Crayfish Procambarus clarkii shows circadian variations in different parameters of the GSH cycle

The present study investigated the rhythmic changes in glutathione status in midgut gland and hemolymph as well as in glutathione reductase (GR) activity in the crayfish Procambarus clarkii. In order to determine the circadian nature of these rhythms different groups of crayfish were submitted to constant-darkness conditions for 24 or 72 h after they had spent 15 days under light-dark 12:12 cycles. The animals of the different batches were killed at 6 h intervals during a 24 h cycle. Reduced glutathione (GSH) and oxidized glutathione (GSSG) in hemolymph and midgut as well as midgut GR activity were determined in midgut gland and hemolymph by fluorometric and spectrophotometric method. Data analysis by chronogram and single Cosinor revealed circadian rhythmicity for GSH and GSSG concentration in both tissues as well as midgut GR activity. The rhythm parameters revealed oxidative stress induced by light. The possible correlation between the glutathione rhythm and other metabolic and behavioral rhythms of crayfish as well as the importance of the glutathione circadian temporal order in the adaptation of crayfish are discussed.     

Endoplasmic reticulum targeted AIE bioprobe as a highly efficient inducer of immunogenic cell death

Focused oxidative stress of the specific organelles(e.g., endoplasmic reticulum(ER) and mitochondrion) of cancer cells can boost the immunogenic cell death(ICD) effect for cancer immunotherapy. Herein, an ER-targeted bioprobe with aggregationinduced emission(AIE) characteristics(TPE-PR-FFKDEL) was rationally designed and synthesized by integrating a new AIE photosensitizer with ER targeting peptide, which has been demonstrated to be able to efficiently induce ER oxidative stress to evoke ICD. Compared with the photosensitizer hypericin that is well-known as an ER-targeted ICD inducer, TPE-PR-FFKDEL can lead to more robust emission of immunostimulatory damage-associated molecular patterns such as surface-exposed calreticulin, ATP secretion, and high-mobility group protein B1(HMGB1) and heat shock protein 70(HSP 70) expression.Furthermore, a range of immune responses are activated to protect mice from the attack of cancer cells in vivo.     

Carotid atherosclerotic plaques: proteomics study after a low-abundance protein enrichment step

Atherosclerosis is one of the most important causes of cardiovascular and cerebrovascular events. Although phenotypic differentiation between stable and unstable plaques is currently possible, proteomic analysis of the atherosclerotic plaque could offer a global view of the atherosclerosis pathology. With the objective to highlight the detection of low-abundance proteins, we reduced the dynamic range of proteins by combinatorial peptide ligand library treatment of human carotid artery atherosclerotic plaques. After enrichment step, abundance of major proteins was decreased, revealing different protein profiles as assessed by both SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis comparative analyses. Identification of proteins that were contained in a spot allowed finding large differences between noncomplicated and complicated plaques from carotid atherosclerotic lesions. Novel low-abundance proteins were detected correlating very well with biological alterations related to atherosclerosis (heat shock protein 27 (HSP27) isoforms, aldehyde dehydrogenase, moesin, Protein kinase C delta-binding protein, and inter-α trypsin inhibitor family heavy chain-related protein (ITIH4)). At the same time, the differential expression of known proteins of interest such as hemoglobin β-chain and heat shock protein 27 between noncomplicated and hemorrhagic complicated plaques was maintained after enrichment step. The detection of different isoforms of a low-abundance protein such as heat shock protein 27 species was actually improved after enrichment of tissue protein extracts. All of these findings clearly support further investigations in view to confirm the role of these proteins as possible biomarkers.     

