Reduced diversity of the human erythrocyte membrane sialic acids in polycythemia vera. Absence of N-glycolylneuraminic acid and characterisation of N-acetylneuraminic acid 1,7 lactone

Sialic acids from the erythrocyte (RBC) membrane of a patient suffering from polycythemia vera, a malignant orphan disorder of hematopoietic cells, was studied using GC/MS. We found that the sialic acid diversity of these membranes was drastically reduced since only four entities were identified: Neu5Ac (91.5%) and its 1,7 lactone Neu5Ac1,7L (7.5%) which is absent in normal RBC, Neu4,5Ac(2) (0.50%) and Neu4,5Ac(2) 9Lt (0.50%); in normal RBC, Neu5,7Ac(2), Neu5,9Ac(2), Neu5Ac9Lt, Neu5Ac8S and Neu, as well as traces of Kdn, were also present. Neu5Gc and its O-alkylated or O-acetylated derivatives, which are considered by various authors as cancer markers, were not detected.     

Continuum simulations of acetylcholine diffusion with reaction-determined boundaries in neuromuscular junction models

The reaction-diffusion system of the neuromuscular junction has been modeled in 3D using the finite element package FEtk. The numerical solution of the dynamics of acetylcholine with the detailed reaction processes of acetylcholinesterases and nicotinic acetylcholine receptors has been discussed with the reaction-determined boundary conditions. The simulation results describe the detailed acetylcholine hydrolysis process, and reveal the time-dependent interconversion of the closed and open states of the acetylcholine receptors as well as the percentages of unliganded/monoliganded/diliganded states during the neuro-transmission. The finite element method has demonstrated its flexibility and robustness in modeling large biological systems.     

Protection induced in BALB/c mice by the high-molecular-mass (hMM) fraction of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by a dimorphic fungus, Paracoccidioides brasiliensis. The present study investigated the protective activity of the P. brasiliensis high-molecular-mass (hMM) fraction (~380 kDa) in experimental murine PCM. In the first step, lymphocyte proliferation and production of IFNγ (but not IL-4) were observed in “in vitro” spleen cells (from female BALB/c mice infected (i.v.) with P. brasiliensis) that were stimulated with hMM fractions. In the second step, female BALB/c mice were previously immunized (s.c.) with hMM fraction (25 μg/protein = F-25 and 50 μg/protein = F-50), and the colony-forming units (CFU) of the lung and spleen, the histopathological characteristics of the granulomatous lesions, and plasmatic gp43 soluble antigens and anti-hMM IgG levels were analyzed at 28 and 56 days after infection. The lung and liver CFU were lower in mice previously immunized with the hMM fraction (P < 0.05). The granulomatous lesions revealed a greater degree of compaction and organization, with no dissemination of the fungus to other organs. Lower soluble antigen levels (P < 0.05) and higher IgG anti-hMM fraction (P < 0.05) were observed in immunized groups. The results for CFU, histopathology and antigenemia suggest that the hMM fraction has a protective effect in experimental paracoccidioidomycosis in BALB/c mice.     

Assisted large fragment insertion by Red/ET-recombination (ALFIRE)--an alternative and enhanced method for large fragment recombineering

Functional genomics require manipulation and modification of large fragments of the genome. Such manipulation has only recently become more efficient due to the discovery of different techniques based on homologous recombination. However, certain limitations of these strategies still exist since insertion of homology arms (HAs) is often based on amplification of DNA sequences with PCR. Large quantities of PCR products longer than 4-5 kb can be difficult to obtain and the risk of mutations or mismatches increases with the size of the template that is being amplified. This can be overcome by adding HAs by conventional cloning techniques, but with large fragments such as entire genes the procedure becomes time-consuming and tedious. Second, homologous recombination techniques often require addition of antibiotic selection genes, which may not be desired in the final construct. Here, we report a method to overcome the size and selection marker limitations by a two- or three-step procedure. The method can insert any fragment into small or large episomes, without the need of an antibiotic selection gene. We have humanized the mouse luteinizing hormone receptor gene (Lhcgr) by inserting a approximately 55 kb fragment from a BAC clone containing the human Lhcgr gene into a 170 kb BAC clone comprising the entire mouse orthologue. The methodology is based on the rationale to introduce a counter-selection cassette flanked by unique restriction sites and HAs for the insert, into the vector that is modified. Upon enzymatic digestion, in vitro or in Escherichia coli, double-strand breaks are generated leading to recombination between the vector and the insert. The procedure described here is thus an additional powerful tool for manipulating large and complex genomic fragments.     

Can population genetic structure be predicted from life-history traits?

