Cloning and expression of a heat shock protein (HSP) 90 gene in the haemocytes of Crassostrea hongkongensis under osmotic stress and bacterial challenge

Heat shock protein 90 (HSP90) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Here, we report the cloning of the HSP90 homologue in Crassostrea hongkongensis (ChHSP90) through SSH in combination with RACE from cDNA of haemocytes. The full-length cDNA of ChHSP90 is 2459 bp in length, consisting of a 3', 5'-untranslated region (UTR) and an open reading frame of 2169 bp encoding 722 amino acids. The identity analysis of the amino acid sequence of HSP90 revealed that ChHSP90 is highly conserved. Distribution of ChHSP90 mRNA in gonad, heart, adductor muscle, mantle, gill, digestive gland, and haemocytes suggested that ChHSP90 is ubiquitously expressed. The mRNA levels of ChHSP90 under salinity and bacterial challenges were analyzed by real-time PCR. Under hypo-osmotic treatment, ChHSP90 mRNA in gonad, heart and haemocytes were significantly up-regulated on day 2 and onwards; while in gill, digestive gland and adductor muscle it was significantly down-regulated; the expression in mantle was decreased significantly on day 2 and 3 (P < 0.01), and then up-regulated on day 4 (P < 0.05). Under hyper-osmotic treatment, the mRNA level in gonad, heart, adductor muscle was increased on day 2 and onwards; in gill, it was firstly increased, and then gradually decreased, reaching a minimum on day 3. On day 4, the expression level in gill recovered to pre-treatment level; in mantle and digestive gland, the expression levels were decreased, reaching to the minimum on day 3. During Vibrio alginolyticus challenge, the mRNA level of ChHSP90 increased 3-fold at 4 h post-infection, returned to its pre-challenge level at 6 h post-infection, then was further up-regulated from 8 to 36 h post-infection. These experiments demonstrate that ChHSP90 mRNA is constitutively expressed in various tissues and apparently inducible in haemocytes under salinity and bacterial challenges, suggesting its important role in response to both osmotic stress and bacterial invasion.     

The involvement of HSP22 from bay scallop <Emphasis Type="Italic">Argopecten irradians</Emphasis> in response to heavy metal stress

Heat shock protein 22 (HSP22) is an important member of small heat shock protein (sHSP) subfamily which plays a key role in the process of protecting cells, facilitating the folding of nascent peptides, and responding to stress. In the present study, the cDNA of HSP22 was cloned from Argopecten irradians (designated as AiHSP22) by rapid amplification cDNA end (RACE) based on the expressed sequence tags (ESTs). The full-length cDNA of AiHSP22 was of 1,112 bp, with an open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The deduced amino acid sequence of AiHSP22 showed high similarity to previously identified HSP22s. The expression patterns of AiHSP22 mRNA in different tissues and in haemocytes of scallops exposed to Cd²⁺, Pb²⁺ or Cu²⁺ were investigated by real-time quantitative RT-PCR. The mRNA of AiHSP22 was constitutively expressed in all examined tissues, including haemocyte, muscle, kidney, gonad, gill and heart. The expression level in heart and muscle was higher than that in other tissues. The mRNA level of AiHSP22 in haemocytes was up-regulated after a 10 days exposure of scallops to Cu²⁺, Pb²⁺ and Cd²⁺. However, the expression of AiHSP22 did not increase linearly along with the rise of heavy metal concentration. Different concentrations of the same metal resulted in different effects on AiHSP22 expression. The sensitive response of AiHSP22 to Cu²⁺, Pb²⁺ and Cd²⁺ stress indicated that it could be developed as an indicator of exposure to heavy metals for the pollution monitoring programs in aquatic environment.     

