The multidrug transporter MATE1 sequesters OCs within an intracellular compartment that has no influence on OC secretion in renal proximal tubules.

Secretion of organic cations (OCs) across renal proximal tubules (RPTs) involves basolateral OC transporter (OCT)2-mediated uptake from the blood followed by apical multidrug and toxin extruder (MATE)1/2-mediated efflux into the tubule filtrate. Whereas OCT2 supports electrogenic OC uniport, MATE is an OC/H(+) exchanger. As assessed by epifluorescence microscopy, cultured Chinese hamster ovary (CHO) cells that stably expressed human MATE1 accumulated the fluorescent OC N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in human OCT2-expressing cells was largely restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of efflux of the prototypic OC [(3)H]1-methyl-4-phenylpyridinium (MPP) from MATE1-expressing CHO cells. Punctate accumulation of NBD-MTMA was markedly reduced by coexposure of MATE1-expressing cells with 5 µM bafilomycin (BAF), an inhibitor of V-type H(+)-ATPase, and accumulation of [(3)H]MPP and [(3)H]NBD-MTMA was reduced by >30% by coexposure with 5 µM BAF. BAF had no effect on the initial rate of MATE1-mediated uptake of NBD-MTMA, suggesting that the influence of BAF was a secondary effect involving inhibition of V-type H(+)-ATPase. The accumulation of [(3)H]MPP by isolated single nonperfused rabbit RPTs was also reduced >30% by coexposure to 5 µM BAF, suggesting that the native expression in RPTs of MATE protein within endosomes can increase steady-state OC accumulation. However, the rate of [(3)H]MPP secretion by isolated single perfused rabbit RPTs was not affected by 5 µM BAF, suggesting that vesicles loaded with OCs(+) are not likely to recycle into the apical plasma membrane at a rate sufficient to provide a parallel pathway for OC secretion.

Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.

Expulsion of gastrointestinal nematodes is associated with pronounced mucosal mast cell (MMC) hyperplasia, differentiation, and activation, accompanied by the systemic release of MMC granule chymases (chymotrypsin-like serine proteases). The beta-chymase mouse mast cell protease-1 (mMCP-1) is expressed predominantly by intraepithelial MMCs, and levels in the bloodstream and intestinal lumen are maximal at the time of worm expulsion in parasitized mice. To address the in vivo functions of MMC-specific beta-chymases, we have generated transgenic mice that lack the mMCP-1 gene. They were backcrossed onto a congenic BALB/c background to investigate the response to nematode infection. The deletion of the mMCP-1 gene is associated with significantly delayed expulsion of Trichinella spiralis and increased deposition of muscle larvae in BALB/c mice despite the presence of normal and sometimes increased numbers of MMCs. Neither worm fecundity nor worm burdens were altered in Nippostrongylus-infected mMCP-1(-/)- BALB/c mice. These data demonstrate, for the first time, that the ablation of an MMC-derived effector molecule compromises the expulsion process.

Characterization of the disposition and toxicokinetics of N-butylpyridinium chloride in male F-344 rats and female B6C3F1 mice and its transport by organic cation transporter 2.

Studies were conducted to characterize the effect of dose and route of administration on the disposition of N-butylpyridinium chloride (NBuPy-Cl), an ionic liquid with solvent properties. Urine was the major route of NBuPy-Cl excretion after intravenous (5 mg/kg), single oral (0.5, 5, or 50 mg/kg), or repeated oral (50 mg/kg/day, 5 days) administration to male F-344 rats and single oral (50 mg/kg) administration to female B6C3F1 mice. Depending on the vehicle, absorption after dermal application (5 mg/kg, 125 microg/cm(2)) was 10 to 35% at 96 h. After the single intravenous dose, the blood concentration of NBuPy-Cl decreased in a biphasic manner with an elimination half-life of 2.2 h and a clearance of 7 ml/min. After single oral administration of NBuPy-Cl (50 mg/kg), maximum blood concentration was reached at 1.3 h, and the bioavailability was determined to be 47% at 6 h based on the blood toxicokinetics and 67% at 72 h based on urinary excretion. In all the urine and blood samples, only the parent compound was detected. Coadministration of NBuPy-Cl and inulin (by intravenous injection) revealed that the clearance of NBuPy-Cl exceeded the rat glomerular filtration rate. After incubation with Chinese hamster ovary cells expressing human organic cation transporter 2 (hOCT2), NBuPy-Cl was transported effectively (K(t) = 18 microM), and also a potent inhibitor of hOCT2 mediated tetraethylammonium transport (IC(50) = 2.3 microM). In summary, NBuPy-Cl is partially absorbed from the gastrointestinal tract and eliminated rapidly in the urine as parent compound most likely by renal glomerular filtration and OCT2-mediated secretion.

Transport of Dissolved Amino Acids by the Mussel, Mytilus edulis: Demonstration of Net Uptake from Natural Seawater.

High-performance liquid chromatography provides direct evidence for substantial removal of naturally occurring specific free amino acids during a single passage of water through the mantle cavity of mussels. This occurs during the few seconds required for passage of the water across the gill, and removal proceeds unabated at ambient substrate concentrations as low as 38 nanomoles per liter.

Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes.

When DNA molecules are injected into Xenopus oocyte nuclei, they can recombine with each other. With bacteriophage lambda DNAs, it was shown that this recombination is stimulated greatly by introduction of double-strand breaks into the substrates and is dependent on homologous overlaps in the recombination interval. With plasmid DNAs it was shown that little or no recombination occurs between circular molecules but both intra- and intermolecular events take place very efficiently with linear molecules. As with the lambda substrates, homology was required to support recombination; no simple joining of ends was observed. Blockage of DNA ends with nonhomologous sequences interfered with recombination, indicating that ends are used directly to initiate homologous interactions. These observations are combined to evaluate possible models of recombination in the oocytes. Because each oocyte is capable of recombining nanogram quantities of linear DNA, this system offers exceptional opportunities for detailed molecular analysis of the recombination process in a higher organism.

Tetrapentylammonium (TPeA): slowly dissociating inhibitor of the renal peritubular organic cation transporter.

The efflux of tetraethylammonium (TEA) from suspensions of rabbit renal proximal tubules is completely blocked by 500 microM tetrapentylammonium (TPeA) in the extracellular medium. The basis of this trans-inhibition of TEA transport by TPeA was examined in tubule suspensions. At TPeA concentrations < 10 microM, efflux of TEA was reduced by approximately 50%, whereas at concentrations > 10 microM, TPeA reduced efflux an additional 50% to produce a near complete block of TEA efflux. Increasing concentrations of TPeA from 0-500 microM were found to produce a biphasic, concentration-dependent trans-inhibition of TEA efflux from tubule suspensions suggesting that TPeA may block efflux by binding to both a high and low affinity TPeA binding site. The trans-inhibition of TEA efflux by TPeA at low concentrations (< 10 microM) may result from a slow carrier turnover when TPeA is bound to the carrier site. To determine whether the inhibitory effectiveness of TPeA was also associated with its slow dissociation from the carrier site, the effect of a 10 s preincubation with 1 microM TPeA on TEA uptake was examined. The uptake of TEA by tubules preincubated for 10 s with TPeA was reduced by approximately 30-50% compared to control tubules not preincubated with TPeA. A 10 s preincubation with 150 microM unlabeled TEA had no effect on TEA uptake compared to control tubules not preincubated with TEA. When the 10 s preincubation with 1 microM TPeA was followed by a 10 min recovery period, TEA uptake returned to control levels, indicating that the prolonged inhibition was reversible. This prolonged inhibition of TEA uptake after a 10 s preincubation with 1 microM TPeA, as suspected, may arise from a slow dissociation of TPeA from the OC transporter following a rapid association to the binding site. TPeA inhibition of TEA uptake into tubules was competitive in nature with a Ki of 1 microM. The ability of TEA to compete with TPeA for binding to the carrier suggests that the binding of TPeA to the carrier can be displaced by large concentrations of TEA. These observations suggest that the interactions of TPeA, and perhaps similarly large hydrophobic OCs, with the OC transporter are complex.

Paraquat2+/H+ exchange in isolated renal brush-border membrane vesicles.

