Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied 'food grade' sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC50 value of 5.521 mg/mL is high compared with 0.2811 mu g/mL (182.5 pM or 1.825 x 10(-10) M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E (R) is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%. 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease. (C) 2009 Elsevier Ltd. All rights reserved

A flow reactor process for the synthesis of peptides utilizing immobilized reagents, scavengers and catch and release protocols

A general flow process for the multi-step assembly of peptides has been developed and this procedure has been used to successfully construct a series of Boc, Cbz and Fmoc N-protected dipeptides in excellent yields and purities, including an extension of the method to enable the preparation of a tripeptide derivative.

In vitro study on the cell adhesion ability of immobilized lactobacilli on natural supports

The aim of the present study was to investigate the effect of probiotic immobilization onto wheat grains, both wet and freeze dried, on the adhesion properties of the probiotic cells and make comparisons with wet and freeze dried free cells. Lactobacillus casei ATCC 393 and Lactobacillus plantarum NCIMB 8826 were used as model probiotic strains. The results showed satisfactory adhesion ability of free cells to a monolayer of Caco-2 cells (> 1000 CFU/100 Caco-2 cells for wet cells). Cell immobilization resulted in a significant decrease in adhesion, for both wet and freeze dried formulations, most likely because immobilized cells did not have direct access to the Caco-2 cells, but it still remained in adequate levels (> 100 CFU/100 Caco-2 cells for wet cells). No clear correlation could be observed between cell adhesion and the hydrophobicity of the bacterial cells, measured by the hexadecane adhesion assay. Most notably, immobilization enhanced the monolayer integrity of Caco-2 cells, demonstrated by a more than 2-fold increase in transepithelial electrical resistance (TEER) compared to free cells. SEM micrographs ascertained the adhesion of both immobilized and free cells to the brush border microvilli. Finally, the impact of the food matrix on the adhesion properties of probiotic bacteria and on the design of novel functional products is discussed.

Contributions of labile and resistant organic materials to the immobilization of inorganic soil N when used in the restoration of abandoned agricultural fields

We have examined the contributions sucrose and sawdust make to the net immobilization of inorganic soil N and assimilation of both C and N into microbial biomass when they are used as part of a restoration plan to promote the establishment of indigenous vegetation on abandoned agricultural fields on the Central Hungarian Plain. Both amendments led to net N immobilization. Sucrose addition also led to mobilization of N from the soil organic N pool and its immobilization into microbial biomass, whereas sawdust addition apparently immobilized soil N into a non-biomass compartment or a biomass component that was not detected by the conventional biomass N assay (CHCl3 fumigation and extraction). This suggests that the N was either cycled through the biomass, but not immobilized within it, or that it was immobilized in a protected biomass fraction different to the fraction into which N was immobilized in response to sucrose addition.

Contributions of labile and resistant organic materials to the immobilization of inorganic soil N when used in the restoration of abandoned agricultural fields

We have examined the contributions sucrose and sawdust make to the net immobilization of inorganic soil N and assimilation of both C and N into microbial biomass when they are used as part of a restoration plan to promote the establishment of indigenous vegetation on abandoned agricultural fields on the Central Hungarian Plain. Both amendments led to net N immobilization. Sucrose addition also led to mobilization of N from the soil organic N pool and its immobilization into microbial biomass, whereas sawdust addition apparently immobilized soil N into a non-biomass compartment or a biomass component that was not detected by the conventional biomass N assay (CHCl3 fumigation and extraction). This suggests that the N was either cycled through the biomass, but not immobilized within it, or that it was immobilized in a protected biomass fraction different to the fraction into which N was immobilized in response to sucrose addition.

Iron-regulated surface determinant protein a mediates adhesion of staphylococcus aureus to human corneocyte envelope proteins

The ability of Staphylococcus aureus to colonize the human nares is a crucial prerequisite for disease. IsdA is a major S. aureus surface protein that is expressed during human infection and required for nasal colonization and survival on human skin. In this work, we show that IsdA binds to involucrin, loricrin, and cytokeratin K10, proteins that are present in the cornified envelope of human desquamated epithelial cells. To measure the forces and dynamics of the interaction between IsdA and loricrin (the most abundant protein of the cornified envelope), single-molecule force spectroscopy was used, demonstrating high-specificity binding. IsdA acts as a cellular adhesin to the human ligands, promoting whole-cell binding to immobilized proteins, even in the absence of other S. aureus components (as shown by heterologous expression in Lactococcus lactis). Inhibition experiments revealed the binding of the human ligands to the same IsdA region. This region was mapped to the NEAT domain of IsdA. The NEAT domain also was found to be required for S. aureus whole-cell binding to the ligands as well as to human nasal cells. Thus, IsdA is an important adhesin to human ligands, which predominate in its primary ecological niche.

Modified rare earth semiconductor oxide as a new nucleotide probe

Recent rapid developments in biological analysis, medical diagnosis, pharmaceutical industry, and environmental control fuel the urgent need for recognition of particular DNA sequences from samples. Currently, DNA detection techniques use radiochemical, enzymatic, fluorescent, or electrochemiluminescent methods; however, these techniques require costly labeled DNA and highly skilled and cumbersome procedure, which prohibit any in-situ monitoring. Here, we report that hybridization of surface-immobilized single-stranded oligonucleotide on praseodymium oxide (evaluated as a biosensor surface for the first time) with complimentary strands in solution provokes a significant shift of electrical impedance curve. This shift is attributed to a change in electrical characteristics through modification of surface charge of the underlying modified praseodymium oxide upon hybridization with the complementary oligonucelotide strand. On the other hand, using a noncomplementary single strand in solution does not create an equivalent change in the impedance value. This result clearly suggests that a new and simple electrochemical technique based on the change in electrical properties of the modified praseodymium oxide semiconductor surface upon recognition and transduction of a biological event without using labeled species is revealed.