Assessment of the state of the Japanese scallopMizuhopecten yessoensis in Alekseeva Bight (Peter the Great Bay, Sea of Japan) based on morphological and biochemical parameters

The results of an integrated examination of the state of the scallopMizuhopecten yessoensis in Alekseeva Bight (Peter the Great Bay, Sea of Japan) are presented. In mollusks of different ages, shell height was measured; in animals of commercial size (over 100 mm), some size and weight characteristics (annual increment of shell and adductor muscle and soft tissue weight) were determined. The morphology of the digestive gland and gills was studied. In the adductor muscle and digestive gland, the concentration of heavy metals (HMs) (Hg, Cu, Zn, Mn, Pb, Cd, Ni, Co, and Cr) was determined. In the digestive gland, metallothionein and reduced glutathione concentration was also determined, as was the activity of glutathione-dependent enzymes (glutathione peroxidase and glutathione reductase). In scallops collected outside Alekseeva Bight, the linear growth rate and adductor muscle weight were on average 1.3 and 1.7 times greater, respectively, than in those collected in the bight. In scallop organs, numerous histomorphological alterations were revealed: digestive cell vacuolization and hemocyte infiltration of the digestive gland, hyperplasia and vacuolization of the respiratory epithelium, and connective tissue hypertrophy in gill filaments. The biochemical parameters of scallops from Alekseeva Bight differed substantially from those of mollusks collected outside the bight. We conclude that one of the factors negatively affecting the state of theM. yessoensis population in Alekseeva Bight is the contamination of the bight with HMs, especially mercury. This is consistent with the results of chemical analysis of bottom sediments and tissues of two mytilid species,Modiolus kurilensis andCrenomytilus grayanus, specimens of which were collected in the bight together with the scallops [3].     

Mitochondrial thioredoxin-2 from disk abalone (Haliotis discus discus): molecular characterization, tissue expression and DNA protection activity of its recombinant protein

Thioredoxin-2 is a mitochondria-specific member of the thioredoxin (TRx) super-family that plays an important role as a component of the mitochondrial antioxidant system. The gene coding mitochondrial TRx-2 was isolated from the disk abalone (Haliotis discus discus) cDNA library, denoted as AbTRx-2. It contains 1214-bp full length with 519-bp open reading frame, encoding 173 amino acids. AbTRx-2 showed characteristic TRx active site at ⁹⁶WCGPC¹⁰⁰ and mitochondrial targeting peptide at the N-terminal amino acid sequence. The deduced amino acid comparison showed that AbTRx-2 shares 43 and 42% identity with Xenopus laevis and human TRx-2, respectively. Purified recombinant AbTRx-2 fusion protein was shown to catalyze insulin reduction and protect supercoiled plasmid DNA from damages induced by metal-catalyzed generation of reactive oxygen species. Constitutive AbTRx-2 mRNA was detected in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes, in a tissue specific manner. The AbTRx-2 mRNA was up-regulated in gill and digestive tract tissues initially at 3 h post-injection of H₂O₂ and maintained higher level at 6 h. Our results suggest that abalone TRx-2 may play an important role in regulating oxidative stress in mitochondria by catalyzing protein disulfide reduction, scavenging of ROS, and minimizing the DNA damage.     

Changes in antioxidant gene expression and induction of oxidative stress in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) under Al stress

Aluminium toxicity has been recognized as a primary growth-limiting factor in acid soil, resulting in a decrease in plant growth and production. In this experiment we have studied the induction of oxidative stress and changes in antioxidant gene expression in pea (var. ALASKA) under aluminium (Al) stress. We have found that Al treatment affected the growth of pea plant and induced oxidative stress with a change in antioxidant gene expression profile. While the expression of glutathione-s-transferase (GST) and catalase (CAT) was more in root, cytosolic Ascorbate peroxidase (cAPX) expression increased in shoot under aluminium stress. Copper- Zinc Superoxide dismutase (Cu-Zn SOD) gene expression was higher after 24 h but decreased after 48 h along with elevated expression of manganese superoxide dismutase (MnSOD) and iron-superoxide dismutase (FeSOD) at higher aluminium contentrations after 24 and 48 h. Aluminium stress elevated hydrogen peroxide (H₂O₂) level and affected the growth. The proline content did not change significantly, whereas glutathione content increased with a decreased ascorbate content under Al stress. The present study indicates that aluminium treatment affected the antioxidant gene expression and induced oxidative stress in pea plant.     