The mechanism(s) by which paraquat (1,1'-dimethyl-4,4'-bipyridinium), a divalent organic cation (OC) and proximal tubule nephrotoxicant, crosses renal cell membranes is unclear. The structurally-related monovalent OC, 1-methyl-4-phenylpyridinium (MPP+), crosses the renal brush border via OC/H+ exchange using the same pathway by which tetraethylammonium (TEA) is transported. We examined whether paraquat shares the TEA(MPP+)/H+ exchanger by examining 14C-paraquat transport in rabbit renal BBMV. Compared to a pH equilibrium condition (pH 7.5in:7.5o), an H-gradient (pH 6in:7.5o) stimulated the 5 s and 60 s uptakes of 230 microM paraquat by 51% and 108%, respectively, and this stimulation was blocked by both 20 mM unlabeled paraquat and TEA. Pre-loading BBMV with 2 mM unlabeled TEA (under conditions of pH equilibrium) stimulated by 3-fold the 60 s uptake of 120 microM paraquat and by 5 min produced a transient intravesicular accumulation of paraquat that exceeded equilibrium (2 h) uptake by 45%. The presence of 200 microM paraquat in the extravesicular solution competitively inhibited H-gradient-stimulated transport of 14C-TEA in renal BBMV, increasing the apparent Kt for TEA transport from 169 microM to 379 microM, without significantly influencing the Jmax (16.0 vs. 15.4 nmol mg-1 min-1). The calculated Ki for paraquat (presumably equal to its Kt for transport) after transport was between 160 and 220 microM (depending upon the method of estimation). Significantly, the Kt for MPP+/H exchange is 12 microM, suggesting that the affinity of the exchanger is profoundly influenced by the presence on paraquat of a second positive charge. We conclude that renal transport of paraquat involves the OC/H+ exchanger of proximal cell luminal membranes and that this pathway may play a role in the renal secretion of polyvalent organic cations.

Effect of pH on the transport of Krebs cycle intermediates in renal brush border membranes.

Lowering extravesicular pH stimulated Na+-dependent citrate transport in renal brush border membrane vesicles: e.g., at pHout = 5.5, the initial rate of citrate uptake was increased 10-fold compared to parallel control experiments at pH 7.5. The same experimental conditions had little effect on succinate uptake. The influence of pH on citrate transport is a product of the extravesicular H+ concentration; pH gradients did not potentiate the effects nor were proton gradients capable of driving transport in the absence of Na+. The effect of pH is adequately explained if only the mono- and divalent species of citrate (Cit1-, Cit2-) are considered acceptable substrates for transport. The stimulatory influence of pH on transport correlated quite well with pH-related increases in the concentrations of Cit1- and Cit2-, and over the same pH range [Cit3-] was inversely related to citrate uptake. A model of the Na+-dependent dicarboxylate transport system is discussed in which three sodium ions are translocated per molecule of dicarboxylic acid.

Na+-dependent transport of tricarboxylic acid cycle intermediates by renal brush border membranes. Effects on fluorescence of a potential-sensitive cyanine dye.

The effect of the transport of tricarboxylic acid cycle intermediates on the membrane potential of renal brush border vesicles was studied using fluorescence of the cyanine dye, 3,3'-dipropylthiadicarbocyanine iodide. The behavior of the dye in the preparation was established with valinomycin-induced K+-diffusion potentials; increases in fluorescence were associated with depolarizing conditions. Addition of 1 mm succinate or citrate to membrane/dye suspensions produced transient increases in fluorescence, indicative of a depolarizing event(s) associated with the transport of these substrates. The transient response in fluorescence was Na+ dependent, of greater magnitude under Na+-gradient as compared to Na+-equilibrium conditions, and was a saturable function of substrate concentration. The specificity of the fluorescence response was identical to that obtained from studies of the competitive inhibition of succinate transport by tricarboxylic acid cycle intermediates and analogs We conclude that the major tricarboxylic acid cycle intermediates are transported via a common Na+-dependent transport system in renal brush border membranes.

High-affinity phlorizin binding in Mytilus gill.

The gill of the marine mussel, Mytilus, contains a high affinity, Na-dependent D-glucose transporter capable of accumulating glucose directly from sea water. We examined the ability of the beta-glucoside, phlorizin, to act as a high-affinity ligand of this process in intact gills and isolated brush border membrane vesicles (BBMV). The time course of association of nanomolar [3H]phlorizin to gills and BBMV was slow, with t50 values between 10 and 30 min, and a half-time for dissociation of approx. 30 min. 1 mM D-glucose reduced equilibrium binding of 1 nM phlorizin by 90-95%, indicating that there was little non-specific binding of this ligand to the gill. In addition, there was little, if any, hydrolysis by the gill of phlorizin to its constituents, glucose and phloretin. Phlorizin binding to gills and BBMV was significantly inhibited by the addition of 50 microM concentrations of D-glucose and alpha-methyl-D-glucose, and unaffected by the addition of L-glucose and fructose. Binding to gills and BBMV was reduced by greater than 90% when Na+ was replaced by K+. Replacement of Na+ by Li+ effectively blocked binding to the intact gill, although Li+ did support a limited amount of glucose-specific phlorizin binding in BBMV. The Kd values for glucose-specific phlorizin binding in intact gills and BBMV were 0.5 nM and 6 nM, respectively. We conclude that phlorizin binds with extremely high affinity to the Na-dependent glucose transporter of Mytilus gill, which may be useful in future efforts to isolate and purify the protein(s) involved in integumental glucose transport.

Genetic recombination of Xenopus laevis 5 S DNA in bacteria.

The behavior in genetic recombination of Xenopus laevis 5 S DNA has been examined, with particular emphasis on the role of 15-base-pair tandem repeats in the A + T-rich spacer. Fragments of 5 S DNA were introduced into Escherichia coli cells as inserts in the recombination vectors, lambda rva and lambda rvb. Intermolecular recombinants were selected in which, because of properties of the phage vectors, the crossover event must have occurred within the 5 S DNA inserts. Inserts from individual recombinants have been characterized in detail. The effects of varying the number (n) of 15-base-pair repeats and the recombination capabilities of the phage and host have been investigated. In these crosses, unequal crossovers can occur, yielding inserts different in size from the parental inserts. When the number of 15-mers is large (n = 12 or 20), most of the unequal crossovers have occurred within the 15-mers, resulting in an altered n value, although other homologies within the 5 S DNA sequence can also support unequal events. Increasing n in the parental inserts modestly increases the overall frequency of recombination and the percentage of altered inserts. We conclude that, in a bacterial setting, the 15-base-pair repeats stimulate recombination only slightly by allowing alternative registers for heteroduplex formation. The degree of stimulation observed is less than predicted by one simple model.

pH and drug resistance. II. Turnover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs.

Resistance to chemotherapeutic agents is a major cause of treatment failure in patients with cancer. The primary mechanism leading to a multidrug-resistant phenotype is assumed to be plasma-membrane localized overexpression of drug efflux transporters, such as P-glycoprotein (P-gp). However, acidic intracellular organelles can also participate in resistance to chemotherapeutic drugs. In this study, we investigated, both experimentally and theoretically, the effect of acidic vesicle turnover on drug resistance. We have developed a general model to account for multiple mechanisms of resistance to weakly basic organic cations, e.g. anthracyclines and Vinca alkaloids. The model predicts that lower cytosolic concentrations of drugs can be achieved through a combination of high endosomal turnover rates, a low endosomal pH, and an alkaline-inside pH gradient between cytosol and the extracellular fluid. Measured values for these parameters have been inserted into the model. Computations using conservative values of all parameters indicate that turnover of acidic vesicles can be an important contributor to the drug-resistant phenotype, especially if vesicles contain an active uptake system, such as H+/cation exchange. Even conservative estimates of organic cation-proton antiport activity would be sufficient to make endosomal drug extrusion a potent mechanism of resistance to weakly basic drugs. The effectiveness of such a drug export mechanism would be comparable to drug extrusion via drug pumps such as P-gp. Thus, turnover of acidic vesicles can be an important factor in chemoresistance, especially in cells that do not overexpress plasma membrane-bound drug pumps like P-glycoprotein.

Nephrotoxicity and uptake of 1-benzyl quinolinium in precision-cut rabbit renal cortical slices.

Nephrotoxicity studies have not always meshed toxicity with transport and uptake, although the two are interdependent. To begin to address this issue, a series of model organic cations (quaternary amines) was examined which revealed differences in toxicity that were not explained by slight structural variations. Thus, a single model organic cation, 1-benzyl quinolinium (BQ), was used to assess organic cations in rabbit renal cortical slices. Histopathological evaluation revealed that BQ produced site-specific injury in the S(3) region of the proximal tubule. Biochemical assays (K(+), qO(2) and ATP) revealed dose- and time-dependent decreases of BQ-exposed slices over 2-12 hr. Cation transport (uptake of tetraethylammonium) was decreased by BQ at sub-toxic doses within 2 hr, although anion transport (uptake of p-aminohippurate) was not affected. Understanding the toxicity and transport of model cations such as BQ will help to identify the mechanisms associated with organic cation nephrotoxicity as well as to facilitate the use of transport parameters to prevent toxicity or prolong drug action of cations.

Seroepidemiological survey of Taenia saginata cysticercosis in Kenya.

A sero-epidemiological study of Taenia saginata cysticercosis was carried out to determine the prevalence and distribution of the infection in three provinces of Kenya. Serum samples and meat inspection records were collected from cattle at slaughter at export and district abattoirs. Cattle origin and the presence of T. saginata cysticerci were noted as was the prevalence of other helminths such as Echinococcus granulosus and Fasciola gigantica. Serum samples were screened for circulating parasite antigen using a monoclonal antibody-based enzyme-linked immunosorbent assay (Ag-ELISA) and for ante-parasite antibody by indirect ELISA (Ab-ELISA). Eighty per cent of the sera were collected from cattle from the Rift Valley Province of Kenya. The prevalence of T. saginata cysticercosis and the other helminth infections varied between districts and was particularly high in Narok. Animal husbandry practices in arid areas such as Narok may be particularly conducive to transmission. The potential value of the Ag-ELISA for use in sero-epidemiological studies was verified by this study. It detected at least twice as many cases as T. saginata cysticercosis as meat inspection and, of the three methods investigated, was considered the most valuable.

