Pig Kidney Epithelial Cells Research Articles

Novel organotin antibacterial and anticancer drugs

From the reaction of 6-( p-methoxyphenyl) fulvene ( 1a), 6-( p- N, N-dimethylaminophenyl) fulvene (1b) and 6-(3,4-dimethoxyphenyl) fulvene (1c) with LiBEt 3H, lithiated cyclopentadienide intermediates (2a– c) were synthesised. These intermediates were then transmetallated to tin with SnCl 4 to yield tetra-substituted bis(cyclopentadienyl)tin dichloride complexes (3a– c). Further reaction with tin tetrachloride yielded the benzyl-substituted derivatives bis-[( p-methoxybenzyl)cyclopentadienyl] tin(IV) dichloride (4a), bis-[( p- N,N-dimethylaminobenzyl)cyclopentadienyl] tin(IV) dichloride (4b), and bis-[(3,4-dimethoxyphenyl)cyclopentadienyl] tin(IV) dichloride ( 4c). Preliminary antibacterial tests were carried out using the Kirby–Bauer disk-diffusion method, in which 4a– c showed little to no activity against the Gram-negative bacterium Escherichia coli, but medium activity against Gram-positive bacteria (MRSA, MSSA). In addition, the organotin complexes had their cytotoxicity investigated through preliminary in vitro testing on the LLC-PK (pig kidney epithelial) cell line in order to determine their IC50 values. Compound 4c showed no cytotoxic activity, while 4a and 4b were found to have IC50 values of 15 and 205 μM, respectively.

Direct contact between boar spermatozoa and porcine oviductal epithelial cell (OEC) cultures is needed for optimal sperm survival in vitro

Oviductal epithelial cell (OEC) co-culture prolongs sperm viability and motility in vitro in a number of species including humans and horses. This study has sought to determine the effects of homologous OEC co-culture on boar sperm function. To determine whether the effects on spermatozoa were specifically caused by co-culture with or by OEC secretions, or by both factors together, a number of co-culture and cell-conditioned medium (CM) experiments were conducted. Firstly, Percoll-washed spermatozoa were co-cultured with OECs and pig kidney epithelial (LLC-PK1) cells, and in medium without cells. Secondly, Percoll-washed spermatozoa were incubated with CM derived from both OECs and LLC-PK1 cells and in unconditioned medium. A number of sperm function parameters were assessed after 5, 30, 60, 90, 120, and 180 min, and 24 h of co-culturing or incubation with CM. Of all the sperm function parameters investigated, the percentage (%) viability data yielded the most interesting results. OECs (mean ± S.E.M.; 31.2 ± 1.10) were better than LLC-PK1 cells (24.3 ± 0.93) at prolonging the viability of unbound spermatozoa after 24 h of co-culturing ( P < 0.05). Also after 24 h, the viability of spermatozoa bound to the OECs (77.6 ± 1.83) was significantly higher than in the case of the LLC-PK1 cells (53.5 ± 1.43; P < 0.001). Other sperm function parameters, e.g., capacitation and motility, were also influenced by OEC co-culturing and incubation with CM, although to a lesser degree. In conclusion, porcine homologous OEC co-culture and CM incubation specifically affect sperm function. However, we propose that it is OEC co-culturing, rather than OEC-CM, that has the greater influence.

Molecular characterization of the porcine GBP1 and GBP2 genes

Experimental evidence indicated that interferon-inducible guanylate-binding proteins (GBPs) are similar with genes in the myxovirus (Mx) resistance protein subfamily of the large GTPases protein family and play important roles in the resistance to intracellular pathogens. As more diseases exert significant influence on pig industry, it is anticipated that more candidate disease-resistance genes could be found in future strategies aimed at improving genetic resistance to infectious diseases. In this study, we cloned cDNA sequences and analyzed the genomic structure of porcine GBP1 (poGBP1) and GBP2 (poGBP2). The two genes were mapped to SSC4q21-q23 and SSC4q24 by the SCHP panel respectively, further IMpRH panel analysis showed both genes were most closely linked to the marker SWR153. The deduced amino acid sequences of these two genes share the same three classical GTP-binding motifs at the amino terminus and are less conserved at the carboxyl termini except for a CaaX motif, compared with human and mouse counterparts. The reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that poGBP1 and poGBP2 were both widely expressed in many tissues, and transient transfection indicated that poGBP1 and poGBP2 proteins were both located in cytoplasms within Pig Kidney Epithelial cells (PK15). Quantitative real-time PCR (Q-RT-PCR) analyses showed poGBP1 and poGBP2 had very similar expression patterns in PK15 cells at different time points after poly I:C stimulation, suggesting that ISRE (interferon-stimulated response element) plays a crucial role in the transcriptional regulation of these two genes. Four single nucleotide polymorphisms (SNPs) and three SNPs were detected in the cDNA sequences of poGBP1 and poGBP2, respectively. Association analyses revealed that the poGBP1 Eco81I and poGBP2 SspI polymorphisms both had significant associations ( p < 0.05) with red blood cell count (RBC), haemoglobin concentration (HGB) and hematocrit (HCT) of 17-day-old pigs.

