M-S vibrational study in three-coordinate thiolato compounds (NEt4)2[M(SC6H4-p-X)3] and (NEt4)

By using p-substituted benzenethiolate ligands, the novel three-coordinate copper(I) and silver(I) thiolato complexes (NEt4)2[Cu(SC6H4-p-X)3] (X=Cl (1) and Br (2)), (NEt4)2[Ag(SC6H4-p-X)3] (X=Cl (3) and Br (4)) and novel clusters (NEt4)2[M4(mu-SC6H4-p-Cl)6] (M=Cu (5) and Ag(6)) have been prepared and structurally characterized by single crystal X-ray diffraction. All the complexes have three-coordinate sites having point-group D3h symmetry. The three-coordinate mononuclear silver(I) complexes 3 and 4 are the first examples. The M-S stretching bands were determined by far-IR and FT-Raman spectroscopies; nu(Cu-S) 363-372 cm(-1) and nu(Ag-S) 353-363 cm(-1). These results indicate that M-S stretching vibration energy in the three-coordinate metal(I) site of the mononuclear compounds or clusters is around 340-380 cm(-1), and it is a useful tool for determining their coordination modes.     

Investigations of the {ReO} core: A '2+2' complex from bidentate and potentially trident ligands: [ReO(η-HOC(6)H(4)-2-CH(2)NC(6)H(4)S)(η-SC(5)H(4)N)(PPh(3))

The reaction of [ReOCl(3)(PPh(3))(2)] with N-(2-hydroxybenzyl)-2-mercaptoaniline (H(3)hbma) (2) and 2-mercaptopyridine in hot CHCl yields [ReO(η(2)-HOC(6)H(4)-2-CH(2)NC(6)H(4)S)(η(2)-SC(5)H(4)N)(PPh(3))] (3). The structure of 3 consists of distorted octahedral Re(V) monomers. The coordination geometry at the rhenium is defined by a terminal oxo-group, the nitrogen and sulfur donors of the chelating mercaptopyridine, the nitrogen and sulfur donors of a bidentate (Hhbma)(2-) ligand, and the phosphorus of the PPh(3) group. The -C(6)H(4)OH arm of (Hhbma)(2-) is pendant, and the coordinated nitrogen of this ligand is present as a deprotonated amido nitrogen.     

(3)H](2S,4R)-4-Methylglutamate: a novel ligand for the characterization of glutamate transporters

[(3)H](2S,4R)-4-Methylglutamate ([(3)H]4MG), used previously as a ligand for low-affinity kainate receptors, was employed to establish a binding assay for glutamate transporters (GluTs), as 4MG has also been shown to have affinity for the glial GluTs, GLT1 and GLAST. In rat brain membrane homogenates in the presence of Na(+) ions at 4 degrees C, specific binding of [(3)H]4MG was rapid and saturable (t(1/2) approximately 15 min), representing > 90% of total binding. Dissociation of [(3)H]4MG occurred in a biphasic manner, however, saturation studies and Scatchard analysis indicated a single site of binding (n(H) = 0.85) and a K(d) of 6.2 +/- 0.8 microM with a B(max) of 111.8 +/- 23.8 pmol/mg protein. Specific binding of [(3)H]4MG was Na(+)-dependent and inhibited by K(+) and HCO(3-). Pharmacological inhibition with compounds acting at GluTs revealed that Glu, D- and L-aspartate, L-serine-O-sulfate and Ltrans-pyrrolidine-2,4-dicarboxylate fully displaced specific binding. Drugs having preferential affinity for GLT1, kainate, dihydrokainate and Lthreo-3-methylglutamate, all inhibited approximately 40% of specific binding. The inhibition pattern of L-serine-O-sulfate in the presence of a saturating concentration of dihydrokainate was suggestive of [(3)H]4MG also labelling GLAST. 6-Cyano-7-nitroquinoxaline, a kainate receptor antagonist, and a range of Glu receptor agonists and antagonists failed to significantly inhibit [(3)H]4MG binding. The pharmacological profile of binding of [(3)H]4MG resembled that found for [(3)H]D-aspartate, a ligand specific for GluTs, reinforcing the hypothesis that [(3)H]4MG was labelling GluTs in this assay. Together, these data illustrate the development of an efficient, economic binding assay that is suitable for the characterization of different subtypes of GLuTs.     

