Sulfhydryl Reduction Research Articles

Consolidating the gelling performance of myofibrillar protein using a novel OSA-modified-starch-stabilized Pickering emulsion filler: Effect of starches with distinct crystalline types

Starch-stabilized Pickering emulsions were employed as a novel particulate filler in myofibrillar protein (MP)-based gels for improving the gelling characteristics. The role of emulsions prepared by native starches (NS) with distinctive crystalline types (i.e., A-type waxy corn starch, B-type potato starch, and C-type pea starch) and their OSA-modified counterparts (A-OS, B-OS, C-OS) in the gelling performance was evaluated and compared with MP-stabilized-emulsion. Compared with MP-emulsion, starch-emulsion caused substantial increases in the gelling properties, notably for OSA-starch emulsions. Herein, A-OS exhibited up to 1.26-, 5.3-, and 2.9-fold increments in storage modulus, gel strength, and water holding capacity relative to pure MP gel, respectively, higher than B-OS and C-OS. Moreover, light microscopy evinced a more compact gel network filled with smaller and uniform oil droplets when A-OS emulsions were incorporated into the gels. The addition of OSA-starch emulsions, especially A-OS emulsion, facilitated the protein conformational conversion from α-helix to β-sheet and caused a marked reduction of free sulfhydryls in the gels; yet, the chemical forces that stabilized the gels altered, where remarkable reinforcements in hydrogen bond and hydrophobic interaction were detected, in support of the construction of splendid MP gels. Hence, OSA-starch emulsions show promise as functional components in meat products.

Increasing trans-cleavage catalytic efficiency of Cas12a and Cas13a with chemical enhancers: Application to amplified nucleic acid detection

The exceptional programable trans-cleavage ability of type V and VI CRISPR/Cas nucleases paved the way for ultrasensitive CRISPR/Cas based sensing of nucleic acid and alternative targets. However, the enhancement of the trans-cleavage activity of Cas effector with organic chemical agents has not been explored thus far. We report here chemically enhanced trans-cleavage activity of Cas12a and Cas13a nucleases which improves sensor performance in CRISPR/Cas biosensing. Improved trans-ssDNA cleavage of Cas12a and trans-ssRNA cleavage of Cas13a were demonstrated by using sulfhydryl reductants and non-ionic surfactants. DTT and PVA were demonstrated to be the most effective chemical enhancers in both cases. By using a fluorescence resonance energy transfer (FRET)-based intramolecular distance measurements, we identified the mechanism of this enhancement to be the conformation change of the ribonucleoprotein and quantified it to be major (about 50% increase of a relevant intramolecular distance). These chemical enhancers have been integrated into the established CRISPR/Cas biosensing protocols without additional modifications. For the detection of Helicobacter Pylori DNA and SARS-CoV-2 RNA, we found a decreased reaction time by 75–83% and 4–6-fold increased sensitivity. These results indicate that chemical enhancers provide a versatile and broadly applicable approach to break through the barriers of long reaction time and sensitivity in CRISPR/Cas sensors.

Improvement of gel properties of mackerel mince by phlorotannin extracts from sporophyll of Undaria pinnatifidai and UVA induced cross-linking.

The effects of phlorotannin extracts (PTE) (from sporophyll of Undaria pinnatifida) added at different concentration (0, 1.6, 8, and 40 mg/g protein) with and without ultraviolet A (UVA) irradiation treatment on gel properties of mackerel (Scomberomorus niphonius) mince (MM) were investigated. The results indicated that both the breaking force and water-holding capacity (WHC) of the MM gel increased with addition of PTE, and the highest breaking force and WHC were observed with 40 mg/g PTE under UVA irradiation. Low-field nuclear magnetic resonance revealed that free water significantly decreased in this PTE-MM gel as well. UVA irradiation treatment led to reduction of total sulfhydryls and free amines in the PTE-MM gel, indicating more cross-linking between proteins. Electrophoresis results indicated that myosin heavy chains were aggregated in the UVA-treated PTE-MM gel. The microstructure of the UVA-treated PTE-MM gel showed that protein formed more evenly distributed cross-linked structures and was more compact than the untreated control gel. As a conclusion, PTE addition with UVA irradiation is shown to improve gelling properties of mackerel mince.

