Internal Sequestration Research Articles

Role of humic acid on benzo[a]anthracene: Insights from aging on adsorption, speciation distribution and bioavailability.

As a crucial component of soil organic matter, humic acid (HA) persists in soil and exert a complex interaction with hydrophobic organic pollutants, yet its specific role still remains unclear. In this study, HA was obtained from weathered coal via alkaline dissolution and acidic precipitation for the adsorption of benzo[a]anthracene (BAA). Subsequently, an aging simulation was employed to assess its long-term performance. The results demonstrated a theoretical maximal adsorption capacity of 0.29mg/g for BAA on HA, and aging led to a 32.7% decline in its adsorption performance. While the speciation distribution of sorbed BAA remained largely unchanged. Combined with the characterization results, it can be inferred that the surface interactions between HA and BAA, including partitioning, van der Waals forces, hydrophobic interactions and hydrogen bonds, could be identified as the mechanisms underlying the desorbing BAA fraction, whereas strong hydrogen bonds, n-π and π-π staking contribute to the formation of non-desorbing BAA. The bound residue fraction of BAA was ascribed to the internal sequestration by porous structure in HA. Upon inoculation with BAA-degrading bacteria, all three fractions of sorbed BAA were decreased, demonstrating their availability. Further applied HA into soil media, a significant passivation effect was obtained as a substantial amount of desorbing and non-desorbing BAA was converted into bound-residue and non-extractable fractions. Overall, this study highlighted the potential of HA as adsorbent for mitigating PAHs environmental activity and its synergistic remediation with microorganisms.

The impact of soil chloride concentration and salt type on the excretions of four recretohalophytes with different excretion mechanisms

Four natives Canadian recretohalophytic species: Atriplex canescens, Armeria maritima, Spartina pectinata, and Distichlis spicata were examined to determine their relative uptake and excretion of chloride in the context of phytoremediation. Adult plants were grown in soils contaminated with either sodium chloride or potassium chloride at various concentrations, then manually washed to collect the excreted salts. Atriplex canescens which has salt bladders, was found to have negligible excretions, suggesting that these structures release minimal amounts of salt onto the leaf’s surface. Chloride excretions of S. pectinata and D. spicata increased with higher soil chloride concentrations. A. maritima showed minimal excretion until a threshold soil salinity was reached. This species shifted from a reliance on internal sequestration to secretion at higher soil salinity. The salt used in the media did not impact these trends, but D. spicata excreted significantly more chloride under sodium chloride conditions. While all four species studied were able to translocate significant amount of salt to their shoots, only S. pectinata, D. spicata, and A. maritima are suitable candidates for remediation by haloconduction. Among these, A. maritima showed the greatest potential and significantly reduced the soil chloride concentration by up to 60% in the highest concentration treatment (4 mg/g).HIGHLIGHTSArmeria maritima, Spartina pectinata, and Distichlis spicata are suitable species for remediation via haloconduction.Armeria maritima had the highest total extraction capacity at high soil chloride.Spartina pectinata had the most consistent excretion capacity and is the most suitable for remediation of soils with lower soil chloride.

Expression of MEP Pathway Genes and Non-volatile Sequestration Are Associated with Circadian Rhythm of Dominant Terpenoids Emission in Osmanthus fragrans Lour. Flowers.

Osmanthus fragrans Lour. is one of the top 10 traditional ornamental flowers in China famous for its unique fragrance. Preliminary study proved that the terpenoids including ionone, linalool, and ocimene and their derivatives are the dominant aroma-active compounds that contribute greatly to the scent bouquet. Pollination observation implies the emission of aromatic terpenoids may follow a circadian rhythm. In this study, we investigated the variation of volatile terpenoids and its potential regulators. The results showed that both volatile and non-volatile terpenoids presented circadian oscillation with high emission or accumulation during the day and low emission or accumulation during the night. The volatile terpenoids always increased to reach their maximum values at 12:00 h, while free and glycosylated compounds continued increasing throughout the day. The depletion of non-volatile pool might provide the substrates for volatile emission at 0:00–6:00, suggesting the sequestration of non-volatile compounds acted like a buffer regulating emission of terpenoids. Further detection of MEP pathway genes demonstrated that their expressions increased significantly in parallel with the evident increase of both volatile and non-volatile terpenoids during the day, indicating that the gene expressions were also closely associated with terpenoid formation. Thus, the expression of MEP pathway genes and internal sequestration both played crucial roles in modulating circadian rhythm of terpenoid emission in O. fragrans.

