Biomarker responses as indication of contaminant effects in Oreochromis niloticus

The current study investigated oxidative stress parameters (enzymes activities, metallothionein content and lipid peroxidation) in freshwater fish, Oreochromis niloticus, tilapia exposure to Monjolinho River (in 4 months of year: January, April, July and November). One critical site in Monjolinho River (site B) was assessed in comparison to a reference site (site A). Water pH and oxygen concentration was lower than that recommended by CONAMA (Brazilian National Environmental Committee), resolution 357/2005 for protection of aquatic communities, and ammonium and the metals Cu, Zn, Mn and Fe (on all months) concentrations were higher than the maximum concentration recommended. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were significantly decreased in liver and muscle in tila. pia from Monjolinho River, throughout the year, in relation to reference except in gills that SOD activity increased. Glutathione S-transferase (GST) activity was significantly increased in liver of the tilapia from Monjolinho River in all sites, in relation to reference except in gills that GST activity increased in July and decreased in November, suggesting that GST activity could be induced to neutralize the pollutants toxicity. On the other hand, GST activity was significantly decreased in white muscle indicating a toxic effect of pollutants, resulting in a decreased ability of tilapia to perform defense reactions associated to GSTs. The decrease of catalase (CAT) activity in gills of the O. niloticus together with the increase of SOD activity, could explain the increased lipid peroxidation (LPO) level in this organ. Metallothionein levels in liver and gills were significantly high in all sites. Results indicate that the exposure to metals caused severe damage to tissues; despite the consensually assumed antioxidant induction as a sign of exposure to contaminants the effects seem in part to be mediated by suppression of antioxidant system with SOD, CAT and GPx as potential candidates for tissues toxicity biomarkers of pollutants. (c) 2012 Elsevier Ltd. All rights reserved.

Assessment of the sublethal toxicity of organochlorine pesticide endosulfan in juvenile common carp (Cyprinus carpio)

This study is aimed at evaluating the sublethal effects of endosulfan (EDS) in juvenile common carp (Cyprinus carpio). For this purpose, fish were exposed for 15 days to the technical EDS (95% pure) diluted in dimethyl sulfoxide (DMSO) 0.1% of the total volume in water solution in a semi-static system at sublethal concentration (1 mu g/L). Subsequently, the liver somatic index (LSI) and factor condition (K) were determined. The total cytocrome P450 (CYP), CYP1A isoform, and the ethoxyresorufin-O-deethylase (EROD) activity were determined from the hepatic microsomal fraction as well as the activity of the oxidative stress enzyme system such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GP(X)), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH). Among the parameters assessed, EDS at the sublethal concentration in subchronic exposure caused significant changes in liver somatic indices as well as induction of the phase I biotransformation system and oxidative stress in juvenile common carp (Cyprinus carpio). Thus, it is seen that the use of biochemical biomarkers of environmental contamination in this study proved to be an extremely important tool for detecting the adverse effects of xenobiotics in the aquatic environment, even at low concentration.

ENVIRONMENTAL TOBACCO SMOKE INDUCES OXIDATIVE STRESS IN DISTINCT BRAIN REGIONS OF INFANT MICE

Environmental tobacco smoke (ETS) leads to the death of 600,000 nonsmokers annually and is associated with disturbances in antioxidant enzyme capacity in the adult rodent brain. However, little is known regarding the influence of ETS on brain development. The aim of this study was to determine levels of malonaldehyde (MDA) and 3-nitrotyrosine (3-NT), as well as enzymatic antioxidant activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD), in distinct brain structures. BALB/c mice were exposed to ETS twice daily for 1 h from postnatal day 5 through postnatal day 18. Acute exposure was performed for 1 h on postnatal day 18. Mice were euthanized either immediately (0) or 3 h after the last exposure. Immediately after an acute exposure there were higher GR and GST activities and MDA levels in the hippocampus, higher GPx and SOD activities in the prefrontal cortex, and higher GST activity and MDA levels in the striatum and cerebellum. Three hours later there was an increase in SOD activity and MDA levels in the hippocampus and a decrease in the activity of all enzymes in the prefrontal cortex. Immediately after final repeated exposure there were elevated levels of GST and GR activity and decreased GPx activity in the hippocampus. Moreover, a rise was found in GPx and GST activities in the prefrontal cortex and increased GST and GPx activity in the striatum and cerebellum, respectively. After 3 h the prefrontal cortex showed elevated GR and GST activities, and the striatum displayed enhanced GST activity. Data showed that enzymatic antioxidant system in the central nervous system responds to ETS differently in different regions of the brain and that a form of adaptation occurs after several days of exposure.

Antioxidative responses of cell suspension cultures of two Coffea arabica varieties to low aluminum levels at pH 5.8

The effects of aluminum (Al) on the activities of antioxidant enzymes and ferritin expression were studied in cell suspension cultures of two varieties of Coffea arabica, Mundo Novo and Icatu, in medium with pH at 5.8. The cells were incubated with 300 µM Al3+, and the Al speciation as Al3+ was 1.45% of the mole fraction. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) were increased in Mundo Novo, whereas glutathione reductase (GR) and guaiacol peroxidase (GPOX) activities remained unchanged. SOD, GR, and GST activities were increased in Icatu, while CAT activity was not changed, and GPOX activity decreased. The expression of two ferritin genes (CaFer1 and CaFer2) were analyzed by Real-Time PCR. Al caused a downregulation of CaFER1 expression and no changes of CaFER2 expression in both varieties. The Western blot showed no alteration in ferritin protein levels in Mundo Novo and a decrease in Icatu. The differential enzymes responses indicate that the response to Al is variety-dependent.

Manipolazione del metabolismo degli xenobiotici da frutta convenzionale ed attività chemiopreventiva

A reduced cancer risk associated with fruit and vegetable phytochemicals initially dictated chemopreventive approaches focused on specific green variety consumption or even single nutrient supplementations. However, these strategies not only failed to provide any health benefits but gave rise to detrimental effects. In parallel, public-health chemoprevention programmes were developed in the USA and Europe to increase whole vegetable consumption. Among these, the National Cancer Institute (NCI) sponsored plan “5 to 9 a day for a better health” was one of the most popular. This campaign promoted wide food choice through the consumption of at least 5 to 9 servings a day of colourful fruits and vegetables. In this study the effects of the diet suggested by NCI on transcription, translation and catalytic activity of both xenobiotic metabolizing (XME) and antioxidant enzymes were studied in the animal model. In fact, the boost of both antioxidant defences and “good” phase-II together with down-regulation of “bad” phase-I XMEs is still considered one of the most widely-used strategies of cancer control. Six male Sprague Dawley rats for each treatment group were used. According to the Italian Society of Human Nutrition, a serving of fruit, vegetables and leafy greens corresponds to 150, 250 and 50 g, respectively, in a 70 kg man. Proportionally, rats received one or five servings of lyophilized onion, tomato, peach, black grape or lettuce – for white, red, yellow, violet or green diet, respectively - or five servings of each green (“5 a day” diet) by oral gavage daily for 10 consecutive days. Liver subcellular fractions were tested for various cytochrome P450 (CYP) linked-monooxygenases, phase-II supported XMEs such as glutathione S-transferase (GST) and UDP-glucuronosyl transferase (UDPGT) as well as for some antioxidant enzymes. Hepatic transcriptional and translational effects were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. dROMs test was used to measure plasmatic oxidative stress. Routine haematochemical parameters were also monitored. While the five servings administration didn’t significantly vary XME catalytic activity, the lower dose caused a complex pattern of CYP inactivation with lettuce exerting particularly strong effects (a loss of up to 43% and 45% for CYP content and CYP2B1/2-linked XME, respectively; P<0.01). “5 a day” supplementation produced the most pronounced modulations (a loss of up to 60% for CYP2E1-linked XME and a reduction of CYP content of 54%; P<0.01). Testosterone hydroxylase activity confirmed these results. RT-PCR and Western blot analysis revealed that the “5 a day” diet XMEs inactivations were a result of both a transcriptional and a translational effect while lettuce didn’t exert such effects. All administrations brought out none or fewer modulation of phase-II supported XMEs. Apart from “5 a day” supplementation and the single serving of lettuce, which strongly induced DT- diaphorase (an increase of up to 141 and 171%, respectively; P<0.01), antioxidant enzymes were not significantly changed. RT-PCR analysis confirmed DT-diaphorase induction brought about by the administration of both “5 a day” diet and a single serving of lettuce. Furthermore, it unmasked a similar result for heme-oxygenase. dROMs test provided insight into a condition of high systemic oxidative stress as a consequence of animal diet supplementation with “5 a day” diet and a single serving of lettuce (an increase of up to 600% and 900%, respectively; P<0.01). Haematochemical parameters were mildly affected by such dietary manipulations. According to the classical chemopreventive theory, these results could be of particular relevance. In fact, even if antioxidant enzymes were only mildly affected, the phase-I inactivating ability of these vegetables would be a worthy strategy to cancer control. However, the recorded systemic considerable amount of reactive oxygen species and the complexity of these enzymes and their functions suggest caution in the widespread use of vegan/vegetarian diets as human chemopreventive strategies. In fact, recent literature rather suggests that only diets rich in fruits and vegetables and poor in certain types of fat, together with moderate caloric intake, could be associated with reduced cancer risk.

Nuclear receptor and target gene mRNA abundance in duodenum and colon of dogs with chronic enteropathies

Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR) and peroxisome proliferator-associated receptors alpha and gamma (PPARalpha, PPARgamma) are mediators of inflammation and may be involved in inflammatory bowel disease (IBD) and food responsive diarrhea (FRD) of dogs. The present study compared mRNA abundance of NR and NR target genes [multi drug-resistance gene-1 (MDR1), multiple drug-resistance-associated proteins (MRD2, MRD3), cytochrome P450 (CYP3A12), phenol-sulfating phenol sulfotransferase (SULT1A1) and glutathione-S-transferase (GST A3-3)] in biopsies obtained from duodenum and colon of dogs with IBD and FRD and healthy control dogs (CON; n=7 per group). Upon first presentation of dogs, mRNA levels of PPARalpha, PPARgamma, CAR, PXR and RXRalpha in duodenum as well as PPARgamma, CAR, PXR and RXRalpha in colon were not different among groups (P>0.10). Although mRNA abundance of PPARalpha in colon of dogs with FRD was similar in both IBD and CON (P>0.10), PPARalpha mRNA abundance was higher in IBD than CON (P<0.05). Levels of mRNA of MDR1 in duodenum were higher in FRD than IBD (P<0.05) or CON (P<0.001). Compared with CON, abundances of mRNA for MRP2, CYP3A12 and SULT1A1 were higher in both FRD and IBD than CON (P<0.05). Differences in mRNA levels of PPARalpha and MRP2 in colon and MDR1, MRP2, CYP3A12 and SULT1A1 in duodenum may be indicative for enteropathy in FRD and (or) IBD dogs relative to healthy dogs. More importantly, increased expression of MDR1 in FRD relative to IBD in duodenum may be a useful diagnostic marker to distinguish dogs with FRD from dogs with IBD.

Genetic variation in genes for the xenobiotic-metabolizing enzymes CYP1A1, EPHX1, GSTM1, GSTT1, and GSTP1 and susceptibility to colorectal cancer in Lynch syndrome.

Individuals with Lynch syndrome are predisposed to cancer due to an inherited DNA mismatch repair gene mutation. However, there is significant variability observed in disease expression likely due to the influence of other environmental, lifestyle, or genetic factors. Polymorphisms in genes encoding xenobiotic-metabolizing enzymes may modify cancer risk by influencing the metabolism and clearance of potential carcinogens from the body. In this retrospective analysis, we examined key candidate gene polymorphisms in CYP1A1, EPHX1, GSTT1, GSTM1, and GSTP1 as modifiers of age at onset of colorectal cancer among 257 individuals with Lynch syndrome. We found that subjects heterozygous for CYP1A1 I462V (c.1384A>G) developed colorectal cancer 4 years earlier than those with the homozygous wild-type genotype (median ages, 39 and 43 years, respectively; log-rank test P = 0.018). Furthermore, being heterozygous for the CYP1A1 polymorphisms, I462V and Msp1 (g.6235T>C), was associated with an increased risk for developing colorectal cancer [adjusted hazard ratio for AG relative to AA, 1.78; 95% confidence interval, 1.16-2.74; P = 0.008; hazard ratio for TC relative to TT, 1.53; 95% confidence interval, 1.06-2.22; P = 0.02]. Because homozygous variants for both CYP1A1 polymorphisms were rare, risk estimates were imprecise. None of the other gene polymorphisms examined were associated with an earlier onset age for colorectal cancer. Our results suggest that the I462V and Msp1 polymorphisms in CYP1A1 may be an additional susceptibility factor for disease expression in Lynch syndrome because they modify the age of colorectal cancer onset by up to 4 years.

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components.

Thiazolides promote apoptosis in colorectal tumor cells via MAP kinase-induced Bim and Puma activation

While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance mechanisms in the tumor cells prevent cell death induction. In particular enzymes of the glutathion-S-transferase (GST) family represent a well-known detoxification mechanism, which limit the effect of chemotherapeutic drugs in tumor cells. Specifically, GST of the class P1 (GSTP1-1) is overexpressed in colorectal tumor cells and renders them resistant to various drugs. Thus, GSTP1-1 has become an important therapeutic target. We have recently shown that thiazolides, a novel class of anti-infectious drugs, induce apoptosis in colorectal tumor cells in a GSTP1-1-dependent manner, thereby bypassing this GSTP1-1-mediated drug resistance. In this study we investigated in detail the underlying mechanism of thiazolide-induced apoptosis induction in colorectal tumor cells. Thiazolides induce the activation of p38 and Jun kinase, which is required for thiazolide-induced cell death. Activation of these MAP kinases results in increased expression of the pro-apoptotic Bcl-2 homologs Bim and Puma, which inducibly bind and sequester Mcl-1 and Bcl-xL leading to the induction of the mitochondrial apoptosis pathway. Of interest, while an increase in intracellular glutathione levels resulted in increased resistance to cisplatin, it sensitized colorectal tumor cells to thiazolide-induced apoptosis by promoting increased Jun kinase activation and Bim induction. Thus, thiazolides may represent an interesting novel class of anti-tumor agents by specifically targeting tumor resistance mechanisms, such as GSTP1-1.

Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction

Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.

Enzyme activity and liver tissue PCB concentrations of chicks of northern fulmars (F. glacialis) and black-legged kittiwakes (R. tridactyla) from Kongsfjorden

