Hsp70 mRNA Research Articles

MicroRNA-570 targets the HSP chaperone network, increases proteotoxic stress and inhibits mammary tumor cell migration

The dynamic network of chaperone interactions known as the chaperome contributes significantly to the proteotoxic cell response and the malignant phenotype. To bypass the inherent redundancy in the network, we have used a microRNA (mir) approach to target multiple members of the chaperome simultaneously. We identified a potent microRNA, miR-570 that could bind the 3′untranslated regions of multiple HSP mRNAs and inhibit HSP synthesis. Transfection of cells with this miR species reduced expression of multiple HSPs, inhibited the heat shock response and reduced tumor cell growth while acted additively in combination with cytotoxic drugs. As overexpression of miR-570 elicited tumor suppressive effects, we inferred that this miR could play a potential role in inhibiting tumorigenesis and cancer cell growth. In accordance with this hypothesis, we determined a significant role for miR-570 in regulating markers of mammary tumor progression, including cell motility and invasion. Our data provide a proof of the principle that the tumor chaperome can be targeted by microRNAs suggesting a potential therapeutic avenue towards cancer therapy.

Dietary Protein Modifies Hepatic Glycolipid Metabolism, Intestinal Immune Response, and Resistance to Streptococcus agalactiae of Genetically Improved Farmed Tilapia (GIFT: Oreochromis niloticus) Exposed to High Temperature

The present study investigates the effects of dietary protein levels on glucolipid metabolism, immune function, and resistance to Streptococcus agalactiae of genetically improved farmed tilapia (GIFT) exposed to high temperature. Six practical diets were prepared to feed 360 fish (initial weight 43.78 ± 0.12 g) with graded protein levels (26.45%, 29.28%, 31.69%, 33.68%, 36.18%, and 38.75% dry matter). The results showed that 26.45% dietary protein significantly improved glycolysis by increasing PK mRNA levels, while the 29.28% and 31.69% dietary protein levels promoted gluconeogenesis by increasing PEPCK and G6Pase mRNA levels. For lipid metabolism, 26.45% dietary protein enhanced lipid synthesis by increasing PPAR-γ, SREBP1c, and FAS mRNA levels, while 31.69% dietary protein enhanced the level of lipolysis by increasing the PPAR-α and CPT1 mRNA levels. The highest plasma TG and TC contents were observed in the 29.28% and 31.69% dietary protein groups, respectively. In terms of antioxidants and immunity, the 31.69% dietary protein level activated the expression levels of HSP90 mRNA, thus increasing the expression levels of antioxidant-related genes (CAT, SOD, and GPx), and upregulating the anti-inflammatory factor IL-10 mRNA levels. In addition, regarding the antioxidant enzymes, the highest GSH content was found in the 29.28% dietary protein group, while the 31.69% dietary protein group had the maximum GSH-Px activity. The lowest plasma ALT and AST activities were observed in the 31.69% dietary protein group. Ultimately, the survival rate of juvenile GIFT fed 31.69% dietary protein was highest after a Streptococcus agalactiae challenge. Overall, 29.28–31.69% dietary protein was recommended in the diet of GIFT in a high-temperature environment.

β-Elemene Promotes Apoptosis Induced by Hyperthermia via Inhibiting HSP70.

Thermotherapy has been presented as a promising strategy to be used as an effective nonsurgical technique for colorectal carcinoma. Although this strategy presents several advantages, including low toxicity and high repeatability, thermotherapy often needs to be combined with other therapies because residual tumor cells that survive hyperthermal treatment often lead to relapse. In this study, we evaluated the effects of β-elemene, which has been proven to have the potential to reverse chemotherapy drug resistance, on promoting the antitumor effects of hyperthermia. β-elemene treatment significantly promoted apoptosis after 2 hours of hyperthermia treatment and blocked cell cycle phases at G1/G0. β-elemene also significantly decreased colony formation and tumor formation abilities after hyperthermia treatment. β-elemene treatment significantly decreased HSP70, but not HSP90 or HSP27, induced by hyperthermia treatment without disturbing HSP70 mRNA. It was also found that β-elemene decreased phosphorylated ERK1/2 induced by hyperthermia. Regain of HSP70 reversed β-elemene-mediated apoptosis, indicating that β-elemene may induce apoptosis by decreasing HSP70. Moreover, β-elemene treatment significantly decreased invasion capacity by decreasing the EMT, which was induced by hyperthermia treatment. Taken together, our results offer a potential strategy for CRC therapy via the combination of hyperthermia and β-elemene.

