Uptake Of L-cysteine Research Articles

Fuzzy optimization for identifying antiviral targets for treating SARS-CoV-2 infection in the heart

In this paper, a fuzzy hierarchical optimization framework is proposed for identifying potential antiviral targets for treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the heart. The proposed framework comprises four objectives for evaluating the elimination of viral biomass growth and the minimization of side effects during treatment. In the application of the framework, Dulbecco’s modified eagle medium (DMEM) and Ham’s medium were used as uptake nutrients on an antiviral target discovery platform. The prediction results from the framework reveal that most of the antiviral enzymes in the aforementioned media are involved in fatty acid metabolism and amino acid metabolism. However, six enzymes involved in cholesterol biosynthesis in Ham’s medium and three enzymes involved in glycolysis in DMEM are unable to eliminate the growth of the SARS-CoV-2 biomass. Three enzymes involved in glycolysis, namely BPGM, GAPDH, and ENO1, in DMEM combine with the supplemental uptake of L-cysteine to increase the cell viability grade and metabolic deviation grade. Moreover, six enzymes involved in cholesterol biosynthesis reduce and fail to reduce viral biomass growth in a culture medium if a cholesterol uptake reaction does not occur and occurs in this medium, respectively.

Redox status in erythrocyte of Northeast Indians suffering from chronic liver disease (CLD)

Chronic liver disease is a hepato-necrotic disease persisting for more than six months. The objective of the study was to assess the redox process in chronic liver disease (CLD). The Redox process has been recognized as a significant regulatory system in various biological processes like cell proliferation, signal transduction and gene expression etc. Glutathione plays a crucial role by acting as a regulatory molecule in the immune system. Here a study has been conducted on 57 indoor patients suffering from CLD and 57 healthy controls to observe the redox potential in the erythrocyte by estimating reduced glutathione and L-cysteine levels. Liver cirrhosis causes a decreased level of glutathione in plasma and hepatic cells due to impaired uptake of L-Cysteine by RBC. Therefore we conclude that L-cysteine uptake by RBC is impaired in CLD which causes the vicious circle of oxidative stress. Keywords: CLD, Redox process, Liver cirrhosis, Reduced glutathione, L-cysteine, RBC.

Dendrimer-mediated delivery of N-acetyl cysteine to microglia in a mouse model of Rett syndrome

BackgroundRett syndrome (RTT) is a pervasive developmental disorder that is progressive and has no effective cure. Immune dysregulation, oxidative stress, and excess glutamate in the brain mediated by glial dysfunction have been implicated in the pathogenesis and worsening of symptoms of RTT. In this study, we investigated a new nanotherapeutic approach to target glia for attenuation of brain inflammation/injury both in vitro and in vivo using a Mecp2-null mouse model of Rett syndrome.MethodsTo determine whether inflammation and immune dysregulation were potential targets for dendrimer-based therapeutics in RTT, we assessed the immune response of primary glial cells from Mecp2-null and wild-type (WT) mice to LPS. Using dendrimers that intrinsically target activated microglia and astrocytes, we studied N-acetyl cysteine (NAC) and dendrimer-conjugated N-acetyl cysteine (D-NAC) effects on inflammatory cytokines by PCR and multiplex assay in WT vs Mecp2-null glia. Since the cysteine-glutamate antiporter (Xc−) is upregulated in Mecp2-null glia when compared to WT, the role of Xc− in the uptake of NAC and l-cysteine into the cell was compared to that of D-NAC using BV2 cells in vitro. We then assessed the ability of D-NAC given systemically twice weekly to Mecp2-null mice to improve behavioral phenotype and lifespan.ResultsWe demonstrated that the mixed glia derived from Mecp2-null mice have an exaggerated inflammatory and oxidative stress response to LPS stimulation when compared to WT glia. Expression of Xc− was significantly upregulated in the Mecp2-null glia when compared to WT and was further increased in the presence of LPS stimulation. Unlike NAC, D-NAC bypasses the Xc− for cell uptake, increasing intracellular GSH levels while preventing extracellular glutamate release and excitotoxicity. Systemically administered dendrimers were localized in microglia in Mecp2-null mice, but not in age-matched WT littermates. Treatment with D-NAC significantly improved behavioral outcomes in Mecp2-null mice, but not survival.ConclusionsThese results suggest that delivery of drugs using dendrimer nanodevices offers a potential strategy for targeting glia and modulating oxidative stress and immune responses in RTT.

