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.

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.

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.

Metabolomic profiles exhibit the influence of endoplasmic reticulum stress on sorghum seedling growth over time.

Environmental stresses disturb the endoplasmic reticulum (ER) protein folding. However, primary metabolic responses induced by ER stress remain unclear. Thus, we investigated the morphophysiological and metabolomic changes under ER stress, induced by dithiothreitol (DTT) and tunicamycin (TM) treatments in sorghum seedlings from 24 to 96 h. The ER stress caused lipid peroxidation and increased the expression of SbBiP1, SbPDI, and SbIRE1. The development impairment was more pronounced in roots than in shoots as distinct metabolomic profiles were observed. DTT decreased root length, lateral roots, and root hair, while TM decreased mainly the root length. At 24 h, under ER stresses, the glutamic acid and o-acetyl-serine were biomarkers in the shoots. While homoserine, pyroglutamic acid, and phosphoric acid were candidates for roots. At the latest time (96 h), kestose and galactinol were key metabolites for shoots under DTT and TM, respectively. In roots, palatinose, trehalose, and alanine were common markers for DTT and TM late exposure. The accumulation of sugars such as arabinose and kestose occurred mainly in roots in the presence of DTT at a later time, which also inhibited glycolysis and the tricarboxylic acid cycle (TCA). Amino acid metabolism was induced, which also contributed TCA components decreasing, such as succinate in shoots and citrate in roots. Thus, our study may provide new insights into primary metabolism modulated by ER stress and seedling development.

A feasible method to extract DNA from the cambium of high-canopy trees: from harvest to assessment

Muitas árvores tropicais possuem dossel alto e folhas não facilmente acessíveis. O uso de tecido de um órgão mais acessível (câmbio) para extração de DNA pode ser uma alternativa para estudos moleculares. Nós adaptamos uma metodologia viável para extrair DNA genômico de tecido cambial coletado no campo para avaliação com PCR. Testamos três condições de armazenamento (dois tampões e sílica gel) e quatro períodos após a coleta. Utilizamos protocolos descritos anteriormente e os testamos em três espécies encontradas em florestas amazônicas e outros biomas: Anadenanthera peregrina var. peregrina, Cedrela fissilis e Ceiba speciosa. Nosso protocolo foi eficaz na obtenção de DNA adequado para sequenciamento e genotipagem de microssatélites. Recomendamos o uso de sílica para armazenamento de longo prazo e o tampão com ácido ascórbico para curto prazo. (AU)

Dithiothreitol-based oxidative potential for airborne particulate matter: an estimation of the associated uncertainty.

Oxidative stress is considered as one of the main mechanisms by which airborne particles produce adverse health effects. Several methods to estimate the oxidative potential (OP) of particulate matter (PM) have been proposed. Among them, the dithiothreitol (DTT) assay has gained popularity due to its simplicity and overall low implementation cost. Usually, the estimations of OPDTT are based on n-replicates of a set of samples and their associated standard deviation. However, interlaboratory comparisons of OPDTT can be difficult and lead to misinterpretations. This work presents an estimation of the total uncertainty for the OPDTT measurement of PM10 and PM2.5 samples collected in Santiago (Chile), based on recommendations by the Joint Committee for Guides in Metrology and Eurachem. The expanded uncertainty expressed as a percentage of the mass-normalized OPDTT measurements was 18.0% and 16.3% for PM10 and PM2.5 samples respectively. The dominating contributor to the total uncertainty was identified (i.e., DTT consumption rate, related to the regression and repeatability of experimental data), while the volumetric operations (i.e., pipettes) were also important. The results showed that, although the OP measured following the DTT assay has been successfully used to estimate the potential health impacts of airborne PM, uncertainty estimations must be considered before interpreting the results.

A blockage monoclonal antibody protocol as an alternative strategy to avoid anti-CD38 interference in immunohematological testing.

