Removal of hexavalent chromium from groundwater using sodium alginate dispersed nano zero-valent iron.

Hexavalent chromium (Cr), one of the most common heavy metals, is widely found in contaminated soil and groundwater. Nano zero-valent iron (nZVI) is used to treat Cr(VI) in polluted groundwater. However, due to agglomeration, rapid sedimentation, and limited mobility of nanoparticles in the aquatic environment, nZVI is not widely used in groundwater treatment. In this study, we used sodium alginate (SA) to modify nZVI to generate dispersed SA-nZVI. SA-nZVI particles were found to embed in the polymer material and exist as an amorphous state with a diameter less than 100 nm. Compared with traditional nZVI and carboxymethyl cellulose (CMC)-nZVI, SA-nZVI had better stability and higher absolute zeta potential. The presence of SA enhanced mobility of nZVI and effectively prevented sedimentation and aggregation. Furthermore, SA-nZVI had a higher Cr(VI) removal rate than (CMC)-nZVI under both acidic and alkaline conditions. XPS analysis showed that Cr(VI) was reduced to Cr(III) and formed Cr(OH)3 as precipitates after treatment with SA-nZVI. In addition, NO3- had no effect on the final removal rate of Cr(VI) by SA-nZVI. These results suggest that SA-nZVI has high penetration and a high removal rate in Cr(VI) removal and can be used to stabilize nZVI to remediate Cr(VI)-contaminated groundwater in the future.

Serum deprivation can suppress receptor-mediated calcium signaling in pterygial-derived fibroblasts.

PURPOSE: Pterygium is characterized as invasive, proliferative fibrovascular altered conjunctival tissue. The extensive vascular network is likely to significantly contribute to the progression of the disease. In the present study, we investigated the effects of reduced serum (to mimic a suppressed blood supply) on cell signaling events and the functional role of the calcium store in cultured, pterygial-derived fibroblasts. METHODS: Pure fibroblast cultures were established from cell outgrowths of pterygial tissue. Growth and migration of pterygial-derived fibroblast was evaluated using a patch growth assay, MTS assay, and a scratch wound assay. Intracellular calcium levels were determined using Fura-2 detection in response to ligand stimulation using a 96-well plate format. RESULTS: A progressive increase in serum concentration resulted in promotion of pterygial cell growth detected using the MTS assay. A significant increase in intracellular calcium level was observed in response to histamine (10 and 100 µM), ATP (10 and 100 µM), acetylcholine (10 and 100 µM) and epidermal growth factor (10 ng/mL) in serum-maintained cells. However, no significant changes were observed when cells were maintained in serum-free medium. Thapsigargin (1 µM), a Ca-ATPase inhibitor, induced a significantly greater increase in intracellular calcium level in the serum-maintained group relative to serum-starved cells. In both cases, elevation of intracellular calcium was reduced when calcium-free bathing medium was used. Preincubation of cells with 1 µM thapsigargin ablated ligand-induced calcium responses. Disruption of calcium signaling through thapsigargin treatment significantly perturbed cell growth and migration. CONCLUSIONS: Receptor-induced calcium signaling activity is suppressed in pterygial-derived fibroblasts in response to serum deprivation. This correlates with reduced growth rates and a depleted endoplasmic reticulum calcium store. The store plays a key role in cell growth and migration of pterygial-derived fibroblasts. Therefore, the strategic reduction of the vascular network in pterygium will affect the calcium store level and in turn affect functional responses associated with pterygia.

Human DNA2 is a mitochondrial nuclease/helicase for efficient processing of DNA replication and repair intermediates.

DNA2, a helicase/nuclease family member, plays versatile roles in processing DNA intermediates during DNA replication and repair. Yeast Dna2 (yDna2) is essential in RNA primer removal during nuclear DNA replication and is important in repairing UV damage, base damage, and double-strand breaks. Our data demonstrate that, surprisingly, human DNA2 (hDNA2) does not localize to nuclei, as it lacks a nuclear localization signal equivalent to that present in yDna2. Instead, hDNA2 migrates to the mitochondria, interacts with mitochondrial DNA polymerase gamma, and significantly stimulates polymerase activity. We further demonstrate that hDNA2 and flap endonuclease 1 synergistically process intermediate 5' flap structures occurring in DNA replication and long-patch base excision repair (LP-BER) in mitochondria. Depletion of hDNA2 from a mitochondrial extract reduces its efficiency in RNA primer removal and LP-BER. Taken together, our studies illustrate an evolutionarily diversified role of hDNA2 in mitochondrial DNA replication and repair in a mammalian system.

Removal of oxidative DNA damage via FEN1-dependent long-patch base excision repair in human cell mitochondria.

Repair of oxidative DNA damage in mitochondria was thought limited to short-patch base excision repair (SP-BER) replacing a single nucleotide. However, certain oxidative lesions cannot be processed by SP-BER. Here we report that 2-deoxyribonolactone (dL), a major type of oxidized abasic site, inhibits replication by mitochondrial DNA (mtDNA) polymerase gamma and interferes with SP-BER by covalently trapping polymerase gamma during attempted dL excision. However, repair of dL was detected in human mitochondrial extracts, and we show that this repair is via long-patch BER (LP-BER) dependent on flap endonuclease 1 (FEN1), not previously known to be present in mitochondria. FEN1 was retained in protease-treated mitochondria and detected in mitochondrial nucleoids that contain known mitochondrial replication and transcription proteins. Results of immunofluorescence and subcellular fractionation studies were also consistent with the presence of FEN1 in the mitochondria of intact cells. Immunodepletion experiments showed that the LP-BER activity of mitochondrial extracts was strongly diminished in parallel with the removal of FEN1, although some activity remained, suggesting the presence of an additional flap-removing enzyme. Biological evidence for a FEN1 role in repairing mitochondrial oxidative DNA damage was provided by RNA interference experiments, with the extent of damage greater and the recovery slower in FEN1-depleted cells than in control cells. The mitochondrial LP-BER pathway likely plays important roles in repairing dL lesions and other oxidative lesions and perhaps in normal mtDNA replication.

Nucleolar localization and dynamic roles of flap endonuclease 1 in ribosomal DNA replication and damage repair.

Despite the wealth of information available on the biochemical functions and our recent findings of its roles in genome stability and cancer avoidance of the structure-specific flap endonuclease 1 (FEN1), its cellular compartmentalization and dynamics corresponding to its involvement in various DNA metabolic pathways are not yet elucidated. Several years ago, we demonstrated that FEN1 migrates into the nucleus in response to DNA damage and under certain cell cycle conditions. In the current paper, we found that FEN1 is superaccumulated in the nucleolus and plays a role in the resolution of stalled DNA replication forks formed at the sites of natural replication fork barriers. In response to UV irradiation and upon phosphorylation, FEN1 migrates to nuclear plasma to participate in the resolution of UV cross-links on DNA, most likely employing its concerted action of exonuclease and gap-dependent endonuclease activities. Based on yeast complementation experiments, the mutation of Ser(187)Asp, mimicking constant phosphorylation, excludes FEN1 from nucleolar accumulation. The replacement of Ser(187) by Ala, eliminating the only phosphorylation site, retains FEN1 in nucleoli. Both of the mutations cause UV sensitivity, impair cellular UV damage repair capacity, and decline overall cellular survivorship.

Altered traffic to the lysosome in an ex vivo lacrimal acinar cell model for chronic muscarinic receptor stimulation.

Evidence suggests that lacrimal and salivary epithelial cells constitutively expose potentially pathogenic autoantigens, but that active regulatory networks normally suppress pathological autoimmune responses . Events that potentially disrupt the regulatory networks include increased exposure of constitutive autoantigens and induced exposure of previously cryptic autoantigen epitopes. Chronic muscarinic receptor (MAChR) stimulation in an ex vivo rabbit lacrimal acinar cell model induces functional and biochemical alterations reminiscent of the functional quiescence associated with Sjogren's syndrome . Chronic MAChR stimulation also elicits changes in the compartmental distribution of beta-hexosaminidase, a product that normally is dually targeted into the lysosomal pathway and the regulated apical secretory pathway. Here, we use subcellular fractionation analyses to further explore the nature of the stimulation-induced traffic changes and to identify effectors that might mediate this change. Overnight stimulation of primary cultured rabbit lacrimal gland acinar cells with 10 microM carbachol (CCh) significantly decreased the abundance of mature cathepsin B in the pre-lysosome and lysosome; decreased the abundance of preprocathepsin B in fractions containing the TGN and late endosome; increased the abundance of procathepsin B in fractions containing the basal-lateral membrane; and increased the accumulation of endocytosed [(125)I]-EGF in the recycling endosome. Alterations in distribution or abundance of traffic effectors included: increased abundances of rab5A and rab6 in the TGN; decreased overall abundance of gamma-adaptin; remarkably increased relative abundance of membrane phase-associated actin; redistribution of cytoplasmic dynein from biosynthetic and proximal endocytic compartments to the lysosome; and redistribution of p150(Glued) from the lysosome to biosynthetic or proximal endocytic compartments. We conclude that chronic MAChR stimulation blocks traffic from the early endosome and the TGN to the lysosome, causing lysosomal proteins to reflux to the TGN, endosomes, and basal-lateral membrane. These traffic alterations may be mediated through action on one or more of the effectors noted.

Role of the microtubule cytoskeleton in traffic of EGF through the lacrimal acinar cell endomembrane network.

We have previously documented a novel biphasic traffic pattern for epidermal growth factor (EGF) in the acinar epithelial cell of the lacrimal gland. Different from the typical paradigm observed in many other cell types, EGF initially accumulates in the acinar basal-lateral recycling endosome, then is re-directed to the prelysosomes and lysosomes and degraded. While the cellular content of intact EGF decreases by 40% between 20 and 120 m of continuous incubation at 37 degrees C, the EGF receptor (EGFR) content decreases only modestly [J. Cell Physiol. 199 (2004) 108]. The purpose of the present study was to investigate the role of the microtubule cytoskeleton in this traffic. Primary cultured rabbit lacrimocytes were incubated with [(125)I]-EGF, lysed, and analyzed by subcellular fractionation on sorbitol density gradients. Nocodazole treatment appeared to slightly decrease the initial uptake rate but to have no significant effect on the total amount of [(125)I] accumulation. However, it enhanced accumulation of [(125)I]-EGF and EGFR in the basal-lateral recycling endosome, and it enhanced accumulation of prepro- and pro- cathepsin B in fractions containing late endosomes and prelysosomes. Nocodazole permitted the time-dependent release of [(125)I]-EGF from the recycling endosome, but it partially inhibited [(125)I]-EGF degradation and decreased accumulation of [(125)I]-labeled degradation products in the lysosome. The microtubule-based molecular motors, cytoplasmic dynein and kinesin, were localized in compartments containing the late endosomes, prelysosomes, and lysosomes, consistent with the suggestion that microtubule-based molecular motors play important roles in traffic within the lysosomal pathway. Confocal fluorescence microscopy imaging of FITC-EGF substantiated the effects observed in biochemical studies by demonstrating that nocodazole increased accumulation in a peripheral compartment and decreased traffic to a perinuclear compartment. These data suggest that initial accumulation in the basal-lateral recycling endosome and subsequent release from the recycling endosome to the late endosomes and prelysosome are not microtubule-dependent. On the other hand, microtubule-based motors are more critical for traffic from the prelysosome to the lysosome.

Novel biphasic traffic of endocytosed EGF to recycling and degradative compartments in lacrimal gland acinar cells.

The purpose of this study was to delineate the traffic patterns of EGF and EGF receptors (EGFR) in primary cultured acinar epithelial cells from rabbit lacrimal glands. Uptake of [(125)I]-EGF exhibited saturable and non-saturable, temperature-dependent components, suggesting both receptor-mediated and fluid phase endocytosis. Accumulation of [(125)I] was time-dependent over a 120-min period, but the content of intact [(125)I]-EGF decreased after reaching a maximum at 20 min. Analytical fractionation by sorbitol density gradient centrifugation and phase partitioning indicated that within 20 min at 37 degrees C [(125)I] reached an early endosome, basal-lateral recycling endosome, pre-lysosome, and lysosome. Small components of the label also appeared to reach the Golgi complex and trans-Golgi network. Intact [(125)I]-EGF initially accumulated in the recycling endosome; the content in the recycling endosome subsequently decreased, and by 120 min increased amounts of [(125)I]-labeled degradation products appeared in the pre-lysosomes and lysosomes. Confocal microscopy imaging of FITC-EGF and LysoTrackerRed revealed FITC enriched in a dispersed system of non-acidic compartments at 20 min and in acidic compartments at 120 min. Both confocal immunofluorescence microscopy and analytical fractionation indicated that the intracellular EGFR pool was much larger than the plasma membrane-expressed pool at all times. Cells loaded with [(125)I]-EGF released a mixture of intact EGF and [(125)I]-labeled degradation products. The observations indicate that in lacrimal acinar cells, EGFR and EGF-EGFR complexes continually traffic between the plasma membranes and a system of endomembrane compartments; EGF-stimulation generates time-dependent signals that initially decrease, then increase, EGF-EGFR traffic to degradative compartments.

Cytoplasmic dynein participates in apically targeted stimulated secretory traffic in primary rabbit lacrimal acinar epithelial cells.

