Production and properties of adhesin-free gingipain proteinase RgpA.

The Arg-specific gingipains of Porphyromonas gingivalis RgpA and RgpB have 97% identical sequences in their catalytic domains yet their propeptides are only 76% identical. RgpA isolates as a proteinase-adhesin complex (HRgpA) which hinders direct kinetic comparison of RgpAcat as a monomer with monomeric RgpB. We tested modifications of rgpA identifying a variant that enabled us to isolate histidine-tagged monomeric RgpA (rRgpAH). Kinetic comparisons between rRgpAH and RgpB used benzoyl-L-Arg-4-nitroanilide with and without cysteine and glycylglycine acceptor molecules. With no glycylglycine, values of Km, Vmax, kcat and kcat/Km for each enzyme were similar, but with glycylglycine Km decreased, Vmax increased and kcat increased ~ twofold for RgpB but ~ sixfold for rRgpAH. The kcat/Km for rRgpAH was unchanged whereas that of RgpB more than halved. Recombinant RgpA propeptide inhibited rRgpAH and RgpB with Ki 13 nM and 15 nM Ki respectively slightly more effectively than RgpB propeptide which inhibited rRgpAH and RgpB with Ki 22 nM and 29 nM respectively (p < 0.0001); a result that may be attributable to the divergent propeptide sequences. Overall, the data for rRgpAH reflected observations previously made by others using HRgpA, indicating rRgpAH fidelity and confirming the first production and isolation of functional affinity tagged RgpA.

Multi-omics analyses reveal new insights into nutritional quality changes of alfalfa leaves during the flowering period.

High-quality alfalfa is an indispensable resource for animal husbandry and sustainable development. Its nutritional quality changes dramatically during its life cycle and, at present, no molecular mechanisms for nutrient metabolic variation in alfalfa leaves at different growth stages have been clearly reported. We have used correlation and network analyses of the alfalfa leaf metabolome, proteome, and transcriptome to explore chlorophyll, flavonoid, and amino acid content at two development stages: budding stage (BS) and full-bloom stage (FBS). A high correlation between the expression of biosynthetic genes and their metabolites revealed significant reductions in metabolite content as the plant matured from BS to FBS. l-Glutamate, the first molecule of chlorophyll biosynthesis, decreased, and the expression of HemA, which controls the transformation of glutamyl-tRNA to glutamate 1-semialdehyde, was down-regulated, leading to a reduction in leaf chlorophyll content. Flavonoids also decreased, driven at least in part by increased expression of the gene encoding CYP75B1: flavonoid 3'-monooxygenase, which catalyzes the hydroxylation of dihydroflavonols and flavonols, resulting in degradation of flavonoids. Expression of NITRILASE 2 (NIT2) and Methyltransferase B (metB), which regulate amino acid metabolism and influence the expression of genes of the glycolysis-TCA pathway, were down-regulated, causing amino acid content in alfalfa leaves to decrease at FBS. This study provides new insights into the complex regulatory network governing the content and decrease of chlorophyll, amino acids, flavonoids, and other nutrients in alfalfa leaves during maturation. These results further provide a theoretical basis for the generation of alfalfa varieties exhibiting higher nutritional quality, high-yield cultivation, and a timely harvest.

Co-transport of Cr(VI) and Bentonite Colloid in Saturated Porous Media.

Transport of Cr(VI) at the presence of bentonite colloid was carried out in saturated porous media of 16-18 mesh and 40-60 mesh sand columns. Effects of flow rate, pH, ion strength, humic acid and bentonite concentrations on Cr(VI) migration were investigated. The results show that the increase of flow rate accelerated the breakthrough of Cr(VI) and BP, but the transport mass of dissolved Cr(VI) decreased by ~ 15.0% when flow rate increased to 2.5 ml min-1. Increasing IS to 10mM resulted in decrease of Cr(VI) transport mass by 6.86%-21.4%. Increase of pH and decrease of bentonite concentration favored the transport of dissolved Cr(VI). Humic acid had little effect on transport amount of Cr at pH7. Cr(VI) transport was dominated by the dissolved Cr(VI). The transport data of dissolved Cr(VI) were well described by the two-site model. The presence of BP reduced total Cr(VI) transport mass in co-transport.

Microwave-assisted catalytic hydrothermal carbonization of Laminaria japonica for hydrochars catalyzed and activated by potassium compounds.

There are limited investigations describing preparation and application of alga-based hydrochars via microwave-assisted catalytic hydrothermal carbonization (MA-CHTC). Therefore, hydrochars were successfully prepared from macroalgae biomass Laminaria japonica impregnated with KH2PO4, KCl, K2CO3, and KOH as acidic, neutral salt, and alkaline catalysts, respectively, via the MA-CHTC. Comprehensive characterization of physicochemical properties of the hydrochars, including yields, elemental and phase composition, specific surface areas, functional groups, and morphology, confirmed different catalytic effects of these catalysts on hydrochar formation. Adsorption kinetics and isotherms of Pb(II) revealed significant improvement of adsorption capacities for Pb(II) due to synergetic chemical activation of the spiked catalysts. Therefore, the synergetic catalytic effects and chemical activation is benefic for tailored design of engineered hydrochars with different properties for special application through selection of catalysts during the MA-CHTC process.

Application of Intravenous Lidocaine in Obese Patients Undergoing Painless Colonoscopy: A Prospective, Randomized, Double-Blind, Controlled Study.

OBJECTIVE: Propofol for procedural sedation and analgesia (PSA) for colonoscopy can result in a high prevalence of severe respiratory depression. Studies have shown that intravenous (IV) infusion of lidocaine can reduce propofol requirements significantly and increase the ventilatory response to carbon dioxide in humans. We tested the hypothesis that IV lidocaine could improve propofol-induced respiratory depression in obese patients during colonoscopy. METHODS: Ninety obese patients scheduled for painless colonoscopy were randomized to receive lidocaine (1.5 mg/kg, then 2 mg/kg/h, IV) or the same volume of 0.9% saline. Intraoperative sedation was provided by propofol. The primary outcome was the number of oxygen-desaturation episodes. Secondary outcomes were: the number of apnea episodes; total propofol consumption; time to the first hypoxia episode; time to consciousness loss; intraoperative hemodynamic parameters; awakening time; adverse events; duration of post-anesthesia care unit (PACU) stay; satisfaction of endoscopists and patients. RESULTS: Demographic characteristics between the two groups were comparable. The number of oxygen-desaturation episodes in group L (1.49±1.12) decreased by 0.622 (P=0.018) compared with that in group N (2.11±1.32), and the number of apnea episodes in group L decreased by 0.533 (P<0.001). Kaplan-Meier curves showed that the median time to the first hypoxia episode was longer in group L (86.78 s) than that in group N (63.83 s) (Log rank P=0.0008). The total propofol consumption, awakening time, and duration of PACU stay were reduced in group L. There was no significant difference in the prevalence of adverse events (P>0.05 for all). Satisfaction scores for endoscopists and patients in group L were higher than that in group N (P<0.001). CONCLUSION: Intravenous infusion of lidocaine could significantly reduce the number of oxygen-desaturation and apnea episodes in obese patients during painless colonoscopy. This method is worthy of clinical promotion. CLINICAL TRIALS REGISTRATION: ChiCTR2000028937.

Extraction, purification, and structure characterization of polysaccharides from Crassostrea rivularis.

Crude polysaccharide was prepared from Crassostrea rivularis by 30% (w/v) potassium hydroxide solution at 90°C for 120 min. Three fractions (OG1, OG2, and OG3) were purified by DEAE-52 cellulose and Sepharose 2B gel column chromatography. The chemical structures were determined using gas chromatography (GC), high-performance gel permeation chromatography (HPGPC), Fourier-transform infrared (FT-IR) spectroscopy, and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The results indicated that OG1 was composed of rhamnose and little mannose (8.71%), the ratio of Rha: Gal: Xyl: Fuc in OG3 were 14:5.5:3:1. And their average molecular weights (Mw) were about 1.66 × 106 and 2.33 × 106 Da, respectively. OG2 was composed only of glucose (98.23%), which means it was glycogen. OG2 was consisted mainly of →4)- α-D-Glc-(1→, with the branch chain every 6.5 glucose residues on average, which is →4,6)-α-D-Glc-(1→ and trace amount of α-D-Glc-(1→ branched units. The Mw was 2.27 × 106 Da. It provides the bases for the bioactivity research.

Porphyromonas gingivalis Gingipains Display Transpeptidation Activity.

Porphyromonas gingivalis is a keystone periodontal pathogen that has been associated with autoimmune disorders. The cell surface proteases Lys-gingipain (Kgp) and Arg-gingipains (RgpA and RgpB) are major virulence factors, and their proteolytic activity is enhanced by small peptides such as glycylglycine (GlyGly). The reaction kinetics suggested that GlyGly may function as an acceptor molecule for gingipain-catalyzed transpeptidation. Purified gingipains and P. gingivalis whole cells were used to digest selected substrates including human hemoglobin in the presence or absence of peptide acceptors. Mass spectrometric analysis of the substrates digested with gingipains in the presence of GlyGly showed that transpeptidation outcompeted hydrolysis, whereas the trypsin-digested controls exhibited predominantly hydrolysis activity. The transpeptidation levels increased with increasing concentration of GlyGly. Purified gingipains and whole cells exhibited extensive transpeptidation activities on human hemoglobin. All hemoglobin cleavage sites were found to be suitable for GlyGly transpeptidation, and this transpeptidation enhanced hemoglobin digestion. The transpeptidation products were often more abundant than the corresponding hydrolysis products. In the absence of GlyGly, hemoglobin peptides produced during digestion were utilized as acceptors leading to the detection of up to 116 different transpeptidation products in a single reaction. P. gingivalis cells were able to digest hemoglobin faster when acceptor peptides derived from human serum albumin were included in the reaction, suggesting that gingipain-catalyzed transpeptidation may be relevant for substrates encountered in vivo. The transpeptidation of host proteins in vivo may potentially lead to the breakdown of immunological tolerance, culminating in autoimmune reactions.

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System.

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a ß-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.

Characterisation of the Porphyromonas gingivalis Manganese Transport Regulator Orthologue.

PgMntR is a predicted member of the DtxR family of transcriptional repressors responsive to manganese in the anaerobic periodontal pathogen Porphyromonas gingivalis. Our bioinformatic analyses predicted that PgMntR had divalent metal binding site(s) with elements of both manganous and ferrous ion specificity and that PgMntR has unusual twin C-terminal FeoA domains. We produced recombinant PgMntR and four variants to probe the specificity of metal binding and its impact on protein structure and DNA binding. PgMntR dimerised in the absence of a divalent transition metal cation. PgMntR bound three Mn(II) per monomer with an overall dissociation constant Kd 2.0 x 10(-11) M at pH 7.5. PgMntR also bound two Fe(II) with distinct binding affinities, Kd1 2.5 x 10(-10) M and Kd2 ≤ 6.0 x 10(-8) M at pH 6.8. Two of the metal binding sites may form a binuclear centre with two bound Mn2+ being bridged by Cys108 but this centre provided only one site for Fe2+. Binding of Fe2+ or Mn2+ did not have a marked effect on the PgMntR secondary structure. Apo-PgMntR had a distinct affinity for the promoter region of the gene encoding the only known P. gingivalis manganese transporter, FB2. Mn2+ increased the DNA binding affinity of PgMntR whilst Fe2+ destabilised the protein-DNA complex in vitro. PgMntR did not bind the promoter DNA of the gene encoding the characterised iron transporter FB1. The C-terminal FeoA domain was shown to be essential for PgMntR structure/function, as its removal caused the introduction of an intramolecular disulfide bond and abolished the binding of Mn2+ and DNA. These data indicate that PgMntR is a novel member of the DtxR family that may function as a transcriptional repressor switch to specifically regulate manganese transport and homeostasis in an iron-dependent manner.

Levo-Tetrahydropalmatine Attenuates Bone Cancer Pain by Inhibiting Microglial Cells Activation.

OBJECTIVE: The present study is to investigate the analgesic roles of L-THP in rats with bone cancer pain caused by tumor cell implantation (TCI). METHODS: Thermal hyperalgesia and mechanical allodynia were measured at different time points before and after operation. L-THP (20, 40, and 60 mg/kg) were administrated intragastrically at early phase of postoperation (before pain appearance) and later phase of postoperation (after pain appearance), respectively. The concentrations of TNF-α, IL-1ß, and IL-18 in spinal cord were measured by enzyme-linked immunosorbent assay. Western blot was used to test the activation of astrocytes and microglial cells in spinal cord after TCI treatment. RESULTS: TCI treatment induced significant thermal hyperalgesia and mechanical allodynia. Administration of L-THP at high doses significantly prevented and/or reversed bone cancer-related pain behaviors. Besides, TCI-induced activation of microglial cells and the increased levels of TNF-α and IL-18 were inhibited by L-THP administration. However, L-THP failed to affect TCI-induced astrocytes activation and IL-1ß increase. CONCLUSION: This study suggests the possible clinical utility of L-THP in the treatment of bone cancer pain. The analgesic effects of L-THP on bone cancer pain maybe underlying the inhibition of microglial cells activation and proinflammatory cytokines increase.

The Porphyromonas gingivalis ferric uptake regulator orthologue binds hemin and regulates hemin-responsive biofilm development.

Porphyromonas gingivalis is a Gram-negative pathogen associated with the biofilm-mediated disease chronic periodontitis. P. gingivalis biofilm formation is dependent on environmental heme for which P. gingivalis has an obligate requirement as it is unable to synthesize protoporphyrin IX de novo, hence P. gingivalis transports iron and heme liberated from the human host. Homeostasis of a variety of transition metal ions is often mediated in Gram-negative bacteria at the transcriptional level by members of the Ferric Uptake Regulator (Fur) superfamily. P. gingivalis has a single predicted Fur superfamily orthologue which we have designated Har (heme associated regulator). Recombinant Har formed dimers in the presence of Zn2+ and bound one hemin molecule per monomer with high affinity (Kd of 0.23 µM). The binding of hemin resulted in conformational changes of Zn(II)Har and residue 97Cys was involved in hemin binding as part of a predicted -97C-98P-99L- hemin binding motif. The expression of 35 genes was down-regulated and 9 up-regulated in a Har mutant (ECR455) relative to wild-type. Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation. A truncated Zn(II)Har bound the promoter region of dnaA (PGN_0001), one of the up-regulated genes in the ECR455 mutant. This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability. ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability. P. gingivalis possesses a hemin-binding Fur orthologue that regulates hemin-dependent biofilm formation.

Receptor-interacting protein kinase 4 and interferon regulatory factor 6 function as a signaling axis to regulate keratinocyte differentiation.

Receptor-interacting protein kinase 4 (RIPK4) and interferon regulatory factor 6 (IRF6) are critical regulators of keratinocyte differentiation, and their mutation causes the related developmental epidermal disorders Bartsocas-Papas syndrome and popliteal pterygium syndrome, respectively. However, the signaling pathways in which RIPK4 and IRF6 operate to regulate keratinocyte differentiation are poorly defined. Here we identify and mechanistically define a direct functional relationship between RIPK4 and IRF6. Gene promoter reporter and in vitro kinase assays, coimmunoprecipitation experiments, and confocal microscopy demonstrated that RIPK4 directly regulates IRF6 trans-activator activity and nuclear translocation. Gene knockdown and overexpression studies indicated that the RIPK4-IRF6 signaling axis controls the expression of key transcriptional regulators of keratinocyte differentiation, including Grainyhead-like 3 and OVO-like 1. Additionally, we demonstrate that the p.Ile121Asn missense mutation in RIPK4, which has been identified recently in Bartsocas-Papas syndrome, inhibits its kinase activity, thereby preventing RIPK4-mediated IRF6 activation and nuclear translocation. We show, through mutagenesis-based experiments, that Ser-413 and Ser-424 in IRF6 are important for its activation by RIPK4. RIPK4 is also important for the regulation of IRF6 expression by the protein kinase C pathway. Therefore, our findings not only provide important mechanistic insights into the regulation of keratinocyte differentiation by RIPK4 and IRF6, but they also suggest one mechanism by which mutations in RIPK4 may cause epidermal disorders (e.g. Bartsocas-Papas syndrome), namely by the impaired activation of IRF6 by RIPK4.

