Experimental infection of house finches with Mycoplasma gallisepticum

Mycoplasma gallisepticum (MG) has caused an endemic upper respiratory and ocular infection in the eastern house finch (Carpodacus mexicanus) after the epidemic first described in 1994. The disease has been studied by a number of investigators at a population level and reports describe experimental infection in group-housed MG-free house finches. Because detailed observation and evaluation of individual birds in group-housed passerines is problematic, we studied individually housed house finches that were experimentally inoculated with the finch strain of MG in a controlled environment. To accomplish this, a study was conducted spanning the period of November 2001-April 2002 with 20 MG-free (confirmed by the rapid plate agglutination assay and polymerase chain reaction [PCR] assay) eastern house finches captured in the Cayuga Basin area of central New York (USA) in the summer of 2001. After a period of acclimatization and observation (12 wk), 20 finches were inoculated with a 0.05-ml aliquot of MG (3.24 x 10(5) colony-forming units/ml) via bilateral conjunctival sac instillations. Two additional finches acted as controls and were inoculated in the same manner with preservative-free sterile saline solution. After inoculation, all finches except the controls exhibited clinical signs of conjunctivitis within 2-6 days. The progression of the disease was evaluated by several methods, including PCR, behavioral observations, and physical examination including eye scoring, body weight, and body condition index. Over a period of 21 wk, MG-infected finches developed signs of disease and recovered (80%), developed signs of disease and progressed to become chronically infected (15%), or died (5%). We hypothesize that the high survival rate and recovery of these finches after infection was associated with the use of controlled environmental conditions, acclimatization, a high plane of nutrition, and low stocking (housing) density, all of which are factors documented to be important in the outcome of MG infections in domestic poultry and other species.     

Synthesis and evaluation of substrate-mimicking cytosolic phospholipase A2 inhibitors--reducing the lipophilicity of the arachidonyl chain isostere

The high lipophilicity of a series of cytosolic phospholipase A(2) inhibitors has been reduced by the modification of a decyloxyphenyl chain designed to mimic the arachidonyl group of the natural substrate. These changes have resulted in an improvement in the whole cell potency of the inhibitors.     

Expression, purification, and characterization of authentic monoferric and apo-human serum transferrins

Transferrin is a bilobal protein with the ability to bind iron in two binding sites situated at the bottom of a cleft in each lobe. We have previously described the production of recombinant non-glycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa-His tag at the amino-terminus. Constructs in this background that contain strategic mutations to completely prevent iron binding in each lobe or in both lobes have now been produced. These monoferric hTFs will allow dissection of the contribution of each lobe to transferrin function. In addition, the construct completely lacking in the ability to bind iron in either lobe provides an opportunity to assess whether hTF has any other functions in addition to iron transport. Following insertion of the His-tagged hTF molecules into the pNUT vector, transfection into baby hamster kidney cells and selection with methotrexate, the secreted recombinant proteins were isolated from the tissue culture medium and characterized with regard to their iron binding properties. Significant improvements over our previous protocol include: (1) addition of butyric acid at a level of 1mM which leads to a substantial increase in protein production (as much as a 65% increase compared to control cells); and (2) elimination of an anion exchange column prior to isolation on a Qiagen Ni-NTA column which makes purification of the His-tagged constructs faster and therefore more efficient. These improvements should be applicable to expression of other recombinant proteins in mammalian cells.     

Insights into the effects on metal binding of the systematic substitution of five key glutamate ligands in the ferritin of Escherichia coli

Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of FeB2+ to FeC2+ enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.     

Regulation of p70 S6 kinase by complex formation between the Rac guanine nucleotide exchange factor (Rac-GEF) Tiam1 and the scaffold spinophilin

Tiam1 is a ubiquitous guanine nucleotide exchange factor (GEF) that activates the Rac GTPase. We have shown previously that the N terminus of Tiam1 contributes to the signaling specificity of its downstream target Rac via association with IB2, a scaffold that promotes Rac activation of a p38 kinase cascade. Here we show that the N terminus of Tiam1 can influence Rac signaling specificity in a different way by interaction with spinophilin, a scaffold that binds to p70 S6 kinase, another protein regulated by Rac. In particular, spinophilin binding promotes the plasma membrane localization of Tiam1 and enhances the ability of Tiam1 to activate p70 S6 kinase. In contrast, spinophilin binding suppresses the ability of Tiam to activate Pak1, a different Rac effector. Finally, a mutant spinophilin that cannot bind to Tiam1 suppresses serum-induced p70 S6 kinase activation in cells, suggesting that a Tiam1/spinophilin complex contributes to p70 S6 kinase regulation by extracellular signals. These findings add to a growing body of evidence supporting the concept that some Rac-GEFs not only activate Rac GTPases but also participate in the selection of Rac effector by binding to particular scaffolds that complex with components of specific Rac effector pathways.     

Junction adhesion molecule is a receptor for reovirus

Virus attachment to cells plays an essential role in viral tropism and disease. Reovirus serotypes 1 and 3 differ in the capacity to target distinct cell types in the murine nervous system and in the efficiency to induce apoptosis. The binding of viral attachment protein sigma1 to unidentified receptors controls these phenotypes. We used expression cloning to identify junction adhesion molecule (JAM), an integral tight junction protein, as a reovirus receptor. JAM binds directly to sigma1 and permits reovirus infection of nonpermissive cells. Ligation of JAM is required for reovirus-induced activation of NF-kappaB and apoptosis. Thus, reovirus interaction with cell-surface receptors is a critical determinant of both cell-type specific tropism and virus-induced intracellular signaling events that culminate in cell death.     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and beta-mercaptoethanol, with concentrations of 10 mM inhibiting by approximately 40%. DTT's inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [(3)H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

Growth and Reproduction of Double-Ended Pipefish, Syngnathoides biaculeatus, in Moreton Bay, Queensland, Australia

Life-history characteristics of the double-ended pipefish, Syngnathoides biaculeatus (Bloch), were investigated to determine growth rate, degree of sexual dimorphism, size at maturity, and reproductive biology. Growth rates of wild juveniles and adults calculated from monthly progression of length-frequency modes ranged from 0.8mmd^–1 (fish lengths 120–145mm standard length (SL)) in summer to 0.2mmd^–1 in winter (185–200mm SL). Growth of laboratory-reared juveniles up to 63d old was greater, ranging from 0.8 to 2.3mmd^-1. The von Bertalanffy growth constant K was estimated at 0.0076d^{- 1}, or 2.8year^–1. Morphological differentiation between the sexes based upon abdominal pattern was possible for fish larger than 120mm SL, with females possessing a zigzag pattern on the abdomen. The association between this pattern and sex was confirmed by histological gonad analysis. Males were significantly longer than females during four of seven seasons examined, and a 1:1 sex ratio was determined for all seasons except autumn when the ratio was female biased. The breeding season was marked by the appearance of pregnant males between October and April, and during courtship both species exhibited increased pigmentation. The minimum paternal size at maturity was 185mm, the maximum length recorded 260mm. Clutch size ranged between 60 and 200 eggs, with a mean of 153. Ovaries had a sequential pattern of egg development, resulting in egg batches that approximated the number of eggs carried by brooding males. Additionally, all eggs in a brood were at the same developmental stage. This suggests that one female provides all of the eggs for one male per breeding event in a monogamous mating system.     

Xanthones from the bark of Garcinia merguensis

The bark of Garcinia merguensis yielded 10 xanthones, merguenone, 1,5-dihydroxy-6'-methyl-6'-(4-methyl-3-pentenyl)-pyrano(2',3':3,2)-xanthone, subelliptenone H, 8-deoxygartanin, rheediaxanthone A, morusignin G, 6-deoxyjacareubin, 1,3,5-trihydroxy-4,8-di(3-methylbut-2-enyl)-xanthone, rheediachromenoxanthone and 6-deoxyisojacareubin. The structure of merguenone was determined using spectroscopic techniques, mainly 1D and 2D NMR spectroscopy.