BACKGROUND: As CD38 is expressed on red blood cells (RBCs), the plasma of patients on daratumumab (DARA) reacts with the panel cells of pretransfusion tests, masking underlying alloantibodies. The treatment of RBCs with dithiothreitol (DTT) is the most disseminated method to overcome DARA effect on immunohematological tests, but it hampers the identification of potentially harmful antibodies. Our goal was to validate a new strategy, the blockage monoclonal antibody protocol (BMAP), to mitigate the DARA interference on RBCs using anti-CD38 and antihuman globulin. METHODS: Samples of patients receiving DARA were included in the study. Sera were tested using both DTT- and BMAP-treated RBCs, which comprised three steps: 1) titration of monoclonal anti-CD38, 2) treatment of RBCs obtained from donors with anti-CD38, and 3) blockage of anti-CD38-adsorbed RBCs with antihuman globulin. RESULTS: Twenty patients were included in the study. Donor RBCs were treated with anti-CD38 and successfully blocked with antihuman globulin. In 19 patients, DARA-mediated agglutination was eliminated using both DTT- and BMAP-treated RBCs. In one patient, agglutination persisted when tested against the BMAP-treated RBCs, and alloantibodies were identified. Patient samples were mixed with commercial anti-D, -C, -e, -K, -Jka, -Kpb and tested against antigen-positive BMAP-treated RBCs, resulting in detection of these antibodies. CONCLUSION: This study validated a new strategy to minimize the interference of DARA on immunohematological tests. The protocol preserves the integrity of RBC antigens, permitting the detection of antibodies from all blood group systems. The BMAP has potential use in other situations where specific antibodies may interfere with pretransfusion screening.

A modified lateral flow assay, using serum, for the rapid identification of human and bovine cysticercosis in the absence of false positives.

Background: Previously we reported the use of a monoclonal antibody-based (HP10) antigen (Ag) detection lateral flow assay (LFA) for the diagnosis of extraparenchymal neurocysticercosis (EP-NCC). The assay performed well when used with cerebrospinal fluid (CSF) samples but not with their paired serum samples, due to false-positive reactions in some known negative control cases. Methods: Our novel modification involves pretreatment of serum samples using a combination of sodium deoxycholate and dithiothreitol. Results: The modification overcomes the problem of false positives when using negative serum samples from clinically characterized cases of EP-NCC and bovine cysticercosis. In general, there was good agreement between HP10 Ag enzyme-linked immunosorbent assay (ELISA) and the HP10 Ag-LFA, but the HP10 Ag-ELISA was marginally more sensitive than the modified HP10 Ag-LFA. Conclusions: The modified HP10 Ag-LFA provides a field test for the rapid identification of endemic human and bovine cysticercosis.

Interaction of metals from group 10 (Ni, Pd, and Pt) and 11 (Cu, Ag, and Au) with human blood δ-ALA-D: in vitro and in silico studies.

Mammalian δ-aminolevulinate dehydratase (δ-ALA-D) is a metalloenzyme, which requires Zn(II) and reduced thiol groups for catalytic activity, and is an important molecular target for the widespread environmental toxic metals. The δ-ALA-D inhibition mechanism by metals of Group 10 (Ni, Pd, and Pt) and 11 (Cu, Ag, and Au) of the periodic table has not yet been determined. The objective of this study was to characterize the molecular mechanism of δ-ALA-D inhibition caused by the elements of groups 10 and 11 using in vitro (δ-ALA-D activity from human erythrocytes) and in silico (docking simulations) methods. Our results showed that Ni(II) and Pd(II) caused a small inhibition (~ 10%) of the δ-ALA-D. Pt(II) and Pt(IV) significantly inhibited the enzyme (75% and 44%, respectively), but this inhibition was attenuated by Zn(II) and dithiothreitol (DTT). In group 11, all metals inhibited δ-ALA-D with great potency (~ 70-90%). In the presence of Zn(II) and DTT, the enzyme activity was restored to the control levels. The in silico molecular docking data suggest that the coordination of the ions Pt(II), Pt(IV), Cu(II), Ag(I), and Au(III) with thiolates groups from C135 and C143 residues from the δ-ALA-D active site are crucial to the enzyme inhibition. The results indicate that a possible mechanism of inhibition of δ-ALA-D by these metals may involve the replacement of the Zn(II) from the active site and/or the cysteinyl residue oxidation.

Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization.

To relieve endoplasmic reticulum (ER) stress, IRE1 splices XBP1 messenger RNA (mRNA) or engages regulated IRE1-dependent decay (RIDD) of other mRNAs. Upon XBP1 deficiency, IRE1 switches to perform RIDD. We examined IRE1 in XBP1-deficient B cells and discovered that IRE1 undergoes phosphorylation at S729. We generated an anti-phospho-S729 antibody to investigate such phosphorylation. Compared with pharmacological ER stress inducers or Toll-like receptor ligands, the bacterial subtilase cytotoxin has an unusual capability in causing rapid and strong phosphorylation at S729 and triggering B cells to express spliced XBP1. To assess the function of S729 in IRE1, we generated S729A knock-in mice and found S729 is critically important for lipopolysaccharide-stimulated plasmablasts to respond to additional ER stress and for antibody production in response to immunization. We further crossed mice carrying an S729A mutation or ΔIRE1 (missing the kinase domain) with B cell-specific XBP1-deficient mice to trigger RIDD and discovered a critical role for S729 in regulating RIDD in B cells.

