hollenbeck 2004). ...
The science of genetics is undergoing a paradigm shift. Recent discoveries, including the activity of retrotransposons, the extent of copy number variations, somatic and chromosomal mosaicism, and the nature of the epigenome as a regulator of DNA expressivity, are challenging a series of dogmas concerning the nature of the genome and the relationship between genotype and phenotype. According to three widely held dogmas, DNA is the unchanging template of heredity, is identical in all the cells and tissues of the body, and is the sole agent of inheritance. Rather than being an unchanging template, DNA appears subject to a good deal of environmentally induced change. Instead of identical DNA in all the cells of the body, somatic mosaicism appears to be the normal human condition. And DNA can no longer be considered the sole agent of inheritance. We now know that the epigenome, which regulates gene expressivity, can be inherited via the germline. These developments are particularly significant for behavior genetics for at least three reasons: First, epigenetic regulation, DNA variability, and somatic mosaicism appear to be particularly prevalent in the human brain and probably are involved in much of human behavior; second, they have important implications for the validity of heritability and gene association studies, the methodologies that largely define the discipline of behavior genetics; and third, they appear to play a critical role in development during the perinatal period and, in particular, in enabling phenotypic plasticity in offspring. I examine one of the central claims to emerge from the use of heritability studies in the behavioral sciences, the principle of minimal shared maternal effects, in light of the growing awareness that the maternal perinatal environment is a critical venue for the exercise of adaptive phenotypic plasticity. This consideration has important implications for both developmental and evolutionary biology.
Each tissue has a dominant set of functional proteins required to mediate tissue-specific functions. Epigenetic modifications, transcription, and translational efficiency control tissue-dominant protein production. However, the coordination of these regulatory mechanisms to achieve such tissue-specific protein production remains unclear. Here, we analyzed the DNA methylome, transcriptome, and proteome in mouse liver and skeletal muscle. We found that DNA hypomethylation at promoter regions is globally associated with liver-dominant or skeletal muscle-dominant functional protein production within each tissue, as well as with genes encoding proteins involved in ubiquitous functions in both tissues. Thus, genes encoding liver-dominant proteins, such as those involved in glycolysis or gluconeogenesis, the urea cycle, complement and coagulation systems, enzymes of tryptophan metabolism, and cytochrome P450-related metabolism, were hypomethylated in the liver, whereas those encoding-skeletal muscle-dominant proteins, such as those involved in sarcomere organization, were hypomethylated in the skeletal muscle. Thus, DNA hypomethylation characterizes genes encoding tissue-dominant functional proteins.
L-arginine and its derivatives, asymmetric and symmetric dimethylarginine (ADMA and SDMA) and L-homoarginine, have emerged as cardiovascular biomarkers linked to cardiovascular outcomes and various metabolic and functional pathways such as NO-mediated endothelial function. Cellular uptake and efflux of L-arginine and its derivatives are facilitated by transport proteins. In this respect the cationic amino acid transporters CAT1 and CAT2 (SLC7A1 and SLC7A2) and the system y + L amino acid transporters (SLC7A6 and SLC7A7) have been most extensively investigated, so far, but the number of transporters shown to mediate the transport of L-arginine and its derivatives is constantly increasing. In the present review, we assess the growing body of evidence regarding the function, expression, and clinical relevance of these transporters and their possible relation to cardiovascular diseases.
