AB5 toxins: structures and inhibitor design

High-resolution crystal structures of AB₅ toxins in their native form or in complex with a variety of ligands have led to the structure-based design and discovery of inhibitors targeting different areas of the toxins. The most significant progress is the development of highly potent multivalent ligands that block binding of the toxins to their receptors.     

Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

Multiple molecular forms of xanthine dehydrogenase and related enzymes. IV. The relationship of aldehyde oxidase to xanthine dehydrogenase

Variants of the enzyme aldehyde oxidase in Drosophila melanogaster are described. In addition to electrophoretic variants, a mutant that causes low levels of the enzyme has been found by screening more than 80 strains for aldehyde oxidase levels. The locus of the mutation maps on the third chromosome near lpo and aldox. The existence of the ry, lpo, and aldox mutants and of the new mutant indicates that xanthine dehydrogenase, pyridoxal oxidase, and aldehyde oxidase are under a separate genetic control, in addition to a common genetic control by ma-l and lxd. The genetic separation is shown to be accompanied by physical separation of the enzymes with DEAE-cellulose column chromatography and (NH ₄)₂SO₄fractionation. Further data on the metabolism of aldehydes by xanthine dehydrogenase and aldehyde oxidase are presented. Although xanthine dehydrogenase requires NAD or a similar cofactor to metabolize purine and pteridine substrates, aldehyde oxidase oxidizes salicylaldehyde to salicylic acid without dissociable cofactors and with the uptake of oxygen.This work was supported in part by Research Grant GM-08202, by a Predoctoral Fellowship (J.C.) and a Genetics Training Grant (J.C. and E.D.), and by a Research Career Development Award (E.G.), all from the National Institutes of Health. Part of this work was submitted by J.C. to the University of North Carolina at Chapel Hill in partial fulfillment of the degree of Doctor of Philosophy.     

Effect of galanin on isolated strips of smooth muscle from the gastrointestinal tract of chickens

The contractile effects of galanin on isolated longitudinal smooth muscle strips of pre-crop esophagus, proventriculus, duodenum, colon, and cecum of chickens were investigated. Application of galanin (5.0-100.0 nM) evoked strong contractions from the colon and cecum (hindgut), but evoked minimal responses from the pre-crop esophagus, proventriculus, and duodenum (foregut). Previous studies have demonstrated that the central administration of galanin stimulates food consumption in rats. Since galanin-like immunoreactivity is present in the chicken brain, we speculate that the central release of galanin may increase food intake and possibly be involved in a hypothalamic-colonic reflex modulating hindgut motility and generating a defecation. Thus, the results of this study demonstrate the presence of galanin receptors in the chicken gut and suggest a possible link with their functional presence in the hindgut to the chicken central nervous system.     

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.     

Nutritional control of xanthine dehydrogenase. II. Effects on xanthine dehydrogenase and aldehyde oxidase of culturing wild-type and mutant Drosophila on different levels of molybdenum

Two new mutants, deficient in aldehyde oxidase and xanthine dehydrogenase, have been isolated from a wild-type stock of Drosophila melanogaster and have been provisionally termed lxd ^c and lxd ^d, respectively, as both mutants appear to be allelic with lxd (low xanthine dehydrogenase). An analysis has been made of the effects of dietary molybdenum on lxd, lxd ^c, lxd^d, lao (low aldehyde oxidase), mal (maroon-like eye color), and pac (Pacific) wild-type flies. On the lower dietary levels of 10 ^?3 M and 10 ^?2 M molybdenum, increases in specific activity of both enzymes were observed only in lxd. Furthermore, two- to three-fold increases in specific activity of both enzymes occurred in all strains, except mal, when cultured on 5×10 ^?2 M molybdenum. The lxd and lxd ^c strains failed to survive on this high concentration of the ion. Similar concentrations of molybdenum had no effect in vitro. An extra electrophoretic band of xanthine dehydrogenase was observed on polyacrylamide gel from extracts of wild-type flies cultured on certain levels of molybdenum, but its appearance was not always correlated with the increases in specific activity.     

Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity

Exosomes are nanometer-scale, cell-derived vesicles that contain various molecules including nucleic acids, proteins, and lipids. These vesicles can release their cargo into adjacent or distant cells and mediate intercellular communication and cellular function. Here we examined the regulation of epithelial sodium channels in mpkCCD cells and distal tubule Xenopus 2F3 cells by exosomes isolated from proximal tubule LLC-PK1 cells. Cultured mpkCCD cells were stained with CTX coupled to a green fluorophore in order to label the cell membranes and freshly isolated exosomes from LLC-PK1 cells were labeled with the red lipophilic dye PKH26 in order to visualize uptake of exosomes into the cells. Single-channel patch clamp recordings showed the open probability of ENaC in Xenopus 2F3 cells and in freshly isolated split-open tubules decreased in response to exogenous application of exosomes derived from LLC-PK1 proximal tubule cells. Active GAPDH was identified within exosomes derived from proximal tubule LLC-PK1 cells. The effect on ENaC activity in Xenopus 2F3 cells was blunted after application of exosomes transfected with the GAPDH inhibitor heptelidic acid. Also, we show GAPDH and ENaC subunits associate in mpkCCD cells. These studies examine a potential role for exosomes in the regulation of ENaC activity and examine a possible mechanism for communication from proximal tubule cells to distal tubule and collecting duct cells.