Ethanol treatment triggers a heat shock-like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.     

Thermal and enzymatic pretreatment of sludge containing phthalate esters prior to mesophilic anaerobic digestion

The present study aimed at investigating the effect of thermal pretreatment of sludge at 70 degrees C on the anaerobic degradation of three commonly found phthalic acid esters (PAE): di-ethyl phthalate (DEP), di-butyl phthalate (DBP), and di-ethylhexyl phthalate (DEHP). Also, the enzymatic treatment at 28 degrees C with a commercial lipase was studied as a way to enhance PAE removal. Pretreatment at 70 degrees C of the sludge containing PAE negatively influenced the anaerobic biodegradability of phthalate esters at 37 degrees C. The observed reduction of PAE biodegradation rates after the thermal pretreatment was found to be proportional to the PAE solubility in water: the higher the solubility, the higher the percentage of the reduction (DEP > DBP > DEHP). PAE were slowly degraded during the pretreatment at 70 degrees C, yet this was probably due to physicochemical reactions than to microbial/biological activity. Therefore, thermal pretreatment of sludge containing PAE should be either avoided or combined with a treatment step focusing on PAE reduction. On the other hand, enzymatic treatment was very efficient in the removal of PAE. The enzymatic degradation of DBP, DEP, and DEHP could be one to two orders of magnitude faster than under normal mesophilic anaerobic conditions. Moreover, the enzymatic treatment resulted in the shortest half-life of DEHP in sludge reported so far. Our study further showed that enzymatic treatment with lipases can be applied to raw sludge and its efficiency does not depend on the solids concentration.     

Heat shock proteins and effects of heat shock in plants

Soybean seedlings when exposed to a heat shock respond in a manner very similar to that exhibited by cultured cells, and reported earlier [2]. Maximum synthesis of heat shock proteins (HSPs) occurs at 40C. The heat shock response is maintained for a relatively short time under continuous high temperature. After 2.5 hr at 40 C the synthesis of HSPs decreases reaching a very low level by 6 hr. The HSPs synthesized by cultured cells and seedlings are identical and there is a large degree of similarity in HSPs synthesized between the taxonomically widely separated species, soybean and corn. Storage protein synthesis in the developing soybean embryo is not inhibited but is actually stimulated during a heat shock, unlike most other non-HSPs, whose synthesis is greatly reduced. Seedlings respond differently to a gradual increase in temperature than they do a sudden heat shock. There is an upward shift of several degrees in the temperature at which maximum protein synthesis occurs and before it begins to be inhibited. In addition, there appears to be a protection of normal protein synthesis from heat shock inhibition when the temperature increase is gradual. An additional function of the heat shock phenomenon might be the protection of seedlings from death caused by extreme heat stress. The heat shock response appears to have relevance to plants in the field.     

Biosorption of di(2-ethylhexyl)phthalate by seaweed biomass

Samples of various Sargassum species were collected in the Hong Kong marine environment and used for studies on biosorption of di(2-ethylhexyl)phthalate (DEHP). Batch adsorption experiments were carried out to determine the removal capacity and removal efficiency of the biosorbents. The DEHP removal ability was similar among beached seaweed and three freshly collected Sargassum species. Different physico-chemical factors were evaluated in order to enhance the performance of the biosorbents. Under optimized conditions (25 mg biomass, initial pH 4, 25 °C, 40 mg L^–1 DEHP), the mean removal capacity of beached seaweed and Sargassum siliquastrum was 5.68 and 6.54 mg g^–1, respectively. Examination of the Langmuir and Freundlich adsorption isotherms showed that the biosorption phenomenon by these biosorbents could well be described by these models. Desorption of DEHP was also assessed with methanol, which showed the most satisfactory desorbing ability. Further study in multiple adsorption–desorption of DEHP by the biosorbents demonstrated the reusability of both beached seaweed and S. siliquastrum for biosorption of DEHP.     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.     

Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

Detailed characterization of heat shock protein synthesis and induced thermotolerance in seedlings of Sorghum bicolor L.

Tolerance of both protein synthesis and seedling growth to apreviously lethal high temperature can be induced by prior exposureto a sub-lethal temperature during which the synthesis of heatshock proteins (HSPs) occurs. In this study, a thermal gradientbar was used to measure the physiological effects of temperatureon seedlings of sorghum (Sorghum bicolor L.) in conjunctionwith studies of gene expression. The duration of HSP synthesis,both during continued high temperature treatment or on returnto normal temperatures, was found to be very finely modulatedand was dependent on the severity of the initial heat shock.The synthesis of heat shock proteins and the induction of thermotolerancewere rapid, reversible and reinducible phenomena. Maximal thermotolerancewas obtained after treatments that induced the full complementof HSPs. Subsequent treatments that repressed HSP synthesis,also abolished thermotolerance. The presence of HSPs, however,was not sufficient for the tissue to be in a thermotolerantstate and the results suggest that either their de novo synthesis,or some other factor, is required for the induction of thermotolerance.Pre-existing HSPs did not inhibit the synthesis of new HSPs.Although the kinetics of the synthesis of HSPs and the developmentof thermotolerance show a tight correlation, the kinetics ofthe decay of thermotolerance and the degradation of HSPs werenot linked. The functional state or distribution of HSPs maywell change during the recovery process. Key words: Heat shock, thermotolerance, Sorghum bicolor, growth, protein synthesis     

Effects of three phthalate esters on the life-table demography of freshwater rotifer <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis> Pallas

Phthalate esters (PAEs) are mainly used in the polymer industry as external plasticizers in PVC, and tend to migrate slowly out of the plastic, either into the air by volatilization or into water or other solvents by dissolution. Di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP) and di-2-ethylhexyl phthalate (DEHP) are three members of PAEs, identified as priority controlled hazardous substances by the United States Environmental Protection Agency, and have been shown to have potential for endocrine disrupting effects on vertebrates and humans. The effects of DBP, BBP and DEHP on survival and reproduction of the freshwater rotifer Brachionus calyciflorus were studied using life-table demographic methods. The results showed that all the life-table demographic parameters of B. calyciflorus were markedly affected by DBP and BBP, but not by DEHP. The net reproductive rate representing the output of reproduction was more affected than all the other parameters representing population growth, development or survival of the rotifers. Compared to the solvent control, DBP and BBP, both at 500 μg l⁻¹, significantly increased the net reproductive rate, and prolonged the generation time and the life expectancy at hatching of the rotifers. DBP at 50 μg l⁻¹ markedly decreased the intrinsic rate of population increase of the rotifers, but the reverse was true for BBP at 50 and 500 μg l⁻¹. Among all the parameters, the intrinsic rate of population increase was the most sensitive to DBP and BBP. The levels of PAEs in water from all the studied rivers and lakes in the world did not affect the population growth of rotifers.     

Identification of DnaJ-like chaperone in Clostridium botulinum type A

Clostridium botulinum type A cells, when challenged to elevated temperature (45°C), increased the expression of at least nine heat shock proteins (HSPs). Simultaneously with the induction of HSPs, changes in the synthesis rates of other cellular proteins were observed. A 40-kDa stress protein was induced and its synthesis rate was enhanced when the cells were shifted to 45°C. Using heterologous antibodies raised against E. coli DnaJ heat shock proteins, the 40-kDa stress protein of C. botulinum type A has been identified as a DnaJ-like chaperone. The DnaJ chaperone might be involved in translocation of the neurotoxin and other cellular proteins across the cell membrane, repair of damaged proteins, and organism survival inside the host. This is the first report of the existence of a DnaJ-like chaperone in this organism.     

Integrated biosorption and photocatalytic oxidation treatment of di(2-ethylhexyl)phthalate

Di(2-ethylhexyl)phthalate (DEHP), a toxic phthalate ester, is a ubiquitous contaminant due to its extensive use and persistence. No available treatment method can cost-effectively remove it from industrial wastewater. In a previous study, DEHP was effectively removed from aqueous solution by adsorption onto the biomass of selected seaweed, i.e., beached seaweed and Sargassum siliquastrum. Since biosorption cannot detoxify DEHP, the degradation (and detoxification) of desorbed DEHP from seaweed biomass by photocatalytic oxidation (PCO) was attempted. The first part of this study was to optimize the conditions for the degradation of desorbed DEHP in aqueous solution by PCO. Under optimized conditions, a total degradation of 20 mg/L of DEHP was achieved within 45 min. The degradation intermediates/products such as phthalic anhydride and 2-ethyhexanol were identified by GC-MS analysis. Total organic carbon analysis was also used to ensure the complete mineralization of DEHP. The Microtox^® test was used to assess the toxicities of the parent and degraded compounds. In the second part of this study, DEHP was first removed and concentrated by adsorption onto seaweed biomass under the conditions optimized in the previous study. It was then desorbed from seaweed biomass and degraded by PCO. Results indicate that the treatment for DEHP by integrating biosorption and PCO is feasible.     

High-performance liquid chromatographic method for the determination of di(2-ethylhexyl) phthalate in total parenteral nutrition and in plasma

A simple, rapid and sensitive reversed-phase high-performance liquid chromatographic method with UV detection was developed for the quantification of di(2-ethylhexyl) phthalate (DEHP) in parenteral nutrition admixtures containing fat emulsion and in plasma samples of children daily treated by total parenteral nutrition. The analyte and the internal standard, di-n-heptyl phthalate, were extracted twice using hexane and the organic layer separated and dried under nitrogen. The residues were reconstituted with acetonitrile and 20 μl was injected into a Waters Spherisorb C₁₈ column, the UV detector was set at 202 nm. The mobile phase was acetonitrile–aqueous buffer (triethylamine 0.08% adjusted to pH 2.8 with 1 M phosphoric acid) mixture (88:12, v/v) and it was pumped at 1 ml/min. Average recoveries were 97% or greater. This method was successfully used to investigate the amounts of DEHP which can leach from bags and tubing into fat emulsion and which could contaminate children under long-term parenteral nutrition. On the other hand, the circulating DEHP concentrations were estimated in four children under regular long-term parenteral nutrition.     

Degradation of bis(2-ethylhexyl) phthalate constituents under methanogenic conditions

The degradation of bis(2-ethylhexyl) phthalate (DEHP) and its intermediary hydrolysis products 2-ethylhexanol (2-EH) and mono(2-ethylhexyl) phthalate (MEHP) was investigated in a methanogenic phthalic acid ester-degrading enrichment culture at 37°C. 2-Ethylhexanoic acid (2-EHA), a plausible degradation product of 2-EH, was also studied. The culture readily degraded 2-EH via 2-EHA to methane which was formed in stoichiometric amounts assuming complete degradation of 2-EH to methane and carbon dioxide. MEHP was degraded to stoichiometric amounts of methane with phthalic acid as a transient intermediate. DEHP remained unaffected throughout the experimental period (330 days).Abbreviations 2-EH 2-ethylhexyl alcohol - 2-EHA 2-ethylhexanoic acid - BBP butylbenzyl phthalate - Be-CoA benzoyl Coenzyme A - CoA Coenzyme A - DEHP bis(2-ethylhexyl) phthalate - MEHP mono(2-ethylhexyl) phthalate - MSW municipal solid waste - PA phthalic acid - PAE phthalic acid ester - TMS trimethylsilyl derivative     

A direct competitive enzyme-linked immunosorbent assay by antibody coated for diethyl phthalate analysis

A direct competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detection of diethyl phthalate (DEP). Protein-hapten conjugate was synthesized to produce polyclonal antibodies against DEP. Experimental parameters were optimized, including immunoreaction conditions, the dilution ratio of horseradish peroxidase (HRP)-antigen conjugate, time of the antibody coated, effect of pH, and ionic strength. The limit of detection was 0.096 ng/ml, and the linear range was 0.1-3500 ng/ml with a regression coefficient (R²) of 0.9957. Recoveries were between 96.4 and 106.2%. The cross-reactivities of the anti-DEP antibody to six structurally related phthalate esters were less than 9%. The method was successfully applied to the determination of DEP in tap water, river water (Yangtze River), and leachate from plastic drinking bottles. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for DEP monitoring. The results obtained were compared with those obtained using the high-performance liquid chromatography method.     

Paclobutrazol- and Hardening-Induced Thermotolerance of Wheat: Are Heat Shock Proteins Involved?

The present report describes the effects of paclobutrazol andheat hardening treatments on the protein synthesis patternsin imbibing and germinating wheat seedlings (Triticum aestivumL. cv Frederick) during heat stress. A heat hardening treatmentgiven during the imbibition period induced the transient expressionof 118, 90, 70 and 18 kDa heat shock proteins (HSPs). However,the hardening and paclobutrazol treatments did not enhance thethermotolerance of imbibed seeds or etiolated seedlings. Bycontrast, the hardening and paclobutrazol treatments enhancedthe thermotolerance of light-grown seedlings. While, both hardenedand unhardened control seedlings synthesized several HSPs duringa high temperature stress period, these proteins were not synthesizedby the paclobutrazol-treated, light-grown seedlings. Thus, HSPsynthesis during heat shock may have been a manifestation ofstress perception by the seedlings and may not have mediatedthe thermotolerance induced by the triazole treatments. Sincedifferential thermotolerance was only apparent in light-grownseedlings, it is suggested that chloroplasts may be requiredfor the expression of paclobutrazol- and hardening-induced thermoprotection.Additional evidence indicating that chloroplasts are an importantsite of injury during high temperature stress was obtained fromchlorophyll fluorescence measurements. (Received July 11, 1994; Accepted October 26, 1994)     

