Monitoring glycosidase activity for clustered sugar substrates,a study on β-glucuronidase

Determination of glycosidase hydrolysis kinetics for a monovalent sugar substrate is relatively straightforward and classically achieved by monitoring the fluorescence signal released from the sugar-conjugated probe after enzymatic hydrolysis. Naturally occuring sugar epitopes are, however, often clustered on biopolymers or at biological surfaces, and previous reports have shown that glycosidase hydrolytic rates can differ greatly with multivalent presentation of the sugar epitopes. New probes are needed to make it easier to interpret the importance of substrate clustering towards a specific enzyme activity. In this work, we developed multivalent glucuronide substrates attached to fluorescent amino-coumarines through self-immolative linkers to enable real time-monitoring of the hydrolysing activity of E.coli β-glucuronidases (GUS) towards clustered substrates. GUS are exoglycosidases of considerable therapeutic interest cleaving β-d-glucuronides and are found in the lysosomes, in the tumoral microenvironment, and are expressed by gut microbiota. GUS showed a much lower catalytic efficiency in hydrolysing clustered glucuronides due to a significantly lower enzymatic velocity and affinity for the substrates. GUS was 52-fold less efficient in hydrolysing GlcA substrates presented on an octameric silsequioxane (COSS) compared with a monovalent GlcA of similar chemical structure. Thus, kinetic and thermodynamic data of GUS hydrolysis towards multivalent glucuronides were easily obtained with these new types of enzymatically-triggered probes. More generally, adapting the substrate nature and valency of these new probes, should improve understanding of the impact of multivalency for a specific enzyme.

Enzymatically-triggered probes to determine glucuronidase hydrolysis kinetics for clustered substrates.     

A new assay for interleukin-1 in the presence of interleukin-2

A simple and reliable assay for interleukin-1 (IL-1) is described which has the advantage over other assays that it is independent of interleukin-2 (IL-2) production by the test cells. The assay makes possible the detection of IL-1 in the supernatants of T cell populations. The ability of IL-1 to induce IL-2 receptor expression in the absence of T cell mitogen is the basis of this assay. Thus, proliferation of mouse thymocytes incubated in the presence of saturating concentrations of IL-2 (10 U/ml) was directly dependent on the concentration of IL-1. The sensitivity of the assay is comparable to the sensitivity of the classical thymocyte co-stimulator assay. Natural and recombinant human and murine IL-1 were measured in this test system with comparable sensitivity.     

Contrast-Enhanced Ultrasound Quantifies the Perfusion Within Tibial Non-Unions and Predicts the Outcome of Revision Surgery

Vascularity is one of the factors determining successful bone regeneration. This prospective study focused on quantifying the microperfusion of tibial non-unions with contrast-enhanced ultrasound (CEUS) 12?wk after revision surgery and comparing it with the osseous consolidation at a maximum of 24?mo assessed with standard radiography and computed tomography. Of 36 patients with tibial non-unions, 28 (77.8%) manifested consolidation, and 8 patients required further revision surgery. CEUS revealed significantly higher perfusion in consolidated versus persistent non-unions for all quantification parameters (e.g., wash-in perfusion index p?=?0.036). Receiver operating characteristic analysis revealed a sensitivity of 82.1% and specificity of 75.0% with a wash-in perfusion index cutoff at 19.9 a.u. for diagnosing persisting non-unions. More than 1 y ahead of the final radiologic diagnostic examination, CEUS could predict eventual consolidation based on the osseous perfusion as soon as 12?wk postoperatively. This information can be crucial for the decision-making process for re-revision at an early stage.     

