EspH,a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. During infection, EPEC and EHEC use a type III secretion system (TTSS) to translocate effector proteins into the infected cells and thereby modify specific host functions. These include transient filopodium formation which is Cdc42-dependent. Filopodia formation is followed by assembly of actin pedestals, the process enhanced by inhibition of Cdc42. We discovered that orf 18 of the enterocyte effacement locus encodes a new effector, which we termed EspH. We show that EspH is translocated efficiently into the infected cells by the TTSS and localizes beneath the EPEC microcolonies. Inactivation of espH resulted in enhanced formation of filopodia and attenuated the pedestals formation. Furthermore, overexpression of EspH resulted in strong repression of filopodium formation and heightened pedestal formation. We also demonstrate that overexpression of EspH by EHEC induces marked elongation of the typically flat pedestals. Similar pedestal elongation was seen upon infection of COS cells overexpressing EspH. EspH transiently expressed by the COS cells was localized to the membrane and disrupted the actin cytoskeletal structure. Our findings indicate that EspH is a modulator of the host actin cytoskeleton structure.     

Voltammetric sensor for vanillylmandelic acid based on molecularly imprinted polymer-modified electrodes

Despite the increasing number of applications of molecularly imprinted polymers (MIPs) in analytical chemistry, the construction of a biomimetic voltammetric sensor remains still challenging. This work investigates the development of a voltammetric sensor for vanillylmandelic acid (VMA) based on acrylic MIP-modified electrodes. Thin layers of MIPs for VMA have been prepared by spin coating the surface of a glassy carbon electrode with the monomers mixture (template, methacrylic acid, a cross-linking agent and solvent), followed by in situ photopolymerisation. After extraction of the template molecule, the peak current recorded with the imprinted sensor after rebinding was linear with VMA concentration in the range 19-350 microg ml(-1), whereas the response of the control electrode is independent of incubation concentration, and was about one-tenth of the value recorded with the imprinted sensor at the maximum concentration tested. Under the conditions used, the sensor is able to differentiate between VMA and other closely structural-related compounds, such as 3-methoxy-4-hydroxyphenylethylene glycol (not detected), or 3,4- and 2,5-dihydroxyphenilacetic acids, which are adsorbed on the bare electrode surface but not at the polymer layer. Homovanillic acid was detected with the imprinted sensors after incubation, indicating that the presence of both methoxy and carboxylic groups in the same position as in VMA is necessary for effective binding in the imprinted sites. Nevertheless, both species can be differentiated by the oxidation potential. It can be concluded that MIP-based voltammetric electrodes are very promising analytical tool for the development of highly selective analytical sensors.     

Chronic Exposure of NG108-15 Cells to Amyloid β Peptide (Aβ1–42) Abolishes Calcium Influx via N-Type Calcium Channels

We investigated whether amyloid--peptide (A_1–42) has an effect on the elevations of the intracellular concentration of Ca²⁺ ions ([Ca²⁺]_i) induced by depolarizations of NG108-15 cells and on related Ca²⁺ channels. A_1–42 (10-1000 nM) had no immediate effect on depolarization-induced [Ca²⁺]_i elevations. [Ca²⁺]_i increases were slightly diminished in cells grown in the presence of 100 or 1000 nM A_1–42. Nifedipine (1 M) reduced these elevations equally in cells grown in the absence or presence of A_1–42. In contrast, the ability of -conotoxin GVIA to diminish the depolarization-induced [Ca²⁺]_i responses became lost in cells grown in the presence of 100 nM A_1–42. This indicates that the influx of calcium through the N-type Ca²⁺ channels was compromised by the chronic exposure of cells to a submicromolar concentration of A_1–42, presumably because of impairement of their function or diminished expression. This may be important in the pathogeny of Alzheimer's dementia in view of the pivotal role of N-type Ca²⁺ channels in neurotransmitter release.     

