Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.     

Syntheses, structures, and properties of lanthanide-organic frameworks with flexible disulfide derivative of carboxylate

Reaction of disulfide-containing ligand 6,6′-dithiodinicotinic acid (H₂cpds) with lanthanide metal salts led to three compounds {[Ln₂(cpds)₃(H₂O)₅]·7H₂O}_n (Ln = Gd, 1; Tb, 2; Er, 3) under hydrothermal conditions. The single-crystal X-ray diffraction analysis revealed that complexes 1-3 are isostructural. Two eight-coordinated metal centers are connected with carboxylate groups of H₂cpds to give a binuclear [Ln₂(CO₂)₄] unit. H₂cpds discloses two independent binding modes resulting in the two types of 1-D chains by the connection of [Ln₂(CO₂)₄] unit. Finally, the 1-D chains are linked by H₂cpds to construct a 3-D network with an uncommon twofold interpenetrating 4-connected CdSO₄ structure type (6⁵,8 topology). It is worth noting that polyrotaxane-like motif is also exhibited in the 3-D network. The Tb(III) compound exhibits strong green photoluminescence in the visible region. Furthermore, the magnetic property of the Gd(III) compound has been investigated in the temperature range of 2-300 K. The thermal behaviors and XPRD patterns of three compounds have also been discussed.     

蚕豆叶片下表皮ABA结合蛋白提取及分离条件的选择

The abaxial epiderm of Vicia faba L. was chosen to prepare the crude extract of ABA binding protein (ABA-BP). The activity of ABA-BP was dependent on different extraction methods. The specific binding capacity of ABA-BP extracted with 0.5% Triton X-100 (binding activity (B) = 0. 487 nmoL/g protein) was higher than that with extracted cold acetone (0. 325 nmol/g protein) or (NH4)2SO4(0. 223 nmol/ g protein). The activity duration of ABA-BP extracted with Triton X-100 (60% Bmax after 40 h) was longer than that with cold acetone (30% Bmax after 10 h). The ABA-BP activity of binding ABA was sensitive to pH change with an optimum pH of 6.5. The ABA-BP specific binding activity decreased quickly under high concentration of NaC1 ( > 300 mmoL/L), but increased 12% with 5 mmol/L KC1. Some divalent cations like Ca2+ and Mg2 + were required for enhancing the ABA-BP activity. These optimum conditions are primordial for ABA-BP purification.     

Lanthanide complexes with disulfide ligand generated in situ: Syntheses, crystal structures and fluorescent properties

Five new lanthanide complexes [Ln₂(DTDN)₄(phen)₄]·7H₂O·2H₃O⁺ (Ln = Nd (1), Sm (2), Eu (3), Tb (4), Dy (5), H₂DTDN = 2,2′-dithiodinicotinic acid, phen = 1,10-phenanthroline) have been solvothermally synthesized and characterized by single-crystal X-ray diffraction, elemental analyses, IR spectra, and TG analyses. By in situ oxidation of 2-mercaptonanicotinic acid (2-H₂MN), the expected ligand H₂DTDN was generated. All crystals are isostructural and crystallize in monoclinic system with space group C2/c. The metal center is eight-coordinated completely by four carboxylic oxygen atoms from four different DTDN²⁻ ligands, and four nitrogen atoms from two phen molecules with a distorted square-antiprismatic geometry. The structures can be considered as two-dimensional (2D) structures and further linked by hydrogen bonds into the final trinodal 4-connected network. Photoluminescence studies revealed that complexes 2-5 exhibit strong fluorescent emission bands in the solid state at room temperature.     

Formation of DNA-adducts and induction of DNA-crosslinks and chromosomal aberrations by the new potent anthracycline antitumor antibiotics: morpholinodaunomycin,cyanomorpholinodaunomycin and cyanomorhpolinoadriamycin