Altered intestinal transport of amino acids in cirrhotic rats: the effect of insulin-like growth factor-I.

The intestine is an important target organ for insulin-like growth factor-I (IGF-I), an anabolic hormone synthesized in the liver upon growth hormone (GH) stimulation. Levels of IGF-I are reduced in cirrhosis, and altered GH/IGF-I axis may contribute to malnutrition in cirrhotic patients. Our aim was to study Na(+)-dependent jejunal transport of amino acids (L-leucine, L-proline, L-glutamic acid, and L-cysteine) in cirrhotic rats and to analyze the effect of IGF-I on this function. IGF-I or saline was administered for 2 wk to rats with CCl(4)-induced cirrhosis and saline was administered to healthy control rats. Transport of amino acids was assessed in brush-border membrane vesicles (BBMV) using (14)C- or (35)S-labeled amino acids, and the kinetic constants V(max) and K(t) were determined. Na(+)-independent uptake of L-leucine, L-proline, L-glutamic acid, and L-cysteine by BBMV was similar in all groups. Na(+)-dependent uptake of all four amino acids was significantly diminished in cirrhotic rats compared with both controls and IGF-I-treated cirrhotic rats. The latter two groups exhibited similar V(max) and K(t), whereas untreated cirrhotic rats had reduced V(max) and increased K(t) compared with normal controls and IGF-I-treated cirrhotic animals. In conclusion, the transport of all four tested amino acids by BBMV is impaired in cirrhotic rats, and low doses of IGF-I can correct this defect.

Enantioselective uptake of amino acid with overoxidized polypyrrole colloid templated with L-lactate.

An overoxidized polypyrrole colloid, which can recognise enantiomers of amino acids has been prepared by a newly developed molecular imprinting technique. A polypyrrole colloid, which had been polymerised from a mixture of pyrrole (monomer), polyvinylpyrrolidone (steric stabiliser), peroxodisulfate (oxidant) and L-lactate (dopant), was overoxidized to create a dopant-complementary cavity. The enantioselectivity of the overoxidized colloid was evaluated by comparing the uptake of L-alanine and L-cysteine with that of the respective D-enantiomers. The L/D uptake ratios for these amino acids were about 2, while phenylalanine showed suppressed uptake for both the enantiomers. The absence of phenylalanine uptake can be explained in terms of the molecular size, which is too large to be accommodated by the cavity created by L-lactate. In contrast, a colloid templated with L-phenyllactate took up L-phenylalanine with a higher enantioselectivity of about 7. A colloid templated with L-lactate was applied to surface chirality analysis through enantioselective adsorption on cysteine-modified gold surfaces. Quartz microbalance experiments and scanning electron microscope observation of the gold surface revealed that the colloidal particle has higher affinity to a surface modified with L-cysteine than to one modified with D-cysteine.

Elevation of Endogenous Nucleophiles in Rat Lung by Cysteine and Glutathione Esters In Vitro