Binding properties and adhesion-mediating regions of the major sheath protein of Treponema denticola ATCC 35405

There is growing evidence that a number of oral Treponema species, in particular Treponema denticola, are associated with the progression of human periodontal disease. The major sheath (or surface) protein (Msp) of T. denticola is implicated in adhesion of bacteria to host cells and tissue proteins and is likely to be an important virulence factor. However, the binding regions of the Msp are not known. We have purified from Escherichia coli recombinant Msp (rMsp) polypeptides corresponding to the following: full-length Msp (rMsp) minus 13 N-terminal amino acid (aa) residues, an amino-terminal fragment (rN-Msp, 189 aa residues), a 57-aa residue segment from the central region (rV-Msp), and a C-terminal fragment (rC-Msp, 272 aa residues). rMsp (530 aa residues) bound to immobilized fibronectin, keratin, laminin, collagen type 1, fibrinogen, hyaluronic acid, and heparin. The N- and V-region polypeptides, but not rC-Msp, also bound to these substrates. Binding of rMsp to fibronectin was targeted to the N-terminal heparin I/fibrin I domain. Antibodies to the N-region or V-region polypeptides, but not antibodies to the rC-Msp fragment, blocked adhesion of T. denticola ATCC 35405 cells to a range of host protein molecules. These results suggest that the N-terminal half of Msp carries epitopes that are surface exposed and that are involved in mediating adhesion. Binding of rMsp onto the cell surface of low-level fibronectin-binding Treponema isolates conferred a 10-fold increase in fibronectin binding. This confirms that Msp functions autonomously as an adhesin and raises the possibility that phenotypic complementation of virulence functions might occur within mixed populations of Treponema species.

Use of miniaturized protein arrays for Escherichia coli O serotyping

Serological typing of Escherichia coli O antigens is a well-established method used for differentiation and identification of O serotypes commonly associated with disease. In this feasibility study, we have developed a novel somatic antibody-based miniaturized microarray chip, using 17 antisera, which can be used to detect bound whole-cell E. coli antigen with its corresponding immobilized antibody, to assess the feasibility of this approach. The chip was tested using the related 17 control strains, and the O types found by the microarray chip showed 100% correlation with the O types found by conventional typing. A blind trial was performed in which 100 E. coli isolates that had been O serotyped previously by the conventional assay were tested by the array approach. Overall, the O serotypes of 88% of isolates were correctly identified by the microarray method. For several isolates, ambiguity of O-type designation by microarray arose due to increased sensitivity of this method, allowing signal intensities of cross-reactions to be quantified. Investigation of discrepancies between conventional and microarray O serotyping indicated that some isolates upon storage had become untypeable and, therefore, gave poor signal intensity when tested by the microarray or retested by conventional means. For all 20 serotype O26 and O157 isolates, the apparent discrepancy in O serotyping was analyzed further by a third independent test, which confirmed the microarray results. Therefore, the use of miniaturized protein arrays increases the speed and efficiency of O serotyping in a cost-effective manner, and these preliminary findings suggest the microarray approach may have a higher accuracy than those of traditional O-serotyping methods.

Platelet protein disulfide isomerase is required for thrombus formation but not essential for hemostasis in mice

Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and ATP secretion induced by thrombin, collagen, and ADP. Such defects were rescued by exogenously-added wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ERp57 and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca2+ mobilization, β3-talin interaction, and platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation, aggregation and ATP secretion of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under arteriolar shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time and blood loss in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.

A bifurcated pathway to thiazoles and imidazoles using a modular flow microreactor

A scalable method for the preparation of 4,5-disubstituted thiazoles and imidazoles as distinct regioisomeric products using a modular flow microreactor has been devised. The process makes use of microfluidic reaction chips and packed immobilized-reagent columns to effect bifurcation of the reaction pathway.

Fibrin activates GPVI in human and mouse platelets

The glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet signaling receptor for collagen. Paradoxically, in a FeCl3 injury model, occlusion, but not initiation of thrombus formation, is delayed in GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that GPVI is a receptor for fibrin and speculate that this contributes to development of an occlusive thrombus. We observed a marked increase in tyrosine phosphorylation, including the FcRγ chain and Syk, in human and mouse platelets induced by thrombin in the presence of fibrinogen and the αIIbβ3 blocker eptifibatide. This was not seen in platelets stimulated by a protease activated receptor (PAR)-4 peptide, which is unable to generate fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar to that induced by activation of GPVI. Consistent with this, thrombin did not induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient mouse platelets. Mouse platelets underwent full spreading on fibrin but not fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in the absence of GPVI. Spreading on fibrin was associated with phosphatidylserine exposure (procoagulant activity), and this too was blocked in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to immobilized monomeric and polymerized fibrin. A marked increase in embolization was seen following FeCl3 injury in GPVI-deficient mice, likely contributing to the delay in occlusion in this model. These results demonstrate that GPVI is a receptor for fibrin and provide evidence that this interaction contributes to thrombus growth and stability.

Syk and Src family kinases regulate C-type lectin receptor 2 (CLEC-2)-mediated clustering of podoplanin and platelet adhesion to lymphatic endothelial cells

The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.

GPVI potentiation of platelet activation by thrombin and adhesion molecules independent of Src kinases and Syk.

We show that Syk is critical for lamellipodia formation on a range of immobilized proteins but that this can be overcome by addition of thrombin. Further, we reveal a novel role for GPVI in supporting thrombin-induced activation, independent of Syk and Src kinases.