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Dimethyl fumarate (DMF), an antioxidant/anti-inflammatory drug approved for the treatment of multiple sclerosis, induces antioxidant enzymes, in part through transcriptional upregulation. We hypothesized that DMF administration to simian immunodeficiency virus (SIV)-infected rhesus macaques would induce antioxidant enzyme expression and reduce oxidative injury and inflammation throughout the brain. Nine SIV-infected, CD8+-T-lymphocyte-depleted rhesus macaques were studied. Five received oral DMF prior to the SIV infection and through to the necropsy day. Protein expression was analyzed in 11 brain regions, as well as the thymus, liver, and spleen, using Western blot and immunohistochemistry for antioxidant, inflammatory, and neuronal proteins. Additionally, oxidative stress was determined in brain sections using immunohistochemistry (8-OHdG, 3NT) and optical redox imaging of oxidized flavoproteins containing flavin adenine dinucleotide (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The DMF treatment was associated with no changes in virus replication, higher expressions of the antioxidant enzymes NQO1, GPX1, and HO-1 in the brain and PRDX1 and HO-2 in the spleen, lower levels of 8-OHdG and 3NT, a lower optical redox ratio. The DMF treatment was also associated with increased expressions of cell-adhesion molecules (VCAM-1, ICAM-1) and no changes in HLA-DR, CD68, GFAP, NFL, or synaptic proteins. The concordantly increased brain antioxidant enzyme expressions and reduced oxidative stress in DMF-treated SIV-infected macaques suggest that DMF could limit oxidative stress throughout the brain through effective induction of the endogenous antioxidant response. We propose that DMF could potentially induce neuroprotective brain responses in persons living with HIV.

Vertical HIV-1 transmission via breastfeeding is the predominant contributor to pediatric infections that are ongoing in this era of highly effective antiretroviral therapy (ART). Remarkably, only ~10% of infants chronically exposed to the virus via breastfeeding from untreated HIV-infected mothers become infected, suggesting the presence of naturally protective factors in breast milk. HIV-specific maternal antibodies are obvious candidates as potential contributors to this protection. This study assessed the protective capacity of common HIV envelope-specific non-broadly neutralizing antibodies isolated from breast milk of HIV-infected women in an infant rhesus monkey (RM), tier 2 SHIV oral challenge model. Prior to oral SHIV challenge, infant RMs were i.v. infused with either a single weakly-neutralizing monoclonal antibody (mAb), a tri-mAb cocktail with neutralizing and ADCC functionalities, or an anti-influenza HA control mAb. Of these groups, the fewest tri-mAb-treated infants developed plasma viremia (2/6, 3/6, and 6/8 animals viremic in tri-mAb, single-mAb, and control mAb groups, respectively). Tri-mAb-treated infants demonstrated significantly fewer transmitted/founder SHIV variants in plasma and decreased peripheral CD4+ T cell proviral loads at 8 week post-challenge compared to control mAb-treated infants. Abortive infection was observed as detectable CD4+ T cell provirus in non-viremic control mAb- and single-mAb-, but not tri-mAb-treated animals. Taken together, these results support the potential viability of maternal or infant vaccine strategies that elicit non-broadly neutralizing antibodies to prevent vertical transmission of HIV through breastfeeding.

