ProDOL: a general method to determine the degree of labeling for staining optimization and molecular counting.

Determining the label to target ratio, also known as the degree of labeling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labeling is beneficial for fluorescence microscopy in general. Yet robust, versatile and easy-to-use tools for measuring cell-specific labeling efficiencies are not available. Here we present a DOL determination technique named protein-tag DOL (ProDOL), which enables fast quantification and optimization of protein-tag labeling. With ProDOL various factors affecting labeling efficiency, including substrate type, incubation time and concentration, as well as sample fixation and cell type can be easily assessed. We applied ProDOL to investigate how human immunodeficiency virus-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adapter protein SLP-76 in signaling microclusters. ProDOL proved to be a versatile and robust tool for labeling calibration, enabling determination of labeling efficiencies, optimization of strategies and quantification of protein stoichiometry.

Substantial Differences in Proanthocyanidin Contents among Ginkgo biloba Leaf Extracts in Herbal Medicinal Products Obtained from the German Market.

Pharmacologic activity of proanthocyanidins in Ginkgo biloba leaf extract has recently been reported. The objective of the present study was to screen proanthocyanidin contents in herbal medicinal products containing Ginkgo extracts. A recently published HPLC method for quantification of proanthocyanidins in G. biloba leaf extract EGb 761 was adopted to also be suitable for finished herbal medicinal products. The method was applied to 14 products from the German market. For each product, a set of three individual batches was purchased and analyzed. Substantial differences in proanthocyanidins contents were found among distinct products, ranging from 0.30 to 5.86%. The batch-to-batch variability within each product was low. The highest concentrations are in a similar range as, for example, the amount of Ginkgo terpene trilactones specified in the monograph for G. biloba leaf extract in the European Pharmacopeia. Although it has not yet been established whether and to what extent proanthocyanidins contribute to the overall pharmacological or clinical efficacy of Ginkgo extracts, a potential impact on the purported benefits of different contents in proanthocyanidins cannot be ruled out. Quality assessment of different Ginkgo extracts in the future may include proanthocyanidins.

Aggregation of N-Heteropolycyclic Aromatic Molecules: The Acridine Dimer and Trimer.

Polycyclic aromatic hydrocarbons and their nitrogen-substituted analogues are of great interest for various applications in organic electronics. The performance of such devices is determined not only by the properties of the single molecules, but also by the structure of their aggregates, which often form via self-aggregation. Gaining insight into such aggregation processes is a challenging task, but crucial for a fine-tuning of the materials properties. In this work, an efficient approach for the generation and characterisation of aggregates is described, based on matrix-isolation experiments and quantum-chemical calculations. This approach is exemplified for aggregation of acridine. The acridine dimer and trimer are thoroughly analysed on the basis of experimental and calculated UV and IR absorption spectra, which agree well with each other. Thereby a novel structure of the acridine dimer is found, which disagrees with a previously reported one. The calculations also show the changes from excitonic coupling towards orbital interactions between two molecules with decreasing distance to each other. In addition, a structure of the trimer is determined. Finally, an outlook is given on how even higher aggregates can be made accessible through experiment.

Isolated Dimers Versus Solid-State Dimers of N-Heteropolycycles: Matrix-Isolation Spectroscopy in Concert with Quantum Chemistry.

In this work, matrix-isolation spectroscopy and quantum-chemical calculations are used together to analyse the structure and properties of weakly bound dimers of the two isomers benzo[a]acridine and benzo[c]acridine. Our measured experimental electronic absorbance spectra agree with simulated spectra calculated for the equilibrium structures of the dimers in gas-phase, but in contrast, disagree with the simulated spectra calculated for the structures obtained by optimising the experimental solid-state structures. This highlights the sensitivity of the electronic excitations with respect to the dimer structures. The comparison between the solid-state and gas-phase dimers shows how far the intermolecular interactions could change the geometric and electronic structure in a disordered bulk material or at device interfaces, imposing consequences for exciton and charge mobility and other material properties.

A Detailed View on the Proanthocyanidins in Ginkgo Extract EGb 761.

The Ginkgo extract EGb 761® manufactured with leaves of Ginkgo biloba has been continuously produced over decades at a large scale and is used as a clinically proven remedy for, among other things, the improvement of age-associated cognitive impairment and quality of life in patients with mild dementia. It belongs to the class of extracts addressed as quantified extracts according to the European Pharmacopeia. Accordingly, several compounds (e.g., flavone glycosides and terpene trilactones) are acknowledged to contribute to its clinical efficacy. Covering only about 30% of the mass balance, these characterized compounds are accompanied by a larger fraction of additional compounds, which might also contribute to the clinical efficacy and safety of the extract. As part of our systematic research to fully characterize the constituents of Ginkgo extract EGb 761, we focus on the structural class of proanthocyanidins in the present study. Structural insights into the proanthocyanidins present in EGb 761 and a quantitative method for their determination using HPLC are shown. The proanthocyanidins were found to be of oligomeric to polymeric structure, which yield delphinidin and cyanidin as main building blocks after acidic hydrolysis. A validated HPLC method for quantification of the anthocyanidins was developed in which delphinidin and cyanidin were detected after hydrolysis of the proanthocyanidins. The content of proanthocyanidins in Ginkgo extract EGb 761 was found to be approximately 7%.

HPLC-UV/HRMS methods for the unambiguous detection of adulterations of Ginkgo biloba leaves with Sophora japonica fruits on an extract level.

CONTEXT: Ginkgo biloba L. (Ginkgoaceae) leaf extract is one of the most frequently sold herbal extracts. There have been reports on poor quality and adulteration of ginkgo leaf extracts or the powdered plant material with extracts or powder of Styphnolobium japonicum (L.) Schott (Fabaceae) (syn. Sophora japonica L.) fruits, which is rich in flavone glycosides. OBJECTIVE: The study investigates whether ginkgo leaves genuinely contain genistein and sophoricoside and whether these two substances could be used as markers to detect adulterations with sophora fruits. MATERIALS AND METHODS: A total of 33 samples of dried ginkgo leaves were sourced from controlled plantations in China, the USA, and France. After extraction, the samples were analyzed using two high-performance liquid chromatography (HPLC) coupled with UV/HRMS methods for the detection of genistein and sophoricoside, respectively. Chromatograms were compared to standard reference materials. RESULTS: In none of the tested ginkgo samples, neither genistein nor sophoricoside could be detected. The applied method was designed to separate genistein from apigenin. The latter is a genuine compound of ginkgo leaves, and its peak may have been previously misidentified as genistein because of the same molecular mass. The method for the detection of sophoricoside allows identification of the adulteration with sophora fruit without prior hydrolysis. By both HPLC methods, it was possible to detect adulterations of ≥2% sophora fruits in the investigated ginkgo extract. CONCLUSION: The methods allow unambiguous detection of adulterations of ginkgo leaves with sophora fruits, using genistein and sophoricoside as marker compounds.

High-Resolution Electronic Excitation and Emission Spectra of Pentacene and 6,13-Diazapentacene Monomers and Weakly Bound Dimers by Matrix-Isolation Spectroscopy.

N-Heteropolycycles are among the most promising candidates for applications in organic devices. For this purpose, a profound understanding of the low-energy electronic absorbance and emission characteristics is of crucial importance. Herein, we report high-resolution absorbance and fluorescence spectra of pentacene (PEN) and 6,13-diazapentacene (DAP) in solid neon obtained using the matrix-isolation technique. Accompanying DFT calculations allow the assignment of specific vibrationally resolved signals to corresponding modes. Furthermore, we present for the first time evidence for the formation of van der Waals dimers of both substances. These dimers exhibit significantly different optical characteristics resulting from the change of electronic properties evoked by the incorporation of sp2 nitrogen into the molecular backbone.

Benzopyranones and their sulfate esters from Pelargonium sidoides.

Investigation of the benzopyranones from the roots of PELARGONIUM SIDOIDES led to the identification of 15 benzopyranones, among them the novel compounds 12 and 14. Furthermore, benzopyranones 4 and 15 were detected in PELARGONIUM SIDOIDES for the first time. Structure determinations were performed by one- and two-dimensional NMR spectroscopy. An HPLC system for the discrimination of these benzopyranones has been developed.

A detailed view on the constituents of EPs 7630.

Extracts of PELARGONIUM SIDOIDES, commonly known as EPs 7630, are produced by extraction of milled roots with 11 % (w/w) ethanol in water. This solvent leads to a spectrum of constituents which differs significantly from extracts obtained by extraction with non-polar solvents. EPs 7630 is composed of six main groups of constituents, namely unsubstituted and substituted oligomeric prodelphinidins, monomeric and oligomeric carbohydrates, minerals, peptides, purine derivatives and highly substituted benzopyranones. The oligoprodelphinidins, frequently supposed to be compounds with unspecific tanning interactions, show, in contrast to other polyphenols, an amazing variety of substructures and connectivities which results in an uncommon diversity even at a low degree of polymerization. Three distinct purine derivatives, second messengers and probably intermediates of DNA synthesis, were identified and characterized by phytochemical means. The main benzopyranones of EPs 7630 are highly oxygenated at the phenyl moiety (three to four oxygens) and in addition sulfated at distinct positions. A disulfate of 6,7,8-trihydoxybenzopyranone has been identified for the first time in plants. Taken together, these constituents amount to about 70 to 80 % of the total weight of EPs 7630, the active ingredient of the phytopharmaceutical Umckaloabo (Iso Arzneimittel Ettlingen).