Impaired integrin‐mediated adhesion contributes to reduced barrier properties in VASP‐deficient microvascular endothelium

Recent studies point to a significant role of vasodilator‐stimulated phosphoprotein (VASP) in the maintenance of endothelial barrier functions in vivo and in vitro. Moreover, it has been reported that VASP is required for activation of the small GTPase Rac 1. However, little is known whether VASP is involved in the regulation of cell adhesion molecules that are critical for maintenance of the endothelial barrier. Here we demonstrate that impaired barrier properties in VASP‐deficient (VASP?/?) microvascular myocardial endothelial cells (MyEnd) correlated with both impaired integrin‐mediated adhesion as revealed by laser tweezer trapping and reduced integrin‐dependent cell migration. This was paralleled by reduction of focal adhesions at the cell periphery as well as of β₁‐integrin and VE‐cadherin cytoskeletal anchorage. Incubation of MyEnd VASP wt with RGD peptide to block interaction of integrins with extracellular matrix (ECM) reduced barrier properties and Rac 1 activity in wt endothelial monolayers mimicking the situation in VASP (?/?) cells under resting conditions. Moreover, cAMP‐mediated Rac 1 activation was reduced under conditions of impaired integrin‐mediated adhesion in wt cells and cAMP‐induced increase in VE‐cadherin cytoskeletal anchorage was abolished in VASP (?/?) endothelium. In summary, these data indicate that VASP is required for integrin‐mediated adhesion which stabilizes endothelial barrier properties at least in part by facilitating Rac 1 activation. J. Cell. Physiol. 220: 357–366, 2009. © 2009 Wiley‐Liss, Inc.     

Computational and Functional Analyses of a Small-Molecule Binding Site in ROMK

The renal outer medullary potassium channel (ROMK, or Kir1.1, encoded by KCNJ1) critically regulates renal tubule electrolyte and water transport and hence blood volume and pressure. The discovery of loss-of-function mutations in KCNJ1 underlying renal salt and water wasting and lower blood pressure has sparked interest in developing new classes of antihypertensive diuretics targeting ROMK. The recent development of nanomolar-affinity small-molecule inhibitors of ROMK creates opportunities for exploring the chemical and physical basis of ligand-channel interactions required for selective ROMK inhibition. We previously reported that the bis-nitro-phenyl ROMK inhibitor VU591 exhibits voltage-dependent knock-off at hyperpolarizing potentials, suggesting that the binding site is located within the ion-conduction pore. In this study, comparative molecular modeling and in silico ligand docking were used to interrogate the full-length ROMK pore for energetically favorable VU591 binding sites. Cluster analysis of 2498 low-energy poses resulting from 9900 Monte Carlo docking trajectories on each of 10 conformationally distinct ROMK comparative homology models identified two putative binding sites in the transmembrane pore that were subsequently tested for a role in VU591-dependent inhibition using site-directed mutagenesis and patch-clamp electrophysiology. Introduction of mutations into the lower site had no effect on the sensitivity of the channel to VU591. In contrast, mutations of Val¹⁶⁸ or Asn¹⁷¹ in the upper site, which are unique to ROMK within the Kir channel family, led to a dramatic reduction in VU591 sensitivity. This study highlights the utility of computational modeling for defining ligand-ROMK interactions and proposes a mechanism for inhibition of ROMK.     

Ethylene Glycol Metabolism by Pseudomonas putida

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.     

Identification and evaluation of novel synovial tissue biomarkers in rheumatoid arthritis by laser scanning cytometry

Introduction

Suitable biomarkers are essential for therapeutic strategies in personalized medicine in terms of diagnosis as well as of prognosis. With highly specific biomarkers, it is possible, for example, to identify patients with poor prognosis, which enables early intervention and intensive treatment. The aim of this study was to identify and validate biomarkers and possible combinations for a prospective use in immunoscintigraphy, which may improve diagnosis of rheumatoid arthritis (RA) patients with consideration of inflammatory activity in the affected joints. Therefore, we tested several monoclonal antibodies (mAbs) directed against cellular-surface molecules on cells likely to be involved in the pathogenesis of RA.

Methods

Synovial tissue from patients with long-standing RA (accompanied by synovitis with varying states of current activity) and patients with acute non-RA arthritis were stained for surface molecules on different cell types by using fluorochrome-labeled antibodies. Tissue analysis was done by laser scanning cytometry (LSC), and statistical evaluation, by discriminant analysis and ROC analysis.