Degradation of environmental endocrine disruptor di-2-ethylhexyl phthalate by a newly discovered bacterium, Microbacterium sp. strain CQ0110Y

In this study di-2-ethylhexyl phthalate (DEHP)-degradation strain CQ0110Y was isolated from activated sludge. According to the biophysical/biochemical characteristics and analysis of 16S rDNA, the strain was identified as Microbacterium sp. The results of this study showed the optimal pH value and optimal temperature which influenced the degradation rate in wastewater: pH 6.5–7.5, 25–35°C. Kinetics of degradation reaction had been performed at different initial concentrations and different time. Analyzed with SPSS10.0 software, the DEHP degradation can be described as the same exponential model when the initial DEHP concentration was lower than 1,350 mg/l. The kinetics equation was ln C = −0.4087t + A, with the degradation half life of DEHP in wastewater (1.59 days). To the best of our knowledge, this is the first reported case of DEHP degradation by Microbacterium sp. strain. Xiang Li and Ji-an Chen contributed equally to this work.     

Induction of Heat Shock Proteins and Acquisition of Thermotolerance in Germinating Pigeonpea Seeds

Heat shock proteins (HSPs) ranging in molecular masses from 14 to 110 kDa were induced in embryonic axes of germinating Cajanus cajan (L.) Millspaugh seeds after exposure to 40 °C for 1 or 2 h. At 45 °C, there was a marked decline in synthesis of HSPs. A close relationship was observed between HSPs induced and the growth of the germinating seeds. Pretreatment of germinating seeds at 40 °C for 1 h or 45 °C for 10 min followed by incubation at 28 °C for 3 h led to considerable thermotolerance (45 °C, 2 h) and the recovery of protein synthesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Aerobic degradation of diethyl phthalate by Sphingomonas sp

An aerobic diethyl phthalate (DEP) degrading bacterium, DEP-AD1, was isolated from activated sludge. Based on its 16S rDNA sequence, this isolate was identified belonging to Sphingomonas genus with 99% similarity to Sphingomonas sp. strain C28242 and 98% similarity to S. capsulate. The specific degradation rate of DEP was concentration dependent with a maximum of 14 mg-DEP/(Lh). Results of degradation tests showed that DEP-AD1 could also degrade monoethyl phthalate (MEP), dimethyl phthalate (DMP), dibutyl phthalate (DBP), and diethylhexyl phthalate (DEHP), but not phthalate and benzoate.     

Heat shock and ceramide have different apoptotic pathways in radiation induced fibrosarcoma (RIF) cells

Heat shock induces various cellular responses including inhibition of protein synthesis, production of heat shock proteins (HSPs) and induction of thermotolerance. The molecular mechanisms of the processes have not been well understood. It has been proposed that ceramide formation during heat shock mediates heat shock induced apoptosis. We examined whether C2-ceramide mimicked the cellular response to heat shock in RIF-1 cells and their thermotolerant derivative TR-RIF-1 cells. Discernible effects between heat shock and C2-ceramide treatments were observed in cellular changes such as total protein synthesis, HSP synthesis, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activity and PARP cleavage. Heat shock immediately inhibited cellular protein synthesis, which was recovered by synthesizing HSPs first and then whole proteins later. Heat shock also activated SAPK/JNK and increased PARP cleavage in dose-dependent manner. Thermotolerant TR-RIF-1 cells responded to heat shock more insensitively than RIF-1 cells. On the other hand, C2-ceramide treatment did not accompany any changes induced by heat shock. No discernible differences between RIF-1 and TR-RIF-1 cells were observed by C2-ceramide treatment. We tried to figure out how C2-ceramide interacts with cellular membrane and found that exogenous C2-ceramide was incorporated into the outer monolayer and flipped into the inner monolayer of human erythrocytes in ATP-dependent manner. However, the rate of C2-ceramide incorporation was similar in control and thermotolerant cells. In summary, thermotolerant cells are resistant to heat shock induced apoptotic signaling but not resistant, rather sensitive to membrane disturbing C2-ceramide mediated apoptosis. These results suggest that heat shock and ceramide have different signal transduction pathways.