Lamp1 mediates lipid transport, but is dispensable for autophagy in Drosophila.

The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1 and LAMP2 strongly affects autophagy and cholesterol trafficking. Here we show that the previously uncharacterized Drosophila Lamp1 is a bona fide ortholog of vertebrate LAMP1 and LAMP2. Surprisingly and in contrast to lamp1 lamp2 double-mutant mice, Drosophila Lamp1 is not required for viability or autophagy, suggesting that fly and vertebrate LAMP proteins acquired distinct functions, or that autophagy defects in lamp1 lamp2 mutants may have indirect causes. However, Lamp1 deficiency results in an increase in the number of acidic organelles in flies. Furthermore, we find that Lamp1 mutant larvae have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Because DAGs are the main lipid species used for transport through the hemolymph (blood) in insects, our results indicate broader functions of Lamp1 in lipid transport. Our findings make Drosophila an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes.Abbreviations: aa: amino acid; AL: autolysosome; AP: autophagosome; APGL: autophagolysosome; AV: autophagic vacuole (i.e. AP and APGL/AL); AVi: early/initial autophagic vacuoles; AVd: late/degradative autophagic vacuoles; Atg: autophagy-related; CMA: chaperone-mediated autophagy; Cnx99A: Calnexin 99A; DAG: diacylglycerol; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; FB: fat body; HDL: high-density lipoprotein; Hrs: Hepatocyte growth factor regulated tyrosine kinase substrate; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LDL: low-density lipoprotein; Lpp: lipophorin; LTP: Lipid transfer particle; LTR: LysoTracker Red; MA: macroautophagy; MCC: Manders colocalization coefficient; MEF: mouse embryonic fibroblast MTORC: mechanistic target of rapamycin kinase complex; PV: parasitophorous vacuole; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; Snap: Synaptosomal-associated protein; st: starved; TAG: triacylglycerol; TEM: transmission electron microscopy; TFEB/Mitf: transcription factor EB; TM: transmembrane domain; tub: tubulin; UTR: untranslated region.

Gastroprotective action of the ethanol extract of Leonurus sibiricus L. (Lamiaceae) in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus sibiricus L. (Lamiaceae) is a medicinal plant known in Brazil as "rubim" or "erva de macaé". It is used for various purposes, including stomach disorders. AIM OF THE STUDY: To evaluate the effect of the ethanol extract of the aerial parts of L. sibiricus (EELs) in models of gastric damage in mice. MATERIAL AND METHODS: The effect of EELs (50, 100 and 300 mg/kg, p.o., 1 h before induction) was tested on acidified ethanol (ACEt)-induced gastric ulcers. Additionally, we tested the effect of EELs (by intraduodenal administration) in the pylorus ligation (PL) model. RESULTS: Pretreatment with EELs, at 300 mg/kg, but not 50 and 100 mg/kg, reduced the relative area of gastric ulcers induced by ACEt (p < 0.01) and lipoperoxidation (p < 0.001), and increased the sulfhydryl content (p < 0.01) in the stomach in comparison with the vehicle group. Pretreatment with N-ethylmaleimide (a blocker of non-protein sulfhydryl groups, 10 mg/kg, i.p.) or glibenclamide (a KATP channel blocker, 10 mg/kg, i.p.) inhibited the gastroprotective response caused by EELs (300 mg/kg; p < 0.001), but there were no alterations due to pretreatments with inhibitors of the synthesis of prostaglandins (indomethacin, 10 mg/kg), nitric oxide (L-NAME, 70 mg/kg) or hydrogen sulfide (DL-propargylglycine, 10 mg/kg). Treatment with EELs (300 mg/kg) reduced mucus production (p < 0.001) and the volume of gastric secretion (p < 0.001) after PL without affecting gastric acidity or pH. CONCLUSIONS: These results provide evidence that EELs exerts gastroprotective action in mice, with the participation of oxidative stress and mediation of NP-SH, KATP channels and mucus production.

Context-dependent monoclonal antibodies against protein carbamidomethyl-cysteine.