Diagnosis of Taenia saginata cysticercosis in Kenyan cattle by antibody and antigen ELISA.

Sera from calves, either experimentally or naturally infected with Taenia saginata, were screened for an antibody response to T. saginata, and for parasite antigen, by enzyme-linked immunosorbent assays (ELISAs). An antibody response was detected by 3 weeks post infection (p.i.), rose to a peak at 10-12 weeks p.i., and was still in evidence 1 year p.i. Parasite antigen was first detected 4-7 weeks p.i. and persisted until the end of the experiment, over 1 year p.i. In the experimentally infected animals, cattle with 14 or more live cysticerci had detectable levels of parasite antigen in their sera at slaughter, while animals with live cyst burdens ranging from 0 to 4 were negative. Furthermore, levels of circulating antigen were positively correlated with live cysticercus burden in the experimental animals. In naturally infected cattle, 83% (5/6) of those with 30 or more live cysts, and 22% (5/23) of those with 1-29 live cysts, could be detected by the ELISA for parasite antigen, although no significant correlation between antigen level and live cyst burden could be detected. Antibody levels were not found to be associated with cyst burdens in either experimentally or naturally infected cattle. In slaughterhouse cattle, the antigen assay was almost three times as sensitive as meat inspection. However, there was no agreement between cattle found positive at meat inspection and those found positive by the antigen detection ELISA. One possible reason is that the ELISA only detects live cysts, while lesions left by dead cysts are more noticeable at meat inspection. The mouse monoclonal antibody-based antigen detection ELISA is of value for the diagnosis of naturally occurring, viable, T. saginata cysticercosis in live cattle and has an immediate application for field based epidemiological studies designed to determine prevalence.

Characteristics of influex and net flux of amino acids in Mytilus californianus.

1. Intact mussels and an in vitro preparation of isolated gill tissue were employed to study characteristics of the influx and net flux of amino acids in Mytilus californianus. 2. The kinetics of influx of 14C-labelled amino acids were complicated by the presence of a rapidly labelled extracellular compartment. 3. Correction of influx data for the extracellular compartment revealed influx of 14C-cycloleucine and 14C-glycine to be mediated by a transport mechanism adequately described by Michaelis-Menten kinetics. Passive diffusion plays no significant role in influx. 4. Influx and net flux of glycine into intact Mytilus were examined. From high concentrations (greater than 20 micronM) the influx of 14C-glycine was equivalent to the net influx of primary amine as determined by fluorescamine. At low ambient concentrations (greater than 20 micronM), influx of 14C-glycine occurred more rapidly than net influx as determined by fluorescamine. The data suggest that influx of labelled substrate is accompanied by efflux of unknown primary amine(s). In the absence of labelled substrate, efflux continues until a steady-state concentration of 2-5 micronM in the medium is achieved.5. The rate of influx of 14C-cycloleucine into isolated gill tissue, and the concentration gradient which can be developed by gill tissue are reduced when sodium is replaced in the medium. 6. The efflux of primary amines from isolated gill tissue is stimulated by the replacement of sodium in the medium with choline. 7. The data are consistent with a sodium-coupled mechanism for the transport of amino acid into gill tissue. However, energetic considerations cast doubt on the adequacy of such a mechanism to account fully for the observed trans-epidermal transport.

Seroepidemiological study of Taenia saginata cysticercosis in Swaziland.

A cross-sectional study of Taenia saginata cysticercosis in Swaziland using a serodiagnostic ELISA for parasite antigen is described. The seroprevalence and the levels of parasite antigen were compared in the sera of cattle from different geographical localities, and from areas of high or low population density. Cattle from the Lowveldt region, which has a hot and dry climate relative to the other areas investigated, exhibited significantly higher serum antigen levels. Seroprevalence was also higher in the Lowveldt but this difference was not found to be significant. Within the Lowveldt, antigen levels were found to be slightly elevated in cattle from more highly populated areas. It is suggested that either human behaviour and/or practices in animal husbandry, or increased susceptibility of cattle to reinfection at certain times of the year, may enhance transmission in the Lowveldt since climatic conditions in this region are not conducive to transmission.