Dithiothreitol reduces oxidative stress and necrosis caused by ultraviolet A radiation in L929 fibroblasts.

Ultraviolet A (UVA) radiation, present in sunlight, can induce cell redox imbalance leading to cellular damage and even cell death, compromising skin health. Here, we evaluated the in vitro antioxidant and photochemoprotective effect of dithiothreitol (DTT). DTT neutralized the free radicals 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+), 2,2-diphenyl-1-picrylhydrazyl (DPPH·), and superoxide anion (O2·-) in in vitro assays, as well as the ferric ion (Fe3+) in the ferric reducing antioxidant power (FRAP) assay. We also evaluated the effect of DTT pre-treatment in L929 dermal fibroblasts and DTT (50 and 100 µM) led to greater cell viability following UVA-irradiation compared to cells that were untreated. Furthermore, the pre-treatment of cells with DTT prevented the increase of intracellular reactive oxygen species (ROS) production, including hydrogen peroxide (H2O2), lipid peroxidation, and DNA condensation, as well as the decrease in mitochondrial membrane potential (Δψm), that occurred following irradiation in untreated cells. The endogenous antioxidant system of cells was also improved in irradiated cells that were DTT pre-treated compared to the untreated cells, as the activity of the superoxide dismutase (SOD) and catalase (CAT) enzymes remained as high as non-irradiated cells, while the activity levels were depleted in the untreated irradiated cells. Furthermore, DTT reduced necrosis in UVA-irradiated fibroblasts. Together, these results showed that DTT may have promising use in the prevention of skin photoaging and photodamage induced by UVA, as it provided photochemoprotection against the harmful effects of this radiation, reducing oxidative stress and cell death, due mainly to its antioxidant capacity.

Dithiothreitol Affects the Fertilization Response in Immature and Maturing Starfish Oocytes.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.

Inactivation of Human Aldehyde Oxidase by Small Sulfhydryl-Containing Reducing Agents.

Human aldehyde oxidase (hAOX1) is a molybdoflavoenzyme that belongs to the xanthine oxidase (XO) family. hAOX1 is involved in phase I drug metabolism, but its physiologic role is not fully understood to date, and preclinical studies consistently underestimated hAOX1 clearance. In the present work, we report an unexpected effect of the common sulfhydryl-containing reducing agents, e.g., dithiothreitol (DTT), on the activity of hAOX1 and mouse aldehyde oxidases. We demonstrate that this effect is due to the reactivity of the sulfido ligand bound at the molybdenum cofactor with the sulfhydryl groups. The sulfido ligand coordinated to the Mo atom in the XO family of enzymes plays a crucial role in the catalytic cycle and its removal results in the total inactivation of these enzymes. Because liver cytosols, S9 fractions, and hepatocytes are commonly used to screen the drug candidates for hAOX1, our study suggests that DTT treatment of these samples should be avoided, otherwise false negative results by an inactivated hAOX1 might be obtained. SIGNIFICANCE STATEMENT: This work characterizes the inactivation of human aldehyde oxidase (hAOX1) by sulfhydryl-containing agents and identifies the site of inactivation. The role of dithiothreitol in the inhibition of hAOX1 should be considered for the preparation of hAOX1-containing fractions for pharmacological studies on drug metabolism and drug clearance.

Long-term storage protocol of reagent red blood cells treated with 0.01M dithiothreitol (DTT) for pre-transfusion testing of patients receiving anti-CD38 therapy, daratumumab.

OBJECTIVE: Use red blood cell stabilizer to store the antibody screening and antibody identification reagent red blood cells (RBCs) treated with 0.01 mol/L DTT and investigate its value in the pre-transfusion examinations of patients treated with daratumumab. METHOD: Determined the optimal incubation time for the 0.01 mol/L DTT-treated RBCs method by evaluating the effect of treatment at different time points. Added ID-CellStab to store DTT-treated RBCs, determined the maximum shelf life of reagent RBCs by monitoring the hemolysis index, and assessed changes in the antigenicity of blood group antigens on the surface of RBCs during storage with antibody reagents. RESULT: A protocol for long-term storage of reagent red blood cells treated with the 0.01 mol/L DTT method was established. The optimal incubation time was 40-50 min. RBCs could be stored stably for 18 days after adding ID-CellStab. The protocol was able to eliminate pan-agglutination caused by daratumumab, with no significant changes in the antigens of most blood group systems, except for some attenuation of K antigen and Duffy blood group system antigens during the storage period. CONCLUSION: The storage protocol of reagent RBCs based on the 0.01 mol/L DTT method does not affect the detection of most blood group antibodies and retains a certain degree of detection ability for anti-K antibodies, allowing patients treated with daratumumab to quickly perform pre-transfusion examinations, making up for the shortcomings of currently commercial reagent RBCs.

