An Atypical Arginine Dihydrolase Involved in the Biosynthesis of Cyclic Hexapeptide Longicatenamides

The incorporation of non-proteinogenic amino acids (NPAAs) enriches the structural diversity of nonribosomal peptides. Recently, four NPAA-containing cyclic hexapeptides, longicatenamides A−D, were isolated using a combined-culture strategy. Based on in silico analysis, we discovered their putative biosynthetic gene cluster (lon) and proposed a possible biosynthetic mechanism. Surprisingly, the lon22 gene encodes an atypical arginine dihydrolase, which can also catalyze the hydrolysis of citrulline to ornithine. Phylogenetic analysis showed that Lon22-like proteins form a novel clade that is separated from other guanidine-modifying enzymes. After rational design, the catalytic efficiencies of a Lon22 Y80F mutant for arginine and citrulline substrates were 2.31- and 4.70-fold that of the wild-type (WT), respectively. In addition, characterization of the Lon20-A4 adenylation domain suggested that it can incorporate both ornithine and lysine into the final products.     

H-NMR波谱潜手性基团质子氢的化学不等价探讨*

对1H-NMR谱中潜手性基团质子氢的化学不等价问题进行了详细归纳总结,并通过实例进行阐述,为系统讲授化学不等价内容提供材料补充和参考。     

Photochemical Reactions Involved in the Phototoxicity of the Anticonvulsant and Antidepressant Drug Lamotrigine (Lamictal®)

Lamotrigine (LTG) [3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine], an anticonvulsant and antidepressant drug Lamictal^®, produces a (photo)toxic response in some patients. LTG absorbs UV light, generating singlet oxygen (¹O₂) with a quantum yield of 0.22 in CH₂Cl₂, 0.11 in MeCN and 0.01 in D₂O. A small production of superoxide radical anion was also detected in acetonitrile. Thus, LTG is a moderate photosensitizer producing phototoxicity and oxidizing linoleic acid. LTG is a weak ¹O₂ quencher ( k _q = 3.2 × 10⁵  m ⁻¹ s⁻¹ in MeCN), but its photodecomposition products in dimethyl sulfoxide (DMSO) quenched ¹O₂ very efficiently. Upon intense UV irradiation from a xenon lamp, LTG was photobleached rapidly in DMSO and slowly in acetonitrile, alcohol and water. The rate increased significantly when laser pulses at 266 nm were employed. The photobleaching products generated ¹O₂ twice as strongly as LTG. Photobleaching was usually accompanied by the release of chloride anions, which increased in the presence of ascorbic acid. This suggests the formation of aryl radicals via dechlorination, a process which may be responsible for the photoallergic response observed in some patients. Our results demonstrate that LTG is a moderate generator of ¹O₂ prone to photodechlorination, especially in a reducing environment, which can contribute to the reported phototoxicity of LTG.     

Biotransformations of steroids to testololactone by a multifunctional strain Penicillium simplicissimum WY134-2

The biotransformations of a range of steroidal compounds, including 17α-hydroxy progesterone, progesterone, testosterone, androst-4-ene-3,17-dione (AD), pregnenolone, and dehydroepiandrosterone (DHEA), by Penicillium simplicissimum WY134-2 have been investigated. In all the cases, testolic acid and testololactone were detected, and the acid was converted to the lactone when pH was adjusted to 1, leading to isolation of testololactone in 25%–96% yields. Especially for progesterone and testosterone, the isolated yields were 93% and 96% with substrate concentration being 3 g/L, suggesting that P. simplicissimum WY134-2 may be used for the synthesis of testololactone. The results revealed the multi-functional catalytic activity of P. simplicissimum WY134-2 toward steroids for the first time. The possible reaction pathways of steroids promoted by this strain were discussed.     

Tuning the Properties of Graphdiyne by Introducing Electron-Withdrawing/Donating Groups

The properties of graphdiyne (GDY), such as energy gap, morphology, and affinity to alkali metals, can be adjusted by including electron-withdrawing/donating groups. The push–pull electron ability and size differences of groups play a key role on the partial property adjusting of GDY derivatives MeGDY, HGDY, and CNGDY. Cyano groups (electron-withdrawing) and methyl groups (electron-donating) decrease the band gap and increase the conductivity of the GDY network. The cyano and methyl groups affects the aggregation of GDY, providing a higher number of micropores and specific surface area. These groups also endow the original GDY additional advantages: the stronger electronegativity of cyano groups increase the affinity of GDY frameworks to lithium atoms, and the larger atomic volume of methyl groups increases the interlayer distance and provides more storage space and diffusion tunnels.     