In this study, we have compared the uptake of l-cysteine ( l-CySH), d-cysteine ( d-CySH), l-cysteine isopropyl ester ( l-CIPE) and d-cysteine isopropyl ester ( d-CIPE) in rat lung slices and tracheal sections and determined the effectiveness of glutathione (GSH), GSH isopropyl monoester, GSH isopropyl diester, γ-glutamylcysteine (γ-glu-cys) isopropyl monoester and γ-glu-cys isopropyl diester to elevate and prolong intracellular GSH concentrations in rat lung slices. Lung slices were incubated with 1.0 mM of thiol and the concentrations determined intracellularly and extracellularly with time. Slices incubated with GSH, GSH isopropyl diester and γ-glu-cys isopropyl diester had cellular GSH concentrations increased by up to 60%, 95% and 58%, respectively, whereas GSH isopropyl monoester and γ-glu-cys isopropyl monoester did not increase the intracellular GSH concentration. Extracellularly, the GSH concentration had decreased by 15%, GSH isopropyl diester by 27%, γ-glu-cys isopropyl diester by 66% and both isopropyl monoesters by over 90% at 120 min. Lung slices and tracheal sections incubated with l- or d-CySH at 37° had increased cellular concentrations of l- and d-CySH which ranged between 0.88–1.25 nmol mg −1 and 1.35–2.25 nmol mg −1, respectively. Reducing the incubation temperature to 4° had little effect on the accumulation of d-CySH; however, l-CySH concentrations increased progressively in the trachea and lung to reach 2.73 and 2.63 nmol mg −1 at 90 min, respectively. Lung slices incubated with l- or d-CIPE had increased l- or d-CySH concentrations up to a max of 13.7 and 11.1 nmol mg −1 and tracheal sections up to a max of 5.56 and 11.09 nmol mg −1. In the lung slice medium, l- and d-CIPE levels had decreased by 75.2% and 74.0% at 90 min, respectively, and from the tracheal section medium, l- and d-CIPE concentrations had decreased by 66.7% and 32.7%, respectively. Preincubation of lung slices and tracheal sections with the carboxylesterase inhibitor, bis ( p-nitrophenyl) phosphate (BNPP), almost completely prevented the disappearance of l- and d-CIPE extracellularly and greatly reduced the appearance of cellular l- and d-CySH. GSH, GSH isopropyl diester and γ-glu-cys isopropyl diester elevated and prolonged GSH concentrations in rat lung slices, but GSH isopropyl monoester and γ-glu-cys isopropyl monoester did not increase GSH levels. The uptake of l-CySH, but not d-CySH, is temperature sensitive in rat lung slices and tracheal sections and carboxylesterases appear to have a major influence on the uptake and metabolism of l- and d-CIPE by rat lung slices and tracheal sections.

Increased uptake of l-cysteine and l-cystine by nerve growth factor in rat pheochromocytoma cells

Nerve growth factor is a neurotrophic factor which promotes cell survival and differentiation in the central and peripheral nervous system. The rat pheochromocytoma (PC12) cell has been frequently used to study the actions of nerve growth factor (NGF). Our previous studies demonstrate that pretreatment with NGF for 24 h protects PC12 cells from oxidative stress by increasing glutathione (GSH) concentrations and the activity of γ-glutamylcysteine synthetase, which is a rate-limiting enzyme in GSH synthesis. The synthesis of intracellular GSH is dependent on the availability of the precursor amino acid, l-cysteine. Cells take up l-cystine from the extracellular fluid and convert it to l-Cysteine intracellularly. l-Cysteine is then released from cells to maintain extracellular l-cysteine. Here we report that NGF increased the uptake of l-cysteine or l-cystine. The increased concentrations of l-Cysteine or l-cystine by NGF was responsible for the enhanced intracellular GSH concentrations. The increased GSH and l-cysteine concentrations by NGF also served as intracellular antioxidants. The protection of PC12 cells by NGF from oxidative stress was due to the stimulated increased levels of intracellular glutathione and l-cysteine or l-cystine.

Comparative study of the uptake of L-cysteine and L-cystine in the renal proximal tubule

1. The transport mechanisms of L-cysteine and L-cystine by luminal membrane vesicles isolated from either the proximal convoluted part (pars convoluta) or the proximal straight part (pars recta) of rabbit proximal tubuli were examined. 2. The uptake of L-cysteine in pars convoluta is characterized by a single sodium-dependent transport system (Km = 0.58 mM), whereas the sodiumdependent influx of L-cysteine in pars recta proceeds via transport systems with different affinities (Km1 = 0.03 mM, Km2 = 5.84 mM). An H(+)-gradient enhanced the uptake of L-cysteine in pars recta and only in the presence of a Na(+)-gradient. 3. The presence of a Na(+) gradient stimulated the influx of L-cystine in both parts of the nephron, but to a lesser extent than for L-cysteine. In addition a considerable amount of binding of L-cystine to membrane vesicles was observed in both pars convoluta and pars recta. 4. Stoichiometric studies indicated a coupling ratio of 1 Na(+): 1 amino acid for the transport components involved in the uptake of L-cysteine and L-cystine along the proximal tubule. 5. Competition experiments demonstrated that neutralα-amino acids inhibited the influx of L-cysteine in the proximal tubule. Basic and acidic amino acids had no effect on uptake of L-cysteine in pars convoluta, whereas a slight inhibitory effect of L-lysine and L-arginine was noted in pars recta. 6. Both basic and neutral amino acids, but not L-glutamate inhibited the uptake of L-cystine in pars convoluta. This was in general also the case in pars recta. However, addition of L-proline did not influenced the uptake of L-cystine, and L-phenylalanine, L-asparagine, L-glutamine, L-leucine and L-methionine only inhibited at a high concentration (5 mM).