Expression of MRJP3 and HSP70 mRNA Levels in Apis mellifera L. Workers after Dietary Supplementation with Proteins, Prebiotics, and Probiotics.

Simple SummaryThe development of honey bees depends on royal jelly. Some of the main components of this glandular secretion are proteins known as major royal jelly proteins (MRJPs). In addition to these, bees also produce proteins that help balance the body in situations of stress. During periods of food shortage, beekeepers can supplement colony food to maintain the health and productivity of honey bees. Supplementation can be provided with feed that contains, in addition to other components, probiotics and prebiotics, which are important to balance bees’ intestinal microbiota and, consequently, to keep colonies healthy and without food stress. In order to verify if the addition of probiotics and prebiotics in food supplementation is efficient for the health of bees, after supplementation, we analyzed the levels of synthesis of one MRJP and the stress protein HSP70. The results show that, with the addition of probiotics and prebiotics in the supplementation, there was an increase in the synthesis of the analyzed MRJP3 and low synthesis of the stress protein. Therefore, we suggest that these components be added to honey bee supplementation in periods of food shortage, as it will contribute to colony health in apiaries.Royal jelly is an essential substance for the development of bees from larval to adult stages. Studies have identified a group of key proteins in royal jelly, denominated major royal jelly proteins (MRJPs). The group currently consists of nine proteins (MRJP1–MRJP9), with MRJP1 being the most abundant and MRJP3 being used as a microsatellite marker for the selection of queens with a greater production of royal jelly. The diet of bees is mostly composed of proteins, and supplementing this intake to encourage a higher production of their primary product is important for producers. It is estimated that, by adding probiotic and prebiotic organisms to their diets, the benefits to bees will be even greater, both for their immune systems and primary responses to stress. Circumstances that are adverse compared to those of the natural habitat of bees eventually substantially interfere with bee behavior. Stress situations are modulated by proteins termed heat shock proteins (HSPs). Among these, HSP70 has been shown to exhibit abundance changes whenever bees experience unusual situations of stress. Thus, we sought to supplement A. mellifera bee colony diets with proteins and prebiotic and probiotic components, and to evaluate the expression levels of MRJP3 and HSP70 mRNAs using qRT-PCR. The results revealed that differences in the expression of MRJP3 can be attributed to the different types of feed offered. Significant differences were evident when comparing the expression levels of MRJP3 and HSP70, suggesting that protein supplementation with pre/probiotics promotes positive results in royal jelly synthesis carried out by honey bee nurses.

Heat stress and hypoxia inhibit the secretion of androgens and induce epithelial-to-mesenchymal transition associated with activated TGF-β/Smad signalling in canine cryptorchidism.

Cryptorchidism, as a common congenital disease of canine testes, is mainly caused by factors leading to endocrine abnormalities in testes and infertility in a heat stress and hypoxia microenvironment. Moreover, heat stress and hypoxia, as critical microenvironmental factors, promote epithelial-mesenchymal transition (EMT), which occurs during adult tissue remodelling responses including carcinogenesis and fibrosis and is the main cause of testicular tumours. In this study, we found by haematoxylin-eosin staining that the canine cryptorchid tissue produced a lot of collagen fibres. Also, the quantitative PCR and Western blot results showed that the mRNA and protein levels of the heat stress makers HSP70 and HO-1 and the hypoxia maker HIF-1α are significant higher compared with normal testes. Moreover, we found the expression levels of TGF-βs and its two receptors TGF-βRI and TGF-βRII increased in case of cryptorchidism. From the study in vitro, we found both heat stress and COCl2 mimic hypoxia inhibited the secretion of testosterone (T) and androstenedione (A4) and promoted the expression of the EMT maker α-SMA and vimentin in Leydig cells, and also that heat stress and COCl2 stimulated with the TGF-β signalling promoted the expression of TGF-βs and its two type receptors and also the active phosphorylation of Smad2 and Smad3. The use of LY2109761, a receptor inhibitor of TGF-βs/Smad signalling pathway, was associated with heat stress and COCl2 suppression of androgens' secretion and stimulated EMT in Leydig cells. These findings characterized a novel pathogenesis of cryptorchidism and provided a new idea for therapeutics.