Study of AcOH/HCOOH/H2O/CHCL3 Solvent System Application to the Separation of Two Large and Hydrophobic Fragments of Bacteriorhodopsin Membrane Protein by Centrifugal Partition Chromatography

Abstract

Centrifugal Partition Chromatography has been found useful for the separation of C₁ and C₂, two large hydrophobic peptides resulting from the chymotryptic cleavage of the membrane protein Bacteriorhodopsin. For this purpose the biphasic system AcOH/HCOOH/H₂O/CHCL₃ has been studied and the role of the two main parameters, i.e. Acetic acid and Formic acid, clearly demonstrated.     

8-Oxodeoxyguanosine Formation in the DNA of Cultured Cells After Exposure to H2O2 Alone or with UVB or UVA Irradiation

Abstract— The purpose of the study was to determine if aluminum phthalocyanine tetrasulfonate (A1PcS₄) photodynamic therapy (PDT) induced the formation of micronuclei in vitro and whether micronuclei formation was dependent on fiuence or cell type. NIH-3T3 and EMT-6 monolayer cultures were incubated in AlPcS₄ (0 or 1 μg/mL) for 24 h, received 0.0, 0.5,1.0 or 1.5 J/cm ² of 675 nm light, then reincubated and harvested at either 24, 48 or 72 h. The micronucleus frequency was determined in binucleated cells using the cytochalasin-block method. Cytotoxicity was assessed by using the 3(4,5-dimenthylthiazoyl-2-yl)2,5(diphenyl-tetrazolium bromide) (MTT) method. The effect of treatment on cell cycle progression was determined by calculating a proliferative index.
Aluminum phthalocyanine tetrasulfonate PDT induced a fluence-dependent increase in the frequency of micro-nuclei in NIH-3T3 and EMT-6 cells. The maximal effect of PDT was obtained in both cell lines 24 h after treatment. NIH-3T3 and EMT-6 cells exposed to a low fiuence of 0.5 J/cm² had a significantly lower number of micro-nuclei per cell 48 h following PDT treatment compared to the number of micronuclei per cell observed 24 h following treatment; however, when cells were exposed to a fluence (1.0 or 1.5 J/cm²) the number of micronuclei per cell did not diminish until 72 h after PDT treatment. The results obtained from the micronucleus assay paralleled those results obtained from the MTT assay.     

Deciphering the Mechanism Involved in the Switch On/Off of Molecular Pistons

Manufacturing machines converting energy to mechanical work at the molecular level is a vital pathway to explore the microscopic world. A kind of operable molecular engines, composed of β‐cyclodextrin (β‐CD), aryl, alkene and amide moiety was investigated using molecular dynamics simulations combined with free‐energy calculations. To understand how the integrated alkene double bond controls the work performed on the engines, two alkene isomers of the prototype were considered as two molecular engines. The free‐energy profiles delineating the binding process of the amide (Z)‐ and (E)‐isomers for each alkene isomer with 1‐adamantanol indicate that for the alkene (E)‐isomer, the apparent work performed on the amide bond is 1.6 kcal/mol, while the alkene (Z)‐isomer is incapable to perform work. Direct switch on/off of engines caused by the isomerization of the alkene bond was, therefore, witnessed, in line with experimental measurements. Decomposition of the free‐energy profile into different components and structural analyses suggest that the isomerization of the alkene bond controls the position of the aryl unit relative to the cavity of the CD, resulting in the difference among the free‐energy profiles and the stark contrast of the work performed on engines.     

中性载体ETH1117推动H+通过水/硝基苯界面传递的电化学研究

用循环伏安法研究中性载体ETH1117推动H⁺通过水/硝基苯界面的转移。通过络合使H⁺穿过水/硝基苯界面转移,是受扩散控制的可逆过程,ETH1117与H⁺在硝基苯相的配合比为1:1,配合物的稳定常数β₀为2.9×10⁷,扩散系数D为4.2×10^-6cm²/s。     

Proton/Mg2+ Co-Insertion Chemistry in Aqueous Mg-Ion Batteries: From the Interface to the Inner