AEG3482 is an antiapoptotic compound that inhibits Jun kinase activity and cell death through induced expression of heat shock protein 70

We describe a group of small-molecule inhibitors of Jun kinase (JNK)-dependent apoptosis. AEG3482, the parental compound, was identified in a screening effort designed to detect compounds that reduce apoptosis of neonatal sympathetic neurons after NGF withdrawal. We show that AEG3482 blocks apoptosis induced by the p75 neurotrophin receptor (p75NTR) or its cytosolic interactor, NRAGE, and demonstrate that AEG3482 blocks proapoptotic JNK activity. We show that AEG3482 induces production of heat shock protein 70 (HSP70), an endogenous inhibitor of JNK, and establish that HSP70 accumulation is required for the AEG3482-induced JNK blockade. We show that AEG3482 binds HSP90 and induces HSF1-dependent HSP70 mRNA expression and find that AEG3482 facilitates HSP70 production while retaining HSP90 chaperone activity. These studies establish that AEG3482 inhibits JNK activation and apoptosis by a mechanism involving induced expression of HSP proteins.     

Differential expression of molecular chaperones in brain of patients with Down syndrome

Heat shock proteins (HSPs) in their molecular capacity as chaperones have been reported to regulate the apoptotic pathway and also play a critical role in protein conformational diseases such as Alzheimer's disease (AD). As all Down syndrome (DS) brains display AD-like neuropathology, neuronal loss in DS was shown to be mediated by apoptosis. We decided to investigate the expression patterns of HSPs in seven brain regions of adults with DS using two-dimensional polyacrylamide gel electrophoresis (2-DE). Following 2-DE, approximately 120 protein spots were successfully identified by matrix-assisted laser desorption/ionization--mass spectrometry (MALDI-MS) followed by quantification of the identified proteins. We unambiguously identified and quantified nine different chaperone proteins. Accordingly, all but three chaperone proteins did exhibit a significant change in expression. HSP 70 RY, heat shock cognate (HSC) 71 and glucose-regulated protein (GRP) 75 showed a significant decrease (P < 0.05) in DS temporal cortex whereas HSP 70.1 and GRP 78 were significantly increased (P<0.05) in cerebellum. Whilst T-complex 1 (TCP-1) epsilon subunit showed a significant decrease (P< 0.05) in parietal cortex, a similar extent of increase (P<0.05) as that observed in cerebellum was obtained in parietal levels of GRP 78. Alpha-crystallin B, HSP 60 and GRP 94 did not show any detectable changes in expression patterns. This report presents the first approach to quantify nine different chaperones simultaneously at the protein level in different brain regions and provides evidence for aberrant chaperone expression patterns in DS. The relevance of this aberrant expression patterns are discussed in relation to the biochemical and neuropathological abnormalities in DS brain.     

A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-kappaB and hypoxia-inducible factor-1alpha

Reactive oxygen species (ROS) play an important role in the pathogenesis of airway inflammation and hyperresponsiveness. Recent studies have demonstrated that antioxidants are able to reduce airway inflammation and hyperreactivity in animal models of allergic airway disease. A newly developed antioxidant, small molecular weight thiol compound, N-acetylcysteine amide (AD4) has been shown to increase cellular levels of glutathione and to attenuate oxidative stress related disorders such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. However, the effects of AD4 on allergic airway disease such as asthma are unknown. We used ovalbumin (OVA)-inhaled mice to evaluate the role of AD4 in allergic airway disease. In this study with OVA-inhaled mice, the increased ROS generation, the increased levels of Th2 cytokines and VEGF, the increased vascular permeability, the increased mucus production, and the increased airway resistance in the lungs were significantly reduced by the administration of AD4. We also found that the administration of AD4 decreased the increases of the NF-kappaB and hypoxia-inducible factor-1alpha (HIF-1alpha) levels in nuclear protein extracts of lung tissues after OVA inhalation. These results suggest that AD4 attenuates airway inflammation and hyperresponsiveness by regulating activation of NF-kappaB and HIF-1alpha as well as reducing ROS generation in allergic airway disease.     

