L-type amino-acid transporter 1 (LAT1): a therapeutic target supporting growth and survival of T-cell lymphoblastic lymphoma/T-cell acute lymphoblastic leukemia.

The altered metabolism of cancer cells is a treasure trove to discover new antitumoral strategies. The gene (SLC7A5) encoding system L amino-acid transporter 1 (LAT1) is overexpressed in murine lymphoma cells generated via T-cell deletion of the pten tumor suppressor, and also in human T-cell acute lymphoblastic leukemia (T-ALL)/lymphoma (T-LL) cells. We show here that a potent and LAT1 selective inhibitor (JPH203) decreased leukemic cell viability and proliferation, and induced transient autophagy followed by apoptosis. JPH203 could also alter the in vivo growth of luciferase-expressing-tPTEN-/- cells xenografted into nude mice. In contrast, JPH203 was nontoxic to normal murine thymocytes and human peripheral blood lymphocytes. JPH203 interfered with constitutive activation of mTORC1 and Akt, decreased expression of c-myc and triggered an unfolded protein response mediated by the C/EBP homologous protein (CHOP) transcription factor associated with cell death. A JPH203-resistant tPTEN-/-clone appeared CHOP induction deficient. We also demonstrate that targeting LAT1 may be an efficient broad spectrum adjuvant approach to treat deadly T-cell malignancies as the molecule synergized with rapamycin, dexamethasone, doxorubicin, velcade and l-asparaginase to alter leukemic cell viability.

The effect of STAT3 inhibition on status epilepticus and subsequent spontaneous seizures in the pilocarpine model of acquired epilepsy.

Pilocarpine-induced status epilepticus (SE), which results in temporal lobe epilepsy (TLE) in rodents, activates the JAK/STAT pathway. In the current study, we evaluate whether brief exposure to a selective inhibitor of the JAK/STAT pathway (WP1066) early after the onset of SE affects the severity of SE or reduces later spontaneous seizure frequency via inhibition of STAT3-regulated gene transcription. Rats that received systemic WP1066 or vehicle at the onset of SE were continuously video-EEG monitored during SE and for one month to assess seizure frequency over time. Protein and/or mRNA levels for pSTAT3, and STAT3-regulated genes including: ICER, Gabra1, c-myc, mcl-1, cyclin D1, and bcl-xl were evaluated in WP1066 and vehicle-treated rats during stages of epileptogenesis to determine the acute effects of WP1066 administration on SE and chronic epilepsy. WP1066 (two 50mg/kg doses) administered within the first hour after onset of SE results in transient inhibition of pSTAT3 and long-term reduction in spontaneous seizure frequency. WP1066 alters the severity of chronic epilepsy without affecting SE or cell death. Early WP1066 administration reduces known downstream targets of STAT3 transcription 24h after SE including cyclin D1 and mcl-1 levels, known for their roles in cell-cycle progression and cell survival, respectively. These findings uncover a potential effect of the JAK/STAT pathway after brain injury that is physiologically important and may provide a new therapeutic target that can be harnessed for the prevention of epilepsy development and/or progression.

Lyophilization of a triply unsaturated phospholipid: effects of trace metal contaminants.

As liquid liposomal formulations are prone to chemical degradation and aggregation, these formulations often require freeze drying (e.g., lyophilization) to achieve sufficient shelf-life. However, liposomal formulations may undergo oxidation during lyophilization and/or during prolonged storage. The goal of the current study was to characterize the degradation of 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (DLPC) during lyophilization and to also probe the influence of metal contaminants in promoting the observed degradation. Aqueous sugar formulations containing DLPC (0.01 mg/ml) were lyophilized, and DLPC degradation was monitored using HPLC/UV and GC/MS methods. The effect of ferrous ion and sucrose concentration, as well as lyophilization stage promoting lipid degradation, was investigated. DLPC degradation increased with higher levels of ferrous ion. After lyophilization, 103.1 ± 1.1%, 66.9 ± 0.8%, and 28.7 ± 0.7% DLPC remained in the sucrose samples spiked with 0.0 ppm, 0.2 ppm, and 1.0 ppm ferrous ion, respectively. Lipid degradation predominantly occurs during the freezing stage of lyophilization. Sugar concentration and buffer ionic strength also influence the extent of lipid degradation, and DLPC loss correlated with degradation product formation. We conclude that DLPC oxidation during the freezing stage of lyophilization dramatically compromises the stability of lipid-based formulations. In addition, we demonstrate that metal contaminants in sugars can become highly active when lyophilized in the presence of a reducing agent.

Apical voltage-driven urate efflux transporter NPT4 in renal proximal tubule.

