Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells

The response ofH⁺-ATPase to lethal acid stress isunknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cyclesof lethal acid stress. Cells were grown to confluence on coverslips,loaded with2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, andmonitored for intracellular pH(pH_i) recovery from an acid load. The rate of Na⁺-independentpH_i recovery from an acid load inmutant cells was approximately fourfold higher than in parent cells(P < 0.001). TheNa⁺-independentH⁺ extrusion was ATP dependent and K⁺ independent and wascompletely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide,or N-ethylmaleimide. Theseresults indicate that theNa⁺-independentH⁺ extrusion in cultured medullarycells is mediated via H⁺-ATPaseand is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H⁺-ATPase remainedunchanged in mutant cells compared with parent cells. We propose thatlethal acid stress results in increased H⁺-ATPase activity in innermedullary collecting duct cells. Upregulation ofH⁺-ATPase could play a protectiverole against cell death in severe intracellular acidosis.

     

Omega 3 Fatty Acids Promote Macrophage Reverse Cholesterol Transport in Hamster Fed High Fat Diet

The aim of this study was to investigate macrophage reverse cholesterol transport (RCT) in hamster, a CETP-expressing species, fed omega 3 fatty acids (ω3PUFA) supplemented high fat diet (HFD). Three groups of hamsters (n = 6/group) were studied for 20 weeks: 1) control diet: Control, 2) HFD group: HF and 3) HFD group supplemented with ω3PUFA (EPA and DHA): HFω3. In vivo macrophage-to-feces RCT was assessed after an intraperitoneal injection of ³H-cholesterol-labelled hamster primary macrophages.Compared to Control, HF presented significant (p<0.05) increase in body weight, plasma TG (p<0.01) and cholesterol (p<0.001) with an increase in VLDL TG and in VLDL and LDL cholesterol (p<0.001).Compared to HF, HFω3 presented significant decrease in body weight. HFω3 showed less plasma TG (p<0.001) and cholesterol (p<0.001) related to a decrease in VLDL TG and HDL cholesterol respectively and higher LCAT activity (p<0.05) compared to HF. HFω3 showed a higher fecal bile acid excretion (p<0.05) compared to Control and HF groups and higher fecal cholesterol excretion (p<0.05) compared to HF. This increase was related to higher gene expression of ABCG5, ABCA1 and SR-B1 in HFω3 compared to Control and HF groups (<0.05) and in ABCG1 and CYP7A1 compared to HF group (p<0.05). A higher plasma efflux capacity was also measured in HFω3 using ³H- cholesterol labeled Fu5AH cells.In conclusion, EPA and DHA supplementation improved macrophage to feces reverse cholesterol transport in hamster fed HFD. This change was related to the higher cholesterol and fecal bile acids excretion and to the activation of major genes involved in RCT.     

New sabre-toothed cats in the Late Miocene of Toros Menalla (Chad)

The Toros Menalla Late Miocene Formation (7 Ma), Djurab desert, Chad, has yielded a large number of vertebrate remains, and among them around twenty taxa of carnivores. Recent collections allow us to describe two sabre-toothed felids assigned to the genera Lokotunjailurus and cf. Megantereon. The former does exist in the Nawata Formation, Lothagam, northern Kenya with a slightly derived species and the TM specimens are considered as belonging to a new species. The latter cannot be identified to the species level, but it could be one of the earliest specimens of Megantereon known in the world and it would reinforce the hypothesis of an Old World origin for this taxon. Both allow us to put forward some biogeographical conclusions.     

Potential antidiabetic activity and molecular docking studies of novel synthesized 3.6-dimethyl-5-oxo-pyrido[3,4-f][1,2,4]triazepino[2,3-a]benzimidazole and 10-amino-2-methyl-4-oxo pyrimido[1,2-a]benzimidazole derivatives

Diabetes affects a large population of the globe and is considered as a leading cause of death. Many synthetic and natural inhibitors have been developed for diabetes treatment. Herein, we report the potential antidiabetic activity of two new heterocyclic systems, namely 3.6-dimethyl-5-oxo-pyrido[3,4f][1,2,4]triazepino[2,3-a]benzimidazole (I) and 10-amino-2-methyl-4-oxo pyrimido[1,2-a]benzimidazole (II) against three related enzymes: α-amylase, α-glucosidase and β-galactosidase. Compounds I and II were synthesized by the action of DMF-DMA and dimethyl sulfate in the presence of water on 2-methyl-3H-benzimidazolo[1,2b][1,2,4]triazepin-4(5H)-one, and are characterized by single X-ray diffraction. The binding interaction modes in the active sites of I and II and targeted enzymes (stable complexes ligand-receptor) are emphasized using the molecular docking approach by applying the Lamarckian genetic algorithm method. Furthermore, plausible mechanisms have been proposed explaining their synthesis. Hirshfeld surface analysis reveals the nature of molecular interactions and fingerprint plots provide information about the percentage contribution from each individual molecular contact to the structure surface.