Propeptide-mediated inhibition of cognate gingipain proteinases.

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism's cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB) and the Lys-specific proteinase (Kgp), which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingivalis mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp) that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with K(i) values of 2.04 µM and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.

Reversible redox regulation of specificity of Arg-gingipain B in Porphyromonas gingivalis.

Arg-gingipain B (RgpB), a major virulence factor secreted by the periodontal pathogen Porphyromonas gingivalis is an Arg-specific cysteine proteinase. By monitoring proteolytic cleavage of a human salivary peptide histatin 5 using MALDI-TOF MS, RgpB purified from P. gingivalis HG66 was found to shift from a dominant Arg-X to dominant Lys-X activity, both in vitro and in vivo, upon reversible cysteine oxidation. Native PAGE analysis revealed the association of novel Lys-X activity with a reversible state change of the oxidized enzyme. The redox-regulated Lys-X activity of RgpB may provide a survival advantage to P. gingivalis against the oxidative host defence.

[Analysis of cortical lateralization during mental task of geometric figure rotation].

Mean Kolmogorov entropy within one second is introduced and is used to analyze and localize the cortical lateralization based on spontaneous EEG and nonlinear dynamics. The results of analysis indicate: The conclusion drawn form the use of this method is almost consistent with that from other methods, but this method is more sensitive to cortical lateralization; this method can identify the differences of cortical lateralization between different brain areas. To analyze and localize cortical lateralization, mean Kolmogorov entropy based on spontaneous EEG is a good method.

Etiology and pathology of epidermal papillomas in allogynogenetic crucian carp Carassius auratus gibelio (female) x Cyprinus carpio var. singuonensis (male).

Allogynogenetic crucian carp Carassius auratus gibelio (female) x Cyprinus carpio var. singuonensis (male) is one of the main freshwater aquaculture species in China. In recent years, epidermal papillomas have been observed on the scales, fins and opercula of adult fish in many fish farms in the Chongming county of Shanghai, China. The disease appears in the late autumn of the first year and becomes more severe in winter. It gradually regresses in the late spring or summer of the second year, as water temperature increases. Our study revealed that the disease pathogen was likely to be a herpes-like virus, as indicated by enveloped viral particles in the cytoplasm, empty capsids in the nucleus and a virus-like morphology of the pathogen. The size of the enveloped herpes-like virus was 118.18 +/- 10.53 (SD) nm (n = 22) and its nucleocapsid was 78.64 +/- 7.74 nm (n = 22) in diameter. Histopathological examination of tumours revealed that both epithelial and stromal cells proliferated to form papillomas. The nuclei of epithelial tumour cells were irregular in shape and in size. Most of the mucous cells were located in clusters near the middle of each papilloma. Cytoplasmic organelles were sparse in tumour cells. Numerous granulocytes and lymphocytes infiltrated into the tumour tissue. There were no inclusion bodies in the cytoplasm and nuclei of tumour cells. The lesions only occurred in adult allogynogenetic crucian carp, even though they were cultured together with other fish species. There was marked variation in incidence: in some ponds, only a few fish were affected, while the incidence was up to 90% in other ponds.

Electron paramagnetic resonance characterization of the copper-resistance protein PcoC from Escherichia coli.

