The anti-estrogen hydroxytamoxifen is a potent antagonist in a novel yeast system

The budding yeast Saccharomyces cerevisiae has been used extensively as a biological 'test tube' to study the regulation of the human estrogen receptor (ER) alpha. However, anti-estrogens, which are of great importance as therapeutic agents and research tools, fail to antagonize the activation by estrogen in yeast. Here, we have surveyed the antagonistic potential of five different anti-estrogens of diverse chemical nature. While they all act as agonists for wild-type ERalpha, we have established a novel yeast assay system for anti-estrogens, in which at least the commonly used anti-estrogen hydroxytamoxifen is a potent antagonist.     

Recombinant glycoproteins that inhibit complement activation and also bind the selectin adhesion molecules

Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo. A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo. In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewisx (sLex) tetrasaccharide (NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) during post-translational glycosylation. The resulting glycoproteins, designated sCR1sLex and sCR1[desLHR-A]sLex, respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCR1sLex and sCR1[desLHR-A]sLex indicated an average incorporation of 10 and 8 mol of sLex/mol of glycoprotein, respectively. sLex is a carbohydrate ligand for the selectin adhesion molecules. sCR1sLex was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLex inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLex inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG. sCR1sLex and sCR1[desLHR-A]sLex have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro.     

Genetic variability of foetal bovine myoblasts in primary culture

Selected strains of adult bovines and those which either have high muscle growth capacity or are double-muscled present particular characteristics of muscle fibres and collagen at slaughter that favour meat tenderness. For double-muscled bovines, it has been shown that these characteristics originated during foetal life. However, no studies have been done to determine the origin of muscle growth superiority in bovine with high muscle growth capacity compared to those with a low muscle growth capacity. Therefore, the objective of this study was to examine the proliferation and differentiation phases of myoblasts in primary culture taken from high and low muscle growth capacity foetuses at 110 days post-conception. These cultures were analysed on 1, 2, 3, 4, 6, 8, 10 days of culture. The proliferation phase was monitored by appropriate marker antibodies. The differentiation was studied by immunocytochemistry with specific antibodies for foetal, I, II (IIa and IIb), I and IIb, I and IIa myosin heavy chains (MHCs) and connectin respectively, and by immunoblotting with desmin antibody. A higher proliferation, a lower fusion and a delayed differentiation of the late markers namely MHCs fast (IIa and IIb) and connectin were shown in high muscle growth capacity foetuses compared to low capacity ones. The results indicate that the muscle growth superiority of high muscle growth capacity bovines seems, therefore, to have a similar foetal origin to that of double-muscled ones.     

Polyamine derivatives as selective RNaseA mimics

Site-selective scission of ribonucleic acids (RNAs) has attracted considerable interest, since RNA is an intermediate in gene expression and the genetic material of many pathogenic viruses. Polyamine-imidazole conjugates for site-selective RNA scission, without free imidazole, were synthesized and tested on yeast phenylalanine transfer RNA. These molecules catalyze RNA hydrolysis non-randomly. Within the polyamine chain, the location of the imidazole residue, the numbers of nitrogen atoms and their relative distances have notable influence on cleavage selectivity. A norspermine derivative reduces the cleavage sites to a unique location, in the anticodon loop of the tRNA, in the absence of complementary sequence. Experimental results are consistent with a cooperative participation of an ammonium group of the polyamine moiety, in addition to it’s binding to the negatively charged ribose-phosphate backbone, as proton source, and the imidazole moiety as a base. There is correlation between the location of the magnesium binding sites and the RNA cleavage sites, suggesting that the protonated nitrogens of the polycationic chain compete with some of the magnesium ions for RNA binding. Therefore, the cleavage pattern is specific of the RNA structure. These compounds cleave at physiological pH, representing novel reactive groups for antisense oligonucleotide derivatives or to enhance ribozyme activity.     

Phase I/II trial of immunogenicity of a human papillomavirus (HPV) type 16 E7 protein–based vaccine in women with oncogenic HPV-positive cervical intraepithelial neoplasia

