Effects of betamethasone,sulindac and quinacrine drugs on the inflammatory neoangiogenesis response induced by polyurethane sponge implanted in mouse

In this study, we showed the effect of the betamethasone, sulindac and quinacrine alone or combined, on the inflammatory angiogenesis promoted by polyurethane sponge on mice. The main finding reported here is that the formation of new blood vessels was strongly inhibited by low concentration of betamethasone, sulindac or quinacrine, whether alone or in combination. It is known that steroidal anti-inflammatory drugs inhibit the enzymes required for the production of prostaglandins through a nuclear glucocorticoid receptor (GR) mediated mechanism. This mechanism may occur in endothelial cells as well. Considering that activity of cyclo-oxigenases 1 and 2 is inhibited by sulindac, and that these enzymes are located in the stromal tissue, we propose that the anti-angiogenic effect of these agents may occur via inhibition of both COX isoforms. On the other hand, quinacrine inhibited PLA2 activity, and we propose here that the anti-angiogenic effect occurs via inhibition of the enzyme PLA2. The potentiated effect of the association of betamethasone, sulindac and quinacrine may have some therapeutic benefit in the control of pathological angiogenesis. Further studies are required to validate these propositions.     

Morbidity and mortality factors in key deer (Odocoileus virginianus clavium)

The population health of endangered Key deer (Odocoileus virginianus clavium) was monitored from 10 February 1986 to 28 September 2000 by necropsy of animals that were killed by vehicles, euthanized because of terminal injuries or disease conditions, or found dead. The predominant mortality factor during the period was collision with motor vehicles; however, several infectious diseases were diagnosed, including infections with Arcanobacterium pyogenes, Haemonchus contortus, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis. During the period monitored, the only infectious disease that was thought to have affected population dynamics was haemonchosis. Nevertheless, several of the observed diseases have potential to impact viability of the Key deer population under appropriate environmental conditions.     

Characterization of genetic markers for in vitro cell line identification of the marine sponge Axinella corrugata

The marine sponge Axinella corrugata is being developed as a model organism for in vitro marine invertebrate research. Molecular genetics methods such as DNA fingerprinting [amplified fragment length polymorphism (AFLP) and single-stranded conformation polymorphism (SSCP)] and single-locus DNA sequence analyses were applied to this model to meet the primary objective of identifying positive A. corrugata-specific molecular markers that will aid in verifying cell identity in vitro and distinguish sponge cells from potential microbial contaminants. The extent of intra- and interspecific variation in these markers from geographically distinct samples of A. corrugata and closely related sponge taxa was also assessed. Two novel nuclear loci along with intervening transcribed spacer (ITS) regions of nuclear rRNA were characterized, although the latter appeared to better meet primary marker criteria, such as taxonomic specificity and high frequency of detection (via polymerase chain reaction [PCR]) from different individuals (n > 40) and cell cultures. Phylogenetic and phylogeographic analyses of ITS DNA sequences helped clarify taxonomies and also suggested species boundaries between and among western Atlantic and eastern Atlantic/Indian Ocean A. corrugata and Axinellidae samples. Patterns of genetic variation have important implications for the systematics, evolution, and chemical ecology of A. corrugata and related axinellids and are discussed.     

Linkage analysis for prenatal diagnosis in a familial case of Stickler syndrome

The Stickler syndrome is among the most common heritable disorders of connective tissue. The syndrome fully expressed clinical phenotype includes the degeneration of the vitreous gel and retina, frequently associated with myopia, accompanied by non-ocular features, such as craniofacial dysmorphisms or malformations, hearing impairment, skeletal dysplasia and progressive arthropathy. So far, mutations at three collagen loci, COL2A1, COL11A1 and COL11A2, have been found in Stickler syndrome patients, with about two thirds of investigated familial cases found to be associated to COL2A1 gene mutations. We report on a three generation family in which a diagnosis of Stickler syndrome was made and linkage analysis suggested COL2A1 to be the causing gene. These data permitted us to perform two prenatal diagnosis analysing the 3'VNTR polymorphism of the involved gene on amniocytes' DNA and to provide the family with genetic counselling and paediatric support at the delivery.     

Parathyroid hormone effects on signaling pathways in endothelial cells vary with peptide concentration

We have previously reported that parathyroid hormone (PTH) has specific effects on a human umbilical vein endothelial cell line. Further studies were performed to characterize the signaling cascades initiated by PTH. We report that PTH induced the appearance of voltage sensitive calcium channels. Furthermore, PTH increased ceramide but not diacylglycerol content. Since elevations in [Ca(2+)](i) and phospholipid turnover are signals for the activation of protein kinase C (PKC), the cells were screened for PKC isoforms. PTH induced a redistribution of the PKCepsilon to the particulate fractions of cell homogenates. In summary, PTH induced PKC translocation through a calcium-phospholipid pathway in an endothelial cell line.     

Exploring the active site of Trypanosoma brucei phosphofructokinase by inhibition studies: specific irreversible inhibition

This work deals with the phosphofructokinase enzyme (PFK) of the parasite Trypanosoma brucei. Inhibitors which are analogues of fructose-6-phosphate (F6P) derived from 2,5-anhydromannitol and therefore blocked in a closed conformation, both nonphosphorylated and phosphorylated, were designed. They provided information on this class of ATP-dependent PFK (structurally more similar to PPi-dependent PFKs revealing (i) an ordered mechanism, ATP binding first, inducing an essential conformational change to increase the affinity for F6P, and (ii) a rather hydrophobic environment at the ATP binding site. Nonphosphorylated mannitol derivatives bind at both the ATP and F6P binding sites, whereas the phosphorylated derivatives only bind at the ATP binding site. The inhibitors bearing an aromatic ring substituted at the meta position indicate a polar interaction with lysine 227, which is specific to T. brucei PFK and is replaced by a glycine in human PFK. This lysine can be irreversibly bound, leading to inhibition when an electrophilic carbon atom is beta to the meta position on the ring. This lysine was identified by site-directed mutagenesis. This first example of a specific irreversible inactivation of T. brucei PFK offers an opportunity to develop biologically active compounds against the sleeping sickness, the causative agent of which is the trypanosome.     

