FGF3 attached to a phosholipid membrane anchor gains a high transforming capacity. Implications of microdomains for FGF3 cell transformation

NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan.     

The cross-reactive calcium-binding pollen allergen,Phl p 7, reveals a novel dimer assembly

The timothy grass pollen allergen Phl p 7 assembles most of the IgE epitopes of a novel family of 2 EF-hand calcium-binding proteins and therefore represents a diagnostic marker allergen and vaccine candidate for immunotherapy. Here we report the first three-dimensional structure of a representative of the 2 EF-hand allergen family, Phl p 7, in the calcium-bound form. The protein occurs as a novel dimer assembly with unique features: in contrast to well known EF-hand proteins such as calmodulin, parvalbumin or the S100 proteins, Phl p 7 adopts an extended conformation. Two protein monomers assemble in a head-to-tail arrangement with domain-swapped EF-hand pairing. The intertwined dimer adopts a barrel-like structure with an extended hydrophobic cavity providing a ligand-binding site. Calcium binding acts as a conformational switch between an open and a closed dimeric form of Phl p 7. These findings are interesting in the context of lipid- and calcium-dependent pollen tube growth. Furthermore, the structure of Phl p 7 allows for the rational development of vaccine strategies for treatment of sensitized allergic patients.     

Nuclear and Nucleomorph SSU rDNA Phylogeny in the Cryptophyta and the Evolution of Cryptophyte Diversity

The plastid-bearing members of the Cryptophyta contain two functional eukaryotic genomes of different phylogenetic origin, residing in the nucleus and in the nucleomorph, respectively. These widespread and diverse protists thus offer a unique opportunity to study the coevolution of two different eukaryotic genomes within one group of organisms. In this study, the SSU rRNA genes of both genomes were PCR-amplified with specific primers and phylogenetic analyses were performed on different data sets using different evolutionary models. The results show that the composition of the principal clades obtained from the phylogenetic analyses of both genes was largely congruent, but striking differences in evolutionary rates were observed. These affected the topologies of the nuclear and nucleomorph phylogenies differently, resulting in long-branch attraction artifacts when simple evolutionary models were applied. Deletion of long-branch taxa stabilized the internal branching order in both phylogenies and resulted in a completely resolved topology in the nucleomorph phylogeny. A comparison of the tree topologies derived from SSU rDNA sequences with characters previously used in cryptophyte systematics revealed that the biliprotein type was congruent, but the type of inner periplast component incongruent, with the molecular trees. The latter is indicative of a hidden cellular dimorphism (cells with two periplast types present in a single clonal strain) of presumably widespread occurrence throughout cryptophyte diversity, which, in consequence, has far-reaching implications for cryptophyte systematics as it is practiced today.     

Differential control of vesicle priming and short-term plasticity by Munc13 isoforms

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca(2+)-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.     

The recent impact of solid-phase synthesis on medicinally relevant benzoannelated nitrogen heterocycles

Benzoannelated heterocycles such as benzodiazepines and indoles can be prepared efficiently through cyclization on solid supports, although no single approach is currently universal for the preparation of all benzoannelated N-heterocycle chemistries. In this review, a number of synthetic strategies for the generation of benzoannelated nitrogen heterocycles using resin-bound substrates have been described. Classical heterocycle forming reactions such as the Fischer indole, the Bischler-Napieralski tetrahydroisoquinoline, the Pictet-Spengler tetrahydro-beta-carboline, the Tsuge, the Nenitzescu and the Richter cinnoline reaction are presented. In addition, the Heck, Sonogashira, Wittig, Diels-Alder, and olefin metathesis reactions have been also used. Multicomponent reactions such as the Grieco three-component assembly have been exploited for the synthesis of heterocycles. Cyclative cleavage from the solid support is particularly suitable for the synthesis of heterocycles while particular emphasis has been focused on the synthesis of libraries and the use of combinatorial chemistry techniques. In addition, the most relevant pharmacological properties of benzoannelated nitrogen heterocycles are included.     

