Tuning the preference of thiodigalactoside- and lactosamine-based ligands to galectin-3 over galectin-1.

Inhibitors for galectin-1 and -3 were synthesized from thiodigalactoside and lactosamine by derivatization of the galactose C3. Introduction of 4-phenyl-1H-1,2,3-triazol-1-yl substituents at the thiodigalactoside C3 by CuAAC, targeting arginine-arene interactions, increased the affinity to 13 nM but yielded little selectivity. The bulkier 4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl substituent, however, increased the preference for galectin-3 over galectin-1 to more than 200-fold. Modeling showed more arginine-arene interactions for galectin-3 than for galectin-1. Introducing 4-phenoxyaryl groups on lactosamine had a similar effect.

Use of tetravalent galabiose for inhibition of streptococcus suis serotype 2 infection in a mouse model.

Streptococcus suis is an important swine pathogen associated with a variety of infections such as meningitis, arthritis and septicemia. The bacterium is zoonotic and has been found to cause meningitis especially in humans occupationally exposed to infected pigs. Since adhesion is a prerequisite for colonization and subsequent infection, anti-adhesion treatment seems a natural alternative to traditional treatment with antibiotics. In order to optimize the inhibitory potency a multivalency approach was taken in the inhibitor design. A synthetic tetravalent galabiose compound was chosen which had previously shown promising anti-adhesion effects with S. suis in vitro. The aim of this study was to evaluate the in vivo effects of the compound using an infection peritonitis mouse model. As such S. suis serotype 2 infection and treatment were tested in vivo and the effects were compared to the effect of treatment with penicillin.

The influence of respiratory acid-base changes on muscle performance and excitability of the sarcolemma during strenuous intermittent hand grip exercise.

Acidification has been reported to provide protective effects on force production in vitro. Thus, in this study, we tested if respiratory acid-base changes influence muscle function and excitability in vivo. Nine subjects performed strenuous, intermittent hand grip exercises (10 cycles of 15 s of work/45 s of rest) under respiratory acidosis by CO(2) rebreathing, alkalosis by hyperventilation, or control. The Pco(2), pH, K(+) concentration ([K(+)]), and Na(+) concentration were measured in venous and arterialized blood. Compound action potentials (M-wave) were elicited to examine the excitability of the sarcolemma. The surface electromyogram (EMG) was recorded to estimate the central drive to the muscle. The lowest venous pH during the exercise period was 7.24 ± 0.03 in controls, 7.31 ± 0.05 with alkalosis, and 7.17 ± 0.04 with acidosis (P < 0.001). The venous [K(+)] rose to similar maximum values in all conditions (6.2 ± 0.8 mmol/l). The acidification reduced the decline in contraction speed (P < 0.001) but decreased the M-wave area to 73.4 ± 19.8% (P < 0.001) of the initial value. After the first exercise cycle, the M-wave area was smaller with acidosis than with alkalosis, and, after the second cycle, it was smaller with acidosis than with the control condition (P < 0.001). The duration of the M-wave was not affected. Acidification diminished the reduction in performance, although the M-wave area during exercise was decreased. Respiratory alkalosis stabilized the M-wave area without influencing performance. Thus, we did not find a direct link between performance and alteration of excitability of the sarcolemma due to changes in pH in vivo.

Clinical fMRI: evidence for a 7T benefit over 3T.

Despite there being an increasing number of installations of ultra high field MR systems (>3T) in clinical environments, no functional patient investigations have yet examined possible benefits for functional diagnostics. Here we performed presurgical localization of the primary motor hand area on 3T and 7T Siemens scanners with identical investigational procedures and comparable system specific sequence optimizations. Results from 17 patients showed significantly higher functional sensitivity of the 7T system measured via percent signal change, mean t-values, number of suprathreshold voxels and contrast to noise ratio. On the other hand, 7T data suffered from a significant increase of artifacts (ghosting, head motion). We conclude that ultra high field systems provide a clinically relevant increase of functional sensitivity for patient investigations.

Rapid screening of lectins for multivalency effects with a glycodendrimer microarray.

Multivalency is an important phenomenon in protein-carbohydrate interactions. In order to evaluate glycodendrimers as multivalent inhibitors of carbohydrate binding proteins, we displayed them on a microarray surface. Valencies were varied from 1 to 8, and corrections were made for the valencies so that all surfaces contained the same amount of the sugar ligand. Five different carbohydrates were attached to the dendrimers. A series of fluorescent lectins was evaluated, and for each of them a binding profile was obtained from a single experiment showing both the specificity of the lectin for a certain sugar and whether it prefers multivalent ligands or not. Very distinct binding patterns were seen for the various lectins. The results were rationalized with respect to the interbinding distances of the lectins.

Detection of pathogenic Streptococcus suis bacteria using magnetic glycoparticles.

Detection of the zoonotic bacterial pathogen Streptococcus suis was achieved using magnetic glycoparticles. The bacteria contain an adhesion protein for the carbohydrate sequence Galalpha1,4Gal. After incubation with various amounts of the pathogen, magnetic concentration and ATP detection, bacterial levels down to 10(5) cfu could be detected. Submicrometer particles were needed, since with the larger microparticles the method did not succeed.

ITAM-derived phosphopeptide-containing dendrimers as multivalent ligands for Syk tandem SH2 domain.

Spleen tyrosine kinase (Syk) is activated when its tandem SH2 domain (tSH2) binds to a diphosphorylated ITAM motif of e.g. the FcepsilonRI receptor. In this divalent interaction each SH2 domain binds to a phosphotyrosine-containing tetrapeptide motif in ITAM. One of those tetrapeptide sequences was synthesized and conjugated to dendrimers via 'click' chemistry to create a series of functional phosphopeptide-containing dendrimers ranging from a monovalent to an octavalent dendrimer. The affinity of the functionalized dendrimers for Syk tSH2 has been assayed in SPR competition experiments. Both the tetra- and octavalent dendrimer had an affinity in the high nanomolar range, which is approximately 100-fold enhanced compared to the monovalent tetrapeptide, indicating a multivalency effect.

Potential scorpionate antibiotics: targeted hydrolysis of lipid II containing model membranes by vancomycin-TACzyme conjugates and modulation of their antibacterial activity by Zn-ions.

The antibiotic vancomycin-that binds lipid II in the bacterial cell membrane-was conjugated to a mono- and tetravalent mimic of the tris-histidine catalytic triad of metalloenzymes. Targeted hydrolysis by the conjugate was observed using model membranes containing lipid II, and in vitro MIC-values of the targeted mimic constructs could be modulated by Zn-ions.

The influence of ligand valency on aggregation mechanisms for inhibiting bacterial toxins.

Divalent and tetravalent analogues of ganglioside GM1 are potent inhibitors of cholera toxin and Escherichia coli heat-labile toxin. However, they show little increase in inherent affinity when compared to the corresponding monovalent carbohydrate ligand. Analytical ultracentrifugation and dynamic light scattering have been used to demonstrate that the multivalent inhibitors induce protein aggregation and the formation of space-filling networks. This aggregation process appears to arise when using ligands that do not match the valency of the protein receptor. While it is generally accepted that multivalency is an effective strategy for increasing the activity of inhibitors, here we show that the valency of the inhibitor also has a dramatic effect on the kinetics of aggregation and the stability of intermediate protein complexes. Structural studies employing atomic force microscopy have revealed that a divalent inhibitor induces head-to-head dimerization of the protein toxin en route to higher aggregates.

Multivalent carbohydrate recognition on a glycodendrimer-functionalized flow-through chip.

Dendrimers were fitted out with up to eight mannose moieties by "click" chemistry. They were subsequently attached to aluminum oxide chips via a spacer that was linked to the dendrimer core; this resulted in a microarray of glycodendrimers. Binding of the glycodendrimers to the fluorescent lectins ConA and GNA was observable in real time. In a single experiment it was possible to observe the multivalency enhancement or cluster effect in the binding event. This effect was small for ConA, in agreement with its widely spaced binding sites, whereas it was large for GNA, with its twelve much more closely spaced binding sites. The dendrimer-fitted chip represents a valuable screening tool for multivalency effects. Furthermore kinetic and thermodynamic data on binding events can be deduced. Inhibition experiments are also possible with the system as was shown for ConA with alpha-methyl mannose as the inhibitor.

Synthesis of multivalent Streptococcus suis adhesion inhibitors by enzymatic cleavage of polygalacturonic acid and 'click' conjugation.

A galabiose disaccharide building block was synthesized by an efficient pectinase cleavage of polygalacturonic acid and subsequent chemical functional group transformations. Besides the disaccharide, the corresponding trisaccharide was also obtained and modified. The compounds were subsequently conjugated to dendrimers with up to eight end groups using 'click' chemistry. The compounds were evaluated as inhibitors of adhesion of the pathogen Streptococcus suis in a hemagglutination assay and strong inhibition was observed for the tetra- and octavalent galabiose compound with MIC values in the low nanomolar range. The corresponding octavalent trisaccharide was a ca. 20-fold weaker inhibitor.

Strong inhibition of cholera toxin binding by galactose dendrimers.

Galactose-containing dendrimers with long spacer arms inhibit cholera toxin binding as strongly as the natural ganglioside GM1 oligosaccharide does.

Reversible photoswitching enables single-molecule fluorescence fluctuation spectroscopy at high molecular concentration.

We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high molecular concentration. Reversible photoswitching allows precise control of the density of fluorescing entities, because the equilibrium between the fluorescent ON- and the dark OFF-state can be shifted through optical irradiation at a specific wavelength. Depending on the irradiation intensity, the concentration of the ON-state markers can be up to 1,000 times lower than the actual concentration of the labeled molecular entity. Photoswitching expands the range of single-molecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range.

Pharmacokinetics of SCH 56592, a new azole broad-spectrum antifungal agent, in mice, rats, rabbits, dogs, and cynomolgus monkeys.

SCH 56592 is a new broad-spectrum azole antifungal agent that is in phase 3 clinical trials for the treatment of serious systemic fungal infections. The pharmacokinetics of this drug candidate were evaluated following its intravenous (i.v.) or oral (p.o.) administration as a solution in hydroxypropyl-beta-cyclodextrin (HPbetaCD) or oral administration as a suspension in 0.4% methylcellulose (MC) in studies involving mice, rats, rabbits, dogs, and cynomolgus monkeys. SCH 56592 was orally bioavailable in all species. The oral bioavailability was higher with the HPbetaCD solution (range, 52 to approximately 100%) than from the MC suspension (range, 14 to 48%) and was higher in mice ( approximately 100% [HPbetaCD] and 47% [MC]), rats ( approximately 66% [HPbetaCD] and 48% [MC]), and dogs (72% [HPbetaCD] and 37% [MC]) than in monkeys (52% [HPbetaCD] and 14% [MC]). In rabbits, high concentrations in serum suggested good oral bioavailability with the MC suspension. The i.v. terminal-phase half-lives were 7 h in mice and rats, 15 h in dogs, and 23 h in monkeys. In rabbits, the oral half-life was 9 h. In species given increasing oral doses (mice, rats, and dogs), serum drug concentrations were dose related. Food produced a fourfold increase in serum drug concentrations in dogs. Multiple daily doses of 40 mg of SCH 56592/kg of body weight for eight consecutive days to fed dogs resulted in higher concentrations in serum, indicating accumulation upon multiple dosing, with an accumulation index of approximately 2.6. Concentrations above the MICs and minimum fungicidal concentrations for most organisms were observed at 24 h following a single oral dose in MC suspension in all five species studied (20 mg/kg for mice, rats, and rabbits and 10 mg/kg for dogs and monkeys), suggesting that once-daily administration of SCH 56592 in human subjects would be a therapeutically effective dosage regimen.

[Intraoperative precision of mechanical, electromagnetic, infrared and laser-guided navigation systems in computer-assisted surgery]. / Intraoperative Präzision mechanischer, elektromagnetischer, infrarot- und lasergeführter Navigationssysteme in der computergestützten Chirurgie.

Intraoperative precision in computer-assisted surgery depends on the characteristics of a navigation system, the precision of correlation between object and data set, the position, number and fixation of landmarks, and the parameters of the data set. The characteristics of a navigation system, in particular the immanent precision, can be detected by the use of the geometric model and navigation analyzer developed at the University of Regensburg with the support of Carl Zeiss, Germany. The precision of five navigation systems of different types and technology was measured: Viewing Wand (ISG, mechanical system), the SMN microscope (Carl Zeiss, infrared system with laser autofocus), the MKM system (Carl Zeiss, robot platform with laser autofocus) and the STP pointer (Leibinger, infrared system). The immanent precision of these systems ranges from 0.1 to 2.0 mm. An electromagnetic system (3-Space Digitizer, Polhemus) was compared; this produces serious spherical deviations of 10.0 to 20.0 mm in the presence of metal, surgical and rotating instruments, and circuits. The application of these different systems for craniomaxillofacial surgery is discussed.

[Development of a surgical simulator for interventions of the paranasal sinuses. Technical principles and initial prototype]. / Entwicklung eines Operationssimulators für Eingriffe an den Nasennebenhöhlen. Technische Grundlagen und erste Realisation.

BACKGROUND: Traditional training methods for surgeons include anatomical studies on human cadavers. Limited availability and ethical problems led to the consideration of alternatives. The surgeon's level of training could be significantly increased by a interactive training system trough computer graphics and virtual reality (VR). METHODS: Two main issues addressed are the 3-D reconstruction process and 3-D interaction to guide the surgical instruments. To provide the virtual environment, a realistic representation of the region of interest with all relevant anatomical parts is required. This model has to be suitable for computer simulation, while preserving as accurately as possible important anatomic features. Therefore the concept of creating a 3-D representation semi-automatically was developed. Textures derived from endoscopic images are superimposed on the virtual anatomic structures and provide better realism. RESULTS: Intuitive handling of the surgical instruments is ensured by using the tracking technique. The system allows navigation via a virtual camera and interaction with the virtual anatomical structures. Currently the use of a force feedback system and the simulation of deformations of tissues is critical. CONCLUSIONS: The VR based simulation system offers an alternative to conventional training methods. The future role of surgical simulation depends on overcoming the current drawbacks to provide greater interactive realism: the integration of a force feedback system to simulate the resistance of anatomical structures and the simulation of tissue deformations--both currently under development.

[Comparative accuracy assessment between a mechanical (viewing wand) and a laser-controlled microscopic positioning system using a geometric test model]. / Vergleichende Genauigkeitsmessung zwischen einem mechanischen (Viewing-Wand) und einem lasergeleiteten mikroskopischen Positionierungssystem (MKM) mit Hilfe eines geometrischen Messobjekts.

The precision of a new laser-guided navigation system (the "MKM") was compared with the "Viewing Wand" mechanical navigation system. We describe the inherent deviations in each positioning system as well as errors caused by data acquisition and referencing between the CT data set used for navigation and the original object. Two thousand individual measurements were performed on geometric test models scanned by CT. The technical accuracy of the mechanical navigation system varied between 0.48 mm in the most favorable area of the working field and 1.8 mm in the more inaccurate areas. For the laser-guided system a precision of 0.27 mm was found for all areas of the working field. After referencing between the CT data set and the original object, the error of measurement increased in both navigation systems: i.e., 0.83 mm for the Viewing Wand and 0.49 mm for the MKM system.

[Intraoperative precision of mechanical, electromagnetical, infrared and laser-guided navigation systems for computer-assisted surgery]. / Intraoperative Präzision mechanischer, elektromagnetischer, infrarot- und lasergeführter Navigationssysteme in der computergestützten Chirurgie.

Intraoperative precision in computer-assisted surgery depends on the characteristics of a navigation system, the precision of correlation between object and data set, the position, number and fixation of landmarks, and the parameters of the data set. The characteristics of a navigation system, in particular the immanent precision, can be detected by the use of the geometric model and navigation analyzer developed at the University of Regensburg with the support of Carl Zeiss, Germany. The precision of five navigation systems of different types and technology was measured: Viewing Wand (ISG, mechanical system), the SMN microscope (Carl Zeiss, infrared system with laser autofocus), the MKM system (Carl Zeiss, robot platform with laser autofocus) and the STP pointer (Leibinger, infrared system). The immanent precision of these systems ranges from 0.1 to 2.0 mm. An electromagnetic system (3-Space Digitizer, Polhemus) was compared; this produces serious spherical deviations of 10.0 to 20.0 mm in the presence of metal, surgical and rotating instruments, and circuits. The application of these different systems for craniomaxillofacial surgery is discussed.