A major function of the acinar cells of the lacrimal gland is the production and stimulated release of tear proteins into ocular surface fluid. We investigate the participation of cytoplasmic dynein in carbachol-stimulated traffic to the apical plasma membrane in primary rabbit lacrimal acinar epithelial cells. Confocal fluorescence microscopy revealed a major carbachol-induced, microtubule-dependent recruitment of cytoplasmic dynein and the dynactin complex into the subapical region. Colocalization studies, sorbitol density gradient/phase partitioning analysis and microtubule-affinity purification of membranes showed that some dynein and dynactin complex were associated with VAMP2-enriched membranes. Adenovirus-mediated overexpression of p50/dynamitin inhibited the recruitment and colocalization of dynein, the dynactin complex and VAMP2 in the subapical region. Nocodazole treatment and p50/dynamitin overexpression also depleted subapical stores of rab3D in resting acini, suggesting that dynein activity was also involved in maintenance of rab3D-enriched secretory vesicles. These data implicate cytoplasmic dynein in stimulated traffic to the apical plasma membrane in these secretory epithelial cells.

Diverse perturbations may alter the lacrimal acinar cell autoantigenic spectra.

Lacrimal gland acinar cell autoantigens in Sjögren's syndrome include both intracellular proteins and plasma membrane proteins, to which the immune system normally must be tolerant. Attention has largely focused on the roles apoptotic cell death may play in exposing sequestered autoantigens and novel surface epitopes. We hypothesize that perturbations of ongoing membrane traffic in intact, functioning cells may also increase autoantigen exposure. We review the vesicular traffic between acinar cell basal-lateral plasma membranes (blm) and endomembrane compartments, then describe experiments in which isolated acinar cells were stimulated with epidermal growth factor (EGF), lysed, and analyzed by sorbitol gradient centrifugation. Whereas the cholinergic agonist, carbachol, impairs traffic from the trans-Golgi network to prelysosomes, causing Golgi, secretory, and lysosomal proteins to reflux into domains of the trans-Golgi network that communicate with the blm and to accumulate in the blm, EGF specifically causes a 2.6-fold (P < 0.05) increase in the beta-hexosaminidase content of the blm fraction, apparently by impairing traffic from early endosomes to prelysosome. We, therefore, suggest that a variety of physiologic stimuli may alter the spectra of autoantigens acinar cells secrete to the interstitium, express in their blm, and present via MHC Class II molecules after proteolytic processing.

Heterotrimeric GTP-binding proteins in the lacrimal acinar cell endomembrane system.

Secretagogues accelerate traffic in the lysosomal and basal-lateral pathways, as well as in the regulated apical secretory pathway, of lacrimal acinar cells. It has been proposed that alterations of protein segregation in compartments where these traffic pathways intersect may influence autoimmune responses. Heterotrimeric GTP-binding proteins couple secretagogue receptor ligand binding to activation of intracellular signaling cascades, but they are also suggested to participate in endomembrane traffic phenomena. Distributions of G(o), G(i3), G(q), G(11), and two G(s)isoforms were mapped in reconstituted lacrimal acini by confocal immunofluorescence microscopy and in lysates of the reconstituted acini by analytical subcellular fractionation. All G proteins examined were detected at low levels in isolated compartments (blm(i,j)) believed to represent the basal-lateral plasma membrane. G(i3), G(11), and the G(s)isoforms were concentrated in a series of isolated compartments believed to be related to domains of a basal-lateral endosome with sorting and recycling functions (ble-s/r(i,j,k)), a distinct endosomal compartment with basal-lateral membrane-like composition (e-blml), and domains of the trans-Golgi network believed to be involved in traffic to and from the basal-lateral membrane (tgn-blmr). G(o)and G(q)were concentrated in compartments believed to represent a mixture of immature and mature secretory vesicle membranes (isvm and svm) and domains of the trans-Golgi network compartment believed to mediate traffic to secretory vesicles (tgn-svr) and to pre-lysosomes (tgn-lr). Confocal fluorescence microscopy confirmed the presence of both basal-lateral membrane and intracellular pools of the G proteins. Stimulation with 10 microM carbachol for 20min caused a component of the G(o)to redistribute away from the isvm+svm; components of the G(i3), G(q), and G(s)to redistribute away from the tgn-svr+tgn-lr; and a component of the G(i3)to redistribute away from the ble-blml+tgn-blmr. Thus, these proteins may participate in endomembrane traffic steps activated by cholinergic stimulation in addition to playing their classical roles in plasma membrane signal transduction.