Acquisition of thermotolerance in bay scallops, Argopecten irradians irradians, via differential induction of heat shock proteins

Many cells and organisms are rendered transiently resistant to lethal heat shock by short exposure to sublethal temperatures. This induced thermotolerance is thought to be related to increased amounts of heat shock proteins (HSPs) which, as molecular chaperones, protect cells from stress-induced damage. As part of a study on bivalve stress and thermotolerance, work was undertaken to examine the effects of sublethal heat shock on stress tolerance of juveniles of the northern bay scallop, Argopecten irradians irradians, in association with changes in the levels of cytoplasmic HSP70 and 40. Juvenile bay scallops heat-shocked at a sublethal temperature of 32 °C survived an otherwise lethal heat treatment at 35 °C for at least 7 days. As determined by ELISA, acquisition of induced thermotolerance closely paralleled HSP70 accumulation, whereas HSP40 accrual appeared less closely associated with thermotolerance. Quantification of scallop HSPs following lethal heat treatment, with or without conditioning, suggested a causal role for HSP70 in stress tolerance, with HSP40 contributing to a lesser, but significant extent. Overall, this study demonstrated that sublethal heat shock promotes survival of A. irradians irradians juveniles upon thermal stress and the results support the hypothesis that HSPs have a role in this induced thermotolerance. Exploitation of the induced thermotolerance response shows promise as a means to improve survival of bay scallops in commercial culture.     

Histone H4 deacetylation down-regulates catalase gene expression in doxorubicin-resistant AML subline

We explored if epigenetic mechanisms could be involved in the down-regulated expression of catalase gene (CAT) in the doxorubicin-resistant acute myelogenous leukemia (AML)-2/DX100 cells. Down-regulated CAT expression in AML-2/DX100 cells was completely recovered after treatment of hydrogen peroxide (H₂O₂) and histone deacetylase inhibitor, trichostatin A (TSA) but was increased slightly by the treatment of DNA methylation inhibitor, 5-aza-2′-deoxycytidine (5-AdC). Bisulfite-sequencing PCR revealed that a CpG island of CAT was not methylated in AML-2/DX100 cells. Chromatin immunoprecipitation assay confirmed that acetylation of histone H4 in AML-2/DX100 cells significantly decreased as compared with that in AML-2/WT cells, which was significantly increased by TSA more than 5-AdC. Meanwhile, overexpression of other up-regulated peroxidase genes appears to make compensation for decreased H₂O₂-scavenging activity for the down-regulated CAT expression in AML-2/DX100 cells. These results suggest that histone H4 deacetylation is responsible for the down-regulated CAT expression in AML-2/DX100 cells, which are well adapted to oxidative stress.     

Variations in the antioxidant system and circadian rhythms of goldfish,Carassius auratus,exposed to ammonia: profile of the effects of green LED spectra

The present study evaluated effects of green light emitting diode (LED) spectra on oxidative stress and circadian rhythms in goldfish exposed to various concentrations (0.25 and 0.5 mg/L) of NH₃, under a white fluorescent bulb (control; simulated natural period) and green LED light. We measured mRNA expression and activity of antioxidant enzymes (superoxide dismutase and catalase) and mRNA expression of circadian rhythms (period 2), in addition to levels of plasma hydrogen peroxide, cortisol and melatonin. Damage to nuclear DNA was assessed using the comet assay. All stress indicators and melatonin were significantly lower in the green LED group than in the control group. With an increase in the concentration of ammonia, the observed effects became even more significant and generally increased with time. Comparatively, damage to the nuclear DNA was greater in the 0.5 mg/L NH₃ group, and lower in the green LED group. The Period 2 mRNA expression reduced as increasing ammonia treatment but increased as green LED exposed. We have suggested that Green LED reduced levels of oxidative stress, which suggests an antioxidant effect against NH₃ toxicity. Additionally, ammonia is affected the circadian rhythms and the green LED wavelength is able to regulate effectively the circadian rhythm.     

