Diclofenac-induced stimulation of SMCT1 (SLC5A8) in a heterologous expression system: A RPE specific phenomenon

SMCT1 is a Na⁺-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na⁺-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, β-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.     

Fluidity,permeability and antioxidant behaviour of model membranes incorporated with α-tocopherol and vitamin E acetate

Magnetic resonance studies reveal a marked difference between the binding of α-tocopherol and that of the corresponding acetate (vitamin E acetate) with dipalmitoylphosphatidylcholine (DPPC) vesicles. This is reflected in differences in the phase-transition curves of the DPPC vesicles incorporated with the two compounds, as well as in the ¹³C relaxation times and line widths. A model for the incorporation of these molecules in lipid bilayers has been suggested. α-Tocopherol binds strongly with the lipids, possibly through a hydrogen bond formation between the hydroxyl group of the former and one of the oxygen atoms of the latter. The possibility of such a hydrogen bond formation is excluded in vitamin E acetate, which binds loosely through the normal hydrophobic interaction. The model for lipid-vitamin interaction explains the in vitro decomposition of H₂O₂ by α-tocopherol. α-Tocopherol in conjuction with H₂O₂ can also act as a free-radical scavenger in the lipid phase. The incorporation of α-tocopherol and vitamin E acetate in DPPC vesicles enhances the permeability of lipid bilayers for small molecules such as sodium ascorbate.     

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg²⁺ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg²⁺ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg²⁺ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the γ-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.     

Somatic Variations in Cervical Cancers in Indian Patients

There are very few reports that describe the mutational landscape of cervical cancer, one of the leading cancers in Indian women. The aim of the present study was to investigate the somatic mutations that occur in cervical cancer. Whole exome sequencing of 10 treatment naïve tumour biopsies with matched blood samples, from a cohort of Indian patients with locally advanced disease, was performed. The data revealed missense mutations across 1282 genes, out of 1831 genes harbouring somatic mutations. These missense mutations (nonsynonymous + stop-gained) when compared with pre-existing mutations in the COSMIC database showed that 272 mutations in 250 genes were already reported although from cancers other than cervical cancer. More than 1000 novel somatic variations were obtained in matched tumour samples. Pathways / genes that are frequently mutated in various other cancers were found to be mutated in cervical cancers. A significant enrichment of somatic mutations in the MAPK pathway was observed, some of which could be potentially targetable. This is the first report of whole exome sequencing of well annotated cervical cancer samples from Indian women and helps identify trends in mutation profiles that are found in an Indian cohort of cervical cancer.     

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.