Arginine (Arg) is derived from dietary intake, body protein breakdown, and endogenous Arg production, and is also present in saliva. Arg has been introduced as an additive in toothpaste and other fluoride‐containing dental care products and is now being promoted as more efficacious than conventional fluoride‐only toothpaste for the prevention of dental caries. Arg inclusion in dentifrice could potentially inhibit oral biofilm formation and destabilize dental plaque. Arg is expected to inhibit oral biofilm formation and destabilize complex aggregates in dental plaque and its inclusion in mouth rinse could facilitate oral biofilm removal. The destabilization of the oral biofilm by Arg may facilitate the removal of dental plaque during routine oral care. Since removing this solid biological plaque from tooth surfaces by water rinsing or brushing can be difficult, Arg metabolism in oral biofilms leading to an increase in pH decreases the risk for caries development. Moreover, clinical data have shown that Arg‐containing toothpaste increased benefits leading to the prevention of dental caries. However, there are concerns regarding the use of Arg dentifrice in caries progression or regression and some studies contradict the claims of Arg benefits in caries progression or regression.
Cerebral ischemia induces neuroinflammation and microglial activation, in which activated microglia upregulate their proliferative activity and change their metabolic states. In activated microglia, l-arginine is metabolized competitively by inducible nitric oxide synthase (iNOS) and arginase (Arg), which then synthesize NO or polyamines, respectively. Our previous study demonstrated that Sema4D deficiency inhibits iNOS expression and promotes proliferation of ionized calcium-binding adaptor molecule 1 (Iba1)-positive (Iba1 +) microglia in the ischemic cortex, although the underlying mechanisms were unclear. Using middle cerebral artery occlusion, we tested the hypothesis that Sema4D deficiency alters the balance of l-arginine metabolism between iNOS and Arg, leading to an increase in the production of polyamines, which are an essential factor for cell proliferation. In the peri-ischemic cortex, almost all iNOS + and/or Arg1 + cells were Iba1 + microglia. In the peri-ischemic cortex of Sema4D-deficient (Sema4D −/− ) mice, the number of iNOS + Arg1 − Iba1 + microglia was smaller and that of iNOS − Arg1 + Iba1 + microglia was greater than those of wild-type (WT) mice. In addition, urea and polyamine levels in the ischemic cortex of Sema4D −/− mice were higher than those of WT mice; furthermore, the presence of Sema4D inhibited polyamine production in primary microglia obtained from Sema4D −/− mice. Finally, microglia cultured under polyamine putrescine-supplemented conditions demonstrated increased proliferation rates over non-supplemented controls. These findings indicate that Sema4D regulates microglial proliferation at least in part by regulating the competitive balance of l-arginine metabolism.
Arginine is derived from dietary intake, body protein breakdown, or endogenous de novo arginine production. Arginine methylation of non-histone proteins is used in transcriptional regulation. Protein-arginine methylation is used for regulation of transcriptional and various physiological pathological processes. Protein methylation may affect protein-protein, protein-DNA, or protein-RNA interaction. Arginine has an effect on the DNA-binding activity of NF-κB, a dominant transcriptional factor in inflammation. Adduct formation results in increased secretion of messenger molecules such as cytokines and chemokines that mediate communication among cells and promote inflammation. Arginine and lysine amino acid-rich histones in nucleosomes on modification by environmental agents form histone-DNA adducts, making it immunogenic. Alteration of DNA resulting from photomodification could lead to the development of antibodies or mutations to modified DNA. Lysine and arginine-rich histones in nucleosomes on modification by environmental agents form histone-DNA adducts, making it immunogenic. Alteration of DNA resulting from photomodification could lead to the development of antibodies or mutations to modified DNA. Therefore, the DNA-arginine photoadduct and modified photoadduct could have important implications in various pathophysiological conditions such as toxicology, carcinogenesis, and autoimmune phenomena.
Abbreviations: Arg: Arginine; SLE: systemic lupus erythematosus; UV: ultraviolet; Tm: thermal melting temperature; NO: nitric oxide; O2.⁻: superoxide anion.