Using Theoretical Protein Isotopic Distributions to Parse Small-Mass-Difference Post-Translational Modifications via Mass Spectrometry

Small-mass-difference modifications to proteins are obscured in mass spectrometry by the natural abundance of stable isotopes such as (13)C that broaden the isotopic distribution of an intact protein. Using a ZipTip (Millipore, Billerica, MA, USA) to remove salt from proteins in preparation for high-resolution mass spectrometry, the theoretical isotopic distribution intensities calculated from the protein's empirical formula could be fit to experimentally acquired data and used to differentiate between multiple low-mass modifications to proteins. We could readily distinguish copper from zinc bound to a single-metal superoxide dismutase (SOD1) species; copper and zinc only differ by an average mass of 1.8Da and have overlapping stable isotope patterns. In addition, proteins could be directly modified while bound to the ZipTip. For example, washing 11mM S-methyl methanethiosulfonate over the ZipTip allowed the number of free cysteines on proteins to be detected as S-methyl adducts. Alternatively, washing with the sulfhydryl oxidant diamide could quickly reestablish disulfide bridges. Using these methods, we could resolve the relative contributions of copper and zinc binding, as well as disulfide reduction to intact SOD1 protein present from <100μg of the lumbar spinal cord of a transgenic, SOD1 overexpressing mouse. Although techniques like ICP-MS can measure total metal in solution, this is the first method able to assess the metal-binding and sulfhydryl reduction of SOD1 at the individual subunit level and is applicable to many other proteins.

Accumulation of β-Conglycinin in Soybean Cotyledon through the Formation of Disulfide Bonds between α′- and α-Subunits

β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α'- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α'/α-subunits were complexes of more than 720 kD. The α'- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α'/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α'- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions.

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

Structural and Functional Diversity of Glutaredoxins in Yeast

Glutaredoxins are defined as thiol disulfide oxidoreductases that reduce disulfide bonds employing reduced glutathione as electron donor. They constitute a complex family of proteins with a diversity of enzymatic and functional properties. Thus, dithiol glutaredoxins are able to reduce disulfide bonds and deglutathionylate mixed disulfides between glutathione and cysteine protein residues. They could act regulating the redox state of sulfhydryl residues of specific proteins, while thioredoxins (another family of thiol disulfide oxidoreductases which employ NADPH as electron donor) would be the general sulfhydryl reductants. Some dithiol glutaredoxins such as human Grx2 form dimers bridged by one iron-sulfur cluster, which acts as a sensor of oxidative stress, therefore regulating the activity of the glutaredoxin. The ability to interact with iron-sulfur clusters as ligands is also characteristic of monothiol glutaredoxins with a CGFS-type active site. These do not display thiol oxidoreductase activity, but have roles in iron homeostasis. The three members of this subfamily in Saccharomyces cerevisiae participate in the synthesis of the iron-sulfur clusters in mitochondria (Grx5), or in signalling the iron status inside the cell for regulation of iron uptake and intracellular iron relocalization (Grx3 and Grx4). Such a role in iron metabolism seems to be evolutionary conserved. Fungal cells also contain membrane-associated glutaredoxins structurally and enzymatically similar to dithiol glutaredoxins, which may act as redox regulators at the early stages of the protein secretory machinery.