The structure of sub-nucleosomal particles. The octameric (H3/H4)4--125-base-pair-DNA complex

Chicken erythrocyte chromatin was depleted of histones H1, H5, H2A and H2B. The resulting (H3/H4)-containing chromatin was digested with micrococcal nuclease to yield monomer, dimer, trimer etc. units, irregularly spaced on the DNA, with even-number multimers being more prominent. Sucrose density gradient centrifugation separated monomers and dimers (7.7 S and 10.5 S). Sodium dodecyl sulphate gel electrophoresis and cross-linking indicated: the monomer contains 50-base-pair (bp), 60-bp and 70-bp DNA and the dimer 125-bp DNA; the monomer contains a tetramer and the dimer an octamer of H3 and H4. Partial association of monomer units to dimers inhibits structural studies of monomers. The internal structure of the dimer, i.e. and (H3/H4)4-125-bp-DNA particle, was studied using circular dichroism, thermal denaturation and nuclease digestion. Both micrococcal nuclease and DNase I digestion indicate that, unlike core particles, accessible sites occur in the centre of the particle and it is concluded that the (H3/H4)4-125-bp-DNA particle is not a 'pseudo-core particle' in which the 'extra' H3 and H4 replace H2A and H2B. It is proposed that the octamer particle is formed by the sliding together of two 'monomer' units, each containing the (H3/H4)2 tetramer and 70 bp of DNA. Excision of this dimer unit with micrococcal nuclease results in the loss of 10 readily digestible base pairs at each end, leaving 125 bp.     

Voltage sensitivities and deactivation kinetics of histamine H(3) and H(4) receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by inhibitory autoreceptors. Likewise, receptor deactivation rates upon agonist removal have been implicated in autoreceptor function. Using G protein-coupled potassium (GIRK) channel activation in Xenopus oocytes as readout of receptor activity, we have investigated the voltage sensitivities and signaling kinetics of the hH(3)(445) and hH(3)(365) isoforms of the human histamine H(3) receptor, which functions as an inhibitory auto- and heteroreceptor in the nervous system. We have also investigated both the human and the mouse homologues of the related histamine H(4) receptor, which is expressed mainly on hematopoietic cells. We found that the hH(3)(445) receptor is the most sensitive to voltage, whereas the hH(3)(365) and H(4) receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH(3)(445) and hH(3)(365) isoforms, with the hH(3)(365) isoform being five to six-fold slower than the hH(3)(445) receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH(4) receptor. The differences in voltage sensitivities and deactivation kinetics between the hH(3)(445), hH(3)(365), and H(4) receptors might be relevant to their respective physiological roles.     

Binding of 4(5)-[2-(4-azido-2-nitroanilino)ethyl]imidazole to histamine receptors in guinea pig cerebral cortical membranes

The interaction of 4(5)-[2-(4-azido-2-nitroanilino)ethyl]imidazole (AAH), a photolabile histamine receptor antagonist, with the binding of histamine, mepyramine, and tiotidine to guinea pig cerebral cortical membranes was examined to evaluate the specificity of AAH for histamine H1 and H2 receptors. Saturable, specific binding of [3H]histamine, [3H]mepyramine, and [3H]tiotidine to the membranes was observed. Competition assays were used to assess the relative affinity of AAH for H1- and H2-receptors. The rank order of IC50 values obtained was (most to least potent) (i) for competing with [3H]histamine binding: histamine greater than AAH much greater than mepyramine approximately equal to tiotidine; (ii) for competing with [3H]mepyramine binding: mepyramine much greater than AAH greater than histamine greater than tiotidine; and (III) for competing with [3H]tiotidine binding: tiotidine much greater than mepyramine greater than histamine approximately equal to AAH. The affinity of AAH for H1 receptors was ca. 14-fold greater than for H2 receptors. These findings support previous evidence obtained in isolated smooth muscle preparations that AAH shows H1-receptor selectivity as an antagonist.     

Cross-linking of T3 (CD3) with T4 (CD4) enhances the proliferation of resting T lymphocytes

Monoclonal antibodies reactive with defined T lymphocyte surface antigens were covalently coupled to protein A-Sepharose beads using the bifunctional imidoester, dimethyl pimelimidate. Sepharose-immobilized antibody reactive with T3 induced the proliferation of resting T lymphocytes in the presence of either recombinant interleukin 2 or phorbol myristate acetate. When monoclonal antibodies reactive with T3 and T4 were coupled to the same Sepharose bead (hereafter designated Sepharose (T3:T4)), proliferation was enhanced an average of three-fold. Similarly prepared Sepharose beads coupled to anti-T3 and anti-T8 also enhanced proliferation over that observed with anti-T3 alone. Sepharose (T3:T4) similarly increased the proliferation of T4+ lymphocytes and a T4+ clone but failed to enhance the proliferation of T8+ lymphocytes. The increased proliferation of T4+ lymphocytes resulted from a preferential activation of the T4+2H4- helper population over the T4+2H4+ suppressor-inducer population. The enhanced proliferation induced by Sepharose (T3:T4) could be completely inhibited by soluble anti-T4. These results suggest that perturbation of T3 may be a minimal signal for T cell activation and that the assembly of a multimeric complex including T3 and T4 may be required for optimal T cell activation.     

Synthesis, characterisation and antimicrobial activity of copper(II) and manganese(II) complexes of coumarin-6,7-dioxyacetic acid (cdoaH2) and 4-methylcoumarin-6,7-dioxyacetic acid (4-MecdoaH2): X-ray crystal structures of [Cu(cdoa)(phen)2].8.8H(2)O and [Cu(4-Mecdoa)(phen)2].13H2O (phen=1,10-phenanthroline)

Two novel coumarin-based ligands, coumarin-6,7-dioxyacetic acid (1) (cdoaH(2)) and 4-methylcoumarin-6,7-dioxyacetic acid (2) (4-MecdoaH(2)), were reacted with copper(II) and manganese(II) salts to give [Cu(cdoa)(H(2)O)(2)].1.5H(2)O (3), [Cu(4-Mecdoa)(H(2)O)(2)] (4), [Mn(cdoa)(H(2)O)(2)] (5) and [Mn(4-Mecdoa)(H(2)O)(2)].0.5H(2)O (6). The metal complexes, 3-6, were characterised by elemental analysis, IR and UV-Vis spectroscopy, and magnetic susceptibility measurements and were assigned a polymeric structure. 1 and 2 react with Cu(II) in the presence of excess 1,10-phenanthroline (phen) giving [Cu(cdoa)(phen)(2)].8.8H(2)O (7) and [Cu(4-Mecdoa)(phen)(2)].13H(2)O (8), respectively. The X-ray crystal structures of 7 and 8 confirmed trigonal bipyramidal geometries, with the metals bonded to the four nitrogen atoms of the two chelating phen molecules and to a single carboxylate oxygen of the dicarboxylate ligand. The complexes were screened for their antimicrobial activity against a number of microbial species, including methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Candida albicans. The metal-free ligands 1 and 2 were active against all of the microbes. Complexes 3-6 demonstrated no significant activity whilst the phen adducts 7 and 8 were active against MRSA (MIC(80)=12.1microM), E. coli (MIC(80)=14.9microM) and Patonea agglumerans (MIC(80)=12.6microM). Complex 7 also demonstrated anti-Candida activity (MIC(80)=22microM) comparable to that of the commercially available antifungal agent ketoconazole (MIC(80)=25microM).     

Yeast CAF-1 assembles histone (H3-H4)2 tetramers prior to DNA deposition

Following acetylation, newly synthesized H3-H4 is directly transferred from the histone chaperone anti-silencing factor 1 (Asf1) to chromatin assembly factor 1 (CAF-1), another histone chaperone that is critical for the deposition of H3-H4 onto replicating DNA. However, it is unknown how CAF-1 binds and delivers H3-H4 to the DNA. Here, we show that CAF-1 binds recombinant H3-H4 with 10- to 20-fold higher affinity than H2A-H2B in vitro, and H3K56Ac increases the binding affinity of CAF-1 toward H3-H4 2-fold. These results provide a quantitative thermodynamic explanation for the specific H3-H4 histone chaperone activity of CAF-1. Surprisingly, H3-H4 exists as a dimer rather than as a canonical tetramer at mid-to-low nanomolar concentrations. A single CAF-1 molecule binds a cross-linked (H3-H4)₂ tetramer, or two H3-H4 dimers that contain mutations at the (H3-H4)₂ tetramerization interface. These results suggest that CAF-1 binds to two H3-H4 dimers in a manner that promotes formation of a (H3-H4)₂ tetramer. Consistent with this idea, we confirm that CAF-1 synchronously binds two H3-H4 dimers derived from two different histone genes in vivo. Together, the data illustrate a clear mechanism for CAF-1-associated H3-H4 chaperone activity in the context of de novo nucleosome (re)assembly following DNA replication.     

