During vertebrate gastrulation, mesodermal and ectodermal cells undergo convergent extension, a process characterised by prominent cellular rearrangements in which polarised cells intercalate along the medio-lateral axis leading to elongation of the antero-posterior axis. Recently, it has become evident that a noncanonical Wnt/Frizzled (Fz)/Dishevelled (Dsh) signalling pathway, which is related to the planar-cell-polarity (PCP) pathway in flies, regulates convergent extension during vertebrate gastrulation. Here we isolate and functionally characterise a zebrafish homologue of Drosophila prickle (pk), a gene that is implicated in the regulation of PCP. Zebrafish pk1 is expressed maternally and in moving mesodermal precursors. Abrogation of Pk1 function by morpholino oligonucleotides leads to defective convergent extension movements, enhances the silberblick (slb)/wnt11 and pipetail (Ppt)/wnt5 phenotypes and suppresses the ability of Wnt11 to rescue the slb phenotype. Gain-of-function of Pk1 also inhibits convergent extension movements and enhances the slb phenotype, most likely caused by the ability of Pk1 to block the Fz7-dependent membrane localisation of Dsh by downregulating levels of Dsh protein. Furthermore, we show that pk1 interacts genetically with trilobite (tri)/strabismus to mediate the caudally directed migration of cranial motor neurons and convergent extension. These results indicate that, during zebrafish gastrulation Pk1 acts, in part, through interaction with the noncanonical Wnt11/Wnt5 pathway to regulate convergent extension cell movements, but is unlikely to simply be a linear component of this pathway. In addition, Pk1 interacts with Tri to mediate posterior migration of branchiomotor neurons, probably independent of the noncanonical Wnt pathway.

ERM (Ezrin-Radixin-Moesin) proteins function as plasma membrane-actin cytoskeleton linkers and participate in the formation of specialized domains of the plasma membrane. We have investigated ezrin function in tubulogenesis of a kidney-derived epithelial cell line, LLC-PK1. Here we show that cells overproducing a mutant form of ezrin in which Tyr-353 was changed to a phenylalanine (Y353F) undergo apoptosis when assayed for tubulogenesis. While investigating the mechanism responsible for this apoptosis, we found that ezrin interacts with p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Two distinct sites of ezrin are involved in this interaction, the amino-terminal domain containing the first 309 aa and the phosphorylated Tyr-353 residue, which binds to the carboxyl-terminal SH2 domain of p85. Cells producing Y353F ezrin are defective in activation of the protein kinase Akt, a downstream target of PI 3-kinase that protects cells against apoptosis. Furthermore, the apoptotic phenotype of these cells is rescued by production of a constitutively activated form of PI 3-kinase. Taken together, these results establish a novel function for ezrin in determining survival of epithelial cells by activating the PI 3-kinase/Akt pathway.

Prions are thought to consist mainly or entirely of misfolded PrP, a constitutively expressed host protein. Prions associated with the same PrP sequence may occur in the form of different strains; the strain phenotype is believed to be encoded by the conformation of the PrP. Some cell lines can be persistently infected by prions and, interestingly, show preference for certain strains. We report that a cloned murine neuroblastoma cell population, N2a-PK1, is highly heterogeneous in regard to its susceptibility to RML and 22L prions. Remarkably, sibling subclones may show very different relative susceptibilities to the two strains, indicating that the responses can vary independently. We have assembled four cell lines, N2a-PK1, N2a-R33, LD9 and CAD5, which show widely different responses to prion strains RML, 22L, 301C, and Me7, into a panel that allows their discrimination in vitro within 2 weeks, using the standard scrapie cell assay (SSCA).

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH(2)- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.

Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.

A heterotrimeric G alpha i subunit, alpha i-3, is localized on Golgi membranes in LLC-PK1 and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence. The alpha i-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The alpha i-3 subunit is found on isolated rat liver Golgi membranes by Western blotting and G alpha i-3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLC-PK1 cells were stably transfected with G alpha i-3 on an MT-1, inducible promoter in order to overexpress alpha i-3 on Golgi membranes. The intracellular processing and constitutive secretion of the basement membrane heparan sulfate proteoglycan (HSPG) was measured in LLC-PK1 cells. Overexpression of alpha i-3 on Golgi membranes in transfected cells retarded the secretion of HSPG and accumulated precursors in the medial-trans-Golgi. This effect was reversed by treatment of cells with pertussis toxin which results in ADP-ribosylation and functional uncoupling of G alpha i-3 on Golgi membranes. These results provide evidence for a novel role for the pertussis toxin sensitive G alpha i-3 protein in Golgi trafficking of a constitutively secreted protein in epithelial cells.