Feline leukemia virus (FeLV) is a naturally transmitted gammaretrovirus that infects domestic cats. FeLV-945, the predominant isolate associated with non-T-cell disease in a natural cohort, is a member of FeLV subgroup A but differs in sequence from the FeLV-A prototype, FeLV-A/61E, in the surface glycoprotein (SU) and long terminal repeat (LTR). Substitution of the FeLV-945 LTR into FeLV-A/61E resulted in pathogenesis indistinguishable from that of FeLV-A/61E, namely, thymic lymphoma of T-cell origin. In contrast, substitution of both FeLV-945 LTR and SU into FeLV-A/61E resulted in multicentric lymphoma of non-T-cell origin. These results implicated the FeLV-945 SU as a determinant of pathogenic spectrum. The present study was undertaken to test the hypothesis that FeLV-945 SU can act in the absence of other unique sequence elements of FeLV-945 to determine the disease spectrum. Substitution of FeLV-A/61E SU with that of FeLV-945 altered the clinical presentation and resulted in tumors that demonstrated expression of CD45R in the presence or absence of CD3. Despite the evident expression of CD45R, a typical B-cell marker, T-cell receptor beta (TCRβ) gene rearrangement indicated a T-cell origin. Tumor cells were detectable in bone marrow and blood at earlier times during the disease process, and the predominant SU genes from proviruses integrated in tumor DNA carried markers of genetic recombination. The findings demonstrate that FeLV-945 SU alters pathogenesis, although incompletely, in the absence of FeLV-945 LTR. Evidence demonstrates that FeLV-945 SU and LTR are required together to fully recapitulate the distinctive non-T-cell disease outcome seen in the natural cohort.

Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxØ trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed ΔGY, leads to a striking perturbation in pathogenesis in rhesus macaques ( Macaca mulatta ). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4 + T cells and an absence of microbial translocation. Here we evaluated ΔGY in pig-tailed macaques ( Macaca nemestrina ), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques, ΔGY also replicated acutely to high peak plasma RNA levels identical to those for SIVmac239 and caused only transient infection of CD4 + T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled ΔGY replication with plasma viral loads of + T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust antiviral CD4 + T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8 + cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by ΔGY's ablation of the GYxxØ trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of simian immunodeficiency virus (SIV) infection. IMPORTANCE The pathogenesis of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) reflects a balance between viral replication, host innate and adaptive antiviral immune responses, and sustained immune activation that in humans and Asian macaques is associated with persistent viremia, immune escape, and AIDS. Among nonhuman primates, pig-tailed macaques following SIV infection are predisposed to more rapid disease progression than are rhesus macaques. Here, we show that disruption of a conserved tyrosine-based cellular trafficking motif in the viral transmembrane envelope glycoprotein cytoplasmic tail leads in pig-tailed macaques to a unique phenotype in which high levels of acute viral replication are followed by elite control, robust cellular responses in mucosal tissues, and no disease. Paradoxically, control of this virus in rhesus macaques is only partial, and progression to AIDS occurs. This novel model should provide a powerful tool to help identify host-specific determinants for viral control with potential relevance for vaccine development.

Cellular Al accumulation has been shown to alter iron metabolism and induce peroxidative injury. Therefore antioxidants could potentially reduce or prevent peroxidative injury in Al-loaded cells. To test this hypothesis we assessed the effect of the antioxidants N -acetyl cysteine (NAC), catalase, superoxide dismutase (SOD), and tetramethylpiperidine 1-oxyl (TEMPO) in abrogating Al-associated cell toxicity and melonyldialdehyde (MDA) accumulation in mouse hepatocytes. Mouse hepatocytes (MH) were grown in media containing the minimum toxic concentration of Al (100 μg/L as Al-transferrin). All antioxidants protected MH from injury as assessed by cell growth and enzyme leakage into media. The antioxidants did not affect Al uptake by MH, protect MH from lipid peroxidation or decrease the reactive iron content of MH. Although antioxidants protected Al loaded MH from injury the mechanisms of this effect are unknown.

The chemokine SDF-1 alpha (CXC12) and its receptor CXCR4 have been shown to play a role in the development of normal cerebellar cytoarchitecture. We report here that SDF-1 alpha both induces chemotactic responses in granule precursor cells and enhances granule cell proliferative responses to Sonic hedgehog. Chemotactic and proliferative responses to SDF-1 alpha are greater in granule cells obtained from cerebella of animals in the first postnatal week, coinciding with the observed in vivo peak in cerebellar CXCR4 expression. SDF-1 alpha activation of neuronal CXCR4 differs from activation of CXCR4 in leukocytes in that SDF-1 alpha-induced calcium flux is activity dependent, requiring predepolarization with KCl or pretreatment with glutamate. However, as is the case in leukocytes, neuronal responses to SDF-1 alpha are all abolished by pretreatment of granule cells with pertussis toxin, suggesting they occur through G(alpha i) activation. In conclusion, SDF-1 alpha plays a role in two important processes of granule cell maturation - proliferation and migration - assisting in the achievement of appropriate cell number and position in the cerebellar cortex.

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 μM induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 μM of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (ΔΨm) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC6(3), a cationic lipophilic dye into mitochondria indicating the disruption of ΔΨm. This drop in ΔΨm was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation → excess ROS production → GSH depletion → oxidative stress → disruption of ΔΨm → release of cytochrome C and other apoptosis related proteins to cytosol → apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.

Chemokines are believed to play a role in the neuropathogenesis of AIDS through their recruitment of neurotoxin-secreting, virally infected leukocytes into the CNS. Levels of chemokines are elevated in brains of patients and macaques with HIV/SIV-induced encephalitis. The chemokine receptors CCR3, CCR5, and CXCR4 are found on subpopulations of neurons in the cortex of human and macaque brain. We have developed an in vitro system using both macaque and human fetal neurons and astrocytes to further investigate the roles of these receptors in neuronal response to inflammation. Here we report the presence of functional HIV/SIV coreceptors CCR3, CCR5, and CXCR4 on fetal human and macaque neurons and CCR5 and CXCR4 on astrocytes immediately ex vivo and after several weeks in culture. Confocal imaging of immunostained neurons demonstrated different patterns of distribution for these receptors, which may have functional implications. Chemokine receptors were shown to respond to their appropriate chemokine ligands with increases in intracellular calcium that, in the case of neurons, required predepolarization with KCl. These responses were blocked by neutralizing chemokine receptor in mAbs. Pretreatment of neural cells with pertussis toxin abolished responses to stromal-derived factor-1α, macrophage inflammatory protein-1β, and RANTES, indicating coupling of CCR5 and CXCR4 to a Giα protein, as in leukocytes. Cultured macaque neurons demonstrated calcium flux response to treatment with recombinant SIVmac239 envelope protein, suggesting a mechanism by which viral envelope could affect neuronal function in SIV infection. The presence of functional chemokine receptors on neurons and astrocytes suggests that chemokines could serve to link inflammatory and neuronal responses.

Caco-2 cells grown in bicameral chambers, a model of intestinal epithelial iron transport (Biochim. Biophys. Acta (1991) 1070, 205–208), were used to study the effect of apo-transferrin (apo-Tf) in the basal chamber on 59 Fe uptake from the apical surface, intracellular 59 Fe distribution, and 59 Fe transport into the basal chamber. Caco-2 cells were grown with varying amounts of iron to achieve cells that were either iron-deficient (FeD), of normal iron status (FeN), or iron-loaded (FeH). The effect of apo-Tf was most marked in FeD cells with the transport of 59 Fe from 1 μM 59 Fe-ascorbate on the apical side to the basal chamber measured as (22.2 ± 3.0)· 10 4 , (8.2 ± 0.6)· 10 4 , and (2.7 ± 0.4)· 10 4 atoms 59 Fe/cell/min in the presence of apo-Tf, BSA, and no added protein, respectively. Unexpectedly in FeD cells total 59 Fe uptake (i.e., both 59 Fe in the cells and that transported into the basal chamber) was decreased by basolateral apo-Tf with total uptake of (2.6 ± 0.3)· 10 5 , (4.8 ± 0.6)· 10 5 , and (4.8 ± 0.7)· 10 5 atoms/cell/min with apo-Tf, BSA, and no additions, respectively. Analysis of intracellular 59 Fe by isoelectrofocusing in polyacrylamide gels demonstrated 59 Fe migrating both with a basic p I and with the p I values of ferritin (Ft) at a ratio of 200:1 (basic p I moiety: ferritin) in FeD cells. The presence of Tf further decreased the small amount of 59 Fe in Ft. These studies demonstrate that basolateral Tf affects the apical uptake of 59 Fe, the intracellular distribution of 59 Fe, and the transport of 59 Fe across intestinal epithelium, the latter effect occurring even when cellular content of ferritin is high.

Caco-2 cells grown as monolayers on porous membranes in bicameral chambers have been used to study the transport of Fe from the apical (lumenal) chamber to the basal (serosal) chamber. The transport of Fe is stimulated by the presence of either apo-transferrin (apo-Tf) or ferri-transferrin (Fe-Tf) in the basal chamber with the stimulation occurring at much lower concentrations of apo-Tf than Fe-Tf. To further explore the involvement of Tf in Fe transport across the basal surface, laser scanning confocal microscopy with 3-dimensional reconstruction of the confocal images was used to visualize the internalization of Texas Red-labeled apo-Tf and Bodipy-labeled Fe-Tf from the basal chamber. These studies show that apo-Tf was readily internalized and routed preferentially to a perinuclear region of the Caco-2 cells while internalized Fe-Tf stayed preferentially below the nuclei. These findings suggest that intestinal cells have a specialized mechanism to recognize and sort apo-Tf. (Blood. 2000;95:721-723)

Caco-2, a human cell line, displays several biochemical and morphological characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concentrations of the metal. High pressure liquid chromatography fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins), were present in the apical and basal media of controls as well as in cells grown in the presence of high concentrations of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling experiments with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concentrations of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells.

The inhibition of gamma-aminobutyric acid (GABA)-gated chloride currents by the protein kinase C (PKC) activator 4beta-phorbol 12-myristate 13-acetate (PMA) was investigated using recombinant human GABAA receptors expressed in Xenopus oocytes. PMA (5 nM) reduced the GABA response in oocytes expressing the alpha1 beta2 gamma2L receptor construct, as measured by the two-electrode voltage-clamp method. GABA responses declined to approximately 25% of their pretreatment value within 45 min. GABA responses in oocytes expressing a receptor construct from which the known PKC phosphorylation sites were absent, alpha1 beta2(S410A), were comparably inhibited. Phorbol 12-monomyristate (PMM; 5 nM), which does not activate PKC, did not alter the GABA response in either construct, while the PKC inhibitor calphostin C (0.5 microM) prevented the PMA effect. To further investigate PMA inhibition of the GABA response, a GABAA receptor alpha1 subunit/green fluorescent protein (GFP) chimera (alpha1GFP) was used to visualize GABAA receptor distribution. Similar to the wild type constructs, PMA robustly decreased GABA responses in oocytes expressing alpha1GFP beta2 gamma2L and alpha1GFP beta2(S410A) receptor constructs. Following PMA treatment, GFP fluorescence in the oocyte plasma membrane was decreased to approximately 45% of the pretreatment values indicating GABAA receptor internalization. This effect of PMA was prevented by calphostin C and was not produced by PMM. Experiments with bd24, a monoclonal antibody which recognizes an extracellular epitope of the alpha1 subunit, were used to demonstrate that PMA, but not PMM, decreases alpha1 subunit immunoreactivity in the plasma membrane of intact oocytes expressing the alpha1 beta2 gamma2L construct, thus confirming the results obtained with the chimeric receptor. It is concluded that, in Xenopus oocytes, PMA induces an internalization of the GABAA receptor through PKC-mediated phosphorylation of an unidentified protein(s) and that this contributes to the decrease in electrophysiological responses to GABA following PKC activation.

The GABAA receptor is a ligand-gated chloride channel belonging to the superfamily of ligand-gated ion channels of which the nicotinic acetylcholine (nACh) receptor is prototypic. In the central nervous system the GABAA receptor mediates fast neuronal inhibition. To facilitate the study of this receptor, a GABAA receptor-green fluorescent protein (GABAAR-GFP) chimera was constructed by fusing green fluorescent protein (GFP) to the C-terminus region of the GABAA receptor alpha1 subunit. When expressed in Xenopus oocytes, this chimera responded in a manner indistinguishable from the wild-type GABAA receptor with respect to agonist potency, receptor desensitization, allosteric modulation, rectification, and ion selectivity of the channel. The addition of GFP to the GABAA receptor alpha1 subunit did not appear to alter the assembly or efficiency of expression of the GABAA receptor complex. The GABAAR-GFP chimera generated a strong fluorescent signal that was restricted to the animal pole of the oocyte plasma membrane. This signal was readily detectable using either epifluorescence or laser confocal microscopy. To confirm the extracellular location of the GFP portion of the chimera, non-permeabilized oocytes were immunolabeled with an anti-GFP antibody. Fluorescence microscopy showed that GFP was located extracellularly since it was accessible to the GFP antibody. These results confirm the predicted extracellular location of the C-terminus of the GABAA receptor alpha1 subunit and also demonstrate that GFP retains its fluorescent property when expressed extracellularly. The usefulness of the GABAAR-GFP chimera in receptor trafficking was investigated using non-hydrolyzable GTP analogues since GTP binding proteins participate in protein transport in oocytes. Microinjections of GTP-gamma-S but not GDP-beta-S reduced both GABA-gated chloride currents and cell surface GFP fluorescence in oocytes expressing the GABAAR-GFP chimera indicating that the chimera undergoes internalization upon stimulation of oocyte GTP-binding proteins. The results of the present study show that the GABAAR-GFP chimera is functionally similar to the wild-type GABAA receptor and can be used to study receptor trafficking in living cells. This is the first demonstration of a ligand-gated ion channel-GFP chimera for an ion channel belonging to this superfamily and also is the first example of the fusion of GFP to an extracellular domain of an integral membrane protein.

Previous studies have demonstrated that diferric transferrin and apotransferrin compete for the binding to basolateral transferrin receptors and that transferrin-mediated iron uptake by Caco-2 cells is inhibited by apotransferrin to a larger extent than that predicted solely by receptor competition. This inhibition can have important implications in determining the net exchange of iron between intestinal cells and the basolateral milieu. Accordingly, we further characterized the endocytic cycles of apotransferrin and diferric transferrin in Caco-2 cells. We found that after internalization both apotransferrin and diferric transferrin recycled to the cell exterior, but that apotransferrin had a protracted endocytic cycle. Confocal microscopy studies revealed a different cellular distribution of apotransferrin and diferric transferrin; both were found in a compartment close to the basal membrane, but apotransferrin reached as well regions closer to the apical membrane. Moreover, the intracellular distribution of transferrin receptors was influenced by the iron load of transferrin; cells incubated with apotransferrin presented a more apical distribution of transferrin receptors than cells incubated with diferric transferrin. These results indicate for the first time that the endocytic cycle of transferrin receptors in intestinal epithelial cells is determined by the iron content of transferrin. They explain also the marked inhibitory effect of apotransferrin on transferrin-mediated iron uptake by Caco-2 cells, since incubation of cells with apotransferrin resulted in the actual sequestration of the receptor in the cell interior.

To study the cell biology of aluminum uptake and transport in intestinal epithelia, an in vitro system based on intestine-derived Caco-2 cells grown in bicameral chambers was used. Aluminum was offered on the apical surface of Caco-2 cell monolayers as either aluminum citrate, aluminum lactate or aluminum nitrilotriacetate at 1:2 molar ratios, and the aluminum uptake into the cells and transport into the basal chamber were measured. The kinetics of cellular uptake of aluminum were different for the three chelators, although with all three chelators a final cellular concentration of approximately 50 nmol/mg cell protein was achieved. The total transport of aluminum into the basal chamber was greater for aluminum citrate and aluminum nitrilotriacetate than for aluminum lactate, suggesting that the chelator may direct aluminum into compartments from which aluminum is more easily transported. The iron status of the Caco-2 cells significantly affected both cellular uptake and transport of aluminum. Both iron-depleted and iron-overloaded cells exhibited significantly lower aluminum transport than cells of normal iron status. Aluminum loading of the Caco-2 cells had adverse effects on 59Fe2+ and 59Fe3+ transport compared with that of normal cells. These findings suggest that the Caco-2 cell line grown in bicameral chambers provides a model for studying aluminum transport, that aluminum uptake and transport to the basal chamber were affected by the chelator used, and that aluminum uptake and transport pathways are similar to those of iron.

Recent concern over the release of Al in the environment has prompted researchers and health officials to assess its effects on biological systems. Aluminum, despite being the most abundant metal in earth`s lithosphere, is normally complexed in soil and is therefore unavailable for biological assimilation. The recent advent of acid rain, however, has prompted Al release due to mobilization from surrounding sediments into the environment. This is of particular concern in aquatic environments because organisms in aquatic food chains can access and concentrate sublethal levels of Al in their tissues relatively quickly. The ingestion of affected organisms by humans may therefore pose a potential health risk. One such organism, is known to concentrate metals in a variety of tissues. In northern Louisiana, many people trap or fish for crayfish in lowland areas which lie adjacent to highways and secondary roadways. Water, soil, and crayfish from these areas are known to contain high levels of Al. Some tissues known to concentrate Al (muscle, hepatopancreas and intestine tissue and contents) are those which humans commonly consume. The ingestion of these tissues may therefore expose humans to elevated Al levels. Many people who eat crayfish often purge them in dilute concentrations (1-2%) ofmore » NaCl to rid them of contaminants and make them more palatable. We are aware of no literature which corroborates the claim that purging removes contaminating metals. The objectives of this study were to (1) document the amount of Al found in water, soil, and gut tissue of crayfish (P. clarkii) collected from a roadside wetland site; (2) determine the affect of NaCl purging on the release of Al in P. clarkii and (3) assess the differences in Al levels found between stomach tissue, stomach tissue contents, intestine tissue, and intestine contents in P. clarkii. 12 refs., 3 figs., 1 tab.« less

Iron transport across polarized intestinal epithelium was studied by using Caco-2 cells grown in bicameral chambers. When cells were grown under conditions of low, normal, or high iron concentration not only was the iron content of the cells markedly altered but the low iron cells exhibited a nearly 2-fold increase in transepithelial electrical resistance (TEER). 59Fe uptake from the apical surface into cells and transport into the basal chamber was affected both by the valency of the iron and the iron status of the cells. Uptake from 59Fe(II)-ascorbate was about 600 pmol 59Fe/h per mg protein, increased about 2-fold in low iron cells, and was about 13-200-fold greater than uptakes from 59Fe(III) chelated to nitrilotriacetic acid, BSA, or citrate. Transport into the basal chamber from 59Fe(II)-ascorbate was 3.7 +/- 1.7 pmol/h per cm2 for Fe-deficient cells vs. 0.72 +/- 0.1 pmol/h per cm2 for normal-Fe cells and from 59Fe(III)-BSA 1.1 +/- 0.2 pmol/h per cm2 vs. 0.3 +/- 0.03 pmol/h per cm2 for deficient vs. normal iron cells, respectively. The greater transport of iron both from Fe(II) and in iron deficient cells supports the use of the Caco-2 cells as a model for iron transport.

The concentration of Pb, Cd, and Al in tissues of crayfish Procambarus clarkii were evaluated from several wetland sites located adjacent to roadways and were compared to crayfish harvested from a commercial site free from roadside influences. Abdominal muscle, hepatopancreas, alimentary tract, exoskeleton and blood were analyzed for metal content. Results indicated that levels of contamination obtained in almost all tissues of crayfish from roadside ditches contained significantly higher amounts of metals than those of the commercially harvested control crayfish (p = less than or equal to .05-.001). Detection limits of Pb, Cd, and Al ranged from 0.04 microgram Pb/g to 16.15 micrograms Pb/g, .001 microgram Cd/g to .13 microgram Cd/g, and 1.22 micrograms Al/g to 981 micrograms Al/g, respectively. Concentrations of Pb, Cd, and Al were highest in the hepatopancreas and alimentary tract. High levels of these elements were also detected in the exoskeleton. In contrast, muscle tissue was the least affected tissue. Several significant correlations among concentrations of metals were found when comparing a variety of tissues in Procambarus clarkii.