Mitigation of therapeutic anti-CD38 antibody interference with fab fragments: How well does it perform?

BACKGROUND: Administration of anti-CD38 antibodies is a state-of-the-art therapy for patients diagnosed with multiple myeloma (MM). However, this treatment frequently leads to pan-agglutination of red blood cells (RBCs) in patients' serological testing making accurate blood typing and timely transfusion of compatible blood a challenging effort. The antigen masking indirect antiglobulin test (AMIAT) is an approach to address this diagnostic challenge. STUDY DESIGN AND METHODS: A new reagent, called DaraEx plus, uses anti-CD38 Fab fragments to mitigate the anti-CD38 antibody interference in serological assays by masking CD38 on the cell surface. Its performance is extensively examined with commercial sera as well as with patient samples, and compared to the current standard method using dithiothreitol (DTT), which denatures the CD38 antigens on test panel erythrocytes. RESULTS: In the Bio-Rad ID System, DaraEx plus effectively mitigated the interference caused by anti-CD38 antibodies in 86% of patient samples tested while DTT was successful in only 68%. Moreover, there was no negative influence on DTT-sensitive blood group systems such as KEL upon DaraEx plus treatment. The agglutination reactions of all tested anti-CD38 antibodies (Daratumumab, Felzartamab, and Isatuximab) were inhibited by DaraEx plus. The treatment was successful only if DaraEx plus was added to the test cells before the sample. Some of the other gel card systems tested showed background reactions with DaraEx plus-treated cells. CONCLUSION: DaraEx plus treatment is straightforward and quick to perform. In the Bio-Rad ID System, it is superior to DTT treatment in the prevention of anti-CD38 antibody interference.

Differential effects of DTT on HEWL amyloid fibrillation and fibril morphology at different pH.

Proteins can transform from their native state to a state having fibrillar aggregates characterized by cross ß sheet structure. The fibrillar aggregates are known as amyloid and have been linked to several disorders. Disulfide bonds in proteins are one of the important factors that determine the propensity of aggregation. Hen Egg White Lysozyme (HEWL) was used by us as a model protein to decipher the role disulfide bonds play in the amyloid fibril formation and fibril morphology by using Dithiothreitol (DTT) as reducing agent at pH 2.7 and pH 7.4. We found that DTT can have different effects on HEWL amyloid depending on pH and the buffer used for preparing the amyloid fibrils. Our studies highlight the critical role of non-native disulfide bonds in amyloidogenesis and how disruption of these bonds can greatly affect the fibrillation process. Overall, these studies throw light on the fibrillation mechanism and can be explored further in designing effective inhibitors against amyloidosis.

Exposure to emamectin benzoate confers cytotoxic effects on human molt-4 T-cells and possible ameliorative role of vitamin E and dithiothreitol.

Emamectin benzoate (EMB) is an avermectin insecticide that is extensively used for pest control, but there are few reports concerning its cytotoxic effects on human lymphocytes. In the current study, the hematotoxicity of EMB was evaluated in Molt-4 T-cells, a human T-lymphoblastic cell line with high motility, and the role of vitamin E (VitE) and dithiothreitol (DTT) in attenuating EMB cytotoxicity was characterized. Exposure of Molt-4 cells to EMB decreased cell viability and proliferation, induced a loss of cell clusters, and significantly increased membrane collapse and chromatin condensation. Moreover, EMB significantly increased cell death and suppressed transglutaminase activity. EMB treatment modulated the NF-κB signaling pathway, decreased the expression of p105, p50, and p65/RelA in cytosolic and nuclear fractions, and increased nuclear IκBα expression. EMB increased oxidative stress, as demonstrated by a significant increase in the levels of reactive oxygen species (ROS). Treatment with non-cytotoxic concentrations of VitE or DTT ameliorated the hematotoxicity induced by pretreatment with EMB, increased Molt-4 cell viability, raised the IC50 values of EMB, limited intracellular ROS generation, and mitigated EMB-mediated effects on NF-κB signaling. The results indicate the potential cytotoxicity of EMB on human lymphocytes, and demonstrate that VitE and DTT treatment can reduce the cytotoxic effects of EMB.