Heat shock protein 70 regulates the abscisic acid-induced antioxidant response of maize to combined drought and heat stress

We investigated the interaction between heat shock protein 70 (HSP70) and abscisic acid (ABA)-induced antioxidant response of maize to the combination of drought and heat stress. First, the increased activities of enzymes, including superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT), induced by drought were less than those by heat or combined drought and heat stress, except some individual cases (e.g. CAT in leaves, GR in roots). Second, both HSP70 synthesis and H₂O₂ production increased prominently under drought, heat or their combination stress; the increase in leaves induced by drought and heat combination was the highest, followed by heat and by drought, while the increase in roots had not visible difference. Third, either in leaves or roots, pretreatment with ABA inhibitor, HSP70 inhibitor and H₂O₂ scavenger, significantly arrested the stress-induced increase of antioxidant enzyme activities, and ABA inhibitor and H₂O₂ scavenger obviously suppressed HSP70 synthesis, while HSP70 inhibitor slightly heightened H₂O₂ accumulation. Finally, 100 μM ABA significantly enhanced the activities of antioxidant enzymes, HSP70 expression and H₂O₂ production under stresses in comparison with ABA-deficient mutant vp5 maize plants without pretreatment. Thus, ABA-induced H₂O₂ production enhances the HSP70 synthesis and up-regulates the activities of antioxidant enzymes, resulting in the suppression of cellular reactive oxygen species (ROS) levels. Our results suggest that HSP70 may play a crucial role in ABA-induced antioxidant defense of maize to drought and heat combination.     

Alternation of immune parameters and cellular energy allocation of Chlamys farreri under ammonia-N exposure and Vibrio anguillarum challenge

The complex interactions among host, pathogen and environment are believed to be the main causes for the mass mortality of cultured scallops. In the present study, the temporal variations of immune parameters and cellular energy allocation (CEA) of Chlamys farreri under ammonia-N, Vibrio anguillarum as well as their combined treatment were investigated to better understand the energetic mechanisms of scallop in immune defense. After 1 d exposure to ammonia-N, V. anguillarum and their combination, the superoxide anion level and superoxide dismutase (SOD) activity in the serum of scallops increased substantially. At 24 d post exposure, the mRNA expression levels of isocitrate dehydrogenase (IDH), heat shock protein 70 (HSP 70), HSP 90 and glutamine synthetase (GS), as well as the malondialdehyde content remarkably increased, while SOD activity was depressed significantly (P < 0.05). The glycogen reserved in the tissues from scallops exposed to the combined stress for 1 d, 12 d and 24 d were significantly lower than those in the control (P < 0.05). The CEA values in all the examined tissues including gonad, gill, hepatopancreas and adductor muscle were significantly lower than those of control (P < 0.05) when exposure to ammonia-N, V. anguillarum and their combined treatment for 12 and 24 d. Furthermore, the combined stress also had a significant impact upon CEA in all the examined tissues in scallops post 1 d exposure (P < 0.05). The above results demonstrated that SOD, IDH, HSPs and GS in hemolymph of treated scallops are necessary, but not sufficient to the complete protection against stress-induced cellular damage along with the treatment duration. Immune defense against the combination of pathogen invasion and environmental stress can impose greater costs on scallop's energy expenditure than a single stressor, and the combined treatment preferentially consumed more available glycogen in scallops for immune defense. Hence, in addition to be used in immunological evaluation, CEA is also a powerful tool to provide valuable insights into possible mechanisms of mass mortalities in cultured scallops.     

Cloning and characterization of two ferritin subunit genes from bay scallop, <Emphasis Type="Italic">Argopecten irradians</Emphasis> (Lamarck 1819)

We have cloned two full-length cDNAs from two ferritin genes (Aifer1 and Aifer2) of the bay scallop, Argopecten irradians (Lamarck 1819). The cDNAs are 1,019 and 827 bp in length and encode proteins of 171 and 173 amino acids, respectively. The 5′ UTR of each contains a conserved iron response element (IRE) motif. Sequence analyses reveal that both proteins belong to the H-ferritin family with seven conserved amino acids in the ferroxidase center. Highest expression of Aifer1 is found in the mantle and adductor muscle, while that of Aifer2 is only in the latter tissue. These Aifer genes are differentially expressed following bacterial challenge of the scallop. The expression level of Aifer1 was acutely up-regulated (over 10 fold) at 6 h post-bacteria injection, whereas Aifer2 expression was not significantly changed by bacterial challenge. Both genes were effectively expressed in E. coli BL21 (DE3), producing proteins of similar molecular weight, approximately 23 kDa. Purified Aifer1 and Aifer2 proteins exhibited iron-chelating activity of 33.1% and 30.4%, respectively, at a concentration of 5 mg/ml. Cations, Mg²⁺, Zn²⁺ and Ca²⁺, depressed iron-chelating activity of both proteins. Additionally, the E. coli cells expressing recombinant Aifer1 and Aifer2 showed tolerance to H₂O₂, providing a direct evidence of the antioxidation function of ferritin. The results presented in this study suggest important roles of Aifer1 and Aifer2 in the regulation of iron homeostasis, immune response, and antioxidative stress in A. irradians.     

