On the use of N‐dicyclopropylmethyl aspartyl‐glycine synthone for backbone amide protection

To prevent aspartimide formation and related side products in Asp‐Xaa, particularly Asp‐Gly‐containing peptides, usually the 2‐hydroxy‐4‐methoxybenzyl (Hmb) backbone amide protection is applied for peptide synthesis according to the Fmoc‐protocols. In the present study, the usefulness of the recently proposed acid‐labile dicyclopropylmethyl (Dcpm) protectant was analyzed. Despite the significant steric hindrance of this bulky group, N‐terminal H‐(Dcpm)Gly‐peptides are quantitatively acylated by potent acylating agents, and alternatively the dipeptide Fmoc‐Asp(OtBu)‐(Dcpm)Gly‐OH derivative can be used as a building block. In contrast to the Hmb group, Dcpm is inert toward acylations, but is readily removed in the acid deprotection and resin‐cleavage step. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.     

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer

The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.     

Analysis of humoral immune responses in rhesus macaques vaccinated with attenuated SIVmac239Δnef and challenged with pathogenic SIVmac251

Background To determine the correlation between protection and humoral immune response against simian immunodeficiency virus (SIVmac251), 11 macaques were immunized with live‐attenuated SIVmac239Δnef either intravenously or via the tonsils and exposed to SIVmac251 after either 6 or 15 months along with unvaccinated controls. Results Independent of the route of vaccine application, viremia was significantly reduced in vaccinees compared with controls 2 weeks post‐challenge. Concomitantly, viremia correlated inversely with SIV‐specific IgG, complement‐mediated lysis and neutralizing antibodies and these parameters seemed to contribute to reduced viremia. During chronic infection, six monkeys controlled viremia in the circulation (two or fewer infectious units per 10⁶ PBMCs) and showed no signs of trapping in lymphatic tissues (Appendix S1). Conclusions As no significant differences were observed throughout the study, with respect to the humoral immune response and viremia control, between the two vaccinated cohorts, mucosal immunization strategies are recommended due to more simplified application.     

Estimation by PLFA of Microbial Community Structure Associated with the Rhizosphere of Lygeum spartum and Piptatherum miliaceum Growing in Semiarid Mine Tailings

The objective of this study was to compare the microbial community composition and biomass associated with the rhizosphere of a perennial gramineous species (Lygeum spartum L.) with that of an annual (Piptatherum miliaceum L.), both growing in semiarid mine tailings. We also established their relationship with the contents of potentially toxic metals as well as with indicators of soil quality. The total phospholipid fatty acid (PLFA) amount was significantly higher in the rhizosphere soil of the annual species than in the rhizosphere soil of the perennial species. The fungal/bacterial PLFA ratio was significantly greater in the perennial species compared to the annual species. The fatty acid 16:1ω5c, the fungal/bacterial PLFA ratio and monounsaturated/saturated PLFA ratio were correlated negatively with the soluble contents of toxic metals. The cyc/prec (cy17:0 + cy19:0/16:1ω7 + 18:1ω7) ratio was correlated positively with the soluble contents of Pb, Zn, Al, Ni, Cd, and Cu. The results of the PLFA analysis for profiling microbial communities and their stress status of both the plant species indicate that perennial and annual gramineous species appear equally suitable for use in programmes of revegetation of semiarid mine tailings.     

The effect of intracerebroventricular administration of substance P fragment and met-enkephalin on the transmission of nervous impulses between the midbrain reticular formation and two generators of the hippocampal theta rhythm in rabbits

The effect of intracerebroventricular administration of Substance P fragment and met-enkephalin on the excitability of two generators of hippocampal theta rhythm was investigated in the experiments performed on chronic rabbits. Substance P had a strong facilitatory effect on the threshold of the generator of the hippocampal theta rhythm of the frequency 4-7 c/s and an inhibitory effect on the threshold of the generator of the 7-12 c/s frequency evoked by stimulation of the midbrain reticular formation. These effects were dose dependent. The effects of met-enkephalin were opposite. They increased the threshold of the 4-7 c/s hippocampal generator and decreased the threshold of the other generator. The effect of these two compounds was evaluated according to the energy of electrical trains of pulses maintaining the continuous arousal pattern in the hippocampus.     