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Graphical abstract Left Molecular packing of 1,4-dimethyl-2-oxo-pyrimido[1,2-a]benzimidazole hydrate. Right Docking active site of α-glucosidase

     

Vitellogenin Titres in Normal and Accelerated Maturation of Gregarious-Phase Schistocerca gregaria

Isolation of vitellogenin of the Schistocerca gregaria (Forskal) in its gregarious phase was achieved by a combination of gel permeation and anion exchange chromatography. Staining for carbohydrate and lipid moieties showed that the vitellogenin is a glycolipoprotein. The vitellogenin of S. gregaria has a native molecular weight of about 700 kDa. On SDS-PAGE, the protein showed nine apoproteins of about 124, 120, 105, 60, 59, 58, 57, 53 and 34 kD. Determination of the levels of vitellogenin by ELISA in the haemolymph of maturing females showed that those exposed to mature males from 1 to 2 days after ecdysis had increased levels of vitellogenin from day 10 (81.1 ± 4.5). In contrast, females exposed to immature males or kept alone showed an increase (107.3 ± 0.9 and 70.2 ± 2.7) not until day 16 or later, respectively. These results are consistent with the accelerating effect of pheromonal emissions from mature males on the maturation of female S. gregaria.     

Mapping Daunorubicin-binding Sites in the ATP-binding Cassette Transporter MsbA Using Site-specific Quenching by Spin Labels