Amino acid transport and glutathione homeostasis: What is the mechanism for cysteine uptake from bile?

Transport of L-cysteine and a cysteine S-conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was investigated in rat liver canalicular plasma membrane (cLPM) vesicles. Cysteine uptake into an osmotically active intravesicular space was temperature sensitive and further enhanced by an inwardly directed Na+ gradient. Na+ -dependent and -independent L-cysteine uptake exhibited saturation kinetics with apparent K m of 53 ± 0.7 and 1300 ± 300 μM and Vmax of 95 ± 21 and 1600 ± 200 pmol ± mg protein−1; 10 sec−1 for the Na+ dependent components, and an apparent K m of 207 ± 48 μM and a Vmax of 355 ± 71 pmol ± mg protein−1 ± 10 sec−1 for the Na+ -independent component. Na+-dependent uptake was inhibited by L-alanine, glycine, L-phenylalanine and L-leucine, whereas Na+-independent uptake was inhibited by L-phenylalanine, L-leucine and 2-amino-2-norbornanecarboxylic acid. Both Na+ -dependent and -independent L-cysteine transport processes were inhibited by several cysteine S-conjugates, with DCVC having the strongest effect. Inhibition of [35S]-cysteine uptake by DCVC was noncompetitive with a K, of 1.2 ± 0.1 mM. On the other hand, uptake of [35 S IDCVC by the rat cLPM vesicles was not stimulated by a Na+-gradient, but was inhibited by several other amino acids, including L-cysteine. Further investigation of [35SlDCVC uptake in rat cLPM vesicles indicated a saturable Na+ -independent process with an apparent K, of 155 f 42 μM, and a V max of 393 f 53 pmol mg protein −1 5 sec −1. Time-dependent nonspecific binding of DCVC to rat cLPM vesicles was diminished by addition of aminooxyacetic acid, an inhibitor of cysteine conjugate β-lyase activity. These findings demonstrate that cysteine and DCVC uptake into cLPM vesicles occurs by both saturable and nonsaturable transport mechanisms. These transport pathways may function in vivo for uptake and recycling of biliary L-cysteine for glutathione turnover and some cysteine S-conjugates for intrahepatic mercapturic acid biosynthesis.

Amino acid transport and glutathione homeostasis: What is the mechanism for cysteine uptake from bile?

Transport of L-cysteine and a cysteine S-conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was investigated in rat liver canalicular plasma membrane (cLPM) vesicles. Cysteine uptake into an osmotically active intravesicular space was temperature sensitive and further enhanced by an inwardly directed Na+ gradient. Na+ -dependent and -independent L-cysteine uptake exhibited saturation kinetics with apparent Km of 53 ± 0.7 and 1300 ± 300 μM and Vmax of 95 ± 21 and 1600 ± 200 pmol ± mg protein−1; 10 sec−1 for the Na+ dependent components, and an apparent Km of 207 ± 48 μM and a Vmax of 355 ± 71 pmol ± mg protein−1 ± 10 sec−1 for the Na+ -independent component. Na+-dependent uptake was inhibited by L-alanine, glycine, L-phenylalanine and L-leucine, whereas Na+-independent uptake was inhibited by L-phenylalanine, L-leucine and 2-amino-2-norbornanecarboxylic acid. Both Na+ -dependent and -independent L-cysteine transport processes were inhibited by several cysteine S-conjugates, with DCVC having the strongest effect. Inhibition of [35S]-cysteine uptake by DCVC was noncompetitive with a K, of 1.2 ± 0.1 mM. On the other hand, uptake of [35 S IDCVC by the rat cLPM vesicles was not stimulated by a Na+-gradient, but was inhibited by several other amino acids, including L-cysteine. Further investigation of [35SlDCVC uptake in rat cLPM vesicles indicated a saturable Na+ -independent process with an apparent K, of 155 f 42 μM, and a Vmax of 393 f 53 pmol mg protein −1 5 sec −1. Time-dependent nonspecific binding of DCVC to rat cLPM vesicles was diminished by addition of aminooxyacetic acid, an inhibitor of cysteine conjugate β-lyase activity. These findings demonstrate that cysteine and DCVC uptake into cLPM vesicles occurs by both saturable and nonsaturable transport mechanisms. These transport pathways may function in vivo for uptake and recycling of biliary L-cysteine for glutathione turnover and some cysteine S-conjugates for intrahepatic mercapturic acid biosynthesis.

Poly(A)+ RNA from rabbit intestinal mucosa induces b0,+ and y+ amino acid transport activities in Xenopus laevis oocytes.

Injection of poly(A)+ RNA (mRNA) isolated from rabbit intestinal mucosa into Xenopus laevis oocytes results in an increase in sodium-independent uptake of L-[3H]leucine, L-[35S]cystine, and L-[3H]arginine. This uptake activity is related to an mRNA species corresponding to the recently isolated rabbit kidney cortex cDNA clone rBAT (related to b0,+ amino acid transporter; Bertran, J., Werner, A., Stange, G., Markovich, D., Moore, M. L., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 281, 717-723) and to a protein involved in amino acid transport via system y+. This conclusion is based on the following observations: 1) mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, induces sodium-independent uptake of L-leucine, L-cystine, and L-arginine. 2) In Northern blot analysis, mRNA isolated from mucosa of duodenum, jejunum, and ileum, but not from colon, hybridizes to an rBAT cDNA probe, with signals of 2.2-2.3 kilobases and 3.7-3.9 kilobases. 3) mRNA isolated from mucosa of jejunum induces sodium-independent uptake of L-leucine and L-cysteine which shows an inhibition pattern corresponding to system b0,+; the inhibition pattern of mRNA-induced uptake of L-arginine is compatible with the contribution of system b0,+ and y+. 4) Hybrid depletion with an rBAT antisense oligonucleotide greatly prevents the mRNA-dependent induction of uptake of L-cystine (greater than 90%) and of L-leucine (approximately 75%); it reduces to about 50% the induction of L-arginine uptake. 5) After separation of mRNA on a sucrose density gradient, the fractions resulting in expression of b0,+ transport activity were also those hybridizing with rBAT cDNA; induction of transport activity from these fractions was also sensitive to hybrid depletion. 6) The mRNA-induced component of L-arginine uptake which is resistant to rBAT hybrid depletion is inhibited by L-homoserine, only in the presence of sodium; thus, it is related to a system y(+)-like activity.

Growth and Hydrogen Sulfide Emission of Photoheterotrophic Tobacco Cells Supplied with L-cysteine as Sole Sulfur Source

Summary Growth of photoheterotrophic tobacco cells ( Nicotiana tabacum L. var. «Samsun») in suspension cultures is inhibited in the presence of 0.8 mM L-cysteine as sole sulfur source, when inoculated at densities up to 9.5 mg dry weight. Under these conditions, hydrogen sulfide is emitted in amounts increasing with increasing inoculation densities. An inoculum of 19 mg dry weight, however, enables a moderate growth of the cells (doubling time 178 h) while reducing the emission of hydrogen sulfide. Feeding of 0.4 mM L-leucine to the suspensions did not only reduce hydrogen sulfide emission at all inoculation densities applied; it also caused exponential growth (doubling time 110 h) at inoculation densities of 9.5 mg and upwards. Initial and final rate of growth of tobacco cells supplied with L-cysteine as sulfur source increased, hydrogen sulfide emission decreased with increasing L-leucine concentrations in the medium. These effects are not specific for L-leucine, but are also observed when L-isoleucine, L-threonine, or Lvaline were added to the suspensions. Support of growth and suppression of hydrogen sulfide emission by L-leucine is accompanied by a reduction of the initial as well as the final rate of uptake of L-cysteine by the cells.