Depleting high-abundant and enriching low-abundant proteins in human serum: An evaluation of sample preparation methods using magnetic nanoparticle, chemical depletion and immunoaffinity techniques.

The efficiency of three different depletion methods to remove the most abundant proteins, enriching those human serum proteins with low abundance is checked to make more efficient the search and discovery of biomarkers. These methods utilize magnetic nanoparticles (MNPs), chemical reagents (sequential application of dithiothreitol and acetonitrile, DTT/ACN), and commercial apparatus based on immunoaffinity (ProteoMiner, PM). The comparison between methods shows significant removal of abundant protein, remaining in the supernatant at concentrations of 4.6±0.2, 3.6±0.1, and 3.3±0.2µgµL-1 (n=3) for MNPs, DTT/ACN and PM respectively, from a total protein content of 54µgµL-1. Using GeLC-MS/MS analysis, MNPs depletion shows good efficiency in removing high molecular weight proteins (>80kDa). Due to the synergic effect between the reagents DTT and ACN, DTT/ACN-based depletion offers good performance in the depletion of thiol-rich proteins, such as albumin and transferrin (DTT action), as well as of high molecular weight proteins (ACN action). Furthermore, PM equalization confirms its efficiency in concentrating low-abundant proteins, decreasing the dynamic range of protein levels in human serum. Direct comparison between the treatments reveals 72 proteins identified when using MNP depletion (43 of them exclusively by this method), but only 20 proteins using DTT/ACN (seven exclusively by this method). Additionally, after PM treatment 30 proteins were identified, seven exclusively by this method. Thus, MNPs and DTT/ACN depletion can be simple, quick, cheap, and robust alternatives for immunochemistry-based protein depletion, providing a potential strategy in the search for disease biomarkers.

RA Differentiation Enhances Dopaminergic Features, Changes Redox Parameters, and Increases Dopamine Transporter Dependency in 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells.

Research on Parkinson's disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.

Partial Characterization of the Proteolytic Properties of an Enzymatic Extract From "Aguama" Bromelia pinguin L. Fruit Grown in Mexico.

Plant proteases are capable of performing several functions in biological systems, and their use is attractive for biotechnological process due to their interesting catalytic properties. Bromelia pinguin (aguama) is a wild abundant natural resource in several regions of Central America and the Caribbean Islands but is underutilized. Their fruits are rich in proteases with properties that are still unknown, but they represent an attractive source of enzymes for biotechnological applications. Thus, the proteolytic activity in enzymatic crude extracts (CEs) from wild B. pinguin fruits was partially characterized. Enzymes in CEs showed high proteolytic activity at acid (pH 2.0-4.0) and neutral alkaline (pH 7.0-9.0) conditions, indicating that different types of active proteases are present. Proteolytic activity inhibition by the use of specific protease inhibitors indicated that aspartic, cysteine, and serine proteases are the main types of proteases present in CEs. Activity at pH 3.0 was stable in a broad range of temperatures (25-50 °C) and retained its activity in the presence of surfactants (SDS, Tween-80), reducing agents (DTT, 2-mercapoethanol), and organic solvents (methanol, ethanol, acetone, 2-propanol), which suggests that B. pinguin proteases are potential candidates for their application in brewing, detergent, and pharmaceutical industries.

Thiols of flagellar proteins are essential for progressive motility in human spermatozoa.

Male infertility is a disorder of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse. The presence of low-motile or immotile spermatozoa is one of many causes of infertility; however, this observation provides little or no information regarding the pathogenesis of the malfunction. Good sperm motility depends on correct assembly of the sperm tail in the testis and efficient maturation during epididymal transit. Thiols of flagellar proteins, such as outer dense fibre protein 1 (ODF1), are oxidised to form disulfides during epididymal transit and the spermatozoa become motile. This study was designed to determine how oxidative changes in protein thiol status affect progressive motility in human spermatozoa. Monobromobimane (mBBr) was used as a specific thiol marker and disruptor of sperm progressive motility. When mBBr was blocked by dithiothreitol it did not promote motility changes. The analysis of mBBr-treated spermatozoa revealed a reduction of progressive motility and an increased number of spermatozoa with non-progressive motility without affecting ATP production. Laser confocal microscopy and western blot analysis showed that one of the mBBr-positive proteins reacted with an antibody to ODF1. Monobromobimane fluorescence intensity of the sperm tail was lower in normozoospermic than asthenozoospermic men, suggesting that thiol oxidation in spermatozoa of asthenozoospermic men is incomplete. Our findings indicate that mBBr affects the thiol status of ODF1 in human spermatozoa and interferes with progressive motility.

