Comparison of Protein-Repellent Behavior of Linear versus Dendrimer-Structured Surface-Immobilized Polymers.

For many biomedical applications, material surfaces should not only prevent unspecific protein adsorption and bacterial attachment as in many other applications in the food, health, or marine industry, but they should also promote the adhesion of tissue cells. In order to take a first step toward the challenging development of protein and bacteria-repelling and cell-adhesion-promoting materials, polyamine and poly(amido amine) surface coatings with terminal amine groups and varying structure (dendrimer, oligomer, polymer) were immobilized on model surfaces via silane chemistry. Physicochemical analysis showed that all modifications are hydrophilic (contact angles <60°) and possess similar surface free energies (SFEs, ∼46-54 mN/m), whereas their amine group densities and zeta potentials at physiological conditions (pH 7.4) varied greatly (-50 to +75 mV). In protein adsorption experiments with single proteins (human serum albumin (HSA) and lysozyme) as well as complex physiological fluids (fetal bovine serum (FBS) and human saliva), the amounts of adsorbed protein were found to correlate strongly with the zeta potential of the surface coatings. Both modifications based on linear polymers exhibited good protein repellency toward all proteins examined and are thus promising for testing in cell adhesion studies.

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, time-lapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/sorting endosomes regulates the postsynaptic clustering of gephyrin and GABAA receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cb-mediated recruitment of gephyrin and GABAA receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations.

Two N-terminally truncated variants of human ß-galactoside α2,6 sialyltransferase I with distinct properties for in vitro protein glycosylation.

Sialic acid groups of protein N-glycans are important determinants of biological activity. Exposed at the end of the glycan chain, they are potential targets for glycan remodeling. Sialyltransferases (STs; EC 2.4.99) are the enzymes that catalyze the sialic acid transfer from a CMP-activated donor on to a carbohydrate acceptor in vivo. Recombinant expression of the full-length human ß-galactoside α2,6 sialyltransferase I (ST6Gal-I) was hampered and therefore variants with truncated N-termini were investigated. We report on the distinct properties of two N-terminally truncated versions of ST6Gal-I, namely Δ89ST6Gal-I and Δ108ST6Gal-I, which were successfully expressed in human embryonic kidney cells. The different properties of these enzymes result most probably from the loss of interactions from helix α1 in the Δ108ST6Gal-I variant, which plays a role in acceptor substrate binding. The Km for N-acetyl-d-lactosamine was 10-fold increased for Δ108ST6Gal-I (84 mM) as compared to Δ89ST6Gal-I (8.3 mM). The two enzyme variants constitute a suitable tool box for the terminal modification of N-glycans. While the enzyme Δ89ST6Gal-I exhibited both ST (di-sialylation) and sialidase activity on a monoclonal antibody, the enzyme Δ108ST6Gal-I showed only ST activity with specificity for mono-sialylation.

PIP3 induces the recycling of receptor tyrosine kinases.

Down-regulation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) is achieved by endocytosis of the receptor followed by degradation or recycling. We demonstrated that in the absence of ligand, increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations induced clathrin- and dynamin-mediated endocytosis of EGFR but not that of transferrin or G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. Endocytosis of the receptor in response to binding of EGF resulted in a decrease in the abundance of the EGFR, but PIP3-induced internalization decreased receptor ubiquitination and phosphorylation and resulted in recycling of the receptor to the plasma membrane. An RNA interference (RNAi) screen directed against lipid-binding domain-containing proteins identified polarity complex proteins, including PARD3 (partitioning defective 3), as essential for PIP3-induced receptor tyrosine kinase recycling. Thus, PIP3 and polarity complex proteins regulate receptor tyrosine kinase trafficking, which may enhance cellular responsiveness to growth factors.

Increasing capillary diameter and the incorporation of gelatin enhance axon outgrowth in alginate-based anisotropic hydrogels.

Substantial recovery of function following peripheral and central nervous system (CNS) injury critically depends on longitudinally directed axon regeneration across the injury site, which requires a mechanical guidance providing scaffold. We have previously shown that anisotropic alginate-based hydrogels with a defined capillary diameter (25 µm), which form via a self-organizing process driven by unidirectional diffusion of divalent cations into sodium alginate sols, promoted longitudinally oriented elongation of CNS axons in vitro and in vivo. In the present study the influence of various capillary diameters and the incorporation of gelatin to promote directed axon outgrowth and Schwann cell migration were assessed in a dorsal root ganglion outgrowth assay in vitro. Superimposing an alginate sol with Cu(2+), Sr(2+), or Zn(2+) ion containing solutions allowed the creation of hydrogels with capillaries 18, 25 and 55 µm in diameter, respectively. Axon outgrowth and Schwann cell migration were analyzed in terms of axon length/density and Schwann cell density within the capillary structures. Axon ingrowth into capillary hydrogels, which was always accompanied by Schwann cells, was enhanced with increasing capillary diameter. The incorporation of gelatin did not influence overall axon density, but promoted the length of axon outgrowth within the hydrogels. The longitudinal orientation of axons decreased in wider capillaries, which suggests that medium-sized capillaries are the optimal substrate to elicit substantial axon growth and longitudinal orientation after axon injury.