Results

CD11b, HLA-DR, CD90, and CD64 revealed significant differences between tissues from patients with RA and acute non-RA arthritis. Especially with the expression of CD64, both patient cohorts could be discriminated with high sensitivity and specificity. RA classification was improved by simultaneously investigating the expression of two or three different surface proteins, such as HLA-DR, CD90, and CD29 in the tissue. The simultaneous analysis of CD64 together with CD304 or the combination of CD11b and CD38 was suitable for the identification of RA patients with high current activity in synovitis.

Conclusions

In this study, we showed that LSC is a novel reliable method in biomarker prevalidation in RA. Hence, identified mAbs in situ may allow their potential use in in vivo approaches. Moreover, we proved that biomarker-combination analysis resulted in better discrimination than did single-marker analysis. Combinations of these markers make a novel and reliable panel for the discrimination between RA and acute non-RA arthritis. In addition, further expedient combinations may be novel promising biomarker panels to identify current activity in synovitis in RA.     

Cloning and characterization of fiber type-specific ryanodine receptor isoforms in skeletal muscles of fish

We have cloned agroup of cDNAs that encodes the skeletal ryanodine receptor isoform(RyR1) of fish from a blue marlin extraocular muscle library. The cDNAsencode a protein of 5,081 amino acids with a calculated molecular massof 576,302 Da. The deduced amino acid sequence shows strong sequenceidentity to previously characterized RyR1 isoforms. An RNA probederived from a clone of the full-length marlin RyR1 isoform hybridizesto RNA preparations from extraocular muscle and slow-twitch skeletalmuscle but not to RNA preparations from fast-twitch skeletal or cardiacmuscle. We have also isolated a partial RyR clone from marlin andtoadfish fast-twitch muscles that shares 80% sequence identity withthe corresponding region of the full-length RyR1 isoform, and a RNAprobe derived from this clone hybridizes to RNA preparations fromfast-twitch muscle but not to slow-twitch muscle preparations. Westernblot analysis of slow-twitch muscles in fish indicates the presence ofonly a single high-molecular-mass RyR proteincorresponding to RyR1. [³H]ryanodine bindingassays revealed the fish slow-twitch muscle RyR1 had a greatersensitivity for Ca²⁺ than thefast-twitch muscle RyR1. The results indicate that, in fish muscle,fiber type-specific RyR1 isoforms are expressed and the two proteinsare physiologically distinct.

     

Raccoons (Procyon lotor) in Germany as potential reservoir species for Lyssaviruses

Raccoons can be found almost everywhere in Germany since their first successful introduction in 1934. Although the animal is a well-known reservoir species for rabies in the USA, during the last European fox rabies epizootic, only a few rabid raccoons were reported from Germany. In recent years, the raccoon population density has increased tremendously, especially in (semi) urban settings. Presently, Germany is free of terrestrial wildlife rabies. To assess the potential risk that the raccoon population in Germany could act as a reservoir species upon reemergence of rabies, the susceptibility of the local raccoon population was investigated. Wild-caught animals were inoculated with the most likely lyssavirus variants to infect the local population. It was shown that the raccoons were fully susceptible for a dog and raccoon rabies virus isolate. Also, five of six raccoons inoculated with a fox rabies virus isolate showed clinical signs. However, none of the raccoons infected with European Bat Lyssavirus type 1 succumbed to rabies; meanwhile, all these raccoons seroconverted. It is concluded that the highest risk for the raccoon population in Germany to become infected with lyssaviruses is through the importation of rabies infected dogs.     

Diffusional mobility of parvalbumin in spiny dendrites of cerebellar Purkinje neurons quantified by fluorescence recovery after photobleaching

Ca(2+)-binding proteins (CaBPs) represent key factors for the modulation of cellular Ca(2+) dynamics. Especially in thin extensions of nerve cells, Ca(2+) binding and buffered diffusion of Ca(2+) by CaBPs is assumed to effectively control the spatio-temporal extend of Ca(2+) signals. However, no quantitative data about the mobility of specific CaBPs in the neuronal cytosol are available. We quantified the diffusion of the endogenous CaPB parvalbumin (PV) in spiny dendrites of cerebellar Purkinje neurons with two-photon fluorescence recovery after photobleaching. Fluorescently labeled PV diffused readily between spines and dendrites with a median time constant of 49 ms (37-61 ms, interquartile range). Based on published data on spine geometry, this value corresponds to an apparent diffusion coefficient of 43 microm(2) s(-1) (34-56 microm(2) s(-1)). The absence of large or immobile binding partners for PV was confirmed in PV null-mutant mice. Our data validate the common but so far unproven assumption that PV is highly mobile in neurons and will facilitate simulations of neuronal Ca(2+) buffering. Our experimental approach represents a versatile tool for quantifying the mobility of proteins in neuronal dendrites.