Monochromatic blue light not green light exposure is associated with continuous light-induced hepatic steatosis in high fat diet fed-mice via oxidative stress

Irregular light exposure is a newly identified environmental factor for the progression of lipid metabolism; however, the specific effect of light color exposure on lipid homeostasis remains unknown. Herein, 4-week-old male C57BL/6 J mice (n = 12) fed a high-fat diet (HFD) were exposed to a standard 12-h light: 12-h dark cycle (LD-WF) and a 24-h continuous monochromatic blue light (LL-BF), green light (LL-GF), or white light (LL-WF) condition for 12 weeks. LL-BF interfered with the expression of circadian genes in the hypothalamus and upregulated the plasma corticosterone (CORT) levels (p < 0.05) compared with LD-WF. Along with elevation of the CORT level, LL-BF enhanced glucocorticoid receptor synthesis, increased the Hsp90 mRNA level, reduced the antioxidant capacity, increased the production of ROS and MDA, and reduced the Pgc-1α mRNA level in the liver (p < 0.05). Furthermore, LL-BF disrupted the hepatic expression levels of genes involved in lipid metabolism, Acc and Hl, which further aggravated the hepatic steatosis status and significantly increased the liver pathological scores, TG, TC, IL-6, and TNF-α levels (p < 0.05). LL-BF consistently increased the body weight and incidence of dyslipidemia and lipid deposition. However, no difference was observed between LL-BF and LL-WF (p > 0.05). Surprisingly, LL-GF did not show any changes induced by LL-BF and LL-WF, and contrary to LL-BF, LL-GF and LD-WF showed no significantly differing changes (p > 0.05). Taken together, exposure to monochromatic blue light but not green light is associated with continuous light-aggravated hepatic steatosis in HFD-fed mice. The effect of continuous blue light exposure may be attributed to the disturbance of biological rhythm, increase in CORT secretion, induction of oxidative stress, and interference of the Acc and Hl levels in the liver.

The effects of Agaricus bisporus polysaccharides enriched diet on growth, nonspecific immunity and disease resistance in crayfish (Procambarus clarkii)

In order to accelerate the application of ABPs in shrimp and crab feed, we evaluated the effect of ABPs on the growth, non-specific immunity and disease resistance of crayfish. Crayfish (Procambarus clarkii) were randomly divided into six groups, the fed diets supplemented with different levels of ABPs: 0, 0.05, 0.15, 0.3, 0.45 and 0.6 g/kg. After 28 days of feeding, ABPs were found to significantly increase FBW and WG of crayfish. The addition of 0.15–0.6 g/kg of ABPs to the feed significantly increased the activities of ACP, AKP and T-SOD, decreased the activity of MDA enzymes in the hepatopancreas of crayfish. In the hemolymph, ABPs at 0.3–0.45 g/kg can significantly increase the activities of ACP, AKP, T-SOD and LZM enzymes, while the addition of 0.15 or 0.6 g/kg has no significant difference with the control group. The results of qPCR showed the addition of 0.15–0.6 g/kg ABPs to the feed could significantly up-regulate the expression levels of gpx, lec and hsp70 mRNA in the hepatopancreas (P < 0.05). Adding 0.15–0.6 g/kg of ABPs could up-regulate the expression of ctsb and lec gene in gills. In addition, the expression of alf was up-regulated in gills only when the addition amount of ABPs was 0.3 g/kg. While the expression of mtMnsod in gills was up-regulated when the addition amount was 0.3–0.6 g/kg. Addition of 0.05–0.6 g/kg of ABPs reduced the mortality of crayfish infected by A.hydrophila. The above results indicated that the addition of ABPs could improve the growth, non-specific immunity and disease resistance of crayfish, and the addition of 0.3 g/kg and 0.45 g/kg of ABPs might be beneficial to the culture of crayfish.

Physiological responses of channel catfish (Ictalurus punctatus) reared at different stocking densities in a recirculating aquaculture system

This study investigated the physiological responses of channel catfish (Ictalurus punctatus) reared under different stocking densities (SDs). Juvenile channel catfish with an average initial body weight of 40.6 ± 2.23 g were reared at three densities of 2.03 (low SD; LSD), 6.09 (medium SD; MSD), and 12.18 (high SD; HSD) kg m−3 for 60 days. Serum biochemistry, antioxidant defense status, immune parameters, and hepatic heat shock proteins (hsps) mRNA expression were analyzed. Serum cortisol levels are significantly elevated in MSD and HSD. A significant increase in lysozyme (LZM) activity and the contents of immunoglobulin M (IgM) and complement component 3 (C3) were observed with the increased SD. Total antioxidant capacity (T-AOC) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) did not significantly affect SD. I. punctatus reared in HSD showed significantly increased catalase (CAT) activity, malonaldehyde (MDA) content, and nitric oxide (NO) levels. Hepatic hsp70 and hsp90 mRNA expression were significantly downregulated in HSD compared to LSD. Overall, HSD caused chronic stress in I. punctatus, leading to biochemical and immune function variations

Mitochondrion Participated in Effect Mechanism of Manganese Poisoning on Heat Shock Protein and Ultrastructure of Testes in Chickens.

Manganese (Mn) poisoning can happen in the case of environmental pollution and occupational exposure. However, the underlying mechanisms of Mn-induced teste toxicity and whether mitochondrion and heat shock proteins (HSPs) are involved in toxic effect of Mn on chicken testes remain poorly understood. To investigate this, MnCl2·4H2O was administered in the diet (600, 900, and 1800mg/kg Mn) of chickens for 30, 60, and 90days. Electron microscopy and qPCR were performed. Results showed that Mn exposure suppressed dose- and time-dependently HSP40 and HSP60 mRNA levels, meanwhile increased does-dependently HSP27, HSP70, and HSP90 mRNA levels at all three time points under three Mn exposure concentrations. Furthermore, Mn treatment damaged myoid cells, spermatocytes, and Sertoli cells through electron microscopic observation, indicating that Mn treatment damaged chicken testes. In addition, abnormal shapes of mitochondria were found, and mitochondria displayed extensive vacuolation. The increase of HSP90 and HSP70 induced by Mn exposure inhibited HSP40 and stimulated HSP27, respectively, in chicken testes, which needs further to be explored. Taken together, our study suggested that there was toxic effect in excess Mn on chickens, and HSPs and mitochondria were involved in the mechanism of dose-dependent injury caused by Mn in chicken testes. This study provided new insights for Mn toxicity identification in animal husbandry production practice.

The expression of HSP70 in skeletal muscle is not associated with glycogen availability during recovery following prolonged exercise in elite endurance athletes.

The 70-kDa heat shock protein (HSP70) is a ubiquitous molecular chaperone which is highly inducible by cellular stress such as exercise. To investigate the role of muscle glycogen content on the HSP70 expression, muscle glycogen was manipulated by consumption of either water (H2O) or a carbohydrate-enriched diet (CHO) during recovery from 4h of glycogen-depleting cycling exercise in fourteen elite endurance athletes. Muscle biopsies were obtained pre- and post-exercise, and after 4 and 24h of recovery, and analyzed for HSP70 mRNA expression, as well as HSP70 protein expression and muscle glycogen within the same skeletal muscle fibers using immunohistochemistry. Exercise reduced glycogen by 59 ± 10% (P < 0.0001). After 4h of recovery, glycogen approached resting levels in the CHO group (86% of pre, P = 0.28) but remained suppressed in the H2O group (41% of pre, P < 0.001) (group × time interaction: P = 0.002). Importantly, both the HSP70 mRNA (+ 1.6-fold (+ 0.28/- 0.24), P = 0.02) and protein expression (+ 147 ± 99%, P < 0.0001) was substantially increased after exercise and remained elevated in both groups after 4h of recovery, despite clear differences in muscle glycogen content. Thus, muscle glycogen content was not related to the variation in single fiber HSP70 expression at the 4-h time-point (r2 = 0.004). In conclusion, muscle HSP70 expression remained elevated during recovery from prolonged exercise in highly trained skeletal muscle, irrespective of muscle glycogen availability.

Physical Activity and Sedentary Time in Pregnancy: An Exploratory Study on Oxidative Stress Markers in the Placenta of Women with Obesity.