Plant chromosomal HMGB proteins efficiently promote the bacterial site-specific beta-mediated recombination in vitro and in vivo

In the presence of an accessory DNA bending protein, the bacterial site-specific beta recombinase catalyzes resolution and DNA inversion. Five different maize high mobility group B (HMGB) proteins were examined for their potential to facilitate beta recombination in vitro using DNA substrates with different intervening distances (73-913 bp) between two directly oriented recombination (six) sites. All analyzed HMGB proteins (HMGB1 to HMGB5) could promote beta recombination, but depending on the DNA substrate with different efficiencies. The HMGB1 protein displayed an activity comparable to that of the natural promoting protein Hbsu, whereas the other HMGB proteins were less effective. Phosphorylation of the HMGB1 protein resulted in an increased efficiency of HMGB1 to promote beta recombination. Analyses of DNA substrates with closely spaced six sites demonstrated that in the presence of HMGB1 the recombination rate was correlated to the distance between the six sites, but independent of the helical orientation of the six sites. Using a Bacillus subtilis strain defective in Hbsu, the coexpression of beta recombinase and HMGB1 (or a truncated HMGB1 derivative) revealed that a plant HMG-box domain protein is sufficient for assisting beta to catalyze recombination in vivo. Our results using beta recombination as a model system suggest that the various plant HMGB proteins (and their posttranslationally modified versions) have the potential of forming a repertoire of different DNA structures, which is compatible with the idea that the HMGB proteins can act as architectural factors in a variety of nucleoprotein structures.     

Novel immunogenicity of Thomsen-Friedenreich disaccharide obtained by a molecular rotation on its carrier linkage

The alpha-anomeric Galbeta1-3GalNAc, called Thomsen-Friedenreich disaccharide (TFD), is overexpressed in epithelial cancer cells by aberrant O-glycosylation. TFD is also the main ligand of Agaricus bisporus lectin (ABL), a reversible noncytotoxic inhibitor of proliferation of epithelial cell lines. In order to obtain anti-TFD antibody response with a fine carbohydrate-binding specificity similar to that of ABL, we designed an immunogen of TFD with a molecular rotation on its carrier linkage that exposes more GalNAc than Gal, since ABL recognizes GalNAc more than Gal in TFD. The synthesis was accomplished by C-6 oxidation of Gal from TFD or its alpha-benzyl derivative (BzlalphaTFD), followed by reductive amination between the C-6 aldehyde yielded and the available amine of protein. Mice immunized with TFD-KLH (keyhole limpet hemocyanin) or BzlalphaTFD-KLH produced antibodies which were then analyzed by ELISA against several target antigens. Both immunogens raised anti-KLH antibody titers; however, TFD-KLH did not raise anti-TFD antibodies showing low TFD immunogenicity. In contrast, BzlalphaTFD-KLH gave much higher anti-TFD antibody response, indicating that benzyl residue helps improve anti-carbohydrate immune response. When IgG and IgM anti-TFD antibodies were analyzed by competitive ELISA using TFD-related carbohydrates as inhibitors, a high specificity to TFD as well as an enhanced binding to GalNAc over Gal were observed. The axial C-4 hydroxyl group of GalNAc interacted with IgG anti-TFD antibody, as evidenced by the lack of inhibitory activity of GlcNAc in contrast to GalNAc. These findings indicate that the anti-TFD antibodies have fine carbohydrate-binding specificity more similar to ABL than to other TFD-binding proteins that stimulate proliferation of epithelial cell lines.     

Human 100-kDa homologous DNA-pairing protein is the splicing factor PSF and promotes DNA strand invasion

Proteins promoting homologous pairing could be involved in various fundamental biological processes. Previously we detected two mammalian nuclear proteins of 100 and 75 kDa able to promote homologous DNA pairing. Here we report isolation and characterisation of the human (h) 100-kDa DNA-pairing protein, hPOMp100, from HeLa nuclei. The peptide sequences of hPOMp100 revealed identity to the human splicing factor PSF and a DNA-binding subunit of p100/p52 heterodimer of unknown function. Bacterially expressed PSF promotes DNA pairing identical to that of hPOMp100. hPOMp100/PSF binds not only RNA but also both single-stranded (ss) and double-stranded (ds) DNA and facilitates the renaturation of complementary ssDNAs. More important, the protein promotes the incorporation of a ss oligonucleotide into a homologous superhelical dsDNA, D-loop formation. A D-loop is the first heteroduplex DNA intermediate generated between recombining DNA molecules. Moreover, this reaction could be implicated in re-establishing stalled replication forks. Consistent with this hypothesis, DNA-pairing activity of hPOMp100/PSF is associated with cellular proliferation. Significantly, phosphorylation of hPOMp100/PSF by protein kinase C inhibits its binding to RNA but stimulates its binding to DNA and D-loop formation and may represent a regulatory mechanism to direct this multifunctional protein to DNA metabolic pathways.