Characterization of the self-assembly of meso-tetra(4-sulfonatophenyl)porphyrin (H(2)TPPS(4-)) in aqueous solutions

The aggregation of meso-tetra(4-sulfonatophenyl)porphyrin (H(2)TPPS(4-)) in phosphate solutions was investigated as a function of pH, concentration, time, ionic strength, and solution preparation (either from dilution of a freshly prepared 2 mM stock or by direct preparation of μM solution concentrations) using a combination of complementary analytical techniques. UV-vis and fluorescence spectroscopy indicated the formation of staggered, side-by-side (J-type) assemblies. Their size and self-associative behavior were determined using analytical ultracentrifugation and small-angle X-ray scattering. Our results indicate that in neutral and basic solutions of H(2)TPPS(4-), porphyrin dimers and trimers are formed at micromolar concentrations and in the absence of NaCl to screen any ionic interactions. At these low concentrations and pH 4, the protonated H(4)TPPS(2-) species self-assembles, leading to the formation of particularly stable aggregates bearing 25 ± 3 macrocycles. At higher concentrations, these structures further organize or reorganize into tubular, rod-like shapes of various lengths, which were imaged by cryogenic and freeze-fracture transmission electron microscopy. Micron-scale fibrillar aggregates were obtained even at micromolar concentrations at pH 4 when prepared from dilution of a 2 mM stock solution, upon addition of NaCl, or both.     

Comparison of [(3)H]-(2S,4R)-4-methylglutamate and [(3)H]D-aspartate as ligands for binding and autoradiographic analyses of glutamate transporters

While studies with [(3)H]D-aspartate ([(3)H]d-Asp) illustrate specific interactions with excitatory amino acid transporters (EAATs), new insights into the pharmacological characteristics and localization of specific EAAT subtypes depend upon the availability of novel ligands. One such ligand is [(3)H]-(2S,4R)-4-methylglutamate ([(3)H]4MG) which labels astrocytic EAATs in homogenate binding studies. This study examined the utility of [(3)H]4MG for binding and autoradiography in coronal sections of rat brain. Binding of [(3)H]4MG was optimal in 5mM HEPES buffer containing 96 mM NaCl, pH 7.5. Specific binding of [(3)H]4MG exhibited two components, but was to a single site when glutamate receptor (GluR) sites were masked with kainate (KA; 1 microM): t(1/2) approximately 5 min, K(d) 250 nM and B(max) 5.4 pmol/mg protein. Pharmacological studies revealed that [(3)H]4MG, unlike [(3)H]d-Asp, labeled both EAAT and ionotropic GluR sites. Further studies employed 6-cyano-7-nitroquinoxaline (30 microM) to block GluR sites, but selective EAAT ligands displayed lower potency than expected for binding to transporters relative to drugs possessing mixed transporter/receptor activities. Autoradiography in conjunction with densitometry with [(3)H]4MG and [(3)H]d-Asp revealed wide, but discrete distributions in forebrain; significant differences in binding levels were found in hippocampus, nucleus accumbens and cortical sub-areas. Although EAAT1 and EAAT2 components were detectable using 3-methylglutamate and serine-O-sulphate, respectively, the majority of [(3)H]4MG binding was to KA-related sites. Overall, in tissue sections [(3)H]4MG proved unsuitable for studying the autoradiographic localization of EAATs apparently due to its inability to selectively discriminate Na(+)-dependent binding to Glu transporters.     

Synthesis of (LysA13)bovine insulin A chain analogs as (Lys(Tfa)A13)A(SO3H)4 and Nalpha-A1-Msc-(LysA13)A(SO3H)4 derivatives using the S-tert-butylmercapto residue for thiol protection (author's transl)

The following paper describes the synthesis of the [LysA13]bovine insulin A chain analog as [Lys(Tfa)A13]A(SO3H)4 and NalphaA1-Msc-[LysA13]A(SO3H)4 derivatives using the S-tert-butylmercapto residue for thiol protection. Although the intermediate S-tert-butylmercaptocysteinyl-peptide derivatives showed a good solubility in organic solvents the resulting fully protected A chain derivatives had a poor solubility in organic solvents and therefore were deblocked converted into the tetra(S-sulfonic acid) derivatives and purified via ionexchange chromatography.     