Regular moderate-to-vigorous physical activity (MVPA) and reduced sedentary time (ST) improve maternal glucose metabolism in pregnancy. More MVPA and less ST outside pregnancy increase antioxidant capacity, hence, are beneficial in preventing oxidative stress. The placenta is the first line of defense for the fetus from an adverse maternal environment, including oxidative stress. However, effects of MVPA and ST on oxidative stress markers in the placenta are unknown. The purpose of this study was to assess the association of MVPA and ST in pregnancy with oxidative stress markers in placentas of overweight/obese women (BMI ≥ 29 kg/m2). MVPA and ST were objectively measured with accelerometers at <20 weeks, 24–27 and 35–37 weeks of gestation. Using linear Bayesian multilevel models, the associations of MVPA and ST (mean and changes) with mRNA expression of a panel of 11 oxidative stress related markers were assessed in 96 women. MVPA was negatively correlated with HSP70 mRNA expression in a sex-independent manner and with GCLM expression only in placentas of female fetuses. ST was positively associated with HO-1 mRNA expression in placentas of male neonates. None of the other markers were associated with MVPA or ST. We speculate that increasing MVPA and reducing ST attenuates the oxidative stress state in placentas of obese pregnant women.

Toxicity response to benzo[α]pyrene exposure: Modulation of immune parameters of the bay scallop, Argopectenirradians

Bay scallops were exposed to four BaP concentrations (0.5, 1.0, 10 and 50 μg/L) for 72 h to elucidate their immune response. Immune parameters were evaluated by measuring nitric oxide (NO) levels in hemolymph. Additionally, we measured peptidoglycan recognition proteins (PGRP), fibrinogen-domain-containing protein (FReDC1), metallothionein (MT), and heat shock protein (HSP) 70 mRNA expression in digestive diverticula. NO as well as FReDC1 and MT expression in each BaP group increased significantly over time except for the BaP 0.5 group. The PGRP and HSP70 mRNA expression in the BaP 50 group increased in the range 6–24 h and then decreased. In situ hybridization also confirmed that there was higher MT mRNA expression in the BaP 50 group than in the control group at 72 h. Our results suggest that higher levels of BaP dampened scallop immune responses, while simultaneously reducing their ability to cope with oxidative stress and DNA damage. BaP exposure can be considered a potential immune inducer in bay scallop.

Eicosapentaenoic acid mitigates palmitic acid-induced heat shock response, inflammation and repair processes in fish intestine

Understanding the metabolic effects of fatty acids on fish intestine is critical to the substitution of fish oil with vegetable oils in aquaculture. In this study, the effects of eicosapentaenoic acid (EPA) and palmitic acid (PA) on fish intestine were evaluated in vitro and in vivo. As the first step for in vitro study, an intestinal cell line (SPIF) was established from silver pomfret (Pampus argenteus). Thereafter, the effects of EPA and PA on cell viability, prostaglandin E2 (PGE2) production, and the expression of genes related to heat shock response, inflammation, extracellular matrix (ECM) formation and degradation were examined in SPIF cells. Finally, these metabolic effects of EPA and PA on the intestine were examined in zebrafish (Danio rerio) larvae. Results showed that all tested fatty acids (PA, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, and docosahexaenoic acid) except EPA reduced SPIF viability to distinct degrees at the same concentrations. PA decreased SPIF viability accompanied by an increase in PGE2 level. Meanwhile, PA increased the expression of genes related to heat shock response (grp78, grp94, hsp70, and hsp90) and inflammation (nf-κb, il-1β, and cox2). Furthermore, PA reduced the expression of collagen type I (col1a1a and col1a1b) and extracellular matrix (ECM) degradation-related gene mmp2, while up-regulating timp2 mRNA expression. In vivo, PA also increased hsp70, il-1β, and cox2 mRNA levels and limited the expression of collagen type I in the larval zebrafish intestine. Interestingly, the combination of EPA and PA partially recovered the PA-induced changes in cell viability, PGE2 production, and mRNA expression in vitro and in vivo. These results suggest that PA may result in heat shock and inflammatory responses, as well as alter ECM formation and degradation in fish intestine, while EPA could at least partially mitigate these negative effects caused by PA.

