Intracellular Aluminum Research Articles

Malonate is relevant to the lung environment and induces genome-wide stress responses in Pseudomonas aeruginosa.

Versatility in carbon source utilization is a major contributor to niche adaptation in Pseudomonas aeruginosa. Malonate is among the abundant carbon sources in the lung airways, yet it is understudied. Recently, we characterized how malonate impacts quorum sensing regulation, antibiotic resistance, and virulence factor production in P. aeruginosa. Herein, we show that malonate as a carbon source supports more robust growth in comparison to glycerol in several cystic fibrosis isolates of P. aeruginosa. Furthermore, we show phenotypic responses to malonate were conserved among clinical strains, i.e., formation of biomineralized biofilm-like aggregates, increased tolerance to kanamycin, and increased susceptibility to norfloxacin. Moreover, we explored transcriptional adaptations of P. aeruginosa UCBPP-PA14 (PA14) in response to malonate versus glycerol as a sole carbon source using transcriptomics. Malonate utilization activated glyoxylate and methylcitrate cycles and induced several stress responses, including oxidative, anaerobic, and metal stress responses associated with increases in intracellular aluminum and strontium. We identified several genes that were required for optimal growth of P. aeruginosa in malonate. Our findings reveal important remodeling of P. aeruginosa gene expression during its growth on malonate as a sole carbon source that is accompanied by several important phenotypic changes. These findings add to the accumulating literature highlighting the role of different carbon sources in the physiology of P. aeruginosa and its niche adaptation.

Comparative study of aluminum (Al) speciation on apoptosis-promoting process in PC12 cells: Correlations between morphological characteristics and mitochondrial kinetic disorder

Aluminum contamination in environment is very serious and the central nervous system is the main target of aluminum toxicity. The neurotoxic of aluminum is closely related to its speciation. PC12 cells were taken as the cell model to compare the morphological characteristics and mitochondrial kinetic disorder of two speciation of aluminum compounds (AlCl3 and aluminum-maltolate (Al(mal)3)). When the concentration of AlCl3 was 3 mM, the intracellular aluminum ion content was 3.87 times that of the 0.5 mM Al(mal)3 treatment group. At the 3 mM AlCl3 treatment group, intracellular ion homeostasis was disrupted. Abnormally elevated Ca2+ levels inhibited protein kinase B (AKT) phosphorylation, resulting in impaired cell morphology. At the 0.5 mM Al(mal)3 treatment group, abnormally high levels of Ca2+ caused mitochondrial kinetic disorder, which led to impaired cellular energy metabolism. Al(mal)3 had shown more cytotoxic in PC12 than AlCl3 at the same concentration. AlCl3 tended to inhibit the phosphorylation of AKT and damages cell morphology. Al(mal)3 mainly affected mitochondrial kinetic disorder, which led to impaired cellular energy metabolism. These findings provided experimental evidence for in-depth research on aluminum-induced neurotoxicity.

Aluminum Enters Mammalian Cells and Destabilizes Chromosome Structure and Number.

Chromosome instability (CIN) consists of high rates of structural and numerical chromosome abnormalities and is a well-known hallmark of cancer. Aluminum is added to many industrial products of frequent use. Yet, it has no known physiological role and is a suspected human carcinogen. Here, we show that V79 cells, a well-established model for the evaluation of candidate chemical carcinogens in regulatory toxicology, when cultured in presence of aluminum—in the form of aluminum chloride (AlCl3) and at concentrations in the range of those measured in human tissues—incorporate the metal in a dose-dependent manner, predominantly accumulating it in the perinuclear region. Intracellular aluminum accumulation rapidly leads to a dose-dependent increase in DNA double strand breaks (DSB), in chromosome numerical abnormalities (aneuploidy) and to proliferation arrest in the G2/M phase of the cell cycle. During mitosis, V79 cells exposed to aluminum assemble abnormal multipolar mitotic spindles and appear to cluster supernumerary centrosomes, possibly explaining why they accumulate chromosome segregation errors and damage. We postulate that chronic aluminum absorption favors CIN in mammalian cells, thus promoting carcinogenesis.

EBER in situ hybridization in subcutaneous aluminum granulomas/lymphoid hyperplasia: A diagnostic clue to differentiate injection-associated lymphoid hyperplasia from other forms of pseudolymphomas and cutaneous lymphomas.

Subcutaneous vaccination or desensitization may induce persistent nodules at the injection sites. Without the knowledge of prior injection, histopathological work-up may be challenging. Aim of this study was to contribute to the histopathological work-up of unclear subcutaneous nodules, especially their differentiation from cutaneous lymphoma. We retrospectively reviewed clinical data and histopathological slides of four patients with subcutaneous nodules, which were suspected to suffer from cutaneous T- or B-cell lymphoma. Sections of these cases and 12 negative controls were stained with hematoxylin and eosin and a standardized immunohistochemical panel of B- and T-cell markers including EBER in situ hybridization as well as electron microscopy. In all cases, large histiocytes with granular cytoplasm compatible with intracellular aluminum hydroxide were present. EBER in situ hybridization revealed positive staining of these granular histiocytes while staining was absent in negative controls. Post hoc completion of medical history revealed that vaccination or specific immunotherapy had been applied before at the biopsy site in only three out of four patients; one patient was lost to follow-up. EBER in situ hybridization is an adjunctive tool to differentiate aluminum-induced granuloma/lymphoid hyperplasia from other forms of pseudolymphoma and cutaneous B- or T-cell lymphomas.

Unequivocal imaging of aluminium in human cells and tissues by an improved method using morin

Aluminium is biologically reactive and its ability to potentiate the immune response has driven its inclusion in both veterinary and human vaccines. Consequently, the need for unequivocal visualisation of aluminium in vivo has created a focused research effort to establish fluorescent molecular probes for this purpose. The most commonly used direct fluorescent labels for the detection of aluminium are morin (2′,3,4′,5,7-pentahydroxyflavone) and lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzene-1-sulphonic acid]. While the former has gained popularity in the detection of aluminium in plants and predominantly within root tips, the latter boasts greater sensitivity and selectivity for the detection of aluminium in human cells and tissues. Herein, we have developed a simplified morin staining protocol using the autofluorescence quenching agent, Sudan Black B. This modified protocol improves tissue morphology and increases analytical sensitivity, which allows intracellular aluminium to be detected in monocytes and when co-localised with senile plaques in human brain tissue of donors diagnosed with familial Alzheimer’s disease. Overall, our results demonstrate a simple approach to minimise false positives in the use of morin to unequivocally detect aluminium in vivo.

A Highly Selective Turn-on Fluorescent Probe for the Detection of Aluminum and Its Application to Bio-Imaging.

Aluminum is the most abundant metallic element in the Earth’s crust and acts as a non-essential element for biological species. The accumulation of excessive amounts of aluminum can be harmful to biological species. Thus, the development of convenient and selective tools for the aluminum detection is necessary. In this work, a highly selective aluminum ion fluorescent probe N’-(2,5-dihydroxybenzylidene)acetohydrazide (Al-II) has been successfully synthesized and systemically characterized. The fluorescence intensity of this probe shows a significant enhancement in the presence of Al3+, which is subject to the strong quench effects caused by Cu2+ and Fe3+. The binding ratio of probe-Al3+ was determined from the Job’s plot to be 1:1. Moreover, the probe was demonstrated to be effective for in vivo imaging of the intracellular aluminum ion in both living Drosophila S2 cells and Malpighian tubules.

Aluminium in brain tissue in autism

Autism spectrum disorder is a neurodevelopmental disorder of unknown aetiology. It is suggested to involve both genetic susceptibility and environmental factors including in the latter environmental toxins. Human exposure to the environmental toxin aluminium has been linked, if tentatively, to autism spectrum disorder. Herein we have used transversely heated graphite furnace atomic absorption spectrometry to measure, for the first time, the aluminium content of brain tissue from donors with a diagnosis of autism. We have also used an aluminium-selective fluor to identify aluminium in brain tissue using fluorescence microscopy. The aluminium content of brain tissue in autism was consistently high. The mean (standard deviation) aluminium content across all 5 individuals for each lobe were 3.82(5.42), 2.30(2.00), 2.79(4.05) and 3.82(5.17) μg/g dry wt. for the occipital, frontal, temporal and parietal lobes respectively. These are some of the highest values for aluminium in human brain tissue yet recorded and one has to question why, for example, the aluminium content of the occipital lobe of a 15year old boy would be 8.74 (11.59) μg/g dry wt.? Aluminium-selective fluorescence microscopy was used to identify aluminium in brain tissue in 10 donors. While aluminium was imaged associated with neurones it appeared to be present intracellularly in microglia-like cells and other inflammatory non-neuronal cells in the meninges, vasculature, grey and white matter. The pre-eminence of intracellular aluminium associated with non-neuronal cells was a standout observation in autism brain tissue and may offer clues as to both the origin of the brain aluminium as well as a putative role in autism spectrum disorder.

Al adjuvants can be tracked in viable cells by lumogallion staining

The mechanism behind the adjuvant effect of aluminum salts is poorly understood notwithstanding that aluminum salts have been used for decades in clinical vaccines. In an aqueous environment and at a nearly neutral pH, the aluminum salts form particulate aggregates, and one plausible explanation of the lack of information regarding the mechanisms could be the absence of an efficient method of tracking phagocytosed aluminum adjuvants and thereby the intracellular location of the adjuvant.In this paper, we want to report upon the use of lumogallion staining enabling the detection of phagocytosed aluminum adjuvants inside viable cells. Including micromolar concentrations of lumogallion in the culture medium resulted in a strong fluorescence signal from cells that had phagocytosed the aluminum adjuvant. The fluorescence appeared as spots in the cytoplasm and by confocal microscopy and co-staining with probes presenting fluorescence in the far-red region of the spectrum, aluminum adjuvants could to a certain extent be identified as localized in acidic vesicles, i.e., lysosomes.Staining and detection of intracellular aluminum adjuvants was achieved not only by diffusion of lumogallion into the cytoplasm, thereby highlighting the presence of the adjuvant, but also by pre-staining the aluminum adjuvant prior to incubation with cells. Pre-staining of aluminum adjuvants resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks and with only a minor reduction of fluorescence upon extensive washing or incubation with cells. Both aluminum oxyhydroxide and aluminum hydroxyphosphate, two of the most commonly used aluminum adjuvants in clinical vaccines, could be pre-stained with lumogallion and were easily tracked intracellularly after incubation with phagocytosing cells.Staining of viable cells using lumogallion will be a useful method in investigations of the mechanisms behind aluminum adjuvants' differentiation of antigen-presenting cells into inflammatory cells. Information will be gained regarding the phagosomal pathways and the events inside the phagosomes, and thereby the ultimate fate of phagocytosed aluminum adjuvants could be resolved.

Disulfide stress-induced aluminium toxicity: molecular insights through genome-wide screening of Saccharomyces cerevisiae

Formation of non-native disulfide bonds within or between proteins can lead to protein misfolding and disruption to cellular metabolism. Such a process is defined as disulfide stress. A marked effect of disulfide stress in cells is the elevated accumulation of the intracellular aluminium ion (Al(3+)) accompanied by increased cytotoxicity. To gain an in-depth understanding of the underlying molecular mechanism for disulfide stress-induced aluminium toxicity, the complete set of Saccharomyces cerevisiae deletion mutants (5047) was screened in this study simultaneously with a combination of the two stressors, diamide and Al(3+). The combined treatment of a benign concentration of diamide (0.8 mM) with a sublethal concentration of aluminium sulfate (0.4 mM) revealed 494 sensitive deletion mutants, distinct from those found when either of the single stressors (0.8 mM diamide or 0.4 mM aluminium sulfate) was used. Hierarchical clustering and functional analyses of the 494 mutants sensitive to the dual stressors indicated a significant enrichment in the genes involved in cell wall homeostasis, signaling cascades, secretory transport machinery and detoxification. The results highlight the process of maintaining cell wall integrity as the central response to the combined exposure of diamide and Al(3+), which is mediated by the signaling pathways and transcription activation via Rlm1p and Swi6p for biosynthesis of the essential cell wall components such as glucan and chitin. Sensitivity of mutants associated with endoplasmic reticulum (ER), vesicle and vacuole functions demonstrates that secretory machinery is essential for surviving the stress conditions, probably due to their roles in transporting polysaccharides to the cell wall and detoxification of accumulated Al(3+). Finally, the phenotype of 100 previously uncharacterized genes against the dual stressors will contribute to their eventual functional annotation.

Intracellular effects of aluminium on receptor-activated cytoplasmic Ca2+ signals in pancreatic acinar cells.

The hypothesis that intracellular aluminium may interfere with cytoplasmic Ca2+ signals evoked by the activation of receptors linked to inositol lipid hydrolysis has been tested. Single mouse pancreatic acinar cells were used, because there is much information in this system on the mechanism by which acetylcholine (ACh) evokes cytoplasmic Ca2+ oscillations (spiking) and these spikes can be monitored in internally perfused cells by measuring the Ca(2+)-dependent chloride current. ACh normally evokes repetitive Ca2+ spikes, but when aluminium (1 microM-1 mM) is present in the internal perfusion solution the responses are reduced or absent. When aluminium is acutely infused into the internal perfusion solution the ACh-evoked Ca2+ signals quickly disappear. Aluminium also inhibits Ca2+ signals evoked by the Ca2+ releasing agent caffeine. Preliminary results suggest that silicic acid may protect against the toxic effects of aluminium. Silicic acid and citrate, in the absence of added Al3+, have the effect of enhancing the ACh-evoked Ca2+ signals. This could be due to binding of traces of Al3+ in the solutions. We conclude that aluminium can disrupt receptor-activated cytosolic Ca2+ signals when present inside cells.

Effects of aluminum in red spruce (Picea rubens) cell cultures: Cell growth and viability, mitochondrial activity, ultrastructure and potential sites of intracellular aluminum accumulation

The effects of Al on red spruce (Picea rubens Sarg.) cell suspension cultures were examined using biochemical, stereological and microscopic methods. Exposure to Al for 24–48 h resulted in a loss of cell viability, inhibition of growth and a significant decrease in mitochondrial activity. Soluble protein content increased in cells treated with Al. Using energy‐dispersive X‐ray microanalysis on sections of freeze‐substituted cells that had no obvious disruption in cytoplasmic or cell wall structure, Al (always in the presence of P) was detected in dense regions in cell walls, cytoplasm, plastids and vacuoles after 48 h exposure to Al. Stereological quantification of spruce cell structure showed that, after 24 h of Al treatment, intact cells had increased vacuolar and total cell volume, but the nuclear volume did not change. In addition, Al treatment resulted in increased surface area of Golgi membranes and endoplasmic reticulum. The biochemical and ultrastructural alterations in red spruce cells, in combination with the presence of Al in cellular organelles of visually intact cells, suggest that Al movement occurred across the plasma membrane without major cellular disruption. Detailed short‐term time course studies are needed to determine if intracellular Al in these cells results from its passage into cells through submicroscopic lesions in the plasma membrane or it is taken up into the symplast through the intact membrane by an active, but slow, process.

Competition between internal AlF(4)(-) and receptor-mediated stimulation of dorsal raphe neuron G-proteins coupled to calcium current inhibition.

Intracellular aluminum fluoride (AlF(4)(-)), placed in a patch pipette, activated a G-protein, resulting in a "tonic" inhibition of the Ca(2+) current of isolated serotonergic neurons of the rat dorsal raphe nucleus. Serotonin (5-HT) also inhibits the Ca(2+) current of these cells. After external bath application and quick removal of 5-HT to an AlF(4)(-) containing cell, there was a reversal or transient disinhibition (TD) of the inhibitory effect of AlF(4)(-) on Ca(2+) current. A short predepolarization of the membrane potential to +70 mV, a condition that is known to reverse G-protein-mediated inhibition, reversed the inhibitory effect of AlF(4)(-) on Ca(2+) current and brought the Ca(2+) current to the same level as that seen at the peak of the TD current. With AlF(4)(-) in the pipette, the TD phenomenon could be eliminated by lowering pipette MgATP, or by totally chelating pipette Al(3+). In the presence of AlF(4)(-), but with either lowered MgATP or extreme efforts to eliminate pipette Al(3+), the rate of recovery from 5-HT on wash was slowed, a condition opposite to that where a TD occurred. The putative complex of AlF(4)(-)-bound G-protein (Galpha.GDP. AlF(4)(-)) appeared to free G-betagamma-subunits, mimicking the effect on Ca(2+) channels of the G.GTP complex. The ON-rate of the inhibition of Ca(2+) current, after a depolarizing pulse, by betagamma-subunits released by AlF(4)(-) in the pipette was significantly slower than that of the agonist-activated G-protein. The OFF-rate of the AlF(4)(-)-mediated inhibition in response to a depolarizing pulse, a measure of the affinity of the free G-betagamma-subunit for the Ca(2+) channel, was slightly slower than that of the agonist stimulated G-protein. In summary, AlF(4)(-) modified the OFF-rate kinetics of G-protein activation by agonists, but had little effect on the kinetics of the interaction of the betagamma-subunit with Ca(2+) channels. Agonist application temporarily reversed the effects of AlF(4)(-), making it a complementary tool to GTP-gamma-S for the study of G-protein interactions.

Role of Ca 2+ and NO in differential effects of extra and intracellular aluminum on acetyl-CoA and acetylcholine metabolism in rat brain nerve terminals

Role of Ca 2+ and NO in differential effects of extra and intracellular aluminum on acetyl-CoA and acetylcholine metabolism in rat brain nerve terminals

Do aluminium and/or glutamate induce Alz-50 reactivity? A light microscopic immunohistochemical study.

Alzheimer's disease is thought to be characterized by conformational and phosphorylation changes in tau protein, leading to the formation of aggregations of paired helical filaments within neurons. Potential agents for inducing conformational changes in tau, namely aluminium and glutamate, were investigated in this study. Explant cultures of cortical neurons were established from embryonic day 17 rat fetuses. Cultures were exposed to aluminium, glutamate, aluminium/glutamate, aluminium/citric acid and citric acid (since aluminium is thought to enter cells via the transferrin receptor by complexing with citric acid) from 7-12 days in vitro. Control explants were exposed to basal medium only. On day 12, explants were paraffin-embedded. Four-six explants were serially sectioned per condition. For each explant, every 10th and adjacent 4 microm section were randomly selected and processed, with controls, for: (1) alcoholic morin histochemistry (to detect intracellular aluminium), (2) Perls' iron histochemistry (to control for the morin stain which also detects iron), (3) neurofilament immunohistochemistry (to estimate total neuronal number per explant) and (4) Alz-50 immunohistochemistry (to detect possible conformational changes in tau). The absolute number of stained/immunoreactive neurons was determined per explant. The percentage incidence was then determined per explant and averaged per condition. Explants in the aluminium conditions contained significant increases in the incidence of morin-positive aluminium containing neurons. There was also a significant increase in the incidence of Alz-50 positive neurons for the aluminium compared with control explants. These results suggest: (1) aluminium enters neurons and (2) aluminium alone induces possible conformational changes in tau as detected by the Alz-50 antibody, while aluminium combined with glutamate, or glutamate alone, do not.

Aluminum Elicits Exocellular Phosphatidylethanolamine Production in Pseudomonas fluorescens

Pseudomonas fluorescens ATCC 13525 was found to grow in a minimal mineral medium supplemented with millimolar amounts of aluminum, a known environmental toxicant. During the stationary phase of growth, the trivalent metal was localized in a phosphatidylethanolamine (PE)-containing residue. The concentration of PE in pellets ranged from 1.7 to 13.9 mg ml of culture(sup-1) in media supplemented with 1 to 30 mM aluminum. Although the gelatinous residue was observed during the stationary phase of growth, ultracentrifugation and dialysis experiments revealed that PE was produced from earlier stages of incubation and was associated with aluminum. A sharp diminution in the levels of PE and aluminum in the spent fluid was concomitant with the formation of the insoluble deposit. The aluminum content of the soluble cellular fraction increased during growth and reached an optimum of 1.85 mM of test metal at 45 h in cultures with 15 mM aluminum. Further incubation, however, led to a marked decrease in the cellular aluminum content, and during the stationary phase of growth, only trace amounts of the trivalent metal were detected in this fraction. When 45-h cells were incubated in fresh citrate medium, most of the intracellular aluminum was secreted in the spent fluid and citrate was rapidly consumed. Aluminum efflux was also observed in cultures in which d-glucose was substituted for citrate. However, no efflux of this trivalent metal was evident in media devoid of either citrate or d-glucose. Scanning electron microscopic studies and X-ray energy-dispersive analyses of the dialyzed supernatant aided in the visualization of nodule-like aluminum- and phosphorus-rich bodies associated with thread-like carbon-, oxygen-, and phosphorus-containing structures. Transmission electron microscopic and electron energy loss spectroscopic analyses revealed the presence of aluminum within bacteria after 45 h of incubation. Cells harvested after aluminum insolubilization did not shown aluminum inclusions. This aluminum-tolerant microbe may have potential application in bioremediation processes.

Comparison of the effects of elevated intracellular aluminum and calcium levels on neuronal survival and tau immunoreactivity

Both calcium and aluminum have been implicated in the cell damage and death that occurs in several neurodegenerative disorders including Alzheimer's disease (AD). We examined the effects of experimentally elevated intraneuronal levels of aluminum ([Al] i) and/or calcium ([Ca 2+] i) on neuronal degeneration and antigenic alterations in the microtubule-associated protein tau in cell cultures of rat hippocampus and human cerebral cortex. Exposure of cultures to Al 3+ alone (200 μM) for up to 6 d did not result in neuronal degeneration. Neurons exposed to the divalent cation ionophore A23187 degenerated within 4 h when Ca 2+ was present in the culture medium whether or not Al 3+ was present. Measurements of [Ca 2+ i] using the calcium indicator dye fura-2 demonstrated a direct relationship between increased [Ca 2+] i and neuronal degeneration. In contrast, neurons did not degenerate when exposed to A231887 in the presence of Al 3+ and the absence of Ca 2+, despite a 10-fold elevation in [Al] i as measured by laser microprobe mass spectrometry. Calcium influx, but not aluminum influx, elicited antigenic changes in tau similar to those seen in AD neurofibrillary tangles. Neurons exposed to glutamate in the presence of Al 3+ but in the absence of Ca 2+ were not vulnerable to injury. Finally, increased [Al] i] occured in neurons that degenerated as the result of exposure to glutamate indicating that aluminum associates with degenerating neurons. Taken together these data indicate that, in contrast to increased [Ca 2+] i, elevated [Al] i may not induce degeneration or antigenic changes in tau.

Enhanced neurite growth in cultured neuroblastoma cells exposed to aluminum

Patients with aluminum-induced encephalopathy syndromes have been shown to have a high level of aluminum concentration in the brain. In the present study, the effects of aluminum were studied in mouse neuroblastoma cells (N-2A) grown in medium supplemented with aluminum (100 μM). It was found that aluminum enhanced neurite growth within 2 days of exposure. The mean total length of neurites in the control after 14 days in culture was 29.8 ± 4.7 μm, whereas the neurite length of cells pre-exposed to aluminum for 2 days and then maintained in normal media for an additional 12 days was 56.4 ± 8.9 μm. Further, the duration of exposure did not significantly promote a greater neurite response. The neurite length of cells exposed to aluminum for 14 days (60.7 ± 9.6 μm) was not statistically different from that of cells exposed to aluminum for 2 days. Using morin stain, intracellular aluminum was detected within 24 h of exposure in the majority of aluminum-exposed cells. Intracellular aluminum did not disappear from those cells even after they were grown for 12 days in control medium. Our finding suggests that a brief exposure (2 days) to low level aluminum (100 μM) is sufficient to cause long-lasting effects on the morphology of neuroblastoma cells in culture. Such neurite behavior associated with aluminum exposure may suggest a morphological basis for the dementia seen in aluminum encephalopathy.

Extracellular Polysaccharide Is Not Responsible for Aluminum Tolerance of Rhizobium leguminosarum bv. Phaseoli CIAT899

Strain UHM-5, a pSym Exo derivative of the aluminum-tolerant Rhizobium leguminosarum bv. phaseoli strain CIAT899, was equally tolerant of aluminum (Al) as the parental culture. Dialyzed culture supernatants of the wild-type cells grown in YEM broth (10 cells ml) contained 185 mug of glucose equivalents ml whereas UHM-5 culture supernatants yielded 2 mug of glucose ml. The Exo derivative and the parental strain gave essentially similar growth in medium containing from 0 to 300 muM Al, indicating that the pSym of CIAT899, and extracellular polysaccharide, were not involved in the aluminum tolerance of this strain. However, increasing the level of Al from 80 to 150 muM increased the lag phase, induced a slight killing of the inoculum, and depressed the final populations by about fivefold. Doubling the aluminum concentration from 150 to 300 muM presented a severe aluminum stress to CIAT899 and UHM-5: the inoculum level dropped 10-fold, indicating killing of the inoculum, and remained depressed for ca. 4 days before continuing to grow slowly; the final population was decreased 15-fold relative to that of cultures grown in medium containing 80 muM Al. The production by CIAT899 of other extracellular or intracellular aluminum tolerance factors was investigated in culture by using aluminum-sensitive rhizobia as stress indicators. These experiments, conducted at 80 muM Al, demonstrated that CIAT899 produced neither extracellular nor intracellular products that could alleviate toxicity for the Al-sensitive indicator rhizobia.

Aluminium effects on Scenedesmus obtusiusculus with different phosphorus status. I. Mineral uptake

Synchronously growing cultures of the unicellular green alga Scenedesmus obtusiusculus were cultivated for 24 and 72 h in the presence or absence of phosphorus. Aluminium chloride (37, 74, 111, 148, 185, or 222 μmol) was added daily to 1 l cell suspension at the end of the cell division phase. As AlCl3 decreases the pH of the growth medium, controls were run in media with low pH in the absence of AlCl3. Samples for analysis of the internal (net uptake) and external (bound to cell surface) levels of Al, Mg, P, Ca, and Fe were taken every second hour during a 24 h period or once after 72 h.The investigation shows that the intracellular aluminium in Scenedesmus affects the nutrient status of the cells. A high intracellular level of Al is in consort with an enhancement of the intracellular fractions of Mg, P, Ca and Fe. The increase in net uptake of the minerals measured in the presence of Al is not due to an Al‐induced lowering of the pH, caused by Al. The concentrations of Al, Mg, P, Ca and Fe in the cells are generally lower during the dark period of the cell cycle, when the cells are dividing, than during the light period. A peak in mineral concentration of the cells could be monitored in the middle of the 24 h life cycle of the cells. The intracellular Al level is higher when the growth medium is low in P than in phosphorus‐rich medium, due to precipitation of aluminium‐phosphate both in medium and at cell surfaces. The extracellular Al and P fractions are thus higher in the presence than in the absence of P. The highest Al content monitored in the cells is about 100 nmol Al (106 cells)−1. A large fraction of Al initially taken up after addition to the medium is subsequently released from the cells during the 24 h cell cycle. The results are interpreted as Al effects on the plasma membrane, thus indirectly affecting various mechanisms for ion transport across the membrane. There are also indications that a surface covered with aluminium‐phosphate, formed at high P level in the medium, may prevent ion uptake.

Aluminum Uptake by Neuroblastoma Cells

Aluminum uptake studies in viable neuroblastoma cells were performed. Aluminum uptake was largely dependent on the pH of the suspension medium. At physiological pH values, cells were apparently unable to incorporate detectable amounts of aluminum in the absence of proper mediators. Aluminum uptake was enhanced as the pH decreased, attaining a plateau at about pH 6.0. In experiments with 2 x 10(6) cells/ml, pH 6.0, and 25 microM aluminum in the medium, aluminum incorporation reached saturation at 5 nmol of aluminum/mg of cellular protein, accounting for 60-70% of aluminum added. At pH 6.0, cells showed a large capacity for accumulating aluminum; about 70% of intracellular aluminum was associated with the postmitochondrial fraction. At neutral pH, application of apotransferrin seemed to facilitate aluminum translocation into cells via membrane receptors. Fatty acids were also capable of mediating aluminum uptake at neutral pH, probably by forming aluminum-fatty acid complexes. Low molecular weight aluminum chelators, e.g., citrate, inhibited aluminum uptake. Treatment of cells with energy metabolism blockers had virtually no influence on aluminum uptake, indicative of passive mechanisms. The results suggest that aluminum uptake occurs via different modes dependent on growth conditions, such as medium pH.