The espin actin-bundling proteins, which are the target of the jerker deafness mutation, caused a dramatic, concentration-dependent lengthening of LLC-PK1-CL4 cell microvilli and their parallel actin bundles. Espin level was also positively correlated with stereocilium length in hair cells. Villin, but not fascin or fimbrin, also produced noticeable lengthening. The espin COOH-terminal peptide, which contains the actin-bundling module, was necessary and sufficient for lengthening. Lengthening was blocked by 100 nM cytochalasin D. Espin cross-links slowed actin depolymerization in vitro less than twofold. Elimination of an actin monomer-binding WASP homology 2 domain and a profilin-binding proline-rich domain from espin did not decrease lengthening, but made it possible to demonstrate that actin incorporation was restricted to the microvillar tip and that bundles continued to undergo actin treadmilling at approximately 1.5 s-1 during and after lengthening. Thus, through relatively subtle effects on actin polymerization/depolymerization reactions in a treadmilling parallel actin bundle, espin cross-links cause pronounced barbed-end elongation and, thereby, make a longer bundle without joining shorter modules.

The sex pheromone system of Enterococcus faecalis is a unique, highly efficient plasmid collection mechanism for this species. A crucial role in this system is played by an adhesin called aggregation substance which enables the cell-cell contact between donor and recipient strains. The existence of the amino acid motif Arg-Gly-Asp-Ser in the adhesin prompted us to look for a possible binding of E. faecalis cells expressing aggregation substance to eucaryotic cells. We were able to show that the adhesin mediated binding to cultured renal tubular cells (porcine cell line LLC-PK1) via light microscopic, electron microscopic, and enzyme-linked immunosorbent assay-based studies. Synthesis of the adhesin was induced by some component(s) of serum. These data are interpreted to mean that aggregation substance is an adhesin mediating not only cell-cell contact between different E. faecalis strains but also binding of E. faecalis to eucaryotic cells, and therefore it might contribute to virulence.

To perform vectorial secretory and transport functions that are critical for the survival of the organism, epithelial cells sort plasma membrane proteins into polarized apical and basolateral domains. Sorting occurs post-synthetically, in the trans Golgi network (TGN) or after internalization from the cell surface in recycling endosomes, and is mediated by apical and basolateral sorting signals embedded in the protein structure. Basolateral sorting signals include tyrosine motifs in the cytoplasmic domain that are structurally similar to signals involved in receptor internalization by clathrin-coated pits. Recently, an epithelial-specific adaptor protein complex, AP1B, was identified. AP-1B recognizes a subset of basolateral tyrosine motifs through its mu 1B subunit. Here, we characterized the post-synthetic and post-endocytic sorting of the fast recycling low density lipoprotein receptor (LDLR) and transferrin receptor (TfR) in LLC-PK1 cells, which lack mu 1B and mis-sort both receptors to the apical surface. Targeting and recycling assays in LLC-PK1 cells, before and after transfection with mu 1B, and in MDCK cells, which express mu 1B constitutively, suggest that AP1B sorts basolateral proteins post-endocytically.

Binding of ouabain to Na(+)/K(+)-ATPase activates tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), Src, and p42/44 mitogen-activated protein kinases (MAPKs) in both cardiac myocytes and A7r5 cells. Here, we explored the roles of Src and the EGFR in the ouabain-invoked pathways that lead to the activation of MAPKs. Exposure of A7r5 and LLC-PK1 cells to ouabain caused a dose-dependent inhibition of Na(+)/K(+)-ATPase activity, which correlated well with ouabain-induced activation of Src and MAPKs in these cells. Immunoprecipitation experiments showed that ouabain stimulated Src binding to Na(+)/K(+)-ATPase in a dose- and time-dependent manner and increased phosphorylation of Src at Tyr(418) but had no effect on Tyr(529) phosphorylation. Ouabain failed to activate MAPKs in A7r5 cells that were pretreated with the Src inhibitor PP2 and in SYF cells in which Src family kinases are knocked out. Preincubation with AG1478, but not AG1295, also blocked the effects of ouabain on p42/44 MAPKs in A7r5 cells. Significantly, both herbimycin A and PP2 abrogated ouabain-induced but not epidermal growth factor-induced Src binding to the EGFR and the subsequent EGFR tyrosine phosphorylation. Ouabain also failed to affect tyrosine phosphorylation of the EGFR in SYF cells. In addition, unlike epidermal growth factor, ouabain did not increase EGFR autophosphorylation at Tyr(1173). These findings clearly indicate that ouabain transactivates the EGFR by activation of Src and stimulation of Src binding to the EGFR. Furthermore, we found that the transactivated EGFR was capable of recruiting and phosphorylating the adaptor protein Shc. This resulted in increased binding of another adaptor protein Grb2 to the Src-EGFR complex and the subsequent activation of Ras and MAPKs. Taken together, these new findings suggest that Src mediates the inter-receptor cross-talk between Na(+)/K(+)-ATPase and the EGFR to transduce the signals from ouabain to the Ras/MAPK cascade.

