Neocarzinostatin chromophore: Presence of a cyclic carbonate subunit and its modification in the structure of other biologically active forms

On the basis of spectroscopic evidence, opening of a five-membered cyclic carbonate ring (1,3-dioxolan-2-one) in the C₁₅-subunit of the previously determined partial structure $\text{1}$ (Fig. 1) of the major neocarzinostatin chromophore (NCS-Chrom $\text{A}$), is proposed to account for its base-catalyzed methanolysis to NCS-Chrom $\text{C}$. NCS-Chrom $\text{B}$, apparently an authentic natural product present as a minor component in all preparations of NCS studied, was found to be formally equivalent to the hydrolysis/decarboxylation product of the cyclic carbonate functionality in NCS-Chrom $\text{A}$. The mercaptan-dependent DNA strand-scission activity, equivalent for NCS-Chrom $\text{A}$, $\text{B}$ and $\text{C}$, is independent of the integrity of the cyclic carbonate ring system and implicates a secondary site in the C₁₅-substructure for mercaptan activation.     

Th1, Th2, and Th17 effector T cell-induced autoimmune gastritis differs in pathological pattern and in susceptibility to suppression by regulatory T cells

Th cells can be subdivided into IFN-gamma-secreting Th1, IL-4/IL-5-secreting Th2, and IL-17-secreting Th17 cells. We have evaluated the capacity of fully differentiated Th1, Th2, and Th17 cells derived from a mouse bearing a transgenic TCR specific for the gastric parietal cell antigen, H(+)K(+)-ATPase, to induce autoimmune gastritis after transfer to immunodeficient recipients. We have also determined the susceptibility of the disease induced by each of the effector T cell types to suppression by polyclonal regulatory T cells (Treg) in vivo. Each type of effector cell induced autoimmune gastritis with distinct histological patterns. Th17 cells induced the most destructive disease with cellular infiltrates composed primarily of eosinophils accompanied by high levels of serum IgE. Polyclonal Treg could suppress the capacity of Th1 cells, could moderately suppress Th2 cells, but could suppress Th17-induced disease only at early time points. The major effect of the Treg was to inhibit the expansion of the effector T cells. However, effector cells isolated from protected animals were not anergic and were fully competent to proliferate and produce effector cytokines ex vivo. The strong inhibitory effect of polyclonal Treg on the capacity of some types of differentiated effector cells to induce disease provides an experimental basis for the clinical use of polyclonal Treg in the treatment of autoimmune disease in humans.     

Chromatographic resolution, characterisation and quantification of VX enantiomers in hemolysed swine blood samples

The present study was initiated to develop a sensitive and highly selective method for the analysis of the enantiomers of the nerve agent VX (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate) in blood samples for toxicokinetic and therapeutic research. To achieve this goal, analytical and semi-preparative enantioseparation of VX were carried out with gas and liquid chromatography. The GC chiral stationary phase was HYDRODEX-beta-TBDAc (beta cyclodextrin), on which VX was baseline-resolved. On the chiral HPLC phase CHIRALCEL OD-H the enantiomers of VX were isolated with enantiomeric excess >99.99%. They were characterised by specific optical rotation (+/-25.8degmldm(-1)g(-1) at 20 degrees C and 589nm) and by determination of cholinesterase inhibition rate constants. For the quantitative chiral detection of VX the enantioresolution was realized on the HPLC chiral phase CHIRAL AGP. A specific procedure was developed to isolate VX from swine blood samples thereby stabilising its enantiomers. The limit of detection was 200fg per enantiomer on column. The absolute recovery of the overall sample preparation procedure was 75%. After an intravenous and percutaneous administration of a supralethal dose of VX in anesthetised swine (+)-VX and (-)-VX could be quantified up to 720min.     

NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII

Messenger RNA is recruited to the eukaryotic ribosome by a complex including the eukaryotic initiation factor (eIF) 4E (the cap‐binding protein), the scaffold protein eIF4G and the RNA helicase eIF4A. To shut‐off host–cell protein synthesis, eIF4G is cleaved during picornaviral infection by a virally encoded proteinase; the structural basis of this reaction and its stimulation by eIF4E is unclear. We have structurally and biochemically investigated the interaction of purified foot‐and‐mouth disease virus (FMDV) leader proteinase (Lb^pro), human rhinovirus 2 (HRV2) 2A proteinase (2A^pro) and coxsackievirus B4 (CVB4) 2A^pro with purified eIF4GII, eIF4E and the eIF4GII/eIF4E complex. Using nuclear magnetic resonance (NMR), we completed ¹³C/¹⁵N sequential backbone assignment of human eIF4GII residues 551–745 and examined their binding to murine eIF4E. eIF4GII_551–745 is intrinsically unstructured and remains so when bound to eIF4E. NMR and biophysical techniques for determining stoichiometry and binding constants revealed that the papain‐like Lb^pro only forms a stable complex with eIF4GII_551–745 in the presence of eIF4E, with K_D values in the low nanomolar range; Lb^pro contacts both eIF4GII and eIF4E. Furthermore, the unrelated chymotrypsin‐like 2A^pro from HRV2 and CVB4 also build a stable complex with eIF4GII/eIF4E, but with K_D values in the low micromolar range. The HRV2 enzyme also forms a stable complex with eIF4E; however, none of the proteinases tested complex stably with eIF4GII alone. Thus, these three picornaviral proteinases have independently evolved to establish distinct triangular heterotrimeric protein complexes that may actively target ribosomes involved in mRNA recruitment to ensure efficient host cell shut‐off.     

The morphological evolution of the Adephaga (Coleoptera)

The evolution of the coleopteran suborder Adephaga is discussed based on a robust phylogenetic background. Analyses of morphological characters yield results nearly identical to recent molecular phylogenies, with the highly specialized Gyrinidae placed as sister to the remaining families, which form two large, reciprocally monophyletic subunits, the aquatic Haliplidae + Dytiscoidea (Meruidae, Noteridae, Aspidytidae, Amphizoidae, Hygrobiidae, Dytiscidae) on one hand, and the terrestrial Geadephaga (Trachypachidae + Carabidae) on the other. The ancestral habitat of Adephaga, either terrestrial or aquatic, remains ambiguous. The former option would imply two or three independent invasions of aquatic habitats, with very different structural adaptations in larvae of Gyrinidae, Haliplidae and Dytiscoidea.     

Contribution to the quantitative and enantioselective determination of kavapyrones by high-performance liquid chromatography on ChiraSpher NT material

A simultaneous HPLC separation of the enantiomers of kavain, dihydrokavain, methysticin and dihydromethysticin, as well as the achiral dienolides yangonin and desmethoxyyangonin was carried out on a ChiraSpher NT column. For quantitative determinations, calibration curves with correlation coefficients between 0.9982 and 0.9996 were established for the genuine kavapyrones. Detection limits between 0.25 μg and 0.5 μg per injection were measured at 240 nm. The defined scopes of work corresponded with the different kavapyrone amounts, depending on growth factors of distinct plant locations. The precision of the method was verified by analysing a phytopharmacon with a nominal value of 40 mg kavapyrones per tablet. The evaluation revealed 39.62 mg per tablet by the sum of single calculated kavapyrones. Relative standard deviations between 1.06% and 2.39% were found for the compounds under investigation. The accuracy of the method was proved by a recovery of 99.7%. To simplify the determination of the total kavapyrone amount, response factors and correlation factors for (+)-dihydrokavain, (+)-methysticin, (+)-dihydromethysticin, yangonin and desmethoxyyangonin were calculated relative to (+)-kavain.