Purpose: Infection with oncogenic human papillomavirus (HPV) and HPV-16 in particular is a leading cause of anogenital neoplasia. High-grade intraepithelial lesions require treatment because of their potential to progress to invasive cancer. Numerous preclinical studies have demonstrated the therapeutic potential of E7-directed vaccination strategies in mice tumour models. In the present study, we tested the immunogenicity of a fusion protein (PD-E7) comprising a mutated HPV-16 E7 linked to the first 108 amino acids of Haemophilus influenzae protein D, formulated in the GlaxoSmithKline Biologicals adjuvant AS02B, in patients bearing oncogenic HPV-positive cervical intraepithelial neoplasia (CIN). Methods: Seven patients, five with a CIN3 and two with a CIN1, received three intramuscular injections of adjuvanted PD-E7 at 2-week intervals. Three additional CIN1 patients received a placebo. CIN3 patients underwent conization 8 weeks postvaccination. Cytokine flow cytometry and ELISA were used to monitor antigen-specific cellular and antibody responses from blood taken before and after vaccine or placebo injection. Results: Some patients had preexisting systemic IFN- CD4⁺ (1/10) and CD8⁺ (5/10) responses to PD-E7. Vaccination, not placebo injection, elicited systemic specific immune responses in the majority of the patients. Five vaccinated patients (71%) showed significantly increased IFN- CD8⁺ cell responses upon PD-E7 stimulation. Two responding patients generated long-term T-cell immunity toward the vaccine antigen and E7 as well as a weak H. influenzae protein D (PD)–directed CD4⁺ response. All the vaccinated patients, but not the placebo, made significant E7- and PD-specific IgG. Conclusions: The encouraging results obtained from this study performed on a limited number of subjects justify further analysis of the efficacy of the PD-E7/AS02B vaccine in CIN patients.     

Characterization of functional bacterial groups in a hypersaline microbial mat community (Salins-de-Giraud, Camargue, France)

A photosynthetic microbial mat was investigated in a large pond of a Mediterranean saltern (Salins-de-Giraud, Camargue, France) having water salinity from 70 per thousand to 150 per thousand (w/v). Analysis of characteristic biomarkers (e.g., major microbial fatty acids, hydrocarbons, alcohols and alkenones) revealed that cyanobacteria were the major component of the pond, in addition to diatoms and other algae. Functional bacterial groups involved in the sulfur cycle could be correlated to these biomarkers, i.e. sulfate-reducing, sulfur-oxidizing and anoxygenic phototrophic bacteria. In the first 0.5 mm of the mat, a high rate of photosynthesis showed the activity of oxygenic phototrophs in the surface layer. Ten different cyanobacterial populations were detected with confocal laser scanning microscopy: six filamentous species, with Microcoleus chthonoplastes and Halomicronema excentricum as dominant (73% of total counts); and four unicellular types affiliated to Microcystis, Chroococcus, Gloeocapsa, and Synechocystis (27% of total counts). Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments confirmed the presence of Microcoleus, Oscillatoria, and Leptolyngbya strains (Halomicronema was not detected here) and revealed additional presence of Phormidium, Pleurocapsa and Calotrix types. Spectral scalar irradiance measurements did not reveal a particular zonation of cyanobacteria, purple or green bacteria in the first millimeter of the mat. Terminal-restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA gene fragments of bacteria depicted the community composition and a fine-scale depth-distribution of at least five different populations of anoxygenic phototrophs and at least three types of sulfate-reducing bacteria along the microgradients of oxygen and light inside the microbial mat.     

Mapping of bovine skeletal muscle proteins using two-dimensional gel electrophoresis and mass spectrometry

The large individual variation in meat quality seen both within and between animals is not fully understood. Consequently, our long-term goal is to identify reliable proteins which control or determine bovine meat quality. Using a proteomic approach, bovine skeletal muscle samples were analyzed by two-dimensional gel electrophoresis (2-DE) using an immobilized pH 4-7 gradient in the first dimension and mass spectrometry. We first tested the reproducibility of the method. These experiments showed slightly greater intersample than intrasample variability. In order to evaluate the type of visualized proteins in 2-DE, we initiated the construction of a protein reference map of bovine Semitendinosus muscle. In total, 129 protein spots corresponding to 75 different gene products were identified. Of these proteins, the largest portion is involved in metabolism (25.5%), cell structure (17%), cell defense (16%) and contractile apparatus (14.5%). One quarter of the identified proteins are represented by two or several protein spots and multiple isoforms of troponin T are present. Peptide mass fingerprint results indicate that these isoforms are partly generated by alternative splicing. The data presented here are an important step for further proteome analyses on bovine muscle. This may lead to progress in understanding the mechanisms controlling postmortem muscle metabolism and meat quality.     

Hsp90 invades the outside

Metalloproteases are required for the invasive nature of cancer cells. Surprisingly, the cytosolic molecular chaperone Hsp90 is now shown to promote maturation of the extracellular metalloprotease MMP2. This finding extends the multiplicity of roles assigned to the Hsp90 family to a new function outside the cell.     

Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins

Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure-conserving solubilization of membrane proteins, and one such polymer, 18C(10), was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrie et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca(2+)-dependent ATPase and Ca(2+)-pumping activity. We found that 18C(10)-mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long-term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions.