Secretion of extracellular proteins by Pseudomonas aeruginosa

Pseudomonas aeruginosa is a bacterial species of commercial value secreting numerous extracellular proteins, involved in pathogenesis. Most strains produce at least a lipase, a phospholipase, an alkaline phosphatase, an exotoxin and 2 proteases (elastase and alkaline protease). Various mechanisms for secretion of exoproteins appear to exist in P aeruginosa. Genetic analysis has led to the identification of 2 secretion pathways: i) a "general" secretion pathway, defined by the xcp mutations, which mediates secretion of most extracellular proteins, and; ii) an independent secretion pathway specific for alkaline protease. Our present knowledge on the pathways and components of the secretion machinery in P aeruginosa is reviewed in this article.     

Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase

The xcp genes are required for the secretion of most extracellular proteins by Pseudomonas aeruginosa. The products of these genes are essential for the transport of exoproteins across the outer membrane after they have reached the periplasm via a signal sequence-dependent pathway. To date, analysis of three xcp genes has suggested the conservation of this secretion pathway in many Gram-negative bacteria. Furthermore, the xcpA gene was shown to be identical to pilD, which encodes a peptidase involved in the processing of fimbrial (pili) subunits, suggesting a connection between pili biogenesis and protein secretion. Here the nucleotide sequences of seven other xcp genes, designated xcpR to -X, are presented. The N-termini of four of the encoded Xcp proteins display similarity to the N-termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstrated in vivo. Furthermore, two other proteins, XcpR and XcpS, show similarity to the PilB and PilC proteins required for fimbriae assembly. Since XcpR and PilB display a canonical nucleotide-binding site, ATP hydrolysis may provide energy for both systems.     

Techniques for encapsulating bioactive agents into liposomes

As a prerequisite for the use of liposomes for delivery of biologically active agents, techniques are required for the efficient and rapid entrapment of such agents in liposomes. Here we review the variety of procedures available for trapping hydrophilic and hydrophobic compounds. Considerations which are addressed include factors influencing the choice of a particular liposomal system and techniques for the passive entrapment of drugs in multilamellar vesicles and unilamellar vesicles. Attention is also paid to active trapping procedures relying on the presence of (negatively) charged lipid or transmembrane ion gradients. Such gradients are particularly useful for concentrating lipophilic cationic drugs inside liposomes, allowing trapping efficiencies approaching 100%.     

Isolation of Azospirillum lipoferum 4T Tn5 Mutants Affected in Melanization and Laccase Activity

Azospirillum lipoferum 4T has original properties such as nonmotility, melanin synthesis, and laccase activity. Following random Tn5 mutagenesis in A. lipoferum 4T, we obtained 10 mutants which were affected in melanization and laccase activity. The class 1 mutants, with intermediate levels of laccase activity, showed some coloration; the class 2 mutants, which were completely negative for laccase activity, were also colorless. The Tn5 localization on the chromosome or on the cryptic 300-MDa plasmid of A. lipoferum 4T was proven by hybridization for all class 1 mutants or for most class 2 mutants, respectively.     

Xcp-mediated protein secretion in Pseudomonas aeruginosa: identification of two additional genes and evidence for regulation of xcp gene expression

In Pseudomonas aeruginosa, several exoproteins synthesized with a signal sequence (elastase, lipase, phospholipases, alkaline phosphatase and exotoxin A) are secreted by a two-step mechanism. They first cross the inner membrane in a signal sequence-dependent way, and are further translocated across the outer membrane in a second step requiring secretion functions encoded by several xcp genes. Ten xcp genes have already been characterized (Bally et al., 1992a). In this study, two additional xcp genes, xcpP and xcpQ, are described. They are located in the 40 min region of the chromosome where they probably define an operon, divergent from the xcpR–Z operon previously characterized in this region. These two genes encode two proteins, XcpP and XcpQ, similar to PulC and PulD of the pul system of Klebsiella oxytoca. Moreover, the two divergent operons share a common regulation which is growth-phase dependent.     

Characterization of two new crystal forms of uteroglobin.

Two new crystal forms of oxidized uteroglobin have been obtained. An orthorhombic one ($\text{P2}{}_1\text{2}{}_1\text{2, Z = 2, a = 44.48 (5)}\text{A}\text{?}\text{, b = 36.93 (5)}\text{A}\text{?}\text{, c = 32·34 (5)}\text{A}\text{?}$) and a monoclinic one ($\text{P2}{}_1\text{, Z = 2, a = 44.56 (5)}\text{A}\text{?}\text{, b = 46.06 (5)}\text{A}\text{?}\text{, c = 37.43 (4)}\text{A}\text{?}\text{, β = 120.92 ° (5)}$). Both were grown at pH ~7.0 and diffract to a resolution of 2·1 to 2·2 Å. Data collections for native crystals have been recorded with an automatic four-circle diffractometer.     

Method for rapid separation of liposome-associated doxorubicin from free doxorubicin in plasma

To understand and predict the efficacy and/or toxicity of liposomal drugs in vivo, it is essential to have rapid, reliable methods of separating and quantitating both the free and the liposomal forms of the drug. A method using solid-phase extraction chromatography columns was developed to separate and quantitate unencapsulated doxorubicin and liposome-associated doxorubicin in plasma following the intravenous injection of liposomal doxorubicin. The method facilitated the recovery and quantitation of free and liposomal drug. The separation and recovery of doxorubicin were linear across the entire range of possible mixtures (0 to 100%) of the two forms of the drug in plasma. Free drug and liposomal drug were readily separated for liposomal doxorubicin systems varying in size (0.1-1.0 microns) and lipid composition (egg yolk phosphatidylcholine/cholesterol and distearylphosphatidylcholine/cholesterol). The method is rapid and allows for multiple samples to be processed simultaneously.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.