Protein sulfhydryl residues participate in key structural and biochemical functions. Alterations in sulfhydryl status, regulated by either reversible redox reactions or by permanent covalent capping, may be challenging to identify. To advance the detection of protein sulfhydryl groups, we describe the production of new Rabbit monoclonal antibodies that react with carbamidomethyl-cysteine (CAM-cys), a product of iodoacetamide (IAM) labeling of protein sulfhydryl residues. These antibodies bind to proteins labeled with IAM (but not N-ethylmaleimide (NEM) or acrylamide) and identify multiple protein bands when applied to Western blots of cell lysates treated with IAM. The monoclonal antibodies label a subset of CAM-cys modified peptide sequences and purified proteins (human von Willebrand Factor (gene:vWF), Jagged 1 (gene:JAG1), Laminin subunit alpha 2 (gene:LAMA2), Thrombospondin-2 (gene:TSP2), and Collagen IV (gene:COL4)) but do not recognize specific proteins such as Bovine serum albumin (gene:BSA) and human Thrombospondin-1 (gene:TSP1), Biglycan (gene:BGN) and Decorin (gene:DCN). Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity. In addition to recognition of in vitro labeled proteins, the antibodies were used to identify selected sulfhydryl-containing proteins from living cells that were pulse labeled with IAM. Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins. Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins.

Gastroprotective Effect of Juanislamin on Ethanol-Induced Gastric Lesions in Rats: Role of Prostaglandins, Nitric Oxide and Sulfhydryl Groups in the Mechanism of Action.

Peptic ulcer disease, the most common gastrointestinal disorder, is currently treated with several types of drugs, but all have severe side effects. The aim of the present study was to evaluate the gastroprotective activity of juanislamin, isolated from Calea urticifolia, in a rat model of ethanol-induced gastric lesions. Thirty minutes after orally administering a given dose of juanislamin (from 1 to 30 mg/kg) or carbenoxolone (the reference drug, at 1-100 mg/kg) to rats, 1 mL of ethanol was applied, and the animals were sacrificed 2 h later. The stomachs were removed and opened to measure the total area of lesions in each. To examine the possible participation of prostaglandins, nitric oxide and/or sulfhydryl groups in the mechanism of action of juanislamin, the rats received indomethacin, NG-Nitro-l-arginine methyl ester hydrochloride (l-NAME) or N-ethylmaleimide pretreatment, respectively, before being given juanislamin and undergoing the rest of the methodology. Juanislamin inhibited gastric lesions produced by ethanol in a non-dose-dependent manner, showing the maximum gastroprotective effect (100%) at 10 mg/kg. The activity of juanislamin was not modified by pretreatment with indomethacin, l-NAME or N-ethylmaleimide. In conclusion, juanislamin protected the gastric mucosa from ethanol-induced damage, and its mechanism of action apparently does not involve prostaglandins, nitric oxide or sulfhydryl groups.

Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1.

The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr1007 of KCC2, and Thr203, Thr207 and Thr212 of NKCC1. Dephosphorylation of Thr1007 of KCC2, and Thr207 and Thr212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr233 and Ser373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser940, as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.

Endogenous Cys-Assisted GSH@AgNCs-rGO Nanoprobe for Real-Time Monitoring of Dynamic Change in GSH Levels Regulated by Natural Drug.

Glutathione (GSH) levels are closely related to the homeostasis of redox state which directly affects human disease occurrence by regulating cell apoptosis. Hence, real-time monitoring of dynamic changes in intracellular GSH levels is urgently needed for disease early diagnosis and evaluation of therapy efficiency. In this study, an endogenous cysteine (Cys)-assisted detection system based on GSH@AgNCs and reduced graphene oxide (rGO) with high sensitivity and specificity was developed for GSH detection. Compared with GSH, GSH@AgNCs with weaker affinity and bonding force was quite easier to extrude from the rGO surface when competing against GSH, leading to the obvious change in fluorescence signal. This phenomenon was termed as "a crowding out effect". Furthermore, the presence of Cys can improve GSH assay sensitivity by enhancing the quenching efficiency of rGO on the GSH@AgNCs. In vitro assay indicated that the efficiency of fluorescence recovery was positively related with GSH concentration in the range from 0 to 10 mM. In addition, the method was employed for real-time monitoring of the dynamic changes in GSH levels regulated by natural drugs. The imaging results showed that the natural compound 3 (C3) can downregulate GSH levels in HepG2 cells, which was accompanied by reactive oxygen species (ROS) release and apoptosis induction. Finally, the method was used to monitor the change of GSH levels in serum samples with chronic hepatitis B (CHB) infection. The results demonstrated that the occurrence and development of CHB may be positively correlated with GSH levels to some extent. Overall, the above results demonstrate the potential application of this new nanosystem in anticancer natural drug screening and clinical assay regarding GSH levels.

Extracellular blebs: Artificially-induced extracellular vesicles for facile production and clinical translation.