The Effect of Dansyl-Modified β-Cyclodextrin on the Chaperone Activity of Heat Shock Proteins

The effect of dansyl modified -cyclodextrin (1)on the chaperone activity of heat shock proteins such as HSP70 and HSP90 hasbeen studied. The fluorescence intensity of 1 was decreased when HSP70 and HSP90 were added to the host solution. This phenomenon suggested that host–guest complexation was occuring. The binding constants of 1 were obtained using a 1:1 complex formation type equation by employing the guest-induced fluorescence variations. Host 1 exhibited a higher binding ability forHSP70 than for HSP90. The effects of 1 on the chaperone activity and degradation of HSP70 and HSP90 were studied by measuring the absorption of aggregation of citrate synthase (C.S.) and sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis of trypsin degradation, respectively. Host1 can contribute to regulate C.S. aggregation andpromote trypsin degradation of HSP70 and HSP90.     

Stoichiometric Analysis of Shifting in Subcellular Compartmentalization of HSP70 within Ischemic Penumbra

The heat shock protein (HSP) 70 is considered the main hallmark in preclinical studies to stain the peri-infarct region defined area penumbra in preclinical models of brain ischemia. This protein is also considered as a potential disease modifier, which may improve the outcome of ischemic damage. In fact, the molecule HSP70 acts as a chaperonine being able to impact at several level the homeostasis of neurons. Despite being used routinely to stain area penumbra in light microscopy, the subcellular placement of this protein within area penumbra neurons, to our knowledge, remains undefined. This is key mostly when considering studies aimed at deciphering the functional role of this protein as a determinant of neuronal survival. The general subcellular placement of HSP70 was grossly reported in studies using confocal microscopy, although no direct visualization of this molecule at electron microscopy was carried out. The present study aims to provide a direct evidence of HSP70 within various subcellular compartments. In detail, by using ultrastructural morphometry to quantify HSP70 stoichiometrically detected by immuno-gold within specific organelles we could compare the compartmentalization of the molecule within area penumbra compared with control brain areas. The study indicates that two cell compartments in control conditions own a high density of HSP70, cytosolic vacuoles and mitochondria. In these organelles, HSP70 is present in amount exceeding several-fold the presence in the cytosol. Remarkably, within area penumbra a loss of such a specific polarization is documented. This leads to the depletion of HSP70 from mitochondria and mostly cell vacuoles. Such an effect is expected to lead to significant variations in the ability of HSP70 to exert its physiological roles. The present findings, beyond defining the neuronal compartmentalization of HSP70 within area penumbra may lead to a better comprehension of its beneficial/detrimental role in promoting neuronal survival.     

Crayfish Procambarus clarkii retina and nervous system exhibit antioxidant circadian rhythms coupled with metabolic and luminous daily cycles

Based on previous work in which we proposed midgut as a putative peripheral oscillator responsible for circadian reduced glutathione (GSH) crayfish status, herein we investigated the retina and optic lobe-brain (OL-B) circadian GSH system and its ability to deal with reactive oxygen species (ROS) produced as a consequence of metabolic rhythms and light variations. We characterized daily and antioxidant circadian variations of the different parameters of the glutathione system, including GSH, oxidized glutathione (GSSG), glutathione reductase (GR) and glutathione peroxidase (GPx), as well as metabolic and lipoperoxidative circadian oscillations in retina and OL-B, determining internal and external GSH-system synchrony. The results demonstrate statistically significant bi- and unimodal daily and circadian rhythms in all GSH-cycle parameters, substrates and enzymes in OL-B and retina, as well as an apparent direct effect of light on these rhythms, especially in the retina. The luminous condition appears to stimulate the GSH system to antagonize ROS and lipid peroxidation (LPO) daily and circadian rhythms occurring in both structures, oscillating with higher LPO under dark conditions. We suggest that the difference in the effect of light on GSH rhythmic mechanisms of both structures for antagonizing ROS could be due to differences in glutathione-system coupling strength with the circadian clock.     