Co-insertion of protons happens widely and enables divalent-ion aqueous batteries to achieve high performances. However, detailed investigations and comprehensive understandings of proton co-insertion are scarce. Herein, we demonstrate that proton co-insertion into tunnel materials is determined jointly by interface derivation and inner diffusion: at the interface, hdrated Mg²⁺ has poor insertion kinetics, and therefore accumulates and hydrolyzes to produce protons; in the tunnels, co-inserted/lattice H₂O molecules block the Mg²⁺ diffusion while facilitate the proton diffusion. When monoclinic vanadium dioxide (VO₂(B)) anode is tested in Mg(CH₃COO)₂ aqueous solution, the formation of Mg-rich solid electrolyte interphase on the VO₂(B) electrode and co-insertion of derived protons are probed; in the tunnels, the diffusion energy barrier of Mg²⁺+H₂O is 2.7 eV, while that of the protons is 0.37 eV. Thus, protons dominate the subsequent insertion and inner diffusion. As a consequence, the VO₂(B) achieves a high capacity of 257.0 mAh g⁻¹ at 1 A g⁻¹, a high rate retention of 59.1 % from 1 to 8 A g⁻¹, and stable cyclability of 3000 times with a capacity retention of 81.5 %. This work provides an in-depth understanding of the proton co-insertion and may promote the development of rechargeable aqueous batteries.     

Tuning the Properties of Graphdiyne by Introducing Electron‐Withdrawing/Donating Groups

The properties of graphdiyne (GDY), such as energy gap, morphology, and affinity to alkali metals, can be adjusted by including electron‐withdrawing/donating groups. The push–pull electron ability and size differences of groups play a key role on the partial property adjusting of GDY derivatives MeGDY, HGDY, and CNGDY. Cyano groups (electron‐withdrawing) and methyl groups (electron‐donating) decrease the band gap and increase the conductivity of the GDY network. The cyano and methyl groups affects the aggregation of GDY, providing a higher number of micropores and specific surface area. These groups also endow the original GDY additional advantages: the stronger electronegativity of cyano groups increase the affinity of GDY frameworks to lithium atoms, and the larger atomic volume of methyl groups increases the interlayer distance and provides more storage space and diffusion tunnels.     

Charge Movements in the 13-c/s Photocycles of the Bacteriorhodopsin Mutants R82K and R82Q*

We have examined light-induced currents in oriented membranes of the bacteriorhodopsin mutants R82K and R82Q. Our results suggest that two photocurrent components found in R82K, with 30 and 300 us lifetimes, are due to the photocycle of the 13-cis rather than the all-trans form of the pigment. We investigated the pH dependence of these components and their correspondence to absorbance changes at 660 nm characteristic of pho-tointermediates of the 13-cis cycle. The presence of a D₂O effect suggests that the charge motions producing these photocurrents are related to proton or protonated amino acid movement within the molecule. The current amplitudes depend on the protonation states of at least two residues, D85 and (probably) E204. In R82Q, a 10 pis photocurrent is observed that also depends on the protonation state of D85 and is similar to the 30 us current in R82K. We attempt to explain these currents in terms of a model for interacting residues in the extracellular half of the bacteriorhodopsin channel.     

Reorientational Motions of the D96N and T46V/D96N Mutants of Bacteriorhodopsin in the Purple Membrane

Abstract— The reorientational motions of the D96N and T46V/D96N mutants of bacteriorhodopsin in purple membrane have been investigated by time-resolved linear dichroism measurements. The reorientations in the early stages of the photocycle are identical to those observed in wild-type bacteriorhodopsin: anisotropics of photocycle intermediates in both D96N and T46V/D96N are r_K= 0.38±0.01, r_L= 0.35±0.01, r_M= 0.35±0.01. The anisotropy of the initial state, r_BR, exhibits decays to zero in D96N and to negative values in T46V/D96N on the time scale of tens of milliseconds. This anisotropy decay can be explained by a model that involves the motion of unexcited or spectator proteins adjacent to a photocycling protein. The amplitude and time constants of spectator reorientational motion are similar to those that have been observed in the wild type. Contributions from the anisotropy of the N-state were detected in measurements of the T46V/D96N mutant, in which a large N-state population accumulates. The value of r_N is estimated to be 0.30±0.05 in T46V/D96N.     

