LLC-PK1 cells in culture do not concentrate alpha-methylglucoside (alpha-meG) during their early growth phase but develop the capacity to concentrate this hexose as the growth rate decreases in confluent cultures. The concentrating ability is dependent on the Na+ electrochemical gradient and is inhibited by phlorizin with KI,0.5 approximately 0.2 microM. The development of the concentrative capacity can be accelerated by the Friend cell inducer hexamethylene bisacetamide (HMBA) and by the phosphodiesterase inhibitors dibutyryl cAMP, theophylline, and 1-methyl-3-isobutylxanthine (MIX). In cultures treated with any of these differentiation-accelerating chemicals, the development of alpha-meG concentrating capacity is severely inhibited by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) but not by inactive (in tumor promotion) analogs of TPA. In all cases, an early event in the development of alpha-meG accumulating capacity is an elevated intracellular cAMP concentration; however the results suggest that this increase in cAMP may be necessary but not sufficient to induce the differentiated hexose-accumulating capacity.

Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. Whereas de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem, we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, the activity of which is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes and can regulate cofilin phosphorylation, we also asked whether they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained, and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and Madin-Darby canine kidney) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM kinase (LIMK) but not the Rac/PAK/LIMK pathway, because 1) dominant negative (DN) Rho and DN-ROCK but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; 2) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; 3) hyperosmolarity induced LIMK-2 phosphorylation, and 4) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation.We thenexamined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by small interfering RNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase.

Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, we expressed FLAG-tagged wild-type V2R and a Y325F mutant V2R in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr325 with Phe325 prevented ligand-induced internalization of V2R determined by [3H]AVP binding and immunofluorescence but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells or for adenylyl cyclase-mediated signal transduction.

Recent studies of Toxoplasma gondii isolates from animals in Brazil have revealed high genetic diversity. Many of these isolates are virulent to mice. It is speculated that these isolates may also be virulent to humans. However, there is very limited data regarding T. gondii strains from human infection. Therefore, it is not clear whether there is any association between parasite genotypes and disease phenotypes. In this study, a total of 27 T. gondii strains were isolated from humans with congenital toxoplasmosis in Minas Gerais state, Brazil. The genetic variability was assessed by restricted fragment length polymorphism in 11 loci (SAG1, 5' plus 3' SAG2, alternative [alt.] SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Genetic analysis of 24 strains revealed 14 different genotypes, including 7 previously identified from animals and 7 new types. The widespread genotype BrII accounted for 29% (7/24) of the isolates and was the dominant genotype involved in this study. This is the first report of genotyping of T. gondii isolates obtained from blood samples from newborns with congenital toxoplasmosis. Genotypic characterization of these isolates suggests high genetic diversity of T. gondii in this human population in Brazil. Future studies are needed to determine the source of contamination of this human population.

Vectorial transport of bile acids across hepatocytes is a major driving force for bile flow, and bile acid retention in the liver causes hepatotoxicity. The basolateral and apical transporters for bile acids are thought to be targets of drugs that induce cholestasis. Previously, we constructed polarized LLC-PK1 cells that express both a major bile acid uptake transporter human Na+/taurocholate cotransporting polypeptide (SLC10A1) (NTCP) and the bile acid efflux transporter human bile salt export pump (ABCB 11) (BSEP) and showed that monolayers of such cells can be used to characterize vectorial transcellular transport of bile acids. In the present study, we investigated whether cholestasis-inducing drugs could inhibit bile acid transport in such cells. Because fluorescent substrates allow the development of a high-throughput screening method, we examined the transport by NTCP and BSEP of fluorescent bile acids as well as taurocholate. The aminofluorescein-tagged bile acids, chenodeoxycholylglycylamidofluorescein and cholylglycylamidofluorescein, were substrates of both NTCP and BSEP, and their basal-to-apical transport rates across coexpressing cell monolayers were 4.3 to 4.5 times those of the vector control, although smaller than for taurocholate. The well known cholestatic drugs, rifampicin, rifamycin SV, glibenclamide, and cyclosporin A, reduced the basal-to-apical transport and the apical efflux clearance of taurocholate across NTCP- and BSEP-coexpressing cell monolayers. Further analysis indicated that the drugs inhibited both NTCP and BSEP. Our study suggests that such coexpressing cells can provide a useful system for the identification of inhibitors of these two transport systems, including potential drug candidates.

We recently demonstrated that the cardiotonic steroid marinobufagenin (MBG) induced fibrosis in rat hearts through direct stimulation of collagen I secretion by cardiac fibroblasts. This stimulation was also responsible for the cardiac fibrosis seen in experimental renal failure. In this study, the effect of MBG on the development of renal fibrosis in rats was investigated. Four weeks of MBG infusion triggered mild periglomerular and peritubular fibrosis in the cortex and the appearance of fibrotic scars in the corticomedullary junction of the kidney. MBG also significantly increased the protein levels and nuclear localization of the transcription factor Snail in the tubular epithelia. It is known that activation of Snail is associated with epithelial-to-mesenchymal transition (EMT) during renal fibrosis. To examine whether MBG alone can trigger EMT, we used the porcine proximal tubular cell line LLC-PK1. MBG (100 nM) caused LLC-PK1 cells grown to confluence to acquire a fibroblast-like shape and have an invasive motility. The expressions of the mesenchymal proteins collagen I, fibronectin, and vimentin were increased twofold. However, the total level of E-cadherin remained unchanged. These alterations in LLC-PK1 cells in the presence of MBG were accompanied by elevated expression and nuclear translocation of Snail. During the time course of EMT, MBG did not have measurable inhibitory effects on the ion pumping activity of its natural ligand, Na(+)-K(+)-ATPase. Our data suggest that the MBG may be an important factor in inducing EMT and, through this mechanism, elevated levels of MBG in chronic renal failure may play a role in the progressive fibrosis.

The purpose of this study was to examine whether in vivo drug distribution to the brain can be reconstructed by integrating P-glycoprotein (P-gp)/mdr1a expression levels, P-gp in vitro activity, and drug unbound fractions in mouse plasma and brain. For 11 P-gp substrates, in vitro P-gp transport activities were determined by measuring transcellular transport across monolayers of mouse P-gp-transfected LLC-PK1 (L-mdr1a) and parental cells. P-gp expression amounts were determined by quantitative targeted absolute proteomics. Unbound drug fractions in plasma and brain were obtained from the literature and by measuring brain slice uptake, respectively. Brain-to-plasma concentration ratios (K(p brain)) and its ratios between wild-type and mdr1a/1b(-/-) mice (K(p brain) ratio) were obtained from the literature or determined by intravenous constant infusion. Unbound brain-to-plasma concentration ratios (K(p,uu,brain)) were estimated from K(p brain) and unbound fractions. Based on pharmacokinetic theory, K(p brain) ratios were reconstructed from in vitro P-gp transport activities and P-gp expression amounts in L-mdr1a cells and mouse brain capillaries. All reconstructed K(p brain) ratios were within a 1.6-fold range of observed values. K(p brain) then was reconstructed from the reconstructed K(p brain) ratios and unbound fractions. K(p,uu,brain) was reconstructed as the reciprocal of the reconstructed K(p brain) ratios. For quinidine, loperamide, risperidone, indinavir, dexamethasone, paclitaxel, verapamil, loratadine, and diazepam, the reconstructed K(p brain) and K(p,uu,brain) agreed with observed and estimated in vivo values within a 3-fold range, respectively. Thus, brain distributions of P-gp substrates can be reconstructed from P-gp expression levels, in vitro activity, and drug unbound fractions.

OBJECTIVE

The presence of single nucleotide polymorphisms (SNPs) has been reported for multidrug resistance-associated protein 2 (MRP2/ABCC2). The purpose of the current study was to characterize the localization, expression level, and function of MRP2 variants.

METHODS

The expression and cellular localization of the wild-type and three kinds of reported SNP variants of MRP2 molecules were analyzed in LLC-PK1 cells after infection with the recombinant Tet-off adenoviruses. Their function was determined by using the isolated membrane vesicles from the infected LLC-PK1 cells.

RESULTS

The transport activity for E217betaG, LTC4, and DNP-SG, normalized by the expression level of MRP2, was similar between the wild-type, V417I, and A1450T MRP2s. The transport activity of S789F MRP2 was slightly higher than that of wild-type MRP2. However, the expression level of S789F and A1450T MRP2 proteins was significantly lower compared with the wild-type and V417I MRP2. In addition, although the wild-type and V417I MRP2 were exclusively localized in the apical membrane, S789F and A1450T MRP2 were located in the apical membrane and also in the intracellular compartment.

CONCLUSIONS

These results suggest that the most frequently observed V417I substitution may not affect the in vivo function of MRP2, whereas the much less frequently observed S789F and A1450T may be associated with the reduced in vivo function.

Cells of confluent cultures of the established pig renal epithelial line, LLC-PK1, accumulate alpha-methyl-D-glucoside against a concentration gradient. This transport system is strongly inhibited by phlorizin and 6-deoxy-D-glucose, moderately inhibited by phloretin, and only weakly inhibited by 3-0-methyl-D-glucose, paralleling the situation in mammalian kidney. The time courses for the uptake of alpha-methyl-D-glucoside and for the carrier-mediated but passive uptake of 3-0-methyl-D-glucose are identical to those seen in mammalian kidney. Subconfluent cultures of LLC-PK1 cells are unable to accumulate alpha-methyl-D-glucoside, and their transport of this glucose analog is less sensitive to phlorizin inhibition than is the transport system in confluent cultures. Transmission electron micrographs show that cells from subconfluent cultures lack the microvillous surface seen in cells from confluent cultures. Cell density is thus a factor in the occurrence of structural and functional differentiated properties related to transport in these cells.

Transforming growth factor (TGF)-beta is a multifunctional growth factor with important roles in development, cell proliferation, and matrix deposition. It signals through the sequential activation of two serine/threonine kinase receptors, the type I and type II receptors. A third cell surface receptor, betaglycan, serves as a co-receptor for TGF-beta in some cell types, enhancing TGF-beta-mediated signaling. We have examined the function of betaglycan in renal epithelial LLC-PK1 cells that lack endogenous betaglycan. We demonstrate that the expression of betaglycan in LLC-PK1 cells results in inhibition of TGF-beta signaling as measured by reporter gene expression, thymidine incorporation, collagen production, and phosphorylation of the downstream signaling effectors Smad2 and Smad3. In comparison, the expression of betaglycan in L6 myoblasts enhances TGF-beta signaling, which is consistent with the published literature. The effects of betaglycan in LLC-PK1 cells are not mediated by ligand sequestration or increased production of a soluble form of the receptor, which has been reported to serve as a ligand antagonist. We demonstrate instead that in LLC-PK1 cells, unlike L6 cells, expression of betaglycan prevents association between the type I and type II TGF-beta receptors, which is required for signaling. This is a function of the glycosaminoglycan modifications of betaglycan. Betaglycan in LLC-PK1 cells exhibits higher molecular weight glycosaminoglycan (GAG) chains than in L6 cells, and a GAG- betaglycan mutant does not inhibit TGF-beta signaling or type I/type II receptor association in LLC-PK1 cells. Our data indicate that betaglycan can function as a potent inhibitor of TGF-beta signaling by a novel mechanism and provide support for an essential but complex role for proteoglycan co-receptors in growth factor signaling.

Studies in various tissues, including the kidney, have demonstrated that toxins elicit apoptosis under certain conditions and necrosis under others. The nature of the response has important consequences for the injured tissue in that necrotic cells elicit inflammatory responses, whereas apoptotic cells do not. Thus, there has been considerable interest in defining the mode of cell death elicited by known cytotoxins. The present studies examined the response of renal epithelial cells to oxalate, a metabolite excreted by the kidney that produces oxidant stress and death of renal cells at pathophysiological concentrations. These studies employed LLC-PK1 cells, a renal epithelial cell line from pig kidney and NRK-52E (NRK) cells, a line from normal rat kidney, and compared the effects of oxalate with those of known apoptotic agents. Changes in cellular and nuclear morphology, in nuclear size, in ceramide production, and in DNA integrity were assessed. The ability of bcl-2, an anti-apoptotic gene product, to attenuate oxalate toxicity was also assessed. These studies indicated that oxalate-induced death of renal epithelial cells exhibits several features characteristic of apoptotic cell death, including increased production of ceramide, increased abundance of apoptotic bodies, and marked sensitivity to the level of expression of the anti-apoptotic gene bcl-2. Oxalate-induced cell death also exhibits several characteristics of necrotic cell death in that the majority of the cells exhibited cellular and nuclear swelling after oxalate treatment and showed little evidence of DNA cleavage by TUNEL assay. These results suggest that toxic concentrations of oxalate trigger both forms of cell death in renal epithelial cells.

Co-localization of the elements calcium, potassium, sodium and magnesium with sequestering organelles has been achieved by application of two microscopy techniques on the same cell. Organelles were first localized by laser scanning confocal microscopy (LSCFM) using fluorescent organelle stains. The same cells were then analyzed for elemental distribution with ion microscopy. This approach has identified a perinuclear region of prominent total calcium concentration with the Golgi apparatus. Live cells were fluorescently stained with C6-NBD-ceramide for labeling the Golgi apparatus prior to cryogenic preparation and freeze-drying, and imaged with LSCFM for Golgi localization; identical cells were then analyzed with ion microscopy to image subcellular distributions of total calcium, potassium, sodium and magnesium. In three cell lines, LLC-PK1 porcine kidney epithelial cells, Swiss 3T3 mouse fibroblast cells and L5 rat myoblast cells, the Golgi regions contained significantly higher total calcium concentrations than any other region of the cell (as measured at the spatial resolution of ion microscopy of about 0.5 micron). Intracellular potassium, sodium and magnesium were homogeneously distributed throughout the cell and did not show this pattern. Measurements of depletion of calcium by exposure to calcium-free medium showed that the Golgi apparatus was substantially more resistant to calcium depletion than all other regions of these cells, but sequestered Ca2+ could be released from the Golgi by exposing the cells to calcium ionophore A23187. The Golgi apparatus appears to sequester about 5% of the total cell calcium in LLC-PK1 cells, about 2.5% in 3T3 cells and L5 cells.

BACKGROUND

Iron plays a role in free radical-mediated tissue injury, including cisplatin-induced nephrotoxicity. However, the source of iron (catalyzing free radical reactions) is not known. We examined the role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity both in vivo and in vitro.

METHODS

Cisplatin-induced acute renal failure was produced in rats by intraperitoneal injection of cisplatin (10 mg/kg body wt). Piperonyl butoxide, a cytochrome P-450 inhibitor, was administered intraperitoneally (400 mg/kg body wt twice at 48-hr intervals) prior to cisplatin injection. The effects of cisplatin in the absence or presence of piperonyl butoxide on the belomycin-detectable iron, cytochrome P-450 content in the kidney, and renal functional and histological changes were evaluated. In an in vitro study, the effect of cytochrome P-450 inhibitors, cimetidine or piperonyl butoxide, on cisplatin-induced cytotoxicity and catalytic iron release from LLC-PK1 cells was examined.

RESULTS

In cisplatin-treated rats, there was a marked decrease in the cytochrome P-450 content specifically in the kidney, accompanied by increased bleomycin-detectable iron content in the kidney. Piperonyl butoxide prevented cisplatin-induced loss of cytochrome P-450 as well as the increase of bleomycin-detectable iron in the kidney, along with both functional and histological protection. Both cimetidine and piperonyl butoxide prevented cisplatin-induced increase in bleomycin-detectable iron and cytotoxicity in LLC-PK1 cells. Treatment of cimetidine did not affect cellular uptake of cisplatin.

CONCLUSIONS

Cytochrome P-450, a group of heme proteins, may serve as a significant source of catalytic iron in cisplatin-induced nephrotoxicity.

We examined the influence of high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs) on the toxicity of amphotericin B (AmpB) to fungal and renal cells. Candida albicans was incubated for 18 h at 37 degrees C with AmpB and deoxycholate (Fungizone) or liposomal AmpB (L-AmpB) (0.1 to 2.0 micrograms of AmpB per ml) in the presence or absence of HDLs or LDLs (0.5 mg of protein per ml). The MICs of AmpB and L-AmpB, whether or not HDLs or LDLs were present, were similar. LLC PK1 renal cells, derived from primary cultures of pig proximal tubular cells, were incubated for 18 h at 37 degrees C in serum-free medium that contained AmpB and deoxycholate or L-AmpB at 20 micrograms of AmpB per ml, HDLs or LDLs at 0.5 mg of protein per ml, mixtures of AmpB with HDLs or LDLs, and mixtures of L-AmpB with HDLs or LDLs. HDL-associated AmpB was less toxic than AmB to LLC PK1 cells (53.0% +/- 2.5% versus 81.3% +/- 3.6% cytotoxicity; P = 0.01), while LDL-associated AmpB was as toxic as AmpB. L-AmpB, HDL-associated L-AmpB, and LDL-associated L-AmpB were less toxic to LLC PK1 cells than was AmpB (48.3% +/- 1.5%, 25.5% +/- 2.2%, and 52.2% +/- 2.5% versus 81.3% +/- 3.6% cytotoxicity; P = 0.02). To further understand why HDL-associated AmpB reduced renal cytotoxic effects, the LLC PK1 cells were examined for the presence of HDL and LDL receptors. LLC PK1 cells expressed high-affinity (K(d) = 0.0538 nanograms/ml; 96,000 sites per cell) and low-affinity (K(d) = 222.22 nanograms/ml; 77 sites per cell) LDL receptors but only a low-affinity HDL receptor (K(d) = 71.43 nanograms/ml; 2 sites per cell). HDL-associated AmpB and LDL-associated AmpB were less toxic than AmpB to trypsinized LLC PK1 cells (46.6% +/- 10.9% and 16.8% +/- 15.98% versus 74.7% +/- 7.7% cytotoxicity; P = 0.02). HDL-associated AmB and LDL-associated L-AmpB were also less toxic than AmpB to the cells (20.4% +/- 6.2% and 13.5% +/- 8.6% versus 74.7% cytotoxicity; P = 0.01). The antifungal activities of AmpB and L-AmpB were not altered in the presence of HDLs or LDLs. We conclude that the reduced nephrotoxicity associated with the use of L-AmpB is related to a decreased uptake of AmpB by renal cells when AmpB is associated with HDLs because of the low level of expression of HDL receptors in these cells.

A 886 bp cDNA encoding the murine rac1 protein has been isolated. The abundance of rac1 mRNA was determined in fourteen tissues from both mouse and pig. The mRNA 5' non-coding sequence is very rich in G + C, and has the potential to form several stable secondary structures. In addition, this region contains a putative open reading frame of 57 amino acids. Disruption of the actin microfilament network by cytochalasin B in LLC-PK1 cells results in down regulation of rac1 mRNA. In agreement with the proposed role of rac1 in exocytosis, these results could explain the inhibitory effect of cytochalasin B on secretory processes.

Recent studies on LLC-PK1 cells demonstrated that oxalate, a simple dicarboxylic acid, acts as a mitogen for these renal epithelial cells. Exposure to oxalate initiates DNA synthesis, induces the expression of one of the early growth response genes c-myc and stimulates proliferation of quiescent cultures of LLC-PK1 cells. The present studies examined the possibility that expression of the c-myc protooncogene is obligatory for this mitogenic response. Specifically we determined whether pretreatment with c-myc antisense oligonucleotides would block the proliferative effects of oxalate in LLC-PK1 cells. Quiescent cultures of LLC-PK1 cells were exposed to oxalate in the presence and absence of c-myc antisense and the effects of oxalate on c-myc protein expression (Myc), DNA synthesis and cell growth were assessed. Exposure of cells to oxalate alone increased the expression of Myc within two hours. Pretreatment with c-myc antisense abolished this response. Further, pretreatment of cells with c-myc antisense but not nonsense oligonucleotides blocked the oxalate-induced initiation of DNA synthesis. Increases in cell number in response to oxalate (measured after 72 hr exposure) were also blocked by exposure to c-myc antisense. These findings suggest that c-myc gene expression is critical for the mitogenic effects of oxalate in LLC-PK1 cells.

The interaction of verotoxins (VTs) with human erythrocytes (RBCs) in vitro was investigated, with particular reference to the role of P blood group glycolipids that are structurally related to the known VT receptors. RBC binding of purified VT1, VT2, VT2c, and VT2e was detected by direct and indirect immunofluorescence. Glycolipids were extracted from defined RBCs, separated by thin-layer chromatography, and assessed for VT binding in an overlay assay by adding toxin and specific antibodies. All VTs bound to P1 phenotype (Pk, P, and P1 antigens) and P2 phenotype (Pk and P antigens) RBCs but not to p phenotype (lacking the Pk, P, and P1 antigens) RBCs. Binding of VT1 and VT2 was approximately 10-fold greater to P1 and the rare Pk2 (Pk antigen but no P1 or P antigen) phenotype cells than to P2 phenotype RBCs, whereas VT2e bound equally well to P1 and P2 phenotype cells. The VT1 and VT2 immunofluorescence results correlated with the detection of P1 and/or increased amounts of Pk (globotriaosylceramide) antigen; VT2e immunofluorescence correlated with the detection of P (globotetraosylceramide) antigen. The Pk band pattern and VT binding observed in the thin-layer chromatogram of human P1 and P phenotype RBC extracts varied from that of human kidney and Pk1 phenotype (Pk and P1 antigens) RBCs. We conclude that each VT binds to human RBCs in vitro by utilizing specific P blood group glycolipids as receptors. On P1 and P phenotype RBCs, the accessibility of the Pk antigen for VTs appeared to be restricted. The occurrence of VT-RBC binding in natural VT-producing Escherichia coli disease and its relevance for the pathophysiology of hemolytic uremic syndrome remain to be established.

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit micro2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.

There are now at least seven polymer-drug conjugates that have entered phase I/II clinical trial as anticancer agents. These include N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (PK1, FCE28068), HPMA copolymer-paclitaxel (PNU 166945), HPMA copolymer-camptothecin, PEG-camptothecin, polyglutamic acid-paclitaxel, an HPMA copolymer-platinate (AP5280) and also an HPMA copolymer-doxorubicin conjugate bearing additionally galactosamine (PK2, FCE28069). The galactosamine is used as a means to target the conjugate to liver for the treatment of primary and secondary liver cancer. Promising early clinical results with lysosomotropic conjugates has stimulated significant interest in this field. Ongoing research is developing (1) conjugates containing drugs that could otherwise not progress due to poor solubility or uncontrollable toxicity; (2) conjugates of agents directed against novel targets; and (3) two-step combinations such as polymer-directed enzyme prodrug therapy (PDEPT) and polymer-enzyme liposome therapy (PELT) that can cause explosive liberation of drug from either polymeric prodrugs or liposomes within the tumour interstitium. Moreover, bioresponsive polymer-based constructs able to promote endosomal escape and thus intracytoplasmic delivery of macromolecular drugs (peptides, proteins and oligonucleotides) are also under study.

Oxalate-induced oxidative stress contributes to cell injury and promotes renal deposition of calcium oxalate crystals. However, we do not know how oxalate stimulates reactive oxygen species (ROS) in renal tubular epithelial cells. We investigated the signaling mechanism of oxalate-induced ROS formation in these cells and found that oxalate significantly increased membrane-associated protein kinase C (PKC) activity while at the same time lowering cytosolic PKC activity. Oxalate markedly translocated PKC-alpha and -delta from the cytosol to the cell membrane. Pretreatment of LLC-PK1 cells with specific inhibitors of PKC-alpha or -delta significantly blocked oxalate-induced generation of superoxide and hydrogen peroxide along with NADPH oxidase activity, LDH release, lipid hydroperoxide formation, and apoptosis. The PKC activator PMA mimicked oxalate's effect on oxidative stress in LLC-PK1 cells as well as cytosol-to-membrane translocation of PKC-alpha and -delta. Silencing of PKC-alpha expression by PKC-alpha-specific small interfering RNA significantly attenuated oxalate-induced cell injury by decreasing hydrogen peroxide generation and LDH release. We believe this is the first demonstration that PKC-alpha- and -delta-dependent activation of NADPH oxidase is one of the mechanisms responsible for oxalate-induced oxidative injury in renal tubular epithelial cells. The study suggests that the therapeutic approach might be considered toward attenuating oxalate-induced PKC signaling-mediated oxidative injury in recurrent stone formers.

The role of N-glycosylation in trafficking of an apical membrane protein, the gastric H,K-ATPase beta subunit linked to yellow fluorescent protein, was analyzed in polarized LLC-PK1 cells by confocal microscopy and surface-specific biotinylation. Deletion of the N-glycosylation sites at N1, N3, N5, and N7 but not at N2, N4, and N6 significantly slowed endoplasmic reticulum-to-Golgi trafficking, impaired apical sorting, and enhanced endocytosis from the apical membrane, resulting in decreased apical expression. Golgi mannosidase inhibition to prevent carbohydrate chain branching and elongation resulted in faster internalization and degradation of the beta subunit, indicating that terminal glycosylation is important for stabilization of the protein in the apical membrane and protection of internalized protein from targeting to the degradation pathway. The decrease in the apical content of the beta subunit was less with mannosidase inhibition compared with that found in the N1, N3, N5, and N7 site mutants, suggesting that the core region sugars are more important than the terminal sugars for apical sorting.

A new family of angiogenic factors named endocrine-gland-derived vascular endothelial growth factors (EG-VEGF)/prokineticins (PK) have been recently described as predominantly expressed in steroidogenic tissues. Whether the normal and malignant epithelial prostate cells and tissues express EG-VEGF/PK1 and PK2 and their receptors is still unknown. We studied the expression of EG-VEGF/PK1 and PK2 and their receptors (PK-R1 and PK-R2) in human prostate and their involvement in cancer. Using immunohistochemistry, Western blot, and RT-PCR, we determined the expression of EG-VEGF/PK1 in normal prostate (NP) and malignant prostate tissues (PCa), in epithelial cell primary cultures from normal prostate (NPEC) and malignant prostate (CPEC) and in a panel of prostate cell lines. In NPEC, CPEC, and in EPN, a nontransformed human prostate epithelial cell line, EG-VEGF/PK1, PK2, PK-R1, and PK-R2 mRNA levels were evaluated by quantitative RT-PCR. EG-VEGF/PK1 transcript was found in PCa, in CPEC, in EPN, and in LNCaP, whereas it was detected at low level in NP and in NPEC. EG-VEGF/PK1 was absent in androgen-independent PC3 and DU-145 cell lines. Immunochemistry confirmed that EG-VEGF/PK1 protein expression was restricted to hyperplastic and malignant prostate tissues, localized in the glandular epithelial cells, and progressively increased with the prostate cancer Gleason score advancement. EG-VEGF/PK1 and PK2 were weakly expressed in NPEC and EPN. On the other hand, their transcripts were highly detected in CPEC. PK-R1 and PK-R2 were found in NPEC, EPN, and CPEC. Interestingly, CPEC showed a significantly (P < 0.05) higher expression of EG-VEGF/PK1, PK2, PK-R1, and PK-R2 compared with NPEC and EPN. We demonstrated that PKs and their receptors are expressed in human prostate and that their levels increased with prostate malignancy. It may imply that EG-VEGF/PK1 could be involved in prostate carcinogenesis, probably regulating angiogenesis. Thus, the level of EG-VEGF/PK1 could be useful for prostate cancer outcome evaluation and as a target for prostate cancer treatment in the future.

The effects of the phenylglycine derivatives, alpha-methyl-4-carboxyphenylglycine (MCPG), 4-carboxyphenylglycine (4CPG), 4-carboxy-3-hydroxyphenylglycine (4C3HPG), 3-hydroxyphenylglycine (3HPG) and 3,5-dihydrohyphenylglycine (DHPG) were tested on LLC-PK1 cells transiently expressing the rat mGluR1a or mGluR5a receptors. As previously reported by others, (S)-3HPG and (RS)-DHPG were found to be partial agonists at mGluR1a, whereas(+)-MCPG,(S)-4CPG and (S)-4C3HPG competitively antagonized the effect of Glu. Surprisingly, the 4-carboxy derivatives of phenylglycine antagonized the effect of 1S,3R-ACPD on mGluR1a with lower KB values. On mGluR5a, (S)-3HPG and (RS)-DHPG are also partial agonists. However, in contrast to their effects on mGluR1a,(S)-4CPG did not inhibit the effect of Glu or 1S,3R-ACPD, and (S)-4C3HPG acted as an agonist at high concentration. Whereas no significant antagonism of the Glu effect on mGluR5a was observed with 1 mM (+)-MCPG, this compound was found to potently and competitively antagonize the effect of 1S,3R-ACPD. Finally, the effect of 4CPG was also examined on cultured cortical and cerebellar neurons that express mGluR5 and mGluR1 mRNA, respectively. 4CPG inhibited 1S,3R-ACPD-stimulated IP production in cerebellar neurons only. These results(1) demonstrate that phenylglycine derivatives can be used to discriminate between effects mediated by mGluR1 and mGluR5 and (2) suggest that the apparent potency of phenylglycine antagonists depends on the agonist used to activate these receptors.

Cats are essential in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in nature. This study was aimed to determine the seropositivity, distribution of genotypes and mouse virulence of T. gondii from stray cats in Beijing, China. A total of 64 serum samples, 23 feces and tissue samples were collected from stray cats in Beijing. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). 57.8% (37/64) of these stray cats had titers of 1:20 or higher and were considered positive with infection. T. gondii oocysts were not found in feces of the 23 cats. Tissues of 23 cats were bioassayed in mice and 11 T. gondii isolates were obtained. The genotype of these isolates were identified by 11 PCR-RFLP markers, including SAG1, (3'+5')SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker, Apico. Only one genotype was identified. This genotype, designated as ToxoDB genotype #9, was previously reported in cats, pigs and human from Guangdong and Gansu provinces in China and animals from a few other countries. To determine mouse virulence of this lineage of parasites, one isolate was randomly selected and inoculated into BABL/c mice, the result showed that it is intermediately virulent to mice. These results indicated that an atypical, intermediately virulent T. gondii lineage is widespread in China. The high seropositivity of T. gondii in stray cats posts potential risk of transmission of the parasite to human population in the region.