Molecular Simulation of HER2/neu Degradation by Inhibiting HSP90

Several flavonoids were investigated for the degradation of the HER2/neu (ErbB‐2), while the mechanism is still unknown. A hypothesis was generated here that the inhibition of HER2/neu expression was blocked by heat shock protein 90α (HSP90α) through 1‐benzyl‐3‐(5‐hydroxymethyl‐2‐furyl)indazole (YC‐1) derivatives and flavonoids. In order to ensure the accuracy of the simulated protein structure, the RMSD value between the ligand in crystal structure from PDB and the ligand docking into HSP90α was 1.13 Å. By molecular simulation, the flavonoids and YC‐1 derivatives were employed to dock into HSP90α. The results showed a good correlation between the evaluation scores of the flavonoids/HSP90α complexes and the IC₅₀ of flavonoids‐induced degradation of HER2/neu. The YC‐1 derivatives showed higher score values and lower interaction energies on average. Especially, the CLC107 got the highest rank in Potential of Mean Force (PMF) and Dock Score. Luteolin showed the highest dock score and quercetin had the lowest interaction energy of all flavonoid derivatives. This study investigated that the YC‐1 derivatives and the flavonoids may be potent inhibitors for HSP90α in antitumor strategies.     

Changes in hemolymph glutathione status after variation in photoperiod and light-irradiance in crayfish Procambarus clarkii and Procambarus digueti

This work studied the effect of light-stressors, irradiance and photoperiod length on the status of hemolymph glutathione in two species of crayfish, Procambarus clarkii and Procambarus digueti. Adult animals of each species were submitted to two experimental approaches: (1) two batches of each species were placed under low or high light irradiant conditions of light-dark (LD) 24 h cycles of two different photoperiod lengths, one normal LD 12: 12 and one extreme LD 20:4 low and high irradiance for 10 weeks. Time-dependent light changes on hemolymph glutathione concentration were determined throughout the entire experimental period; and (2) three batches of the two species were submitted to independent treatments consisting of the same LD 12:12 cycles of high and low irradiance and 20:4 high-irradiance LD cycles. Reduced and oxidized glutathione hemolymph concentrations were determined and total glutathione was calculated. In addition midgut glutathione reductase activity in both species was determined. The two species showed different hemolymph glutathione reactivity and glutathione status for the two light parameters. Dissimilar responses of both species, as well as the rate of mortality of P. digueti represent specific differences in the metabolic responses, as well as tolerance to photo-oxidative stress produced by light. The role of glutathione in the tolerance of crayfish to photo-oxidative stress is discussed.     

Structural and energetic insight into the interactions between the benzolactam inhibitors and tumor marker HSP90α

The heat shock protein 90α (HSP90α) provides a promising molecular target for cancer therapy. A series of novel benzolactam inhibitors exhibited distinct inhibitory activity for HSP90α. However, the structural basis for the impact of distinct R₁ substituent groups of nine benzolactam inhibitors on HSP90α binding affinities remains unknown. In this study, we carried out molecular docking, molecular dynamics (MD) simulations, and molecular mechanics and generalized Born/surface area (MM–GBSA) binding free energy calculations to address the differences. Molecular docking studies indicated that all nine compounds presented one conformation in the ATP-binding site of HSP90α N-terminal domain. MD simulations and subsequent MM–GBSA calculations revealed that the hydrophobic interactions between all compounds and HSP90α contributed the most to the binding affinity and a good linear correlation was obtained between the calculated and the experimental binding free energies (R = 0.88). The per residue decomposition revealed that the most remarkable differences of residue contributions were found in the residues Ala55, Ile96, and Leu107 defining a hydrophobic pocket for the R₁ group, consistent with the analysis of binding modes. This study may be helpful for the future design of novel HSP90α inhibitors.     

High activity of Mj HSP16.5 under acidic condition

Small heat shock proteins (sHSPs) exist ubiquitously among all organisms, with a variety of functions. All small heat shock proteins assemble into a native large oligomeric state containing 9–40 monomers. The sHSPs show chaperone-like activity to prevent the aggregation of nonnative proteins under stressful cellular conditions such as non-optimal temperatures, pH changes, osmotic pressure, and exposure to toxic chemicals. It was found that a common dimeric subunit of sHSPs might be the major active species, but whether the native large oligomeric state is only a storage state or a state crucial to its molecular chaperone activity is still under debate. The native large oligomeric state of the small heat shock protein from a hyperthermophilic methanarchaeon, Methanococcus jannaschii (Mj HSP 16.5), is a stable icositetramer, which is a symmetric hollow sphere that is very stable even at 85°C, and no small active subunit has been detected till now. Our results show that Mj sHSP 16.5 changes into small and active oligomeric state at pH 3, likely as octamers (average result) at 25°C, and dimers at 65°C. The dimer of Mj HSP 16.5 at pH 3.0 and 65°C is very active and efficient, even 7-fold more efficient than the high-temperature-activated icositetramer at neutral pH. Monomer exchange can be observed between dimers of Mj HSP 16.5 at pH 3.0 and 65°C. These results not only demonstrate that the icositetramer structure of Mj sHSP16.5 is not necessary for its molecular chaperone activity, but also suggest that Mj sHSP16.5 is a very efficient chaperone acting at high temperature and under the acidic condition. Even though it is not clear whether the native environment of Methanococcus jannaschii is acidic or not, given its ability to excrete acidic compounds, it is likely that Methanococcus jannaschii will encounter acidic internal or external environments at high temperature. Our results demonstrate that Mj HSP 16.5 may help Methanococcus jannaschii to survive better under those extreme environmental conditions. Supported by the National Natural Science Foundation of China (Grant Nos. 20203001, 20673003, and 30490245) and Ministry of Science and Technology of China (Grant No. 2006AA02Z301)     

Towards single cell heat shock response by accurate control on thermal confinement with an on-chip microwire electrode

Metal electrodes with micron scale width enable the heating of less than a dozen cells in a confluent layer at predictable temperatures up to 85 °C with an accuracy of ±2 °C. Those performances were obtained by a preliminary robust temperature calibration based on biotin-rhodamine fluorescence and by controlling the temperature map on the substrate through thermal modeling. The temperature accuracy was proved by inducing the expression of heat shock proteins (HSP) in a few NIH-3T3 cells through a confined and precise temperature rise. Our device is therefore effective to locally induce a heat shock response with almost single-cell resolution. Furthermore, we show that cells heated at a higher temperature than the one of heat shock remain alive without producing HSP. Electrode deposition being one of the most common engineering processes, the fabrication of electrode arrays with a simple control circuit is clearly within reach for parallel testing. This should enable the study of several key mechanisms such as cell heat shock, death or signaling. In nanomedicine, controlled drug release by external stimuli such as for example temperature has attracted much attention. Our device could allow fast and efficient testing of thermoactivable drug delivery systems.     

Low‐Level Laser Therapy Restores the Oxidative Stress Balance in Acute Lung Injury Induced by Gut Ischemia and Reperfusion

It remains unknown if the oxidative stress can be regulated by low‐level laser therapy (LLLT) in lung inflammation induced by intestinal reperfusion (i‐I/R). A study was developed in which rats were irradiated (660 nm, 30 mW, 5.4 J) on the skin over the bronchus and euthanized 2 h after the initial of intestinal reperfusion. Lung edema and bronchoalveolar lavage fluid neutrophils were measured by the Evans blue extravasation and myeloperoxidase (MPO) activity respectively. Lung histology was used for analyzing the injury score. Reactive oxygen species (ROS) was measured by fluorescence. Both expression intercellular adhesion molecule 1 (ICAM‐1) and peroxisome proliferator‐activated receptor‐y (PPARy) were measured by RT‐PCR. The lung immunohistochemical localization of ICAM‐1 was visualized as a brown stain. Both lung HSP70 and glutathione protein were evaluated by ELISA. LLLT reduced neatly the edema, neutrophils influx, MPO activity and ICAM‐1 mRNA expression. LLLT also reduced the ROS formation and oppositely increased GSH concentration in lung from i‐I/R groups. Both HSP70 and PPARy expression also were elevated after laser irradiation. Results indicate that laser effect in attenuating the acute lung inflammation is driven to restore the balance between the pro‐ and antioxidants mediators rising of PPARy expression and consequently the HSP70 production.