Comprehensive and accurate tracking of carbon origin of LC-tandem mass spectrometry collisional fragments for 13C-MFA.

In recent years the benefit of measuring positionally resolved 13C-labeling enrichment from tandem mass spectrometry (MS/MS) collisional fragments for improved precision of 13C-Metabolic Flux Analysis (13C-MFA) has become evident. However, the usage of positional labeling information for 13C-MFA faces two challenges: (1) The mass spectrometric acquisition of a large number of potentially interfering mass transitions may hamper accuracy and sensitivity. (2) The positional identity of carbon atoms of product ions needs to be known. The present contribution addresses the latter challenge by deducing the maximal positional labeling information contained in LC-ESI-MS/MS spectra of product anions of central metabolism as well as product cations of amino acids. For this purpose, we draw on accurate mass spectrometry, selectively labeled standards, and published fragmentation pathways to structurally annotate all dominant mass peaks of a large collection of metabolites, some of which with a complete fragmentation pathway. Compiling all available information, we arrive at the most detailed map of carbon atom fate of LC-ESI-MS/MS collisional fragments yet, comprising 170 intense and structurally annotated product ions with unique carbon origin from 76 precursor ions of 72 metabolites. Our 13C-data proof that heuristic fragmentation rules often fail to yield correct fragment structures and we expose common pitfalls in the structural annotation of product ions. We show that the positionally resolved 13C-label information contained in the product ions that we structurally annotated allows to infer the entire isotopomer distribution of several central metabolism intermediates, which is experimentally demonstrated for malate using quadrupole-time-of-flight MS technology. Finally, the inclusion of the label information from a subset of these fragments improves flux precision in a Corynebacterium glutamicum model of the central carbon metabolism.

Distinct purinergic signaling pathways in prepubescent mouse spermatogonia.

Spermatogenesis ranks among the most complex, yet least understood, developmental processes. The physiological principles that control male germ cell development in mammals are notoriously difficult to unravel, given the intricate anatomy and complex endo- and paracrinology of the testis. Accordingly, we lack a conceptual understanding of the basic signaling mechanisms within the testis, which control the seminiferous epithelial cycle and thus govern spermatogenesis. Here, we address paracrine signal transduction in undifferentiated male germ cells from an electrophysiological perspective. We identify distinct purinergic signaling pathways in prepubescent mouse spermatogonia, both in vitro and in situ. ATP-a dynamic, widespread, and evolutionary conserved mediator of cell to cell communication in various developmental contexts-activates at least two different spermatogonial purinoceptor isoforms. Both receptors operate within nonoverlapping stimulus concentration ranges, display distinct response kinetics and, in the juvenile seminiferous cord, are uniquely expressed in spermatogonia. We further find that spermatogonia express Ca(2+)-activated large-conductance K(+) channels that appear to function as a safeguard against prolonged ATP-dependent depolarization. Quantitative purine measurements additionally suggest testicular ATP-induced ATP release, a mechanism that could increase the paracrine radius of initially localized signaling events. Moreover, we establish a novel seminiferous tubule slice preparation that allows targeted electrophysiological recordings from identified testicular cell types in an intact epithelial environment. This unique approach not only confirms our in vitro findings, but also supports the notion of purinergic signaling during the early stages of spermatogenesis.

Chemoenzymatic synthesis of the chiral side-chain of statins: application of an alcohol dehydrogenase catalysed ketone reduction on a large scale.

The chemoenzymatic synthesis of the tert-butyl (S)-6-chloro-5-hydroxy-3-ketohexanoate is described. Our approach relies on a highly regio- and enantioselective reduction of a beta,delta-diketohexanoate ester catalysed by NADP(H)-dependent alcohol dehydrogenase of Lactobacillus brevis (LBADH). A detailed description of the scale-up of the enzymatic synthesis of the hydroxyketo ester is given which includes a scale-up of the substrate synthesis as well, i.e. the preparation of diketo ester on a 100 g scale. Furthermore, studies directed towards improving the co-catalyst [NADP(H)] consumption of the enzymatic key step by kinetic studies and application of a biphasic reaction medium were performed.

Separation of genomic DNA, RNA, and open circular plasmid DNA from supercoiled plasmid DNA by combining denaturation, selective renaturation and aqueous two-phase extraction.

In the current study we developed a process for the capture of pDNA exploiting the ability of aqueous two-phase systems to differentiate between different forms of DNA. In these systems scpDNA exhibits a near quantitative partitioning in the salt-rich bottom phase. The successive recovery from the salt rich bottom phase is accomplished by a novel membrane step. The polish operation to meet final purity demands is again based on a system exploiting a combination of the denaturation of the nucleic acids present, specific renaturation of scpDNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as ocpDNA and genomic host DNA. This polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids which up to date otherwise only can be accomplished with much more cumbersome chromatographic methods. In a benchmark comparison, it could be shown that the newly developed process exhibits a comparable yield to an industrial standard process while at the same time showing superior performance in terms of purity and process time. Additionally it could be shown that the developed polish procedure can be applied as a standalone module to support already existing processes.

Exploitation of the coil-globule plasmid DNA transition induced by small changes in temperature, pH salt, and poly(ethylene glycol) compositions for directed partitioning in aqueous two-phase systems.

In this study, the interplay of two linked equilibria is examined, one concerning an aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and salt employed to partition plasmid DNA (pDNA), and the other a potential structural transition of pDNA depending on PEG and salt concentration and other system parameters. The boundary conditions for pDNA partitioning are set by PEG and salt concentrations, PEG molecular weight, pH, and temperature. While investigating these parameters, it was found that a small increase/decrease of the respective values led to a drastic and significant change in pDNA behavior. This behavior could be attributed to a coil-globule transition of the pDNA triggered by the respective phase conditions. The combination of this structural change, aggregation effects linked to the transition process, and the electrostatic potential difference found in PEG-salt systems thus offers a sensitive way to separate nucleic acid forms on the basis of their unique property to undergo coil-globule transitions under distinct system properties.