Identification of Tyr residues that enhance folate substrate binding and constrain oscillation of the proton-coupled folate transporter (PCFT-SLC46A1).

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption and transport of folates across the choroid plexus. This study focuses on the role of Tyr residues in PCFT function. The substituted Cys-accessibility method identified four Tyr residues (Y291, Y362, Y315, and Y414) that are accessible to the extracellular compartment; three of these (Y291, Y362, and Y315) are located within or near the folate binding pocket. When the Tyr residues were replaced with Cys or Ala, these mutants showed similar (up to 6-fold) increases in influx Vmax and Kt/Ki for [(3)H]methotrexate and [(3)H]pemetrexed. When the Tyr residues were replaced with Phe, these changes were moderated or absent. When Y315A PCFT was used as representative of the mutants and [(3)H]pemetrexed as the transport substrate, this substitution did not increase the efflux rate constant. Furthermore, neither influx nor efflux mediated by Y315A PCFT was transstimulated by the presence of substrate in the opposite compartment; however, substantial bidirectional transstimulation of transport was mediated by wild-type PCFT. This resulted in a threefold greater efflux rate constant for cells that express wild-type PCFT than for cells that express Y315 PCFT under exchange conditions. These data suggest that these Tyr residues, possibly through their rigid side chains, secure the carrier in a high-affinity state for its folate substrates. However, this may be achieved at the expense of constraining the carrier's mobility, thereby decreasing the rate at which the protein oscillates between its conformational states. The Vmax generated by these Tyr mutants may be so rapid that further augmentation during transstimulation may not be possible.

Properties of the Arg376 residue of the proton-coupled folate transporter (PCFT-SLC46A1) and a glutamine mutant causing hereditary folate malabsorption.

The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and is mutated in the autosomal recessive disorder, hereditary folate malabsorption (HFM). This report characterizes properties and requirements of the R376 residue in PCFT function, including a R376Q mutant associated with HFM. Gln, Cys, and Ala substitutions resulted in markedly impaired transport of 5-formyltetrahydrofolate (5-FTHF) and 5-methyltetrahydrofolate (5-MTHF) due to an increase in K(m) and decrease in V(max) in HeLa R1-11 transfectants lacking endogenous folate transport function. In contrast, although the influx K(m) for pemetrexed was increased, transport was fully preserved at saturating concentrations and enhanced for the like-charged R376K- and R376H-PCFT. Pemetrexed and 5-FTHF influx mediated by R376Q-PCFT was markedly decreased at pH 5.5 compared with wild-type PCFT. However, while pemetrexed transport was substantially preserved at low pH (4.5-5.0), 5-FTHF transport remained very low. Electrophysiological studies in Xenopus oocytes demonstrated that 1) the R376Q mutant, like wild-type PCFT, transports protons in the absence of folate substrate, and in this respect has channel-like properties; and 2) the influx K(m) mediated by R376Q-PCFT is increased for 5-MTHF, 5-FTHF, and pemetrexed. The data suggest that mutation of the R376 residue to Gln impairs proton binding which, in turn, modulates the folate-binding pocket and depresses the rate of conformational alteration of the carrier, a change that appears to be, in part, substrate dependent.

Membrane topological analysis of the proton-coupled folate transporter (PCFT-SLC46A1) by the substituted cysteine accessibility method.

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption. Loss-of-function mutations in this gene are the molecular basis for the autosomal recessive disorder, hereditary folate malabsorption. In this study, the substituted cysteine accessibility method was utilized to localize extra- or intracellular loops connecting predicted PCFT transmembrane domains. Cysteine-less PCFT was generated by replacement of all seven cysteine residues with serine and was shown to be functional, following which cysteine residues were introduced into predicted loops. HeLa cells, transiently transfected with these PCFT mutants, were then labeled with an impermeant, cysteine-specific biotinylation reagent (MTSEA-biotin) with or without permeabilization of cells. The biotinylated proteins were precipitated by streptavidin beads and assessed by Western blotting analysis. The biotinylation of PCFT was further confirmed by blocking cysteine residues with impermeant 2-sulfonatoethyl methanethiosulfonate. Two extracellular cysteine residues (66, 298) present in WT-PCFT were not biotinylated; however, in the absence of either one, biotinylation occurred. Likewise, biotinylation occurred after treatment with beta-mercaptoethanol. Taken together, these analyses establish a PCFT secondary structure of 12 transmembrane domains with the N- and C- termini directed to the cytoplasm. The data indicate further that there is a disulfide bridge, which is not required for function, between the native C66 and C298 residues in the first and fourth transmembrane domains, respectively.

Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1.

The proton-coupled folate transporter (PCFT) SLC46A1 mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This study addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains with a detailed analysis of the PCFT E185 residue. D156A-, E185A-, E232A-, R148A-, and R376A-PCFT mutants lost function at pH 5.5, as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an eightfold decrease in the [(3)H]methotrexate (MTX) influx V(max); the MTX influx K(t) was identical to that of wild-type (WT)-PCFT (1.5 microM). Consistent with the intrinsic functionality of E185A-PCFT, [(3)H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (approximately 38% of WT) for the similarly charged E185D-PCFT at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with augmentation of a rate-limiting step in the PCFT transport cycle.

N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT).

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~55 kDa), higher than predicted (~50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 degrees C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~47 kDa protein; substitution of both sites gave a smaller (~35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH(2) or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.

Does the complication rate increase in laparoscopic cholecystectomy for acute cholecystitis?

BACKGROUND: Laparoscopic cholecystectomy (LC) has replaced open cholecystectomy for the treatment of gallbladder disease. Despite the well-accepted success of LC in chronic cholecystitis, the efficacy of this technique has been subject to some debate in acute cholecystitis (AC). This study was designed to evaluate our institution's experience with LC for AC and chronic symptomatic calculous cholecystitis (CC), based on complication and conversion rates to open surgery. PATIENTS AND METHODS: The records of 1158 patients with LC from September 1991 to December 2001 were analyzed. The parameters of age, gender, early and late complication rates, and conversion rates from LC to open cholecystectomy were compared in patients with AC and CC. RESULTS: During the study period, LC was performed in 1158 patients. Of these, 162 patients had AC (group 1) and 996 patients had CC (group 2). The conversion rates were 4.3% (7/162) in group 1 and 2.4% (24/996) in group 2. The complication rates were not significantly different (5.6% in group 1, 5.1% in group 2, P > 0.05). Difficulty in dissection around Calot's triangle and obscure anatomy were the main reasons for conversion to conventional open surgery. The mortality rate was 1.2% in group 1 and 0.01% in group 2. CONCLUSION: LC appears to be a reliable, safe, and effective treatment modality for AC and CC. The surgical approach should be performed carefully because of the spectrum of potential hazards of the laparoscopic procedure. Conversion and complication rates are similar in both AC and CC groups, and improve as surgeons gain experience.

Laparoscopic treatment of a bilobed gallbladder: a case report and review of the literature.

A double or bilobed gallbladder is a rare congenital anomaly. Because other congenital vascular and biliary duct anomalies may accompany this pathology, open cholecystectomy was thought to be the best treatment of symptomatic patients. In this paper, we report a patient with a bilobed gallbladder whose symptoms were successfully treated with laparoscopic cholecystectomy. We also discuss the characteristics and the embryology of this rare anomaly.

Does the early ligation of the splenic artery reduce hemorrhage during laparoscopic splenectomy?

The aim of this study was to investigate whether early ligation of the splenic artery before splenic lysis has an effect on the amount of intraoperative bleeding and conversion rate during laparoscopic splenectomy. Laparoscopic splenectomy was performed in 34 patients with hematological diseases or splenic cysts between January 1993 and January 2003. The splenic artery was ligated before manipulation of the spleen in 22 patients (group 1) and laparoscopic splenectomy was performed with no previous ligation of the splenic artery in 12 patients (group 2). Prospective data was collected and the groups compared regarding intraoperative blood loss, platelet count, operative time, hospital stay, and conversion rate. Laparoscopic splenectomy was successfully completed in 30 (88%) patients. One patient in group 1 (5%) and 3 patients in group 2 (25%) required conversion due to bleeding. Estimated average blood loss was 161 mL (range 70-450 mL) in group 1, and 292 mL (range 100-700 mL) in group 2 (P < 0.001). The average operative time was 140 minutes (range 80-240) in group 1, and 155 minutes (range 80-200) in group 2 (P > 0.05). There were no statistically significant differences between the two groups comparing splenic size, conversion rate, hospital length of stay and platelet count. Early ligation of the splenic artery is feasible, safe and effective and may provide easy dissection and manipulation of the spleen during laparoscopic splenectomy with decreased intraoperative blood loss and no extension of the operative time.

The impact of 5-formyltetrahydrofolate on the anti-tumor activity of pralatrexate, as compared to methotrexate, in HeLa cells in vitro.

PURPOSE: To investigate the impact of 5-formyltetrahydrofolate on the activities of pralatrexate, as compared to methotrexate (MTX), in vitro. METHODS: Cells were exposed to (6S)5-formyltetrahydrofolate (5-formylTHF) for 24 h, before or after a 6-h exposure to antifolates following which the cellular accumulation and activities of the drugs were evaluated in HeLa cells. RESULTS: A 24-h delay between a 6-h exposure to antifolates and a subsequent 24-h exposure to 4 µM 5-formylTHF sustained the full activities of both antifolates. A 72-h interval was required between a single exposure of up to 4 µM 5-formylTHF and subsequent exposure to drugs to sustain activities of the antifolates. When cells were incubated with 4 µM 5-formylTHF for 24 h weekly, for 4 weeks, there was no significant increase in the IC50 for pralatrexate, but the MTX IC50 increased 2.5-fold as compared to cells growing continuously in 25 nM 5-formylTHF. This cyclical exposure to 5-formylTHF increased the cell folate pool by 16 %, had no significant effect on the intracellular pralatrexate level, but decreased intracellular MTX by 15 %. An extracellular concentration of MTX 50-fold higher than that of pralatrexate was required to achieve an intracellular level, and growth inhibition, comparable to that of pralatrexate. CONCLUSIONS: Cyclical exposures to 5-formylTHF at levels in excess of what is achieved in most clinical "rescue" regimens do not affect pralatrexate accumulation nor antitumor activity in HeLa cells, in contrast to MTX. An important element in preserving pralatrexate activity is achieving a sufficient interval between exposure to 5-formylTHF and the next dose of antifolate.

The membrane transport and polyglutamation of pralatrexate: a new-generation dihydrofolate reductase inhibitor.

PURPOSE: To characterize, directly and for the first time, the membrane transport and metabolism of pralatrexate, a new-generation dihydrofolate reductase inhibitor approved for the treatment for peripheral T-cell lymphoma. EXPERIMENTAL DESIGN: [(3)H]pralatrexate transport was studied in unique HeLa cell lines that express either the reduced folate carrier (RFC) or the proton-coupled folate transporter (PCFT). Metabolism to active polyglutamate derivatives was assessed by liquid chromatography. These properties were compared to those of methotrexate (MTX). RESULTS: The pralatrexate influx K t, mediated by RFC, the major route of folate/antifolate transport at systemic pH, was 0.52 µΜ, 1/10th the MTX influx K i. The electrochemical potential of pralatrexate within HeLa cells far exceeded the extracellular level and was greater than for MTX. In contrast, MTX transport mediated by PCFT, the mechanism of folate/antifolate absorption in the small intestine, exceeded that for pralatrexate. After a 6 h exposure of HeLa cells to 0.5 µM pralatrexate, 80 % of intracellular drug was its active polyglutamate forms, predominantly the tetraglutamate, and was suppressed when cells were loaded with natural folates. There was negligible formation of MTX polyglutamates. The difference in pralatrexate and MTX growth inhibition was far greater after transient exposures (375-fold) than continuous exposure (25-fold) to the drugs. CONCLUSIONS: Pralatrexate's enhanced activity relative to MTX is due to its much more rapid rate of transport and polyglutamation, the former less important when the carrier is saturated. The low affinity of pralatrexate for PCFT predicts a lower level of enterohepatic circulation and increased fecal excretion of the drug relative to MTX.