Heat-stable Proteins Research Articles

The induction of Metallothionein by Cadmium in sea anemone (Aiptasia pulchella)

The induction of Metallothionein by Cadmium in sea anemone (Aiptasia pulchella) Metallothioneins(MT) are cysteine rich, heat stable and low molecular weight proteins. They have been widely used as biomarkers to reflect the existence of heavy metal pollution. MT also has the potential being used in well-designed environmental monitoring programmes. Sea anemones(Aiptasia pulchella) should reflect the real condition of its surroundings due to its sedentary. In lab, they are normally reproduced by pedal laceration as asexual reproduction. After exposure to different concentrations of cadmium(0, 0.5, 1.0 and 2.0mg/L) for 24, 48 and 96 hours, obvious morphological alterations in Aiptasia pulchella were recorded. Their tentacles turned dark and their body shrinked. By using atomic absorption to measure the Cd concentration, those Cd-exposed sea anemones showed a time-dependent and a dose-dependent cadmium accumulation. From the results of immunoblotting, the amount of metallothioneins in those cd-exposed A. pulchella significantly increased followed by a time-course. The molecular weight of the induced MT is between 11 and 17.

39: Incidence of human herpesvirus-6 (HHV-6) and human cytomegalovirus (HCMV) infections in donated bone marrow and umbilical cord blood hematopoietic stem cells

Staphylococcus aureus causes a broad range of infections, food poisoning, and toxic shock syndrome. This variety of diseases is related to a number of virulence factors, including pathogenic antigens localized on/in the bacterial cells, enzymes and toxins, such as staphylococcal enterotoxins (SEs). SEs are the important toxins of food poisoning. These toxins have become to be a superfamily with 23 types including SEs and SE-like toxins (SEls). The genes of SEs and SEls are encoded by diverse accessory genetic elements, and the molecular of the toxins share certain structural and biological properties. SEs and SEls are also heat-stable and multifunctional proteins that trigger food poisoning outbreaks, pyrogenicity, superantigenicity, and the capacity to induce lethal hypersensitivity to endotoxin. Understanding the complex biology and the relationship of the different functions of SEs and SEls superfamily will be key to combat S. aureus foodborne diseases.

Proteins of cotyledons of mature horse chestnut seeds

This is the first characterization of proteins from storage parenchyma of cotyledons of mature dormant recalcitrant horse chestnut (Aesculus hippocastanum L.) seeds and evaluation the cell protein-synthesizing capacity. It was established that the content of protein in cotyledons did not exceed 0.5% of tissue fresh weight. Soluble proteins (the proteins of the postmitochondrial supernatant or cytosol) comprised the bulk (up to 90%) of total proteins. Protein of subcellular structures (20000 g-pellet) comprised 5–7% of total protein. Cotyledon proteins were heterogenous in their charges and molecular weights of subunits. Cotyledon protein was easily extracted with a salt (1 M NaCl); they comprised 90% of water-soluble albumin-like proteins. The proportion of globulins was insignificant; it did not exceed 5%. Most water-soluble proteins (more than 80%) were tolerant to heat denaturing. Among these heat-stable proteins, two major groups of polypeptides dominated: an electrophoretically homogeneous component with a mol wt of 24–25 kD and a complex group from three to five polypeptides with mol wts in the range between 6 and 12 kD. Native heat-stable proteins had disulfide bonds. Four fractions of heat-stable proteins were obtained by ammonium sulfate fractionation; three of them were alike in their polypeptide composition and contained major components with mol wts of 24–25 and 5–12 kD. It was established that the active translational machinery functioned in the cells of storage parenchyma in cotyledons of mature dormant horse chestnut seeds. During each stage of stratification, cotyledon fragments incorporated 35S-methionine into TCA-insoluble material more actively than axial organs. We discuss cotyledon protein composition, their function as a storage organ, and a possible role of heat-stable proteins.

Partial purification of human parathyroid hormone 1-84 as a thioredoxin fusion form in recombinant Escherichia coli by thermoosmotic shock

A modified purification method, thermoosmotic shock (osmotic shock coupled with heat-treatment) for heat-stable proteins, was devised in the purification of Trx-hPTH (1-84) (human parathyroid hormone coupled with thioredoxin as a fusion partner) from E. coli. Thermoosmotic shock can integrate the functions of extraction and crude separation of fusion protein Trx-hPTH (1-84). To improve the purification efficiency, thermoosmotic shock conditions were optimized and achieved as follows: the optimized high osmotic solution containing 20 mM Tris–HCl buffer (pH 8.0), 1 mM EDTA, and 25% sucrose; the low osmotic solution containing 20 mM Tris–HCl buffer (pH 8.0), 1 mM EDTA, and the heat-treatment temperature of 100 °C for 10 min. Using this method, the purity of Trx-hPTH (1-84) was up to 73% and the yield was up to 72%, respectively. In addition, the two separation methods of both thermoosmotic shock and affinity chromatography have been compared, indicating that thermoosmotic shock is an economical and feasible way for the fusion protein separation. Besides, the thermoosmotic shock method may be used for the purification of some proteins of thermal stability without N-terminal His-tag.

Pretreatment of seed with H 2O 2 improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins

Increased salinity is a stringent problem to crop production while seed pretreatment can effectively induce salt tolerance in plants. Hydrogen peroxide (H(2)O(2)), a stress signal molecule, was evaluated as seed treatment to produce the metabolic changes, which could lead to improved salt tolerance in wheat. Soaking in 1, 40, 80 and 120 microM H(2)O(2) revealed a low penetration, reaching maximum at 5h (2.58+/-0.23 micro mol g(-1) fresh seeds at 120 microM) and declining thereafter to the level of water control by 8h. This revealed the activation of antioxidants and H(2)O(2) scavenging in seed after 5h. Seeds treated with 1-120 microM H(2)O(2) for 8h and germinated in saline (150 mM NaCl) medium curtailed the mean germination time (MGT) being even less than water controls. Level of H(2)O(2) in seedlings arising from H(2)O(2)-treated seeds grown under salinity was markedly lower than salinized controls, suggesting the operation of antioxidant system in them. These seedlings exhibited better photosynthetic capacity, particularly the stomatal conductance (gs), thus improving the leaf gas exchange due to stomatal component of photosynthesis. Moreover, H(2)O(2) treatment improved leaf water relations and maintained turgor. Although Na(+) and Cl(-) content increased due to salinity, H(2)O(2)-treated seedlings displayed greater tissue K(+), Ca(2+), NO(3)(-) PO(4)(3-) levels and improved K(+):Na(+) ratio. H(2)O(2) treatment enhanced the membrane properties, as revealed from greatly reduced relative membrane permeability (RMP) and less altered ion leakage pattern (comparable to water controls). Seedlings exhibited the expression of two heat-stable (stress) proteins with apparent molecular masses of 32 and 52 kDa. Results suggest that H(2)O(2) signals the activation of antioxidants in seed, which persists in the seedlings to offset the ion-induced oxidative damage. These changes led to the expression of stress proteins and improved physiological attributes, which supported the seedling growth under salinity.

Biological significance of metals partitioned to subcellular fractions within earthworms (Aporrectodea caliginosa)

Metal ions in excess of metabolic requirements are potentially toxic and must be removed from the vicinity of important biological molecules to protect organisms from adverse effects. Correspondingly, metals are sequestrated in various forms, defining the accumulation pattern and the magnitude of steady-state levels reached. To investigate the subcellular fractions over which Ca, Mg, Fe, Cu, Zn, Cd, Pb, Ni, and As are distributed, earthworms (Aporrectodea caliginosa) collected from the field were analyzed by isolating metal-rich granules and tissue fragments from intracellular microsomal and cytosolic fractions (i.e., heat-stable proteins and heat-denatured proteins). The fractions showed metal-specific binding capacity. Cadmium was mainly retrieved from the protein fractions. Copper was equally distributed over the protein fraction and the fraction comprising tissue fragments, cell membranes, and intact cells. Zinc, Ca, Mg, and As were mainly found in this fraction as well. Lead, Fe, and Ni were mainly isolated from the granular fraction. To study accumulation kinetics in the different fractions, three experiments were conducted in which earthworms were exposed to metal-spiked soil and a soil contaminated by anthropogenic inputs and, indigenous earthworms were exposed to field soils. Although kinetics showed variation, linear uptake and steady-state types of accumulation patterns could be understood according to subcellular compartmentalization. For risk assessment purposes, subcellular distribution of metals might allow for a more precise estimate of effects than total body burden. Identification of subcellular partitioning appears useful in determining the biological significance of steady-state levels reached in animals.

A field study examining the relative importance of food and water as sources of cadmium for juvenile yellow perch (Perca flavescens)

To determine the relative importance of water and food as cadmium (Cd) sources for juvenile yellow perch (Perca flavescens, age 1+), fish were caged for up to 30 days in either a reference (Opasatica) or a Cd-contaminated (Dufault) lake and offered prey (zooplankton) from one of these lakes. We established four Cd-exposure regimes: a control, Cd-contaminated water only, Cd-contaminated food only, Cd-contaminated food and water. The 64 µm mesh cages used allowed the free exchange of water with the surrounding lake while retaining the added zooplankton prey for the perch. Cd in the gills and kidney of caged perch was taken up largely from lake water, whereas liver and gut Cd appeared to come from both dietary and aqueous sources. At the subcellular level (liver), the majority of the background Cd in control perch was associated with metal-sensitive fractions (organelles and heat-denatured proteins), whereas in perch exposed to waterborne or diet-borne Cd, the majority of the Cd was found in a metal-detoxified fraction (heat-stable proteins, metallothionein). For perch from all treatment levels, the higher the concentration of hepatic Cd, the greater the proportion of the Cd burden in this organ that was associated with the fraction containing metallothionein-like proteins.

Sub-cellular partitioning of cadmium, copper, nickel and zinc in indigenous yellow perch ( Perca flavescens) sampled along a polymetallic gradient

Sub-cellular metal distributions were studied in indigenous yellow perch ( Perca flavescens) collected from eight lakes located along a cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) concentration gradient. Ambient dissolved metal concentrations were measured to evaluate exposure and total hepatic metal concentrations were determined as a measure of metal bioaccumulation. Metal partitioning among potentially metal-sensitive fractions (cytosolic enzymes, organelles) and detoxified metal fractions (metallothionein) was determined after differential centrifugation of fish liver homogenates. Major proportions of hepatic Cd and Cu were found in the heat-stable cytosolic peptides and proteins fraction (HSP), a fraction including metallothioneins, whereas the potentially metal-sensitive heat-denaturable proteins fraction (HDP) was the largest contributor to the total Ni and Zn burdens. The concentrations of Cd, Cu and Ni (but not Zn) in each sub-cellular fraction increased along the metal contamination gradient, but the relative contributions of each fraction to the total burden of each of these metals remained generally constant. For these chronically exposed fish there was no threshold exposure concentration below which binding of Cd or Ni to the heat-denaturable protein fraction did not occur. The presence of Cd and Ni in the HDP fraction, even for low chronic exposure concentrations, suggests that metal detoxification was imperfect, i.e. that P. flavescens was subject to some metal-related stress even under these conditions.

Subcellular Partitioning and the Prediction of Cadmium Toxicity to Aquatic Organisms

Environmental Context. There is a considerable interest in predicting cadmium (Cd) toxicity to aquatic organisms, largely stemming from environmental Cd pollution and the need to establish water quality criteria to protect aquatic ecosystems. Chemistry-orientated models have been developed over the past decades to predict Cd toxicity, focusing on identifying which Cd forms are present in the aquatic environment, and investigating their interaction with the biological site of action. Understanding the cellular fates of Cd may provide an alternative method to predict Cd toxicity, as the complex cellular interactions of Cd within the organisms can, in this way, be addressed. Abstract. The internal metal sequestration strategies of different aquatic organisms are complex and variable; thus it is a formidable task to predict metal toxicity. Metals accumulated by aquatic organisms are associated with different subcellular compartments (i.e. heat-sensitive proteins, heat-stable proteins (metallothioneins), granules, cellular debris, and organelles). Such subcellular partitioning is dynamic in response to metal exposure and other environmental conditions, and is metal- and organism-specific. Previous models predicting metal toxicity have relied on the free ion metal activity (i.e. the free ion activity model) or more recently on the metal binding with the proposed toxicological site of action (i.e. the biotic ligand model). Neither of these models considers the complexity of internal metal subcellular fractionation, which may significantly affect metal toxicity in aquatic organisms and subsequent trophic transfer of metals to consumers. Recent studies in small aquatic organisms have revealed that the subcellular partitioning model (SPM) may provide an improved method to predict Cd toxicity, but more studies are needed in the future.