The DNA-interaction of three newly developed semisynthetic anthracyclines with high antitumor potency MoDNM³, CNMoDNM, and CNMoADM, was investigated. When primary rat hepatocytes were incubated with tritium labeled MoDNM and CNMoDNM and their DNA was purified and enzymatically hydrolized, the formation of DNA-adducts could be demonstrated by the HPLC chromatography of the resulting mononucleoside mixtures. The parent compound, daunomycin (DNM), also formed covalent adducts with hepatocyte DNA, but to a lesser extent. These findings correlate well earlier observaitons that MoDNM and CNMoDNM are potent inducers of DNA-repair in primary rat hepatocytes, whereas DNM is only weakly active in this regard. Aklaline elution studies were performed with L 1210 mouse leukemia cells and V79 Chinese hamster fibroblasts. The cyanomorpholinyl derivatives showed dose-dependant DNA crosslinking activities in both cell lines at concentrations 5 nMol/l. The formation of crosslinks began a few minutes after treatment of the cells and reached a maximum after 1 hr. In contrast, MoDNM, at concentrations of up to 10 Mol/l, had only a limited capacity to induce single strand breaks in L 1210 cells but did not induce DNA-crosslinks. In addition, chromosomal aberrations (chromatid breaks and translocations) were induced by the treatment of Friend and L 1210 leukemia cells with CNMoADM at concentrations between 0.07–0.6 n Mol/l. At higher doses, chromosome clumping was observed. These results indicate that the high capacity of MoDNM, CNMoDNM and CNMoADM to induce DNA repair in primary rat hepatocytes is due to the formation of covalent adducts with DNA. The cyanomorpholino compounds have alkylating capacities also in cell lines such as L 1210 and V79, whereas MoDNM requires rat hepatocytes for activation. The ready formation of DNA crosslinks and chromosomal aberrations could be responsible for the high cytotoxicity of these compounds.Abbreviations ADM adriamycin - CNMoADM cyanomorpholinoadriamycin - CNMoDNM cyanomorpholinodaunomycin - DNM daunomycin - FLC Friend leukemia cells - (G³H) generally tritium labeled - HPLC high performance liquid chromatography - MoDNM morpholinodaunomycin - Rf retention factor - (Mo³H) tritium labelled at morpholinyl site - Rad radiation unit - RT retention time - SDS sodium dodecylsulphate - Tris tris (hydroxymethyl)aminomethan     

Rosmarinic acid and its derivatives: biotechnology and applications

Rosmarinic acid (RA) is one of the first secondary metabolites produced in plant cell cultures in extremely high yields, up to 19% of the cell dry weight. More complex derivatives of RA, such as rabdosiin and lithospermic acid B, later were also obtained in cell cultures at high yields. RA and its derivatives possess promising biological activities, such as improvement of cognitive performance, prevention of the development of Alzheimer’s disease, cardioprotective effects, reduction of the severity of kidney diseases and cancer chemoprevention. The TNF-α-induced NF-κB signaling pathway has emerged as a central target for RA. Despite these impressive activities and high yields, the biotechnological production of these metabolites on an industrial scale has not progressed. We summarized data suggesting that external stimuli, the Ca²⁺-dependent NADPH oxidase pathway and processes of protein phosphorylation/dephosphorylation are involved in the regulation of biosynthesis of these substances in cultured plant cells. In spite of growing information about pathways regulating biosynthesis of RA and its derivatives in cultured plant cells, the exact mechanism of regulation remains unknown. We suggest that further progress in the biotechnology of RA and its derivatives can be achieved by using new high-throughput techniques.     

Hydrodynamic characterization of the SufBC and SufCD complexes and their interaction with fluorescent adenosine nucleotides

Bacteria, as well as the plastid organelles of algae and higher plants, utilize proteins of the suf operon. These are involved in Fe-S cluster assembly, particularly under conditions of iron limitation or oxidative stress. Genetic experiments in some organisms found that the ATPase SufC is essential, though its role in Fe-S biogenesis remains unclear. To ascertain how interactions with other individual Suf proteins affect the activity of SufC we coexpressed it with either SufB or SufD from Thermotoga maritima and purified the resulting SufBC and SufCD complexes. Analytical ultracentrifuge and multiangle light-scattering measurements showed that the SufBC complex exists in solution as the tetrameric SufB(2)C(2) species, whereas SufCD exists as an equilibrium mixture of SufCD and SufC(2)D(2). Transient kinetic studies of the complexes were made using fluorescent 2'(3')-O-(N-methylanthraniloyl-(mant) analogues of ATP and ADP. Both SufBC and SufCD bound mantATP and mantADP much more tightly than does SufC alone. Compared to the cleavage step of the mantATPase of SufC alone, that of SufBC was accelerated 180-fold and that of SufCD only fivefold. Given that SufB and SufD have 20% sequence identity and similar predicted secondary structures, the different hydrodynamic properties and kinetic mechanisms of the two complexes are discussed.     