SDS-coated atovaquone nanosuspensions show improved therapeutic efficacy against experimental acquired and reactivated toxoplasmosis by improving passage of gastrointestinal and blood-brain barriers.

Toxoplasmic encephalitis (TE) is the most common clinical manifestation of reactivated infection with Toxoplasma gondii in immunocompromised patients that is lethal if untreated. The combination of pyrimethamine plus sulfadiazine or clindamycin is the standard therapy for the treatment of TE, but these combinations are associated with hematologic toxicity and/or life-threatening allergic reactions. Therefore, alternative treatment options are needed. Atovaquone is safe and highly effective against T. gondii in vitro, but the oral micronized solution shows poor bioavailability. We synthesized atovaquone nanosuspensions (ANSs) coated with poloxamer 188 (P188) and sodium dodecyl sulfate (SDS) to improve oral bioavailability and passage through the blood-brain barrier (BBB). Coating of ANSs with SDS resulted in enhanced oral bioavailability and enhanced brain uptake of atovaquone compared to Wellvone(®) in murine models of acute and reactivated toxoplasmosis as measured by high performance liquid chromatography (HPLC). Parasite loads and inflammatory changes in brains of mice treated with SDS-coated ANS were significantly reduced compared to untreated controls and to Wellvone(®)-treated mice. In conclusion, nanosuspensions coated with SDS may ultimately lead to improvements in the treatment of TE and other cerebral diseases.

Streptococcal adhesion to various luting systems and the role of mixing errors.

OBJECTIVE: This study aims at ranking various luting systems according to their susceptibility to adhering Streptococcus mutans and at evaluating the influence of incongruent mixing ratios on adhesion quantities. MATERIAL AND METHODS: Circular specimens measuring 8 mm in diameter were made of nine widely used dental cements -- three of them mixed in different ratios -- and then incubated with S. mutans. Adhering streptococci were quantified using a biofluorometric assay in combination with an automated plate reader for cell quantification. Surface roughness (R(a)) was determined by perthometer measurements. RESULTS: Meron plus revealed the highest R(a) (0.90 microm) and glass the lowest R(a) (<0.01 microm). In regular cement mixtures, the highest mean fluorescence intensities indicated the presence of many viable bacteria [Meron Plus (35,533 relative fluorescence units (rfu)), Maxcem (13,374 rfu), and Panavia F 2.0 (11,701 rfu)]. Moderate fluorescence intensities were found in Harvard (4,171 rfu), Ketac cem (3,766 rfu), Durelon (3,276 rfu), Calibra (3,259), Rely X Unicem (4,358 rfu), and Bifix SE (3,102 rfu). A medium correlation between R(a) and S. mutans adhesion was found. Changes in regular cement proportions (powder/liquid and base/catalyst, respectively) had a significant influence on relative fluorescence intensities, which linearly increased with a higher proportion of liquid in Harvard and with a higher proportion of catalyst in Calibra and Maxcem. CONCLUSIONS: Various luting systems revealed considerable differences in their potential to adhere S. mutans. Variations from recommended cement proportions led to significant changes in the amount of adhering streptococci.

Rapid diagnostic method for quantitative testing of <100 microbes in water.

A robust, real-time polymerase chain reaction (RT-PCR) system to universally detect microbes at a limit of 10 to 50 colony-forming units within 5-6 h was developed. Pre-treatment of RT-PCR master mixes with ethidiumbromide monoacide (EMA) facilitates the development of an RT-PCR assay with appropriate sensitivity, reproducibility, and recovery.The system is useful to replace conventional microbial plating techniques for the analysis of microbial contamination in liquids like water. This was statistically confirmed for eight different bacteria and two different fungi species. Finally a complete procedure including microbial lysis, DNA extraction, EMA treatment, and RT-PCR was developed and evaluated for three different bacteria and two fungi species.

Influence of different collagen species on physico-chemical properties of crosslinked collagen matrices.

Collagen-based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present study investigated the species-related differences of collagen scaffolds with and without 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-crosslinking. Resistance against collagenase digestion, swelling ratio, amino acid sequence, shrinkage temperature, ultrastructural matrix morphology, crosslinking density and stress-strain characteristics were determined to evaluate the physico-chemical properties of equine- and bovine-collagen-based scaffolds. Three-factor ANOVA analysis revealed a highly significant effect of collagen type (p=0.0001), crosslinking (p=0.0001) and time (p=0.0001) on degradation of the collagen samples by collagenase treatment. Crosslinked equine collagen samples showed a significantly reduced swelling ratio compared to bovine collagen samples (p< 0.0001). The amino acid composition of equine collagen revealed a higher amount of hydroxylysine and lysine. Shrinkage temperatures of non-crosslinked samples showed a significant difference between equine (60 degrees C) and bovine collagen (57 degrees C). Three-factor ANOVA analysis revealed a highly significant effect of collagen type (p=0.0001), crosslinking (p=0.0001) and matrix condition (p=0.0001) on rupture strength measured by stress-strain analysis. The ultrastructure, the crosslinking density and the strain at rupture between collagen matrices of both species showed no significant differences. For tissue engineering purposes, the higher enzymatic stability, the higher form stability, as well as the lower risk of transmissible disease make the case for considering equine-based collagen. This study also indicates that results obtained for scaffolds based on a certain collagen species may not be transferable to scaffolds based on another, because of the differing physico-chemical properties.