Hyperammonaemia is a metabolic emergency and prompt treatment is paramount to optimize neurological outcome. Ammonia is extremely neurotoxic and increased levels can arise from an inherited or acquired defect in hepatic detoxification. Inborn errors of metabolism leading to hyperammonaemia mainly affect the hepatic urea cycle due to single enzyme deficiencies, transporter defects or mitochondrial dysfunction. Primary hyperammonaemia is a consequence of direct urea cycle dysfunction whereas secondary hyperammonaemia can result from disturbance of the urea cycle by toxic metabolites or substrate deficiencies. Immediate recognition and early initiation of specific treatment are of utmost importance. Prognostic factors include duration of hyperammonaemic coma and the extent of ammonia accumulation (Häberle et al. Orphanet J Rare Dis 7:32, 2012). The principles of management in the acute situation aim to rapidly remove ammonia, decrease production and replace rate limiting amino acids.
DOI: http://dx.doi.org/10.3329/bjch.v36i1.13039Bangladesh J Child Health 2012; Vol 36 (1): 46-48
Arginine is one of the amino acids produced in the human body by the digestion or hydrolysis of proteins. Arginine is derived from dietary intake, body protein breakdown, or endogenous de novo arginine production. The biochemical characterization of various DNA and polynucleotides modified with arginine has been studied in our laboratory by using UV spectroscopic analysis, and thermal denaturation studies of DNA–arginine adduct showed a decrease in melting temperature (Tm). A strong recognition of photoadducts was observed with anti-DNA autoantibodies found in the sera of systemic lupus erythematosus (SLE) patients. Two hundred base pair DNA–arginine and polydeoxyribonucleotode–arginine photoadduct appears to provide an immunodominant epitope for SLE autoantibody recognition. The result suggests the possible involvement of these photoadducts as a potential trigger for anti-DNA autoantibody production. Lysine- and arginine-rich histones in nucleosomes on modification by environmental agents form histone–DNA adducts, making it immunogenic. Alteration of DNA resulting from photomodification could lead to the development of antibodies or mutations to modified DNA. Therefore, the DNA–arginine photoadduct and modified photoadduct could have important implications in various pathophysiological conditions such as toxicology, carcinogenesis, and autoimmune phenomena.
Because of the constant turnover of proteins, protein-bound and free amino acids exist in a dynamic equilibrium. The intracellular pool of free amino acids, which is replenished by the hydrolysis of existing proteins, by uptake from the intercellular space and by de novo synthesis, is available for protein synthesis and for the many other metabolic processes dependent upon amino acids. The concentration of free amino acids is always lower than that of the protein-bound residues, one limiting factor being the strong osmotic effects of such low molecular weight compounds. Thus, there is no specific amino acid store in an organism; it is more the case that enzymes and structural proteins themselves represent the reserve of amino acids. Insect larvae, which require large amounts of amino acids for metamorphosis and sclerotization of the cuticula, make use of extracellular proteins as a source of amino acids; examples include the larval haemolymph proteins of some dipterans (p. 191) and possibly the haemoglobins of the non-biting midges (Chironomidae) (p. 273).
Die Aminosäuren besitzen im Stoffwechsel der Zelle 3 Funktionen:
1.
Sie stellen die 20 Bausteine bei der Biosynthese der Proteine. Über den Mechanismus und die Regulation des enzymatischen Aufbaus, die Proteinbiosynthese, wurde in Kap.9 (S.225) berichtet. Wie bei allen Körperbausteinen mit Ausnahme der Desoxyribonucleinsäuren besteht auch bei den Proteinen ein dynamisches Gleichgewicht zwischen Auf-und Abbau. Die Halbwertszeit der einzelnen Proteine schwankt zwischen Stunden bei einigen Enzymen, Tagen bis Wochen bei Plasmaproteinen und Monaten bei Strukturproteinen (z. B. Kollagen). Die Halbwertszeit wird auch durch das Organ, in dem das betreffende Protein zu finden ist, bestimmt: So beträgt die Halbwerts-zeit der Alanintransaminase, eines Enzyms des Aminosäurenstoffwechsels, in der Leber 3 Tage, während das Enzym in der Muskulatur erst nach 20 Tagen zur Hälfte durch neues Enzymprotein ersetzt ist. Während wir den Vorgang der Proteinbiosynthese schon in seinen Einzelheiten kennen, liegen über den Mechanismus und die Regulation des enzymatischen Abbaus der Proteine, die Proteolyse, bisher wesentlich weniger Erkenntnisse vor (S. 237).
2.
Sie wirken als Stickstoff- bzw. Aminogruppendonatoren bei der Biosynthese anderer stickstoffhaltiger Verbindungen.
3.
Sie spielen eine große Rolle bei der Glucosehomöostase. Da im Säugetiergewebe die Enzyme fehlen, die für die Biosynthese von Glucose aus geradzahligen Fettsäuren erforderlich sind, und die ungeradzahligen Fettsäuren, bei deren Abbau Propionyl-CoA — das zur Glucosesynthese herangezogen werden kann — entsteht, nur etwa 5% des Gesamtfettsäurenpools ausmachen, verbleiben für die Glucosebildung neben der Freisetzung aus Glykogen und Recyclisierung von Metaboliten, die in der Glykolyse bzw. Lipolyse (Lactat und Pyruvat bzw. Glycerin) entstehen, nur einzelne — als glucogen bezeichnete — Aminosäuren als Vorstufen.
Nitric oxide synthase produces NO, citrulline, water, and NADP at the expense of arginine, NADPH, and dioxygen. While citrulline has been considered to be an inert by-product of the high output inducible isoform of NO synthase (iNOS), we show here that immunostimulants induce a metabolic pathway in vascular smooth muscle cells, which enables them to regenerate arginine from citrulline. Regeneration of arginine from citrulline is accomplished by two urea cycle enzymes: argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). Whereas AL is constitutive to vascular smooth muscle cells, AS mRNA and enzyme activity is markedly induced in cells by treatment with bacterial lipopolysaccharide (LPS). The induction of AS mRNA and activity by LPS follows a time course which mirrors that for iNOS but lags 1-2 h behind. As shown for iNOS, interferon-gamma does not itself induce AS but is synergistic with LPS. AS induction is suppressed by glucocorticoids, actinomycin D, and, to a lesser extent, cycloheximide. On the other hand, AS induction is unaffected by an excess of citrulline or the inhibitor of iNOS, N(omega)-methyl-L-arginine. Our results show the urea cycle enzymes AS and AL confer cells with the capacity to produce NO without a need for exogenous arginine. In conjunction with NOS, citric acid cycle enzymes that covert fumarate to oxaloacetate (fumarase and malate dehydrogenase) and oxaloacetate to aspartate (aspartate transaminase), AS and AL form a novel arginine-citrulline cycle that enables high output NO production by cells.
Urea cycle disorders (UCDs) are inborn errors of liver metabolism that often result in hyperammonemia and failure of arginine synthesis due to the deficiencies of one of the six enzymes or two mitochondrial transporters involved. Despite current treatment options, including dietary therapy, stimulation of alternative routes of nitrogen disposal and cell therapies (hepatocyte and orthotopic liver transplantation), significant morbidity and mortality still remain. Gene therapy has emerged as an attractive alternative strategy for providing a definitive cure for patients with metabolic diseases. Successful phenotype correction in pre- clinical animal models is encouraging and research effort is increasingly being focused on translation from the laboratory bench to proof-of-concept human therapy. Gene therapy for UCDs must target the periportal hepatocytes, as these are the only liver cells that express all six enzymes required for full ammonia detoxification. This review explores current efforts to develop and translate liver-directed gene therapy approaches to UCDs caused by each of the defects of the six enzymes. The prominent issues that will need to be addressed and overcome for the future development of clinical trials, including alternatives to mouse mutants and to vectors that are not necessarily conditioned to rodents are also discussed.