Claudin-16 and claudin-19 interact and form a cation-selective tight junction complex

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an inherited disorder caused by mutations in the genes encoding the TJ proteins claudin-16 (CLDN16) and CLDN19; however, the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney are not understood. Here we showed that in pig kidney epithelial cells, CLDN19 functioned as a Cl(-) blocker, whereas CLDN16 functioned as a Na(+) channel. Mutant forms of CLDN19 that are associated with FHHNC were unable to block Cl(-) permeation. Coexpression of CLDN16 and CLDN19 generated cation selectivity of the TJ in a synergistic manner, and CLDN16 and CLDN19 were observed to interact using several criteria. In addition, disruption of this interaction by introduction of FHHNC-causing mutant forms of either CLDN16 or CLDN19 abolished their synergistic effect. Our data show that CLDN16 interacts with CLDN19 and that their association confers a TJ with cation selectivity, suggesting a mechanism for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC.

Tacrine-induced liver damage: an analysis of 19 candidate genes

Tacrine, the first acetylcholinesterase inhibitor used in the treatment of Alzheimer's disease, is associated with transaminase elevation in up to 50% of patients. The mechanism of tacrine-induced liver damage is not fully understood, but earlier studies have suggested that genetic factors may play a role. Our aim was to investigate whether single-nucleotide polymorphisms (SNPs) in 19 candidate genes were associated with tacrine-induced liver damage. Sixty-nine patients of Caucasian origin treated with tacrine for Alzheimer's disease were investigated by genotyping 241 SNPs in 19 candidate genes potentially related to hepatotoxicity. The association with ABCB4 [which encodes MultiDrug Resistance Protein 3 (MDR3)] was explored in transepithelial transport studies using the ABCB4-transfected pig kidney epithelial cell line (LLC-PK1). The strongest association between alanine aminotransferase levels and three SNPs within ATP-binding cassette, subfamily B (MDR/TAP), member 4 (ABCB4) (uncorrected P=0.0005) was not significant after adjusting for multiple testing. No association was demonstrated with ATP-binding cassette, subfamily B (MDR/TAP), member 1 (ABCB1) or carnitine O-octanoyltransferase (CROT) which are located adjacent to ABCB4. Using the transepithelial transport system we failed to show a difference in tacrine accumulation between ABCB4-transfected and parental cell lines. The association with ABCB4 warrants further testing using either another population and/or functional studies.

Synthesis and cytotoxicity studies of methoxy benzyl substituted titanocenes

From the reaction of 6(2-methoxy-phenyl)fulvene ( 1a), 6(3-methoxy-phenyl)fulvene ( 1b), 6(3,4-dimethoxy-phenyl)fulvene ( 1c) and 6(3,4,5-trimethoxy-phenyl)fulvene ( 1d) with LiBEt 3H, lithiated cyclopentadienide intermediates 2a– d were synthesised. These intermediates were then transmetallated to titanium with TiCl 4 to give benzyl substituted titanocenes bis-[(2-methoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride ( 3a), bis-[(3-methoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride ( 3b), bis-[(3,4-dimethoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride ( 3c) and bis-[(3,4,5-trimethoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride ( 3d). The three titanocenes 3a– c were characterised by single crystal X-ray diffraction, while the structure of the fourth titanocene 3d was elucidated through a DFT calculation. All four titanocenes had their cytotoxicity investigated through preliminary in vitro testing on the LLC-PK (pig kidney epithelial) cell line in order to determine their IC 50 values. Titanocenes 3a– d were found to have IC 50 values of 97, 159, 88 and 253 μM, respectively. All four titanocene derivatives show significant cytotoxicity improvement when compared to unsubstituted titanocene dichloride.

