Functional and structural characterization of a prokaryotic peptide transporter with features similar to mammalian PEPT1

The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.

Enemy damage of exotic plant species is similar to that of natives and increases with productivity

1 .In their colonized ranges, exotic plants may be released from some of the herbivores or pathogens of their home ranges but these can be replaced by novel enemies. It is of basic and practical interest to understand which characteristics of invaded communities control accumulation of the new pests. Key questions are whether enemy load on exotic species is smaller than on native competitors as suggested by the enemy release hypothesis (ERH) and whether this difference is most pronounced in resource-rich habitats as predicted by the resource–enemy release hypothesis (R-ERH). 2. In 72 populations of 12 exotic invasive species, we scored all visible above-ground damage morphotypes caused by herbivores and fungal pathogens. In addition, we quantified levels of leaf herbivory and fruit damage. We then assessed whether variation in damage diversity and levels was explained by habitat fertility, by relatedness between exotic species and the native community or rather by native species diversity. 3. In a second part of the study, we also tested the ERH and the R-ERH by comparing damage of plants in 28 pairs of co-occurring native and exotic populations, representing nine congeneric pairs of native and exotic species. 4. In the first part of the study, diversity of damage morphotypes and damage levels of exotic populations were greater in resource-rich habitats. Co-occurrence of closely related, native species in the community significantly increased the probability of fruit damage. Herbivory on exotics was less likely in communities with high phylogenetic diversity. 5. In the second part of the study, exotic and native congeneric populations incurred similar damage diversity and levels, irrespective of whether they co-occurred in nutrient-poor or nutrient-rich habitats. 9. Synthesis. We identified habitat productivity as a major community factor affecting accumulation of enemy damage by exotic populations. Similar damage levels in exotic and native congeneric populations, even in species pairs from fertile habitats, suggest that the enemy release hypothesis or the R-ERH cannot always explain the invasiveness of introduced species.

Non-pathogenic Actinobacillus isolates antigenically and biochemically similar to Actinobacillus pleuropneumoniae: a novel species?

Two unusual Actinobacillus isolates were recovered from pigs with no clinical signs, no lesions and no history of swine pleuropneumonia. Two representative strains (9953L55 and 0347) analyzed in this study were initially biochemically and antigenically identified as A. pleuropneumoniae serotypes 1 and 9, respectively, by traditional identification methods. Both strains presented, however, negative results with three A. pleuropneumoniae-specific PCR tests and revealed in particular the absence of the apxIV toxin genes. However, both strains produced and secreted ApxII toxin although they only harbored the toxin genes apxIICA, which is an uncommon feature for any of the known A. pleuropneumoniae serotypes. Upon experimental inoculation of pigs, these strains proved to be totally non-pathogenic. Animals infected with one of the strains produced antibodies that cross-react with A. pleuropneumoniae serotypes 1-9-11-specific LC-LPS ELISA. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that these strains form a separate phylogenetic group that is distinct from other Actinobacillus species and is particularly different from A. pleuropneumoniae.

The Effect of Heavy Metal Stress on Avian Proximal Tubule Urate Secretion

In both humans and birds, urate is an important antioxidant when maintained at normal plasma concentrations. Though human kidneys primarily reabsorb filtered urate, while those of birds perform mostly secretion, both maintain urate levels at ~300microM. The importance of maintaining urate levels within the homeostatic range was observed when the study of several prominent diseases revealed an association with hyperuricemia. This study examined the effect of elevated zinc concentration on avian urate secretion. Here, acute exposure of chicken proximal tubule epithelial cells (cPTCs) to zinc stress had no effect on urate secretion, but prolonged zinc-induced cellular stress inhibited active transepithelial urate secretion with no change in Mrp4 expression, glucose transport, or transepithelial resistance. Moreover, zinc had no effect on urate transport by isolated brush border membrane vesicles, suggesting involvement of a more complex cellular stress adaptation. Previous work has demonstrated that AMP-activated protein kinase (AMPK), a critical metabolic regulator, conserves energy during cellular stress by shutting down ATP-utilizing processes and activating ATP-generating processes. Pharmacological activation of AMPK by AICAR produced decreased urate secretion by cPTCs similar to the effect seen with prolonged exposure to zinc, while the AMPK inhibitor Compound C prevented both AICAR and zinc inhibition of urate secretion, suggesting a stress induced mechanism of regulation. Supported by NSF. IACUC #A08-046.

Plausibility of a subglacial lake under Amundsenisen Icefield (Svalbard): spatially variable water content and sensitivity to thermal effect of snow and firn layers

Within our study of the plausibility of a subglacial lake under the Amundsenisen Icefield in Southern Spitzbergen, Svalbard achipelago (Glowacki et al., 2007), here we focus on the sensitivity of the system to the thermal effect of the firn and snow layers. Rough heat balance analysis shows that the firn layer plays an important role by driving the heat release to the atmosphere, so that its influence on the ice-water phase transition cannot be neglected (Bucchignani et al., 2012).

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal “clip” domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.

A cell surface receptor defined by a mAb mediates a unique type of cell death similar to oncosis

Cell death is mediated by distinct pathways including apoptosis and oncosis in response to various death signals. To characterize molecules involved in cell death, a panel of mAbs was raised by immunizing mice with apoptotic cells. One of these antibodies, designated anti-Porimin (for pro-oncosis receptor inducing membrane injury), was found to directly induce a unique type of cell death in Jurkat cells. Anti-Porimin defines a 110-kDa cell surface receptor on Jurkat cells. Functionally, anti-Porimin alone rapidly mediates pore formation on the plasma membrane and induces cell death without participation of complement. Both the cellular expression and functional characteristics of the Porimin antigen indicate that it is distinct from the CD95 (Fas/Apo-1) and other cell receptors known to induce apoptosis. Anti-Porimin-mediated cell death was preceded by cell aggregation, formation of plasma membrane pores, and the appearance of membrane blebs. More important, these cells show neither DNA fragmentation nor apoptotic bodies, but display lethal damage of the cell membrane. Cell death by anti-Porimin is distinct from complement-dependent cytolysis or complement-independent apoptosis but is similar to that described for oncosis, a form of cell death accompanied by the membrane damage followed by karyolysis. The induction of cell death by anti-Porimin may represent a unique cell surface receptor-mediated pathway of cell death in the human lymphoid system.

The silver gene of Drosophila melanogaster encodes multiple carboxypeptidases similar to mammalian prohormone-processing enzymes.

The silver (svr) gene of Drosophila melanogaster is required for viability, and severe mutant alleles result in death prior to eclosion. Adult flies homozygous or hemizygous for weaker alleles display several visible phenotypes, including cuticular structures that are pale and silvery in color due to reduced melanization. We have identified and cloned the DNA encoding the svr gene and determined the sequence of several partially overlapping cDNAs derived from svr mRNAs. The predicted amino acid sequence of the polypeptides encoded by these cDNAs indicates that the silver proteins are members of the family of preprotein-processing carboxypeptidases that includes the human carboxypeptidases E, M, and N. One class of svr mRNAs is alternatively spliced to encode at least two polyproteins, each of which is composed of two carboxypeptidase domains.

Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF.

We show that the higher plant Arabidopsis thaliana has a serine-arginine-rich (SR) protein family whose members contain a phosphoepitope shared by the animal SR family of splicing factors. In addition, we report the cloning and characterization of a cDNA encoding a higher-plant SR protein from Arabidopsis, SR1, which has striking sequence and structural homology to the human splicing factor SF2/ASF. Similar to SF2/ASF, the plant SR1 protein promotes splice site switching in mammalian nuclear extracts. A novel feature of the Arabidopsis SR protein is a C-terminal domain containing a high concentration of proline, serine, and lysine residues (PSK domain), a composition reminiscent of histones. This domain includes a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2.