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(1/365) Suramin and suramin analogs activate skeletal muscle ryanodine receptor via a calmodulin binding site.

Contraction of skeletal muscle is triggered by the rapid release of Ca2+ from the sarcoplasmic reticulum via the ryanodine receptor/calcium-release channel. The trypanocidal drug suramin is an efficient activator of the ryanodine receptor. Here, we used high-affinity [3H]ryanodine binding to sarcoplasmic reticulum from rabbit skeletal muscle to screen for more potent analogs of suramin. This approach resulted in the identification of NF307, which accelerates the association rate of [3H]ryanodine binding with an EC50 = 91 +/- 7 microM at 0.19 microM calculated free Ca2+. In single-channel recordings with the purified ryanodine receptor, NF307 increased mean open probability at 0.6 microM Ca2+ from 0.020 +/- 0.006 to 0.53 +/- 0.07 with no effect on current amplitude and unitary conductance. Like caffeine, NF307 exerts a very pronounced Ca2+-sensitizing effect (EC50 of Ca2+ shifted approximately 10-fold by saturating NF307 concentrations). Conversely, increasing concentrations of free Ca2+ sensitized the receptor for NF307 (EC50 = 14.6 +/- 3.5 microM at 0.82 microM estimated free Ca2+). The effects of NF307 and caffeine on [3H]ryanodine binding were additive, irrespective of the Ca2+ concentration. In contrast, the effects of calmodulin, which activates and inhibits the ryanodine receptor in the absence and presence of Ca2+, respectively, and of NF307 were mutually antagonistic. If the purified ryanodine receptor was prebound to a calmodulin-Sepharose matrix, 100 microM NF307 and 300 microM suramin eluted the purified ryanodine receptor to an extent that was comparable to the effect of 10 microM calmodulin. We conclude that NF307 and suramin interact directly with a calmodulin binding domain of the ryanodine receptor. Because of its potent calcium-sensitizing effect, NF307 may represent a lead compound in the search of synthetic ryanodine receptor ligands.  (+info)

(2/365) Description of Pseudaminobacter gen. nov. with two new species, Pseudaminobacter salicylatoxidans sp. nov. and Pseudaminobacter defluvii sp. nov.

An aerobic bacterium, strain BN12T, which degrades substituted naphthalenesulfonates and substituted salicylates was isolated from a 6-aminonaphthalene-2-sulfonate-degrading microbial consortium originating from the River Elbe, Germany. Chemotaxonomic investigations of quinones, polyamines and polar lipids allowed allocation of this strain to the alpha-subclass of the Proteobacteria and revealed similarity to species of the genera Aminobacter, Chelatobacter and Mesorhizobium. This was confirmed by typing with 16S rRNA-targeted oligonucleotide probes and 16S rDNA sequencing and phylogenetic analysis, indicating that BN12T clusters most closely with a strain 'Thiobacillus' THI 051T and with the above genera but comprising a separate branch. DNA-DNA hybridizations demonstrated that strain BN12T is different from all species of Aminobacter currently described and recognized. The fatty acid patterns, substrate utilization profile and biochemical characteristics displayed no obvious similarity to the characteristics of Aminobacter and Chelatobacter species. 'Thiobacillus' THI 051T, however, revealed phenotypic similarities to BN12T. Furthermore, 16S rRNA sequences of Chelatobacter heintzii showed a high similarity to the 16S rRNA sequences of all currently recognized Aminobacter species. On the basis of these and previously published results, the new genus Pseudaminobacter is proposed, harbouring the two new species Pseudaminobacter salicylatoxidans sp. nov. and Pseudaminobacter defluvii sp. nov. The type strains are BN12T (= DSM 6986T) and THI 051T (= IFO 14570T), respectively.  (+info)

(3/365) Synthesis and characterization of stacked and quenched uridine nucleotide fluorophores.