Synthesis and cytotoxicity studies of titanocene C analogues.

From the carbolithiation of 6--dimethylamino fulvene (3) and 2,4[bis(-dimethylamino)methyl]--methylpyrrolyl lithium (2a), -(-dimethylaminomethyl)benzimidazolyl lithium (2b), or -(-dimethylamino)methylphenyl lithium (2c), the corresponding lithium cyclopentadienide intermediate (4a–c) was formed. These three lithiated intermediates underwent a transmetallation reaction with TiCl4' resulting in -dimethylamino-functionalised titanocenes 5a–c. When these titanocenes were tested against a pig kidney epithelial cell line (LLC-PK), the values obtained were of 23, and 52 M for titanocenes 5a and 5b, respectively. The most cytotoxic titanocene in this paper, 5c with an value of 13 M, was found to be approximately two times less cytotoxic than its analogue Titanocene C ( M) and almost four times less cytotoxic than cisplatin, which showed an value of 3.3 M when tested on the LLC-PK cell line.

In vitro P-Glycoprotein-Mediated Transport of (R)-, (S)-, (R,S)-Methadone, LAAM and Their Main Metabolites

Methadone and L-α-acetylmethadol (LAAM) are used as treatment for opiate addiction. Using a cellular model, we aimed to determine if methadone, LAAM and their main metabolites are substrates of the human P-glycoprotein transporter (P-gp), which is encoded by the ABCB1 gene, and whether methadone transport exhibits stereoselectivity. Pig kidney epithelial cells (control) and human ABCB1-transfected cells were incubated with methadone, LAAM and their metabolites, and their intra- and extracellular concentrations were measured. The intra- to extracellular ratios of methadone, LAAM and their metabolites were all decreased in ABCB1-transfected cells compared to controls (p < 0.05), thus indicating that they are substrates of P-gp. A weak stereoselectivity in methadone transport was observed towards the (S)-enantiomer. P-gp may therefore affect the pharmacokinetics and pharmacodynamics of methadone and LAAM.

Diheteroarylmethyl substituted titanocenes: A novel class of possible anti-cancer drugs

From the reaction of tert-butyl lithium or n-butyl lithium with N-methylpyrrole ( 1a), furan ( 1b) or 2-bromo-thiophen ( 1c), 2- N-methylpyrrolyl lithium ( 2a), 2-furyl lithium ( 2b) or 2-thiophenyl lithium ( 2c), respectively, was obtained. When reacted with 6-(2- N-methylpyrrolyl) fulvene ( 3a), 6-(2-furyl) fulvene ( 3b) or 6-(2-thiophenyl) fulvene ( 3c), the corresponding lithiated intermediates were formed ( 4a– c). Titanocenes ( 5a– c) were obtained through transmetallation with titanium tetrachloride. When these titanocenes were tested against pig kidney epithelial (LLC-PK) cells, inhibitory concentrations (IC 50) of 32 μM, 140 μM, and 240 μM, respectively, were observed. These values represent improved cytotoxicity against LLC-PK, compared to their ansa-analogues.

Characterization of different expression patterns of calsarcin-1 and calsarcin-2 in porcine muscle

Calsarcins comprise a novel family of muscle-specific calcineurin-interacting proteins and play an important role in modulating both the function and substrate specificity of calcineurin in muscle cells. In this study, we cloned and characterized calsarcins from pig muscle. The deduced amino acid sequences of porcine calsarcin-1 (CS-1), calsarcin-2 (CS-2), and calsarcin-3 (CS-3) share the same putative calcineurin and α-actinin binding regions. Radiation hybrid mapping data indicate that CS-1 and CS-2 map to q2.1–2.5 of pig chromosome 8 (SSC8) and q2.4 of pig chromosome 14 (SSC14), respectively. The mRNA expressions of both CS-1 and CS-2 are regulated in skeletal muscle similarly during postnatal development but not during prenatal development, indicating differences in function, additionally demonstrated by minute differences in cellular localization within Pig Kidney Epithelial cells (PK15). We provide the first evidence that CS-1 is abundantly expressed in porcine heart and has an expression pattern similar to the human gene. This result suggests that the pig may be a suitable animal model to study the function of calsarcins in human heart disease.

Glucose activates H(+)-ATPase in kidney epithelial cells.