Characterization of HSP70 expression in drones of Apis cerana (Hymenoptera: Apidae)

• The full-length HSP70 gene of the Eastern honeybee Apis cerana was cloned. • The expression patterns of the HSP70 gene in drones at different developmental stages and ages were investigated. • The expression levels of the HSP70 gene in drones at different reproductive statuses were analyzed. Heat shock proteins (HSPs) constitute a superfamily of molecular chaperones that are rapidly biosynthesized in response to various biotic and abiotic factors. In this study, we first cloned the full-length HSP70 gene of the Eastern honeybee Apis cerana . Then, using real-time quantitative PCR, we explored HSP70 expression profiles in drones at different developmental stages, ages, and reproductive statuses (with and without semen). The full-length HSP70 cDNA is 2421 bp, including a 1953-bp open reading frame (ORF) that encodes a polypeptide of 650 amino acids. The HSP70 gene consists of one intron and two exons. The phylogenetic analysis revealed that the HSP70 genes of A. cerana and Apis mellifera are the most closely related. We observed HSP70 expression at all selected developmental stages and detected the highest expression in pupae with an unpigmented body cuticle and brown eyes (Pb) and much lower expression in larvae hatched within 72 h. In adult drones of different ages, the highest expression level of HSP70 was observed in 16-day-old drones; significantly lower accumulation of HSP70 mRNA was detected in 4-day-old drones. There was no significant difference in HSP70 expression between drones with and without semen captured at the entrance, while the HSP70 gene expression level strikingly differed between drones captured at the entrance and the drones collected within the hive. Our study suggests that HSP70 might play a critical role in drone development and during reproductive mating events.

Topical Spray of dsRNA Induces Mortality and Inhibits Chilli Leaf Curl Virus Transmission by Bemisia tabaci Asia II 1.

Chilli leaf curl virus (ChiLCV; genus: Begomovirus), transmitted by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in a persistent-circulative manner, is a major constraint in chilli production. The present study demonstrates for the first time that a topical spray of naked double-stranded RNA (dsRNA) on chilli plants causes mortality and inability to acquire and transmit ChiLCV in B. tabaci. dsRNA targeting heat shock protein 70 (hsp70) and fasciclin 2 (fas2) of B. tabaci Asia II 1 was first assessed under controlled conditions through oral delivery. Hsp70 and fas2 dsRNA resulted in up to 82.22% and 72% mortality of B. tabaci and around 12.4- and 8.5-fold decreases in mRNA levels, respectively, 24 h post-ingestion. ChiLCV copies in hsp70 dsRNA-fed B. tabaci steadily decreased with an increase in dsRNA concentration and were undetectable at a higher concentration of dsRNA. However, ChiLCV copies significantly increased in fas2 dsRNA-fed B. tabaci. Transmission of ChiLCV by B. tabaci was completely inhibited post-24 h feeding on hsp70 dsRNA at 3 μg/mL. Naked hsp70 dsRNA was topically sprayed on ChiLCV-infected chilli plants like an insecticide. 67.77% mortality of B. tabaci, 4.6-fold downregulation of hsp70 mRNA, and 1.34 × 1015-fold decreased ChiLCV copies in B. tabaci were recorded when adults were exposed to the dsRNA-treated plants under semi-field conditions. Foliar application of naked dsRNA reduced the ChiLCV transmission by 75% without any visible symptoms in the inoculated plants. A total of 2 consecutive sprays of dsRNA provided significant protection to B. tabaci for up to 20 days under semi-field conditions.

Thermal acclimation alters both basal heat shock protein gene expression and the heat shock response in juvenile lake whitefish (Coregonus clupeaformis).

Long-term temperature shifts associated with seasonal variability are common in temperate regions. However, these natural shifts could place significant strain on thermal stress responses of fishes when combined with mean increases in water temperatures predicted by climate change models. We examined the relationship between thermal acclimation, basal expression of heat shock protein (hsp) genes and the activation of the heat shock response (HSR) in lake whitefish (LWF; Coregonus clupeaformis), a cold water species of cultural and commercial significance. Juveniles were acclimated to either 6, 12, or 18°C water for several months prior to the quantification of hsp mRNA levels in the presence or absence of acute heat shock (HS). Acclimation to 18°C increased basal mRNA levels of hsp70 and hsp47, but not hsc70 or hsp90β in gill, liver and white muscle, while 6°C acclimation had no effect on basal hsp transcription. Fish in all acclimation groups were capable of eliciting a robust HSR following acute HS, as indicated by the upregulation of hsp70 and hsp47. An increase of only 2°C above the 18°C acclimation temperature was required to trigger these transcriptional changes, suggesting that the HSR may be frequently initiated in LWF populations living at mildly elevated temperatures. Collectively, these expression profiles show that environmental temperature influences both basal hsp levels and the HSR in LWF, and indicate that these fish may have a greater physiological and ecological susceptibility to elevated temperatures than to cooler temperatures.

Gene expression of glutathione S-transferase alpha, glutathione S-transferase rho, glutathione peroxidase, uncoupling protein 2, cytochrome P450 1A, heat shock protein 70 in liver of Oreochromis niloticus upon exposure of microcystin-LR, microcystin-RR and toxic cyanobacteria crude

ObjectiveMicrocystins (MCs) are considered to be one of the most hazard groups of cyanobacteria and are known as a group of cyclic polypeptide hepatotoxins of varying potency. The study determined the variation of gene expression of GSTA, GSTR, GPX, UCP2, CYP1A1 and HSP70 genes in liver of tilapia fish (Oreochromis niloticus) under the effect of MC-LR or MC-RR toxins. MethodsTilapia fish adult male fishes (n = 20; 120–178 g) obtained from fish farm. Fishes injected i.p. with a single dose of 50 μg/kg MC-LR, and 20 fishes injected with 500 μg/kg MC-RR. Whereas the control group was only received the vehicle solution. Total mRNA was isolated from 50 mg of hepatic sections. The relative levels of GSTA, GSTR, GPX, UCP2, HSP70/β actin mRNA were determined by RT-real time PCR. ResultsSignificantly increased in GSTA mRNA expression was observed in the liver of fish after exposure to all exposure groups. Variable alterations in the levels of GPx, GSTR, UCP2 and CYP1A mRNA expression were observed post exposure groups. Whereas, the relative level of HSP70 mRNA was increased at 12 h after MCs exposure and was elevated gradually at 24 h until 7 days post treatment. ConclusionThe presence of inducible expression of GSTA, GSTR and GPx, and no significant changes of CYP1A mRNA expression in the liver of tilapia post MCs exposure, indicates that it should be the phase II detoxification enzyme in the liver of tilapia, but not the phase I detoxification enzyme. In addition to restrain over production of ROS, increasing of UCP2 and HSP70 expression may play an important role as a molecular chaperone against oxidative stress consequently, preventing hepatic cells from apoptosis.

Specific locations and amounts of denatured collagen and collagen-specific chaperone HSP47 in the oviducts and uteri of old cows as compared with those of heifers.

Collagen, the most abundant extra-cellular matrix in oviducts and uteri, performs critical roles in pregnancies. We hypothesised that the locations and amounts of both denatured collagen and the collagen-specific molecular chaperone 47-kDa heat shock protein (HSP47) in the oviducts and uteri of old cows are different compared with those of young heifers because of repeated pregnancies. Since detecting damaged collagen in tissues is challenging, we developed a new method that uses a denatured collagen detection reagent. Then, we compared damaged collagen in the oviducts and uteri between post-pubertal growing nulliparous heifers (22.1±1.0months old) and old multiparous cows (143.1±15.6months old). Further, we evaluated the relationship between denatured collagen and HSP47 by combining this method with fluorescence immunohistochemistry. Picro-sirius red staining showed collagen in almost all parts of the oviducts and uteri. Expectedly, damaged collagen was increased in the oviducts and uteri of old cows. However, damaged collagen and HSP47 were not located in the same area in old cows. The number of fibroblasts increased, suggesting the presence of fibrosis in the oviducts and uteri of old cows. These organs of old cows showed higher HSP47 protein amounts than those of heifers. However, the uteri, but not oviducts, of old cows had lower HSP47 mRNA amounts than those of heifers. These findings revealed the specific location and amounts of denatured collagen and HSP47 in the oviducts and uteri of old cows compared with those of heifers.