Intramolecular aromatic interactions in aqueous solution often lead to stacked conformation for model organic molecules. This designing principle was used to develop stacked and folded uridine nucleotide analogs that showed highly quenched fluoroscence in aqueous solution by attaching the fluorophore 1-aminonaphthalene-5-sulfonate (AmNS) to the terminal phosphate via a phosphoramidate bond. Severalfold enhancement of fluorescence could be observed by destacking the molecules in organic solvents, such as isopropanol and dimethylsulfoxide or by enzymatic cleavage of the pyrophosphate bond. Stacking and destacking were confirmed by 1-H NMR spectroscopy. The extent of quenching of the uridine derivatives correlated very well with the extent of stacking. Taking 5-H as the monitor, temperature-variable NMR studies demonstrated the presence of a rapid interconversionary equilibrium between the stacked and open forms for uridine-5'-diphosphoro-beta-1-(5-sulfonic acid) naphthylamidate (UDPAmNS) in aqueous solution. DeltaH was calculated to be -2.3 Kcal/mol, with 43-50% of the population in stacked conformation. Fluorescence lifetime for UDPAmNS in water was determined to be 2.5 ns as against 11 ns in dimethyl sulfoxide or 15 ns for the pyrophosphate adduct of AmNS in water. Such a greatly reduced lifetime for UDPAmNS in water suggests collisional interaction between the pyrimidine and thefluorophore moieties to be responsible for quenching. The potential usefulness of such stacked and quenched nucleotide fluorophores as probes for protein-ligand interaction studies has been briefly discussed.  (+info)

(4/365) An arginine residue is essential for stretching and binding of the substrate on UDP-glucose-4-epimerase from Escherichia coli. Use of a stacked and quenched uridine nucleotide fluorophore as probe.

In the previous paper we demonstrated that uridine-5'-beta-1-(5-sulfonic acid) naphthylamidate (UDPAmNS) is a stacked and quenched fluorophore that shows severalfold enhancement of fluorescence in a stretched conformation. UDPAmNS was found to be a powerful competitive inhibitor (Ki = 0.2 mM) for UDP-glucose-4-epimerase from Escherichia coli. This active site-directed fluorophore assumed a stretched conformation on the enzyme surface, as was evidenced by full enhancement of fluorescence in saturating enzyme concentration. Complete displacement of the fluorophore by UDP suggested it to bind to the substrate binding site of the active site. Analysis of inactivation kinetics in presence of alpha,beta-diones such as phenylglyoxal, cyclohaxanedione, and 2,3-butadione suggested involvement of the essential arginine residue in the overall catalytic process. From spectral analysis, loss of activity could also be directly correlated with modification of only one arginine residue. Protection experiments with UDP showed the arginine residue to be located in the uridyl phosphate binding subsite. Unlike the native enzyme, the modified enzyme failed to show any enhancement of fluorescence with UDPAmNS clearly demonstrating the role of the essential arginine residue in stretching and binding of the substrate. The potential usefulness of such stacked and quenched nucleotide fluorophores has been discussed.  (+info)

(5/365) Naturally occurring CCR5 extracellular and transmembrane domain variants affect HIV-1 Co-receptor and ligand binding function.

Analysis of CCR5 variants in human immunodeficiency virus, type 1 (HIV-1), high risk cohorts led to the identification of multiple single amino acid substitutions in the amino-terminal third of the HIV-1 co-receptor CCR5 suggesting the possibility of protective and permissive genotypes; unfortunately, the low frequency of these mutations did not led to correlation with function. Therefore, we used analytical methods to assess the functional and structural significance of six of these variant receptors in vitro. These studies showed three categories of effects on CCR5 function. 1) Mutations in the first extracellular domain of CCR5 severely reduce specific ligand binding and chemokine-induced chemotaxis. 2) An extracellular domain variant, A29S, when co-expressed with CD4, supported HIV-1 infection whereas the others do not. 3) The transmembrane region variants of CCR5 support monotropic HIV-1 infection that is blocked by addition of some receptor agonists. Mutations in the first and second transmembrane domains increase RANTES (regulated on activation normal T-cell expressed) binding affinity but did not affect MIP1beta binding affinity presumably based on differences in ligand-receptor interaction sites. Furthermore, the CCR5 transmembrane mutants do not respond to RANTES with the classical bell-shaped chemotactic response curve, suggesting that they are resistant to RANTES-induced desensitization. These data demonstrate that single amino acid changes in the extracellular domains of CCR5 can have profound effects on both HIV-1 co-receptor and specific ligand-induced functions, whereas mutations in the transmembrane domain only affect the response to chemokine ligands.  (+info)

(6/365) The topical microbicide PRO 2000 protects against genital herpes infection in a mouse model.

