The effects of aluminum loading on the renal response to parathyroid hormone in the vitamin D-replete rat

Aluminum (Al) may cause vitamin D-resistant osteomalacia and depress the serum levels of immunoreactive parathyroid hormone (iPTH) in patients treated with maintenance dialysis and those on total parental nutrition (TPN). Both conditions have been associated with low serum levels of 1,25(OH)2-vitamin D (1,25(OH)2D). Al may inhibit PTH secretion in vitro; however, induction of hypocalcemia can enhance endogenous PTH secretion in Al-loaded dogs and TPN patients. Despite hypocalcemia and/or increased endogenous iPTH levels, Al-loaded TPN patients fail to show the expected rise in serum 1,25(OH)2D levels. Such observations suggest that Al may impair the renal response to PTH. We studied vitamin D-replete rats given Al or saline vehicle IP for 5 days. Al and control rats then received a saline infusion with an IV bolus of PTH 1-34. Urinary cyclic AMP and P excretion rose in Al and control rats by 1 hr post-PTH, without differences between the groups. Serum P and ionized Ca levels were not different between Al and control rats. In other Al and control rats, serum 1,25(OH)2D levels were measured after saline without PTH. Serum 1,25(OH)2D levels were higher in controls given PTH than in those without, but 1,25(OH)2D levels were not different between Al rats given PTH and those with none. Thus, aluminum does not affect cyclic AMP or P excretion but may impair 25(OH)D-1 alpha-hydroxylase activity in response to PTH.     

Freezing of dCMP aminohydrolase in the activated conformation by glutaraldehyde.

dCMP aminohydrolase, which is an allosteric enzyme, was reacted with glutaraldehyde in the presence of the allosteric activator deoxycytidine-5′-triphosphate and of the competitive inhibitor deoxyadenosine-5′-monophosphate. The isolated modified enzyme is no longer sensitive to the effect of the allosteric ligands and shows kinetics typical of the activated enzyme. Gel electrophoresis demonstrated that glutaraldehyde, under our experimental conditions, does not produce intermolecular cross-links but fixes 80% of the enzyme in a stable hexameric form by intramolecular cross-links.The kinetic and molecular data are explained assuming that glutaraldehyde freezes the enzyme in the activated conformation.     

The reversion of trp frameshift mutations in mut,polA, lig and dnaE mutant strains of Escherichia coli

Mutator mutations mutL25, mutR34, and mutU4 had similar effects on the reversion of 4 trp frameshift mutations of known sequence. The mutation trpE9777, which resulted from the addition of an A–T base-pair to a run of 5 A–T base-pairs, was most strongly reverted by the 4 mutators. Reversion of trpE9777 was also increased by mutation polA1 (DNA polymerase I) and dnaE486 and dnaE511 (DNA polymerase III). No effect was found with the ligase mutations, lig-4 or lig-ts7. Mutations polAex1 and polA107, both deficient in the 5′ → 3′ exonuclease activity of DNA polymerase I, had different mutator effects; the factor increase in reversion of trpE9777 was 28-fold for polAex1, 6-fold for polA107, and 21-fold for polA1. The trpE9777 mutation is a useful indicator of frameshift mutator activity.     

VMP1 is a new player in the regulation of the autophagy-specific phosphatidylinositol 3-kinase complex activation

We have elucidated a novel mechanism through which the autophagy-specific class III phosphatidylinositol 3-kinase (PtdIns3K) complex can be recruited to the PAS in mammalian cells, through the interaction between BECN1 and the vacuole membrane protein 1 (VMP1), an integral autophagosomal membrane protein. This interaction involves the binding between the C-terminal 20 amino acids of the VMP1 hydrophilic domain, which we have named the VMP1 autophagy-related domain (VMP1-AtgD), and the BH3 domain of BECN1. The association between these two proteins allows the formation of the autophagy-specific PtdIns3K complex, which activity favors the generation of phosphatidylinositol-3-phosphate (PtdIns3P) and the subsequent association of the autophagy-related (ATG) proteins, including ATG16L1, with the phagophore membranes. Therefore, VMP1 regulates the PtdIns3K activity on the phagophore membrane through its interaction with BECN1. Our data provide a novel model describing one of the key steps in phagophore assembly site (PAS) formation and autophagy regulation, and positions VMP1 as a new interactor of the autophagy-specific PtdIns3K complex in mammalian cells.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Involvement of phenylalanine 272 of DNA polymerase beta in discriminating between correct and incorrect deoxynucleoside triphosphates

DNA polymerase beta is a small monomeric polymerase that participates in base excision repair and meiosis [Sobol, R., et al. (1996) Nature 379, 183-186; Plug, A., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1327-1331]. A DNA polymerase beta mutator mutant, F272L, was identified by an in vivo genetic screen [Washington, S., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1321-1326]. Residue 272 is located within the deoxynucleoside triphosphate (dNTP) binding pocket of DNA polymerase beta according to the known DNA polymerase beta crystal structures [Pelletier, H., et al. (1994) Science 264, 1891-1893; Sawaya, M., et al. (1997) Biochemistry 36, 11205-11215]. The F272L mutant produces errors at a frequency 10-fold higher than that of wild type in vivo and in the in vitro HSV-tk gap-filling assay. F272L shows an increase in the frequency of both base substitution mutations and frameshift mutations. Single-enzyme turnover studies of misincorporation by wild type and F272L DNA polymerase beta demonstrate that there is a 4-fold decrease in fidelity of the mutant as compared to that of the wild type enzyme for a G:A mismatch. The decreased fidelity is due primarily to decreased discrimination between the correct and incorrect dNTP during ground-state binding. These results suggest that the phenylalanine 272 residue is critical for maintaining fidelity during the binding of the dNTP.     

Saposins and Their Interaction with Lipids

The lysosomal degradation of several sphingolipids requires the presence of four small glycoproteins called saposins, generated by proteolytic processing of a common precursor, prosaposin. Saposins share several structural properties, including six similarly located cysteines forming three disulfide bridges with the same cysteine pairings. Recently it has been noted that also other proteins have the same polypeptide motif characterized by the similar location of six cysteines. These saposin-like (SAPLIP) proteins are surfactant protein B (SP-B), Entamoeba histolytica poreforming peptide, NK-lysin, acid sphingomyelinase and acyloxyacyl hydrolase. The structural homology and the conserved disulfide bridges suggest for all SAPLIPs a common fold, called saposin fold. Up to now a precise fold, comprising five -helices, has been established only for NK-lysin. Despite their similar structure each saposin promotes the degradation of specific sphingolipids in lysosomes, e.g. Sap B that of sulfatides and Sap C that of glucosylceramides. The different activities of the saposins must reside within the module of the -helices and/or in additional specific regions of the molecule. It has been reported that saposins bind to lysosomal hydrolases and to several sphingolipids. Their structural and functional properties have been extensively reviewed and hypotheses regarding their molecular mechanisms of action have been proposed. Recent work of our group has evidenced a novel property of saposins: some of them undergo an acid-induced change in hydrophobicity that triggers their binding to phospholipid membranes. In this article we shortly review recent findings on the structure of saposins and on their interactions with lipids, with special attention to interactions with phospholipids. These findings offer a new approach for understanding the physiological role of saposins in lysosomes.