ATP-binding cassette (ABC) transporters transduce the free energy of ATP hydrolysis to power the mechanical work of substrate translocation across cell membranes. MsbA is an ABC transporter implicated in trafficking lipid A across the inner membrane of Escherichia coli. It has sequence similarity and overlapping substrate specificity with multidrug ABC transporters that export cytotoxic molecules in humans and prokaryotes. Despite rapid advances in structure determination of ABC efflux transporters, little is known regarding the location of substrate-binding sites in the transmembrane segment and the translocation pathway across the membrane. In this study, we have mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster at the cytoplasmic end of helices 3 and 6 at a site accessible from the membrane/water interface and extending into an aqueous chamber formed at the interface between the two transmembrane domains. Binding of a nonhydrolyzable ATP analog inverts the transporter to an outward-facing conformation and relieves DNR quenching by spin labels suggesting DNR exclusion from proximity to the spin labels. The simplest model consistent with our data has DNR entering near an elbow helix parallel to the water/membrane interface, partitioning into the open chamber, and then translocating toward the periplasm upon ATP binding.ATP-binding cassette (ABC)² transporters transduce the energy of ATP hydrolysis to power the movement of a wide range of substrates across the cell membranes (1, 2). They constitute the largest family of prokaryotic transporters, import essential cell nutrients, flip lipids, and export toxic molecules (3). Forty eight human ABC transporters have been identified, including ABCB1, or P-glycoprotein, which is implicated in cross-resistance to drugs and cytotoxic molecules (4, 5). Inherited mutations in these proteins are linked to diseases such as cystic fibrosis, persistent hypoglycemia of infancy, and immune deficiency (6).The functional unit of an ABC transporter consists of four modules. Two highly conserved ABCs or nucleotide-binding domains (NBDs) bind and hydrolyze ATP to supply the active energy for transport (7). ABCs drive the mechanical work of proteins with diverse functions ranging from membrane transport to DNA repair (3, 5). Substrate specificity is determined by two transmembrane domains (TMDs) that also provide the translocation pathway across the bilayer (7). Bacterial ABC exporters are expressed as monomers, each consisting of one NBD and one TMD, that dimerize to form the active transporter (3). The number of transmembrane helices and their organization differ significantly between ABC importers and exporters reflecting the divergent structural and chemical nature of their substrates (1, 8, 9). Furthermore, ABC exporters bind substrates directly from the cytoplasm or bilayer inner leaflet and release them to the periplasm or bilayer outer leaflet (10, 11). In contrast, bacterial importers have their substrates delivered to the TMD by a dedicated high affinity substrate-binding protein (12).In Gram-negative bacteria, lipid A trafficking from its synthesis site on the inner membrane to its final destination in the outer membrane requires the ABC transporter MsbA (13). Although MsbA has not been directly shown to transport lipid A, suppression of MsbA activity leads to cytoplasmic accumulation of lipid A and inhibits bacterial growth strongly suggesting a role in translocation (14-16). In addition to this role in lipid A transport, MsbA shares sequence similarity with multidrug ABC transporters such as human ABCB1, LmrA of Lactococcus lactis, and Sav1866 of Staphylococcus aureus (16-19). ABCB1, a prototype of the ABC family, is a plasma membrane protein whose overexpression provides resistance to chemotherapeutic agents in cancer cells (1). LmrA and MsbA have overlapping substrate specificity with ABCB1 suggesting that both proteins can function as drug exporters (18, 20). Indeed, cells expressing MsbA confer resistance to erythromycin and ethidium bromide (21). MsbA can be photolabeled with the ABCB1/LmrA substrate azidopine and can transport Hoechst 33342 ({"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342) across membrane vesicles in an energy-dependent manner (21).The structural mechanics of ABC exporters was revealed from comparison of the MsbA crystal structures in the apo- and nucleotide-bound states as well as from analysis by spin labeling EPR spectroscopy in liposomes (17, 19, 22, 23). The energy harnessed from ATP binding and hydrolysis drives a cycle of NBD association and dissociation that is transmitted to induce reorientation of the TMD from an inward- to outward-facing conformation (17, 19, 22). Large amplitude motion closes the cytoplasmic end of a chamber found at the interface between the two TMDs and opens it to the periplasm (23). These rearrangements lead to significant changes in chamber hydration, which may drive substrate translocation (22).Substrate binding must precede energy input, otherwise the cycle is futile, wasting the energy of ATP hydrolysis without substrate extrusion (7). Consistent with this model, ATP binding reduces ABCB1 substrate affinity, potentially through binding site occlusion (24-26). Furthermore, the TMD substrate-binding event signals the NBD to stimulate ATP hydrolysis increasing transport efficiency (1, 27, 28). However, there is a paucity of information regarding the location of substrate binding, the transport pathway, and the structural basis of substrate recognition by ABC exporters. In vitro studies of MsbA substrate specificity identify a broad range of substrates that stimulate ATPase activity (29). In addition to the putative physiological substrates lipid A and lipopolysaccharide (LPS), the ABCB1 substrates Ilmofosine, {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342, and verapamil differentially enhance ATP hydrolysis of MsbA (29, 30). Intrinsic MsbA tryptophan (Trp) fluorescence quenching by these putative substrate molecules provides further support of interaction (29).Extensive biochemical analysis of ABCB1 and LmrA provides a general model of substrate binding to ABC efflux exporters. This so-called “hydrophobic cleaner model” describes substrates binding from the inner leaflet of the bilayer and then translocating through the TMD (10, 31, 32). These studies also identified a large number of residues involved in substrate binding and selectivity (33). When these crucial residues are mapped onto the crystal structures of MsbA, a subset of homologous residues clusters to helices 3 and 6 lining the putative substrate pathway (34). Consistent with a role in substrate binding and specificity, simultaneous replacement of two serines (Ser-289 and Ser-290) in helix 6 of MsbA reduces binding and transport of ethidium and taxol, although {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342 and erythromycin interactions remain unaffected (34).The tendency of lipophilic substrates to partition into membranes confounds direct analysis of substrate interactions with ABC exporters (35, 36). Such partitioning may promote dynamic collisions with exposed Trp residues and nonspecific cross-linking in photo-affinity labeling experiments. In this study, we utilize a site-specific quenching approach to identify residues in the vicinity of the daunorubicin (DNR)-binding site (37). Although the data on DNR stimulation of ATP hydrolysis is inconclusive (20, 29, 30), the quenching of MsbA Trp fluorescence suggests a specific interaction. Spin labels were introduced along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent quenching of DNR fluorescence. Residues that quench DNR cluster along the cytoplasmic end of helices 3 and 6 consistent with specific binding of DNR. Furthermore, many of these residues are not lipid-exposed but face the putative substrate chamber formed between the two TMDs. These residues are proximal to two Trps, which likely explains the previously reported quenching (29). Our results suggest DNR partitions to the membrane and then binds MsbA in a manner consistent with the hydrophobic cleaner model. Interpretation in the context of the crystal structures of MsbA identifies a putative translocation pathway through the transmembrane segment.