Addition Of L-cysteine Research Articles

Purification and characterization of a NAD +-dependent sorbitol dehydrogenase from Japanese pear fruit

NAD +-dependent sorbitol dehydrogenase NAD-SDH, EC 1.1.1.14) from Japanese pear fruit was purified to apparent homogeneity (single band by SDS-PAGE with silver staining), and had a specific activity of 916.7 nKatal/mg protein. The molecular of the native enzyme was calculated to be 160 kDa by gel filtration, whereas SDS-PAGE gave a subunit size of 40 kDa, indicating that the native enzyme is a homotetramer. The protein immunologically reacted with an antibody raised in rabbit against the fusion protein expressed in E. coli harboring an apple NAD-SDH cDNA. The K m values for sorbitol and fructose were 96.4±8.60 and 4239±33.5 mM, respectively, and optimum pH for sorbitol oxidation was 9.0 and 7.0 for fructose reduction. Pear NAD-SDH had a very narrow substrate specificity, that is, sorbitol, L-iditol, xylitol and L-threitol were oxidized but not any of the other alcohols tested. These data suggest the structural importance of an S configuration at C-2 and an R configuration at C-4 in the substrate(s). Its enzymatic activity was strongly inhibited both by heavy metal ions such as mercury, and by thiol compounds, such as L-cysteine. However, the addition of zinc ion reversed the enzyme inactivation caused by addition of L-cysteine.

Taenia solium: A Cysteine Protease Secreted by Metacestodes Depletes Human CD4 Lymphocytes in Vitro

Molinari, J. L., Mejia, H., White, A. C. Jr., Garrido, E., Borgonio, V. M., Baig, S., and Tato, P., 2000. Taenia solium: A cysteine protease secreted by metacestodes depletes human CD4 Lymphocytes in Vitro. Experimental Parasitology94, 133–142. Excreted/secreted products from Taenia solium metacestodes cultured in vitro were analyzed for peptidase activity using peptide substrates Z-Phe-Arg-AFC, Arg-AFC, and Z-Gly-Gly-Arg-AFC and zymography studies. Specific inhibitor profiles revealed mainly cysteine and metalloprotease activities. Hydrolysis of substrate Z-Phe-Arg-AFC was augmented by the addition of L-cysteine and acid pH, consistent with cysteine protease activity. Cysteine protease activity was more prominent in supernatants from living metacestodes cultured in PBS than in either RPMI or RPMI plus fetal calf serum and was proportional to the number of metacestodes. Flow cytometry analysis showed depletion of human T lymphocytes cultured with living T. solium metacestodes. CD4+ expression was significantly decreased when metacestode E/S products and L-cysteine were added to lymphocyte cultures (P = 0.027). This peptidase activity was inhibited by E-64 indicating that the depletion of CD4+ cells was due to cysteine protease activity. Thus, T. solium metacestodes produce excretory/secretory proteases. These enzymes may cleave molecules critical for the host immune response allowing the parasites to survive in the host tissues.

Genetic and biochemical properties of a hemolysin (pyolysin) produced by a swine isolate of Arcanobacterium (Actinomyces) pyogenes.

Arcanobacterium (Actinomyces) pyogenes, a causative agent of various pyogenic diseases in domestic animals, produces a hemolysin which is thought to be an important virulence factor. This hemolysin was purified from the culture supernatant of A. pyogenes swine isolate. The purified hemolysin showed a single band with a molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis, and its isoelectric point was 9.2. The activity of this hemolysin was not enhanced by the addition of L-cysteine or sodium thioglycolate, but it was inhibited by cholesterol. The gene encoding the hemolysin was cloned, sequenced and expressed in Escherichia coli by means of ZAP Express vector. Analysis by SDS-polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E. coli is the same as that of the hemolysin purified from A. pyogenes. Nucleotide sequence analysis revealed an open reading frame of 1,605 bp encoding a 534 amino acid protein of 57,989 Da. The nucleotide sequence of the hemolysin gene from A. pyogenes swine isolate differed only slightly (97.6% identity) from the sequence of plo gene from A. pyogenes strain BBR1 reported by Billington et al (J. Bacteriol. 179: 6100-6106, 1997). The cysteine residue existed in the undecapeptide region of the hemolysin, which is highly conserved in thiol-activated cytolysins (cholesterol-binding cytolysins), and is replaced with alanine. Therefore, the hemolysin of A. pyogenes seems to be a novel member of the thiol-activated cytolysin family.

