Antibody activation of mutant human fructosediphosphate aldolase B in liver extracts of patients with hereditary fructose intolerance

Antiserum to crystallized fructosediphosphate aldolase B from human liver precipitated/inhibited the antigen in solution. It activated the mutant enzyme in liver extracts of 3 patients with hereditary fructose intolerance but not in 2 others. It was concluded that genetic variability existed between these patients. In vitro activation of a defective human enzyme, demonstrated here for the first time, indicates that in vivo restoration of activity of mutant enzymes may become feasible.     

Structure and refinement of the oxidized P21 form of uteroglobin at 1.64 A resolution

One of the monoclinic P21 forms of uteroglobin, a progesterone-binding protein secreted by the rabbit uterus, was crystallized and subjected to X-ray diffraction analysis at 1.64 A resolution. The analysis was refined to an R factor of 0.19 and the 1096 non-hydrogen atomic positions are known to an accuracy of about 0.18 A. The average isotropic temperature factor B was 10.4 A2. Uteroglobin is a dimer of two independent polypeptide chains of 70 residues linked by two disulfide bridges and related by a pseudo binary axis. Each monomer is folded into four alpha-helices. An oblong hydrophobic pocket is observed inside the dimer, and the possibility that it represents a progesterone-binding site is discussed. The present model includes 165 possible sites for water molecules, of which six are located in the hydrophobic pocket. Polar groups are involved in hydrogen bonding (intramolecular, intermolecular or with water molecules).     

Modular optical topometric sensor for 3D acquisition of human body surfaces and long-term monitoring of variations.

Optical topometric 3D sensors such as laser scanners and fringe projection systems allow detailed digital acquisition of human body surfaces. For many medical applications, however, not only the current shape is important, but also its changes, e.g., in the course of surgical treatment. In such cases, time delays of several months between subsequent measurements frequently occur. A modular 3D coordinate measuring system based on the fringe projection technique is presented that allows 3D coordinate acquisition including calibrated color information, as well as the detection and visualization of deviations between subsequent measurements. In addition, parameters describing the symmetry of body structures are determined. The quantitative results of the analysis may be used as a basis for objective documentation of surgical therapy. The system is designed in a modular way, and thus, depending on the object of investigation, two or three cameras with different capabilities in terms of resolution and color reproduction can be utilized to optimize the set-up.     

A phase variant of Azospirillum lipoferum lacks a polar flagellum and constitutively expresses mechanosensing lateral flagella.

Flagellation of a nonswimming variant of the mixed flagellated bacterium Azospirillum lipoferum 4B was characterized by electron microscopy, and polyclonal antibodies were raised against polar and lateral flagellins. The variant cells lacked a polar flagellum due to a defect in flagellin synthesis and constitutively expressed lateral flagella. The variant cells were able to respond to conditions that restricted the rotation of lateral flagella by producing more lateral flagella, suggesting that the lateral flagella, as well as the polar flagellum, are mechanosensing.     

A multi-step lipid mixing assay to model structural changes in cationic lipoplexes used for in vitro transfection.

Formation of liposome/polynucleotide complexes (lipoplexes) involves electrostatic interactions, which induce changes in liposome structure. The ability of these complexes to transfer DNA into cells is dependent on the physicochemical attributes of the complexes, therefore characterization of binding-induced changes in liposomes is critical for the development of lipid-based DNA delivery systems. To clarify the apparent lack of correlation between membrane fusion and in vitro transfection previously observed, we performed a multi-step lipid mixing assay to model the sequential steps involved in transfection. The roles of anion charge density, charge ratio and presence of salt on lipid mixing and liposome aggregation were investigated. The resonance-energy transfer method was used to monitor lipid mixing as cationic liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na(2)HPO(4). Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentration increased, lipid mixing and aggregation increased, but with Na(2)HPO(4) only aggregation occurred. NaCl (150 mM) reduced the extent of lipid mixing. Transfection studies suggest that the presence of salt during complexation had minimal effects on in vitro transfection. These data give new information about the effects of polynucleotide binding to cationic liposomes, illustrating the complicated nature of anion induced changes in liposome morphology and membrane behavior.     

Identification of the C1q-binding Sites of Human C1r and C1s: A REFINED THREE-DIMENSIONAL MODEL OF THE C1 COMPLEX OF COMPLEMENT*

The C1 complex of complement is assembled from a recognition protein C1q and C1s-C1r-C1r-C1s, a Ca²⁺-dependent tetramer of two modular proteases C1r and C1s. Resolution of the x-ray structure of the N-terminal CUB₁-epidermal growth factor (EGF) C1s segment has led to a model of the C1q/C1s-C1r-C1r-C1s interaction where the C1q collagen stem binds at the C1r/C1s interface through ionic bonds involving acidic residues contributed by the C1r EGF module (Gregory, L. A., Thielens, N. M., Arlaud, G. J., Fontecilla-Camps, J. C., and Gaboriaud, C. (2003) J. Biol. Chem. 278, 32157–32164). To identify the C1q-binding sites of C1s-C1r-C1r-C1s, a series of C1r and C1s mutants was expressed, and the C1q binding ability of the resulting tetramer variants was assessed by surface plasmon resonance. Mutations targeting the Glu¹³⁷-Glu-Asp¹³⁹ stretch in the C1r EGF module had no effect on C1 assembly, ruling out our previous interaction model. Additional mutations targeting residues expected to participate in the Ca²⁺-binding sites of the C1r and C1s CUB modules provided evidence for high affinity C1q-binding sites contributed by the C1r CUB₁ and CUB₂ modules and lower affinity sites contributed by C1s CUB₁. All of the sites implicate acidic residues also contributing Ca²⁺ ligands. C1s-C1r-C1r-C1s thus contributes six C1q-binding sites, one per C1q stem. Based on the location of these sites and available structural information, we propose a refined model of C1 assembly where the CUB₁-EGF-CUB₂ interaction domains of C1r and C1s are entirely clustered inside C1q and interact through six binding sites with reactive lysines of the C1q stems. This mechanism is similar to that demonstrated for mannan-binding lectin (MBL)-MBL-associated serine protease and ficolin-MBL-associated serine protease complexes.The classical pathway of complement, a major component of innate immune defense against pathogens and altered self, is triggered by C1, a 790-kDa Ca²⁺-dependent complex assembled from a recognition protein C1q and C1s-C1r-C1r-C1s, a tetramer of two modular proteases, C1r and C1s, that respectively mediate activation and proteolytic activity of the complex (1–3). C1q has the overall shape of a bunch of tulips and comprises six heterotrimeric collagen-like triple helices that assemble through their N-terminal moieties to form a “stalk” and then diverge to form individual “stems,” each prolonged by a C-terminal globular recognition domain (4). C1r and C1s are homologous modular proteases each comprising, starting from the N-terminal end, a C1r/C1s, sea urchin EGF² (uEGF), bone morphogenetic protein (CUB) module (5), an EGF-like module (6), a second CUB module, two complement control protein modules (7), and a serine protease domain. This modular structure is shared by the mannan-binding lectin-associated serine proteases (MASPs), a group of enzymes that associate with mannan-binding lectin (MBL) and the ficolins and thereby trigger activation of the lectin pathway of complement (8).Assembly of the C1s-C1r-C1r-C1s tetramer involves Ca²⁺-dependent heterodimeric C1r-C1s interactions between the CUB₁-EGF segments of each protease (9–12). Similarly, MASP-1, MASP-2, MASP-3, and mannan-binding lectin-associated protein 19 (MAp19), an alternative splicing product of the MASP-2 gene comprising the N-terminal CUB₁-EGF segment of MASP-2, all associate as homodimers through their N-terminal CUB₁-EGF moieties (13–15). The structures of human C1s CUB₁-EGF, human MAp19, human MASP-1/3 CUB₁-EGF-CUB₂, and rat MASP-2 CUB₁-EGF-CUB₂ have been solved by x-ray crystallography (16–19), revealing that these domains all associate as head-to-tail homodimers through a highly conserved interface involving interactions between the CUB₁ module of one monomer and the EGF module of its counterpart. In addition, all CUB modules contained in these structures were found to contain a hitherto unrecognized Ca²⁺-binding site involving three conserved acidic residues (Glu⁴⁵, Asp⁵³, and Asp⁹⁸ in C1s), defining a novel CUB module subset diverging from the type originally described in the spermadhesins (20).Mutagenesis studies have recently established that assembly of the MBL- and ficolin-MASP complexes involves a major electrostatic interaction between two acidic Ca²⁺ ligands from the MASP CUB modules and a conserved lysine located in the collagen fibers of MBL and ficolins (16, 18, 21, 22). In the case of C1, a hypothetical model of the C1q/C1r/C1s interface, involving interaction between acidic residues mainly contributed by the C1r EGF module and unmodified lysine residues also located in the collagen-like stems of C1q, was derived from the x-ray structure of the C1s CUB₁-EGF interaction domain (16, 23). The aim of this work was to use site-directed mutagenesis to delineate the sites of C1r and C1s involved in the interaction between C1s-C1r-C1r-C1s and C1q. Our data rule out our previous interaction model and provide evidence that C1 assembly involves the same basic Ca²⁺-dependent mechanism as demonstrated in the case of MBL-MASP and ficolin-MASP complexes.     

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 degrees C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.     

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.     

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.