Assessment of bioavailability of heavy metal pollutants using soil isolates of Chlorella sp.

Biotests conducted with plants are presently used to estimate metal bioavailability in contaminated soils. But when plants are grown in soils, especially the plants with fine roots, root collection is easily biased and tedious. Indeed, at harvest, small amounts of soil can adhere to roots, resulting in overestimation of root metal content, and the finest roots are often discarded from the analysis because of their difficult and almost impossible recovery. This report presents a novel method for assessing the bioavailability of heavy metals in soils using microalgae. Two species of green unicellular microalgae were isolated from two highly contaminated soils and identified by phylogenetic and molecular evolutionary analyses as Chlorella sp. RBM and Chlorella sp. RHM. These two cultures were used to determine the metal uptake from metal-contaminated soils of South Australia as a novel, cost-effective, simple and rapid method for assessing the bioavailability of heavy metals in soils. The suggested method is an attempt to achieve a realistic estimate of bioavailability which overcomes the inherent drawback of root metal contamination in the bioavailability indices so far reported.

Manganese(II)-catalyzed and clay-minerals-mediated reduction of chromium(VI) by citrate.

Unlike lower valent iron (Fe), the potential role of lower valent manganese (Mn) in the reduction of hexavalent chromium (Cr(VI)) in soil is poorly documented. In this study, we report that citrate along with Mn(II) and clay minerals (montmorillonite and kaolinite) reduce Cr(VI) both in aqueous phase and in the presence of dissolved organic carbon (SDOC) extracted from a forest soil. The reduction was favorable at acidic pH (up to pH 5) and followed the pseudo-first-order kinetic model. The citrate (10 mM) + Mn(II) (182.02 µM) + clay minerals (3% w/v) system in SDOC accounted for complete reduction of Cr(VI) (192.32 µM) in about 72 h at pH 4.9. In this system, citrate was the reductant, Mn(II) was a catalyst, and the clay minerals acted as an accelerator for both the reductant and catalyst. The clay minerals also serve as a sink for Cr(III). This study reveals the underlying mechanism of the Mn(II)-induced reduction of Cr(VI) by organic ligand in the presence of clay minerals under certain environmental conditions.

Remediation of hexavalent chromium through adsorption by bentonite based Arquad® 2HT-75 organoclays.

Unlike hydrophobic organic pollutants, the potential of organoclays to adsorb inorganic ionic contaminants is relatively underexplored. The present study attempts to characterise bentonite (QB) based organoclays synthesised from a commercially available, low-cost alkyl ammonium surfactant Arquad® 2HT-75 (Aq) and test their ability to adsorb hexavalent chromium (Cr (VI)) in aqueous solution. XRD, FTIR and TGA characterisation techniques prove successful modification of the bentonite structure and reveal that higher surfactant loadings gives rise to more ordered surfactant conformation in the organoclays. The zeta potential values indicate that higher surfactant loadings also create positive charges on the organoclay surfaces. Detailed isothermal and kinetic studies show that the organoclays effectively remove hexavalent chromium (Cr (VI)) from aqueous solution by both physical and chemical adsorption processes. Higher surfactant loadings provide better adsorption efficiency. The adsorption performance is reasonably efficient under the levels of pH, temperature, electrolyte concentration and natural organic matter concentration that generally prevail in contaminated soil and water. This study shows that organoclay sorbents offer good potential for remediating Cr (VI) under real environmental conditions.

Synthesis and characterisation of novel organopalygorskites for removal of p-nitrophenol from aqueous solution: isothermal studies.

Organopalygorskites were synthesised by using dimethyldioctadecylammonium bromide (DMDOA) and cetylpyridinium chloride (CP) with surfactant loadings equivalent to 100% and 200% CEC of the palygorskite. The four organopalygorskites, thus produced, were characterised by Fourier Transform Infrared Spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and zeta potential measurement. FTIR and TGA data demonstrated that higher surfactant loadings as well as long branched chain DMDOA produced highly ordered surfactant conformation. SEM morphological results showed that the organopalygorskites had less entangled fibres than the unmodified palygorskite. The zeta potential values showed positive charge formation on the organopalygorskites surface when they were synthesised with surfactant loadings equivalent to 200% CEC of the palygorskite. The organopalygorskites were tested for adsorption of p-nitrophenol (PNP) with a special focus on the adsorption isotherms. The adsorption data could be fitted with multiple isothermal models indicating that the adsorption was controlled by multiple mechanisms. Sorbent loading rate, initial pH, temperature and ionic strength might all affect the adsorption process. Also, DMDOA modified organopalygorskites reduced desorption/redispersal of adsorbed PNP back into the environment to a great extent. This study will be helpful in designing palygorskite-based organoclay adsorbents for remediating organic environmental contaminants which are ionic in nature.

Bioavailability of cadmium-organic complexes to soil alga--an exception to the free ion model.

It is generally considered that cadmium bioavailability shows a considerable dependence on chemical speciation of Cd in solution, correlates best with the activity of free metal ion (Cd2+) in solution, and is largely indifferent to soluble metal complexes. The role of soluble organic matter (DOM) and soluble metal-organic complexes in metal bioavailability and toxicity, however, is not clear. Growth studies with a soil alga (Chlorococcum sp.) were conducted on a growth medium and pore water of Cookes Plain soil (Paleuxeralf), spiked with Cd as Cd(NO3)2. Speciation of the Cd in pore water, and in growth medium with and without citrate, was performed using the MINTEQA2 computer model incorporating updated values of the stability constants of Cd-DOM complexes, as well as using anode stripping voltammetry. Analysis of the toxicity data showed that Cd-citrate, as well as the Cd-DOM complexes, is bioavailable and contributes toward the toxicity to alga. These data contradict the long-held notion that Cd-DOM complexes are not bioavailable to soil biota although they may increase the mobility of Cd.

Solid-solution speciation and phytoavailability of copper and zinc in soils.

The soil solution speciation and solid-phase fractionation of copper (Cu) and zinc (Zn) in 11 typical uncontaminated soils of South Australia were assessed in relation to heavy metal phytoavailability. The soils were analyzed for pH (4.9-8.4), soil organic matter content (3.5 to 23.8 g of C kg(-1)), total soil solution metal concentrations, Cu8 (49-358 microg kg(-1)) and Zn8 (121-582 microg kg(-1)), and dissolved organic matter (DOM) (69-827 mg of C L(-1)). The solid-liquid partition coefficient (Kd) ranged from between 13.9 and 152.4 L kg(-1) for Cu and 22.6 to 266.3 L kg(-1) for Zn. The phytoavailability of Cu and Zn could be predicted significantly using an empirical model with the solid-phase fractions of Cu and Zn, as obtained from selective sequential extraction scheme, as components. Phytoavailable Cu and Zn were found to significantly correlate with fulvic complex Cu (r= 0.944, P < 0.0001) and exchangeable Zn (r = 0.832, P = 0.002), respectively. The fulvic complex Cu was found to explain 89.2% of the variation in phytoavailable Cu, where as, the exchangeable Zn together with fulvic complex Zn could explain 78.9% of the variation in phytoavailable Zn. The data presented demonstrate the role of solid-phase metal fractions in understanding the heavy metal phytoavailability. The assessment of the role of solid-phase fractions in heavy metal phytoavailability is a neglected area of study and deserves close attention.

An in vitro rapid-turnover assay for human immunodeficiency virus type 1 replication selects for cell-to-cell spread of virus.

We have developed a rapid-turnover culture system where the life span of a human immunodeficiency virus type 1-infected cell is controlled by periodic addition of a cytotoxic agent, mitomycin C. These mitomycin C-exposed cells are cocultured with a constant number of uninfected cells as new targets for the virus. Passage of the virus-infected cells under these conditions led to the emergence of a viral variant that was able to replicate efficiently in this culture system. After biologic and molecular cloning, we were able to identify a single frameshift mutation in the vpu open reading frame that was sufficient for growth of the mutant virus in the rapid-turnover assay. This virus variant spread more efficiently by cell-to-cell transfer than the parental virus did. Electron micrographs of cells infected with the delta vpu virus revealed a large number of mature viral capsids attached to the plasma membrane. The presence of these mature virus particles on the cell surface led to enhanced fusion and formation of giant syncytia with uninfected cells. Enhanced cell-to-cell transfer of the delta vpu virus provides an explanation for the survival of this mutant virus in the rapid-turnover culture system. The in vitro rapid-turnover culture system is a good representation of the in vivo turnover kinetics of infected cells and their continual replacement by host lymphopoietic mechanisms.

Cell cycle- and Vpr-mediated regulation of human immunodeficiency virus type 1 expression in primary and transformed T-cell lines.

Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) transiently arrests cells in the G2 phase of the cell cycle and is a weak transcriptional transactivator. We found that Vpr increased HIV-1 long terminal repeat (LTR) activity in all cells examined but, when expressed at high levels, decreased HIV-1 LTR expression due to cytotoxic effects. Moreover, Vpr-mediated enhancement of HIV-1 LTR-driven transcription was observed in cycling primary human CD4(+) T cells but not in terminally differentiated, noncycling primary human macrophages. In single-round infection experiments using primary human CD4(+) T cells, proviral clones expressing either wild-type Vpr or Vpr mutants that retained the ability to cause a G2 arrest replicated to higher levels than proviruses lacking Vpr or expressing mutants of Vpr that did not cause an arrest. In support of the hypothesis that enhancement of HIV-1 LTR transcription by Vpr is an indirect effect of the ability of Vpr to delay cells in G2, counterflow centrifugal elutriation of cells into different phases of the cell cycle demonstrated that HIV-1 LTR expression was highest in G2. Finally, the ability of Vpr to upregulate viral transcription was dependent on a minimal promoter containing a functional TATA box and an enhancer.

Costimulatory pathways in lymphocyte proliferation induced by the simian immunodeficiency virus SIVsmmPBj14.

The PBj14 isolate of the simian immunodeficiency virus SIVsmmPBj14 is unique among primate lentiviruses in its ability to induce lymphocyte proliferation and acutely lethal disease. The studies reported here show that viral induction of T-cell proliferation requires accessory cells, such as primary monocytes or Raji B-lymphoma cells, as well as the presence of a putative immunoreceptor tyrosine-based activation motif within the viral Nef protein. Addition of CTLA4-immunoglobulin fusion protein or anti-B7 antibodies to virally infected T cells led to substantial, but not complete, inhibition of monocyte-costimulated T-cell proliferation-suggesting that both CD28/B7-dependent and non-CD28-dependent pathways may contribute to the costimulation of virally induced lymphoproliferation. Finally, cyclosporin A, a specific inhibitor of the calcium-calmodulin-regulated phosphatase activity of calcineurin, which influences activation of the transcription factor nuclear factor of activated T cells, was shown to block virally mediated T-cell proliferation. Taken together, these findings suggest that the effect of SIVsmmPBj14 on T-cell activation may be functionally analogous, at least in part, to the effect of engagement of the T-cell receptor.

Expression of pro- and anti-apoptosis gene products in brains from paediatric patients with HIV-1 encephalitis.

We have previously demonstrated the presence of DNA fragmentation in neurons, macrophages and microglia consistent with apoptosis, but not in reactive astrocytes in brain tissue from paediatric patients with HIV-1 encephalitis (HIVE). To further understand the underlying mechanism(s) for these findings as they relate to gene-directed neural cell death, we studied the in-situ expression of the Bcl-2 family of proteins, including the pro-apoptosis gene product Bax, the anti-apoptosis gene product Bcl-2, and Bcl-x. We demonstrate significantly elevated numbers of Bax-positive microglia and macrophages immunoreactive in basal ganglia and cerebral cortex of children who had HIVE, in comparison to HIV-1 infected children without encephalitis or children who were seronegative for HIV-1. In contrast, patients with HIVE, but not HIV-1 without encephalitis, or seronegative controls, had increased expression of Bcl-2 and Bcl-x in reactive astrocytes in cortex and basal ganglia. In vitro studies using Western blot analysis demonstrated an up-regulation in the levels of Bax, and phosphorylated (i.e. inactive) Bcl-2 in HIV-1 infected macrophages, and in LPS-activated macrophages, relative to levels in virus-negative unstimulated macrophages. These results suggest that productive HIV-1 infection, or cellular activation, renders macrophages more vulnerable to apoptosis. Taken together, these findings suggest that brain-resident macrophages and microglia in patients with HIV-1 encephalitis are more prone to undergo apoptosis and that astrocytes in contrast may be resistant to apoptosis. This may represent a mechanism to limit microglial activation and the spread of productive HIV-1 infection in the CNS of children with HIV-1 encephalitis.

Apoptosis correlates with immune activation in intestinal lymphoid tissue from macaques acutely infected by a highly enteropathic simian immunodeficiency virus, SIVsmmPBj14.

The PBj14 isolate of simian immunodeficiency virus, SIVsmmPBj14, induces an acutely lethal disease in experimentally inoculated pigtailed macaques, that is characterized by severe enteropathy and extensive immune activation, particularly within gut-associated lymphoid tissue (GALT). Experiments were conducted to determine whether virally induced immune activation might promote the induction of apoptosis in GALT during acute SIVsmmPBj14 infection. In situ labeling studies revealed a significant increase in the number of apoptotic cells within GALT from macaques with acute SIVsmmPBj14 infection, compared to (i) other tissues from the same animals, (ii) GALT from virus-negative animals, or (iii) GALT from macaques that were sacrificed soon after infection with SIVmac239, which does not cause acutely lethal enteropathy. These findings were confirmed by biochemical assays of DNA fragmentation, using DNA laddering and ELISA techniques. Immunostaining experiments revealed a strong positive correlation between the extent of apoptosis and the degree of immune activation, as assessed either by the number of cells which contained nuclear (activated) RelA or by the number of cells immunoreactive for CD25, a T-cell activation marker. Additional analyses of SIV antigen expression revealed that the majority of the apoptotic cells were not productively infected by SIV (i.e., that they were bystander cells). Taken together, these findings support the hypothesis that SIVsmmPBj14 efficiently induces immune activation and that this results in extensive apoptosis within gut-associated lymphoid tissue during acute viral infection.