Role of sugars and proteins in development of desiccation tolerance in fresh and shade-dried onion seeds

In a 2-year field study, the umbels of open-pollinated onion cv. ‘Pusa Red’ were tagged on the day of anthesis and were sampled at different intervals until harvest maturity. Each umbel was cut in half and bulked with others in the respective replication. Seeds were immediately taken from half of the umbels for analysis of seed water content, leachate conductivity, seed capsule chlorophyll, seed dry weight and seed germination. They were also subjected to rapid desiccation to identify the stage at which desiccation tolerance occurred. The remaining umbel halves were shade dried and subjected to the same quality analyses except chlorophyll content. With seed maturation, there were rapid declines in chlorophyll content, leachate conductivity and seed water content, whereas seed dry weight and germination increased. In fresh seeds, maximum germination occurred around 50 days after anthesis, when physiological maturity and maximum seed weight were attained. In shade-dried seed, maximum germination occurred at 42 days after anthesis. This coincided with drastic reductions in seed capsule chlorophyll, leachate conductivity and seed water content in fresh seeds. At this stage, reducing sugar concentration declined and non-reducing sugar concentration increased many fold in shade-dried seeds. SDS-PAGE analysis of the heat-stable proteins extracted from the seeds also showed prominent bands in the lower molecular weight region at 42 DAA. Therefore, the shade-dried seeds attained membrane stability at 42 DAA. In cases of adverse weather conditions or disease attack, seed umbels can be harvested as early as 42 DAA and shade dried without compromising seed germination. Desiccation tolerance in fresh onion seeds occurred around 46 DAA and was a gradual event.

Seasonal variations in hepatic Cd and Cu concentrations and in the sub-cellular distribution of these metals in juvenile yellow perch ( Perca flavescens)

Temporal fluctuations in metal (Cd and Cu) concentrations were monitored over four months (May to August) in the liver of juvenile yellow perch ( Perca flavescens) sampled from four lakes situated along a metal concentration gradient in northwestern Quebec: Lake Opasatica (reference lake, low metal concentrations), Lake Vaudray (moderate metal concentrations) and lakes Osisko and Dufault (high metal levels). The objectives of this study were to determine if hepatic metal concentrations and metal-handling strategies at the sub-cellular level varied seasonally. Our results showed that Cd and Cu concentrations varied most, in both absolute and relative values, in fish with the highest hepatic metal concentrations, whereas fish sampled from the reference lake did not show any significant variation. To examine the sub-cellular partitioning of these two metals, we used a differential centrifugation technique that allowed the separation of cellular debris, metal detoxified fractions (heat-stable proteins such as metallothionein) and metal sensitive fractions (heat-denaturable proteins (HDP) and organelles). Whereas Cd concentrations in organelle and HDP fractions were maintained at low concentrations in perch from Lakes Opasatica and Vaudray, concentrations in these sensitive fractions were higher and more variable in perch from Lakes Dufault and Osisko, suggesting that there may be some liver dysfunction in these two fish populations. Similarly, Cu concentrations in these sensitive fractions were higher and more variable in perch from the two most Cu-contaminated lakes (Dufault and Osisko) than in perch from the other two lakes, suggesting a breakdown of homeostatic control over this metal. These results suggest not only that metal concentrations vary seasonally, but also that concentrations vary most in fish from contaminated sites. Furthermore, at the sub-cellular level, homeostatic control of metal concentrations in metal-sensitive fractions is difficult to maintain in perch with high hepatic metal concentrations.

Exchange rates of cadmium between a burrowing mayfly and its surroundings in nature