Hepatocyte transplantation is an attractive process for replacing deficient cells in an anatomically normal liver. In metabolic liver diseases, cell therapy could be an interesting alternative to orthotopic liver transplantation. The replacement of a small percentage (5-10%) of deficient hepatocytes by normal hepatocytes could restore the metabolic defect at a long term. Data from clinical studies of hepatocyte autotransplantation or allotransplantation, genetically modified or not, provided poor results, insufficient cell engraftment in the liver parenchyma and, in the majority of cases, a transient therapeutic effect. The limited efficacy of hepatocyte transplantation in metabolic liver diseases is mainly due to the poor percentage of engrafted and finally functional hepatocytes. Numerous animal models have been developed in order to study the factors that could increase the number and the percentage of transplanted and engrafted hepatocytes. However, the majority of these models cannot be used in patients since they present important risks for them. The aim of this work was to evaluate less invasive procedures for inducing liver regeneration and significant hepatocyte engraftment in order to develop a new approach of transplantation of ex vivo genetically modified hepatocytes for the treatment of familial hypercholesterolemia. The effect of reversible portal vein embolization (PVE) on liver regeneration and hepatocyte proleferation was evaluated in monkeys. In contrast to PVE by a permanent embolizing agent, reversible PVE has not a long term deleterious effect on embolized liver. A more complete venous occlusion was obtained by using the powdered form of an absorbable gelatin sponge (Curaspon®). We showed for the first time in the literature the safe and successful use of reversible PVE for inducing significant hepatocyte proliferation and liver regeneration. Our data support that an initial occlusion of the portal branch, even if not permanent, is sufficient to start the mechanisms of liver regeneration in the contralateral lobe. Embolization with Curaspon® powder could be considered to be the ultimate form of embolization: very distal, reversible and lasting sufficiently in order to induce substantial liver hypertrophy. Our findings suggest that this method could reliably be used for clinical purposes, particularly in situations in which short-term regeneration is required (i.e. multi-step management of hepatic malignancies) or in cases where resection of the liver is not finally necessary, such as in hepatocyte transplantation for the treatment of metabolic liver diseases. These promising results on reversible PVE allowed us to evaluate this approach in our preclinical study of gene therapy for the treatment of familial hypercholesterolemia in macaques. Our protocol consisted of an autotransplantation of ex vivo genetically modified hepatocytes by a lentiviral vector. We showed that reversible PVE induces liver regeneration of the non-embolized liver segments and improves considerably hepatocyte transplantation of genetically modified cells expressing Green Fluorescent Protein (GFP). Sixteen weeks after transplantation, transduced engrafted hepatocytes expressed the transgene, which was under control of the human apo-AII promoter. Our protocol showed for the first time in a big animal that PVE by an absorbable agent leads safely to an important and long-term repopulation of the liver by lentivirally transduced hepatocytes. The extremely encouraging results of this work opened our way advancing in our preclinical study and preparing a phase I/II clinical trial for the treatment of familial hypercholesterolemia based on our protocol of autotransplantation of ex vivo genetically modified hepatocytes by a lentiviral vector.
Citrullinemia type I is an urea cycle defect caused by mutations in the argininosuccinate synthetase (ASS1) gene. We report a novel argininosuccinate synthetase gene mutation in a Korean family with type I citrullinemia. Metabolic evaluation revealed significant hyperammonemia. Amino acid/acylcarnitine screening using tandem mass spectrometry showed high level of citrulline. Plasma amino acid analysis showed high level of citrulline and the urine organic acid analysis showed makedly increased level of orotic acid. To confirm diagnosis of citrullinemia we did mutation analysis of the ASS1 gene. The patient was found to have mutations of c.689G>C (p.G230A) and c.892G>A (p.E298K), which were new types of argininosuccinate synthetase gene mutation have never been reported in Korea. We report a novel case of argininosuccinate synthetase 1 gene mutation and suggest that the gene study to the family members is necessary to carry out when a patient is diagnosed as citrullinemia.