Activation of stress response genes can impart cellular tolerance to environmental stress. Iodoacetamide (IDAM) is an alkylating toxicant that up-regulates expression of hsp70 (Liu, H., Lightfoot, D. L., and Stevens, J. L. (1996) J. Biol. Chem. 271, 4805-4812) and grp78 in LLC-PK1 renal epithelial cells. Therefore, we used IDAM to determine the role of these genes in tolerance to toxic chemicals. Prior heat shock did not protect cells from IDAM but pretreatment with trans-4,5-dihydroxy-1,2-dithiane (DTTox), thapsigargin, or tunicamycin enhanced expression of the endoplasmic reticulum (ER) chaperones GRP78 and GRP94 and rendered cells tolerant to IDAM. Cells expressing a 524-base pair antisense grp78 fragment (pkASgrp78) had a diminished capacity to up-regulate grp78 and grp94 expression after ER stress. Protection against IDAM due to prior ER stress was also attenuated in pkASgrp78 cells suggesting that ER chaperones of the GRP family are critical for tolerance. Covalent binding of IDAM to cellular macromolecules and depletion of cellular thiols was similar in tolerant and naïve cells. However, DTTox pretreatment blocked the increases in cellular Ca2+ and lipid peroxidation observed after IDAM treatment. Overexpressing the ER Ca2+-binding protein calreticulin prevented IDAM-induced cell death, the rise in cytosolic Ca2+, and oxidative stress. Although activation of the ER stress response did not prevent toxicity due to Ca2+ influx, EGTA-AM and ruthenium red both blocked cell death suggesting that redistribution of intracellular Ca2+ to the mitochondria may be important in toxicity. The data support a model in which induction of ER stress proteins prevents disturbances of intracellular Ca2+ homeostasis, thus uncoupling toxicant exposure from oxidative stress and cell death. Multiple ER stress proteins are likely to be involved in this tolerance response.

Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ∼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (∼0.5 kb) derived from the 3' untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5'-3' exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3' UTR. Mutations or deletions of various secondary structures in the 3' UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNV(KUN)) 3' UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.

Claudins are tight junction proteins that play a key selectivity role in the paracellular conductance of ions. Numerous studies of claudin function have been carried out using the overexpression strategy to add new claudin channels to an existing paracellular protein background. Here, we report the systematic knockdown of endogenous claudin gene expression in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells using small interfering RNA against claudins 1-4 and 7. In MDCK cells (showing cation selectivity), claudins 2, 4, and 7 are powerful effectors of paracellular Na+ permeation. Removal of claudin-2 depressed the permeation of Na+ and resulted in the loss of cation selectivity. Loss of claudin-4 or -7 expression elevated the permeation of Na+ and enhanced the proclivity of the tight junction for cations. On the other hand, LLC-PK1 cells express little endogenous claudin-2 and show anion selectivity. In LLC-PK1 cells, claudin-4 and -7 are powerful effectors of paracellular Cl- permeation. Knockdown of claudin-4 or -7 expression depressed the permeation of Cl- and caused the tight junction to lose the anion selectivity. In conclusion, claudin-2 functions as a paracellular channel to Na+ to increase the cation selectivity of the tight junction; claudin-4 and -7 function either as paracellular barriers to Na+ or as paracellular channels to Cl-, depending upon the cellular background, to decrease the cation selectivity of the tight junction.

