Synthesis of novel 4- and 5-substituted benzyl ether derivatives of vesamicol and in vitro evaluation of their binding properties to the vesicular acetylcholine transporter site

Detection of the central cholinergic deficits, a consistent feature of Alzheimer's disease, is essential to allow preventive measures and/or symptomatic treatment already at a very early stage of the disease. The vesicular acetylcholine transporter (VAChT) represents an appropriate target to establish PET radiotracer that are adequate for brain imaging the loss of cholinergic terminals. Here we describe the synthesis and binding characteristics of novel derivatives of vesamicol, known to represent a specific antagonist of VAChT sites. Novel benzyl ether derivatives of vesamicol either 4- or 5-substituted at the cyclohexylring have been synthesized by different regioselective ring opening reactions of a same epoxide precursor. The affinity and selectivity of the novel compounds to VAChT sites were analyzed by competitive radioligand binding studies in rat brain and liver membrane preparations using tritium labeled radioligands. The 4-substituted fluorobenzylether of vesamicol 10b was shown to exhibit a high affinity to VAChT sites (K(i)-value(10b)=10.7+/-1.7 nM), but demonstrated also binding capacities to sigma receptors (K(i-)value(10b)=18.5+/-6.9 nM, [(3)H]DTG; K(i)-value(10b)=30.6+/-9.6 nM, [(3)H]haloperidol). The data suggest the potential of vesamicol derivatives to design appropriate radiotracer for PET imaging of central cholinergic deficits.     

Dual 5-HT1A agonists and 5-HT re-uptake inhibitors by combination of indole-butyl-amine and chromenonyl-piperazine structural elements in a single molecular entity

The dual serotonin (5-HT) re-uptake inhibitor and 5-HT(1A) receptor agonist vilazodone was found to increase central serotonin levels in rat brain. In the course of structural modifications of vilazodone 3-[4-[4-(2-oxo-2H-1-benzopyran-6-yl)-1-piperazinyl]-butyl]-1H-indole-5-carbonitrile 8i and its fluorine analogue 6-[4-[4-(5-fluor-3-indolyl)-butyl]-1-piperazinyl]-2H-1-benzopyran-2-one have been identified. These unsubstituted chromenones are equally potent at the 5-HT(1A) receptor and 5-HT transporter. The implementation of nitrogen functionalities in position 3 of the chromenones resulted in compounds acting as agonists at the 5-HT(1A) receptor and as 5-HT re-uptake inhibitors like vilazodone. Ex vivo 5-HT re-uptake inhibition and in vitro 5-HT agonism were determined in the PCA- and GTPgammaS-assay, respectively. The potential of these chromenones to increase central 5-HT levels was measured in microdialysis studies and especially the derivatives 3-[4-[4-(3-amino-2-oxo-2H-chromen-6-yl)-piperazin-1-yl]-butyl]-1H-indole-5-carbonitrile 8f, ethyl (6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-carbamate 8h and N-(6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-acetamide 8k give rise to rapid development of increased serotonin levels in rat brain cortex, lasting longer than 3h.     

Localization of a symbiosis-related protein, Sym32, in the Anthopleura elegantissima-Symbiodinium muscatinei Association

Cnidarian-dinoflagellate symbioses are widespread in the marine environment. Growing concern over the health of coral reef ecosystems has revealed a fundamental lack of knowledge of how cnidarian-algal associations are regulated at the cellular and molecular level. We are interested in identifying genes that mediate interactions between the partners, and we are using the temperate sea anemone Anthopleura elegantissima as a model. We previously described a host gene, sym32, encoding a fasciclin domain protein, that is differentially expressed in symbiotic and aposymbiotic A. elegantissima. Here, we describe the subcellular localization of the sym32 protein. In aposymbiotic (symbiont-free) hosts, sym32 was located in vesicles that occur along the apical edges of gastrodermal cells. In symbiotic hosts, such vesicles were absent, but sym32 was present within the symbiosome membranes. Sym32 (or a cross-reactive protein) was also present in the accumulation bodies of the symbionts. Although the anti-sym32 antiserum was not sufficiently specific to detect the target protein in cultured Symbiodinium bermudense cells, Western blots of proteins from two Symbiodinium species revealed a protein doublet of 45 and 48 kDa, suggesting that the symbionts may also produce a fasciclin domain protein. We suggest that host sym32 is relocated from gastrodermal vesicles to the symbiosome membrane when symbionts are taken into host cells by phagocytosis.     

On translocation through a membrane channel via an internal binding site: kinetics and voltage dependence

Here we present a model for maltodextrin translocation through maltoporin channels. In a first step, our theoretical analysis does consider the case of a single binding site for a given substrate in a structurally unaffected channel with a possibly different entrance barrier on either side. It is shown how by means of conventional electrical conductance measurements (including current noise analysis) the basic equilibrium and rate constants can be determined as functions of the applied voltage. Then also the net translocation rate of the substrate becomes accessible quantitatively. This most simple model mechanism has been extended to include a voltage-dependent fast conformational change of the channel that prevents the binding process. The so developed approach has been tested with experimental data for a single maltoporin trimer being reconstituted in black lipid membranes when studied in the presence of maltohexaose as the substrate. The experimental results turned out to be clearly incompatible with binding alone. They are, however, very satisfactorily fitted by pertinent theoretical curves if also inhibition of binding by a conformational transition is taken into account. Accordingly, quantitative evaluations of the underlying parameters and eventually of the translocation rate have been carried out successfully. Our analysis reveals a set of parameters necessary for an optimal translocation that nicely corresponds to natural conditions.     

Evidence for glycosylphosphatidylinositol (GPI)-anchored eosinophil-derived neurotoxin (EDN) on human granulocytes

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST–SLAP-130–SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells.     

Process formation of podocytes: morphogenetic activity of microtubules and regulation by protein serine/threonine phosphatase PP2A

Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases. MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based peripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One- and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after okadaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases.     

Cosecretion of chaperones and low-molecular-size medium additives increases the yield of recombinant disulfide-bridged proteins

Attempts were made to engineer the periplasm of Escherichia coli to an expression compartment of heterologous proteins in their native conformation. As a first approach the low-molecular-size additive L-arginine and the redox compound glutathione (GSH) were added to the culture medium. Addition of 0.4 M L-arginine and 5 mM reduced GSH increased the yield of a native tissue-type plasminogen activator variant (rPA), consisting of the kringle-2 and the protease domain, and a single-chain antibody fragment (scFv) up to 10- and 37-fold, respectively. A variety of other medium additives also had positive effects on the yield of rPA. In a second set of experiments, the effects of cosecreted ATP-independent molecular chaperones on the yields of native therapeutic proteins were investigated. At optimized conditions, cosecretion of E. coli DnaJ or murine Hsp25 increased the yield of native rPA by a factor of 170 and 125, respectively. Cosecretion of DnaJ also dramatically increased the amount of a second model protein, native proinsulin, in the periplasm. The results of this study are anticipated to initiate a series of new approaches to increase the yields of native, disulfide-bridged, recombinant proteins in the periplasm of E. coli.