Fe2+-catalyzed non-enzymatic glycosylation alters collagen conformation during AGE-collagen formation in vitro

Advanced glycation end-products (AGEs) are one of the major factors of hyperglycemia related complications for diabetic patients. We studied the formation of AGEs in type I collagen after Fe²⁺-catalyzed non-enzymatic glycosylation in vitro. Type I collagen isolated from rat tail tendon was incubated with glucose and increasing concentrations of iron ions Fe²⁺. After 4 weeks incubation, cytotoxity of AGEs was indicated by the cytotoxity assay of primary human umbilical vein endothelial cells and primary human monocytes cultured with glycosylated collagen AGEs. Fourier transform infrared spectroscopy analysis revealed that structural changes of functional groups in glycosylated collagen are accelerated by the catalyst Fe²⁺. Using two-dimensional Fourier-transform infrared correlation spectroscopy analyses, for the first time, we demonstrated that the order of structural changes of these functional groups is -CH- > Amide I > Amide II > Amide III > ν(CO) the carboxylic group of Asn, Gln or polyproline amino acid residue in the course of AGE-collagen formation. Knowing the positions of these functional groups in collagen, this order of changes indicates that during glycation of collagen, the structure of the main chain residues in collagen changed first, and then the side chain changed gradually, which may lead to more carboxylic groups exposed to glucose for further formation of AGE-collagen irreversibly. The findings presented may support the design of new therapeutic strategies to prevent or slow down the Fe²⁺-catalyzed glycosylation of collagen and other matrix proteins.     

Evidence that late-endosomal SNARE multimerization complex is promoted by transmembrane segments

Assembly of SNARE proteins into quaternary complexes is a critical step in membrane docking and fusion. Here, we have studied the influence of the transmembrane segments on formation of the late endosomal SNARE complex. The complex was assembled in vitro from full-length recombinant SNAREs and from mutants, where the transmembrane segments were either deleted or replaced by oligo-alanine sequences. We show that endobrevin, syntaxin 7, syntaxin 8, and vti1b readily form a complex. This complex forms a dimer as well as multimeric structures. Interestingly, the natural transmembrane segments accelerate the conversion of the quaternary complex to the dimeric form and are essential for multimerization. These in vitro results suggest that the transmembrane segments are responsible for supramolecular assembly of the endosomal SNARE complex.     

Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba delta-endotoxin complex revealed by electron crystallography: implications for toxin-pore formation

The insecticidal nature of Cry delta-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore.     

Effect of surfactant protein A on the physical properties and surface activity of KL4-surfactant

SP-A, the major protein component of pulmonary surfactant, is absent in exogenous surfactants currently used in clinical practice. However, it is thought that therapeutic properties of natural surfactants improve after enrichment with SP-A. The objective of this study was to determine SP-A effects on physical properties and surface activity of a new synthetic lung surfactant based on a cationic and hydrophobic 21-residue peptide KLLLLKLLLLKLLLLKLLLLK, KL(4). We have analyzed the interaction of SP-A with liposomes consisting of DPPC/POPG/PA (28:9:5.6, w/w/w) with and without 0.57 mol % KL(4) peptide. We found that SP-A had a concentration-dependent effect on the surface activity of KL(4)-DPPC/POPG/PA membranes but not on that of an animal-derived LES. The surface activity of KL(4)-surfactant significantly improved after enrichment with 2.5-5 wt % SP-A. However, it worsened at SP-A concentrations > or =10 wt %. This was due to the fluidizing effect of supraphysiological SP-A concentrations on KL(4)-DPPC/POPG/PA membranes as determined by fluorescence anisotropy measurements, calorimetric studies, and confocal fluorescence microscopy of GUVs. High SP-A concentrations caused disappearance of the solid/fluid phase coexistence of KL(4)-surfactant, suggesting that phase coexistence might be important for the surface adsorption process.     

Structural response of Photosystem 2 to iron deficiency: Characterization of a new Photosystem 2-IdiA complex from the cyanobacterium Thermosynechococcus elongatus BP-1

Iron deficiency triggers various processes in cyanobacterial cells of which the synthesis of an additional antenna system (IsiA) around photosystem (PS) 1 is well documented [T.S. Bibby, J. Nield, J. Barber, Iron deficiency induces the formation of an antenna ring around trimeric photosystem I in cyanobacteria, Nature 412 (2001) 743-745, E.J. Boekema, A. Hifney, A.E. Yakushevska, M. Piotrowski, W. Keegstra, S. Berry, K.P. Michel, E.K. Pistorius, J. Kruip, A giant chlorophyll-protein complex induced by iron deficiency in cyanobacteria, Nature 412 (2001) 745-748]. Here we show that PS2 also undergoes prominent structural changes upon iron deficiency: Prerequisite is the isolation and purification of a PS2-IdiA complex which is exclusively synthesized under these conditions. Immunoblotting in combination with size exclusion chromatography shows that IdiA is only bound to dimeric PS2. Using single particle analysis of negatively stained specimens, IdiA can be localized in averaged electron micrographs on top of the CP43 subunit facing the cytoplasmic side in a model derived from the known 3D structure of PS2 [B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II, Nature 438 (2005) 1040-4]. The presence of IdiA as integral part of PS2 is the first example of a new PS2 protein being expressed under stress conditions, which is missing in highly purified PS2 complexes isolated from iron-sufficient cells.     