The vacuolar H(+)-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H(+)-ATPase remain largely unknown. The present study examines the effect of glucose on H(+)-ATPase activity in the pig kidney epithelial cell line LLC-PK(1). Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH(3)/NH(4)(+) prepulse, and rates of intracellular pH (pH(i)) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 microM ethylisopropylamiloride and were K(+) free to eliminate Na(+)/H(+) exchange and H(+)-K(+)-ATPase activity. After NH(3)/NH(4)(+)-induced acidification, LLC-PK(1) cells had a significant pH(i) recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pH(i) recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pH(i) recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H(+)-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-d-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 microM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification.

Interaction of imatinib mesilate with human P-glycoprotein.

The interaction of imatinib mesilate with P-glycoprotein (P-gp) was examined using pig kidney epithelial LLC-PK1 cells versus L-MDR1 cells, which overexpress human P-gp on the apical membrane. The basal-to-apical transport of imatinib mesilate in L-MDR1 cells significantly exceeded that in the parental LLCPK1 cells. The intracellular accumulation of imatinib mesilate after its basal application to LLC-PK1 and L-MDR1 cells was 35% and 15%, respectively. A P-gp modulator, cyclosporin A, inhibited the basal-to-apical transport in L-MDR1 cells. The intracellular accumulation of imatinib mesilate in L-MDR1 cells was also increased by cyclosporin A. The rhodamine 123 efflux assay showed that the efflux of rhodamine 123 in K562/DXR cells, which overexpress human P-gp, could be blocked markedly by imatinib mesilate in a dose-dependent fashion. The Ki values for the inhibition of P-gp function by cyclosporin A and imatinib mesilate were estimated to be 6.1 and 18.3 muM, respectively, using a calcein-AM efflux assay. These observations demonstrate that imatinib mesilate is a substrate as well as a modulator of human P-gp, suggesting that imatinib mesilate drug interactions may occur via P-gp. It is necessary to consider the pharmacokinetic and pharmacodynamic interactions of imatinib mesilate with other drugs via P-gp.

Pharmacological antagonism of fumonisin B1 cytotoxicity in porcine renal epithelial cells (LLC-PK1): a model for reducing fumonisin-induced nephrotoxicity in vivo.

Fumonisin B1 is a mycotoxin commonly found on corn. It is hepatotoxic and nephrotoxic in domestic and experimental animals, and causes equine leukoencephalomalacia and porcine pulmonary oedema. It is a potent inhibitor of ceramide synthase. Inhibition leads to accumulation of free sphingoid bases in cells and tissues. In pig kidney epithelial cells (LLC-PK1), fumonisin B1 induces increased tumour necrosis factor alpha (TNFalpha) expression independent of the accumulation of sphingoid bases. The objective of this study was to investigate pharmacological approaches for intervening in fumonisin B1 toxicity using the LLC-PK1 cell model. The toxicity of fumonisin B1 was assayed using cell viability and lactate dehydrogenase (lactate dehydrogenase) release. Pretreatment of cells with myriocin, preventing sphinganine accumulates, prevented the fumonisin B1-induced decrease in cell viability and increased lactate dehydrogenase release. Modulation of adenosine receptor activity did not reduce the fumonisin B1 cytotoxicity. As with myriocin, silymarin pretreatment prevented the fumonisin B1-induced effects on cell viability and lactate dehydrogenase release. When added 6 or 24 hr after treatment of cells with fumonisin B1, both myriocin and silymarin reversed the decreased cell viability and suppressed the increased lactate dehydrogenase release. Myriocin, but not silymarin, blocked the accumulation of sphinganine in fumonisin B1-treated cells. Silymarin, unlike myriocin, induced expression of TNFalpha to an extent similar to fumonisin B1, but pretreatment with silymarin decreased the fumonisin B1-induced TNFalpha expression in LLC-PK1 cells. Results suggest that the mechanisms by which myriocin and silymarin protect renal cells are different, and silymarin potentially prevents fumonisin B1-induced toxicity by modulating TNFalpha expression or signals downstream of the inhibition of ceramide synthase.

Prostaglandin D2 synthase induces apoptosis in pig kidney LLC-PK1 cells

Prostaglandin D(2) synthase (PGD(2)S), a unique member of the lipocalin family, is found at elevated levels in the serum of patients with renal impairment and has recently been implicated as a new biochemical marker of renal insufficiency. The aim of this study was to investigate the apoptotic effects of PGD2S on a pig kidney epithelial cell line (LLC-PK1) and to investigate the effects of prostaglandins and growth factors on this process. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL), annexin V staining, and electron microscopy. A four- to fivefold increase in apoptosis was observed in PGD(2)S-treated cells as compared with controls and the apoptosis appeared to act via caspase-3. A cyclooxygenase-2 inhibitor, anti-PGD(2)S antibody, and selenium all significantly inhibited the apoptosis induced by PGD(2)S; however, none had any effect on the apoptosis induced by the known apoptotic inducer camptothecin. Furthermore, prostaglandins E(1) and E(2), known to induce mitogen-activated protein (MAP) kinase phosphorylation and exhibit cytoprotective effects, both inhibited PGD(2)S-induced apoptosis, while prostaglandin H(2) had no significant effect. Growth factors such as insulin, insulin-like growth factor-1, and platelet-derived growth factor also decreased PGD(2)S-induced apoptosis. In addition, PGD(2)S isolated from human serum seemed slightly more effective at inducing apoptosis than recombinantly expressed protein. We report on the induction of apoptosis by PGD(2)S in LLC-PK1 pig kidney epithelial cells, and speculate that the accumulation of PGD(2)S in the serum of kidney failure patients may further exacerbate renal problems and is most likely regulated by other prostaglandins and growth factors.

Multidrug-resistance P-glycoprotein (MDR1) secretes platelet-activating factor

The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed. Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell. In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane. We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF). PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. [14C]PAF synthesized intracellularly from exogenous alkylacetylglycerol and [14C]choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport. Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A. Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1). It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity. While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth.

Multidrug-resistance P-glycoprotein (MDR1) secretes platelet-activating factor.

The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed. Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell. In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane. We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF). PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. [(14)C]PAF synthesized intracellularly from exogenous alkylacetylglycerol and [(14)C]choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport. Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A. Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1). It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity. While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth.

Functional cystic fibrosis transmembrane conductance regulator tagged with an epitope of the vesicular stomatis virus glycoprotein can be addressed to the apical domain of polarized cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel apically localized in epithelial cells. In cystic fibrosis patients, the gene encoding this N-linked glycoprotein is mutated. About 70% of CF patients express a mutated form of CFTR, deleted at the phenylalanine residue at position 508 (deltaF508). CFTR-deltaF508 fails to exit the endoplasmic reticulum; it remains incompletely glycosylated and is rapidly degraded. To optimize CFTR detection for membrane localization studies and biochemical studies, we tagged wild-type and deltaF508 CFTR with the VSV-G epitope at their carboxy-terminal ends. We have generated pig kidney epithelial cell clones (LLCPK1) expressing VSV-G-tagged human wild-type and deltaF508-CFTR. In CFTR-expressing cells, the transfected protein is maturated and transported to the apical membrane where it is concentrated. The cells exhibit a strong anion channel activity after stimulation by cAMP, as demonstrated by a halide sensitive fluorescent dye assay (6-methoxy-N-ethylquinominium, SPQ), and whole-cell patch-clamp approach. This activity of CFTR-VSV-G is indistinguishable from the wild-type CFTR. In contrast, in cells expressing tagged deltaF508-CFTR or in non-transfected cells, no anion channel activity could be detected after stimulation by cAMP. In deltaF508-CFTR-VSV-G-expressing cells, the mutated CFTR remained in the incompletely glycosylated form and was localized in the endoplasmic reticulum. These cell lines reproduce the cellular fate of wild-type and mutated CFTR-deltaF508. To our knowledge, they are the first differentiated epithelial cell lines stably expressing tagged CFTR and CFTR-deltaF508 in which cellular processing and functional activity of these two proteins are reproduced. Thus the addition of the VSV-G epitope does not impair the localization and function of CFTR, and these cell lines can be used to examine CFTR function in vitro.