Interactions between Allosteric Modulators and 4-DAMP and Other Antagonists at Muscarinic Receptors: Potential Significance of the Distance between the N and Carboxyl C Atoms in the Molecules of Antagonists

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M₂ receptors, and strychnine did so also at the M₄ receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M₂) and scopolamine (M₂), between strychnine and butylscopolamine (M₄), L-hyoscyamine (M₂ and M₄) and scopolamine (M₄), and between brucine and scopolamine (M₂). Positive effects of alcuronium, strychnine and brucine on the affinity of the M₂ receptors for 4-DAMP have been confirmed by direct measurements of the binding of [³H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M₂ receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.     

Structure of the Homer EVH1 domain-peptide complex reveals a new twist in polyproline recognition

Homer EVH1 (Ena/VASP Homology 1) domains interact with proline-rich motifs in the cytoplasmic regions of group 1 metabotropic glutamate receptors (mGluRs), inositol-1,4,5-trisphosphate receptors (IP3Rs), and Shank proteins. We have determined the crystal structure of the Homer EVH1 domain complexed with a peptide from mGluR (TPPSPF). In contrast to other EVH1 domains, the bound mGluR ligand assumes an unusual conformation in which the side chains of the Ser-Pro tandem are oriented away from the Homer surface, and the Phe forms a unique contact. This unusual binding mode rationalizes conserved features of both Homer and Homer ligands that are not shared by other EVH1 domains. Site-directed mutagenesis confirms the importance of specific Homer residues for ligand binding. These results establish a molecular basis for understanding the biological properties of Homer-ligand complexes.     

Involvement of ERK in BMP-2 induced osteoblastic differentiation of mesenchymal progenitor cell line C3H10T1/2

The signaling mechanisms responsible for bone morphogenetic protein (BMP) induced osteoblast differentiation remains poorly understood. Previous research demonstrated that Smad proteins are the substrates and the mediators of BMP bound serine/threonine receptor kinase. In the present study, we examined the possible involvement of extracellular signal-regulated kinase (Erk) in the BMP induced osteoblast differentiation of mesenchymal progenitor cell C3H10T1/2. Our results indicate that BMP-2 inducement increased MAP kinase activity in mesenchymal progenitor cell line C3H10T1/2. Contrary to previous reports, this increased MAP kinase activity showed a latent but sustained pattern. Elevation of Erk1 and Erk2 protein levels was observed simultaneously. RT-PCR results demonstrated that the elevation of Erk protein level in BMP-2 induced cells was from the upregulation of mRNA expression. Furthermore, upregulated Erk proteins present enhanced phosphorylation. By using a dominant-negative Erk2 cell line, we demonstrated that nonfunctional Erk2 partially eliminated BMP-2 induced cell proliferation and ALP activity in the C3H10T1/2 cell. These results indicate that Erk is involved in BMP-2 induced osteoblast differentiation. The results also demonstrate that a latent and sustained signaling pattern exists in BMP induced signaling cascade.     

Action mechanism of antitubercular isoniazid. Activation by Mycobacterium tuberculosis KatG, isolation, and characterization of inha inhibitor

Activation of the antitubercular isoniazid (INH) by the Mycobacterium tuberculosis KatG produces an inhibitor for enoyl reductase (InhA). The mechanism for INH activation remains poorly understood, and the inhibitor has never been isolated. We have purified the InhA-inhibitor complex generated in the M. tuberculosis KatG-catalyzed INH activation. The complex exhibited a 278-nm absorption peak and a shoulder around 326 nm with a characteristic A(326)/A(278) ratio of 0.16. The complex was devoid of enoyl reductase activity. The inhibitor noncovalently binds to InhA with a K(d) < 0.4 nM and can be dissociated from denatured InhA for chromatographic isolation. The free inhibitor showed absorption peaks at 326 (epsilon(326) 6900 M(-1) cm(-1)) and 260 nm (epsilon(260) 27,000 M(-1) cm(-1)). The inactive complex can be reconstituted from InhA and the isolated inhibitor. The InhA inhibitor from the KatG-catalyzed INH activation was identical to that from a slow, KatG-independent, Mn(2+)-mediated reaction based on high pressure liquid chromatography analysis and absorption and mass spectral characteristics. By monitoring the formation of the InhA-inhibitor complex, we have found that manganese is not essential to the INH activation by M. tuberculosis KatG. Furthermore, the formation of the InhA inhibitor in the KatG reaction was independent of InhA.