Antimicrobial susceptibility and characterization of Salmonella isolates from processed bison carcasses

Seventeen Salmonella enterica serovar Hadar isolates recovered from bison were found to possess a range of virulence genes and resistance to tetracycline, gentamicin, sulfamethoxazole, and streptomycin simultaneously. A 1-kb class 1 integron containing the aadA1 gene was identified in all isolates. Pulsed-field gel electrophoresis found that all isolates were closely related, indicating the possibility of cross-contamination during processing.     

Immune-related, lectin-like receptors are differentially expressed in the myeloid and lymphoid lineages of zebrafish

The identification of C-type lectin (Group V) natural killer (NK) cell receptors in bony fish has remained elusive. Analyses of the Fugu rubripes genome database failed to identify Group V C-type lectin domains (Zelensky and Gready, BMC Genomics 5:51, 2004) suggesting that bony fish, in general, may lack such receptors. Numerous Group II C-type lectin receptors, which are structurally similar to Group V (NK) receptors, have been characterized in bony fish. By searching the zebrafish genome database we have identified a multi-gene family of Group II immune-related, lectin-like receptors (illrs) whose members possess inhibiting and/or activating signaling motifs typical of Group V NK receptors. Illr genes are differentially expressed in the myeloid and lymphoid lineages, suggesting that they may play important roles in the immune functions of multiple hematopoietic cell lineages.     

Metabolic consequences of susceptibility and resistance (race-specific and broad-spectrum) in barley leaves challenged with powdery mildew

In a compatible interaction biotrophic fungi often lower the yield of their hosts by reducing photosynthesis and altering the fluxes of carbon within the infected leaf. In contrast, comparatively little is known about the metabolic consequences of activating resistance responses. In this study we investigated the hypothesis that the activation of both race-specific (Mla12) and broad-spectrum (mlo) resistance pathways in barley leaves infected with Blumeria graminis represents a cost to the plant in terms of carbon production and utilization. We have shown, using quantitative imaging of chlorophyll fluorescence, that during a susceptible interaction, photosynthesis was progressively reduced both in cells directly below fungal colonies and in adjacent cells when compared with uninoculated leaves. The lower rate of photosynthesis was associated with an increase in invertase activity, an accumulation of hexoses and a down-regulation of photosynthetic gene expression. During both Mla12- and mlo-mediated resistance, photosynthesis was also reduced, most severely inhibited in cells directly associated with attempted penetration of the fungus but also in surrounding cells. These cells displayed intense autofluorescence under ultraviolet illumination indicative of the accumulation of phenolic compounds and/or callose deposition. The depression in photosynthesis was not due only to cell death but also to an alteration in source-sink relations and carbon utilization. Apoplastic (cell wall-bound) invertase activity increased more rapidly and to a much greater extent than in infected susceptible leaves and was accompanied by an accumulation of hexoses that was localized to areas of the leaf actively exhibiting resistance responses. The accumulation of hexoses was accompanied by a down-regulation in the expression of Rubisco (rbcS) and chlorophyll a/b binding protein (cab) genes (although to a lesser extent than in a compatible interaction) and with an up-regulation in the expression of the pathogenesis-related protein 1 (PR-1). These results are consistent with a role for invertase in the generation of hexoses, which may supply energy for defence reactions and/or act as signals inducing defence gene expression.     

Molecular cloning and characterization of patellin1, a novel sec14-related protein, from zucchini (Cucurbita pepo)

A full-length patellin1 (PATL1) cDNA was cloned and characterized from zucchini (Cucurbita pepo). PATL1, originally discovered in the higher plant Arabidopsis thaliana, is a plant Sec14-related protein that localizes to the cell plate during the late stages of cytokinesis. PATL1 is related in sequence to other eukaryotic proteins involved in membrane trafficking and is thought to participate in vesicle trafficking events associated with cell plate maturation. The zucchini PATL1 (CpPATL1) cDNA predicts a 605 amino acid protein which consists of an acidic N-terminal domain (pI=4.2) followed by a Sec14 lipid-binding domain and a C-terminal Golgi dynamics domain (GOLD). The predicted CpPATL1 protein sequence shows a high degree of similarity to Arabidopsis PATL1, especially in the Sec14 (84%) and GOLD domains (87%). A phylogenetic analysis of all available full-length PATL sequences revealed that the PATLs belong to four distinct clades; CpPATL1 is a member of the PATL1/2 clade. RT-PCR analysis showed that the CpPATL1 gene is highly expressed throughout the plant. The domain structure, as well as biochemical fractionation studies, which demonstrated that CpPATL1 is a peripheral membrane protein, support a role in membrane trafficking events.     

Involvement of the LlaKR2I methylase in expression of the AbiR bacteriophage defense system in Lactococcus lactis subsp. lactis biovar diacetylactis KR2

The native lactococcal plasmid, pKR223, from Lactococcus lactis subsp. lactis biovar diacetylactis KR2 encodes two distinct bacteriophage-resistant mechanisms, the LlaKR2I restriction and modification (R/M) system and the abortive infection (Abi) mechanism, AbiR, that impedes bacteriophage DNA replication. This study completed the characterization of AbiR, revealing that it is the first Abi system to be encoded by three genes, abiRa, abiRb, and abiRc, arranged in an operon and that it requires the methylase gene from the LlaKR2I R/M system. An analysis of deletion and insertion clones demonstrated that the AbiR operon was toxic in L. lactis without the presence of the LlaKR2I methylase, which is required to protect L. lactis from AbiR toxicity. The novelty of the AbiR system resides in its original gene organization and the unusual protective role of the LlaKR2I methylase. Interestingly, the AbiR genetic determinants are flanked by two IS982 elements generating a likely transposable AbiR composite. This observation not only substantiated the novel function of the LlaKR2I methylase in the AbiR system but also illustrated the evolution of the LlaKR2I methylase toward a new and separate cellular function. This unique structure of both the LlaKR2I R/M system and the AbiR system may have contributed to the evolution of the LlaKR2I methylase toward a novel role comparable to that of the cell cycle-regulated methylases that include Dam and CcrM methylases. This new role for the LlaKR2I methylase offers a unique snapshot into the evolution of the cell cycle-regulated methylases from an existing R/M system.     

Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.     

Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants

In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [(125)I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/I172M had a synergistic impact on (RS)-CIT recognition ( approximately 10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.     

A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites

Apicomplexan parasites constitute one of the most significant groups of pathogens infecting humans and animals. The liver stage sporozoites of Plasmodium spp. and tachyzoites of Toxoplasma gondii, the causative agents of malaria and toxoplasmosis, respectively, use a unique mode of locomotion termed gliding motility to invade host cells and cross cell substrates. This amoeboid-like movement uses a parasite adhesin from the thrombospondin-related anonymous protein (TRAP) family and a set of proteins linking the extracellular adhesin, via an actin-myosin motor, to the inner membrane complex. The Plasmodium blood stage merozoite, however, does not exhibit gliding motility. Here we show that homologues of the key proteins that make up the motor complex, including the recently identified glideosome-associated proteins 45 and 50 (GAP40 and GAP50), are present in P. falciparum merozoites and appear to function in erythrocyte invasion. Furthermore, we identify a merozoite TRAP homologue, termed MTRAP, a micronemal protein that shares key features with TRAP, including a thrombospondin repeat domain, a putative rhomboid-protease cleavage site, and a cytoplasmic tail that, in vitro, binds the actin-binding protein aldolase. Analysis of other parasite genomes shows that the components of this motor complex are conserved across diverse Apicomplexan genera. Conservation of the motor complex suggests that a common molecular mechanism underlies all Apicomplexan motility, which, given its unique properties, highlights a number of novel targets for drug intervention to treat major diseases of humans and livestock.     

Mechanics of cellular adhesion to artificial artery templates

We are using polymer templates to grow artificial artery grafts in vivo for the replacement of diseased blood vessels. We have previously shown that adhesion of macrophages to the template starts the graft formation. We present a study of the mechanics of macrophage adhesion to these templates on a single cell and single bond level with optical tweezers. For whole cells, in vitro cell adhesion densities decreased significantly from polymer templates polyethylene to silicone to Tygon (167, 135, and 65 cells/mm(2)). These cell densities were correlated with the graft formation success rate (50%, 25%, and 0%). Single-bond rupture forces at a loading rate of 450 pN/s were quantified by adhesion of trapped 2-microm spheres to macrophages. Rupture force distributions were dominated by nonspecific adhesion (forces <40 pN). On polystyrene, preadsorption of fibronectin or presence of serum proteins in the cell medium significantly enhanced adhesion strength from a mean rupture force of 20 pN to 28 pN or 33 pN, respectively. The enhancement of adhesion by fibronectin and serum is additive (mean rupture force of 43 pN). The fraction of specific binding forces in the presence of serum was similar for polystyrene and polymethyl-methacrylate, but specific binding forces were not observed for silica. Again, we found correlation to in vivo experiments, where the density of adherent cells is higher on polystyrene than on silica templates, and can be further enhanced by fibronectin adsorption. These findings show that in vitro adhesion testing can be used for template optimization and to substitute for in-vivo experiments.     

D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily

The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (beta/alpha)(8)-barrel ancestor. The superfamily includes d-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice [Jelakovic, S., Kopriva, S., Suss, K. H., and Schulz, G. E. (2003) J. Mol. Biol. 326, 127-35] and Plasmodium falciparum [Caruthers, J., Bosch, J., Bucker, F., Van Voorhis, W., Myler, P., Worthey, E., Mehlin, C., Boni, E., De Titta, G., Luft, J., Kalyuzhniy, O., Anderson, L., Zucker, F., Soltis, M., and Hol, W. G. J. (2006) Proteins 62, 338-42], the RPE from Streptococcus pyogenes is activated by Zn(2+) which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn(2+) and inactive apoenzyme cannot be prepared, the affinity for Zn(2+) is decreased by alanine substitutions for the two histidine residues that coordinate the Zn(2+) ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn(2+). The crystal structure of the RPE was solved at 1.8 A resolution in the presence of d-xylitol 5-phosphate, an inert analogue of the d-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn(2+) that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn(2+) and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif located at the ends of the seventh and eighth beta-strands of the (beta/alpha)(8)-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis that the members of the "ribulose phosphate binding" (beta/alpha)(8)-barrel "superfamily" as defined by SCOP have not evolved by evolutionary processes involving the intact (beta/alpha)(8)-barrel. Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel.