Pseudomonas aeruginosa AlgZ, a ribbon-helix-helix DNA-binding protein, is essential for alginate synthesis and algD transcriptional activation.

The Pseudomonas aeruginosa algD gene is the first gene of an operon encoding most of the enzymes necessary for biosynthesis of the exopolysaccharide alginate. Transcriptional activation of algD results in the high-level synthesis of alginate, an important P. aeruginosa virulence factor with antiphagocytic and adherence properties. Previously, we have identified a protein(s), AlgZ, expressed in mucoid P. aeruginosa CF isolates that specifically bound to sequences located 280 bp upstream of the algD promoter. Mutagenesis of the AlgZ DNA binding site and transcription assays were used to show that AlgZ was an activator of algD transcription. In the current study, the monomeric size of AlgZ was estimated to be between 6 kDa and 15 kDa by electroelution of a protein preparation from an SDS-PAGE gel and analysis of the fractions via protein staining and electrophoretic mobility shift assays. A biochemical enrichment procedure, resulting in a 130-fold enrichment for AlgZ, was devised, the protein identified and a partial amino-terminal sequence obtained. Using the P. aeruginosa Genome Project database, a complete sequence was obtained, and algZ was cloned and expressed in Escherichia coli. Expression of algZ was sufficient for the observed AlgZ DNA binding previously observed from extracts of P. aeruginosa. A protein database search revealed that AlgZ is homologous to the Mnt and Arc repressors of the ribbon-helix-helix family of DNA-binding proteins. An algZ deletion mutant was constructed in the mucoid CF isolate FRD1. The resulting strain was non-mucoid and exhibited no detectable algD transcription. As an indirect role in transcription would probably result in some residual algD transcription, these data suggest that AlgZ is an integral activator of algD and support the hypothesis that both AlgZ and the response regulator AlgR are involved in direct contact with RNA polymerase containing the alternative sigma factor, AlgT. The cloning of algZ is a crucial step in determining the mechanism of algD activation.

Ease of insertion of the laryngeal mask airway by inexperienced personnel when using an introducer.

The Portex introducer for the laryngeal mask airway was designed as an aid to successful insertion, acting as an idealised 'artificial hard palate' to guide the tip of the laryngeal mask into the correct position. A number of authors have investigated laryngeal mask insertion by unskilled personnel in certain situations, one example being nurses during in-hospital cardiopulmonary resuscitation. We investigated whether the introducer had any effect on the incidence of first-time successful LMA placement by unskilled personnel. These were nonanaesthetist doctors, randomly assigned to have one attempt at LMA insertion in an anaesthetised patient, with and without the introducer. In 44 patients with the LMA being inserted according to the manufacturer's instructions, there was a 68% success rate (14 failures). In 45 patients with the LMA being inserted with the aid of a Portex introducer, there was a 96% success rate (two failures). This was a highly significant improvement (p < 0.001).

Identification and characterization of AlgZ, an AlgT-dependent DNA-binding protein required for Pseudomonas aeruginosa algD transcription.

Transcriptional activation of the Pseudomonas aeruginosa algD gene results in high-level synthesis of the capsular polysaccharide alginate, an important P. aeruginosa virulence factor expressed in cystic fibrosis (CF) patients with chronic pulmonary disease. In this study, electrophoretic mobility-shift assays were used to identify a novel protein (AlgZ), which binds specifically to a sequence located 280 bp upstream of the algD promoter. While AlgZ-binding activity did not require the response regulators AlgB or AlgR, expression of AlgZ was found to be absolutely dependent on the alternative sigma factor AlgT. Electrophoretic mobility-shift assays and copper-phenanthroline footprinting localized AlgZ binding to a 36 bp algD region, which includes several helical repeats. A collection of alginate-producing (mucoid) and non-mucoid P. aeruginosa strains, derived from CF patients, was characterized for AlgZ-binding activity. In all cases, AlgZ binding to algD sequences was observed when extracts derived from mucoid P. aeruginosa CF isolates were examined. However, this binding activity was not present when extracts from non-mucoid P. aeruginosa CF isolates were tested. Oligonucleotide mutagenesis was employed to create an algD allele with a 4 bp mutation in the predicted AlgZ-binding site (algD38) and a heterologous substitution allele (algD40), in which the entire AlgZ-binding site was replaced with a non-specific DNA sequence of identical size. When the algD38 mutation was cloned into an algD-cat transcriptional fusion, this resulted in a 28-fold reduction in algD expression, whereas the algD40 mutation abolished algD transcription, indicating that AlgZ acts as an activator of algD transcription. These results support the hypothesis that activation of algD involves the formation of a high-order looped structure allowing for multivalent contacts between AlgZ, AlgR and RNA polymerase containing the alternative sigma factor AlgT. Characterization of the molecular details of algD activation will provide insights into the control of other prokaryotic and eukaryotic promoters that utilize multiple activators.

Interaction of Lp(a) with plasminogen binding sites on cells.

Lp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 microM), but Lp(a) bound to fewer sites (approximately 10-fold less). Both gangliosides and cell surface proteins with carboxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).

Lipoproteins inhibit the secretion of tissue plasminogen activator from human endothelial cells.

We studied the effect of lipoprotein(a) [Lp(a)], low-density lipoprotein (LDL), and high-density lipoprotein (HDL) on tissue plasminogen activator (TPA) secretion from human endothelial cells. At 1 mumol/L, Lp(a) inhibited constitutive TPA secretion by 50% and phorbol myristate acetate- and histamine-enhanced TPA secretion by 40%. LDL and HDL also depressed TPA secretion by 45% and 35% (constitutive) and 40% to 60% (stimulated). TPA mRNA levels were also examined and found to change in parallel with antigen secretion. In contrast to TPA, plasminogen activator inhibitor type-1 secretion and mRNA levels were not affected by any of the three lipoproteins. These results suggest that the interaction of lipoproteins with certain cell-surface binding sites may interfere with the proper production and/or secretion of TPA.

Disruption of microtubules inhibits the stimulation of tissue plasminogen activator expression and promotes plasminogen activator inhibitor type 1 expression in human endothelial cells.

The expression of certain proteolytic enzymes involved in cell migration (collagenase, urokinase) can be enhanced by the disruption of cellular cytoskeletal organization, suggesting an association between cell shape and gene expression. We have examined the effect of cytoskeleton-disrupting agents on the production and secretion of another proteolytic enzyme, tissue plasminogen activator (tPA), and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), in human endothelial cells. Addition of 1 x 10(-6) M colchicine, 5 x 10(-6) M cytochalasin B, 10(-6) M nocodazole, or 10(-6) M tubulazole had no effect on the constitutive rate of release of tPA. However, the three microtubule-disrupting agents--colchicine, nocodazole, and tubulazole--depressed the stimulation of tPA secretion by phorbol myristate acetate (PMA) by 50- to 65%. Disruption of microfilament structure by cytochalasin B had no effect. In contrast, microtubule disruption in the absence or presence of PMA stimulated PAI-1 secretion by 2.5 and 2 times, respectively. The depression of tPA secretion was not due to inhibition of the secretory function since tPA did not accumulate intracellularly during colchicine treatment. Nor did colchicine affect the PMA activation of protein kinase C-alpha, upon which stimulation of tPA is dependent; neither translocation of the kinase nor phosphorylation of the protein kinase C substrate protein, P80, was inhibited. Measurement of tPA mRNA levels demonstrated that the increase which precedes PMA-enhanced tPA secretion was also inhibited by colchicine by 50%. However, tPA gene transcriptional activity was only reduced 13%, suggesting that a post-transcriptional event was affected by microtubule disruption. PAI-1 mRNA levels and transcription rates were elevated 3.5 times. This study suggests that the changes that occur in endothelial cells during PMA-induced signal transmission leading to enhanced tPA mRNA levels and tPA antigen production can be partly blocked by agents that disrupt microtubule organization.