In vitro gibberellin a(4) binding to extracts of cucumber hypocotyls

Cucumber hypocotyls were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [(3)H]-gibberellin(4) (GA(4)) to soluble macromolecular components present in the cytosol was demonstrated at 0 C by Sephadex chromatography. Binding assays performed with cytosol that had been preheated or incubated with protease, DNase, RNase, or phospholipase A or C indicated that heat and protease treatments disrupted the binding, which suggests that binding occurred to a protein. Equilibrium dialysis of a protein-enriched fraction prepared by ammonium sulfate precipitation also indicated binding of [(3)H]GA(4) to macromolecular components. [(3)H]GA(4) binding was pH-sensitive, saturable, reversible, and significantly affected by biologically active gibberellins, but not by inactive gibberellins or other plant hormones such as indoleacetic acid, abscisic acid, or kinetin. Thin layer chromatography indicated that [(3)H]GA(4), and not a metabolite, was the species bound. A kinetic analysis indicated that specific binding of [(3)H]GA(4) was due to a single class of binding sites having an estimated K(d) of 10(-7) molar and a concentration of 0.8 x 10(-12) moles gram(-1) fresh weight or 0.4 x 10(-12) moles milligram(-1) soluble protein.     

Density functional studies on the endohedral complex of fullerene C70 with tetrahedrane (C4H4): C4H4@C70

B3LYP/6-31G(d) hybrid HF/DFT calculations were carried out to determine the structural and electronic properties of the endohedral complex of a C(70) cage with tetrahedrane (C(4)H(4)). It was demonstrated that the formation of the complex is endothermic, with a destabilization energy of 72.56 kcal mol(-1). C(4)H(4) is seated in the center of the C(70) cage and exists in molecular form inside the fullerene. C(4)H(4) endohedral doping slightly perturbs the molecular orbitals of C(70). The calculated HOMO-LUMO gaps, the electron affinity (EA), and the ionization potential (IP) indicate that C(4)H(4)@C(70) is more chemically reactive than C(70). The IR active modes and harmonic vibrational frequencies of C(4)H(4)@ C(70) are also discussed.     

Introduction of the 4-(4-bromophenyl)benzenesulfonyl group to hydrazide analogs of Ilomastat leads to potent gelatinase B (MMP-9) inhibitors with improved selectivity

Hydrazide derivatives of Ilomastat, carrying either aryl groups or distinct alkyl and arylsulfonyl moieties were synthesized and evaluated for their MMP inhibitory activity. Potent and selective MMP-9 inhibition (IC(50)=3 nM) was observed for compound 3m (arylsulfonyl group: 4-(4-Br-C6H4)-C6H4-SO(2)-). Interaction with the S2 enzyme subsite is mainly responsible for the inhibitory properties of this derivative as confirmed by molecular docking computation.     

Synthesis and characterization of bis(mu-hydroxo)diiron(III) complex of N-(4-nitro-2-hydroxy)phenylmethyl-N-(2-pyridylethyl)-N-(2-pyridylmethyl)amine and hydroxylation reaction of alkane

Bis(mu-hydroxo)diiron(III) complex, [Fe2III(mu-OH)2(NE)2](NO3)2 x 3H2O (3) (N-(4-nitro-2-hydroxy)phenylmethyl-N-(2-pyridylethyl)-N-(2-pyridylmethyl)amine = HNE, where H denotes a dissociable proton of the p-nitrophenol group), has been prepared and characterized by X-ray crystallography, electronic and magnetic spectroscopies.     

Structural signatures of the complex formed between 3-nitro-4-hydroxybenzoate and the Zn(II)-substituted R(6) insulin hexamer

3-Nitro-4-hydroxybenzoate (3N4H) is a probe of the structure and dynamics of the metal-centered His B10 assembly sites of the insulin hexamer. Each His B10 site consists of a approximately 12 A-long cavity situated on the threefold symmetry axis. These sites play an important role in the storage and release of insulin in vivo. The allosteric behavior of the insulin hexamer is modulated by ligand binding to the His B10 zinc sites and to the phenolic pockets. Binding to these sites drives transitions among three allosteric states, designated T(6), T(3)R(3), and R(6). Although a wide variety of mono anions bind to the His B10 zinc sites of R(3), X-ray structures of ligands complexed to this site exist only for H(2)O, Cl(-), and SCN(-). This work combines one- and two-dimensional (1)H NMR and UV-Vis absorbance studies of the structure and dynamics of the 3N4H complex, which establish the following: (1). relative to the NMR time scale, 3N4H exchange between free and bound states is slow, while flipping among three equivalent orientations about the site threefold axis is fast; (2). binding of 3N4H perturbs resonances within the His B10 zinc site and generates NOEs between ligand resonances and the insulin C-alpha and side chain resonances of ValB2, AsnB3, LeuB6, and CysB7; and (3).3N4H exchange for other ligands is limited by a protein conformational transition. These results are consistent with coordination of the 3N4H carboxylate to the His B10 zinc ion and van der Waals interactions with Val B2, Asn B3, Leu B6, and Cys A7.