We conducted a reciprocal transfer of nymphs of the burrowing mayfly Hexagenia limbata between two rivers harboring populations of this insect that differed in their cadmium (Cd) concentrations. We measured Cd uptake and loss rates simultaneously in the field at several levels of biological organization: in the gut and body of whole nymphs as well as in three subcellular fractions. The most marked changes in Cd concentrations occurred in the insect's gut, where Cd was largely associated with heat-stable proteins, a fraction that includes the metal-binding protein metallothionein. Because most of the Cd that entered nymphs was detoxified in this manner, we would not expect it to have direct toxic effects; however, it would be readily transferred to higher trophic levels. Our data on subcellular partitioning suggest that there is a fast-exchanging pool of Cd bound to heat-stable proteins such as metallothionein and a slow-exchanging pool bound to either or both of the remaining fractions (heat-denatured proteins; remainder, which includes exoskeleton and granules). Nymphs of H. limbata responded rapidly to changes in bioavailable Cd, which suggests that they would be useful for monitoring changes in ambient Cd for weeks or months in the field. A one-compartment model successfully explained the changes we observed in nymph Cd values. Furthermore, values of the model constant for Cd loss were close to those reported for Hexagenia rigida in the laboratory, which suggests that accumulation patterns observed in the laboratory can be representative of those measured in nature.

Defining Genes in the Genome of the Hyperthermophilic Archaeon Pyrococcus furiosus : Implications for All Microbial Genomes

The original genome annotation of the hyperthermophilic archaeon Pyrococcus furiosus contained 2,065 open reading frames (ORFs). The genome was subsequently automatically annotated in two public databases by the Institute for Genomic Research (TIGR) and the National Center for Biotechnology Information (NCBI). Remarkably, more than 500 of the originally annotated ORFs differ in size in the two databases, many very significantly. For example, more than 170 of the predicted proteins differ at their N termini by more than 25 amino acids. Similar discrepancies were observed in the TIGR and NCBI databases with the other archaeal and bacterial genomes examined. In addition, the two databases contain 60 (NCBI) and 221 (TIGR) ORFs not present in the original annotation of P. furiosus. In the present study we have experimentally assessed the validity of 88 previously unannotated ORFs. Transcriptional analyses showed that 11 of 61 ORFs examined were expressed in P. furiosus when grown at either 95 or 72 degrees C. In addition, 7 of 54 ORFs examined yielded heat-stable recombinant proteins when they were expressed in Escherichia coli, although only one of the seven ORFs was expressed in P. furiosus under the growth conditions tested. It is concluded that the P. furiosus genome contains at least 17 ORFs not previously recognized in the original annotation. This study serves to highlight the discrepancies in the public databases and the problems of accurately defining the number and sizes of ORFs within any microbial genome.

Probing heat-stable water-soluble proteins from barley to malt and beer

Proteins determine the quality of barley in malting and brewing end-uses. In this regard, water-soluble barley proteins play a major role in the formation, stability, and texture of head foams. Our objective was to survey the barley seed proteins that could be involved in the foaming properties of beer. Therefore, two-dimensional (2-D) electrophoresis and mass spectrometry were combined to highlight the barley proteins that could resist the heating treatments occurring during malting and brewing processes. As expected, from barley to malt and to beer, most of the heat-stable proteins are disulfide-rich proteins, implicated in the defense of plants against their bio-aggressors, e.g., serpin-like chymotrypsin inhibitors (protein Z), amylase and amylase-protease inhibitors, and lipid transfer proteins (LTP1 and LTP2). For LTP1s, the complex pattern displayed in 2-D electrophoresis could be related to some chemical modifications already described elsewhere, such as acylation or glycation through Maillard reactions, which occur on malting. Our proteomics approach allowed the identification of the numerous proteins present in beer in addition to the major ones already described. The involvement of these proteins in the quality of beer foam can now be evaluated.

Dynamics of Cd, Cu and Zn accumulation in organs and sub-cellular fractions in field transplanted juvenile yellow perch ( Perca flavescens)

Juvenile yellow perch ( Perca flavescens) were caught in a reference lake and transplanted to cages held within a lake impacted by mining activities, with elevated levels of aqueous bioavailable copper (Cu 2+), zinc (Zn 2+) and cadmium (Cd 2+). Fish were sampled from the cages over 70 d and changes in metal concentrations were followed over time in the gills, gut, liver and kidney. In addition, the hepatic sub-cellular partitioning of the three metals was determined by differential centrifugation of liver samples, yielding the following fractions: cellular debris; organelles; heat-denaturable proteins (HDP); and heat-stable proteins (HSP) (including metallothionein). In transplanted fish, Cd concentrations increased in all the organs sampled, whereas Cu mainly increased in the gills, gut and liver but not the kidney; some slight accumulation of Zn occurred in the kidneys and gills of the transplanted fish. The sub-cellular partitioning results demonstrated that metal-handling strategies in juvenile yellow perch differed among metals. Cellular sequestration in the HSP fraction was an important strategy used by these fish in response to increased ambient Cd. Accumulation of Zn was not seen in the organs examined, indicating that transplanted perch were able to either reduce influx, or increase efflux rates of this metal. The response of yellow perch to elevated ambient Cu appeared to combine the strategies used for Cd and Zn, as both cellular sequestration and reduced accumulation were observed in transplanted fish.