Inhibition of lysinoalanine formation in alkali-pickled duck egg (Pidan)

Duck eggs were pickled in 4.2% NaOH/5% NaCl solution for 20 days to prepare the traditional Chinese Pidan. Influence of some additives, such as 0.2% CuSO 4 0.2% ZnSO 4 0.2% PbO, 10 mM l-cysteine or 10 mM reduced glutathione solution, in the pickle solution on the formation of lysinoalanine (LAL) as well as the degradation of lysine in albumen and egg yolk were investigated. It was observed that CuSO 4 exhibited the most prevalent inhibition effect on LAL formation in both Pidan albumen and egg yolk, followed by ZnSO 4 and PbO. Addition of l-cysteine or reduced glutathione in the pickling solution marked a decreased LAL formation in Pidan.

Proteases involved in the metabolic degradation of human interleukin-1beta by rat kidney lysosomes.

The in vitro metabolic degradation of human interleukin (IL)-1beta was studied using lysates of rat kidney lysosomes, and proteases involved in the degradation were identified. In the study of IL-1beta degradation, fluorescein isothiocyanate (FITC)-labeled IL-1beta was used as a substrate. The maximal degradation of IL-1beta occurred at pH 3.0, and the reaction was proportional to the lysosomal protein concentration and time of incubation. The degradation was stimulated by the addition of L-cysteine. The reaction was not inhibited by phenylmethanesulfonyl fluoride or EDTA, indicating that serine proteases or metalloproteases do not play a major role in the degradation process. N-Ethylmaleimide, leupeptin and E-64, inhibitors of thiol protease, inhibited the degradation of IL-1beta, by 59%-70%. Pepstatin A, an inhibitor of carboxyl protease, inhibited the degradation by 58%. Combinations of thiol and carboxyl protease inhibitors nearly completely inhibited the degradation. Bio-Gel P-10 gel filtration chromatography of in vitro reactants confirmed the ability of lysosomal proteases to degrade IL-1beta and revealed four to five peaks of degradation products. Taken together, these results indicate that thiol protease and carboxyl protease play an important role in the IL-1beta degradation process by kidney lysosomes. Leupeptin and E-64 dose dependently inhibited both cathepsin B and cathepsin L activities, and pepstatin A strongly inhibited cathepsin D activity in rat kidney lysosomes. The present results suggest that cathepsin B, cathepsin L, and cathepsin D in kidney lysosomes are involved in the metabolic degradation of human IL-1beta.

Determination of inorganic mercury in biological tissues by cold vapor atomic absorption spectrometry following tetramethylammonium hydroxide solubilization

A rapid and simple method for the quantification of inorganic and total mercury in biological tissues using flow injection cold vapor generation atomic absorption spectrometry is presented. Samples were solubilized using tetramethylammonium hydroxide. The inorganic Hg was released by on-line addition of L-cysteine and then reduced to metallic Hg by SnCl2. The detection limit for inorganic mercury was 0.1 µg l–1 and the precision of determination was better than 2% (RSD) at 20 µg l–1. The proposed method was validated by the analyses of a suite of certified marine biological reference materials, DORM-2 (dogfish muscle), DOLT-2 (dogfish liver) and TORT-2 (lobster hepatopancreas).

New antioxidant mixture for long term stability of serotonin, dopamine and their metabolites in automated microbore liquid chromatography with dual electrochemical detection.

An automated microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin, dopamine and their metabolites, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Due to the chemical instability of the compounds, the addition of an antioxidant is required for automated analysis over a long period of time (e.g., 20 h). Therefore, the time stability of these substances was tested with different antioxidants. The stability for serotonin and 5-hydroxyindoleacetic acid was poor in acidic medium containing Na2EDTA but could greatly be improved by the addition of L-cysteine and ascorbic acid. Using this assay, the neurotransmitters and their metabolites could easily be determined in microdialysates obtained from different rat brain areas.

Characterization of leukotriene production in vivo and in vitro in resident and elicited peritoneal macrophages in chickens and mice

Previously, we reported differences in arachidonic acid metabolism in elicited chicken peritoneal macrophages when compared with murine resident and elicited peritoneal macrophages. 1 We now describe leukotriene (LT) production in the same systems, using resident (murine) and inflammatory macrophages (from both species). Inflammatory (4- or 42-h Sephadex-elicited) peritoneal macrophages from chickens lacked the capacity to produce LT in vivo (following opsonized zymosan [OZ] stimulation) or in vitro, in response to A23187. In addition, chicken macrophages were unable to metabolize exogenously added LTC 4 or LTD 4 in vitro. In contrast, resident murine peritoneal macrophages produced measurable quantities of LTs (in vivo) within 5 min with an 8-fold increase after 45 min. LTC 4 was effectively converted to LTE 4 in vivo in a time-dependent manner (65% LTC 4/35% LTE 4 after 5 min stimulation with OZ and 6% LTC 4/94% LTE 4 after 60 min stimulation), but not in vitro. The lack of LTC 4 metabolism to LTE 4 in vitro could not be explained by cell-cell interaction between adherent and nonadherent cells. LTD 4 was not detected under any experimental condition. Murine peritoneal cells incubated with LTD 4 (with or without agonist) produced LTE 4 in a time-dependent fashion. Addition of l-cysteine (a dipeptidase inhibitor) did not explain the lack of detectable levels of LTD 4 following intraperitoneal stimulation with OZ. These results suggest that elicited chicken peritoneal macrophages are incapable of producing LTs compared to murine peritoneal macrophages. In addition, these studies fail to explain the different product profiles associated with in vivo stimulation of murine peritoneal macrophages as compared to in vitro stimulation.

L-Cysteine Improves Growth of Human Peritoneal Mesothelial CellsIn Vitro

To improve the growth characteristics of human peritoneal mesothelial cells (HPMC). The effect of commonly used agents, L-cysteine and epidermal growth factor (EGF), added individually ("single") or mixed with hydrocortisone and apo-transferrin ("admixture") in the culture medium (M199) on cultured HPMC, was investigated. Growth agents were added to M199 medium along with 2% fetal bovine serum and L-glutamine. Growth was determined by the analysis of thymidine ([methyl-3H] thymidine) incorporation into deoxyribonucleic acid, total cell protein, and by cell counts. Morphology was assessed by phase contrast light microscopy and scanning electron microscopy. HPMC exposed to L-cysteine 0.25 x 10(-3) mol/L (30 micrograms/mL) exhibited significantly improved attachment and growth. Attached cells appeared flat and well spread out shortly after seeding, and produced a tight polygonal monolayer in 14 days, in contrast to the growth of HPMC in control M199 medium, which failed to reach confluence. After an initial lag period in cell growth, EGF (0.01 microgram/mL) produced a greater increase in cell growth than L-cysteine did; however, this was associated with changes in HPMC morphology. During the growth period (14 days), EGF-stimulated HPMC appeared distorted and irregular compared to L-cysteine-treated cells, which had the characteristic tight "cobblestone" appearance. L-cysteine improved cell attachment with preservation of the characteristic morphology of HPMC. Epidermal growth factor improved cell growth but produced changes in morphology. The addition of L-cysteine to the culture medium has an important cell growth enhancement role due to the improved cell attachment and cell viability.

Formation of the alpha-aminoacrylate immediate limits the overall reaction catalyzed by O-acetylserine sulfhydrylase.

O-Acetylserine sulfhydrylase-A (OASS-A) catalyzes the final step in the synthesis of L-cysteine, viz., the beta-substitution of acetate in O-acetyl-L-serine (OAS) by sulfide via a ping-pong kinetic mechanism . Rapid-scanning stopped-flow and single-wavelength absorbance and fluorescence stopped-flow experiments were carried out to obtain information on the location and amount of limitation of rate-determining steps for the overall reaction and the individual half-reactions of OASS-A. The first half-reaction, conversion of OAS to the alpha-aminoacrylate intermediate and acetate, is rate-limiting for the overall reaction catalyzed by OASS-A. No intermeidates are detected within the second half-reaction, and thus rate constants for all steps must be > or = 1000s-1 at the lowest sulfide concentration used. Within the first half reaction, formation of the extrernal Schiff base (Kassociation = 0.2 mM-1) is observed in the first milliseconds, followed by its slower conversion to the alpha-aminocacrylate intermediate with a rate constant of 300 s-1, close to the value of 130 s-1 obtained for V/Et [Tai, C.H., Nalabolu, S.R., Jacobson, T.M., Minter D.E., & Cook, P.F. (1993) Biochemistry 32, 6433-6442]. Addition of L-cysterine ot OASS-A results in a rapid formation of the external Schiff base (Kassociation = 6.7 mM-1), followed by transient formation of the alpha-aminoacylate intermediate with a slightly lower rate (70-100 s-1) compared to OAS. The alpha-aminoacrylate intermediate decays to generate a species absorbing maximally at 418 nm, resulting from attack of the cysteine thiol to give ether in external Schiff base linkage with the active site PLP.

Application and optimization of capillary zone electrophoresis in vitamin analysis

The application of capillary zone electrophoresis to the determination of ascorbic acid and biotin in vitamin mixtures (pharmaceutical formulations and also native citrus juice and fruit beverage) was investigated. A method was developed for determining them together with thiamine, nicotinamide and nicotinic acid in a single run. For ascorbic acid, addition of L-cysteine as antioxidant was necessary to obtain reliable results. The performance of the method was tested according to sensitivity, detection limit and standard deviation.

Identification and spectral characterization of the external aldimine of the O-acetylserine sulfhydrylase reaction.

The O-acetylserine sulfhydrylase (OASS) reaction has been studied using a number of spectral probes including UV--visible, fluorescence, circular dichroism, and 31P NMR spectroscopy. The addition of L-cysteine, L-alanine, and glycine to OASS results in a shift in lambda max of 412 nm for the internal Schiff base to 418 nm resulting from the formation of the external Schiff base. The addition of L-serine or O-methyl-D,L-serine gives decreases of the absorbance of unliganded enzyme at 412 nm of about 50% and 20%, respectively, concomitant with an increase in the absorbance at 320 nm and a shift in the lambda max of the remaining visible absorbance to 418 nm. The spectral shifts observed in the presence of L-serine are suggestive of establishing an equilibrium between different forms of external Schiff base. The concentration dependence of the changes at 440 (L-cysteine) and 320 nm (L-serine) provides an estimate of the dissociation constant for the external aldimine. The pH dependence of the dissociation constant suggests the alpha-amine of the amino acid must be unprotonated for nucleophilic attack at C4' of PLP, and an enzyme side chain must be unprotonated to hydrogen-bond the thiol or hydroxyl side chain of the amino acid. When L-cysteine is the amino acid, the thiol side chain must be protonated to hydrogen-bond to the unprotonated enzyme side chain. The 31P NMR chemical shift is increased from 5.2 ppm for unliganded enzyme to 5.3 ppm in the presence of L-cysteine, signaling a tighter interaction at the 5'-phosphate upon formation of the external Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)

CD38-mediated ribosylation of proteins.