L-arginine is a nutritionally important amino acid that controls a wide spectrum of cellular functions and physiological processes, acting by itself or through its various metabolites. There are several factors that determine overall L-arginine homeostasis: dietary supplementation, endogenous de novo synthesis, whole-body protein turnover and its extensive metabolism. The destiny of L-arginine is determined by the complex network of enzymes and pathways differentially expressed according to health and disease status. Diabetes is characterized by reduced concentrations of L-arginine in plasma and many tissues, and failure of its metabolic effects. Emerging data suggest that oral supplementation of L-arginine exerts multiple beneficial effects on the complex etiological and pathophysiological basis of diabetes including: i) β-cell function and mass and ii) obesity and peripheral insulin resistance. This review emphasizes important aspects of L-arginine action which classifies this amino acid as a promising therapeutic approach in the treatment of diabetes.
The citrulline-nitric oxide (NO) cycle, comprised of the enzymes argininosuccinate synthase (AS), argininosuccinate lyase (AL) and endothelial nitric oxide synthase (eNOS), is responsible for the regulated production of endothelial NO. Although most studies have focused on eNOS to uncover important regulatory mechanisms, we and others have determined that AS is an essential and regulated step in endothelial NO production. AS is rate limiting for endothelial NO production and is the primary source of arginine, the substrate for eNOS-mediated NO production, despite saturating intracellular levels of arginine and available arginine transport systems. AS is essential for endothelial cell viability and its expression is regulated coordinately with eNOS by TNF and thiazolidenediones with concomitant effects on NO production. Given the importance of AS for endothelial health, we explored three independent regulatory mechanisms.
In Chapter One, the functional consequences of altered AS expression due to overexpression, insulin, VEGF and ceramide were studied. We demonstrated that overexpression of AS leads to enhanced NO production and that insulin, VEGF and ceramide coordinately regulate the expression of AS and eNOS. In Chapter Two, the first post-translational modifications of AS in the endothelium were characterized. We determined that AS is an endogenous phosphoprotein in the endothelium, described several levels of biological significance of AS phosphorylation, identified 7 sites of AS phosphorylation and began to uncover the direct impact of phosphorylation on AS function. Finally, in Chapter Three, endothelial AS subcellular localization was defined and important protein interactions were identified including caveolin-1 and HSP90.
The work presented in this dissertation demonstrates that multiple mechanisms regulate the function of AS, often coordinately with eNOS, and have a direct impact on nitric oxide production. Our findings suggest that the global understanding of the citrulline-NO cycle as a metabolic unit will unravel new paradigms that will re-define our understanding of the regulation of vascular function by NO.
Adult onset type II citrullinemia is an autosomal recessive disorder of the amino acid metabolism caused by a deficiency of liver specific argininosuccinate synthetase activity. This disease can occur at any age in life with recurrent episodes of neurological signs and symptoms such as disorientation, abnormal behaviors (aggression, irritability and hyperactivity), seizures, coma and potential death from brain edema, which are resulted from hyperammonemia. We should consider this rare metabolic disease for the adult patient who exhibits mental change and hyperammonemia without liver or brain disease. Recently. SLC25A13 gene, encoding the mitochondrial aspartate glutamate carrier protein named citrin, is demonstrated to be responsible for adult onset type II citrullinemia. We experienced a 39-year-old female who suffered from generalized weakness, dizziness and lethargy, and diagnosed as adult onset type II citrullinemia by highly elevated plasma citrulline and ammonia and the SLC25A13 gene mutation. We report here on this unusual case of adult onset type II citrullulinemia with a brief review of the related literature.
