Introduction of axial chirality in a planar aromatic ligand results in chiral recognition with DNA

Dimerization of a hydroxycarbazole produces an axially chiral biaryl, BICOL ( 2 ). One enantiomer (R)‐ 2 , is capable of enantioselective binding to different polymorphs of DNA. The biaryl (R)‐ 2 was shown by fluorescence and circular dichroism to induce a shift of Z‐DNA to B‐DNA. The opposite enantiomer (S)‐ 2 shows no specific binding. The significant difference in behaviour between the two enantiomers (S)‐ 2 and (R)‐ 2 is in line with molecular modelling studies which show two very different binding geometries between the enantiomers with each polymorph of DNA. Copyright © 2010 John Wiley & Sons, Ltd.     

Chromatographic separation and spectroscopic characterization of the e/z isomers of acrivastine

A reverse phase high performance liquid chromatography (HPLC) method has been developed for the separation of two geometric isomers of Acrivastine using crude reaction mixture. The resolution between two isomers was found more than 2.9. The geometric isomers have been isolated by preparative HPLC and characterized by spectroscopic techniques, such as NMR, infrared, and MS. The developed method has been validated for the determination of Z‐isomer in Acrivastine. The limit of detection and limit of quantification of the Z‐isomer were 0.05 and 0.2 μg/ml, respectively. The developed method is precise, linear, accurate, rugged and robust for its intended use. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

四川天南星属(天南星科)一新种——川西南星

描述了四川西部天南星属(天南星科)植物一新种,即川西天南星.     

Glycan analysis of glycoprotein pharmaceuticals: Evaluation of analytical approaches to Z number determination in pharmaceutical erythropoietin products

N-Glycosylation of many glycoprotein drugs is important for biological activity and should therefore be the target of specific and quantitative analytical methods. In this study, we focus on the two N-glycan mapping approaches that are used in pharmacopoeial monograph to analyse N-glycans released from fifteen preparations of recombinant human erythropoietin supplied by ten Chinese manufacturers. Underivatised N-glycans were analysed by high performance anion-exchange chromatography with pulsed amperometric detection and fluorophore-labelled N-glycans were analysed by weak anion-exchange and normal-phase high performance liquid chromatography. N-glycans were also analysed by matrix assisted laser desorption ionisation mass spectrometry. The release of N-glycans by PNGase F was shown to be consistent. Z number, a mathematical expression of the total negatively charged N-glycans composition has provided a convenient way to summarise the complex dataset and it might be suitable for product consistency monitoring. However, this Z number reduces the information of individual acidic N-glycan structure and is also found to be method dependent. Therefore, its use requires clear specification and validation. In this study, we only found weak but positive correlation between the Z number and its bioactivity. Wide range of N-glycans yields were obtained from the fifteen preparations but the significance of their differences is unclear.     

Chemical synthesis and evaluation of a backbone‐cyclized minimized 2‐helix Z‐domain

The Z‐molecule is a small, engineered IgG‐binding affinity protein derived from the immunoglobulin‐binding domain B of Staphylococcus aureus protein A. The Z‐domain consists of 58 amino acids forming a well‐defined antiparallel three‐helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three‐helix bundle. The small size and the stable three‐helix structure are two attractive properties comprised in the Z‐domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein‐based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z‐domain and joining the N‐ and C‐termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two‐helix Z‐domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N‐ and C‐termini. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

The functional sites of chlorophylls in D1 and D2 subunits of Photosystem II identified by pulsed EPR

The functional site of Chl_Z, an auxiliary electron donor to P680⁺, was determined by pulsed ELDOR applied to a radical pair of Y_D^• and Chlz⁺ in oriented PS II membranes from spinach. The radical-radical distance was determined to be 29.5 Å and its direction was 50° from the membrane normal, indicating that a chlorophyll on the D2 protein is responsible for the EPR Chlz⁺ signal. Spin polarized ESEEM (Electronin Spin Echo Envelop Modulation) of a ³Chl and Q_A⁻ radical pair induced by a laser flash was observed in reaction center D1D2Cytb₅₅₉ complex, in which Q_A was functionally reconstituted with DBMIB and reduced chemically. Q_A⁻ESEEM showed a characteristic oscillating time profile due to dipolar coupling with ³Chl. By fitting with the dipolar interaction parameters, the distance between ³Chl and Q_A⁻ was determined to be 25.9 Å, indicating that the accessory chlorophyll on the D1 protein is responsible for the ³Chl signal.     