SIVsmmPBj14 induces expression of a mucosal integrin on macaque lymphocytes.

The PBj14 isolate of simian immunodeficiency virus, SIVsmmPBj14, is an acutely pathogenic lentivirus that causes severe gastrointestinal disease in macaque monkeys. The studies reported here examine the basis for the enteropathic phenotype of SIVsmmPBj14, using flow cytometric analysis of cultured macaque lymphocytes and immunohistochemical staining of tissue specimens from virus-infected macaques. The data show that enteropathic molecular clones of SIVsmmPBj14 induce expression of the alpha E beta 7 integrin, which is believed to mediate mucosal retention of T-cells, whereas molecular clones from nonenteropathic derivatives of SIVsmmPBj14 do not do so. Thus, elevated expression of alpha E beta 7 may ber responsible, at least in part, for the accumulation of abnormally large numbers of T-cells within the intestinal mucosa during acute SIVsmmPBj14 infection.

Direct sequence analysis of human herpesvirus 6 (HHV-6) sequences from infants and comparison of HHV-6 sequences from mother/infant pairs.

Direct sequence analysis of polymerase chain reaction-amplified DNA fragments from the large tegument protein (LTP) gene of human herpesvirus 6 (HHV-6) was performed with use of uncultured peripheral blood mononuclear cells (PBMCs) from four mother/infant pairs. In two cases, LTP gene sequences were identical in paired mother/infant specimens, thus suggesting that mother-to-infant transmission of HHV-6 may have occurred. The genetic stability of HHV-6 strains was confirmed by the fact that there was no difference between amplified DNA fragments from sequential PBMC samples from two of two infants analyzed. In contrast, a change in the amplified viral strain was detected in an infant who had reinfection with HHV-6 variant B (HHV-6B). Furthermore, HHV-6B strains concurrently amplified from saliva and PBMCs from an adult were found to be different. The data suggest that HHV-6 may be frequently transmitted from mother-to-infant and that reinfection with HHV-6B may occur.

Tumor necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA.

Tumor necrosis factor alpha (TNF-alpha) is a candidate human immunodeficiency virus type 1-induced neurotoxin that contributes to the pathogenesis of AIDS dementia complex. We report here on the effects of exogenous TNF-alpha on SK-N-MC human neuroblastoma cells differentiated to a neuronal phenotype with retinoic acid, TNF-alpha caused a dose-dependent loss of viability and a corresponding increase in apoptosis in differentiated SK-N-MC cells but not in undifferentiated cultures. Importantly, intracellular signalling via TNF receptors, as measured by activation of the transcription factor NF-kappa B, was unaltered by retinoic acid treatment. Finally, overexpression of bcl-2 or crmA conferred resistance to apoptosis mediated by TNF-alpha, as did the addition of the antioxidant N-acetylcysteine. These results suggest that TNF-alpha induces apoptosis in neuronal cells by a pathway that involves formation of reactive oxygen intermediates and which can be blocked by specific genetic interventions.

Enhanced responsiveness to nuclear factor kappa B contributes to the unique phenotype of simian immunodeficiency virus variant SIVsmmPBj14.

Infection with a variant of simian immunodeficiency virus, SIVsmmPBj14, leads to severe acute disease in macaques. This study was designed to investigate the functional significance of previously described mutations in the viral long terminal repeat (LTR) and to elucidate their contribution to the unique phenotype of SIVsmmPBj14. LTR-directed transcription was measured by using luciferase reporter constructs that were transiently transfected into cultured cells. In a wide range of cell types, the basal transcriptional activity of the LTR from SIVsmmPBj14 was found to be 2- to 4.5-fold higher than that of an LTR from a non-acutely pathogenic strain. These LTRs differ by five point mutations and a 22-bp duplication in SIVsmmPBj14, which includes a nuclear factor kappa B (NF kappa B) site. Transcriptional differences between these LTRs were further enhanced by two- to threefold upon treatment of cells with phorbol ester or tumor necrosis factor alpha or by cotransfection with plasmids expressing NF kappa B subunits. Mutagenesis studies, and the use of a reporter construct containing an enhancerless promoter, indicate that these transcriptional effects are due principally to the 22-bp sequence duplication and the NF kappa B site contained within it. Finally, infectious virus stocks that were isogenic except for the LTR were generated. The LTR from SIVsmmPBj14 was found to confer an increase in the kinetics of virus replication in cultured cells. Inclusion of this LTR in recombinant SIVs also resulted in a two- to threefold rise in the extent of cellular proliferation that was induced in quiescent simian peripheral blood mononuclear cells. These studies are consistent with the hypothesis that LTR mutations assist SIVsmmPBj14 in responding efficiently to cellular stimulation and allow it to replicate to high titers during the acute phase of viral infection.