Isolation, Purification, and Characterization of Catalase from the Methylotrophic Yeast Pichia pastoris

Catalase (CAT_pp) with molecular weight 223 kD was isolated from the methylotrophic yeast Pichia pastoris and purified 90-fold by ion-exchange chromatography and gel filtration. Quantitative parameters of absorption and CD spectra of CAT_pp solutions and of its membrane-concentrated form (CAT_pp-conc) were studied. Rates of H₂O₂ decomposition and kinetic characteristics K _m and k _cat of CAT_pp and CAT_pp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30°C, as well as the effective constant k _in of the enzyme inactivation rate during the catalysis and the constant k ₂ of the interaction rate of the Complex I catalases with H₂O₂. Thermal inactivation of CAT_pp in solutions at 45°C was characterized by the effective rate constant k _in ^*, and the low-frequency (27 kHz) ultrasonic inactivation of CAT_pp at 20°C was characterized by the firstorder rate constant k _in (US). All spectral and kinetic characteristics of CAT_pp and CAT_pp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CAT_cb). All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CAT_pp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of k _cat/K _m, thermal stability comparable with the thermal stability of CAT in terms of k _in ^*, the minimal k _in, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm².     

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

Changes in gene expression, by application of H₂O₂, O₂°^–generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H₂O₂ and gamma irradiation, but not to O₂°^– generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I–IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H₂O₂ and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H₂O₂ action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H₂O₂ pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.     

Heat shock protein 27 plays a protective role in thoracic aortic dissection by promoting cell proliferation and inhibiting apoptosis

Background

Thoracic aortic dissection (TAD) is one of the most severe aortic diseases. The study aimed to explore the potential role of heat shock protein 27 (HSP27) in the pathogenesis of TAD using an in vitro model of oxidative stress in vascular smooth muscle cells (VSMCs).

Methods

HSP27 was analyzed in aortic surgical specimens from 12 patients with TAD and 8 healthy controls. A lentiviral vector was used to overexpress HSP27 in rat aortic VSMCs. Cell proliferation and apoptosis were measured under oxidative stress induced by H₂O₂.

Results

HSP27 expression was significantly higher in aortic tissue from patients with TAD and VSMCs in the aortic media were the main cell type producing HSP27. Elevated oxidative stress was also detected in the TAD samples. Overexpression of HSP27 significantly attenuated H₂O₂-induced inhibition of cell proliferation. Furthermore, HSP27 was found to decrease H₂O₂-induced cell apoptosis and oxidative stress.

Conclusions

These results suggest that HSP27 expression promotes VSMC viability, suppresses cell apoptosis, and confers protection against oxidative stress in TAD.

     

An aqueous extract of Zingiber officinale Roscoe protects mouse primary hepatic cells against hydrogen peroxide-induced oxidative stress

Hepatocytes exposed to an oxidative stressor such as hydrogen peroxide (H₂O₂) are potentially sensitized to cell death; thus, reactive oxygen species (ROS) are considered to be critical mediators of liver damage. Zingiber officinale Roscoe (ZO), also known as ginger, is cultivated commercially in China, India, Korea, and other parts of the world. In addition, it is used as a spice and flavoring agent and is also purported to possess a number of medicinal properties. In the present study, we examined the protective effect of ZO against cell damage caused by H₂O₂-induced oxidative stress. ZO reduced H₂O₂-induced apoptotic signals and the levels of intracellular ROS. ZO pretreatment also increased the phosphorylation of c-Jun, and JNK kinase. The expression of heme oxygenase-1 (HO-1) and heat shock protein 72 (HSP72) were increased by ZO pretreatment more than H₂O₂ treatment. In addition, siRNA-mediated knockdown of HO-1 and HSP72 decreased protective effect of ZO pretreatment. Our data suggest that ZO decreases ROS levels and the expressions of HO-1 and HSP72 are involved in the hepatocyte protective function of ZO against H₂O₂.