Using hydroxymethylphenoxy derivates with the SPOT technology to generate peptides with authentic C-termini

The SPOT technology can fulfill most requirements for highly parallel, multiple peptide synthesis of soluble peptides within the upper microgram range. Here, we report on an improved method using hydroxymethylphenoxyacetic acid (HMPA) for 19 amino acids and 4-(4-hydroxymethyl-3-methoxyphenoxy)-butyric acid (HMPB) for proline as acidic labile linkers in SPOT synthesis. Using this approach we could reduce side-chain reactions normally occurring during conventional alkaline peptide cleavage from cellulose membranes. All synthesis steps were adapted to fully-automated SPOT synthesis and therefore represent a time- and cost-saving procedure. Furthermore, the improved cleavage and washing steps resulted in peptides with authentic C-termini in a purity range of 60–95%. Our improved method is ideal for synthesizing many thousand different peptides subsequently used directly for different biological assays requiring authentic C-termini, such as CD8 T-cell epitope screening, vaccine immunization, or tumor imaging.     

Siderotyping of fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis>: molecular mass determination by mass spectrometry as a powerful pyoverdine siderotyping method

The numerous pyoverdines so far characterized as siderophores of fluorescent Pseudomonas could be usually differentiated one from each others by the two physico-chemical and physiological methods of siderotyping, i.e., siderophore-isoelectrofocusing and siderophore-mediated iron uptake. As shown in the present paper, the structural diversity of the peptide chain characterizing these molecules results in a very large panel of molecular masses representing 64 different values ranging from 889 to 1,764 Da for the 68 compounds included in the study, with only a few structurally different compounds presenting an identical molecular mass. Thus, the molecular mass determination of pyoverdines through mass spectrometry could be used as a powerful siderotyping method.     

In vivo optical imaging of neurogenesis: watching new neurons in the intact brain

Adult neurogenesis is a highly dynamic process modulated by several pathologic and environmental factors, as well as by various compounds. So far, available techniques to study neurogenesis are lengthy and personnel and cost intensive. We developed a new tool based on the doublecortin promoter driving the expression of the luciferase reporter gene (DCX-promo-luciferase) in transgenic mice to perform in vivo imaging of neurogenesis. Indeed, the DCX-promo-luciferase mice allowed optical in vivo imaging of the onset of and increase in neurogenesis in developing fetal brains, as well as imaging of neurogenesis in the intact adult mouse central nervous system. Moreover, the capacity to specifically detect a small number of migrating neuronal precursors in vivo after transplantation is for the first time feasible using this DCX-promo-luciferase transgenic tool. The present imaging approach offers several crucial advantages over methods currently available, such as bromodeoxyuridine incorporation or labeling using iron oxide nanoparticles. Hence, it allows longitudinal study of neurogenesis in intact animals without the requirement of cellular prelabeling. Moreover, it guarantees that detection is specific for neuronal precursors and restricted to viable cells. Hence, our DCX-promo-luciferase transgenic model constitutes an effective tool that answers the pressing need for rapid investigation of the impact on neurogenesis of a large number of candidate compounds waiting to be tested.     

Oxygen transfer in intensive microbial culture

Oxygen transfer performances in intensive microbial cultures are compared with those occuring in coalescing and non-coalescing mineral media. E. coli fed-batch cultures are carried out in a 22 L bioreactor. Biomass concentrations of 80 g(DW) L(-1) are reached, with oxygen consumption rates of up to 0.6 mol L(-1) h(-1). To achieve these high transfer performances, dissipated power e reaches 35 kW m(-3). The hold-up in the culture broth and in the corresponding supernatant matches the non-coalescing mineral medium. Oxygen transfer coefficients, K (L) a in mineral media, and K (T) in the culture broth, are compared. K (T), calculated online from a gas balance method, excesses 1 s(-1). Yet, for given values of e, K (T) is 4-8 times lower than K (L) a determined in the non-coalescing mineral medium. The cell activity modifies the chemical medium properties and reduces the oxygen transfer conductance, as in a non-coalescing ionic medium containing surfactant.     

Environmental Indication: A Field Test of an Ecosystem Approach to Quantify Biological Self-Organization

In this study, we take an ecosystem approach to examine the degree of biological self-organization at the ecosystem level. An integrated set of indicators is derived from a theoretical framework and tested by field data from an ecosystem research project focusing on the Bornhöved Lake district in northern Germany. This field test is based on a comparison of the self-organized phenomena that comprise the carbon, water, and energy budgets of two adjacent edaphically and climatically similar ecosystems, that have vastly different levels of human interference—a crop field and a beech forest. In terms of biomass storage, biologically incorporated nitrogen and phosphorus, species number, total ecosystem respiration per total biomass (qCO₂), total ecosystem assimilation per available nutrients, and transpiration per total evapotranspiration, we found clear differences between the systems. Ecosystem surface temperature and R_n/K* were found to be of limited utility in characterizing the two systems. The study is rooted in the concept of ecological integrity, an influential idea at the interface of ecological and environmental debate that has acquired a number of different meanings. Among other interpretations, it can be viewed as a guiding principle for sustainable land use that aims at long-term protection of ecological life-support systems. Effective use of any interpretation of this concept requires a theoretically consistent and applicable set of indicators. Therefore, we also discuss the integration of the indicator set and its potential use in monitoring programs.     

Sarmatian (Late Middle Miocene) gastropod assemblages of the Central Paratethys

Summary A rather diverse gastropod fauna from Sarmatian deposits of the Austrian/Hungarian Eisenstadt-Sopron Basin was studied. The fauna derives from two layers of clay and silt within a siliciclastic section at St. Margarethen in Burgenland (Austria). These layers are interpreted as littoral mudflats which formed during the Sarmatian (Late Middle Miocene) along the western coast of the Central Paratethys. Strong shifts in the composition of the gastropod fauna, dominated by Potamididae (Cenogastropoda: Cerithioidea), within each layer indicate successions of limnic-fluvial to oligohaline, brackish-littoral, and marine-littoral environments. These shifts in facies are reflected by an alternational of thePotamides hartbergensis assemblage,Granulolabium bicinctum assemblage, and thePotamides disjunctus assemblage. The speciesJujubinus turriculus (Eichwald, 1850),Gibbula buchi (Dubois, 1831), andCylichnina elongata (Eichwald, 1830) are reported for the first time from the Sarmatian of the Paratethys.Mitrella agenta nov. sp. (Neogastropoda: Columbellidae) is introduced as a new species. These species might represent relics of the diverse Badenian fauna but could also prove a minor ingression of marine species from an adjacent bioprovince in the Late SarmatianMactra “Zone”.     

Autocatalytic peptide cyclization during chain folding of histidine ammonia-lyase.

Histidine ammonia-lyase requires a 4-methylidene-imidazole-5-one group (MIO) that is produced autocatalytically by a cyclization and dehydration step in a 3-residue loop of the polypeptide. The crystal structures of three mutants have been established. Two mutants were inactive and failed to form MIO, but remained unchanged elsewhere. The third mutant showed very low activity and formed MIO, although it differed from an MIO-less mutant only by an additional 329-C(beta) atom. This atom forms one constraint during MIO formation, the other being the strongly connected Asp145. An exploration of the conformational space of the MIO-forming loop showed that the cyclization is probably enforced by a mechanic compression in a late stage of chain folding and is catalyzed by a well-connected internal water molecule. The cyclization of the respective 3-residue loop of green fluorescent protein is likely to occur in a similar reaction.