Arctic seabirds are exposed to a wide range of halogenated organic contaminants (HOCs). Exposure occurs mainly through food intake, and many pollutants accumulate in lipid-rich tissues. Little is known about how HOCs are biotransformed in arctic seabirds. In this study, we characterized biotransformation enzymes in chicks of northern fulmars (Fulmarus glacialis) and black-legged kittiwakes (Rissa tridactyla) from Kongsfjorden (Svalbard, Norway). Phase I and II enzymes were analyzed at the transcriptional, translational and activity levels. For gene expression patterns, quantitative polymerase chain reactions (qPCR), using gene-sequence primers, were performed. Protein levels were analyzed using immunochemical assays of western blot with commercially available antibodies. Liver samples were analyzed for phase I and II enzyme activities using a variety of substrates including ethoxyresorufin (cytochrome (CYP)1A1/1A2), pentoxyresorufin (CYP2B), methoxyresorufin (CYP1A), benzyloxyresorufin (CYP3A), testosterone (CYP3A/CYP2B), 1-chloro-2,4-nitrobenzene (CDNB) (glutathione S-transferase (GST)) and 4-nitrophenol (uridine diphosphate glucuronyltransferase (UDPGT)). In addition, the hydroxylated (OH-) polychlorinated biphenyls (PCBs) were analyzed in the blood, liver and brain tissue, whereas the methylsulfone (MeSO2-) PCBs were analyzed in liver tissue. Results indicated the presence of phase I (CYP1A4/CYP1A5, CYP2B, and CYP3A) and phase II (GST and UDPGT) enzymes at the activity, protein and/or mRNA level in both species. Northern fulmar chicks had higher enzyme activity than black-legged kittiwake chicks. This in combination with the higher XOH-PCB to parent PCB ratios suggests that northern fulmar chicks have a different biotransformation capacity than black-legged kittiwake chicks.

(Table 1) Polychlorinated biphenyl (PCB) and PCB metabolite concentrations in ringed seals (Phoca hispida) from Svalbard and the Baltic Sea

Polychlorinated biphenyls (PCBs) may induce activity of hepatic enzymes, mainly Phase I monooxygenases and conjugating Phase II enzymes, that catalyze the metabolism of PCBs leading to formation of metabolites and to potential adverse health effects. The present study investigates the concentration and pattern of PCBs, the induction of hepatic phase I and II enzymes, and the formation of hydroxy (OH) and methylsulfonyl (CH3SO2=MeSO2) PCB metabolites in two ringed seal (Phoca hispida) populations, which are contrasted by the degree of contamination exposure, that is, highly contaminated Baltic Sea (n = 31) and less contaminated Svalbard (n = 21). Phase I enzymes were measured as ethoxyresorufin-O-deethylation (EROD), benzyloxyresorufin-O-dealkylation (BROD), methoxyresorufin-O-demethylation (MROD), and pentoxyresorufin-O-dealkylation (PROD) activities, and phase II enzymes were measured as uridine diphosphophate glucuronosyl transferase (UDPGT) and glutathione-S-transferase (GST). Geographical comparison, multivariate, and correlation analysis indicated that sum-PCB had a positive impact on Phase I enzyme and GST activities leading to biotransformation of group III (vicinal ortho-meta-H atoms and <=1 ortho-chlorine (Cl)) and IV PCBs (vicinal meta-para-H atoms and <=2 ortho-Cl). The potential precursors for the main OH-PCBs detected in plasma in the Baltic seals were group III PCBs. MeSO2-PCBs detected in liver were mainly products of group IV PCB metabolism. Both CYP1A- and CYP2B-like enzymes are suggested to be involved in the PCB biotransformation in ringed seals.

GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP

We have identified a mammalian protein called GIPC (for GAIP interacting protein, C terminus), which has a central PDZ domain and a C-terminal acyl carrier protein (ACP) domain. The PDZ domain of GIPC specifically interacts with RGS-GAIP, a GTPase-activating protein (GAP) for Gαi subunits recently localized on clathrin-coated vesicles. Analysis of deletion mutants indicated that the PDZ domain of GIPC specifically interacts with the C terminus of GAIP (11 amino acids) in the yeast two-hybrid system and glutathione S-transferase (GST)-GIPC pull-down assays, but GIPC does not interact with other members of the RGS (regulators of G protein signaling) family tested. This finding is in keeping with the fact that the C terminus of GAIP is unique and possesses a modified C-terminal PDZ-binding motif (SEA). By immunoblotting of membrane fractions prepared from HeLa cells, we found that there are two pools of GIPC–a soluble or cytosolic pool (70%) and a membrane-associated pool (30%). By immunofluorescence, endogenous and GFP-tagged GIPC show both a diffuse and punctate cytoplasmic distribution in HeLa cells reflecting, respectively, the existence of soluble and membrane-associated pools. By immunoelectron microscopy the membrane pool of GIPC is associated with clusters of vesicles located near the plasma membrane. These data provide direct evidence that the C terminus of a RGS protein is involved in interactions specific for a given RGS protein and implicates GAIP in regulation of additional functions besides its GAP activity. The location of GIPC together with its binding to GAIP suggest that GAIP and GIPC may be components of a G protein-coupled signaling complex involved in the regulation of vesicular trafficking. The presence of an ACP domain suggests a putative function for GIPC in the acylation of vesicle-bound proteins.

