Characterisation of the thiol–disulphide chemistry of desmopressin by LC, μ-LC, LC-ESI-MS and Maldi-Tof

Summary. To date, the majority of therapeutic peptides and proteins have to be administered via parenteral routes, which are painful and inconvenient. In order to gain sufficient high blood concentrations after oral application, various barriers in the gastrointestinal tract have to be overcome. Apart from a poor membrane uptake and intense enzymatic degradation, this study will demonstrate that thiol–disulphide reactions are an underestimated essential part of the presystemic metabolism. Glutathione, integrative part of the antioxidant defence system in the gastrointestinal tract, may play an important role in the inactivation of orally given peptides and proteins. In order to verify this hypothesis, desmopressin which bears a single disulphide bond was used as model peptide drug. Desmopressin was incubated with GSH in various concentrations, and the extent of thiol/disulphide exchange reactions between the peptide drug and GSH was investigated in dependence on pH and ratio of reactants determined as a function of time via HPLC, LC-MS and Maldi-Tof-MS analyses. Results showed that desmopressin is degraded by 1% reduced glutathione at pH 4 and pH 5.5. In presence of 0.01%, 0.1% and 1% of reduced glutathione 6.1%, 19.4% and 52.1% of desmopressin, respectively, were degraded. The masses of the conjugates after deconvolution measured by liquid chromatography and electrospray ionisation mass spectrometric detection were m/z 1069.67, m/z 1376.50, m/z 1683.40 and m/z 2138. These molecular masses, confirmed by Maldi-Tof-MS analysis, correspond with the masses of conjugates expected in theory. Under defined conditions, these results reveal that thiol–disulphide exchange reactions have a considerable impact on the alteration of peptide drugs and proteins.     

Identification of a novel chromosomal passenger complex and its unique localization during cytokinesis in Trypanosoma brucei

Aurora B kinase is a key component of the chromosomal passenger complex (CPC), which regulates chromosome segregation and cytokinesis. An ortholog of Aurora B was characterized in Trypanosoma brucei (TbAUK1), but other conserved components of the complex have not been found. Here we identified four novel TbAUK1 associated proteins by tandem affinity purification and mass spectrometry. Among these four proteins, TbKIN-A and TbKIN-B are novel kinesin homologs, whereas TbCPC1 and TbCPC2 are hypothetical proteins without any sequence similarity to those known CPC components from yeasts and metazoans. RNAi-mediated silencing of each of the four genes led to loss of spindle assembly, chromosome segregation and cytokinesis. TbKIN-A localizes to the mitotic spindle and TbKIN-B to the spindle midzone during mitosis, whereas TbCPC1, TbCPC2 and TbAUK1 display the dynamic localization pattern of a CPC. After mitosis, the CPC disappears from the central spindle and re-localizes at a dorsal mid-point of the mother cell, where the anterior tip of the daughter cell is tethered, to start cell division toward the posterior end, indicating a most unusual CPC-initiated cytokinesis in a eukaryote.     

Activation and route of administration both determine the ability of bone marrow-derived dendritic cells to accumulate in secondary lymphoid organs and prime CD8<Superscript>+</Superscript> T cells against tumors

Aims

To examine the effects of route of administration and activation status on the ability of dendritic cells (DC) to accumulate in secondary lymphoid organs, and induce expansion of CD8⁺ T cells and anti-tumor activity.

Methods

DC from bone marrow (BM) cultures were labeled with fluorochromes and injected s.c. or i.v. into naïve mice to monitor their survival and accumulation in vivo. Percentages of specific CD8⁺ T cells in blood and delayed tumor growth were used as readouts of the immune response induced by DC immunization.

Results

The route of DC administration was critical in determining the site of DC accumulation and time of DC persistence in vivo. DC injected s.c. accumulated in the draining lymph node, and DC injected i.v. in the spleen. DC appeared in the lymph node by 24 h after s.c. injection, their numbers peaked at 48 h and declined at 96 h. DC that had spontaneously matured in vitro were better able to migrate compared to immature DC. DC were found in the spleen at 3 h and 24 h after i.v. injection, but their numbers were low and declined by 48 h. Depending on the tumor cell line used, DC injected s.c. were as effective or more effective than DC injected i.v. at inducing anti-tumor responses. Pre-treatment with LPS increased DC accumulation in lymph nodes, but had no detectable effect on accumulation in the spleen. Pre-treatment with LPS also improved the ability of DC to induce CD8⁺ T cell expansion and anti-tumor responses, regardless of the route of DC administration.

Conclusions

Injection route and activation by LPS independently determine the ability of DC to activate tumor-specific CD8⁺ T cells in vivo.

     

Habitat suitability,corridors and dispersal barriers for large carnivores in Poland

Carnivores are often particularly sensitive to landscape fragmentation. Ecological corridors may help to connect local populations, ensuring gene flow and retaining viable meta-populations. We aimed to establish habitat suitability models for two large carnivores in Poland, the grey wolf Canis lupus Linnaeus, 1758 and the Eurasian lynx Lynx lynx Linnaeus, 1758, based on ecological niche factor analysis (ENFA). Secondly, we calculated least cost paths (LCPs) based on cost values obtained from ENFA. Thirdly, we determined structures that might act as barriers, thus diminishing the value of the corridor unless appropriate conservation measures are taken. We compared some of the results with actual dispersal data of four lynx in eastern Poland. Results indicate that both species are highly marginalised. Less habitat that is currently available in Poland is suitable for lynx than for wolves. We determined a total of 76 LCPs. Comparison of these theoretical corridors with actual dispersal routes suggests that the traits of calculated LCPs are mostly within the range of those of real routes. We highlight a variety of features that might act as barriers, such as major roads (including planned highways), urbanized areas, and large un-forested areas. We give suggestions where concerted conservation efforts (eg wildlife passages) might be particularly well-directed.     

Polystyrene/Divinylbenzene Based Monolithic and Encapsulated Capillary Columns for the Analysis of Nucleic Acids by High‐Performance Liquid Chromatography‐Electrospray Ionisation Mass Spectrometry

Monolithic capillary columns are prepared by copolymerization of styrene and divinylbenzene, encapsulated capillary columns by immobilizing silica particles with different pore sizes inside a 200 μm i.d. fused silica capillary by encapsulation of the derivatized silica sorbent in a poly(styrene/divinylbenzene) (PS/DVB) matrix. Both allow the rapid and highly efficient separation of single‐ and double‐stranded DNA by ion‐pair reversed‐phase high‐performance liquid chromatography (IP‐RP‐HPLC). The high resolving power of monolithic and encapsulated capillary columns can be utilized for mutation screening in polymerase chain reaction (PCR) amplified polymorphic loci by denaturing HPLC (DHPLC). Recognition of mutations is based on the separation of homo‐ and heteroduplex species by IP‐RP‐HPLC under denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Separations can be readily hyphenated to electrospray ionization‐mass spectrometry.     

Plasma membrane H+-ATPase is involved in auxin-mediated cell elongation during wheat embryo development

Previous investigations suggested that specific auxin spatial distribution due to auxin movements to particular embryonic regions was important for normal embryonic pattern formation. To gain information on the molecular mechanism(s) by which auxin acts to direct pattern formation in specific embryonic regions, the role of a plasma membrane (PM) ATPase was evaluated as downstream target of auxin in the present study. Western-blot analysis revealed that the PM H(+)-ATPase expression level was significantly increased by auxin in wheat (Triticum aestivum) embryos (two-three times increase). In bilaterally symmetrical embryos, the spatial expression pattern of the PM H(+)-ATPase correlates with the distribution pattern of the auxin analog, tritiated 5-azidoindole-3-acetic acid. A strong immunosignal was observed in the abaxial epidermis of the scutellum and in the epidermal cells at the distal tip of this organ. Pseudoratiometric analysis using a fluorescent pH indicator showed that the pH in the apoplast of the cells expressing the PM H(+)-ATPase was in average more acidic than the apoplastic pH of nonexpressing cells. Cellulose staining of living embryos revealed that cells of the scutellum abaxial epidermis expressing the ATPase were longer than the scutellum adaxial epidermal cells, where the protein was not expressed. Our data indicate that auxin activates the proton pump resulting in apoplastic acidification, a process contributing to cell wall loosening and elongation of the scutellum. Therefore, we suggest that the PM H(+)-ATPase is a component of the auxin-signaling cascade that may direct pattern formation in embryos.     

Poly(glycidyl methacrylate/divinylbenzene)-IDA-FeIII in phosphoproteomics

The study of protein phosphorylation has grown exponentially in recent years, as it became evident that important cellular functions are regulated by phosphorylation and dephosphorylation of proteins on serine, threonine and tyrosine residues. The use of immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides from peptide mixtures has been shown to be useful especially prior to mass spectrometric analysis. For the selective enrichment applying solid-phase extraction (SPE) of phosphorylated peptides, we introduce poly(glycidyl methacrylate/divinylbenzene) (GMD) derivatized with imino-diacetic acid (IDA) and bound Fe(III) as a material. GMD is rapidly synthesized and the resulting free epoxy groups enable an easy access to further derivatization with, e.g., IDA. Electron microscopy showed that the synthesized GMD-IDA-Fe(III) for SPE has irregular agglomerates of spherical particles. Inductively coupled plasma (ICP) analysis resulted in a metal capacity of Fe(III) being 25.4 micromol/mL. To enable on-line preconcentration and desalting in one single step, GMD-IDA-Fe(III) and Silica C18 were united in one cartridge. Methyl esterification (ME) of free carboxyl groups was carried out to prevent binding of nonphosphorylated peptides to the IMAC function. The recovery for a standard phosphopeptide using this SPE method was determined to be 92%. The suitability of the established system for the selective enrichment and analysis of model proteins phosphorylated at different amino acid residues was evaluated stepwise. After successful enrichment of beta-casein deriving phosphopeptides, the established system was extended to the analysis of in vitro phosphorylated proteins, e.g. deriving from glutathione-S-transferase tagged extracellular signal regulated kinase 2 (GST-ERK2).     

The identification and optimization of a N-hydroxy urea series of flap endonuclease 1 inhibitors

Flap endonuclease-1 (FEN1) is a key enzyme involved in base excision repair (BER), a primary pathway utilized by mammalian cells to repair DNA damage. Sensitization to DNA damaging agents is a potential method for the improvement of the therapeutic window of traditional chemotherapeutics. In this paper, we describe the identification and SAR of a series of low nanomolar FEN1 inhibitors. Over 1000-fold specificity was achieved against a related endonuclease, xeroderma pigmentosum G (XPG). Two compounds from this series significantly potentiate the action of methyl methanesulfonate (MMS) and temozolamide in a bladder cancer cell line (T24). To our knowledge, these are the most potent endonuclease inhibitors reported to date.