Evidence for complement activation in type 1 diabetes

Type 1 diabetes is caused by immune mediated destruction of the beta cells in the pancreatic islets of Lmgerhans. There is evidence that complement activation plays a role in this immune mediated cell death. We have demonstrated a trend towards increased terminal complement complex (TCC, SC5b-9) concentrations in cell culture supernatents of BRIN BDI 1 cells (a rat beta cell line) incubated with 10% v/v serum from patients with newly diagnosed type I diabetes for 24 hours than in supmatents from cells incubated with non-diabetic serum (mean 10,765 (sd 4640) n g / d vs 7939 (sd 1935) ng/mL). Cell viability, assessed by the internal LDH assay, was no different between cells incubated with diabetic and non-diabetic serum. This effect could be abolished by heat treating serum to inactivate complement. TCC levels measured in culture supernatent of pig kidney epithelial cell line (LLC PKI) with 10% diabetic serum were 1007 (sd 95) ng/mL after 24 hours and was 1572 (sd 197) ng/mL with 10 % normal human serum when LLC PKI cells were cultured at the same density as the BDl I cells. This indicates complement activation is cell specific. These results suggest that diabetic serum contains factors that specifically fix complement on beta cells (most likely complement fixing antiislet cell antibodies). Evidence for complement activation in type 1 diabetes 30 Immuno-modulatory mechanisms of E.coli heat labile toxin

Residues of the Fourth Transmembrane Segments of the Na,K-ATPase and the Gastric H,K-ATPase Contribute to Cation Selectivity

We have generated protein chimeras to investigate the role of the fourth transmembrane segments (TM4) of the Na,K- and gastric H, K-ATPases in determining the distinct cation selectivities of these two pumps. Based on a helical wheel analysis, three residues of TM4 of the Na,K-ATPase were changed to their H,K-counterparts. A construct carrying three mutations in TM4 (L319F, N326Y, and T340S) and two control constructs were heterologously expressed in Xenopus laevis oocytes and in the pig kidney epithelial cell line LLC-PK(1). Biochemical ATPase assays demonstrated a large sodium-independent ATPase activity at pH 6.0 for the pump carrying the TM4 substitutions, whereas the control constructs exhibited little or no activity in the absence of sodium. Furthermore, at pH 6.0 the K(1/2)(Na(+)) shifted to 1.5 mM for the TM4 construct compared with 9.4 and 5.9 mM for the controls. In contrast, at pH 7.5 all three constructs had characteristics similar to wild type Na,K-ATPase. Large increases in K(1/2)(K(+)) were observed for the TM4 construct compared with the control constructs both in two-electrode voltage clamp experiments in Xenopus oocytes and in ATPase assays. ATPase assays also revealed a 10-fold shift in vanadate sensitivity for the TM4 construct. Based on these findings, it appears that the three identified TM4 residues play an important role in determining both the specific cation selectivities and the E(1)/E(2) conformational equilibria of the Na,K- and H,K-ATPase.

Serine palmitoyltransferase inhibition reverses anti-proliferative effects of ceramide synthase inhibition in cultured renal cells and suppresses free sphingoid base accumulation in kidney of BALBc mice

The purpose of this study was to determine the ability of the fungal serine palmitoyltransferase (SPT) inhibitor, myriocin, to prevent the anti-proliferative and cytotoxic effects of fumonisin B 1 in cultured pig kidney epithelial cells, LLC-PK 1. In an earlier study with LLC-PK 1 cells, β-chloroalanine (a nonspecific SPT inhibitor) was found to inhibit the fumonisin-induced accumulation of free sphinganine by >90% but only partially reversed (50–60%) fumonisin’s antiproliferative and cytotoxic effects. β-Chloroalanine is not the ideal SPT inhibitor for this type of study because it also inhibits other pyridoxal 5′-phosphate-dependent enzymes. A potent and selective fungal SPT inhibitor (myriocin) was partially purified from liquid cultures of Isaria (= Cordyceps) sinclairii by a combination of organic extraction and column chromatography. The various fractions were bioassayed for their ability to inhibit fumonisin-induced sphinganine accumulation in LLC-PK 1 cells. The activity in partially purified material was compared to the activity of highly purified myriocin and the results expressed as myriocin equivalents. The estimated IC 50 and IC 95 for inhibition of fumonisin-induced sphinganine accumulation were approximately 1.8 and 22 nM, respectively. The IC 95 concentration of the fungal SPT inhibitor reversed the antiproliferative effects and prevented fumonisin-induced apoptosis after 48 h exposure to 50 μM fumonisin B 1. The SPT inhibitor was also effective at reducing free sphinganine in vivo. Free sphinganine concentration was reduced 60% in kidney of mice injected i.p. with SPT inhibitor plus fumonisin B 1 when compared to fumonisin B 1 alone. The ability of SPT inhibition to reduce fumonisin B 1-induced sphinganine accumulation in vivo may be useful in the development of therapeutic agents for treatment of animals suspected to have been exposed to toxic levels of fumonisin in feeds.