B cell receptor-associated protein α4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A

Recently, TAP42 was isolated as a high copy suppressor of sit4−, a yeast phosphatase related to protein phosphatase 2A (PP2A). TAP42 is related to the murine α4 protein, which was discovered independently by its association with Ig-α in the B cell receptor complex. Herein we show that a glutathione S-transferase (GST)–α4 fusion protein bound the catalytic subunit (C) of human PP2A from monomeric or multimeric preparations of PP2A in a “pull-down” assay. In an overlay assay, the GST–α4 protein bound to the phosphorylated and unphosphorylated forms of C that were separated in two-dimensional gels and immobilized on filters. The results show direct and exclusive binding of α4 to C. This is unusual because all known regulatory B subunits, or tumor virus antigens, bind stably only to the AC dimer of PP2A. The α4–C form of PP2A had an increased activity ratio compared with the AC form of PP2A when myelin basic protein phosphorylated by mitogen-activated protein kinase and phosphorylase a were used as substrates. Recombinant α4 cleaved from GST was phosphorylated by p56lck tyrosine kinase and protein kinase C. A FLAG-tagged α4 expressed in COS7 cells was recovered as a protein containing phosphoserine and coimmunoprecipitated with the C but not the A subunit of PP2A. Treatment of cells with rapamycin prevented the association of PP2A with FLAG-α4. The results reveal a novel heterodimer α4–C form of PP2A that may be involved in rapamycin-sensitive signaling pathways in mammalian cells.

Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in Escherichia coli enhances thermotolerance

A gene encoding the rice 16.9-kDa class I low-molecular-mass (LMM) heat-shock protein (HSP), Oshsp16.9, was introduced into Escherichia coli using the pGEX-2T expression vector to analyze the possible function of this LMM HSP under heat stress. It is known that E. coli does not normally produce class I LMM HSPs. We compared the survivability of E. coli XL1-Blue cells transformed with a recombinant plasmid containing a glutathione S-transferase (GST)–Oshsp16.9 fusion protein (pGST-FL cells) with the control E. coli cells transformed with the pGEX-2T vector (pGST cells) under heat-shock (HS) after isopropyl β-d-thiogalactopyranoside induction. The pGST-FL cells demonstrated thermotolerance at 47.5°C, a treatment that was lethal to the pGST cells. When the cell lysates from these two E. coli transformants were heated at 55°C, the amount of protein denatured in the pGST-FL cells was 50% less than that of the pGST cells. Similar results as pGST-FL cells were obtained in pGST-N78 cells (cells produced a fusion protein with only the N-terminal 78 aa in the Oshsp16.9 portion) but not in pGST-C108 cells (cells produced a fusion protein with C-terminal 108 aa in the Oshsp16.9 portion). The acquired thermotolerant pGST-FL cells synthesized three types of HSPs, including the 76-, 73-, and 64-kDa proteins according to their abundance at a lethal temperature of 47.5°C. This finding indicates that a plant class I LMM HSP, when effectively expressed in transformed prokaryotic cells that do not normally synthesize this class of LMM HSPs, may directly or indirectly increase thermotolerance.