Reduced sialylation status in UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE)-deficient mice

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. They are involved in a variety of cellular functions, such as cell adhesion or signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. Previously, we have shown that inactivation of the GNE by gene targeting causes early embryonic lethality in mice, whereas heterozygous GNE-deficient mice are vital. In this study we compared the amount of membrane-bound sialic acids of wildtype mice with those of heterozygous GNE-deficient mice. For that we quantified membrane-bound sialic acid concentration in various organs of wildtype- and heterozygous GNE-deficient mice. We found an organ-specific reduction of membrane-bound sialic acids in heterozygous GNE-deficient mice. The overall reduction was 25%. Additionally, we analyzed transferrin and polysialylated neural cell adhesion molecule (NCAM) by one- or two-dimensional gel electrophoresis. Transferrin-expression was unchanged in heterozygous GNE-deficient mice; however the isoelectric point of transferrin was shifted towards basic pH, indicating a reduced sialylation. Furthermore, the expression of polysialic acids on NCAM was reduced in GNE-deficient mice. Daniel Gagiannis and André Orthmann have contributed equally to this work.     

Formation of giant spicules in the deep-sea hexactinellid <Emphasis Type="Italic">Monorhaphis chuni</Emphasis> (Schulze 1904): electron-microscopic and biochemical studies

The siliceous sponge Monorhaphis chuni (Hexactinellida) synthesizes the largest biosilica structures on earth (3 m). Scanning electron microscopy has shown that these spicules are regularly composed of concentrically arranged lamellae (width: 3–10 μm). Between 400 and 600 lamellae have been counted in one giant basal spicule. An axial canal (diameter: ~2 μm) is located in the center of the spicules; it harbors the axial filament and is surrounded by an axial cylinder (100–150 μm) of electron-dense homogeneous silica. During dissolution of the spicules with hydrofluoric acid, the axial filament is first released followed by the release of a proteinaceous tubule. Two major proteins (150 kDa and 35 kDa) have been visualized, together with a 24-kDa protein that cross-reacts with antibodies against silicatein. The spicules are surrounded by a collagen net, and the existence of a hexactinellidan collagen gene has been demonstrated by cloning it from Aphrocallistes vastus. During the axial growth of the spicules, silicatein or the silicatein-related protein is proposed to become associated with the surface of the spicules and to be finally internalized through the apical opening to associate with the axial filament. Based on the data gathered here, we suggest that, in the Hexactinellida, the growth of the spicules is mediated by silicatein or by a silicatein-related protein, with the orientation of biosilica deposition being controlled by lectin and collagen. Carsten Eckert was previously with the Museum für Naturkunde, Invalidenstrasse 43, 10115 Berlin, Germany. The collagen sequence from Aphrocallistes vastus reported here, viz., [COL_APHRO] APHVACOL (accession number AM411124), has been deposited in the EMBL/GenBank data base. This work was supported by grants from the European Commission, the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany (project: Center of Excellence BIOTECmarin), the National Natural Science Foundation of China (grant no. 50402023), and the International Human Frontier Science Program.     

N9-benzyl-substituted 1,3-dimethyl- and 1,3-dipropyl-pyrimido[2,1-f]purinediones: synthesis and structure-activity relationships at adenosine A1 and A2A receptors

Synthesis and physicochemical properties of N-benzyl pyrimido[2,1-f]purinediones are described. These derivatives were synthesized by the cyclization of 7-chloropropylo-8-bromo-1,3-dimethyl- or 1,3-dipropyl xanthine derivatives with corresponding (un)substituted benzylamines. Dipropyl derivatives were obtained under microwave irradiation conditions either. The obtained compounds (1-20) were evaluated for their affinity to adenosine A1 and A2A receptors, selected compounds were additionally investigated for affinity to the A3 receptor subtype. The results of the radioligand binding assays to A1 and A2A adenosine receptors showed that most of the 1,3-dimethyl-9-benzylpyrimidopurinediones exhibited selective affinity to A2A receptors at micromolar or submicromolar concentrations (for example, derivative 9 with o-methoxy substituent displayed a Ki value of 0.699 microM at rat A2A receptor with more than 36-fold selectivity). Contrary to previously described arylpyrimido[2,1-f]purinediones dipropyl derivatives (compounds 15-20) showed affinity to both kinds of receptors increased, however A1 affinity increased to a larger extent, with the result that A2A selectivity was abolished. The best adenosine A1 receptor ligand was m-chlorobenzyl derivative 18 (Ki=0.089 microM and 5-fold A1 selectivity). Structure-activity relationships were discussed with the analysis of lipophilic and spatial properties of the investigated compounds. Pharmacophore model of adenosine A1 receptor antagonist was adopted for this purpose.