Positional and Quantitative Characterization of the Hydroxyethyl Groups in Hydroxyethyl Starch by GC/MS or NMR

Hydroxyethyl starches (HES) have been used medically as plasma volume expanders. They are composed of a starch backbone and substituted hydroxyethyl groups. The backbone is ?(1,4)-glycosidic-linked anhydroglucose units, which has branches formed by ?(1,6)-glycosidic bonds. Hydroxyethylation can take place at the C-2, C-3 and C-6 sites of the glucose rings, as well as at the hydroxyl groups of the substituted hydroxyethyl groups. Variation of the position and quantity of the substituted …     

Aggregation of the Light-Harvesting Complex in Intact Leaves of Tobacco Plants Stressed by CO2 Deficit

Pronounced aggregation of the photosystem II light-harvesting complex (LHC II) was observed in low-lightgrown tobacco plants stressed with a strong CO₂ deficit for 2–3 days. The LHC II aggregates showed a typical band at 697–700 nm (F₆₉₉) in low-temperature emission spectra. Its excitation spectrum corresponded to that of detergent-solubilized LHC II. Formation of F₆₉₉ in stressed plants was not reversed in the dark and leaves did not contain any zeaxanthin showing that neither a light-induced transthylakoid pH gradient nor zeaxanthin was required for LHC II aggregation. The CO₂-stressed plants showed clear signs of photodamage: depression of the potential yield of photosystem II photochemistry (F,/F_M) by 50–70% and a decline in chlorophyll content by 10–15%. Therefore, we propose that the photodamage to the photosynthetic apparatus is the cause of the LHC II aggregation in plants. The F₆₉₉ exhibited a reversible decrease of its intensity upon irradiation of leaves with intensive light. There was no or only slight decrease around 700 nm in unstressed plants. The nonphotochemical quenching of chlorophyll fluorescence showed the opposite relation, being higher before than after the strong CO₂ deficit. This discrepancy was likely related to the different LHC II aggregation state in control and stressed plants.     

The Infestation of Olive Fruits by Bactrocera oleae (Rossi) Modifies the Expression of Key Genes in the Biosynthesis of Volatile and Phenolic Compounds and Alters the Composition of Virgin Olive Oil

Bactrocera oleae, the olive fruit fly, is one of the most important pests affecting the olive fruit, causing serious quantitative and qualitative damage to olive oil production. In this study, the changes induced by B. oleae infestation in the biosynthesis of volatile and phenolic compounds in olive (cvs. Picual, Manzanilla, and Hojiblanca) have been analyzed. Despite cultivar differences, the oils obtained from infested fruits showed a significant increase in the content of certain volatile compounds such as (E)-hex-2-enal, ethanol, ethyl acetate, and β-ocimene and a drastic decrease of the phenolic contents. The impact of those changes on the inferred quality of the oils has been studied. In parallel, the changes induced by the attack of the olive fly on the expression of some key genes in the biosynthesis of volatile and phenolic compounds, such as lipoxygenase, β-glucosidase, and polyphenol oxidase, have been analyzed. The strong induction of a new olive polyphenol oxidase gene (OePPO2) explains the reduction of phenolic content in the oils obtained from infested fruits and suggest the existence of a PPO-mediated oxidative defense system in olives.     

Characterization of Diacylglycerylphosphocholine Molecular Species by FAB-CAD-MS/MS: A General Method Not Sensitive to the Nature of the Fatty Acyl Groups

The use of negative ion mode fast-atom bombardment-collision-activated dissociation-tandem mass spectrometry (FAB-CAD-MS/MS) for diacylglycerylphosphocholine molecular species determinations was investigated for 24 naturally occurring and synthetic compounds. The previously proposed method of selecting [M-15]^? as the parent ion and using the relative abundance of the carboxylate daughter ions to distinguish the positions of esterification was found to be unreliable in cases where the fatty acyl group at sn1 was much larger than that at sn2. The predicted greater abundance of the sn2 carboxylate daughter, relative to the sn1 carboxylate daughter, was also violated when polyunsaturated fatty acyl groups were esterifred at sn2. In addition, several marginal cases were found where the ratio of intensities of the sn2/sn1 carboxylate daughters followed the expected pattern (sn2 > sn1) initially, but reversed over extended scanning time. The use of an alternative FAB-CAD-MS/MS method is proposed where the [M-B6]^? ion is selected as the precursor and the relative intensities of the daughters resulting from loss of the free fatty acids at snl and sn2 are determined. In every case examined to date, the ion formed by loss of the free acid from the sn2 position was always more abundant. Because the parent ion is equivalent to the phosphatidic acid ion, this technique should be equally applicable to all other phospholipid classes where this fragment ion is present in the spectrum.