Human pancreatic adenocarcinoma cell lines PK1 and PK8 are resistant to the clinically relevant chemotherapy agent gemcitabine. Cell cycle analysis demonstrated an accumulation of cells in the early S phase during treatment with 20 microM gemcitabine, consistent with its mode of action as a DNA chain terminator. However, apoptosis was evident in only a small percentage of cells. Similar to pancreatic cancers in the clinic, PK1 and PK8 cells carry constitutively active Ki-Ras and overexpress multiple receptor tyrosine kinases. Both genetic abnormalities may potentially up-regulate the activity of the phosphatidylinositide 3-kinase (P13K)-protein kinase B (PKB)/Akt cell survival pathway. The current study examined the relevance of this pathway in the modulation of drug resistance in PK1 and PK8 cells. After exposure to 20 microM gemcitabine for 48 h and in the continuous presence of the drug, treatment with the P13K inhibitors wortmannin (50-200 nM) and LY294002 (15-120 microM) for 4 h substantially enhanced apoptosis in a concentration-dependent manner as compared with treatment with gemcitabine alone, as determined by the loss of mitochondrial membrane potential and the increase in propidium iodide uptake using flow cytometry. Furthermore, Western blotting showed that the reduction of phosphorylated PKB/Akt levels correlated with the enhancement of gemcitabine-induced apoptosis, suggesting that the PI3K-PKB/Akt pathway plays a significant role in mediating drug resistance in human pancreatic cancer cells. PI3K inhibitors may have therapeutic potential when combined with gemcitabine in the treatment of pancreatic cancers.

Vacuolar H+-ATPases (V-ATPases) are a family of ATP-driven proton pumps. They maintain pH gradients between intracellular compartments and are required for proton secretion out of the cytoplasm. Mechanisms of extrinsic control of V-ATPase are poorly understood. Previous studies showed that glucose is an important regulator of V-ATPase assembly in Saccharomyces cerevisiae. Human V-ATPase directly interacts with aldolase, providing a coupling mechanism for glucose metabolism and V-ATPase function. Here we show that glucose is a crucial regulator of V-ATPase in renal epithelial cells and that the effect of glucose is mediated by phosphatidylinositol 3-kinase (PI3K). Glucose stimulates V-ATPase-dependent acidification of the intracellular compartments in human proximal tubular cells HK-2 and porcine renal epithelial cells LLC-PK1. Glucose induces rapid ATP-independent assembly of the V1 and Vo domains of V-ATPase and extensive translocation of the V-ATPase V1 and Vo domains between different membrane pools and between membranes and the cytoplasm. In HK-2 cells, glucose stimulates polarized translocation of V-ATPase to the apical plasma membrane. The effects of glucose on V-ATPase trafficking and assembly can be abolished by pretreatment with the PI3K inhibitor LY294002 and can be reproduced in glucose-deprived cells by adenoviral expression of the constitutively active catalytic subunit p110alpha of PI3K. Taken together these data provide evidence that, in renal epithelial cells, glucose plays an important role in the control of V-ATPase-dependent acidification of intracellular compartments and V-ATPase assembly and trafficking and that the effects of glucose are mediated by PI3K-dependent signaling.

Tight junctions play a key selectivity role in the paracellular conductance of ions. Paracellin-1 is a member of the tight junction claudin protein family and mutations in the paracellin-1 gene cause a human hereditary disease, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) with severe renal Mg2+ wasting. The mechanism of paracellin-1 function and its role in FHHNC are not known. Here, we report that in LLC-PK1 epithelial cells paracellin-1 modulated the ion selectivity of the tight junction by selectively and significantly increasing the permeability of Na+ (with no effects on Cl-) and generated a high permeability ratio of Na+ to Cl-. Mutagenesis studies identified a locus of amino acids in paracellin-1 critical for this function. Mg2+ flux across cell monolayers showed a far less-pronounced change (compared to monovalent alkali cations) following exogenous protein expression, suggesting that paracellin-1 did not form Mg2+-selective paracellular channels. We hypothesize that in the thick ascending limb of the nephron, paracellin-1 dysfunction, with a concomitant loss of cation selectivity, could contribute to the dissipation of the lumen-positive potential that is the driving force for the reabsorption of Mg2+.

From this study, we predicted that the human calcitonin gene-related peptide (hCGRP) fragment hCGRP-(8-37) would be a selective antagonist for CGRP receptors but an agonist for calcitonin (CT) receptors. In rat liver plasma membrane, where CGRP receptors predominate and CT appears to act through these receptors, hCGRP-(8-37) dose dependently displaced 125I-[Tyr0]rat CGRP binding. However, hCGRP-(8-37) had no effect on adenylate cyclase activity in liver plasma membrane. Furthermore, hCGRP-(8-37) inhibited adenylate cyclase activation induced not only by hCGRP but also by hCT. On the other hand, in LLC-PK1 cells, where calcitonin receptors are abundant and CGRP appears to act via these receptors, the bindings of 125I-[Tyr0]rat CGRP and 125I-hCT were both inhibited by hCGRP-(8-37). In contrast to liver membranes, interaction of hCGRP-(8-37) with these receptors led to stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) production in LLC-PK1 cells, and moreover, this fragment did not inhibit the increased production of cAMP induced not only by hCT but also by hCGRP. Thus hCGRP-(8-37) appears to be a useful tool for determining whether the action of CGRP as well as that of CT is mediated via specific CGRP receptors or CT receptors.

We have constructed stable cell lines expressing transporters for dopamine (DA), norepinephrine (NE), and serotonin (5-HT) by transfection with cloned cDNAs. The parental LLC-PK1 cell does not express any of these neurotransmitter transporters. Therefore, monoamine transport activities in each of these cell lines are due to the transfected DNA only, allowing comparison in the same background. Drug inhibition profiles for each cell line are distinct and as expected for each transporter. LLC-NET and LLC-DAT cells transported both NE and DA and both cell types exhibited a lower KM for DA transport than for NE transport. Analysis of Vmax data for LLC-NET cells suggests that substrate is bound to the NE transporter during the rate-limiting step(s) in transport. The cocaine analog 2-beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binds to each cell type, and is displaced by transport substrate in each case. Binding and transport measurements on parallel cell cultures allowed estimation of turnover numbers for norepinephrine, dopamine, and serotonin transporters. All three transporters require external Na+ and Cl-. The Na+ concentration dependence suggests that a single Na+ ion is involved in transport catalyzed by norepinephrine and serotonin transporters while more than one Na+ ion participate in transport mediated by the dopamine transporter.

Members of the large claudin family of tight junction (TJ) proteins create the differences in paracellular conductance and charge selectivity observed among different epithelia. Previous studies demonstrated that ionic charge selectivity is influenced by acidic or basic amino acids on the first extracellular domain of claudins. We noted two alternatively spliced variants of claudin-10 in the database, 10a and 10b, which are predicted to encode two different first extracellular domains and asked whether this might be a novel mechanism to generate two different permselectivities from a single gene. Using quantitative PCR, we found that claudin-10b is widely expressed among tissues including the kidney; however, claudin-10a is unique to the kidney. Using a nondiscriminating antibody, we found that claudin-10 (a plus b) is expressed in most segments of the nephron. In situ hybridization, however, showed that mRNA for 10a is concentrated in the cortex, and mRNA for 10b is more highly expressed in the medulla. Expression in Madin-Darby canine kidney (MDCK) II and LLC-PK1 cells reveals that both variants form low-resistance pores, and that claudin-10b is more selective for cations than claudin-10a. Charge-reversing mutations of cationic residues on 10a reveal positions that contribute to its anion selectivity. We conclude that alternative splicing of claudin-10 generates unique permselectivities and might contribute to the variable paracellular transport observed along the nephron.

ABCG2/MXR/ABCP1/BCRP is a member of the ATP-binding cassette membrane transporter, which consists of six transmembrane regions and one ATP-binding cassette. The transporter is known to be involved in the efflux of various anticancer compounds such as mitoxantrone, doxorubicin and topoisomerase I inhibitor. In this study, we analyzed the effects of polymorphisms in ABCG2, V12M and Q141K on transporter function. When polarized LLC-PK1 cells were transfected with variant ABCG2, drug-resistance to topoisomerase I inhibitor of cells expressing V12M or Q141K was less than 1/10 that of wild-type ABCG2 transfected cells, and was accompanied by increased drug accumulation and decreased drug efflux in the variant ABCG2-expressing cells. We further elucidated the molecular mechanisms of the transport dysfunction by investigating membrane localization and ATPase activity. Confocal microscopic analysis revealed that apical plasma membrane localization of V12M was disturbed, while the localization of wild-type transporters occurred specifically in the apical plasma membrane of polarized LLC-PK1 cells. Also, ATPase activities measured in the membrane of SF9 cells infected with variant ABCG2 showed that Q141K decreased activity by 1.3 below that of wild-type ABCG2. In addition, kinetic analysis of ATPase activity showed that the K(m) value in Q141K was 1.4-fold higher than that of wild-type ABCG2. These results indicated that naturally occurring SNPs alter transport functions of ABCG2 transporter and analysis of SNPs in ABCG2 may hold great importance in understanding the response/metabolism of chemotherapy compounds that act as substrates for ABCG2.

We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC-PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain.

Recent studies found that isolates of Toxoplasma gondii from Brazil were biologically and genetically different from those in North America and Europe. However, to date only a small number of isolates have been analysed from different animal hosts in Brazil. In the present study DNA samples of 46 T. gondii isolates from cats in 11 counties in São Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. An additional marker, CS3, that locates on chromosome VIIa and has previously been shown to be linked to acute virulence of T. gondii was also used to determine its association to virulence in mice. Genotyping of these 46 isolates revealed a high genetic diversity with 20 genotypes but no clonal Type I, II or III lineage was found. Two of the 46 isolates showed mixed infections. Combining genotyping data in this study with recent reported results from chickens, dogs and cats in Brazil (total 125 isolates) identified 48 genotypes and 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates identified from different hosts and locations are considered the common clonal lineages in Brazil. These lineages are designated as Types BrI, BrII, BrIII and BrIV. These results indicate that the T. gondii population in Brazil is highly diverse with a few successful clonal lineages expanded into wide geographical areas. In contrast to North America and Europe, where the Type II clonal lineage is overwhelmingly predominant, no Type II strain was identified from the 125 Brazil isolates. Analysis of mortality rates in infected mice indicates that Type BrI is highly virulent, Type BrIII is non-virulent, whilst Type BrII and BrIV lineages are intermediately virulent. In addition, allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. Thus, T. gondii has an epidemic population structure in Brazil and the major lineages have different biological traits.

Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin. Their proposed protective role in tumor development may prevail especially in the intestinal tract due to direct exposure of intestinal epithelia to these dietary ingredients. We have screened more than 30 flavonoids for their effects on cell proliferation and potential cytotoxicity in the human colon cancer cell lines Caco-2, displaying features of small intestinal epithelial cells, and HT-29, resembling colonic crypt cells. In addition, for selected compounds we assessed whether they induce apoptosis by determining caspase-3 activation. Studies on the dose dependent effects of the flavonoids showed antiproliferative activity of all compounds with EC50 values ranging between 39.7 +/- 2.3 microM (baicalein) and 203.6 +/- 15.5 microM (diosmin). In almost all cases, growth inhibition by the flavonoids occurred in the absence of cytotoxicity. There was no obvious structure-activity relationship in the antiproliferative effects either on basis of the subclasses (i.e., isoflavones, flavones, flavonols, flavonones) or with respect to kind or position of substituents within a class. In a subset of experiments we examined the antiproliferative activities of the most potent compound of each flavonoid subgroup in addition in LLC-PK1, a renal tubular cell line, and the human breast cancer cell line MCF-7. Out of four flavonols tested, three displayed almost equal antiproliferative activities in all cell lines but fisetin was less potent in MCF-7 cells. The flavanones bavachinin and flavanone inhibited growth of Caco-2 and HT-29 cells with lower EC50 values than that obtained in LLC-PK1 and MCF-7 cells. The lower susceptibility of LLC-PK1 and MCF-7 cells towards growth arrest was even more pronounced in the case of the flavone baicalein. Half maximal growth-inhibition in LLC-PK1 and MCF-7 required 2.5 and 6.6 fold higher concentrations than that needed in the intestinal cell lines. The flavonoids failed to affect apoptosis in LLC-PK1 and MCF-7, whereas baicalein and myricetin were able to induce apoptosis in HT-29 and Caco-2 cells. In conclusion, flavonoids of the flavone, flavonol, flavanone, and isoflavone classes possess antiproliferative effects in different cancer cell lines. The capability of flavonoids for growth inhibition and induction of apoptosis can not be predicted on the basis of their chemical composition and structure.

Using the yeast two-hybrid system and an in vitro binding assay, we have identified a novel protein termed vinexin as a vinculin-binding protein. By Northern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length. Screening for full-length cDNA clones and sequencing indicated that the two mRNA encode 82- and 37-kD polypeptides termed vinexin alpha and beta, respectively. Both forms of vinexin share a common carboxyl-terminal sequence containing three SH3 domains. The larger vinexin alpha contains an additional amino-terminal sequence. The interaction between vinexin and vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin. When expressed, vinexin alpha and beta localized to focal adhesions in NIH 3T3 fibroblasts, and to cell-cell junctions in epithelial LLC-PK1 cells. Furthermore, expression of vinexin increased focal adhesion size. Vinexin alpha also promoted upregulation of actin stress fiber formation. In addition, cell lines stably expressing vinexin beta showed enhanced cell spreading on fibronectin. These data identify vinexin as a novel focal adhesion and cell- cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organization and cell spreading.