Uptake Kinetics and Subcellular Compartmentalization Explain Lethal but Not Sublethal Effects of Cadmium in Two Closely Related Amphipod Species.

Eulimnogammarus cyaneus and Eulimnogammarus verrucosus, closely related amphipod species endemic to Lake Baikal, differ with respect to body size (10- to 50-fold lower fresh weights of E. cyaneus) and cellular stress response (CSR) capacity, potentially causing species-related differences in uptake, internal sequestration, and toxic sensitivity to waterborne cadmium (Cd). We found that, compared to E. verrucosus, Cd uptake rates, related to a given exposure concentration, were higher, and lethal concentrations (50%; LC50) were 2.3-fold lower in E. cyaneus (4 weeks exposure; 6 °C). Upon exposures to species-specific subacutely toxic Cd concentrations (nominal LC1; E. cyaneus: 18 nM (2.0 μg L-1); E. verrucosus: 115 nM (12.9 μg L-1); 4 weeks exposure; 6 °C), Cd amounts in metal sensitive tissue fractions (MSF), in relation to fresh weight, were similar in both species (E. cyaneus: 0.25 ± 0.06 μg g-1; E. verrucosus: 0.26 ± 0.07 μg g-1), whereas relative Cd amounts in the biologically detoxified heat stable protein fraction were 35% higher in E. cyaneus. Despite different potencies in detoxifying Cd, body size appears to mainly explain species-related differences in Cd uptake and sensitivities. When exposed to Cd at LC1 over 4 weeks, only E. verrucosus continuously showed 15-36% reduced oxygen consumption rates indicating metabolic depression and pointing to particular sensitivity of E. verrucosus to persisting low-level toxicant pressure.

Zinc Isotope Fractionation in the Hyperaccumulator Noccaea caerulescens and the Nonaccumulating Plant Thlaspi arvense at Low and High Zn Supply.

On the basis of our previous field survey, we postulate that the pattern and degree of zinc (Zn) isotope fractionation in the Zn hyperaccumulator Noccaea caerulescens (J. & C. Presl) F. K. Mey may reflect a relationship between Zn bioavailability and plant uptake strategies. Here, we investigated Zn isotope discrimination during Zn uptake and translocation in N. caerulescens and in a nonaccumulator Thlaspi arvense L. with a contrasting Zn accumulation ability in response to low (Zn-L) and high (Zn-H) Zn supplies. The average isotope fractionations of the N. caerulescens plant as a whole, relative to solution (Δ(66)Znplant-solution), were -0.06 and -0.12‰ at Zn-L-C and Zn-H-C, respectively, indicative of the predominance of a high-affinity (e.g., ZIP transporter proteins) transport across the root cell membrane. For T. arvense, plants were more enriched in light isotopes under Zn-H-A (Δ(66)Znplant-solution = -0.26‰) than under Zn-L-A and N. caerulescens plants, implying that a low-affinity (e.g., ion channel) transport might begin to function in the nonaccumulating plants when external Zn supply increases. Within the root tissues of both species, the apoplast fractions retained up to 30% of Zn mass under Zn-H. Moreover, the highest δ(66)Zn (0.75‰-0.86‰) was found in tightly bound apoplastic Zn, pointing to the strong sequestration in roots (e.g., binding to high-affinity ligands/precipitation with phosphate) when plants suffer from high Zn stress. During translocation, the magnitude of isotope fractionation was significantly greater at Zn-H (Δ(66)Znroot-shoot = 0.79‰) than at Zn-L, indicating that fractionation mechanisms associated with root-shoot translocation might be identical to the two plant species. Hence, we clearly demonstrated that Zn isotope fractionation could provide insight into the internal sequestration mechanisms of roots when plants respond to low and high Zn supplies.