We describe the hepatopancreas arginase activity of freshwater prawn (Macrobrachium rosenbergii). The enzyme was isolated using reactive blue 2-agarose affinity chromatography and gel filtration on Sephadex G-150. The enzyme had a specific activity of 5.70 µmol/min/mg of protein. The enzyme exhibited a maximal activity at pH 8.5 and Km of 12.5 mM. The enzyme was capable of hydrolysing L-arginine and to a lesser extent, L-arginine monohydrochlorate and L-arginine monohydrate. The optimum temperature of the enzyme was 35 0 C. The molecular weight as determined by gel filtration was approximately 160,000 dalton and SDS-PAGE, was 22,000 dalton. The different amino acids (L-lysine, L-cysteine, L-valine, L-proline, L-aspartic acid, L-glutamic acid and L-serine) and metal ions (Ni 2+ , Co 2+ , Zn 2+ , Mn 2+ and Mg 2+) did not show any inhibition on the enzyme activity. The enzyme was activated with Mn 2+ and different concentration of Mn 2+ had no effect on the enzyme activity. EDTA, citrate and urea showed considerable inhibition on the enzyme activity. INTRODUCTION Arginase (L-arginine ureahydrolase, or amidinohydrolase, EC 3.5.3.1) is the terminal enzyme of the urea cycle among the six enzymes [1]. The enzyme was found to exist in two forms and has a broad tissue distribution [1,2]. The arginase type I form is highly expressed in the liver or hepatic cells and is important in ureogenesis. Extra-hepatic arginase type II form is thought to be involved in the biosynthesis of polyamines, the amino acids ornithine, proline and glutamate and in the inflammatory process [1]. The function of arginase in microbes and invertebrates is mostly unknown. It is speculated that the urea cycle evolved from a biosynthetic pathway for L-arginine and appeared for the first time in amphibians as an adaptation to air-breathing in a terrestrial environment [3]. Many invertebrates are uricotelic organisms and eliminate excess nitrogen in the form of solid uric acid [1]. Nevertheless, a large number of uricotelic organisms possess arginase. It is thought that their generation of L-ornithine by arginase feeds into the production of L-proline, L-glutamate and polyamines used for collagen synthesis, energy metabolism and cell proliferation [1]. Nitrogen excretion had been studied extensively in vertebrates while less is known for invertebrates. Insects and many other invertebrates independently evolved the ability to excrete ammonium ions as uric acid. Such organisms are collectively designated uricotelic. Spiders and other arachnids also use the purine biosynthetic pathway, but stop at guanine as the disposal product [1]. Most studies have concentrated on mammalian liver arginases [1, 4]. In addition to urea synthesis in the liver of ureotelic species, arginase is also involved in biosynthesis of polyamines and proline [5], conversion of arginine into α-ketoglutarate for oxidation in the Krebs cycle [6], adaptive responses to anoxia in some invertebrates [7] and production of urea for osmoregulatory purposes [8]. It should be noted that arginase is found in various tissues of non-ureotelic organisms, including liver, but is not part of a functional urea cycle [9]. It had previously been thought that there were significant kinetic and structural differences between ureotelic and non-ureotelic arginases [10], but many studies suggested that the characteristics of arginases are not consistent with a particular mode of nitrotelism [11, 12]. Non-ureotelic arginases are generally similar to ureotelic arginases but can be distinguished immunologically [12,13]. In uricotelic organisms, that include bacteria, fungi, invertebrates, reptiles and birds [1, 14,] and
It is worthy to supplement Charney with two historical issues: (1) There were two rival trends in the rebirth of genetic thought in the 1960s: the universal and the variation related. This traditional duality suggested that heredity cannot be equated with genetic determinism. (2) The classical debates and reinterpretation of adoption/twin studies in the 1980s regarding intelligence suggested that the environment had a more active role in unfolding the genetic program.
Traditional lay perceptions of genetics are plagued with essentialist biases leading to some unfortunate consequences. Changes in the scientific understanding of heredity in general, and in genotype-phenotype relationships more specifically, provide a vital basis for shifting public understanding of genetics. Facilitating postgenomic literacy among the public has the potential to have translational implications in diminishing deleterious attitudes, beliefs, and behaviors.
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