Mapping of origin of replication in Themococcales

Genome replication is a crucial and essential process for the continuity of life.In all organisms it starts at a specific region of the genome known as origin of replication (Ori) site. The number of Ori sites varies in prokaryotes and eukaryotes. Replication starts at a single Ori site in bacteria, but in eukaryotes multiple Ori sites are used for fast copying across all chromosomes. The situation becomes complex in archaea, where some groups have single and others have multiple origins of replication. Themococcales, are a hyperthermophilic order of archaea. They are anaerobes and heterotrophs-peptide fermenters, sulphate reducers, methanogens being some of the examples of metabolic types. In this paper we have applied a combination of multiple in silico approaches - Z curve, the cell division cycle (cdc6) gene location and location of consensus origin recognition box (ORB) sequences for location of origin of replication in Thermococcus onnurineus, Thermococcus gammatolerans and other Themococcales and compared the results to that of the well-documented case of Pyrococcus abyssi. The motivation behind this study is to find the number of Ori sites based on the data available for members of this order. Results from this in silico analysis show that the Themococcales have a single origin of replication.     

Feeding and hemolymph trehalose concentration influence sex pheromone production in virgin Heliothis virescens moths

Previously, we demonstrated that sex pheromone production in mated female Heliothis virescens moths is dependent upon hemolymph trehalose concentration (HTC), which is influenced by activities such as the feeding of adults on sucrose. In this paper we demonstrate, for the first time, that this effect also occurs in starved (i.e., sugar-stressed) virgin females. Females allowed to feed on sugar for 6 days, following eclosion, had significantly greater titers than females that had fed only on water (i.e., were starved). No differences in pheromone titer were observed between sugar- and water-fed females at shorter (1 or 3 days) periods following eclosion. The relatively short-term effects of HTC on sex pheromone titer of virgins, were demonstrated by feeding experiments, in which starved (for 4 days) virgins fed on 10% sucrose solution had significantly greater HTC and pheromone titers than ones fed only on water; an increase in HTC was apparent within an hour, while the increase in pheromone titer was apparent within 2.5 h, of sugar feeding. Starvation also showed similar effects on titers of pheromone gland fatty acids (pheromone intermediates) and HTC. Over 6 days of starvation, fatty acid titers and HTC declined gradually. After feeding on sucrose, titers of hexadecanoic, (Z)-9-hexadecanoic, (Z)-11-hexadecanoic and (Z)-9-octadecanoic, acids, as well as HTC, increased significantly 24 h later, but titers of octadecanoic and (Z,Z)-9,12-octadecanoic (linoleic) acids did not. Lepidoptera cannot biosynthesize polyunsaturated acids, but the lack of change in octadecanoic acid titer suggests this acid may not participate in pheromone biosynthesis. In addition to these short-term changes in pheromone and fatty acid production, mediated by HTC, a longer-term effect of age, regardless of HTC, on pheromone titer was observed. Overall, these results are consistent with hemolymph trehalose and glandular fatty acids acting as twin metabolite reservoirs for pheromone biosynthesis. Hemolymph trehalose, able to be refilled through feeding on exogenous sugars, has a one-way flow of metabolites for synthesis of glandular free fatty acids (FFAs) and pheromone, while glandular glycerolipids provide a reversible reservoir for metabolites, accepting surplus FFAs when glandular concentrations are high, and providing FFAs for pheromone biosynthesis when concentrations are low.     

New milliliter‐scale stirred tank bioreactors for the cultivation of mycelium forming microorganisms

A novel milliliter‐scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter‐scale. A newly designed one‐sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface‐to‐volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k_La) > 0.15 s^?1 were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter‐scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory‐scale stirred tank bioreactor with six‐bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter‐scale stirred tank bioreactor was reduced compared to the laboratory‐scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale‐up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter‐scale (standard deviation < 8%) with up to 48 stirred tank bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear‐thinning non‐Newtonian behavior. The newly developed one‐sided paddle impellers operated in unbaffled reactors on a 10 milliliter‐scale with a magnetic inductive drive for up to 48 parallel bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically. Biotechnol. Bioeng. 2010; 106: 443–451. © 2010 Wiley Periodicals, Inc.     

贵州悬钩子属(蔷薇科)一新种--务川悬钩子

报道了贵州省务川县发现的悬钩子属Rubus（蔷薇科Rosaceae）一新种——务川悬钩子R．wuchuanensis S．Z．He。该种在体态上与锯叶悬钩子R．wuzhianus L．T．Lu＆Boufford相近,区别在于其叶片卵状披针形,边缘疏生小锯齿;叶柄较短,长0．7-1cm;花序为顶生稀疏总状花序,具花8-10朵：花梗长3．55cm;花瓣先端具骤突尖头。