Thermal Responses in Japanese Pearl Oysters, Pinctada fucata martensii

In order to examine the thermal responses in pearl oysters, both the mRNA expression levels of heatshock protein (Hsp) in their tissues and changes in the mineral levels of their hemolymph were analyzed using heattreated and heat-untreated pearl oysters. Pearl oysters that live in Tsukumo Bay on the Noto Peninsula were used as experimental materials. Pearl oysters kept at 25 C in artificial seawater were exposed to high temperatures (30 C or 35 C) for 1 h. After exposure, the pearl oysters were transferred into the seawater at 25 C and kept for 4 h. Thereafter, the hemolymph was collected by inserting a syringe into the adductor muscle of the heat-treated pearl oyster, and then hemocytes were separated from hemolymph by centrifugation. In addition to hemocytes, the mantles and gills were taken from heat-treated pearl oysters to analyze Hsp mRNA expression. The collected hemolymph was used to analyze its mineral concentration. The data of pearl oysters treated at a high temperature (30 C or 35 C) were compared with those of pearl oysters kept at the initial temperature (25 C) as a heat-untreated control. First, the mRNA expressions of several Hsps (Hsp20, Hsp40, Hsp70, and Hsp90) were analyzed using the hemocytes, mantles, and gills to examine the thermal responses of pearl oysters. With heat treatment, the mRNA expressions of all Hsps (Hsp20, Hsp40, Hsp70, and Hsp90) used in the present study were significantly increased. Especially, Hsp20 mRNA expression in hemocytes, mantles, and gills was remarkably increased by heat treatment. The responsiveness of Hsps mRNA expression was greatly higher at the rate of increase at 35 C than at the rate of increase at 30 C. Among the tissues used in the present study, the mRNA expression levels of Hsp20, Hsp40, Hsp70, and Hsp90 were higher in gills than in hemocytes and mantles. Second, we analyzed the mineral concentrations of hemolymph in the heat-treated pearl oysters. The calcium, magnesium, sodium, chlorine, and potassium ion levels in the hemolymph of pearl oysters kept at 35 C increased significantly as compared with those in the initial hemolymph of pearl oysters kept at 25 C. This suggests that heat treatment influences excretory organs such as gills and kidneys. The fact that the expression level of Hsps was highest in gills, which function as respiratory and International Journal of Zoological Investigations Contents available at Journals Home Page: www.ijzi.net Editor-in-Chief: Prof. Ajai Kumar Srivastav Published by: Saran Publications, Gorakhpur, India 372 excretory organs, supported the influences of the excretory system. This study is the first report to demonstrate changes in both the monovalent and divalent ions of hemolymph in response to heat treatment in Japanese pearl oysters. We plan to investigate the detailed mechanism of the above mineral regulation together with HSP expressions in the near future.

Effects of tricaine methanesulfonate (MS-222) on sedation and responses of yellow catfish (Pelteobagrus fulvidraco) subjected to simulated transportation stress

This study evaluated the anesthetic and sedative effects of tricaine methanesulfonate (MS-222) on yellow catfish (Pelteobagrus fulvidraco), and determined its effectiveness for the simulated transportation. In the anesthesia induction and recovery experiment, the adult fish (87.5 ± 13.9 g) were treated with five concentrations of MS-222 (0, 40, 60, 90 and 110 mg L−1), respectively. The results showed that the effective concentration of MS-222 for short-term anesthesia induction was between 90 and 110 mg L−1, according to the principle of anesthesia within 3 min and recovery within 10 min. In the simulated transportation experiment, the fish (86.7 ± 11.4 g) were stored in tanks containing 40 mg L−1 MS-222 solution for 12 h. The results revealed that the content of water ammonia nitrogen in the MS-222 group was significantly lower than that of control group . The fish in the anesthetized transportation group showed a significant decrease in serum cortisol levels (43.11 ± 3.74 ng mL−1) compared with the control group (50.12 ± 0.65 ng mL−1), whereas the levels of serum glucose in fish exposed to MS-222 significantly increased . The hsp70 mRNA expression in the liver of anesthetized yellow catfish was found to significantly upregulate after transportation . Thus, it is concluded that the effective concentrations of MS-222 for sedation and anesthesia in adult yellow catfish were from 90 to 110 mg L−1, and while 40 mg L−1 MS-222 has the potential to be used for the transportation of yellow catfish within 12 h.