Synthesis, solution structure, binding activity, and cGMP activation of human guanylin and its disulfide isomer

Guanylin is a recently isolated peptide consisting of 15 amino acid residues with four cysteines, which may form two intramolecular disulfide bridges, and stimulates intestinal membrane guanylate cyclase. The position of the disulfide linkages of guanylin was predicted from its structural similarity to a heat stable enterotoxin which is thought to be responsible for secretory diarrhoea. Both guanylin, with disulfide positions 4–12 and 7–15, and its disulfide isomer, with disulfides positions 4–15 and 7–12, were chemically synthesized by the solid-phase method and purified. Two specific disulfides were selectively formed and confirmed by sequencing, mass spectrometry and high-performance liquid chromatography in combination with enzymatic cleavage. The structure of both isomers has been investigated in solution by ¹H nuclear magnetic resonance spectroscopy. Guanylin exists as a mixture of two stable conformations which have compact spiral structures, from comparison with literature data. In contrast, the disulfide isomer of guanylin shows only a single conformation with an elongated curved plate-like structure. Binding assays were performed using labelled guanylin with membranes obtained from rat jejunum. Both disulfide isomers were investigated by the cGMP assay. Both binding and cGMP assays indicated that the relevant form of disulfide bridges in the intact guanylin was as predicted.     

Syntheses and biological activities of the proposed structure of apteniol A and its derivatives

We describe the syntheses of the proposed structure of diphenyl ether oxyneolignan, apteniol A and its derivatives. The diphenyl ether moiety of proposed apteniol A was formed via Ullmann ether synthesis, but the spectral data of the synthesized apteniol A did not agree with that in previous studies. The dimethyl ester derivative of the proposed apteniol A was found to enhance neurite outgrowth in PC12 cells and inhibit antigen-induced degranulation in RBL-2H3 cells.     

Structure and dynamics of UBA5-UFM1 complex formation showing new insights in the UBA5 activation mechanism

Ubiquitin fold modifier 1 (UFM1) is an ubiquitin-like protein (Ubl) involved especially in endoplasmic stress response. Activation occurs via a three-step mechanism like other Ubls. Data obtained reveal that UFM1 regulates the oligomeric state of ubiquitin activating enzyme 5 (UBA5) to initiate the activation step. Mixtures of homodimers and heterotrimers are observed in solution at the equilibrium state, demonstrating that the UBA5-UFM1 complex undergoes several concentration dependent oligomeric translational states to form a final functional complex. The oligomerization state of unbound UBA5 is also concentration dependent and shifts from the monomeric to the dimeric state. Data describing different oligomeric states are complemented with binding studies that reveal a negative cooperativity for the complex formation and thereby provide more detailed insights into the complex formation mechanism.     

肽合成中多对二硫键的形成策略及分析方法

二硫键的正确配对是富含二硫键多肽合成的关键。本文综述了含两对二硫键以上的多肽二硫键的形成策略,优化方法、以及二硫键配对方式的测定方法。     

A click chemistry approach to pleuromutilin derivatives. Part 3: Extended footprinting analysis and excellent MRSA inhibition for a derivative with an adenine phenyl side chain

Five promising pleuromutilin derivatives from our former studies, all containing adenine on various linkers, were supplemented with two new compounds. The binding to Escherichia coli ribosomes was verified by extensive chemical footprinting analysis. The ability to inhibit bacterial growth was investigated on two Staphylococcus aureus strains and compared to the pleuromutilin drugs tiamulin and valnemulin. Three of the compounds show an effect similar to tiamulin and one compound shows an excellent effect similar to valnemulin.     

Novel derivatives of deoxycholic acid bearing aliphatic or cyclic diamine moieties at the C-3 position: Synthesis and evaluation of anti-proliferative activity

A new library of deoxycholic acid derivatives bearing nitrogen-containing moieties at the C-3 position was synthesised from epoxy derivative 1 via an epoxide ring-opening reaction promoted by aliphatic or cyclic diamines and fully characterised by NMR and mass-spectroscopy. The synthesised compounds were screened for cytotoxicity against four human tumour cell lines. The results showed that some of the novel diamine-bearing derivatives displayed improved anti-proliferative activities over the parent compound DCA. Among them, a 1-methylpiperazine containing compound (6) showed promising activity and the highest selectivity against tumour cells of enterohepatic origin (HepG2: IC₅₀ = 3.6 µM, SI = 9.0; HuTu-80: IC₅₀ = 4.6 µM, SI = 6.9) and was identified as a lead molecule.     

On the structural basis of peptide-bond formation and antibiotic resistance from atomic structures of the large ribosomal subunit

The atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with substrates and antibiotics have provided insights into the way the 3000 nucleotide 23S rRNA folds into a compact, specific structure and interacts with 27 ribosomal proteins as well as the structural basis of the peptidyl transferase reaction and its inhibition by antibiotics. The structure shows that the ribosome is indeed a ribozyme.     