Disulfide reduction and sulfhydryl uptake by Streptococcus mutans.

Incubation of Streptococcus mutans cells with certain disulfide compounds resulted in accumulation of reduced sulfhydryl compounds in the extracellular medium or in both the medium and the cells. Oxidized lipoic acid and lipoamide competed for reduction. At high concentrations, these compounds were reduced at rates comparable to that of glucose metabolism, and all of the increase in sulfhydryls was in the medium. Cystamine did not compete with these compounds for reduction but was also reduced at high rates and low apparent affinity, and all of the cysteamine produced from cystamine accumulated in the medium. In contrast, glutathione disulfide (GSSG) and L-cystine were reduced slowly but with high apparent affinity, and 60 to 80% of the increase in sulfhydryls was intracellular. NADH-dependent lipoic acid or lipoamide reductase activity was present in the particulate (wall-plus-membrane) fraction, whereas NADPH-dependent GSSG reductase activity was present in the soluble (cytoplasmic) fraction. Two transport systems for disulfide and sulfhydryl compounds were distinguished. GSSG, L-cystine, and reduced glutathione competed for uptake. L-Cysteine was taken up by a separate system that also accepted L-penicillamine and D-cysteine as substrates. Uptake of glutathione or L-cysteine, or the uptake and reduction of GSSG or L-cystine, resulted in up to a 10-fold increase in cell sulfhydryl content that raised intracellular concentrations to between 30 and 40 mM. These reductase and transport systems enable S. mutans cells to create a reducing environment in both the extracellular medium and the cytoplasm.

The transport of alanine, serine, and cysteine in cultured human fibroblasts.

The transport of L-alanine, L-serine, and L-cysteine has been studied in skin-derived diploid human fibroblasts in culture. Competition analysis, mathematical discrimination by nonlinear regression, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of these amino acids. All the adopted criteria yielded results showing that L-alanine, L-serine, and L-cysteine enter the cell by two Na+-dependent systems, System A and System ASC, and by a Na+-independent route, whose major component has been identified as System L. The apparent affinity of L-alanine, L-serine, and L-cysteine for the putative carrier was higher for System ASC than for System A. The transport Vmax for System A increased in response to cell starvation; after 12 h, its values were similar or higher than those exhibited by System ASC. At amino acid concentrations approaching those present in human plasma, System ASC appeared to be the primary mediation for the inward transport of L-alanine, L-serine, and L-cysteine in human fibroblasts. The contribution of System A was negligible in nonstarved cells and became appreciable under conditions of cell starvation. The Na+-independent System L made no substantial contribution to the uptake of L-alanine and L-serine and accounted for approximately one-fourth of the total uptake of L-cysteine.

Characteristics of an amino acid transport system in rat liver for glutamine, asparagine, histidine, and closely related analogs.

In the rat hepatocyte, whether freshly separated or in primary culture, we do not find L-glutamine entry by Systems A and ASC as seen in cells previously studied. Instead the mediated entry of glutamine appears to occur exclusively by a Na+-dependent system ("N") apparently specific to amino acid amides and L-histidine; however, a portion of asparagine uptake occurs by System A. The simplest evidence for the separateness of the added system is the failure of model substrates for System A (e.g. N-methylalanine) to inhibit glutamine uptake significantly, and the failure of glutamine to inhibit the uptake of L-cysteine, model substrate for System ASC, at least in this cell. As is the case for cysteine, glutamine inhibits transport by System A (although not competitively), even though showing no transport by that system. Our finding confirms an earlier inference that glutamine uptake by this cell may follow a route not taken by alanine or serine, and explains the apparently erroneous companion inference that glutamine also shares a route with these two amino acids. Its uptake has now been characterized to show a series of differences from Systems A and ASC. Especially significant in view of the importance of glutamine metabolism are an insensitivity of the new system to stimulation by either insulin or glucagon, and its distinct enhancement (not as large as that for System A) on starvation of the cells with respect to amino acids. Hence, a second system has been found to show adaptive regulation.