Vaginal gel formulations containing the naphthalene sulfonate polymer PRO 2000 are being developed as topical microbicides to protect against infection with sexually transmitted disease (STD) pathogens. A mouse model was used to determine whether PRO 2000 could protect against genital herpes in vivo. Animals received a single intravaginal application of 15 microL of a 10% PRO 2000 aqueous solution or a 4.0% or 0.5% PRO 2000 vaginal gel formulation 20 s prior to intravaginal challenge with 4.0 log10 pfu of herpes simplex virus type 2. Treatment with the 4.0% gel provided complete protection against infection; treatment with the 0.5% gel or 10% solution provided 81% and 80% protection, respectively. Furthermore, the 4% gel provided significant protection even when viral challenge was delayed until 60 min after treatment. This is the first report to show that PRO 2000 can protect against infection with an STD pathogen in vivo.  (+info)

(7/365) Colicin E1 forms a dimer after urea-induced unfolding.

Unfolding of the soluble colicin E1 channel peptide was examined with the use of urea as a denaturant; it was shown that it unfolds to an intermediate state in 8.5 M urea, equivalent to a dimeric species previously observed in 4 M guanidinium chloride. Single tryptophan residues, substituted into the peptide at various positions by site-directed mutagenesis, were employed as fluorescent probes of local unfolding. Unfolding profiles for specific sites within the peptide were obtained by quantifying the shifts in the fluorescence emission maxima of single tryptophan residues on unfolding and plotting them against urea concentration. Unfolding reported by tryptophan residues in the C-terminal region was not characteristic of complete peptide denaturation, as evidenced by the relatively blue-shifted values of the fluorescence emission maxima. Unfolding was also monitored by using CD spectroscopy and the fluorescent probe 2-(p-toluidinyl)-naphthalene 6-sulphonic acid; the results indicated that unfolding of helices is concomitant with the exposure of protein non-polar surface. Unfolding profiles were evaluated by non-linear least-squares curve fitting and calculation of the unfolding transition midpoint. The unfolding profiles of residues located in the N-terminal region of the peptide had lower transition midpoints than residues in the C-terminal portion. The results of unfolding analysis demonstrated that urea unfolds the peptide only partly to an intermediate state, because the C-terminal portion of the channel peptide retained significant structure in 8.5 M urea. Characterization of the peptide's global unfolding by size-exclusion HPLC revealed that the partly denatured structure that persists in 8.5 M urea is a dimer of two channel peptides, tightly associated by hydrophobic interactions. The presence of the dimerized species was confirmed by SDS/PAGE and intermolecular fluorescence resonance energy transfer.  (+info)

(8/365) Conformations of vertebrate striated muscle myosin monomers in equilibrium with filaments.

Porcine cardiac myosin monomers in equilibrium with filaments under physiological conditions were observed to have two conformations, extended and folded forms, upon electron microscopy and gel filtration HPLC. The conformational state was independent of ATP and the phosphorylation of regulatory light chain. The folded monomers of cardiac myosin were mainly in an open conformation with only one bend in the tail, and may not trap the hydrolysis products of ATP, as assessed by single turnover experiments. These properties are similar to those of the folded monomers of rabbit skeletal myosin [Katoh, T., Konishi, K., and Yazawa, M. (1998) J. Biol. Chem. 273, 11436-11439]. The conformational states of skeletal and cardiac myosin monomers were not affected by pH between 7.0 and 8.5. Although significant disassembly of filaments and thus an increase in the monomer concentration were observed with an increase in pH. The results indicate that the pH-dependent change in filament assembly is due to a shift of equilibrium between the filaments and extended monomers toward filament disassembly. The Mg2+-ATPase activity of these myosin monomers decreased with a decrease in the salt concentration below approximately 0.1 M, suggestive of the formation of a closed conformation similar to the conformation of 10S smooth myosin. The results suggest that the conformational change from the extended to the folded form is a common property of various myosin IIs.  (+info)