[Dithiothreitol promotes apoptosis in rat hepatocytes by inhibiting family with sequence similarity 134 member B (FAM134B)-mediated endoplasmic reticulophagy].

Objective To investigate the effect of family with sequence similarity 134 member B (FAM134B)-mediated endoplasmic reticulophagy on apoptosis of hepatocytes induced by endoplasmic reticulum stress (ERS) and identify its potential regulatory mechanism. Methods BRL-3A cells were treated with 0, 0.5, 1.0, 2.0, 4.0, 6.0 mmol/L dithiothreitol (DTT) for 48 hours. The effect of DTT treatment on the proliferation and apoptosis was analyzed using real time cellular dynamic analysis (RTCA) and flow cytometry. The level of proteins related to ERS, endoplasmic reticulophagy, mitochondria-endoplasmic reticulum contact sites (MERCs), and mitochondrial apoptosis pathway were determined using Western blot analysis. Co-localization of ER and lysosomes were detected using ER and lysosomal fluorescence probes. A Ca2+ fluorescence probe was used to detect the level of Ca2+ in mitochondria. Results DTT treatment significantly inhibited cell proliferation and promoted apoptosis in hepatocytes. The levels of proteins related to ERS and endoplasmic reticulophagy, MERCs and the mitochondrial apoptosis pathway significantly increased in BRL-3A cells treated with DTT. DTT treatment decreased the ER-lysosome co-localization and enhanced the fluorescence intensity of Ca2+ in mitochondria. Conclusion DTT aggravates hepatocyte apoptosis by inhibiting FAM134B-mediated endoplasmic reticulophagy and enhancing the level of mitochondrial Ca2+.

Hydrogel Dressing Containing Basic Fibroblast Growth Factor Accelerating Chronic Wound Healing in Aged Mouse Model.

Due to the decreasing self-repairing ability, elder people are easier to form chronic wounds and suffer from slow and difficult wound healing. It is desirable to develop a novel wound dressing that can accelerate chronic wound healing in elderly subjects to decrease the pain of patients and save medical resources. In this work, Heparin and basic fibroblast growth factor(bFGF) were dissolved in the mixing solution of 4-arm acrylated polyethylene glycol and dithiothreitol to form hydrogel dressing in vitro at room temperature without any catalysts, which is convenient and easy to handle in clinic application. In vitro re-lease test shows the bFGF could be continuously released for at least 7 days, whereas the dressing surface integrity maintained for 3 days degradation in PBS solution. Three groups of treatments including bFGF-Gel, bFGF-Sol and control without any treatment were applied on the full-thickness wound on the 22 months old mice back. The wound closure rate and histological and immunohistochemical staining all illustrated that bFGF-Gel displayed a better wound healing effect than the other two groups. Thus, as-prepared hydrogel dressing seems supe-rior to current clinical treatment and more effective in elderly subjects, which shows promising potential to be applied in the clinic.

Bovine ICSI: limiting factors, strategies to improve its efficiency and alternative approaches.

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique mainly used to overcome severe infertility problems associated with the male factor, but in cattle its efficiency is far from optimal. Artificial activation treatments combining ionomycin (Io) with 6-dimethylaminopurine after piezo-ICSI or anisomycin after conventional ICSI have recently increased the blastocyst rate obtained. Compounds to capacitate bovine spermatozoa, such as heparin and methyl-ß-cyclodextrin and compounds to destabilize sperm membranes such as NaOH, lysolecithin and Triton X-100, have been assessed, although they have failed to substantially improve post-ICSI embryonic development. Disulfide bond reducing agents, such as dithiothreitol (DTT), dithiobutylamine and reduced glutathione, have been assessed to decondense the hypercondensed head of bovine spermatozoa, the two latter being more efficient than DTT and less harmful. Although piezo-directed ICSI without external activation has generated high fertilization rates and modest rates of early embryo development, other studies have required exogenous activation to improve the results. This manuscript thoroughly reviews the different strategies used in bovine ICSI to improve its efficiency and proposes some alternative approaches, such as the use of extracellular vesicles (EVs) as 'biological methods of oocyte activation' or the incorporation of EVs in the in vitro maturation and/or culture medium as antioxidant defence agents to improve the competence of the ooplasm, as well as a preincubation of the spermatozoa in estrous oviductal fluid to induce physiological capacitation and acrosome reaction before ICSI, and the use of hyaluronate in the sperm immobilization medium.

Effective Neutralization of Daratumumab Effects on Pre-Transfusion Testing: a Method Modification.

BACKGROUND: The anti-CD38 antibody daratumumab is a common multiple myeloma treatment. As the erythrocyte's membrane expresses CD38, Daratumumab-treated samples show agglutination in serological pre-transfusion tests, hindering detection of erythrocyte alloantibodies. Dithiothreitol interferes with erythrocyte antigens, affecting investigation of unexpected antibodies. DARAEx®, an anti-CD38 neutralizing agent, overcomes daratumumab-induced effects, without dithiothreitol's interferences. DARAEx® is applied only in Biorad columns. This study aimed to provide a DARAEx® protocol for application with the Grifols platform. METHODS: We introduced a modified DARAEx® protocol (AssutaBB protocol) and performed antibody screenings on samples from nineteen daratumumab-treated patients. RESULTS: The AssutaBB protocol provided antibody screen results for all patients, exactly as established in the default manufacturing protocol. Eleven patients presented natural negative antibody screens; eight presented positive K/E antibodies. CONCLUSIONS: AssutaBB allows the use of the more widespread Grifols platform in daratumumab-treated patients.

Family with sequence similarity 134 member B-mediated reticulophagy ameliorates hepatocyte apoptosis induced by dithiothreitol.

BACKGROUND: Endoplasmic reticulum (ER) stress-related hepatocyte apoptosis is responsible for multiple hepatic diseases. Previous studies have revealed that endoplasmic reticulophagy (ER-phagy) promotes the selective clearance of damaged ER fragments during ER stress, playing a crucial role in maintaining ER homeostasis and inhibiting apoptosis. Family with sequence similarity 134 member B (FAM134B) is a receptor involved in ER-phagy that can form a complex with calnexin (CNX) and microtubule-associated protein 1 light chain 3 (LC3). The complex can mediate the selective isolation of ER fragments to attenuate hepatocyte apoptosis. However, the precise regulatory mechanisms remain unclear. AIM: To elucidate the effect of FAM134B-mediated ER-phagy on ER stress-induced apoptosis in buffalo rat liver 3A (BRL-3A) rat hepatocytes and the potential regulatory mechanisms. METHODS: ER stress-related hepatocyte apoptosis was induced using dithiothreitol (DTT). Proteins related to ER stress and autophagy were measured with western blotting. Protein complex interactions with FAM134B were isolated by co-immunoprecipitation. ER-phagy was evaluated in immunofluorescence experiments. Cell cycle distribution and apoptosis were measured by flow cytometry. Mitochondrial Ca2+ levels were evaluated by the co-localization of intracellular Ca2+-tracker and Mito-tracker. The small interfering RNA against FAM134B was used to knockdown FAM134B in BRL-3A cells. RESULTS: ER stress-related and autophagy-related proteins in BRL-3A cells were elevated by both short and long-term DTT treatment. Furthermore, co-immunoprecipitation confirmed an interaction between FAM134B, CNX, FAM134B, and LC3 in BRL-3A cells. Immunofluorescence assays revealed that autolysosomes significantly decreased following short-term DTT treatment, but increased after long-term treatment. Mitochondrial Ca2+ levels and apoptotic rates were dramatically elevated, and more cells were arrested in the G1 stage after short-term DTT treatment; however, these decreased 48 h later. Moreover, FAM134B downregulation accelerated mitochondrial apoptotic pathway activation and aggravated hepatocyte apoptosis under ER stress. CONCLUSION: FAM134B-mediated ER-phagy attenuates hepatocyte apoptosis by suppressing the mitochondrial apoptotic pathway. Our findings provide new evidence highlighting the importance of FAM134B-mediated ER-phagy in attenuating hepatocyte apoptosis.