Extracellular vesicles (EVs) have emerged as promising biologic and comprehensive therapies for precision medicine. Despite their potential demonstrated at the benchtop, few EV formulations have made it to the clinic due to challenges in regulatory compliant scalable production; including purity, homogeneity, and reproducibility. For translation of this technology, there is a strong need for novel production methods that can meet clinical production criteria. Initial research aimed to address these challenges by taking advantage of natural pathways to increase EV yields. Such "conventional" approaches moderately increased yields but produced inhomogeneous EVs. Additionally, as there are currently no standard methods for isolation, characterization, or quantification, isolated EVs were often impure, contaminated with proteins and other biomacromolecules, and highly diverse in function. The use of shear stress and extrusion methods for EV-like vesicle production has also been investigated. While these processes can produce large EV-like vesicle yields nearly immediately, the harsh processes still result in inhomogeneous loading, and still suffer from poor purity. Chemically-induced membrane blebbing is a promising alternative production method that has the potential to overcome the previously insurmountable barriers of these current methods. This technique produces pure, and well defined EV-like vesicles, termed extracellular blebs (EBs), in clinically relevant scales over the course of minutes to hours. Furthermore, blebbing agents act on the cell in a way which locks the current surface properties and contents, preventing change, allowing for homogeneous EB production, and further preventing post-production changes. EBs may provide a promising pathway for clinical translation of EV technology.

Oxidative Modification of Redox Proteins: Role in the Regulation of HBL-100 Cell Proliferation.

HBL-100 breast epithelial cells were cultured with a blocker (N-ethylmaleimide) and protector (1,4-dithioerythritol) of SH groups. The study assessed changes in redox potential of glutathione and thioredoxin systems, intensity of oxidative modification of proteins, ROS production, and cell proliferation. The roles of thioredoxin system and protein oxidative modification in HBL-100 cell proliferation under redox status modulation were established. The role of carbonylated thioredoxin in arrest of the cell cycle in S-phase was demonstrated, which could be used for targeted therapy of the diseases accompanied by oxidative stress and disturbed redox status.

Pharmacological analysis of transmission activation of two aphid-vectored plant viruses, turnip mosaic virus and cauliflower mosaic virus.

Turnip mosaic virus (TuMV, family Potyviridae) and cauliflower mosaic virus (CaMV, family Caulimoviridae) are transmitted by aphid vectors. They are the only viruses shown so far to undergo transmission activation (TA) immediately preceding plant-to-plant propagation. TA is a recently described phenomenon where viruses respond to the presence of vectors on the host by rapidly and transiently forming transmissible complexes that are efficiently acquired and transmitted. Very little is known about the mechanisms of TA and on whether such mechanisms are alike or distinct in different viral species. We use here a pharmacological approach to initiate the comparison of TA of TuMV and CaMV. Our results show that both viruses rely on calcium signaling and reactive oxygen species (ROS) for TA. However, whereas application of the thiol-reactive compound N-ethylmaleimide (NEM) inhibited, as previously shown, TuMV transmission it did not alter CaMV transmission. On the other hand, sodium azide, which boosts CaMV transmission, strongly inhibited TuMV transmission. Finally, wounding stress inhibited CaMV transmission and increased TuMV transmission. Taken together, the results suggest that transmission activation of TuMV and CaMV depends on initial calcium and ROS signaling that are generated during the plant's immediate responses to aphid manifestation. Interestingly, downstream events in TA of each virus appear to diverge, as shown by the differential effects of NEM, azide and wounding on TuMV and CaMV transmission, suggesting that these two viruses have evolved analogous TA mechanisms.

Gastroprotection of Calein D against Ethanol-Induced Gastric Lesions in Mice: Role of Prostaglandins, Nitric Oxide and Sulfhydryls.

Peptic ulcers are currently treated with various drugs, all having serious side effects. The aim of this study was to evaluate the gastroprotective activity of calein D (from Calea urticifolia), a sesquiterpene lactone with a germacrane skeleton. Gastric lesions were induced in mice by administering ethanol (0.2 mL) after oral treatment with calein D at 3, 10 and 30 mg/kg, resulting in 13.15 ± 3.44%, 77.65 ± 7.38% and 95.76 ± 2.18% gastroprotection, respectively, to be compared with that of the control group. The effect found for 30 mg/kg of calein D was not reversed by pretreatment with NG-nitro-l-arginine methyl ester (l-NAME, 70 mg/kg, ip), indomethacin (10 mg/kg, sc) or N-ethylmaleimide (NEM, 10 mg/kg, sc). Hence, the mechanism of action of calein D does not involve NO, prostaglandins or sulfhydryl compounds. Calein D was more potent than carbenoxolone, the reference drug. The findings for the latter are in agreement with previous reports.

The role of redox-dependent mechanisms in heme release from hemoglobin and erythrocyte hemolysates.

Toxicity mediated by free heme has emerged as an important element of end organ injuries and adverse outcomes in critically ill disease states. Free heme is thought to be derived from oxidative denaturation of free hemoglobin, secondary to red cell hemolysis. In this study, we evaluated the ability of oxidants (H2O2, nitrite, peroxynitrite and hypochlorous acid) formed during inflammation to cause heme release from purified hemoglobin and hemolysates, at pH 7.4 and 6.8. Supraphysiological concentrations of nitrite, peroxynitrite or hypochlorous acid were required to cause appreciable heme release from either free hemoglobin or hemolysates. However, H2O2 administered as a bolus or generated in situ, was more potent at promoting free heme release with free hemoglobin. With hemolysates, only in situ H2O2 formation resulted in significant free heme release. In all cases, free heme release was higher at lower pH and required oxidation of ferrous heme, but was not dependent on ferrylHb formation. Moreover, ligating ferric heme with cyanide or blocking the ß93Cys did not prevent, but in fact increased free heme release. The salient observations from this study are that free heme release is likely mediated by continuous generation of H2O2 versus other heme oxidants, and facilitated at low pH.

Involvement of the cAMP-Dependent Pathway in Dextromethorphan-Induced Inhibition of Spontaneous Glutamate Transmission in the Nucleus Tractus Solitarius Neurons of Guinea Pigs.

Dextromethorphan (DEX) presynaptically decreases glutamatergic transmission in second-order neurons of the nucleus tractus solitarius (TS). To clarify the inhibitory mechanism of DEX, the present study examined the interaction of DEX with cAMP. The effects of DEX on miniature and TS-evoked excitatory postsynaptic currents (mEPSCs and eEPSCs) were recorded under activation of the cAMP-dependent pathway using the brainstem slices. An increase in cAMP by forskolin counteracted the inhibitory effect of DEX on mEPSCs. Eight-Bromo-cAMP and N-ethylmaleimide also attenuated the DEX effect. However, forskolin had negligible effects on the DEX-induced inhibition of eEPSCs. This suggests that DEX decreases spontaneous glutamate release by inhibiting the cAMP-dependent pathway and synchronous release by another unknown mechanism.

Pseudomonas aeruginosa glutathione biosynthesis genes play multiple roles in stress protection, bacterial virulence and biofilm formation.

Pseudomonas aeruginosa PAO1 contains gshA and gshB genes, which encode enzymes involved in glutathione (GSH) biosynthesis. Challenging P. aeruginosa with hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide increased the expression of gshA and gshB. The physiological roles of these genes in P. aeruginosa oxidative stress, bacterial virulence, and biofilm formation were examined using P. aeruginosa ΔgshA, ΔgshB, and double ΔgshAΔgshB mutant strains. These mutants exhibited significantly increased susceptibility to methyl viologen, thiol-depleting agent, and methylglyoxal compared to PAO1. Expression of functional gshA, gshB or exogenous supplementation with GSH complemented these phenotypes, which indicates that the observed mutant phenotypes arose from their inability to produce GSH. Virulence assays using a Drosophila melanogaster model revealed that the ΔgshA, ΔgshB and double ΔgshAΔgshB mutants exhibited attenuated virulence phenotypes. An analysis of virulence factors, including pyocyanin, pyoverdine, and cell motility (swimming and twitching), showed that these levels were reduced in these gsh mutants compared to PAO1. In contrast, biofilm formation increased in mutants. These data indicate that the GSH product and the genes responsible for GSH synthesis play multiple crucial roles in oxidative stress protection, bacterial virulence and biofilm formation in P. aeruginosa.

N-Ethylmaleimide differentiates between the M2- and M4-autoreceptor-mediated inhibition of acetylcholine release in the mouse brain.

