2-DE proteomic analysis of HSP70 in mollusc Chamelea gallina

Bidimensional electrophoresis (2-DE) protocols were adapted on Chamelea gallina digestive glands studies by the analysis of Heat Shock Proteins (HSP) compared with monodimensional electrophoresis (1-DE) results. Because polycyclic aromatic hydrocarbons (PAH) act on HSPs, C. gallina specimens were exposed to 0.5 mg/L of benzo[a]pyrene (B[a]P) for 24 h, 7 and 12 days. Immunoblotting after 1-DE showed a single band of 70 kDa significantly induced after 7 days of B[a]P exposure. After 2-DE, eight major high-resolved spots between 17 and 98 kDa were revealed. Three spots fell within the range of 62–98 kDa and of 5–6 pI, parameters which could include HSP70. Two spots of 77 and 72 kDa, obtained after 2-DE immunoblotting, could correspond to constitutive HSC70 and to inducible HSP70 forms respectively. Changes observed in inducible and in constitutive forms might be related to an adaptation to stress and to a normal protein synthesis capability, respectively. Employment of 2-DE and relationship between HSP70 and HSC70 may be useful to clarify their role in molluscs subjected to stress events.     

Cloning,characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): Response to benzo[α]pyrene exposure

Glutathione S-transferases (GSTs) are phase II enzymes involved in major detoxification reactions of xenobiotics in many organisms. In this study, a full-length cDNA of GST-pi was cloned from the gill of Venerupis philippinarum by rapid amplification of cDNA ends (RACE) method for the first time. The full-length cDNA of V. philippinarum GST-pi (denoted as VpGSTp) was 1142 bp, with a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 438 bp, and an open reading frame (ORF) of 618 bp encoding a protein of 205 amino acid residues with an estimated molecular mass of 23.9 kDa and an predicted isoelectric point (pI) of 7.9. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzyme belongs to the pi-class, and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. Tissue distribution analysis of the VpGSTp mRNA revealed that the GST-pi expression level was observed higher in gill, adductor muscle, mantle and foot while lower in digestive gland. Using quantitative real-time PCR, the dose- and time-related effects of benzo[α]pyrene (B[α]P) on VpGSTp mRNA expression were investigated in gills and digestive gland. The results showed that a time-dependant increase in the expression of VpGSTp was induced by B[α]P and appeared a good linear relationship with B[α]P concentrations. All these results suggested that GST-pi in bivalve had an antioxidant role and VpGSTp expression may be a useful biomarker candidate for monitoring environmental contaminants such as PAHs.     

The combined influence of sub-optimal temperature and salinity on the in vitro viability of Perkinsus marinus, a protistan parasite of the eastern oyster Crassostrea virginica

Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25 ppt) to 10 °C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 °C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7 ppt cultures acclimated to each temperature and then transferred to 3.5 ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30 days (3.5 ppt, 2 °C: 0% viability), 60 days (3.5 ppt, 10 °C: 0% viability) and 90 days (7 ppt, 2 °C: 0.6 ± 0.7%; 7 ppt, 10 °C: 0.2 ± 0.2%).     

Salinities, not diets, affect strontium/calcium ratios in otoliths of Anguilla japonica

Although otolith Strontium (Sr)/calcium (Ca) ratios have been widely used to reconstruct the past salinity environmental history of anguillid eels, factors affecting the Sr/Ca ratios in otoliths are incompletely understood. Japanese Eel (Anguilla japonica) elvers (mean length 54.7 ± 2.1 mm) were collected in the estuary during their upstream migration and reared at 5 different salinities (0, 5, 15, 25, and 35 psu) and 3 types of feeding conditions (formulated feed, tubifex, and starvation) for 30 days to evaluate the effects of salinity and diets on otolith Sr/Ca ratios. Ca and Sr concentrations in the ambient water significantly increased with salinity (SAL) as [Ca] _water = 15.50SAL − 5.56, and [Sr] _water = 0.21SAL + 0.03, respectively. Sr/Ca ratios in otoliths increased with salinity (SAL) of the rearing water as [(Sr/Ca) × 1000] _otolith = 0.091SAL + 3.790. In diets, Sr/Ca ratios were 4 times higher in tubifex than in formulated feed. However, in otoliths, ANOVA indicated that Sr/Ca ratios did not differ significantly between groups fed on tubifex or formulated feed (p = 0.118). Otolith Sr/Ca ratios were negatively correlated with fish growth rates while the growth rates differed significantly among rearing conditions with different salinities and diets. Partition coefficients of the Sr/Ca ratios from ambient water to fish tissues and otoliths significantly increased with salinity. The Sr/Ca ratios of Japanese Eel otoliths thus were positively correlated with the ambient salinity and decreased with increasing fish growth rate, but was not affected by fish diet.     

Association of heat shock protein70-2 (HSP70-2) gene polymorphism with coronary artery disease in an Iranian population

Background

Coronary artery disease (CAD) is an inflammatory process and a major cause of mortality and morbidity. The (heat shock protein70-2) HSP70-2 gene is reported to be associated with coronary artery disease possibly by affecting the regulation of pro-inflammatory cytokines such as TNF-α. The association between CAD and the HSP70-2 gene + 1267A>G polymorphism has been studied in some populations but there are no data about this association in the Iranian population.

Aim

We have investigated the association between the HSP70-2 gene + 1267A>G polymorphism and angiographically defined CAD within an Iranian population.

Methods

We determined the presence of the HSP70-2 gene + 1267A>G polymorphism in 628 patients with CAD and 307 healthy individuals using PCR-RFLP. Of the patients, 433 (68%) had > 50% stenosis (CAD +) and the remaining 195 patients had < 50% stenosis (CAD −), based on coronary angiography. Angiogram positive patients were subdivided into three groups: those with single (n = 113), double (n = 134), and triple vessels (n = 186) disease.

Results

A significant higher frequency of AG + GG genotypes (G allele carriers) was observed in angiogram positive and angiogram negative groups compared to controls in a dominant analysis model of the HSP70-2 gene + 1267A>G position (51.2 vs. 43.2, P = 0.002, OR = 1.37) (51.0 vs. 43.2, P = 0.01, OR = 1.37). The allele frequency of the HSP70-2 G was also significantly higher in angiogram positive and angiogram negative groups compared to the control group (51.2 vs. 43.2, P = 0.002, OR = 1.37) (51.0 vs. 43.2, P = 0.01, OR = 1.37).

Conclusion

These results suggest that HSP70-2 + 1267 polymorphism may influence the risk of CAD in Iranian population, however further studies are needed to clarify the role of other HSP70-2 gene polymorphisms in the pathogenesis of the CAD.     

Antioxidant defenses and oxidative stress parameters in tissues of mud crab (Scylla serrata) with reference to changing salinity

The effects of salinity (10, 17 and 35 ppt) on O₂ consumption, CO₂ release and NH₃ excretion by crabs and oxidative stress parameters and antioxidant defenses of its tissues were reported. An increase in salinity caused a decrease in O₂ consumption and CO₂ release and an increase in ammonia excretion by crabs. Lipid peroxidation, protein carbonyl, H₂O₂ levels and total antioxidant capacity of the tissues elevated significantly at 35 ppt salinity except in abdominal muscle where H₂O₂ content was low. Ascorbic acid content of tissues was higher at 17 ppt salinity than at 10 and 35 ppt salinities. With increasing salinity, a gradual decrease in SOD, an increase in catalase, no change in GPx and a decrease followed by an increase in GR activities were recorded for abdominal muscle. While for hepatopancreas, an increase followed by a decrease in SOD and catalase, decrease in GPx and GR activities were noticed with increasing salinity. In the case of gills, a decrease followed by an increase in SOD, a decrease in catalase and GPx and an increase in GR activities were noted when the salinity increased from 10 ppt to 35 ppt. These results suggest that salinity modulation of oxidative stress and antioxidant defenses in Scylla serrata is tissue specific.     

Gentamicin inhibits HSP70-assisted protein folding by interfering with substrate recognition

We previously reported that gentamicin (GM) specifically binds to heat-shock protein with subunit molecular masses of 70 kDa (HSP70). In the present study, we have investigated the effects of GM binding on HSP70-assisted protein folding in vitro. The C-terminal, and not the N-terminal of HSP70 was found to bind to GM. GM significantly suppressed refolding of firefly luciferase in the presence of HSP70 and HSP40, although the ATPase activity of HSP70 was unaffected by GM. A surface plasmon resonance analysis revealed that GM specifically interferes with the binding of HSP70 to a model peptide that mimics the exposed hydrophobic surface of the folding intermediates. These results indicated that GM inhibits the chaperone activity of HSP70 and may suppress protein folding via inhibition of HSP70 in vivo.

Structured summary

MINT-7384283: HSP40 (uniprotkb:P25685) binds (MI:0407) to HSP70 (uniprotkb:P34930) by surface plasmon resonance (MI:0107)MINT-7384430: RNaseA (uniprotkb:P61823) binds (MI:0407) to HSP70 (uniprotkb:P34930) by surface plasmon resonance (MI:0107)     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

Study on the biochemical basis of mefloquine resistant Plasmodium falciparum

Increase in drug detoxification and alteration of drug uptake and efflux of Plasmodium falciparum were investigated for their possible association with mefloquine (MQ) resistance in five different clones of P. falciparum from Thailand (T994b₃, K1CB₂, PR70CB₁, PR71CB₂ and TM₄CB8-2.2.3). Fifty percent inhibitory concentration (IC₅₀) values from these five clones varied between 30- and 50-fold. Regarding the detoxification mechanism, the ability of P. falciparum clones to biotransform MQ was shown in vitro by parasite microsomal protein prepared from parasite infected red blood cells protein (30 μg), NADPH (1 nM) and phosphate buffer pH 7.4, carried out at 37 °C with agitation. Radiolabelled unmetabolized MQ and possible metabolite(s) generated from the reaction was extracted into ethylacetate and separated by radiometric-HPLC after 1 h. All clones were capable of converting MQ into carboxymefloquine (CMQ), which is the main metabolite in human plasma. In addition, another unidentified metabolite eluted at 4.2 min on the chromatograph could be detected from the incubation reaction. This metabolite has never been detected in human liver microsomes before. There was no significant difference in the percentages of CMQ formed in the resistant (T994_b3, PR₇₀CB₁, PR₇₁CB₂) and sensitive (TM₄CB8-2.2.3, K1CB₂) clones. Another possible mechanism, i.e., alteration in the accumulation of MQ in the parasites was investigated in vitro using [¹⁴C]MQ as a tracer. The time courses of [¹⁴C]MQ uptake and efflux were generally characterized by two phases. A trend of increased efflux of [¹⁴C]MQ was observed in the resistant compared with sensitive clones.     

The influence of nutritional conditions on metal uptake by the mixotrophic dual symbiosis harboring vent mussel Bathymodiolus azoricus

The vent mussel Bathymodiolus azoricus, host thioautotrophic and methanotrophic bacteria, in their gills and complementary, is able to digest suspended organic matter. But the involvement of nutritional status in metal uptake and storage remains unclear. The influence of B. azoricus physiological condition on its response to the exposure of a mixture of metals in solution is addressed. Mussels from the Menez Gwen field were exposed to 50 μg L^− 1 Cd, plus 25 μg L^− 1 Cu and 100 μg L^− 1 Zn for 24 days. Four conditions were tested: (i) mussels harboring both bacteria but not feed, (ii) harboring only methanotrophic bacteria, (iii) without bacteria but fed during exposure and (iv) without bacteria during starvation. Unexposed mussels under the same conditions were used as controls. Eventual seasonal variations were assessed. Metal levels were quantified in subcellular fractions in gills and digestive gland. Metallothionein levels and condition indices were also quantified. Gill sections were used for fluorescence in situ hybridization (FISH) to assess the temporal distribution of symbiotic associations. Starvation damages metal homeostasis mechanisms and increase the intracellular Zn and MT levels function. There is a clear metallic competition for soluble and insoluble intracellular ligands at each condition. Seasonal variations were observed at metal uptake and storage.     

Cloning HSP70 and HSP90 genes of kaluga (<Emphasis Type="Italic">Huso dauricus</Emphasis>) and the effects of temperature and salinity stress on their gene expression

The genes encoding HSP70 and HSP90 proteins were isolated from kaluga by homologous cloning and rapid amplification of complementary DNA (cDNA) ends (RACE). HSP70 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KP050541","term_id":"756558159","term_text":"KP050541"}}KP050541) and HSP90 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KP050542","term_id":"756558161","term_text":"KP050542"}}KP050542) cDNAs were composed of 2275 and 2718 bp and encoded polypeptides of 650 and 725 amino acids, respectively. Basic Local Alignment Search Tool (BLAST) analysis showed that HSP70 and HSP90 of kaluga shared high identities with those of Acipenser ruthenus, Acipenser schrenckii, and Acipenser baerii (98–99 %). Fluorescent real-time RT-PCR under unstressed conditions revealed that HSP70 and HSP90 were expressed in 11 different tissues of kaluga. Messenger RNA (mRNA) expressions of both HSP70 and HSP90 were highest in the intestine and lowest in the muscle. In addition, the patterns of mRNA expression of HSP70 and HSP90 were similar, although the level of expression was more in HSP90 than in HSP70 (P < 0.05).We also analyzed patterns of HSP70 and HSP90 expression in the muscle, gill, and liver of kaluga under different combinations of temperature and salinity stress, including temperatures of 4,10, 25, and 28 °C at 0 ppt salinity, and salinities of 10, 20, 30, and 40 ppt at 16 °C, where 16 °C at 0 ppt (parts per thousand) served as the control. We found that levels of mRNA expression of both HSP70 and HSP90 were highest at 4 °C in the muscle, gill, and liver and changed little with salinity stress. These results increase understanding of the mechanisms of stress response of cold freshwater fish.     

Isolation and characterization of a new Cu-Fe protein from Desulfovibrio aminophilus DSM12254

The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14 ± 1 subunits of 15254.3 ± 7.6 Da. Mössbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S]²⁺ centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S]⁺ spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Furthermore, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance.