Conformational Itinerary of Pseudomonas aeruginosa 1,6-Anhydro-N-acetylmuramic Acid Kinase during Its Catalytic Cycle

Anhydro-sugar kinases are unique from other sugar kinases in that they must cleave the 1,6-anhydro ring of their sugar substrate to phosphorylate it using ATP. Here we show that the peptidoglycan recycling enzyme 1,6-anhydro-N-acetylmuramic acid kinase (AnmK) from Pseudomonas aeruginosa undergoes large conformational changes during its catalytic cycle, with its two domains rotating apart by up to 32° around two hinge regions to expose an active site cleft into which the substrates 1,6-anhydroMurNAc and ATP can bind. X-ray structures of the open state bound to a nonhydrolyzable ATP analog (AMPPCP) and 1,6-anhydroMurNAc provide detailed insight into a ternary complex that forms preceding an operative Michaelis complex. Structural analysis of the hinge regions demonstrates a role for nucleotide binding and possible cross-talk between the bound ligands to modulate the opening and closing of AnmK. Although AnmK was found to exhibit similar binding affinities for ATP, ADP, and AMPPCP according to fluorescence spectroscopy, small angle x-ray scattering analyses revealed that AnmK adopts an open conformation in solution in the absence of ligand and that it remains in this open state after binding AMPPCP, as we had observed for our crystal structure of this complex. In contrast, the enzyme favored a closed conformation when bound to ADP in solution, consistent with a previous crystal structure of this complex. Together, our findings show that the open conformation of AnmK facilitates binding of both the sugar and nucleotide substrates and that large structural rearrangements must occur upon closure of the enzyme to correctly align the substrates and residues of the enzyme for catalysis.     

Relationships of the Escherichia coli O157, O111, and O55 O-antigen gene clusters with those of Salmonella enterica and Citrobacter freundii, which express identical O antigens

Escherichia coli O157, Salmonella enterica O30, and Citrobacter freundii F90 have identical O-antigen structures, as do E. coli O55 and S. enterica O50. The O-antigen gene cluster sequences for E. coli O157 and E. coli O55 have been published, and the genes necessary for O-antigen biosynthesis have been identified, although transferase genes for glycosidic linkages are only generic and have not been allocated to specific linkages. We determined sequences for S. enterica O30 and C. freundii F90 O-antigen gene clusters and compared them to the sequence of the previously described E. coli O157 cluster. We also determined the sequence of the S. enterica O50 O-antigen gene cluster and compared it to the sequence of the previously described E. coli O55 cluster. For both the S. enterica O30-C. freundii F90-E. coli O157 group and the S. enterica O50-E. coli O55 group of O antigens, the gene clusters have identical or nearly identical organizations. The two sets of gene clusters had comparable overall levels of similarity in their genes, which were lower than the levels determined for housekeeping genes for these species, which were 55 to 65% for the genes encoding glycosyltransferases and O-antigen processing proteins and 75 to 93% for the nucleotide-sugar pathway genes. Nonetheless, the similarity of the levels of divergence in the five gene clusters required us to consider the possibility that the parent gene cluster for each structure was in the common ancestor of the species and that divergence is faster than expected for the common ancestor hypothesis. We propose that the identical O-antigen gene clusters originated from a common ancestor, and we discuss some possible explanations for the increased rate of divergence that is seen in these genes.     

Host-range and cold temperature tolerance of Floracarus perrepae Knihinicki & Boczek (Acari: Eriophyidae), a potential biological-control agent of Lygodium microphyllum (Pteridophyta: Lygodiaceae)

Abstract The leaf galling eriophyid mite Floracarus perrepae is a widespread and damaging herbivore of Old World climbing fern, Lygodium microphyllum , across its native range in tropical and subtropical Asia and Australia; and was therefore selected as a candidate biological-control agent for the invasive fern in Florida, USA. The host testing of F. perrepae focused on Lygodium species from North America and the Neotropics, along with threatened or endangered ferns from Florida. Sporeling ferns were used for the initial no-choice screening and F. perrepae showed normal development on the Florida genotype of L. microphyllum with 33.0 ± 4.6 mites per marginal leaf roll gall, modest development on the North American native Lygodium palmatum with 29.0 ± 9.3 mites per roll, and minor development on six other fern species (4.0–0.3). Leaf rolls were induced only on Lygodium species and full rolls were common only on L. microphyllum . These same six non-target plant species were tested again as more mature plants in both no-choice and choice tests and F. perrepae developed only on L. microphyllum . Lethal minimum temperature and cold stress tests were also conducted on F. perrepae . These tests revealed that it would not likely establish north of USDA hardiness zone 8a (−9.5 to −12.2°C), which means it would not overlap with L. palmatum from temperate North America. Our studies conclude that F. perrepae is specific to L. microphyllum , poses little or no risk to native or cultivated ferns in North and South America, and therefore should be considered for release in Florida.     

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.     

Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family.

A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI). A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full-length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis. The authenticity of full-length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E. coli cells and in conditioned media of mouse Ltk- cells, transfected with PAI cDNA inserted into vector pSV2. Analysis of the de novo synthesized anti-plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk- cells synthesize a polypeptide with a mol. wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E. coli an unglycosylated, active product with a mol. wt of 43,000 is made. The amino acid sequence, derived from the determined nucleotide sequence, shows that pre-PAI consists of 402 amino acids. It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues. The amino acid sequence of mature PAI includes three potential asparagine-linked glycosylation sites and lacks cysteine residues. The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g. alpha 1-proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin. Inhibition of kringle-2 binding to fibrin by epsilon-amino caproic acid

The binding of tissue-type plasminogen activator (t-PA) to fibrin is mediated both by its finger domain and by its kringle-2 domain. In this report, we investigate the relative affinities of these domains for lysine. Human recombinant t-PA deletion-mutant proteins were prepared and their ability to bind to lysine-Sepharose was investigated. Mutants containing the kringle-2 domain bound to lysine-Sepharose, whereas mutants lacking this domain but containing the finger domain, the epidermal growth factor domain or the kringle-1 domain did not bind to lysine-Sepharose. Mutant proteins containing the kringle-2 domain could be specifically eluted from lysine-Sepharose with epsilon-amino caproic acid. This lysine derivative also abolished fibrin binding by the kringle-2 domain but had no effect on the fibrin-binding property of the finger domain. Thus, a lysine-binding site is involved in the interaction of the kringle-2 domain with fibrin but not in the interaction of the finger domain with fibrin. The implications of the nature of these two distinct interactions of t-PA with fibrin on plasminogen activation by t-PA will be discussed.     

Inducibility of carbamoylphosphate synthetase (ammonia) in cultures of embryonic hepatocytes: ontogenesis of the responsiveness to hormones

Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is present as soon as the cells become recognizable as hepatocytes. Immunochemical staining of the cultures shows that hepatocytes do not acquire or express the capacity to accumulate CPS at high rates synchronously. The average levels of CPS per hepatocyte that are observed upon hormone treatment are approx 50-fold lower in embryonic than in adult hepatocytes, corresponding with an approx 10-fold lower synthetic capacity (per gram hepatocytes) and an approx 5-fold smaller size of embryonic compared to adult hepatocytes. Carbamoylphosphate synthetase levels are therefore a good parameter in studies that aim to establish the mechanisms that underly the ontogenesis of the hepatic phenotype.