Sulfhydryl-Based Inhibition of δ-ALA-D and Na+ , K+ -ATPase Activities Depends on the Organoselenium Group Bonded to the Isoquinoline.

Organoselenium compounds and isoquinoline derivatives have their toxicity linked to induction of pro-oxidant situations. δ-Aminolevulinate dehydratase (δ-ALA-D) and Na+ , K+ -ATPase have sulfhydryl groups susceptible to oxidation. Thus, we investigated toxicological effects of 4-organoseleno-isoquinoline derivatives, cerebral monoamine oxidase B inhibitors, on rat cerebral δ-ALA-D and Na+ , K+ -ATPase activities and the involvement of sulfhydryl groups in vitro. Compounds substituted with fluoro (4-(4-fluorophenylseleno)-3-phenylisoquinoline), chloro (4-(4-chlorophenylseleno)-3-phenylisoquinoline) and trifluoro (4-(3-trifluoromethylphenylseleno)-3-phenylisoquinoline) at the selenium-bonded aromatic ring inhibited δ-ALA-D (IC50 values: 78.42, 92.27, 44.98 µM) and Na+ , K+ -ATPase (IC50 values: 41.36, 89.43, 50.66 µM) activities, possibly due to electronic effects induced by these groups. 3-Phenyl-4-(phenylseleno) isoquinoline (without substitution at the selenium-bonded aromatic ring) and 4-(4-methylphenylseleno)-3-phenylisoquinoline (with a methyl group substituted at the selenium-bonded aromatic ring) did not alter the activity of these enzymes. Dithiothreitol, a reducing agent, restored the enzymatic activities inhibited by 4-(4-fluorophenylseleno)-3-phenylisoquinoline, 4-(4-chlorophenylseleno)-3-phenylisoquinoline and 4-(3-trifluoromethylphenylseleno)-3-phenylisoquinoline, suggesting the involvement of sulfhydryl residues in this effect. However, the release of essential zinc seems not to be related to the δ-ALA-D inhibition by these compounds. According to these data, the effect of oral administration (300 mg/kg, intragastric) of 3-phenyl-4-(phenylseleno) isoquinoline on markers of systemic toxicity in Wistar rats was evaluated. None signs of toxicity was observed during or after treatment. This study suggests that the insertion of electron-withdrawing groups in the aromatic ring bonded to the selenium atom of isoquinolines tested increased its inhibitory effect on sulfhydryl enzymes in vitro. 3-Phenyl-4-(phenylseleno) isoquinoline, which has documented pharmacological properties, had no toxicological effects on the parameters evaluated in this study. J. Cell. Biochem. 118: 1144-1150, 2017. © 2016 Wiley Periodicals, Inc.

Dithiothreitol treatment of red blood cells.

CONCLUSIONS: Dithiothreitol (DTT), a reducing reagent, has multiple applications in blood bank testing. DTT disrupts the bridging of the disulfide bonds between amino acid residues necessary for structural conformation of some proteins and the bonds holding an IgM molecule in the pentameric formation. DTT treatment of red blood cells (RBCs) can denature or modify certain blood group antigens-in particular, those in the Kell, Lutheran, YT, JMH, LW, Cromer, Indian, Dombrock, and Knops systems-and prevent recognition by the corresponding antibodies. It also destroys RBC CD38, allowing DTT-treated RBCs to be used to avoid testing interference by therapeutic anti-CD38 preparations. DTT treatment can be used to disperse spontaneous agglutination of RBCs caused by heavy IgM autoantibody coating that invalidates ABO/Rh cell grouping and direct antiglobulin tests.

Chromatographic fractionation and molecular mass characterization of Cercidium praecox (Brea) gum.