Redox-Regulated Heterogeneous Thresholds for Ligand Recruitment among InsP3R Ca2+-Release Channels

To clarify the molecular mechanisms behind quantal Ca2+ release, the graded Ca2+ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP3R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP3R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Progressively more channels were activated by more favorable conditions in patches from insect cells with only one InsP3R gene or from cells solely expressing one recombinant InsP3R isoform, demonstrating that channels with identical primary sequence have different ligand recruitment thresholds. Such heterogeneity was largely abrogated, in a fully reversible manner, by treatment of the channels with sulfhydryl reducing agents, suggesting that it was mostly regulated by different levels of posttranslational redox modifications of the channels. In contrast, sulfhydryl reduction had limited effects on channel open probability. Thus, sulfhydryl redox modification can regulate various aspects of intracellular Ca2+ signaling, including quantal Ca2+ release, by tuning ligand sensitivities of InsP3R channels. No intrinsic termination of channel activity with a timescale comparable to that for quantal Ca2+ release was observed under any steady ligand conditions, indicating that this process is unlikely to contribute.

Disulfide S-Monoxides Convert Xanthine Dehydrogenase into Oxidase in Rat Liver Cytosol More Potently Than Their Respective Disulfides

Xanthine oxidase (XO)/xanthine dehydrogenase (XD) oxidizes oxypurines to uric acid, with only the XO form producing reactive oxygen species. In the present study, the effects of cystamine S-monoxide and cystine S-monoxide (disulfide S-monoxides) on the conversion of XD to XO in rat liver were examined. A partially purified enzyme fraction from the rat liver was incubated with xanthine in the presence or absence of NAD+, and the uric acid formed was measured by HPLC. Under basal conditions, XO activity represented about 15% of the total XO plus XD activity. Cystamine S-monoxide and cystine S-monoxide converted XD into XO in a dose-dependent manner, and the concentrations required to increase XO activity by 50% were approximately 1 and 2 microM, respectively. Their respective thiols (cysteamine and cysteine) and disulfides (cystamine and cystine) up to 10 microM showed weak or no effects on the activities of XO and XD and their conversion. Experiments utilizing a sulfhydryl reducing reagent (dithiothreitol) and sulfhydryl modifiers (4,4'-dithiodipyridine and 1-fluoro-2,4-dinitrobenzene) indicated that disulfide S-monoxides-induced conversion of XD to XO occurs via disulfide bridge formation in XD, but not the modification of sulfhydryl groups. These results suggest that disulfide S-monoxides have the potential to increase the generation of reactive oxygen species through the conversion of XD to XO in liver.

Role of Sulfhydryl Groups in Transfection? A Case Study with Chitosan−NAC Nanoparticles

This study investigated the use of chitosan-N-acetylcysteine (NAC) as a non-viral gene carrier. In particular, we aimed to elucidate whether the advantage of thiolation was more pronounced in the stabilization of particles or in the effect of nonspecific sulfhydryl reduction of the target cells. Low-viscosity chitosan was modified by covalent binding of NAC. The resulting conjugate displayed 1.35 mM SH/g polymer. Particles produced via self-assembly of chitosan conjugate and pDNA had a mean particle size of 113.7 nm and a positive zeta-potential. Sulfhydryl group content on the particle surface was investigated by Ellman's test and papain reactivation assay, with the result of about 100 nM SH groups/mL nanoparticle suspension. An oxidation step was performed to stabilize polyplexes via disulfide bonds. The enhanced stability of oxidized particles against both polyanion heparin and alkaline pH was proven by a gel retardation assay. The stabilization was demonstrated to be reversible by treatment with glutathione. Further, the effect of immobilized SH groups and of supplementation with free NAC on transfection efficacy on Caco-2 cells was investigated. The expression of the transgene was raised 2.5-fold and 10-fold with nonoxidized thiomer polyplexes in comparison to polyplexes of unmodified chitosan and oxidized chitosan-NAC, respectively. The impact of sulfhydryl reduction on transfection was assessed via thiol group inactivation with 5,5'-dithiobis-(2-nitrobenzoic acid) (DNTB). This inactivation resulted in a decrease of transfection efficacy. In conclusion, chitosan-NAC conjugate was demonstrated to be beneficial for transfection, either for stabilization via disulfide bonds or for raising the expression of transgene via shifting the redox potential of the target cells.