Muscarinic M2 and M4 receptors resemble each other in brain distribution, function, and Gi/o protein signaling. However, there is evidence from human recombinant receptors that the M4 receptor also couples to Gs protein whereas such an alternative signaling is of minor importance for its M2 counterpart. The question arises whether this property is shared by native receptors, e.g., the murine hippocampal M2- and the striatal M4-autoreceptor. To this end, the electrically evoked tritium overflow was studied in mouse hippocampal and striatal slices pre-incubated with 3H-choline. 3H-Acetylcholine release in either region was inhibited by the potent muscarinic receptor agonist iperoxo (pIC50 8.6-8.8) in an atropine-sensitive manner (apparent pA2 8.6-8.8); iperoxo was much more potent than oxotremorine (pIC50 6.5-6.6). In hippocampal slices, N-ethylmaleimide (NEM) 32 µM, which inactivates Gi/o proteins, tended to shift the concentration-response curve of iperoxo (pIC50 8.8) to the right (pIC50 8.5) and depressed its maximum from 85 to 69%. In striatal slices, the inhibitory effect of iperoxo declined at concentrations higher than 0.1 µM, yielding a biphasic curve with a pIC50 of 8.6 for the falling part and a pEC50 of 6.4 for the rising part of the curve. The inhibitory effect of iperoxo 10 µM (47%) after NEM pre-treatment was lower by about 35% compared to the maximum (74%) obtained without NEM. In conclusion, our data, which need to be confirmed by pertussis toxin, might suggest that in the striatum, unlike the hippocampus, stimulatory Gs protein comes into play at high concentrations of a muscarinic receptor agonist.

The effect of xanthine oxidase and hypoxanthine on the permeability of red cells from patients with sickle cell anemia.

Red cells from patients with sickle cell anemia (SCA) are under greater oxidative challenge than those from normal individuals. We postulated that oxidants generated by xanthine oxidase (XO) and hypoxanthine (HO) contribute to the pathogenesis of SCA through altering solute permeability. Sickling, activities of the main red cell dehydration pathways (Psickle , Gardos channel, and KCl cotransporter [KCC]), and cell volume were measured at 100, 30, and 0 mmHg O2 , together with deoxygenation-induced nonelectrolyte hemolysis. Unexpectedly, XO/HO mixtures had mainly inhibitory effects on sickling, Psickle , and Gardos channel activities, while KCC activity and nonelectrolyte hemolysis were increased. Gardos channel activity was significantly elevated in red cells pharmacologically loaded with Ca2+ using the ionophore A23187, consistent with an effect on the transport system per se as well as via Ca2+ entry likely via the Psickle pathway. KCC activity is controlled by several pairs of conjugate protein kinases and phosphatases. Its activity, however, was also stimulated by XO/HO mixtures in red cells pretreated with N-ethylmaleimide (NEM), which is thought to prevent regulation via changes in protein phosphorylation, suggesting that the oxidants formed could also have direct effects on this transporter. In the presence of XO/HO, red cell volume was better maintained in deoxygenated red cells. Overall, the most notable effect of XO/HO mixtures was an increase in red cell fragility. These findings increase our understanding of the effects of oxidative challenge in SCA patients and are relevant to the behavior of red cells in vivo.

Intracellular acidification reduces l-arginine transport via system y+L but not via system y+/CATs and nitric oxide synthase activity in human umbilical vein endothelial cells.

l-Arginine is taken up via the cationic amino acid transporters (system y+/CATs) and system y+L in human umbilical vein endothelial cells (HUVECs). l-Arginine is the substrate for endothelial NO synthase (eNOS) which is activated by intracellular alkalization, but nothing is known regarding modulation of system y+/CATs and system y+L activity, and eNOS activity by the pHi in HUVECs. We studied whether an acidic pHi modulates l-arginine transport and eNOS activity in HUVECs. Cells loaded with a pH-sensitive probe were subjected to 0.1-20 mmol/L NH4Cl pulse assay to generate pHi 7.13-6.55. Before pHi started to recover, l-arginine transport (0-20 or 0-1000 µmol/L, 10 s, 37 °C) in the absence or presence of 200 µmol/L N-ethylmaleimide (NEM) (system y+/CATs inhibitor) or 2 mmol/L l-leucine (systemy+L substrate) was measured. Protein abundance for eNOS and serine1177 or threonine495 phosphorylated eNOS was determined. The results show that intracellular acidification reduced system y+L but not system y+/CATs mediated l-arginine maximal transport capacity due to reduced maximal velocity. Acidic pHi reduced NO synthesis and eNOS serine1177 phosphorylation. Thus, system y+L activity is downregulated by an acidic pHi, a phenomenon that may result in reduced NO synthesis in HUVECs.