BACKGROUND: Brea gum (BG) is an exudate from the Cercidium praecox tree that grows in semi-arid regions of Argentina. Some previous studies on BG have shown physicochemical characteristics and functional features similar to those of gum arabic. However, there is a need to elucidate the molecular structure of BG to understand the functionality. In this sense, BG was fractionated using hydrophobic interaction chromatography and the obtained fractions were analyzed by size exclusion chromatography. RESULTS: Analysis of the fractions showed that the bulk of the gum (approx. 84% of the polysaccharides) was a polysaccharide of 2.79 × 10(3) kDa. The second major fraction (approx. 16% of the polysaccharides) was a polysaccharide-protein complex with a molecular mass of 1.92 × 10(5) kDa. A third fraction consisted of protein species with a wide range of molecular weights. The molecular weight distribution of the protein fraction was analyzed by size exclusion chromatography. Comparison of the elution profiles of the exudates in native and reducing conditions revealed that some of the proteins were forming aggregates through disulfide bridges in native conditions. Further analysis of the protein fraction by SDS-PAGE showed proteins with molecular weight ranging from 6.5 to 66 kDa. CONCLUSIONS: The findings showed that BG consists of several fractions with heterogeneous chemical composition and polydisperse molecular weight distributions. © 2016 Society of Chemical Industry.

The dynamic of the splicing of bZIP60 and the proteins encoded by the spliced and unspliced mRNAs reveals some unique features during the activation of UPR in Arabidopsis thaliana.

The unfolded protein response (UPR) is a signaling pathway that is activated when the workload of the endoplasmic reticulum (ER) is surpassed. IRE1 is a sensor involved in triggering the UPR and plays a key role in the unconventional splicing of an mRNA leading to the formation of a transcription factor that up-regulates the transcription of genes that play a role in restoring the homeostasis in the ER. In plants, bZIP60 is the substrate for IRE1; however, questions such as what is the dynamics of the splicing of bZIP60 and the fate of the proteins encoded by the spliced and unspliced forms of the mRNA, remain unanswered. In the present work, we analyzed the processing of bZIP60 by determining the levels of the spliced form mRNA in plants exposed to different conditions that trigger UPR. The results show that induction of ER stress increases the content of the spliced form of bZIP60 (bZIP60s) reaching a maximum, that depending on the stimuli, varied between 30 min or 2 hrs. In most cases, this was followed by a decrease in the content. In contrast to other eukaryotes, the splicing never occurred to full extent. The content of bZIP60s changed among different organs upon induction of the UPR suggesting that splicing is regulated differentially throughout the plant. In addition, we analyzed the distribution of a GFP-tagged version of bZIP60 when UPR was activated. A good correlation between splicing of bZIP60 and localization of the protein in the nucleus was observed. No fluorescence was observed under basal conditions, but interestingly, the fluorescence was recovered and found to co-localize with an ER marker upon treatment with an inhibitor of the proteasome. Our results indicate that the dynamics of bZIP60, both the mRNA and the protein, are highly dynamic processes which are tissue-specific and stimulus-dependent.

bZIP17 and bZIP60 Regulate the Expression of BiP3 and Other Salt Stress Responsive Genes in an UPR-Independent Manner in Arabidopsis thaliana.

Plants can be severely affected by salt stress. Since these are sessile organisms, they have developed different cellular responses to cope with this problem. Recently, it has been described that bZIP17 and bZIP60, two ER-located transcription factors, are involved in the cellular response to salt stress. On the other hand, bZIP60 is also involved in the unfolded protein response (UPR), a signaling pathway that up-regulates the expression of ER-chaperones. Coincidentally, salt stress produces the up-regulation of BiP, one of the main chaperones located in this organelle. Then, it has been proposed that UPR is associated to salt stress. Here, by using insertional mutant plants on bZIP17 and bZIP60, we show that bZIP17 regulate the accumulation of the transcript for the chaperone BiP3 under salt stress conditions, but does not lead to the accumulation of UPR-responding genes such as the chaperones Calnexin, Calreticulin, and PDIL under salt treatments. In contrast, DTT, a known inducer of UPR, leads to the up-regulation of all these chaperones. On the other hand, we found that bZIP60 regulates the expression of some bZIP17 target genes under conditions were splicing of bZIP60 does not occur, suggesting that the spliced and unspliced forms of bZIP60 play different roles in the physiological response of the plant. Our results indicate that the ER-located transcription factors bZIP17 and bZIP60 play a role in salt stress but this response goes through a signaling pathway that is different to that triggered by the unfolded protein response.