Self-immobilizing recombinant antibody fragments for immunoaffinity chromatography: generic, parallel, and scalable protein purification

We present the directed immobilization of recombinant antibody fragments as ligands for general immunoaffinity chromatography methods. It is based on fusion proteins of scFv fragments with several chitin-binding domains which can be immobilized directly from a crude bacterial lysate on inexpensive chitin beads for the purification of proteins without any gradient or detector. It has been used with a positive pressure manifold, allowing the parallel processing of 24 different samples on a milligram scale, as convenient as plasmid isolation. The method is demonstrated with several anti-protein antibodies. In addition, methods are presented of using an anti-His tag antibody either alone or directly coupled to IMAC to obtain very pure protein. As those methods are scalable, they should prove very useful in the parallel purification of natural and recombinant proteins on small scales (for proteomics), medium scales (for crystallography and NMR), and very large scales (for therapeutic proteins).     

A new concept for DNA-arrays

We will insert a cleavage site in an oligodeoxynucleotide, which can be used for a selective and quantitative cleavage. For that reason we synthesized the four 5'-S-(4,4'-dimethoxytrityl)-mercapto-2'-deoxynucleotide-3'-O-(2-cyanoethoxydiisopropylamino)-phosphites (5a-d). The cleavage of P-S and C-S bonds is described (Mag, M.; Lücking, S.; Engels, J.W. Synthesis and selective cleavage of an oligodeoxy-nucleotide containing a bridged internucleotide 5'-phosphorthioate linkage. Nucleic Acids Res. 1991, 19 (7), 1437-1441; Marriott, J.H.; Mottahedeh, M.; Reese, C.B. 9-(4-methoxyphenyl)xanthen-9-thiol: A useful reagent for the preparation of thiols. Tetrahedron Lett. 1990, 31 (51), 7485-7488; Divakar, K.J.; Mottoh, A.; Reese, C.B.; Shanghvi, Y.S. Approaches to the synthesis of 2' thio analogues of pyrimidine ribosides. J. Chem. Sc., Perkin Trans. 1 1990, 969-974). The oligodeoxynucleotides with an achiral bridged 5'-phosphorothioate linkage 5'-O-P-S-3' are synthesized by the phosphoramidite procedure.     

P2Y(1) receptor activation inhibits NMDA receptor-channels in layer V pyramidal neurons of the rat prefrontal and parietal cortex

In the 1st part of this study, monosynaptic excitatory postsynaptic potentials (EPSPs) in layer V of the rat prefrontal cortex (PFC) were evoked by electrical stimulation of layer I. Recordings with intracellular sharp, microelectrodes showed a concentration-dependent inhibition of the EPSP by adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S). Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), when given alone depressed the EPSP and in addition antagonized the effect of ADP-beta-S. Exclusion of the N-methyl-D-aspartate (NMDA) component of the EPSP by D(.)-amino-5-phosphonopentanoic acid (AP-5) abolished the ADP-beta-S-induced depression. The pressure-application of both NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) caused reproducible depolarizations. ADP-beta-S inhibited the effect of NMDA, but did not alter that of AMPA. PPADS was also under these conditions antagonistic with ADP-beta-S. In the 2nd part of the study, NMDA-induced currents were measured by whole-cell patch-clamp pipettes. ADP-beta-S caused a concentration-dependent inhibition of the responses to NMDA. PPADS alone did not alter the NMDA-currents but again antagonized the action of ADP-beta-S; 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS 2179) also abolished the NMDA effect. The ADP-beta-S-induced inhibition persisted in the presence of tetrodotoxin (TTX) or guanosine 5'-O-(3-thiodiphosphate) (GDP-beta-S) applied to the external medium and the pipette solution, respectively. The 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) moderately decreased the ADP-beta-S effect. The inhibitory function of ADP-beta-S on EPSPs and the interaction with PPADS was observed also in layer V pyramidal neurons of the parietal somatosensory cortex. In conclusion, metabotropic P2Y(1) receptors appear to exert a new modulatory influence on fast excitatory amino acid transmission in the cerebral cortex.