Population genetic structure is a key parameter in evolutionary biology. Earlier comparative studies have shown that genetic structure depends on species ecological attributes and life-history traits, but species phylogenetic relatedness had not been accounted for. Here we reevaluate the relationships between genetic structure and species traits in seed plants. Each species is characterized by a set of life-history and ecological features as well as by its geographic range size, its heterozygote deficit, and its genetic structure at nuclear and organelle markers to distinguish between pollen- and seed-mediated gene flow. We use both a conventional regression approach and a method that controls for phylogenetic relationships. Once phylogenetic conservatism and covariation among traits are taken into account, genetic structure is shown to be related with only a few synthetic traits, such as mating system for nuclear markers and seed dispersal mode or geographic range size for organelle markers. Along with other studies on invasiveness or rarity, our work illustrates the fact that predicting the fate of species across a broad taxonomic assemblage on the basis of simple traits is rarely possible, a testimony of the highly contingent nature of evolution.     

The role of tryptophan residues in the function and stability of the mechanosensitive channel MscS from Escherichia coli

Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched in protein surfaces involved in protein docking and are often found in membrane proteins close to the lipid head groups. However, they are usually absent from the membrane domains of mechanosensitive channels. Three Trp residues occur naturally in the Escherichia coli MscS (MscS-Ec) protein: W16 lies in the periplasm, immediately before the first transmembrane span (TM1), whereas W240 and W251 lie at the subunit interfaces that create the cytoplasmic vestibule portals. The role of these residues in MscS function and stability were investigated using site-directed mutagenesis. Functional channels with altered properties were created when any of the Trp residues were replaced by another amino acid, with the greatest retention of function associated with phenylalanine (Phe) substitutions. Analysis of the fluorescence properties of purified mutant MscS proteins containing single Trp residues revealed that W16 and W251 are relatively inaccessible, whereas W240 is accessible to quenching agents. The data point to a significant role for W16 in the gating of MscS, and an essential role for W240 in MscS oligomer stability.     

The inner cavity of Escherichia coli DegP protein is not essential for molecular chaperone and proteolytic activity

The Escherichia coli DegP protein is an essential periplasmic protein for bacterial survival at high temperatures. DegP has the unusual property of working as a chaperone below 28 degrees C, but efficiently degrading unfolded proteins above 28 degrees C. Monomeric DegP contains a protease domain and two PDZ domains. It oligomerizes into a hexameric cage through the staggered association of trimers. The active sites are located in a central cavity that is only accessible laterally, and the 12 PDZ domains act as mobile sidewalls that mediate opening and closing of the gates. As access to the active sites is restricted, DegP is an example of a self-compartmentalized protease. To determine the essential elements of DegP that maintain the integrity of the hexameric cage, we constructed several deletion mutants of DegP that formed trimers rather than hexamers. We found that residues 39 to 78 within the LA loops, as well as the PDZ2 domains are essential for the integrity of the DegP hexamer. In addition, we asked whether an enclosed cavity or cage of specific dimensions is required for the protease and chaperone activities in DegP. Both activities were maintained in the trimeric DegP mutants without an enclosed cavity and in deletion DegP mutants with significantly reduced dimensions of the cage. We conclude that the functional unit for the protease and chaperone activities of DegP is a trimer and that neither a cavity of specific dimensions nor the presence of an enclosed cavity appears to be essential for the protease and chaperone activities of DegP.     

The platelet-derived growth factor receptor alpha is destabilized by geldanamycins in cancer cells

The heat shock protein HSP90 serves as a chaperone for receptor protein kinases, steroid receptors, and other intracellular signaling molecules. Targeting HSP90 with ansamycin antibiotics disrupts the normal processing of clients of the HSP90 complex. The platelet-derived growth factor receptor alpha (PDGFRalpha) is a tyrosine kinase receptor up-regulated and activated in several malignancies. Here we show that the PDGFRalpha forms a complex with HSP90 and the co-chaperone cdc37 in ovarian, glioblastoma, and lung cancer cells. Treatment of cancer cell lines expressing the PDGFRalpha with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) promotes degradation of the receptor. Likewise, phospho-Akt, a downstream target, is degraded after treatment with 17-AAG. In contrast, PDGFRalpha expression is not affected by 17-AAG in normal human smooth muscle cells or 3T3 fibroblasts. PDGFRalpha degradation by 17-AAG is inhibited by the proteasome inhibitor MG132. High molecular weight, ubiquitinated forms of the receptor are detected in cells treated with 17-AAG and MG132. Degradation of the receptor is also inhibited by a specific neutralizing antibody to the PDGFRalpha but not by a neutralizing antibody to PDGF or by imatinib mesylate (Gleevec). Ultimately, PDGFRalpha-mediated cell proliferation is inhibited by 17-AAG. These results show that 17-AAG promotes PDGFRalpha degradation selectively in transformed cells. Thus, not only mutated tyrosine kinases but also overexpressed receptors in cancer cells can be targeted by 17-AAG.