Overcoming cancer multidrug resistance through inhibition of microparticles

AbstractOne of the main obstacles to success of chemotherapy agents is the development of cancer resistance. Cancer multi-drug resistance (MDR) is thought to arise from over-expression of efflux transporters on cancer cells’ plasma membranes. Recently, microparticles (MP) were found to play a major role in mediating the resistance to antineoplastic agents. Microparticles can confer MDR phenotype to cancer cells though 3 complimentary pathways: 1) Intercellular transfer of P-gp and MRP1; 2) Intercellular transfer of regulatory nucleic acids that ensure acquisition of MDR phenotype; and 3) Internal sequestration of anticancer drugs to reduce the amount of free active drug. Compounds that inhibit MP formation that are currently under investigation include calpain inhibitors, RhoA inhibitors, ROCK inhibitors, calcium channel blockers, pantethine, glutaminase inhibitors, some anti-platelet drugs and some lipid-lowering agents. This area of research requires further development to select, improve and test those compounds that show the most promise in providing safe and effective treatment against MDR.

Bioorthogonal Calcium Modulation by Direct Intracellular Access using Nanostraws

Ionic calcium plays a ubiquitous role in cell signaling. Calcium signaling uses numerous mechanisms for regulation including coordinated transport across cell membranes, internal sequestration and storage, and various specific and nonspecific buffers. These mechanisms make the generation of ectopic calcium signals difficult, especially if minimal cell perturbation is required. Nevertheless, manipulating calcium effectively is necessary to study how the cell signals using calcium. Patch clamp pipettes can control intracellular ionic content in single cells for a limited time. To increase the number of cells studied, chemical methods for controlling intracellular calcium can be used such as phorbol ester stimulation or thapsigargin release of calcium storage. These indirect methods can conflate different signaling pathways with calcium signaling. To isolate intracellular calcium from other cell functions, techniques which do not rely on biological mechanisms of release are needed, such as calcium photouncaging, which uses external light. Here, we propose an alternate strategy to introduce calcium to cells in a bioorthogonal fashion to minimize the biological footprint. Based on a gap junction-like architecture, we fabricated a system of supported nanotubes, called nanostraws. Due to their high aspect ratio (10:1), the nanostraws penetrate into the cell to allow fluidic exchange between the cell and an external reservoir, which we use to deliver calcium. We focus on generating temporal signal patterns and controlling the quantity of calcium delivered to the cell. To demonstrate the bioorthogonality of nanostraw calcium modulation, we also show that calcium signals can be elicited through normal stimulation methods. This technique represents a microfluidic technique to manipulate intracellular calcium which requires no chemical modification of cells, and the fluidic nature of the delivery can be leveraged to expand the delivery cargo to other biological ions such as zinc or more complex biomolecules.

Oral bioaccessibility of toxic metals in contaminated oysters and relationships with metal internal sequestration

The Hong Kong oysters Crassostrea hongkongensis are widely farmed in the estuarine waters of Southern China, but they accumulate Cu and Zn to alarmingly high concentrations in the soft tissues. Health risks of seafood consumption are related to contaminants such as toxic metals which are bioaccessible to humans. In the present study, we investigated the oral bioaccessibility of five toxic metals (Ag, Pb, Cd, Cu and Zn) in contaminated oysters collected from different locations of a large estuary in southern China. In all oysters, total Zn concentration was the highest whereas total Pb concentration was the lowest. Among the five metals, Ag had the lowest oral bioaccessibility (38.9–60.8%), whereas Cu and Zn had the highest bioaccessibility (72.3–93.1%). Significant negative correlation was observed between metal bioaccessibility and metal concentration in the oysters for Ag, Cd, and Cu. We found that the oral bioaccessibility of the five metals was positively correlated with their trophically available metal fraction (TAM) in the oyster tissues, and negatively correlated with metal distribution in the cellular debris. Thus, metal partitioning in the TAM and cellular debris controlled the oral bioaccessibility to humans. Given the dependence of oral bioaccessibility on tissue metal contamination, bioaccessibility needs to be incorporated in the risk assessments of contaminated shellfish.

Effects of Molecular Size and Surface Hydrophobicity on Oligonucleotide Interfacial Dynamics

Single-molecule total internal reflection fluorescence microscopy was used to observe the dynamic behavior of polycytosine single-stranded DNA (ssDNA) (1-50 nucleotides long) at the interface between aqueous solution and hydrophilic (oligoethylene glycol-modified fused silica, OEG) and hydrophobic (octadecyltriethoxysilane-modified fused silica, OTES) solid surfaces. High throughput molecular tracking was used to determine >75,000 molecular trajectories for each molecular length, which were then used to calculate surface residence time and squared displacement (i.e., "step-size") distributions. On hydrophilic OEG surfaces, the surface residence time increased systematically with ssDNA chain length, as expected due to increasing molecule-surface interactions. Interestingly, the residence time decreased with increasing ssDNA length on the hydrophobic OTES surface, particularly for longer chains. Similarly, the interfacial mobility of polynucleotides slowed with increasing chain length on OEG, but became faster on OTES. On OTES surfaces, the rates associated with desorption and surface diffusion exhibited the distinctive anomalous temperature dependence that is characteristic of hydrophobic interactions for short-chain species but not for longer chains. These combined observations suggest that long oligonucleotides adopt conformations minimizing hydrophobic interactions, e.g., by internal sequestration of hydrophobic nucleobases.

Biochemical changes and adaptive strategies of plants under heavy metal stress

AbstractHeavy metal contamination of soil, aqueous waste stream and ground water causes major environmental and human health problems. Heavy metals are major environmental pollutants when they are present in high concentration in soil and show potential toxic effects on growth and development in plants. Due to unabated, indiscriminate and uncontrolled discharge of hazardous chemicals including heavy metals into the environment, plant continuously have to face various environmental constraints. In plants, seed germination is the first exchange interface with the surrounding medium and has been considered as highly sensitive to environmental changes. One of the crucial events during seed germination entails mobilization of seed reserves which is indispensable for the growth of embryonic axis. But, metabolic alterations by heavy metal exposure are known to depress the mobilization and utilization of reserve food by affecting the activity of hydrolytic enzymes. Some plants possess a range of potential mechanisms that may be involved in the detoxification of heavy metals by which they manage to survive under metal stress. High tolerance to heavy metal toxicity could rely either on reduced uptake or increase planned internal sequestration which is manifested by an interaction between a genotype and its environment. Such mechanism involves the binding of heavy metals to cell wall, immobilization, exclusion of the plasma membrane, efflux of these toxic metal ions, reduction of heavy metal transport, compartmentalization and metal chelation by tonoplast located transporters and expression of more general stress response mechanisms such as stress proteins. It is important to understand the toxicity response of plant to heavy metals so that we can utilize appropriate plant species in the rehabilitation of contaminated areas. Therefore, in the present review attempts have been made to evaluate the effects of increasing level of heavy metal in soils on the key behavior of hydrolytic and nitrogen assimilation enzymes. Additionally, it also provides a broad overview of the strategies adopted by plants against heavy metal stress.

Effects of Interactions Between Cadmium and Lead on Growth, Nitrogen Fixation, Phytochelatin, and Glutathione Production in Mycorrhizal Cajanus cajan (L.) Millsp.

Heavy metals (HM) are a unique class of toxicants because they cannot be broken up into nontoxic forms. Excess HM causes stunted growth, upsets mineral nutrition, and affects membrane structure and permeability. High tolerance to HM toxicity is based on reduced metal uptake or increased internal sequestration in a genotype. Arbuscular mycorrhizal (AM) fungi are important rhizospheric microorganisms that occur in metal-contaminated soils and perhaps detoxify the potential effects of metals. The aim of this work was to study the role of the AM fungus Glomus mosseae in the alleviation of cadmium (Cd) and lead (Pb) toxicities in Cajanus cajan (L.) Millsp. (pigeonpea) genotypes. The effects of interactions between Cd (25 and 50 mg/kg) and Pb (500 and 800 mg/kg) on plant dry mass, nitrogen metabolism, and production of phytochelatins (PCs) and glutathione (GSH) were monitored with and without AM fungus in genotypes Sel-85N (relatively tolerant) and Sel-141-97 (sensitive). Cd treatments were more toxic than Pb, and their combinations led to synergistic inhibitions to growth and nitrogen-fixing potential (acetylene reduction activity [ARA]) in both genotypes. However, the effects were less deleterious in Sel-85N than in Sel-141-97. Exposure to Cd and Pb significantly increased the levels of PCs in a concentration- and genotype-dependent manner, which could be directly correlated with the intensity of mycorrhizal infection (MI). Stimulation of GSH production was observed under Cd treatments, although no obvious effects on GSH levels were observed under Pb treatments. The metal contents (Cd, Pb) were higher in roots and nodules when compared with that in shoots, which was significantly reduced in the presence of AM fungi. The results indicated that PCs and GSH might function as potential biomarkers for metal toxicity, and microbial inoculations showed bioremediation potential by helping pigeonpea plants to grow in multimetal contaminated soils.

Phytoremediation of Heavy Metals: Physiological and Molecular Mechanisms

Heavy metals (HM) are a unique class of toxicants since they cannot be broken down to non-toxic forms. Concentration of these heavy metals has increased drastically, posing problems to health and environment, since the onset of the industrial revolution. Once the heavy metals contaminate the ecosystem, they remain a potential threat for many years. Some technologies have long been in use to remove, destroy and sequester these hazardous elements. Even though effective techniques for cleaning the contaminated soils and waters are usually expensive, labour intensive, and often disturbing. Phytoremediation, a fast-emerging new technology for removal of toxic heavy metals, is cost-effective, non-intrusive and aesthetically pleasing. It exploits the ability of selected plants to remediate pollutants from contaminated sites. Plants have inter-linked physiological and molecular mechanisms of tolerance to heavy metals. High tolerance to HM toxicity is based on a reduced metal uptake or increased internal sequestration, which is manifested by interaction between a genotype and its environment. The growing interest in molecular genetics has increased our understanding of mechanisms of HM tolerance in plants and many transgenic plants have displayed increased HM tolerance. Improvement of plants by genetic engineering, i.e., by modifying characteristics like metal uptake, transport and accumulation and plant’s tolerance to metals, opens up new possibilities of phytoremediation. This paper presents an overview of the molecular and physiological mechanisms involved in the phytoremediation process, and discusses strategies for engineering plants genetically for this purpose.

Understanding molecular mechanisms for improving phytoremediation of heavy metal-contaminated soils

Heavy metal pollution of soil is a significant environmental problem with a negative potential impact on human health and agriculture. Rhizosphere, as an important interface of soil and plants, plays a significant role in phytoremediation of contaminated soil by heavy metals, in which, microbial populations are known to affect heavy metal mobility and availability to the plant through release of chelating agents, acidification, phosphate solubilization and redox changes, and therefore, have potential to enhance phytoremediation processes. Phytoremediation strategies with appropriate heavy metal-adapted rhizobacteria or mycorrhizas have received more and more attention. In addition, some plants possess a range of potential mechanisms that may be involved in the detoxification of heavy metals, and they manage to survive under metal stresses. High tolerance to heavy metal toxicity could rely either on reduced uptake or increased plant internal sequestration, which is manifested by an interaction between a genotype and its environment.A coordinated network of molecular processes provides plants with multiple metal-detoxifying mechanisms and repair capabilities. The growing application of molecular genetic technologies has led to an increased understanding of mechanisms of heavy metal tolerance/accumulation in plants and, subsequently, many transgenic plants with increased heavy metal resistance, as well as increased uptake of heavy metals, have been developed for the purpose of phytoremediation. This article reviews advantages, possible mechanisms, current status and future direction of phytoremediation for heavy-metal–contaminated soils.

