Incorporation of Sed1p into the cell wall of Saccharomyces cerevisiae involves KRE6

KRE6 (YPR159W) encodes a Golgi membrane protein required for normal beta-1,6-glucan levels in the cell wall. A functional Kre6p is necessary for cell wall protein accumulation in response to changing metabolic conditions. The product of the SED1 (YDR077W) gene is a stress-induced GPI-cell wall protein. Successful incorporation of HA-tagged Sed1p into the cell wall involves KRE6. The double-mutant sed1 kre6 has a reduced growth rate, increased flocculation and increased sensitivity to Zymolyase. A similar phenotype is found in mutants defective in glycosyl-phosphatidyl-insositol (GPI) anchor assembly. These findings support the theory that Kre6p could function as a transglucosylase that allows the incorporation of proteins with a GPI anchor into the cell wall.     

The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases

Glucanohydrolases, especially mutanase [alpha-(1-->3) glucanase; EC 3.2.1.59] and dextranase [alpha-(1-->6) glucanase; EC 3.2.1.11], which are present in the biofilm known as dental plaque, may affect the synthesis and structure of glucans formed by glucosyltransferases (GTFs) from sucrose within dental plaque. We examined the production and the structure of glucans synthesized by GTFs B (synthesis of alpha-(1-->3)-linked glucans) or C [synthesis of alpha-(1-->6)- and alpha-(1-->3)-linked glucans] in the presence of mutanase and dextranase, alone or in combination, in solution phase and on saliva-coated hydroxyapatite beads (surface phase). The ability of Streptococcus sobrinus 6715 to adhere to the glucan, which was formed in the presence of the glucanohydrolases was also explored. The presence of mutanase and/or dextranase during the synthesis of glucans by GTF B and C altered the proportions of soluble to insoluble glucan. The presence of either dextranase or mutanase alone had a modest effect on total amount of glucan formed, especially in the surface phase; the glucanohydrolases in combination reduced the total amount of glucan. The amount of (1-->6)-linked glucan was reduced in presence of dextranase. In contrast, mutanase enhanced the formation of soluble glucan, and reduced the percentage of 3-linked glucose of GTF B and C glucans whereas dextranase was mostly without effect. Glucan formed in the presence of dextranase provided fewer binding sites for S. sobrinus; mutanase was devoid of any effect. We also noted that the GTFs bind to dextranase and mutanase. Glucanohydrolases, even in the presence of GTFs, influence glucan synthesis, linkage remodeling, and branching, which may have an impact on the formation, maturation, physical properties, and bacterial binding sites of the polysaccharide matrix in dental plaque. Our data have relevance for the formation of polysaccharide matrix of other biofilms.     

Gold(I) and silver(I) complexes of 2,3-bis(diphenylphosphino)maleic acid: Structural studies and antitumour activity

The 2:1 and 1:2 adducts of Au(I) and 1:2 adducts of Ag(I) with the diphosphine 2,3-bis(diphenylphosphino)maleic acid (dpmaa) have been prepared in high yields. Crystal structures have been determined for the neutral digold complex (AuCl)₂(dpmaa) · 2thf (1) and the bis-chelated complex [Au(dpmaa)₂]Cl · H₂O · CH₃OH (2). For 1, conformational rigidity imposed by the ethylenic bridge facilitates the formation of short intramolecular Au-Au contacts with no evidence of similar intermolecular contacts. Complex 2 crystallizes with [Au(dpmaa)₂]⁺ cations hydrogen bonded through the carboxyl groups to a water molecule and chloride anion to form a H-bonded chain along the a axis. ³¹P NMR titration of 1 with dpmaa in acetone shows conversion to 2 at Au:P-P ratios less than 1:1 indicating similar high thermodynamic and kinetic stabilities to other bis-chelated [Au(P-P)₂]⁺ complexes containing 5- or 6-membered chelate rings. The ionic Au(I) complex 2 and the analogous Ag(I) complex [Ag(dpmma)₂]NO₃ (3) are highly water soluble. The in vitro cytotoxic activity of 2 was assessed against eight different cell lines and no significant activity was found. The solubility properties and solution behaviour of the complexes are compared to the analogous 1,2-bis(diphenylphosphino)ethane (dppe) complexes and the potential significance of these results to the antitumour properties of chelated 1:2 Au(I)diphosphine complexes are discussed.     