Continuous-wave and pulsed electron paramagnetic resonance have been applied to the study of the Cu(II) site of the copper-resistance protein PcoC from Escherichia coli and certain variant forms. Electron spin echo envelope modulation (ESEEM) experiments confirm the presence of two histidine ligands, His1 and His92, at the Cu(II) site of wild-type PcoC, consistent with the available X-ray crystallographic data for the homolog CopC (67% sequence identity) from Pseudomonas syringae pv. tomato. The variants H1F and H92F each lack one of the histidine residues close to the Cu(II) site. The ESEEM data suggest that the surviving histidine residue remains as a ligand. The nA variant features an extra alanine residue at the N terminus, which demotes the His1 ligand to position 2. At least one of the two histidine residues is bound at the Cu(II) site in this form. Simulation of the (14)N superhyperfine structure in the continuous-wave spectra confirms the presence of at least three nitrogen-based ligands at the Cu(II) sites of the wild-type, H92F and nA forms, while the H1F variant has two nitrogen ligands. The spectra of wild-type form can be fitted adequately with a 3N or a 4N model. The former is consistent with the crystal structure of the CopC homolog, where His1 acts as a bidentate ligand. The latter raises the possibility of an additional unidentified nitrogen ligand. The markedly different spectra of the H1F and nA forms compared with the wild-type and H92F proteins further highlight the integral role of the N-terminal histidine residue in the high-affinity Cu(II) site of PcoC.

Surface selective deposition of Mo(IV) on Ni/TiO2 particles in aqueous solutions.

The selective depositions of MoS2 and MoO2 over Ni surfaces are demonstrated on Ni/TiO2 particles in a mild electroless deposition process. High-resolution transmission electron microscopy (HRTEM) images show the uniform distribution of 10-30 nm spherical and hemispherical Ni particles on TiO2 surface, and three to six layers of MoS2 on the surface of Ni particles. The as-prepared MoS2-Ni/TiO2 is used as a catalyst for the hydrodesulfurization (HDS) reaction of dibenzothiophene (DBT), and shows a significant increase over commercial catalysts in turnover frequencies as the result of unique distribution of active components in the binary catalyst. The selective material deposition is explained in the context of Ni catalyzed KBH4 decomposition, which produces strong reducing species responsible for the site selective deposition of Mo. The synthetic method can be potentially used to prepare bimetallic materials with similar nanostructures such as those of Mo-Co, Mo-Pd, and Mo-Rh.

Intermolecular transfer of copper ions from the CopC protein of Pseudomonas syringae. Crystal structures of fully loaded Cu(I)Cu(II) forms.

CopC is a small soluble protein expressed in the periplasm of Pseudomonas syringae pathovar tomato as part of its copper resistance response (cop operon). Equilibrium competition reactions confirmed two separated binding sites with high affinities for Cu(I) (10(-7) > or = K(D) > or = 10(-13) M) and Cu(II) (K(D) = 10(-13(1)) M), respectively. While Cu(I)-CopC was converted cleanly by O2 to Cu(II)-CopC, the fully loaded form Cu(I)Cu(II)-CopC was stable in air. Variant forms H1F and H91F exhibited a lower affinity for Cu(II) than does the wild-type protein while variant E27G exhibited a higher affinity. Cation exchange chromatography detected each of the four different types of intermolecular copper transfer reactions possible between wild type and variant forms: Cu(I) site to Cu(II) site; Cu(II) site to Cu(I) site; Cu(I) site to Cu(I) site; Cu(II) site to Cu(II) site. The availability of an unoccupied site of higher affinity induced intermolecular transfer of either Cu(I) or Cu(II) in the presence of O2 while buffering concentrations of cupric ion at sub-picomolar levels. Crystal structures of two crystal forms of wild-type Cu(I)Cu(II)-CopC and of the apo-H91F variant demonstrate that the core structures of the molecules in the three crystal forms are conserved. However, the conformations of the amino terminus (a Cu(II) ligand) and the two copper-binding loops (at each end of the molecule) differ significantly, providing the structural lability needed to allow transfer of copper between partners, with or without change of oxidation state. CopC has the potential to interact directly with each of the four cop proteins coexpressed to the periplasm.