Antimicrobial Actions of Human and Macaque Sperm Associated Antigen (SPAG) 11 Isoforms: influence of the N-terminal peptide

In addition to their role in sperm maturation, recent evidence has indicated that epididymal proteins have a role in male reproductive tract innate immunity. Herein we demonstrate that human and macaque epididymal protein isoforms in the SPAG (sperm associated antigen) 11 family, full length SPAG11C, K and L exhibit potent antibacterial activity against E. coli. Analysis of activities of the N- and C-terminal domains revealed that the human N-terminal peptide is bactericidal, while the C-terminal domains that contain the defensin-like 6 cysteine array in SPAG11C and partial arrays in SPAG11K and SPAG11L, lack antibacterial activity. The N-terminal peptide does not appear to contain all the determinants of activity since full-length human SPAG11C is more active than the isolated N-terminal peptide and since sulfhydryl reduction and alkylation, which would affect primarily the C-terminal peptides, completely abolished activities of the whole proteins. These results suggest that the structure conferred by the disulfide bonds in human SPAG11C contributes to the antibacterial activity of the whole molecule. The activities of the N-terminal peptide and of full length human SPAG11C were somewhat reduced in increasing NaCl concentrations. In contrast, the antibacterial activities of full length macaque SPAG11C, K and L were unaffected by the presence of NaCl suggesting a mechanism in the macaque that is less dependent upon electrostatic interactions. SPAG11C, K and L disrupted E. coli membranes but had no effect on erythrocyte membranes. Inhibition of E. coli RNA, DNA and protein synthesis by nonlethal concentrations of SPAG11 isoforms indicated an additional mechanism of bacterial killing.

Regulation of K–Cl cotransport by protein phosphatase 1α in mouse erythrocytes

The K–Cl cotransport (KCC) is an electroneutral-gradient-driven-membrane transport system, which is involved in regulation of red cell volume. Although the regulatory cascade of KCC is largely unknown, a signaling pathway involving phosphatases and kinases has been proposed. Here, we investigated the expression and the activity of protein phosphatase 1(PP-1) isoforms in mouse red cells, focusing on two models of abnormally activated KCC: mice genetically lacking the two Src-family tyrosine kinases, Hck and Fgr, (hck-/-fgr-/-) and the SAD transgenic sickle-cell-mice. The PP-1α, PP-1γ, PP-1δ isoforms were expressed at similar levels in wild-type, hck-/-fgr-/- and SAD mouse erythrocytes and in each case were predominantly localized to cytoplasm. The PP-1α activity was significantly higher in both membrane and cytosol fractions of hck-/-fgr-/- and of SAD erythrocytes than in those of wild-type red cells, suggesting PP-1α as a target of the Hck and Fgr kinases. The PP2, a specific inhibitor of Src-family kinase, significantly increased KCC activity in wild-type mouse red cells, but failed to modify the already increased KCC activity in SAD erythrocytes. The lag-time for activation of KCC was considerably reduced in both hck-/-fgr-/- and SAD erythrocytes, suggesting that the rate limiting activation steps in both strains are freed from their tonic inhibition. Sulfhydryl reduction by dithiothreitol (DTT) lowered KCC activity only in SAD red cells, but did not affect the PP2-treated erythrocytes. These data suggest up-regulation of KCC in SAD red cells is mainly secondary to oxidative damage, which most likely reduces or removes the tonic KCC inhibition resulting from PP-1α activity controlled in turn by Src-family kinases.