Direct ANP inhibition of hypoxia-induced inflammatory pathways in pulmonary microvascular and macrovascular endothelial monolayers

Atrial natriuretic peptide (ANP) has been shown to reduce hypoxia-induced pulmonary vascular leak in vivo, but no explanation of a mechanism has been offered other than its vasodilatory and natriuretic actions. Recently, data have shown that ANP can protect endothelial barrier functions in TNF-alpha-stimulated human umbilical vein endothelial cells. Therefore, we hypothesized that ANP actions would inhibit pulmonary vascular leak by inhibition of TNF-alpha secretion and F-actin formation. Bovine pulmonary microvascular (MVEC) and macrovascular endothelial cell (LEC) monolayers were stimulated with hypoxia, TNF-alpha, or bacterial endotoxin (LPS) in the presence or absence of ANP, and albumin flux, NF-kappa B activation, TNF-alpha secretion, p38 mitogen-activated protein kinase (MAPK), and F-actin (stress fiber) formation were assessed. In Transwell cultures, ANP reduced hypoxia-induced permeability in MVEC and TNF-alpha-induced permeability in MVEC and LEC. ANP inhibited hypoxia and LPS increased NF-kappa B activation and TNF-alpha synthesis in MVEC and LEC. Hypoxia decreased activation of p38 MAPK in MVEC but increased activation of p38 MAPK and stress fiber formation in LEC; TNF-alpha had the opposite effect. ANP inhibited an activation of p38 MAPK in MVEC or LEC. These data indicate that in endothelial cell monolayers, hypoxia activates a signal cascade analogous to that initiated by inflammatory agents, and ANP has a direct cytoprotective effect on the pulmonary endothelium other than its vasodilatory and natriuretic properties. Furthermore, our data show that MVEC and LEC respond differently to hypoxia, TNF-alpha-stimulation, and ANP treatment.     

Interaction of thiostrepton and elongation factor-G with the ribosomal protein L11-binding domain

Ribosomal protein L11 and the L11 binding region of ribosomal RNA constitute an important domain involved in active functions of the ribosome during translation. We studied the effects of L11 knock-out and truncation mutations on the structure of the rRNA in this region and on its interactions with a translation elongation factor and the antibiotic thiostrepton. The results indicated that the structure of the L11-binding rRNA becomes conformationally flexible when ribosomes lack the entire L11 protein, but not when the C-terminal domain is present on ribosomes. Probing wild type and mutant ribosomes in the presence of the antibiotic thiostrepton and elongation factor-G (EF-G) rigorously localized the binding cleft of thiostrepton and suggested a role for the rRNA in the L11-binding domain in modulating factor binding. Our results also provide evidence that the structure of the rRNA stabilized by the C-terminal domain of L11 is necessary to stabilize EF-G binding in the post-translocation state, and thiostrepton may modulate this structure in a manner that interferes with the ribosome-EF-G interaction. The implications for recent models of thiostrepton activity and factor interactions are discussed.     