Promiscuity comes at a price: Catalytic versatility vs efficiency in different metal ion derivatives of the potential bioremediator GpdQ

The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) is a highly promiscuous dinuclear metallohydrolase with respect to both substrate specificity and metal ion composition. While this promiscuity may adversely affect the enzyme's catalytic efficiency its ability to hydrolyse some organophosphates (OPs) and by-products of OP degradation have turned GpdQ into a promising candidate for bioremedial applications. Here, we investigated both metal ion binding and the effect of the metal ion composition on catalysis. The prevalent in vivo metal ion composition for GpdQ is proposed to be of the type Fe(II)Zn(II), a reflection of natural abundance rather than catalytic optimisation. The Fe(II) appears to have lower binding affinity than other divalent metal ions, and the catalytic efficiency of this mixed metal center is considerably smaller than that of Mn(II), Co(II) or Cd(II)-containing derivatives of GpdQ. Interestingly, metal ion replacements do not only affect catalytic efficiency but also the optimal pH range for the reaction, suggesting that different metal ion combinations may employ different mechanistic strategies. These metal ion-triggered modulations are likely to be mediated via an extensive hydrogen bond network that links the two metal ion binding sites via residues in the substrate binding pocket. The observed functional diversity may be the cause for the modest catalytic efficiency of wild-type GpdQ but may also be essential to enable the enzyme to evolve rapidly to alter substrate specificity and enhance k_cat values, as has recently been demonstrated in a directed evolution experiment. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.     

生态记忆及其在生态学中的潜在应用

生态记忆指群落过去的状态或经验影响其目前或未来生态响应的能力.作为研究生态系统结构和功能的一个新视角,生态记忆在群落演替、生态恢复、生物入侵和自然资源管理等多个领域中受到重视.本文在综述生态记忆的概念、组成、类别的基础上,进一步探讨了生态记忆的可能机制和影响因素,并指出了其潜在应用,以期为理解演替机理和指导生态恢复提供参考.     

Synthesis, characterization and reactivity of polypyridyl ruthenium(II) carbonyl complexes with phosphine derivatives: Ruthenium-carbon bond labilization based on steric and electronic effects

Ruthenium phosphine complexes with a CO ligand [Ru(tpy)(PR₃)(CO)Cl]⁺ (tpy = 2,2′:6′,2″-terpyridine, R = Ph or p-tolyl), were prepared by introduction of CO gas to the corresponding dichloro complexes at room temperature. New carbonyl complexes were characterized by various methods including structural analyses. They were shown to release CO following the addition of several N-donors to form the corresponding substituted complexes. The kinetic data and structural results observed in this study indicated that the CO release reactions proceeded in an interchange mechanism. The molecular structures of [Ru(tpy)(PPh₃)(CO)Cl]PF₆, [Ru(tpy)(P(p-tolyl)₃)(CO)Cl]PF₆ and [Ru(tpy)(PPh₃)(CH₃CN)Cl]PF₆ were determined by X-ray crystallography.     

Interaction of amphotericin B and its N-fructosyl derivative with murine thymocytes: a comparative study using fluorescent membrane probes

The polyene antibiotics amphotericin B (AmB) and N-(1-deoxy-D-fructos-1-yl)amphotericin (N-Fru-AmB) have different activity towards murine thymocytes (N-Fru-AmB is less toxic but is a potent immunomodulator). The interactions of the drugs with these cells have been studied by fluorescence methods. Fluorescence energy transfer from 1-[4-(trimethylammonio) phenyl]-6-phenylhexa-1,3,5-triene, p-toluenesulfonate (TMA-DPH) to polyenes was used to follow the binding of the two drugs to the plasma membrane. The results, confirmed by circular dichroism measurements, indicate that at saturation the ratio AmB bound/plasma membrane lipid is low (less than 1 molecule of polyene for 170 lipids). The slightly higher binding of AmB as compared to N-Fru-AmB demonstrates that affinity of the antibiotic for plasma membrane does not account for the activity of the polyenes towards lymphocytes. The effect of the two polyenes on membrane fluidity was studied by steady-state fluorescence anisotropy. The results suggest that AmB strongly perturbs the structure of the membrane whereas only a slight decrease of the anisotropy is observed with N-Fru-AmB in the range of concentration where the biological activity has been demonstrated. Polyene location was further investigated by comparing the energy transfer efficiency obtained with TMA-DPH and with the parental compound 1,6-diphenylhexa-1,3,5-triene, p-toluene sulfonate (DPH). While AmB binds to plasma membrane, as well as to intracellular structures, N-Fru-AmB seems to accumulate into the cell and bind to intracellular membrane structures.