Neuroprotective Effect of Sulfhydryl Reduction in a Rat Optic Nerve Crush Model

The signaling of retinal ganglion cell (RGC) death after axotomy is partly dependent on the generation of reactive oxygen species. Shifting the RGC redox state toward reduction is protective in a dissociated mixed retinal culture model of axotomy. The hypothesis for the current study was that tris(2-carboxyethyl)phosphine (TCEP), a sulfhydryl reductant, would protect RGCs in a rat optic nerve crush model of axotomy. RGCs of postnatal day 4 to 5 Long-Evans rats were retrogradely labeled with the fluorescent tracer DiI. At approximately 8 weeks of age, the left optic nerve of each rat was crushed with forceps and, immediately after, 4 muL of TCEP (or vehicle alone) was injected into the vitreous at the pars plana to a final concentration of 6 or 60 microM. The right eye served as the control. Eight or 14 days after the crush, the animals were killed, retinal wholemounts prepared, and DiI-labeled RGCs counted. Bandeiraea simplicifolia lectin (BSL-1) was used to identify microglia. The mean number of surviving RGCs at 8 days in eyes treated with 60 microM TCEP was significantly greater than in the vehicle group (1250 +/- 156 vs. 669 +/- 109 cells/mm(2); P = 0.0082). Similar results were recorded at 14 days. Labeling was not a result of microglia phagocytosing dying RGCs. No toxic effect on RGC survival was observed with TCEP injection alone. The sulfhydryl-reducing agent TCEP is neuroprotective of RGCs in an optic nerve crush model. Sulfhydryl oxidative modification may be a final common pathway for the signaling of RGC death by reactive oxygen species after axotomy.

Gene Expression Analysis of Human Epidermal Keratinocytes after N-Acetyl L-Cysteine Treatment Demonstrates Cell Cycle Arrest and Increased Differentiation

Objectives: Several cancer prevention programmes have previously been executed using treatment of antioxidant compounds. The antioxidant N-acetyl L-cysteine (NAC), a membrane-permeable aminothiol, is a sulfhydryl reductant reducing oxidised glutathione, as well as being a precursor of intracellular cysteine and glutathione. A previous report based on the cellular response to NAC treatment showed that NAC induced a 10-fold more rapid differentiation in normal primary keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation. In order to investigate molecular events underlying thechanges in proliferation and differentiation induced by NAC treatment, we performed global gene expression analysis of normal human epidermal keratinocytes in a time series. Methods: Treated samples were compared to untreated samples through a reference design using a spotted cDNA array comprising approximately 30,000 features. B statistics was used to identify differentially expressed genes, and RT-PCR of a selected set of genes was performed to verify differential expression. Results: The number of differentially expressed genes increased over time, starting with 0 at 30 min, 73 at 3 h and increasing to 952 genes at 48 h. Results of the expression analysis showed arrest of the cell cycle and an upregulation of cytoskeletal reorganisation, implicating increased differentiation. A comparison to gene ontology groups indicated downregulation of a large number of genes involved in cell proliferation and regulation of the cell cycle. Conclusions: A significant fraction of the differentially expressed genes could be classified according to their role in the differentiation process, demonstrating that NAC regulates the conversion from proliferation to differentiation at a transcriptional level.

Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.

Coupling of Metal Containing Homing Devices to Liposomes via a Maleimide Linker: Use of TCEP to Stabilize Thiol-groups without Scavenging Metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe3+), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe3+). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2–3 h coupling to the liposomes, Fe3+ is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes.Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe3+ from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself.In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe3+ from Tf. This method can be applied to other metal-containing homing devices as well.

Activation of KCI Cotransport by Urea Induces Dehydration in Both Sickle and Normal Reticulocytes.

KCl Cotransport (KCC) is highly expressed in sickle red blood cells (SS RBC) and recent data have demonstrated its abnormal response to cell swelling and acid pH. We showed that the final MCHC achieved by SS reticulocytes upon activation of KCC by these stimuli was higher than that of normal (AA) reticulocytes (Joiner et al, Blood, in Press). Here we report studies examining the sensitivity of KCC to activation by urea at concentrations present in the kidney and the effect of urea stimulation of KCC on reticulocyte MCHC. KCC fluxes were assayed as Rb uptake over 20 min in isotonic saline solutions buffered with HEPES to pH 7.4 (37°C) containing 0.1 mM ouabain, and 0.01 mM bumetanide, with 27 mM RbCl replacing equimolar NaCl. Under these conditions > 95 % of Rb uptake was Cl-dependent (assessed by sulfamate replacement of Cl). The maximal volume-stimulated KCC flux (VSmax KCC) was measured for each sample in cells swollen isotonically to MCHC < 27 gm/dl (nystatin method). Urea (100 to 1000 mM) increased osmolality of buffers, but did not alter initial MCHC. MCHC of reticulocytes (detected by flow cytometry) was tracked by measuring density changes on calibrated OPTIprep® gradients. Cl-dependent, ouabain- and bumetanide-insensitive Rb influx in both AA and SS RBC was increased by urea, reaching a plateau at 1000 mM urea that was similar to VSmax KCC. SS RBC were more sensitive to urea stimulation than AA RBC: 50% VSSmax KCC was achieved at 330 mM urea in SS RBC vs 550 mM in AA RBC. This effect was sulfhydryl dependent: exposure to the reducing agent dithiothreitol (preincubation for 30 min at 37°C with 10 mM DTT, then 1 mM in flux media) normalized the response to urea in SS RBC, with no effect in AA RBC. When swollen to MCHC 30 gm/dl, SS and AA retics exhibited Regulatory Volume Decrease (RVD) which increased MCHC. RVD in both AA and SS retics was abolished by incubation in sulfamate media, indicating mediation by KCC. As previously reported, final MCHC achieved after two hours incubation by SS retics was greater than AA retics (see Table, Control).Final Reticulocyte MCHC, gm/dl [mean (SD), n = 3]AASSp (AA vs SS)Control31.9 (0.7)34.7 (1.20.03Urea35.3 (0.5)37.8 (0.3)0.002p (Control vs Urea)0.0050.03Urea (600 mM) enhanced RVD in both AA retics and SS retics. Sulfhydryl reduction with DTT had no effect on urea-stimulated RVD in SS reticulocytes. RVD stimulated by urea was complete within 60 minutes, and was irreversible: additional incubation without urea did not lower MCHC. The partially dehydrating effect of brief (10 min) exposure to urea was also irreversible, and was cumulative: cells exposed to two 10 min exposures to high urea (600 mM), separated by 10 min at a low, non-stimulating concentration of urea (100 mM), yielded the same MCHC as a continuous 20 min exposure. These data demonstrate that urea, at concentrations found in the renal medulla, is a powerful stimulant of KCC and intiates a striking RVD in reticulocytes. To the extent that intermittent stimulation of KCC by urea in the kidney occurs in vivo, this could contribute an exaggerated RVD resulting in dehydration of SS reticulocytes.

KCl cotransport mediates abnormal sulfhydryl-dependent volume regulation in sickle reticulocytes

AbstractKCl cotransport (KCC) activation by cell swelling and pH was compared in sickle (SS) and normal (AA) red blood cells (RBCs). KCC fluxes had the same relationship to mean corpuscular hemoglobin concentration (MCHC) in SS and AA RBCs when normalized to the maximal volume-stimulated (VSmax) flux (MCHC < 270 g/L [27 g/dL]). Acid-stimulated (pH 6.9) KCC flux in SS RBCs was 60% to 70% of VSmax KCC versus 20% in AA RBCs. Density gradients were used to track changes in reticulocyte MCHC during KCC-mediated regulatory volume decrease (RVD). Swelling to MCHC of 260 g/L (26 g/dL) produced Cl-dependent RVD that resulted in higher MCHC in SS than AA reticulocytes. In acid pH, RVD was also greater in SS than AA reticulocytes. Sulfhydryl reduction by dithiothreitol (DTT) lowered VSmax KCC flux in AA and SS RBCs by one third but did not alter swelling-induced RVD. DTT lowered acid-activated KCC in SS RBCs by 50% and diminished acid-induced RVD in SS reticulocytes. Thus, swelling activation of KCC is normal in SS RBCs but KCC-mediated RVD produces higher MCHC in SS than AA reticulocytes. Acid activation of KCC is exaggerated in SS RBCs and causes dehydration in SS reticulocytes. KCC response to acid stimulation was mitigated by DTT, suggesting that it arises from sulfhydryl oxidation.

Mice lacking thioredoxin-interacting protein provide evidence linking cellular redox state to appropriate response to nutritional signals.

Thioredoxin-interacting protein (Txnip) is a ubiquitous protein that binds with high affinity to thioredoxin and inhibits its ability to reduce sulfhydryl groups via NADPH oxidation. HcB-19 mice contain a nonsense mutation in Txnip that eliminates its expression. Unlike normal animals, HcB-19 mice have approximately 3-fold increase in insulin levels when fasted. The C-peptide/insulin ratio is normal, suggesting that the hyperinsulinemia is due to increased insulin secretion. Fasted HcB-19 mice are hypoglycemic, hypertriglyceridemic, and have higher than normal levels of ketone bodies. Ablation of pancreatic beta-cells with streptozotocin completely blocks the fasting-induced hypoglycemia/hypertriglyceridemia, suggesting that these abnormalities are due to excess insulin secretion. This is supported by increased hepatic mRNA levels of the insulin-inducible, lipogenic transcription factor sterol-responsive element-binding protein-1c and two of its targets, acetyl-CoA carboxylase and fatty acid synthase. During a prolonged fast, the hyperinsulinemia up-regulates lipogenesis but fails to down-regulate hepatic phosphoenolpyruvate carboxykinase mRNA expression. Hepatic ratios of reduced:oxidized glutathione, established regulators of gluconeogenic/glycolytic/lipogenic enzymes, were elevated 30% in HcB-19 mice, suggesting a loss of Txnip-enhanced sulfhydryl reduction. The altered hepatic enzymatic profiles of HcB-19 mice divert phosphoenolpyruvate to glyceroneogenesis and lipogenesis rather than gluconeogenesis. Our findings implicate Txnip-modulated sulfhydryl redox as a central regulator of insulin secretion in beta-cells and regulation of many of the branch-points of gluconeogenesis/glycolysis/lipogenesis.

Polyphenolic antioxidants mimic the effects of 1,4-dihydropyridines on neurotensin receptor function in PC3 cells.

This study aimed to determine the mechanism(s) by which 1,4-dihydropyridine Ca2+ channel blockers (DHPs) enhance the binding of neurotensin (NT) to prostate cancer PC3 cells and inhibit NT-induced inositol phosphate formation. Earlier work indicated that these effects, which involved the G protein-coupled NT receptor NTR1, were indirect and required cellular metabolism or architecture. At the micromolar concentrations used, DHPs can block voltage-sensitive and store-operated Ca2+ channels, K+ channels, and Na+ channels, and can inhibit lipid peroxidation. By varying [Ca2+] and testing the effects of stimulators and inhibitors of Ca2+ influx and internal Ca2+ release, we determined that although DHPs may have inhibited inositol phosphate formation partly by blocking Ca2+ influx, the effect on NT binding was Ca2+-independent. By varying [K+] and [Na+], we showed that these ions did not contribute to either effect. For a series of DHPs, the activity order for effects on NTR1 function followed that for antioxidant ability. Antioxidant polyphenols (luteolin and resveratrol) mimicked the effects of DHPs and showed structural similarity to DHPs. Antioxidants with equal redox ability, but without structural similarity to DHPs (such as alpha-tocopherol, riboflavin, and N-acetyl-cysteine) were without effect. A flavoprotein oxidase inhibitor (diphenylene iodonium) and a hydroxy radical scavenger (butylated hydroxy anisole) also displayed the effects of DHPs. In conclusion, DHPs indirectly alter NTR1 function in live cells by a mechanism that depends on the drug's ability to donate hydrogen but does not simply involve sulfhydryl reduction.