RSV-infected airway epithelial cells cause biphasic up-regulation of CCR1 expression on human monocytes

Respiratory syncytial virus (RSV) infection can cause extensive airway inflammation, which is orchestrated by chemokines and their receptors. RSV-infected epithelial cells secrete many cytokines and chemokines, but little is known about regulation of chemokine receptors on target cells. We investigated the effects of conditioned media (CM) from RSV-infected epithelial cells on monocyte CCR1, CCR2, and CCR5 expression. RSV-CM but not control-CM stimulated a biphasic increase in cell-surface CCR1, and levels peaked at 36 h and 96 h poststimulation. Similar CCR1 up-regulation occurred on monocyte-derived macrophages. Cytochlasin D and colchicine blocked both peaks of expression, demonstrating requirement of a functional cytoskeleton. Intracellular staining revealed little internal sequestration of CCR1 protein, and CCR1 up-regulation was inhibited by actinomycin D and cycloheximide, indicating that both waves of RSV-CM-induced surface CCR1 expression were dependent on de novo transcription and protein synthesis. Cytokine-neutralizing experiments showed that the effects of RSV-CM were decreased by blocking TNF-alpha (percent inhibition=51+/-2.3% at 36 h peak and 42+/-7.7% at 96 h peak) and to a lesser extent, IL-1 (percent inhibition=32+/-7.2% at 36 h and 23+/-2.9% at 96 h). In summary, RSV-CM causes a biphasic up-regulation of surface CCR1 on monocytes, which is dependent on an intact cytoskeleton, requires new gene transcription and protein synthesis, and is mediated in part by the proinflammatory cytokines TNF-alpha and IL-1.

The v-SNARE Vti1a Regulates Insulin-stimulated Glucose Transport and Acrp30 Secretion in 3T3-L1 Adipocytes

Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and trans-Golgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNA-based gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.

Molecular Mechanisms and Genetic Basis of Heavy Metal Tolerance/Hyperaccumulation in Plants

Abstract: Phytoremediation has gained increased attention as a cost-effective method for the remediation of heavy metal-contaminated sites. Because some plants possess a range of potential mechanisms that may be involved in the detoxification of heavy metals, they manage to survive under metal stresses. High tolerance to heavy metal toxicity could rely either on reduced uptake or increased plant internal sequestration, which is manifested by an interaction between a genotype and its environment. The growing application of molecular genetic technologies has led to increased understanding of mechanisms of heavy metal tolerance/accumulation in plants and, subsequently, many transgenic plants with increased heavy metal resistance, as well as increased uptake of heavy metals, have been developed for the purpose of phytoremediation. In the present review, our major objective is to concisely evaluate the progress made so far in understanding the molecular/cellular mechanisms and genetic basis that control the uptake and detoxification of metals by plants. (Managing editor: Ping HE)

Genes encoding proteins of the cation diffusion facilitator family that confer manganese tolerance.

The yeast Saccharomyces cerevisiae expressing a cDNA library prepared from Stylosanthes hamata was screened for enhanced Mn(2+) tolerance. From this screen, we identified four related cDNAs that encode membrane-bound proteins of the cation diffusion facilitator (CDF) family. One of these cDNAs (ShMTP1) was investigated in detail and found to confer Mn(2+) tolerance to yeast by internal sequestration rather than by efflux of Mn(2+). Expression of ShMTP1 in a range of yeast mutants suggested that it functions as a proton:Mn(2+) antiporter on the membrane of an internal organelle. Similarly, when expressed in Arabidopsis, ShMTP1 conferred Mn(2+) tolerance through internal sequestration. The ShMTP1 protein fused to green fluorescent protein was localized to the tonoplast of Arabidopsis cells but appeared to localize to the endoplasmic reticulum of yeast. We suggest that the ShMTP1 proteins are members of the CDF family involved in conferring Mn(2+) tolerance and that at least one of these proteins (ShMTP1) confers tolerance by sequestering Mn(2+) into internal organelles.

The extremely high Al resistance of Penicillium janthineleum F-13 is not caused by internal or external sequestration of Al.

Penicillium janthinellum F-13 has been isolated in previous work as a fungus tolerating the presence of high concentrations of Al (as high as 100 mM AlCl3). Here its growth rate and yield in three acidic (pH 3.0) media of different composition with varying concentrations of Al are reported. The presence of Al did not affect these parameters. except that the growth yield was somewhat lower in GM (a glucose/peptone/yeast extract-containing medium) with the highest concentration tested (100 mM AlCl3). The amount of Al found in the mycelium was so low that it cannot lead to a significant decrease in the medium for the higher Al concentrations applied. Although citric acid was excreted at growth on GM, and the presence of Al even promoted this, the concentration of this was far too low to diminish (by chelation) the high Al concentrations in the medium to a non-toxic level, i.e. the level (of approx. 1 mM) that is tolerated by low-resistance fungi. At growth on SLBM (a peptone/yeast extract/soil extract-containing medium), a rise in pH occurred. The same was found for SM (a glucose/mineral salts-containing medium), although in this case the picture was more complicated because the initial rise in pH was followed by a lowering due to the excretion of oxalic acid. Although both phenomena can diminish Al toxicity (by decreasing the external concentration of monomeric Al, regarded to be the toxic species), again the decrease is far too low to attain a non-toxic level when high Al concentrations are applied. Therefore, although in principal the metabolic phenomena observed for P. janthinellum F-13 at growth on different media can diminish Al toxicity, the tolerance of this organism for high external Al concentrations must be caused by another mechanism.

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification.

Wild-type Dictyostelium discoideum cells growing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2-3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100 kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved.

Influence of insulin on sodium efflux in barnacle muscle fibers.

The efflux of radiosodium in single muscle fibers from the barnacleBalanus nubilus was irresponsive to internal or external application of insulin. However, this was not the case with fibers isolated from a barnacle specimen pre-exposed overnight to a large dose of insulin. External application of insulin to pre-exposed fibers caused a decrease in the rate of decline of the radiosodium efflux and stopped the decline in the fractional rate constant for Na efflux. Such kinetics were interpreted as indicating that insulin acts either by releasing sequestered Na or abolishing the process of sequestration. Internal application of saline slowed the rate of decline but failed to completely abolish the mechanism of sequestration. Only in the presence of insulin was the fractional loss of Na each second constant. Internal application of insulin caused a prompt step-up in the rate of Na efflux, followed by a reduced efflux rate constant. This meant that injected insulin caused the release of sequestered Na, leading to partial saturation of the efflux. The response of the Na efflux to injected denatured insulin, though resembling that to native insulin was much smaller in size. Internal application of lysozyme produced a transitory step-up in the rate of Na efflux but failed to produce the kinetics observed with native or denatured insulin. Overnight exposure of the barnacle to a dose of denatured insulin failed to render the fiber sensitive to external and internal application of denatured or native insulinin vitro. Experiments with ouabain-poisoned fibers showed that external or internal application of native insulin caused stimulation of the remaining Na efflux. They also showed that a 10-fold increase in the concentration of ouabain failed to further reduce the ouabaininsensitive Na efflux. Microinjection of GTP into ouabain-poisoned fibers pre-exposed to insulin resulted in a striking rise in the remaining Na efflux. The magnitude of this effect was considerably greater than that in unexposed fibers. The response which was dose-dependent could be blunted by prior injection of CaCl2. Similarly, the response to CaCl2 injection could be blunted by prior injection of GTP. The evidence brought forward is compatible with the view that insulin acts by abolishing the mechanism of internal Na sequestration and by increasing the activity of the guanylate cyclase system.