Staphylococcus aureus adherence to nasal epithelial cells in a physiological in vitro model

Summary Nasal carriage of Staphylococcus aureus represents a risk factor for subsequent invasive infections and interpatient transmission of strains. No physiological in vitro model of nasal epithelial cells is available to study both patient- and bacteria-related characteristics and their interaction, leading to adherence and colonization. Starting with tissues from human nasal polyps, a confluent, squamous, nonkeratinized epithelium in collagen-coated 96-well microtiter plates was obtained after 14 d. This in vitro cell-layer was characterized histologically, ultrastructurally, and immunohistochemically and showed features that were indistinguishable from those observed in the squamous nonkeratinized epithelium found in the posterior part of the vestibulum nasi. Adherence experiments were performed with four different ³H-thymidine-labeled Staphylococcus aureus strains. The effect of bacterial inoculum size, temperature of incubation, and incubation medium were studied. The adherence results were found to be reproducible, reliable and sensitive, allowing detection of small quantitative differences in adherence between the Staphylococcus aureus strains. There was no significant difference in adherence at 23° C and 37° C, nor between the incubation medium M199 and phosphate-buffered saline. Plastic adherence could be reduced and standardized with use of siliconized tips and a constant bacterial inoculum volume of 100 μl/well. This physiological and reliable in vitro cell-culture model offers a unique opportunity to study Staphylococcus aureus adherence to squamous, nonkeratinized nasal epithelial cells and both patient and bacterial characteristics involved in this interaction.     

Dissociable and nondissociable forms of platelet-activating factor acetylhydrolase in human plasma LDL: implications for LDL oxidative susceptibility

Platelet-activating factor acetylhydrolase (PAF-AH) is transported by lipoproteins in plasma and is thought to possess both anti-inflammatory and anti-oxidative activity. It has been reported that PAF-AH is recovered primarily in small, dense LDL and HDL following ultracentrifugal separation of lipoproteins. In the present studies, we aimed to further define the distribution of PAF-AH among lipoprotein fractions and subfractions, and to determine whether these distributions are affected by the lipoprotein isolation strategy (FPLC versus sequential ultracentrifugation) and LDL particle distribution profile. When lipoproteins were isolated by FPLC, the bulk (～85%) of plasma PAF-AH activity was recovered within LDL-containing fractions, whereas with ultracentrifugation, there was a redistribution to HDL (which contained ～18% of the activity) and the d>1.21 g/ml fraction (which contained ～32%). Notably, re-ultracentrifugation of isolated LDL did not result in any further movement of PAF-AH to higher densities, suggesting the presence of dissociable and nondissociable forms of the enzyme on LDL. Differences were noted in the distribution of PAF-AH activity among LDL subfractions from subjects exhibiting the pattern A (primarily large, buoyant LDL) versus pattern B (primarily small, dense LDL) phenotype. In the latter group, there was a relative depletion of PAF-AH activity in subfractions in the intermediate to dense range (d=1.039–1.047 g/ml) with a corresponding increase in enzyme activity recovered within the d>1.21 g/ml ultracentrifugal fraction. Thus, there appears to be a greater proportion of the dissociable form of PAF-AH in pattern B subjects. In both populations, most of the nondissociable activity was recovered in a minor small, dense LDL subfraction. Based on conjugated dienes as a measure of lipid peroxidation, variations in PAF-AH activity appeared to contribute to variations in oxidative behavior among ultracentrifugally isolated LDL subfractions. The physiologic relevance of PAF-AH dissociability and the minor PAF-AH-enriched oxidation-resistant LDL subpopulation remains to be determined.     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands from Haemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C_8 reverse phase HPLC. The purified activity, named longieornin, is a protein of moleeular weight 16 000 on SDS-PAGE under both reduced and nonredueed conditions. Collagen-mediated aggregation of platelets in plasma and of washed platelets (IC_(50) was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effeetors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longieonin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intraeellar Ca~(2 ) level of platelets in response to collagen were com     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands fromHaemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C₈ reverse phase HPLC. The purified activity, named longicomin, is a protein of molecular weight 16 000 on SDS-PAGE under both reduced and nonreduced conditions. Collagen-mediated aggegation of platelets in plasma and of washed platelets (IC₅₀ was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longiconin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intracellar ca²⁺ level of platelets in response to collagen were completely eliminated by longicomin. Increasing amounts of collagen are able to overcome the inhibition of aggregation by longicomin, indicating that longicomin probably shares with collagen a common receptor. In addition, collagen fibers did not emit fluorescence after incubation with isothocyanate-conjugated longicornin, indicating that longicomin did not bind directly to collagen fibers. The identification and isolation of longicomin demonstrates the existence of a new type of platelet inhibitor that should be useful to better undentand the mechanism of collagen stimulation of platelets. Project supported by the National Natural Science Foundation of China (Grant No. 39170591).     

Characteristics and Distributions of Isozymes of Superoxide Dismutase in Leaf Cella of Peanut and wheat

The isozymes of superoxide dismutase (SOD) in leaves of wheat and peanut were investigated and identified by isoelectric focusing gel electrophoresis. The total four bands were found in wheat leaves, among them the band with the highest mobility was proved to be the chloroplastic SOD, the others with intermediate mobilities were probably cytoplasmic SODs. It was observed that there was apparent difference between patterns of SOD isozymes from leaves of wild and cultivated peanut. Leaves of wild peanut contain three different types of SOD isozyme (band A, band B and band C). Band A and B were cyanide-sensitive, hence they should be Cu-Zn-SOD. Band C was cyanide- insensitive and might be Mn-SOD located in mitochondria. The major part of SOD activity in cultivated peanut leaves was concentrated in chloroplasts, and it was cyanide-sensitive. The experiments of SOD activity inhibition by KCN showed that leaves of cultivated peanut contained more Cu-Zn-SOD. On the contrary, the percentage of non-Cu-Zn-SOD was highter in wild species than in cultivated. The PI values of distinct bands were determined and the significance of this study for the further evaluation of resistance of peanut plant is discussed.