Phylogenetic variation in the shoot mineral concentration of angiosperms

The calcium (Ca) concentration of plant shoot tissues varies systematically between angiosperm orders. The phylogenetic variation in the shoot concentration of other mineral nutrients has not yet been described at an ordinal level. The aims of this study were (1) to quantify the shoot mineral concentration of different angiosperm orders, (2) to partition the phylogenetic variation in shoot mineral concentration between and within orders, (3) to determine if the shoot concentration of different minerals are correlated across angiosperm species, and (4) to compare experimental data with published ecological survey data on 81 species sampled from their natural habitats. Species, selected pro rata from different angiosperm orders, were grown in a hydroponic system under a constant external nutrient regime. Shoots of 117 species were sampled during vegetative growth. Significant variation in shoot carbon (C), calcium (Ca), potassium (K), and magnesium (Mg) concentration occurred between angiosperm orders. There was no evidence for systematic differences in shoot phosphorus (P) or organic-nitrogen (N) concentration between orders. At a species level, there were strong positive correlations between shoot Ca and Mg concentration, between shoot P and organic-N concentration, and between shoot K concentration and shoot fresh weight:dry weight ratio. Shoot C and cation concentration correlated negatively at a species level. Species within the Poales and the Caryophyllales had distinct shoot mineralogies in both the designed experiment and in the ecological survey.     

Repeated exposure to prolactin is required to induce luteal regression in the hypophysectomized rat

We investigated whether prolactin (PRL) treatments resembling the intermittent PRL surges of estrous cycles could induce luteal regression in hypophysectomized rats. Immature female rats were stimulated to ovulate and form corpora lutea with exogenous gonadotropins, and were hypophysectomized following ovulation. A single s.c. injection of either vehicle (VEH) or PRL was administered to each rat on post-hypophysectomy Day 8 and again on Day 11. The four resulting treatment groups consisted of rats that received two injections of VEH, VEH followed by PRL, PRL followed by VEH, or two injections of PRL. Rats were killed 24 or 72 h following the second injection. Plasma 20alpha-dihydroprogesterone, luteal weight, and total luteal protein were determined. One ovary was sectioned for immunohistochemistry for monocytes/macrophages, apoptotic nuclei, and major histocompatibility class II (MHC II) molecules. No effect of time (following injection) was observed on any endpoint, indicating that PRL does not have an ongoing regressive action. Time groups from within each treatment group were therefore pooled for analysis. Significant declines (P: < 0.05) in plasma concentrations of 20alpha-dihydroprogesterone, luteal weight, and protein per corpus luteum occurred only after two injections of PRL. Numbers of luteal monocytes/macrophages, apoptotic nuclei, and MHC II-positive cells were low in all groups; numbers of luteal monocytes/macrophages increased following two injections of PRL (P: < 0.05). We conclude that PRL has a cumulative regressive effect on the corpus luteum of the hypophysectomized rat. Drawing a parallel with the estrous cycle, we suggest that continued exposure to PRL, over several cycles, is necessary to induce full luteal regression.     

Metabolic compensation during high energy output in fasting, lactating grey seals (Halichoerus grypus): metabolic ceilings revisited

Lactation is the most energetically expensive period for female mammals and is associated with some of the highest sustained metabolic rates (SusMR) in vertebrates (reported as total energy throughput). Females typically deal with this energy demand by increasing food intake and the structure of the alimentary tract may act as the central constraint to ceilings on SusMR at about seven times resting or standard metabolic rate (SMR). However, demands of lactation may also be met by using a form of metabolic compensation such as reducing locomotor activities or entering torpor. In some phocid seals, cetaceans and bears, females fast throughout lactation and thus cannot offset the high energetic costs of lactation through increased food intake. We demonstrate that fasting grey seal females sustain, for several weeks, one of the highest total daily energy expenditures (DEE; 7.4 x SMR) reported in mammals, while progressively reducing maintenance metabolic expenditures during lactation through means not explained by reduction in lean body mass or behavioural changes. Simultaneously, the energy-exported in milk is progressively increased, associated with increased lipoprotein lipase activity in the mammary gland, resulting in greater offspring growth. Our results suggest that females use compensatory mechanisms to help meet the extraordinary energetic costs of lactation. Additionally, although the concepts of SusMR and ceilings on total DEE may be somewhat different in fasting lactating species, our data on phocid seals demonstrate that metabolic ceilings on milk energy output, in general, are not constrained by the same kind of peripheral limitations as are other energy-consuming tissues. In phocid seals, the high ceilings on DEE during lactation, coupled with metabolic compensation, are undoubtedly important factors enabling shortened lactation.