Activation interferes with the APO-1 pathway in mature human T cells

One of the mechanisms to terminate a specific immune responsemay involve elimination of antigen activated T cells by programmedcell death, apoptosis. Apoptosis in activated T cells may beinduced via the TCR-CD3 complex or/and cell surface moleculeslike the APO-1 (Fas) antigen, a new member of the nerve growthfactor/tumor necrosis factor receptor superfamily. To investigateapoptosis in activated T cells we studied expression of APO-1and sensitivity to APO-1 mediated apoptosis in human peripheralT lymphocytes. APO-1 is not expressed on cord blood and themajority of resting T cells, but on activated T cells. One dayactivated T cells in culture showed activation induced resistanceto apoptosis (ARA). However, after prolonged in vitro culture,6 day activated T cells acquired sensitivity to activation inducedsensitivity to apoptosis (ASA). Restimulation of the ASA⁺ activatedT cells by triggering TCR-CD3 or CD2 induced prollferation andapoptosis in a fraction of the cells. In the surviving fractionof ASA⁺ activated T cells, however, this treatment reinduceda transient ARA⁺ phenotype. Thus, activation of resting matureT cells or restimulation of activated T cells may induce a translentresistance to apoptotic signals. Activation signals may interferewith the APO-1 pathway and may prevent elimination of activatedT cells in the periphery (peripheral selection).     

Epidermolysis bullosa - a group of skin diseases with different causes but commonalities in gene expression

Epidermolysis bullosa (EB) is a group of hereditary skin disorders. Although each subtype is caused by mutations in genes encoding differentially located components of the skin, the resulting phenotype is similar. In this study, we investigated similarities in the gene expression profiles of each subtype on mRNA level. Type XVI collagen (COL16A1), G0/G1 switch 2 (G0S2), fibronectin (FN1), ribosomal protein S27A (RPS27A) and low density lipoprotein receptor (LDLR) were shown to exhibit corresponding changes in gene expression in all three EB subtypes. While COL16A1, G0S2 and FN1 are up-regulated, LDLR and RPS27A mRNA levels are decreased. These data indicate that EB cells seem to take measures increasing their mechanical stability. Apoptosis is likely to be exacerbated, and migratory potential appears to be elevated. Protein degradation is hampered, and the release of fatty acids and glycerol is restricted, probably to save energy. These commonalities might benefit existing EB treatment strategies or could help to reveal new starting points for the treatment of EB in the future.     

Dislocation tendency,stabilizing effect and sintering tendency of different lumbar vertebrae cages in an in vitro experiment

For biomechanical purposes, interbody fusion cages should not dislocate, should provide high stability, and should have a low subsidence risk. Zientek (Marquardt Medzintechnik), Stryker (Stryker Implants), and Ray lumbar interbody fusion cages (Surgical Dynamics) were tested in this study. They were implanted by pairs from a posterior approach without further stabilization. In a first step, each cage design was implanted into four human L3-4 segments and extracted posteriorly under an axial preload of 200 N. In a second step, standard flexibility tests were carried out with 24 human L2-3 and L4-5 specimens in an intact condition, directly after cage implantation, and after cyclic axial compression loading (200-1000 N, 40,000 cycles, 5 Hz). In a third step, a destructive axial compression test was carried out. Maximum pullout force was highest with Ray cages (median 945 N), followed by Zientek (605 N) and Stryker cages (130 N). With all three cage designs, primary stability was higher in lateral bending and flexion than in extension and axial rotation. Implantation of Ray cages caused a decreased range of motion in all three loading directions ranging between 49% and 99%. Zientek cages only stabilized in lateral bending, flexion, and extension (45-78%) and Stryker cages in none of the three loading directions. Cyclic loading caused an increased range of motion in all cases up to 190%. Axial compression force at failure was 8413 N with Ray cages, 8359 N with Stryker cages, and 5486 N with Zientek cages. The cage design seems to influence the dislocation tendency. In this regard, threaded cages or cages with anchorage systems seem to provide more security. The stabilizing effect seems to be mainly influenced by factors such as the degree of distraction or destruction of the facet joints rather than by the cage design.     

Pruritus in pediatric non-Hodgkin's lymphoma

A two-year-old girl presented with a 3-month history of generalized pruritus. One week before hospitalization she developed a superior vena cava syndrome and obstruction of the upper airways. Clinical and laboratory findings included generalized lymphadenopathy, a mediastinal mass compressing the tracheal lumen to the point of near closure, hepatomegaly and moderate eosinophilia. The diagnosis of an anaplastic large cell lymphoma (ALCL) was made by the histologic examination of a mediastinal lymph node. The history of generalized pruritus without diagnostic skin lesions was as uncommon as age at presentation. In conclusion, this case illustrates that generalized pruritus in a toddler can be an early sign of ALCL.