Molecular mechanism and characterization of self-assembly of feather keratin gelation.

Protein gels with controlled viscoelastic properties could find numerous material and biomedical applications. Feather keratin is naturally abundant protein while its gelation property has not been explored. In this study hydrogel from fully reduced feather keratin was prepared by dialysis. The objectives of this work were to study the molecular mechanism of self-assembly of feather keratin gel and to characterize the structural and viscoelastic properties of hydrogels prepared under various pHs (3-9) and temperatures (50-90°C). Re-oxidation of free cysteine thiols and formation of hydrophobic interactions and hydrogen bond were determined as the main stabilizing forces in self-assembly of feather keratin gel. Adding thiol blocking agent of N-ethylmaleimide leads to reduced storage modulus of keratin gel; gelation was completely inhibited at 82% blockage of free thiols. Increasing temperature decreased storage modulus, while gelation at pH 3 resulted in stiffer gels compared to pHs of 5, 7 and 9. Feather keratin gels with tunable viscoelastic properties could find applications as engineered scaffolds for different tissues.

Negative modulation of the GABAA ρ1 receptor function by l-cysteine.

l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABAA ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABAA ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABAA ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABAA ρ1 receptors.

Endogenous nitric oxide inhibits spinal NMDA receptor activity and pain hypersensitivity induced by nerve injury.

The role of nitric oxide (NO) in nociceptive transmission at the spinal cord level remains uncertain. Increased activity of spinal N-methyl-d-aspartate (NMDA) receptors contributes to development of chronic pain induced by peripheral nerve injury. In this study, we determined how endogenous NO affects NMDA receptor activity of spinal cord dorsal horn neurons in control and spinal nerve-ligated rats. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited NMDA receptor currents of spinal dorsal horn neurons in both sham control and nerve-injured rats. Inhibition of neuronal nitric oxide synthase (nNOS) or blocking the S-nitrosylation reaction with N-ethylmaleimide abolished the inhibitory effects of l-arginine on NMDA receptor currents recorded from spinal dorsal horn neurons in sham control and nerve-injured rats. However, bath application of the cGMP analog 8-bromo-cGMP had no significant effects on spinal NMDA receptor currents. Inhibition of soluble guanylyl cyclase also did not alter the inhibitory effect of l-arginine on spinal NMDA receptor activity. Furthermore, knockdown of nNOS with siRNA abolished the inhibitory effects of l-arginine, but not SNAP, on spinal NMDA receptor activity in both groups of rats. Additionally, intrathecal injection of l-arginine significantly attenuated mechanical or thermal hyperalgesia induced by nerve injury, and the l-arginine effect was diminished in rats treated with a nNOS inhibitor or nNOS-specific siRNA. These findings suggest that endogenous NO inhibits spinal NMDA receptor activity through S-nitrosylation. NO derived from nNOS attenuates spinal nociceptive transmission and neuropathic pain induced by nerve injury.

[Redox-dependent mechanisms of regulation of breast epithelial cell proliferation]. / Redoks-zavisimye mekhanizmy reguliatsii proliferatsii kletok épiteliia molochnoi zhelezy.

Activation of free radical oxidation in different cell types, including breast epithelial cells, may result in damage to macromolecules, in particular, proteins taking part in regulation of cell proliferation and apoptosis. The glutathione, glutaredoxin and thioredoxin systems play an essential role in maintaining intracellular redox homeostasis. Due to this fact, modulation of cellular redox status under the effect of an SH group inhibitor and an SH group protector may be used as a model for studying the role of redox proteins and glutathione in regulating cell proliferation in different pathological processes. In this study we have evaluated the state of the thioredoxin, glutaredoxin and glutathione systems as well as their role in regulating proliferation of HBL-100 breast epithelial cells under redox status modulation with N-ethylmaleimide (NEM) and 1,4-dithioerythriol (DTE). Modulating the redox status of breast epithelial cells under the effect of NEM and DTE influences the functional activity of glutathione-dependent enzymes, glutaredoxin, thioredoxin, and thioredoxin reductase through changes in the GSH and GSSG concentrations. In HBL-100 cells under redox-status modulation, we have found an increase in the number of cells in the S-phase of the cell cycle and a decrease in the number of cells in the G0/G1 and G2/М phases, as opposed to the values in the intact culture. The proposed model of proliferative activity of cells under redox status modulation may be used for development of new therapeutic approaches for treatment of diseases accompanied by oxidative stress generation.