[Evaluation of Low Concentration Dithiothreitol for Negating the Monoclonal Anti-CD38 Interference with Transfusion Compatibility Testing].

OBJECTIVE: To investigate the effectiveness and safety of low concentration dithiothreitol (DTT) in removing the interference of monoclonal anti-CD38 on transfusion compatibility testing, and develop a reasonable clinical transfusion strategy. METHODS: The blood type, direct antiglobulin testing (DAT) and antibody screening were tested according to standard methods. Antibody screening cells and donor's red blood cells were treated by DTT 0.2, 0.1, 0.05, 0.02, 0.01 and 0.005 mol/L, and antibody screening and cross-matching of serums after monoclonal anti-CD38 treatment were performed by anti-human globulin card. RESULTS: The 0.01 mol/L DTT at 37℃ for 30 minutes could remove the effect of monoclonal anti-CD38 on antibody screening and cross-matching, meanwhile retain their effectiveness in detecting anti-K, anti-LW, anti-JMH, anti-Lub, anti-e, anti-Dia and anti-Jka alloantibodies. All the 10 patients had no acute or delayed haemolytic transfusion reactions and their routine blood tests showed that the red blood cells transfusion was effective. CONCLUSION: The 0.01 mol/L DTT is a safe and effective method for removing the interference of monoclonal anti-CD38 with transfusion compatibility testing, while retaining the ability to detect most alloantibodies.

Effect of addition of ascorbate, dithiothreitol or a caspase-3 inhibitor to cryopreservation medium on post-thaw survival of bovine embryos produced in vitro.

Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase-3 (z-DEVD-fmk) could improve post-thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled-rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1-3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 µM) and z-DEVD-fmk (0, 50, 100 or 200 µM), respectively. Post-thaw survival was assessed at 24, 48 and 72 h. For experiments 4-5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post-thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated. Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p < .05) re-expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post-thaw cryosurvival was not affected by the addition of either DTT or z-DEVD-fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p < .001) levels of intracellular ROS and fewer (p < .001) cells with DNA fragmentation. In conclusion, post-thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate.

Revealing the Effects of Endoplasmic Reticulum Stress on Ferroptosis by Two-Channel Real-Time Imaging of pH and Viscosity.

Endoplasmic reticulum (ER) is sensitive to changes in the intracellular environment such as pH and viscosity, and slight changes may trigger stress response. Besides, different from apoptosis and necrosis, ferroptosis is the result of lipid peroxidation accumulation. There is evidence that ferroptosis is closely related to endoplasmic reticulum stress (ERS). However, the possible changes in the pH and viscosity of the ER during the ferroptosis process have not yet been studied. Therefore, we used a new type of ER-targeted dual-excitation fluorescent probe (DSPI-3) to investigate the possible changes of pH and viscosity of ER during the ferroptosis. The novel probe DSPI-3 exhibited a highly sensitive and selective response to pH and viscosity. During the bioimaging process, it was found that the ER acidified and viscosity increased during the ferroptosis process induced by erastin, while the cells treated with ferrostatin-1 did not alter significantly. In addition, when dithiothreitol (DTT) and erastin stimulated the cells at the same time, we discovered that ER was acidified considerably at short notice, but the pH was slightly increased in the later stage. Besides, the change of the viscosity enhanced slowly with the passage of time, and there was a noteworthy decline in the later stage, demonstrating that the DTT-induced ERS accelerated the process of ferroptosis. We hope that this unique fluorescent probe can provide an effective method for studying the relationship between ERS and ferroptosis.

[Effects of Sodium Selenite and Dithiothreitol on Expression of Endoplasmic Reticulum Selenoproteins and Apoptosis Markers in MSF7 Breast Adenocarcinoma Cells].

