Investigation of the abundance and subunit composition of GABAA receptor subtypes in the cerebellum of alpha1-subunit-deficient mice

In cerebellum, 75% of all GABAA receptors contain alpha1 subunits. Here, we investigated compensatory changes in GABAA receptor subunit expression and composition in alpha1 subunit-knockout mice. In these mice the total number of cerebellar GABAA receptors was reduced by 46%. Whereas the number of receptors containing alpha6 subunits was unchanged, the total amount of alpha6 subunits was significantly elevated. RT-PCR showed no increase of alpha6 mRNA levels, arguing against increased biosynthesis of these subunits. Elevated levels of alpha6 subunits in alpha1 -/- mice might thus have been caused by an increased incorporation of unassembled alpha6 subunits, replacing alpha1 subunits in alpha1alpha6betagamma2 or alpha1alpha6betadelta receptors, thus rescuing alpha6 subunits from degradation. Elevated levels of alpha3 and alpha4 subunits in the cerebellum of alpha1 -/- mice possibly can be explained similarly. Finally, a small amount of receptors containing no gamma or delta subunits was identified in these mice. Results suggest a total loss of GABAA receptors in cell types where alpha1 was the only alpha subunit expressed and a partial compensation for receptor loss in cell types containing other alpha subunits. Our results do not support a significant compensatory synthesis of other GABAA receptor subunits in the cerebellum of alpha1 -/- mice.     

Vibration training: benefits and risks

The main results of our recent several studies, i.e. the measurements of vibration training results for single case and group studies as well as the cardiovascular parameter measurements during vibrations and the corresponding hydrodynamic analysis, are summarized. Our studies and previous work all confirm that vibration training is an effective training method in order to improve maximal strength and flexibility as well as various other factors if the training is properly designed. Some recommendations regarding the proper ranges of frequencies, amplitudes and exposure duration of vibration training are made based on the existing vibration training practice and mechanism analysis, although much work remains to be carried out in order to set up clear rules for various groups of people so that maximal training results could be expected and in the meantime potential dangerous effects could be avoided. Cardiovascular parameter measurements confirm that total peripheral resistance (TPR) to the blood flow is increased during body vibration. Hydrodynamic analysis offers the mechanism for the increase of TPR through the deformation of vessels. As a reaction of compensation, more capillaries are probably opened in order to keep a necessary level of cardiac output needed for the body, resulting in more efficient gas and material metabolism between the blood and muscle fibres. This might be one of the reasons for the various potential beneficial effects of vibration training.     

CD1 antigen presentation by human dendritic cells as a target for herpes simplex virus immune evasion

In contrast to MHC molecules, which present peptides, the CD1 molecules have been discovered to present lipid Ags to T cells. CD1-restricted T lymphocytes have been recently associated with resistance to virus infection. The mechanisms underlying activation of CD1-restricted T cells in the course of virus infection are not defined. In this study, we wanted to investigate the interaction of HSV with the antiviral CD1 Ag presentation system in human dendritic cells (DC). In response to low titers of HSV, the surface expression of CD1b and CD1d on human DC was up-regulated. These phenotypic changes enhanced the capacity of infected DC to stimulate proliferation of CD1-restricted T lymphocytes. High titers of HSV, however, lead to strong down-regulation of all surface CD1 molecules. This modulation of surface expression was associated with intracellular accumulation, colocalization with viral proteins, and disruption of the CD1 recycling machinery. Finally, even at low titers HSV interfered with the capacity of infected DC to stimulate the release of important cytokines by CD1d-restricted NKT cells. Thus, we demonstrate both the existence of a CD1 pathway allowing human DC to react to viral infection, as well as its blockage by a human herpesvirus.     

Functional specialization of memory Th cells revealed by expression of integrin CD49b

Infection or immunization induces heterogeneous memory T cell subsets, but their origin and protective value against infection are unclear. In this study, we report the functional characterization of two memory Th subsets, defined by expression of integrin CD49b. Stable CD49b expression is induced in up to one-half of all memory Th cells. More importantly, the CD49b- and CD49b+ subsets display distinct helper activities, typified by the production of IL-10 and TNF-alpha, respectively. Although the inflammatory properties of the CD49b+ subset are protective against intracellular bacterial infection, they are associated with immunopathology in acute viral infection. Modulation of the CD49b-defined memory Th subsets may provide infection type-specific interventions, where either enhancement of the inflammatory response or reduction of immunopathology is essential.     

Blood phagocyte activation during open heart surgery with cardiopulmonary bypass

Open heart surgery with a cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response which significantly contributes to adverse postoperative complications. The purpose of this study was to characterize the activation of blood phagocytes during open heart surgery with CPB. Blood samples were collected during and up to 24 h after surgery. The production of reactive oxygen species (ROS) in whole blood, the expression of surface molecules by blood phagocytes and complement activity in the plasma were determined. A cDNA microarray analysis of leukocyte RNA profile of genes was performed related to the inflammatory response. Activation of the complement was already observed at the beginning of CPB. This was followed by an increase in the neutrophil number and in both spontaneous and opsonized zymosan-activated ROS production after the onset of reperfusion. The activation of blood phagocytes was affirmed by changes in surface receptors involved in the adhesion and migration of leukocytes (CD11b, CD62L and CD31). Gene arrays also confirmed the activation of leukocytes 4 h after reperfusion. In conclusion, open heart surgery with a cardiopulmonary bypass was found to be associated with a rapid and pronounced activation of blood phagocytes and complement activation which was partly independent at the onset of CPB.     

Construction, database integration, and application of an Oenothera EST library

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.     

Inhibition of protein-protein interactions: the discovery of druglike beta-catenin inhibitors by combining virtual and biophysical screening

The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions.     

Combining functional and topological properties to identify core modules in protein interaction networks

Advances in large-scale technologies in proteomics, such as yeast two-hybrid screening and mass spectrometry, have made it possible to generate large Protein Interaction Networks (PINs). Recent methods for identifying dense sub-graphs in such networks have been based solely on graph theoretic properties. Therefore, there is a need for an approach that will allow us to combine domain-specific knowledge with topological properties to generate functionally relevant sub-graphs from large networks. This article describes two alternative network measures for analysis of PINs, which combine functional information with topological properties of the networks. These measures, called weighted clustering coefficient and weighted average nearest-neighbors degree, use weights representing the strengths of interactions between the proteins, calculated according to their semantic similarity, which is based on the Gene Ontology terms of the proteins. We perform a global analysis of the yeast PIN by systematically comparing the weighted measures with their topological counterparts. To show the usefulness of the weighted measures, we develop an algorithm for identification of functional modules, called SWEMODE (Semantic WEights for MODule Elucidation), that identifies dense sub-graphs containing functionally similar proteins. The proposed method is based on the ranking of nodes, i.e., proteins, according to their weighted neighborhood cohesiveness. The highest ranked nodes are considered as seeds for candidate modules. The algorithm then iterates through the neighborhood of each seed protein, to identify densely connected proteins with high functional similarity, according to the chosen parameters. Using a yeast two-hybrid data set of experimentally determined protein-protein interactions, we demonstrate that SWEMODE is able to identify dense clusters containing proteins that are functionally similar. Many of the identified modules correspond to known complexes or subunits of these complexes.