Crystallization and preliminary X-ray crystallographic studies of glutaredoxin 2 from Saccharomyces cerevisiae in different oxidation states.

Glutaredoxins are small (9-12 kDa) heat-stable proteins that are highly conserved throughout evolution; the glutaredoxin active site (Cys-Pro-Tyr-Cys) is conserved in most species. Five glutaredoxin genes have been identified in Saccharomyces cerevisiae; however, Grx2 is responsible for the majority of oxidoreductase activity in the cell, suggesting that its primary function may be the detoxification of mixed disulfides generated by reactive oxygen species (ROS). Recombinant Grx2 was expressed in Escherichia coli as a 6xHis-tagged fusion protein and purified by nickel-affinity chromatography. Prior to crystallization trials, the enzyme was submitted to various treatments with reducing agents and peroxides. Crystals suitable for X-ray diffraction experiments were obtained from untreated protein and protein oxidized with t-butyl hydroperoxide (10 mM). Complete data sets were collected to resolutions 2.15 and 2.05 A for untreated and oxidized Grx2, respectively, using a synchrotron-radiation source. The crystals belong to space group P4(1)2(1)2, with similar unit-cell parameters.

Sub-cellular partitioning of Cd, Cu and Zn in tissues of indigenous unionid bivalves living along a metal exposure gradient and links to metal-induced effects

We studied organ and sub-cellular distributions of several trace metals in a freshwater bivalve that has been proposed for use as a metal biomonitor. Specimens of Pyganodon grandis were collected from nine lakes located along a Cd, Cu and Zn concentration gradient (Rouyn-Noranda area, Québec). Gills and digestive gland were isolated, homogenized and six sub-cellular fractions were separated by differential centrifugation and analysed for their Cd, Cu and Zn content. Metallothionein was quantified independently. Gill tissues contained abundant calcium concretions that accounted for over 60% of the total gill burden of each metal. Cadmium and Zn concentrations in this granule fraction reflected ambient metal concentrations. Metal concentrations in the digestive gland also responded to the metal contamination gradient, but to a lesser extent than the gills, reflecting the lower abundance of granules in the digestive gland. Metals (Cd, Cu) in this organ were present largely in the “heat-stable proteins” fraction, and metal concentrations in this fraction were strongly correlated with those of both metallothionein and, to a lesser extent, the “lysosomes + microsomes” and “mitochondria” fractions. In both organs, Cd concentrations in the “heat-denaturable protein” fraction remained low and constant, suggesting reasonably effective metal detoxification. Some evidence for oxidative stress was noted in the gills but not in the digestive gland. Overall, we conclude that in nature metals in P. grandis are bound differently in the gills and in the digestive gland and that metal detoxification in the former organ may be less effective than in the latter.

Sub-cellular partitioning of metals (Cd, Cu, Zn) in the gills of a freshwater bivalve, Pyganodon grandis: role of calcium concretions in metal sequestration

Indigenous unionid molluscs, Pyganodon grandis, were collected from nine lakes in the Rouyn–Noranda area (Québec, Canada) along a polymetallic concentration gradient (Cd, Cu, Zn). After excision, the gills were gently homogenised and the cellular compartments were separated by a differential centrifugation procedure that yielded the following particulate fractions: “nuclei + cellular debris”, “mitochondria”, “lysosomes + microsomes” and “granules”. The supernatant remaining after the final ultracentrifugation step, i.e., the operationally-defined cytosol, was separated into a “heat-denaturable proteins” (HDP) fraction and a “heat-stable proteins” (HSP) fraction containing metallothionein (MT). The Cd, Cu and Zn content of each particulate and cytosolic fraction was determined and gill metallothionein was quantified independently by a mercury saturation assay. Cytosolic Cd concentrations were significantly related to the dissolved Cd concentrations at each site, but cytosolic Cu and Zn (essential metals) were not related to their respective ambient dissolved metal concentrations. Metallothionein concentrations increased along the metal contamination gradient and were related to cytosolic Cd (and Zn) in a concentration-dependent manner. However mass balance calculations showed that binding to metallothionein could only account for a small proportion of total gill metal (∼10% Cd; ∼3% Cu; ∼1% Zn). Under these chronic exposure conditions, the three metals (Cd, Cu and Zn) were mainly located in calcium concretions present in the gills (respectively 58 ± 13% of the total gill Cd, 64 ± 6% of the total gill Cu and 73 ± 6% of the total gill Zn). The overall contribution of granules to the total gill dry weight remained relatively constant among the different lakes, suggesting that lake-to-lake variations in granule synthesis were independent of the metal contamination gradient, i.e., these constituent elements of unionid gills act as non-inducible metal sinks at the cellular level. Metal concentrations increased proportionally in both the granules and the MT pool along the polymetallic gradient, suggesting a constant partitioning between these two compartments. Overall, despite an increase in Cd in the “mitochondria” fraction, metal sequestration mechanisms seem to be reasonably effective in detoxifying cadmium: in the cytosol, Cd concentrations in the potentially metal-sensitive HDP fraction remained relatively low and constant, even in specimens collected from the most contaminated lakes.

Direct proteolysis-based purification of an overexpressed hyperthermophile protein from Escherichia coli lysate: a novel exploitation of the link between structural stability and proteolytic resistance

The susceptibility of a peptide bond to cleavage by a protease is determined by: (a) the flexibility of the protein chain region in which it is located, (b) the extent to which the bond is exposed, and (c) the nature of the local interactions made by the sidechains of its flanking residues. Each of these parameters is known to be influenced by the overall structural stability of the protein; thus, proteins of higher structurally stability commonly show higher resistance to proteolysis. Extrapolating this relationship to ‘ultrastable’ proteins, our intention here was to investigate whether a hyperthermophile protein expressed and folded within Escherichia coli could prove to be so resistant to proteolysis as to allow direct purification from complex mixtures of E. coli cytoplasmic and/or membrane proteins, through proteolytic means. Thus, we cloned the gene encoding the triosephosphate isomerase enzyme of Pyrococcus furiosus (PfuTIM) and overexpressed it in E. coli in fusion with glutathione S-transferase (GST). The GST–PfuTIM fusion product partitioned mainly into the insoluble fraction of the whole cell lysate. Upon exposure of the E. coli cell lysate precipitate fractions to the non-specific protease, subtilisin, all polypeptides barring PfuTIM (including the GST affinity tag cloned in fusion with PfuTIM) were found to be degraded to undetectable levels. Trace residual amounts of an E. coli protein, OmpF, survived proteolytic digestion, together with an extremely pure population of PfuTIM. Either autonomously or in combination with the more conventional method of heating solutions to enrich heat-stable proteins through the thermal unfolding and aggregation of all other proteins, such proteolysis-based purification could prove to be useful.

Helicobacter pylori antibodies and gastric cancer: a gender-related difference

Helicobacter pylori has been proposed as a causative agent of gastric cancer. The aim of this study was to define serum antibodies response against different H. pylori antigens in patients with gastric cancer. Serum samples were collected from 115 Lithuanian patients with non-cardia gastric cancer and 110 age- and sex-matched controls without cancer. Heat-stable, low-molecular-mass, and outer membrane proteins were used as antigens to analyze serum IgG antibody response against H. pylori by enzyme-linked immunosorbent assay. Seroprevalence of H. pylori using low-molecular-mass antigen was significantly higher in gastric cancer patients, compared to controls (77% versus 57%, p < 0.05). Significant differences in the prevalence of H. pylori infection between gastric cancer patients and controls were found in females using all three studied antigens: heat-stable (98% versus 84%, p < 0.05), low-molecular-mass (88% versus 48%, p < 0.05) and outer membrane proteins (78% versus 57%, p < 0.05). In males, no significant differences were revealed between gastric cancer patients and controls. There may be other cofactors in addition to H. pylori that are important for the development of gastric cancer. H. pylori seems, however, to be a more important for development of gastric cancer in females than in males or males may have more confounding risk factors for gastric cancer than females.