The lymphocyte cell-surface Ag CD38 catabolizes NAD to adenosine 5' diphosphoribose (ADPR) and cyclic ADPR (cADPR). We show here that the soluble extracellular domain of CD38 (sCD38) mediates ADP ribosylation of several proteins. This was demonstrated by mass spectrometric analyses which revealed the addition of mass in units of 541.1 Da to these proteins, presumably corresponding to the covalent attachment of one or more ADPR moieties. Separate experiments showed that the same proteins became specifically radiolabeled following incubation with [32P]NAD plus sCD38. Additionally, it is shown that sCD38 can autoribosylate. Moreover, sCD38-mediated protein ribosylation was found to occur specifically at cysteine residues, since it was effectively blocked by addition of L-cysteine but not by other amino acids, and CD38-mediated protein ribosylation could be reversed by the addition of HgCl2, which specifically cleaves thiol-glycosidic bonds. ADPR purified from the reaction of sCD38 with NAD could itself be covalently transferred to target proteins at rates similar to the sCD38-mediated reaction, indicating that the ribosylation proceeds via the generation of this reactive intermediate. In vitro mutagenesis of a catalytic Glu residue that is conserved in numerous ADP-ribosyl transferases revealed that this amino acid is also important for catalysis in CD38. These data suggest that CD38 has the potential to cause ribosylation of experimental proteins, and raises the possibility that its specific ribosylation of a currently unidentified lymphocyte protein may contribute to its array of immunoregulatory activities.

Formation of the l-cysteine-glyoxylate adduct is the mechanism by which l-cysteine decreases oxalate production from glycollate in rat hepatocytes

Formation of thiazolidine-2,4-dicarboxylic acid, the L-cysteine-glyoxylate adduct, is the putative mechanism by which L-cysteine reduces hepatic oxalate production from glycollate [Bais, Rofe and Conyers (1991) J. Urol. 145, 1302-1305]. This was investigated in isolated rat hepatocytes by the simultaneous measurement of both adduct and oxalate formation. Different diastereoisomeric ratios of cis- and trans-adduct were prepared and characterized to provide both standard material for the enzymic analysis of adduct in hepatocyte supernatants and to investigate the stability and configuration of the adduct under physiological conditions. In the absence of L-cysteine, hepatocytes produced oxalate from 2 mM glycollate at a rate of 822 +/- 42 nmol/30 min per 10(7) cells. The addition of L-cysteine to the incubation medium at 1.0, 2.5 and 5.0 mM lowered oxalate production by 14 +/- 2, 25 +/- 3 (P < 0.05) and 38 +/- 3% (P < 0.01) respectively. These reductions were accompanied by almost stoichiometric increases in the levels of the adduct: 162 +/- 6, 264 +/- 27 and 363 +/- 30 nmol/30 min per 10(7) cells. Adduct formation is therefore confirmed as the primary mechanism by which L-cysteine decreases oxalate production from glycollate. As urinary oxalate excretion is a prime risk factor in the formation of calcium oxalate stones, any reduction in endogenous oxalate production is of clinical significance in the prevention of this formation.

Tolerance to the vascular effect of a novel nitric oxide-donating vasodilator, FK409

We investigated whether tolerance develops to the vasorelaxant effects of a new vasodilator, (±)-( E)-4-ethyl-2-[( E)-hydroxyimino]-5-nitro-3-hexenamide (FK409), in isolated canine coronary artery strips and to its hypotensive effect in rats, and whether FK409 activates soluble guanylate cyclase isolated from vascular tissues in the absence of L-cysteine. No tolerance to FK409 (0.46 nM to 0.46 μM or 1–1000 μg/kg, i.v.) or cross-tolerance between FK409 and glyceryl trinitrate was demonstrated in vitro and in vivo experiments, whereas the tolerance to glyceryl trinitrate (0.44 nM to 4.4 μM or 1–1000 μg/kg, i.v.) was marked in both conditions. In addition, FK409 (0.1–10 μM) activated soluble guanylate cyclase without L-cysteine, but glyceryl trinitrate (1–100 μM) required the addition of L-cysteine (5 mM) for the activation of the enzyme. The results suggest that FK409 may be advantageous compared to tolerance-producing nitrates currently in clinical use, and that this property of FK409 is probably due to its independence of a sulfhyhydryl donor.

Inhibition by ethacrynic acid of NO-mediated relaxations of the rat anococcygeus muscle.

1. The effects of ethacrynic acid were studied on relaxations elicited by nitric oxide (NO), the NO-donors sodium nitroprusside (SNP) and glyceryl trinitrate (GTN), nitrergic nerve stimulation and the NO-independent agent papaverine in isolated preparations of rat anococcygeus muscles. 2. Ethacrynic acid (100 mumol/L) produced complete relaxation of partially contracted anococcygeus muscles, but the tone recovered after the ethacrynic acid was washed out. Following exposure to ethacrynic acid, the relaxant responses to NO, SNP, GTN and nitrergic nerve stimulation were abolished or markedly reduced; however, the response to papaverine was only slightly reduced. 3. The presence of 3 mmol/L L-cysteine during the period of exposure to ethacrynic acid prevented the inhibition of the relaxing effects of SNP, GTN and nitrergic nerve stimulation almost completely, but did not affect the slight reduction in responses to papaverine. 4. The addition of L-cysteine (3 mmol/L) after incubation with ethacrynic acid did not significantly affect the inhibited responses to SNP and GTN; however, the inhibited responses to nitrergic nerve stimulation were slightly but significantly increased. 5. The results suggest that endogenous sulphydryl groups are required for the actions of NO, NO-donating drugs and the nitrergic transmitter in the rat anococcygeus muscle and possibly for the synthesis or release of the nitrergic transmitter.

Effects of visible light and other environmental factors on the production of oxygen radicals by hamster embryos

Previous studies have demonstrated that developing hamster embryos are very sensitive to visible light. In order to elucidate why visible light exerts a toxic effect on hamster embryos, we examined the effect of visible light on the production of hydrogen peroxide (H 2O 2) within individual embryos, using a fluorimetric method. In addition, we examined the H 2O 2 generating capacity of other factors which are known to be related to the in vitro developmental capacity of hamster embryos. One-cell hamster embryos were cultured with 2′,7′-dichlorodihydrofluorescin diacetate, and the fluorescence emissions of the H 2O 2-dependent oxidative product in the embryos were measured using an Olympus microscopic photometry system. When embryos were exposed to visible light (14,000 lux) for a specified period (0, 0.5, 1, 2 or 3 min) prior to measurement, the fluorescence emissions from embryos increased with the time of exposure to visible light. An exposure of even 0.5 min resulted in a significant increase in hydrogen peroxide. This increase was more rapid in embryos cultured under 20% O 2 than in those cultured under 5% O 2, and the response was quicker than that observed in mouse embryos. The fluorescence emissions from embryos cultured under 5% O 2 were significantly (P<0.001) lower than those from embryos cultured under 20% O 2 in TLP medium. However, the effects of different oxygen tensions on fluorescence emissions were medium-dependent, and were not significant in embryos cultured in HECM-1 medium. The addition of L-cysteine to or elimination of phenol red from the media decreased the fluorescence emissions from embryos (P<0.001), but glucose and phosphate did not affect them. These results suggest that the toxic effect of visible light on the in vitro development of hamster embryos might be due to increased generation of reactive oxygen species, induced by the visible light. This could be one of the explanations for the strict conditions required for overcoming the in vitro developmental block. It is also suggested that the promotive effects of low oxygen culture and L-cysteine on embryo development seem to be derived from their ability to reduce reactive oxygen species.

Analysis of In Vitro Developmental Block of Rat Embryos: Assessment from the View Point of Oxygen Toxicity.

We have already reported that the block to development in cultured 1-cell rat embryos was overcome using HECM-1, a protein-free chemically defined medium without glucose nor phosphate. In addition, we showed the beneficial effect of low oxygen tension in rat embryos. In this study, in order to clarify the reason why in vitro developmental block of rat embryos is over-come in HECM-1, we examined the effects of phosphate, glucose and L-cysteine on the in Vitro development of rat embryos. Furthermore, we examined their effects on the production of H2O2 in embryos by the fluorimetric method. Addition of KH2PO4 even at only 1 μM completely inhibited the development of rat (Wistar) embryos either from the 1-cell stage or the 2-cell stage. Addition of glucose at the concentration of 1000 μg/ml significantly decreased the blastulation rate. The pro-duction of H2O2 in embryos cultured under 5% O2 was significantly (P<0.01) lower than that of embryos cultured under 20% O2 in HECM-1. H2O2 production in embryos cultured in mKRB was significantly (P<0.01) higher than that in embryos cultured in HECM-1, irrespective of the oxygen tension. Addition of neither phosphate nor glucose to HECM-1 affected the H2O2 production at any concentration examined. L-cysteine significantly decreased the H2O2 production in embryos in mKRB, but developmental block could not be overcome by addition of L-cysteine to mKRB. These results revealed that glucose and phosphate independently inhibited the development of rat em-bryos in vitro, and suggest that the mechanism of in vitro developmental block of rat embryos could not be explained simply in terms of oxygen toxicity.

Requirement of thiol compounds as reducing agents for IL-2-mediated induction of LAK activity and proliferation of human NK cells.

Thiol-related compounds, such as L-cystine, 2-ME or reduced glutathione (GSH), are important in many lymphoid cell activation pathways. We investigated their role in IL-2-generated lymphokine-activated killer (LAK) activity in human NK (CD16+ and/or CD56+) cells. Depletion of GSH and L-cystine, but not L-leucine or glycine, from medium used during culture of NK cells with IL-2 inhibited LAK and proliferative activities, whereas IL-2-independent lytic function of NK cells remained intact. Addition of L-cysteine, 2-ME or GSH, but not methylated analogs of these compounds (which cannot function as proton donors) to L-cystine/GSH-depleted medium restored proliferative response of NK cells and LAK generation. In the presence of L-buthionine-(S,R)-sulfoximine, an inhibitor of GSH synthesis, IL-2-induced LAK activity and proliferation of NK cells in medium without L-cystine and GSH, could be restored, at least in part, by addition of GSH, but not 2-ME or L-cystine. Furthermore, intracellular GSH levels were depressed in cells cultured in L-cystine/GSH-depleted medium but could be restored by the addition of 2-ME. The results suggest that (1) L-cystine or thiol containing compounds such as L-cysteine, 2-ME, or GSH are necessary for effective IL-2-activation of human NK cells, (2) these compounds must be functional proton-donors, i.e., reducing agents, implying regulation of the IL-2 activation pathway by oxidation-reduction, and (3) GSH synthesis is necessary for the activation. Experiments were performed to begin to dissect the redox-sensitive step(s) in LAK development. Depletion of reducing agents had no effect on internalization of rIL-2. In contrast, intracellular granzyme A activity was significantly depressed in NK cells cultured with rIL-2 in L-cystine/GSH-depleted medium compared with those cultured in medium in which L-cystine levels had been replenished. The findings suggest that step(s) in the transduction of the IL-2 signal in NK cells, between the internalization of IL-2 and the maturation of the lytic mechanism, are subject to regulation by oxidation-reduction.

Inhibition by thiol compounds of off-flavor formation in stored orange juice. 1. Effect of L-cysteine and N-acetyl-L-cysteine on 2,5-dimethyl-4-hydroxy-3(2H)-furanone formation

Orange juice stored at 45 o C for 1 and 14 days resulted in increased browning and increased concentration of sugar degradation products such as 5-(hydroxymethyl)furfural (HMF) and 2,5-dimethyl-4-hydroxy-3(2H-furanone (DMHF). Addition of L-cysteine (2.5-12.3 mM) significantly reduced browning and HMF and DMHF formation in the 14-day-stored orange juice in a dose-related manner. Ascorbic acid retention increased with increasing L-cysteine concentrations. Low levels (2.5 mM and below) of L-cysteine and N-acetyl-L-cysteine reduced browning and DMHF formation, but only L-cysteine reduced ascorbic acid degradation