Uric acid (urate) is the end product of purine metabolism in humans. Human kidneys reabsorb a large proportion of filtered urate. This extensive renal reabsorption, together with the fact that humans do not possess uricase that catalyzes the biotransformation of urate into allantoin, results in a higher plasma urate concentration in humans compared to other mammals. A major determinant of plasma urate concentration is renal excretion as a function of the balance between reabsorption and secretion. We previously identified that renal urate absorption in proximal tubular epithelial cells occurs mainly via apical urate/anion exchanger, URAT1/SLC22A12, and by facilitated diffusion along the trans-membrane potential gradient by the basolateral voltage-driven urate efflux transporter, URATv1/SLC2A9/GLUT9. In contrast, the molecular mechanism by which renal urate secretion occurs remains elusive. Recently, we reported a newly characterized human voltage-driven drug efflux transporter, hNPT4/SLC17A3, which functions as a urate exit pathway located at the apical side of renal proximal tubules. This transporter protein has been hypothesized to play an important role with regard to net urate efflux. An in vivo role of hNPT4 is supported by the fact that missense mutations in SLC17A3 present in hyperuricemia patients with urate underexcretion abolished urate efflux capacity in vitro. Herein, we report data demonstrating that loop diuretics and thiazide diuretics substantially interact with hNPT4. These data provide molecular evidence for loop and thiazide-diuretics-induced hyperuricemia. Thus, we propose that hNPT4 is an important transepithelial proximal tubular transporter that transports diuretic drugs and operates functionally with basolateral organic anion transporters 1/3 (OAT1/OAT3).

Human uric acid transporter 1 (hURAT1): an inhibitor structure-activity relationship (SAR) study.

The current study describes the chemical synthesis of a series of (2-ethylbenzofuran-3-yl)(substituted-phenyl)methanone compounds and their subsequent in vitro testing via oocytes expressing hURAT1. The experimental data support the notion that a potent hURAT1 inhibitor requires an anion (i.e., a formal negative charge) to interact with the positively charged hURAT1 binding pocket. An anion appears to be a primary requirement in order to be a hURAT1 substrate (i.e., urate) or inhibitor. We discuss the inhibitor structure-activity relationship and how electronically donating or withdrawing groups attached to the B-ring can decrease or increase inhibitory potency, respectively.

Mercapturic acids (N-acetylcysteine S-conjugates) as endogenous substrates for the renal organic anion transporter-1.

Mercapturic acids are N-acetyl-L-cysteine S-conjugates that are formed from a range of endogenous and exogenous chemicals. Although the kidney is a major site for elimination of mercapturic acids, the transport mechanisms involved have not been identified. The present study examined whether mercapturic acids are substrates for the renal basolateral organic anion transporter-1 (Oat1) from rat kidney. This carrier mediates uptake of organic anions from the bloodstream in exchange for intracellular alpha-ketoglutarate. Uptake of [(3)H]p-aminohippuric acid (PAH) in Oat1-expressing Xenopus laevis oocytes was strongly inhibited by S-(2,4-dinitrophenyl)-N-acetyl-L-cysteine (DNP-NAC) and by all other mercapturic acids tested, including the endogenous mercapturic acid N-acetyl-leukotriene E(4). Inhibition by the mercapturic acids was competitive, which is consistent with the hypothesis that these compounds are substrates for Oat1. This conclusion was supported by the direct demonstration of saturable [(35)S]DNP-NAC uptake in Oat1-expressing oocytes. [(35)S]DNP-NAC uptake was inhibited by PAH and other mercapturic acids and was stimulated in oocytes preloaded with glutarate. The apparent K(m) value for DNP-NAC uptake was only 2 microM, indicating that this mercapturic acid is a high affinity substrate for Oat1. Together, these data indicate that clearance of endogenous mercapturic acids is an important function of the renal organic anion transporter.

Identification of the precipitate in alkalinized solutions of mepivacaine and bupivacaine at 37 degrees C.

Commercial and control solutions of bupivacaine (0(.)75%) and mepivacaine (1(.)5%) were alkalinized with bicarbonate until cloudy at room temperature. The solutions were heated to 37 degrees C for 2(.)5 h. The precipitates were filtered, lyophylized and analysed by fast atom bombardment mass spectrometry. Analysis showed the precipitates to be predominantly the free base of the local anaesthetic. The precipitate of the commercial bupivacaine solution also contained a small amount of the hydrochloride salt. The mepivacaine control crystals contained an unknown at molecular weight 528, which may represent a dimer of the free base and hydrochloride salt.

Glutathione transferase zeta-catalyzed biotransformation of deuterated dihaloacetic acids.

Glutathione transferase zeta (GSTZ) catalyzes the biotransformation of alpha-haloalkanoic acids. Treatment of rats or humans with dichloroacetic acid prolongs its elimination half-life, and preliminary studies in this laboratory show that fluorine-lacking, but not fluorine-containing dihaloacetic acids inactivate GSTZ. In the present study, the GSTZ-catalyzed biotransformation of unlabeled and deuterated dihaloacetic acids was investigated. With [(2)H]dichloroacetic acid and [(2)H]chlorofluoroacetic acid as substrates, the deuterium present in the [(2)H]dihaloacetic acid was retained in the [(2)H]glyoxylic acid formed. This finding indicates that the enol of the dihaloacetic acid does not serve as the substrate for the enzyme. The data afford an explanation of the failure of fluorine-containing dihaloacetic acids to inactivate GSTZ: dichloroacetic acid is converted to glyoxylic acid and inactivates GSTZ, whereas chlorofluoroacetic acid is biotransformed to glyoxylic acid, but produces negligible inactivation. Mechanisms are presented indicating that this difference may be attributed to the nucleofugicity of the leaving group.

Rabies in a pronghorn antelope, Antilocapra americana.

A ten week old antelope died from rabies 35 days after being bitten on the left hock by a skunk. The result of the direct fluorescent antibody test for rabies was positive.