The endoplasmic reticulum (ER) stress inducers dithiothreitol (DTT) and sodium selenite (SS) were tested for effect on expression of ER selenoproteins and apoptosis markers in MCF7 breast adenocarcinoma cells. DTT used at 1 or 5 mM did not affect the survival of MCF7 cells. Based on the real-time PCR data and the protein expression levels of ER stress markers, ER stress was assumed to evolve along an adaptation pathway in MCF7 cells treated with 1 or 5 mM DTT, involving mainly the transcription factors IRE1 and ATF6 and the selenoproteins SELS, SELK, SELT, SELM, and SELN. Cell treatment with 0.01 µM SS decreases the mRNA levels of all genes examined. When the SS concentration was increased to 0.1 µM, an increase in expression was observed for key ER stress genes and apoptosis markers, including CHOP, GADD34, PUMA, BIM, ATF4, sXBP, uXBP, AKT1, BAX, and BAK. Higher SS concentrations were assumed to trigger the unfolded protein response (UPR) via a proapoptic signaling pathway involving PERK and an alternative IRE1 signaling pathway. Used at 1 µM, SS increased the mRNA levels of apoptosis markers, upregulated expression of a spliced form of XBP1, and substantially decreased the cell survival. SS (1 µM) was assumed to trigger apoptosis in MCF7 cells. The results indicate that both adaptive and proapoptic UPR signaling pathways are activated in cells, depending on the nature and concentration of the ER stress inducer.

N-acetylcysteine facilitates extinction of cued fear memory in rats via reestablishing basolateral amygdala glutathione homeostasis.

Individual differences in the development of uncontrollable fear in response to traumatic stressors have been observed in clinic, but the underlying mechanisms remain unknown. In the present study we first conducted a meta-analysis of published clinical data and found that malondialdehyde, an oxidative stress biomarker, was significantly elevated in the blood of patients with fear-related anxiety disorders. We then carried out experimental study in rats subjected to fear conditioning. We showed that reestablishing redox homeostasis in basolateral amygdale (BLA) after exposure to fear stressors determined the capacity of learned fear inhibition. Intra-BLA infusion of buthionine sulfoximine (BSO) to deplete the most important endogenous antioxidant glutathione (GSH) blocked fear extinction, whereas intra-BLA infusion of dithiothreitol or N-acetylcysteine (a precursor of GSH) facilitated extinction. In electrophysiological studies conducted on transverse slices, we showed that fear stressors induced redox-dependent inhibition of NMDAR-mediated synaptic function, which was rescued by extinction learning or reducing agents. Our results reveal a novel pharmacological strategy for reversing impaired fear inhibition and highlight the role of GSH in the treatment of psychiatric disorders.

Effects of Dithiothreitol on Fertilization and Early Development in Sea Urchin.

The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca2+ signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg's fertilization response.

Recruitment of endoplasmic reticulum-targeted and cytosolic mRNAs into membrane-associated stress granules.

Stress granules (SGs) are membraneless organelles composed of mRNAs and RNA binding proteins which undergo assembly in response to stress-induced inactivation of translation initiation. In general, SG recruitment is limited to a subpopulation of a given mRNA species and RNA-seq analyses of purified SGs revealed that signal sequence-encoding (i.e., endoplasmic reticulum [ER]-targeted) transcripts are significantly underrepresented, consistent with prior reports that ER localization can protect mRNAs from SG recruitment. Using translational profiling, cell fractionation, and single molecule mRNA imaging, we examined SG biogenesis following activation of the unfolded protein response (UPR) by 1,4-dithiothreitol (DTT) and report that gene-specific subsets of cytosolic and ER-targeted mRNAs can be recruited into SGs. Furthermore, we demonstrate that SGs form in close proximity to or directly associated with the ER membrane. ER-associated SG assembly was also observed during arsenite stress, suggesting broad roles for the ER in SG biogenesis. Recruitment of a given mRNA into SGs required stress-induced translational repression, though translational inhibition was not solely predictive of an mRNA's propensity for SG recruitment. SG formation was prevented by the transcriptional inhibitors actinomycin D or triptolide, suggesting a functional link between gene transcriptional state and SG biogenesis. Collectively these data demonstrate that ER-targeted and cytosolic mRNAs can be recruited into ER-associated SGs and this recruitment is sensitive to transcriptional inhibition. We propose that newly transcribed mRNAs exported under conditions of suppressed translation initiation are primary SG substrates, with the ER serving as the central subcellular site of SG formation.