Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability.

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogue of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor, Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate approximately 95% incorporation of 5HW into position 140 of nuclease, and we estimate approximately 98% incorporation of 7AW, CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.     

Inhibition of δ8 → δ7-sterol isomerase and of cycloeucalenol—obtusifoliol isomerase by N-benzyl-8-aza-4α, 10-dimethyl-trans-decal-3β-ol,an analogue of a carbocationic high energy intermediate

An enzymatic assay for the δ⁸ → δ⁷-sterol isomerase, an enzyme involved in sterol biosynthesis, has been developed in higher plants. This assay has been used in the study of various inhibitors. N-Benzyl-8-aza-4α, 10-dimethyl- trans-decal-3β-ol was designed to mimic the C-8 and the C-9 carbocationic high energy intermediates occurring during the reactions catalysed by the δ⁸ → δ⁷-sterol isomerase and the cycloeucalenol obtusifoliol isomerase, respectively. In accordance with the ‘transition state analogues’ theory, this analogue of a high energy intermediate was found to be a very potent and specific inhibitor of the two enzymatic reactions both in vitro and in vivo.     

基于能值代数的湿地生态系统服务评价去重复性计算

在对生态系统服务进行评价时,重复计算一直是困扰学者们的一个难题。目前大部分对湿地生态系统服务价值评估的方法都集中于将服务进行简单的分类后,通过经济学的方法计算和评估湿地生态系统服务的价值。而生态系统服务的产生、传输、消费和再生产其实是一个循环的过程,将其生硬的分类进行评估,势必会对结果的精确度造成影响,如果不能正确地去除湿地生态系统服务价值评价中重复计算的部分,会使评估结果的可信度降低。本文通过建立能值流动流程图,对湿地生态系统服务间"分离、反馈、共产物"这三个基本机制进行整体性的预先分析,将计算结果与未经预先分析的计算结果对比,得到结果误差范围为12%~38%。研究表明,经过对系统的预先分析,可以有效地去除重复性计算所带来的误差,使评价的结果更加精确。     

Binding thermodynamics of paromomycin,neomycin, neomycin‐dinucleotide and ‐diPNA conjugates to bacterial and human rRNA

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target‐drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A‐site, with two well‐known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin‐dinucleotide and ‐diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin‐diPNA conjugates as the best candidates for antimicrobial activity. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis and Potent Anti-HCMV Activity of 2′,5′,5′-trifluoro-3′-hydroxy-apiosyl Nucleoside Phosphonic Acid Analogues

As antiviral nucleosides containing a fluorine atom at 2′-position are endowed with increased stabilization of glycosyl bond, it was of interest to investigate the influence of three fluorine atoms at 2′- and 5′-positions of apiosyl nucleoside phosphonate analogues. Various pyrimidine and purine 2′,5′,5′-trifluoro-3′-hydroxy-apiose nucleoside phosphonic acid analogues were synthesized from 1,3-dihydroxyacetone. Electrophilic fluorination of lactone was performed using N-fluorodibenzenesulfonimide. Difluorophosphonation was performed by direct displacement of triflate intermediate with diethyl(lithiodifluoromethyl) phosphonate to give the corresponding (α,α-difluoroalkyl) phosphonate. Condensation successfully proceeded from a glycosyl donor with persilylated bases to yield nucleoside phosphonate analogues. Deprotection of diethyl phosphonates provided the final phosphonic acid sodium salts. The synthesized nucleoside analogues were subjected to antiviral screening against various viruses.     

Relaxant effects of selected sildenafil analogues in the rat aorta

A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine with chiral amino group has been synthesized and characterized. The compounds were tested for their relaxant effects in the rat aorta. Evaluation of prepared derivatives demonstrated that compound (8a) is probably a non-selective phosphodiesterase (PDE) inhibitor, as it induced aortic relaxation through endothelium-independent mechanism.     

Toward chelerythrine optimization: Analogues designed by molecular simplification exhibit selective growth inhibition in non-small-cell lung cancer cells

A series of novel chelerythrine analogues was designed and synthesized. Antitumor activity was evaluated against A549, NCI-H1299, NCI-H292, and NCI-H460 non-small-cell lung cancer (NSCLC) cell lines in vitro. The selectivity of the most active analogues and chelerythrine was also evaluated, and we compared their cytotoxicity in NSCLC cells and non-tumorigenic cell lines, including human umbilical vein endothelial cells (HUVECs) and LL24 human lung fibroblasts. In silico studies were performed to establish structure–activity relationships between chelerythrine and the analogues. The results showed that analogue compound 3f induced significant dose-dependent G₀/G₁ cell cycle arrest in A549 and NCI-H1299 cells. Theoretical studies indicated that the molecular arrangement and electron characteristics of compound 3f were closely related to the profile of chelerythrine, supporting its activity. The present study presents a new and simplified chelerythrinoid scaffold with enhanced selectivity against NSCLC tumor cells for further optimization.     

The impact of β‐azido(or 1‐piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ and its epimer were analysed by ¹H and ¹³C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C^α in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Multinuclear magnetic resonance studies on serine protease transition state analogues

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.     

Characterization of sulfate transport in Desulfovibrio desulfuricans

Uptake of ³⁵S-labelled sulfate was studied with a new isolate of Desulfovibrio desulfuricans, strain CSN. Micromolar additions of sulfate (1–10 M or nmol/mg protein) to cell suspensions incubated in 150 mM KCl at-1°C were almost completely taken up and accumulated about 5,000-fold. Accumulation was not influenced by incubation in NaCl instead of KCl, by acidic pH (5.5) or by incubation under air for 10 min. In alkaline milieu (pH 8.5), after prolonged contact with air (2 h), or after growth with excess sulfate or thiosulfate as electron acceptor, the amount taken up was diminished approximately by half. Pasteurization inhibited sulfate uptake completely. With increasing concentrations of added sulfate (0.1 to 2.5 mM) the intracellular concentration increased only slowly up to 25 mM, and the accumulation factor decreased down to 8. Sulfate transport was reversible. Accumulated sulfate was rapidly lost from the cells after addition of excess non-labelled sulfate or after addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The ATPase inhibitor dicyclohexylcarbodiimide (DCCD) specifically inhibited sulfate reduction but had no immediate influence on sulfate accumulation. Addition of the phosphate analogue arsenate (5 mM) was without effect. These results were not in favour of an ATP-dependent transport system. The K⁺-H⁺-antiporter nigericin (in 150 mM KCl) and the Na⁺-H⁺-antiporter monensin (in 150 mM NaCl) caused partial inhibition of sulfate accumulation, whereas the K⁺-transporter valinomycin (in 150 mM KCl) and the Na⁺-H⁺ exchange inhibitor amiloride (2 mM) were without effect. The permeant thiocyanate anion (150 mM) inhibited sulfate uptake by 60% at pH 7, and completely at pH 8.5. Although the effects of the different ionophores on the chemiosmotic gradients have not been studied so far, the results indicated that probably both, pH and drive sulfate accumulation and that sulfate is taken up electrogenically in symport with more than 2 protons. The structural sulfate analogues tungstate and molybdate (0.1 mM, each) did not affect sulfate accumulation, although molybdate inhibited sulfate reduction. Chromate completely blocked both of these activities. Sulfite and selenite caused little or no decrease of sulfate accumulation, whereas with thiosulfate and selenate significant inhibition was observed.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide