Implication of an Outer Surface Lipoprotein in Adhesion of Bifidobacterium bifidum to Caco-2 Cells

We found that the human intestinal isolate Bifidobacterium bifidum MIMBb75 strongly adhered to Caco-2 cells. Proteinase K and lithium chloride treatments showed that proteins play a key role in MIMBb75 adhesion to Caco-2 cells. By studying the cell wall-associated proteins, we identified a surface protein, which we labeled BopA. We purified the protein chromatographically and found that it functioned as an adhesion promoter on Caco-2 cells. In silico analysis of the gene coding for this protein and globomycin experiments showed that BopA is a cysteine-anchored lipoprotein expressed as a precursor polypeptide. A database search indicated that BopA appears to function biologically as an oligopeptide/tripeptide-solute-binding protein in the ABC transport system. We discovered a protein corresponding to BopA and its gene in eight other highly adherent B. bifidum strains. Finally, we found that B. bifidum MIMBb75 and BopA affected the production of interleukin-8 in Caco-2 epithelial cells. BopA is the first protein described to date to be directly involved in the adhesion of bifidobacteria to Caco-2 cells and to show immunomodulatory activity.     

In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver

Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5′-dimethylpyrazole, DMP, 12 mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis.     

Ebolavirus and Marburgvirus: insight the Filoviridae family

Ebolavirus and Marburgvirus (belonging to the Filoviridae family) emerged four decades ago and cause epidemics of haemorrhagic fever with high case-fatality rates. The genome of filoviruses encodes seven proteins. No significant homology is observed between filovirus proteins and any known macromolecule. Moreover, Marburgvirus and Ebolavirus show significant differences in protein homology. The natural maintenance cycle of filoviruses is unknown, the natural reservoir, the mode of transmission, the epidemic disease generation, and temporal dynamics are unclear. Lastly, Ebolavirus and Marburgvirus are considered as potential biological weapons. Vaccine appears the unique therapeutic frontier. Here, molecular and clinical aspects of filoviral haemorrhagic fevers are summarized.     

Structural perturbation of alphaB-crystallin by zinc and temperature related to its chaperone-like activity

alphaB-crystallin is a small heat shock protein that shows chaperone-like activity, as it protects the aggregation of denatured proteins. In this work, the possible relationships between structural characteristics and the biological activity of alphaB-crystallin were investigated on the native protein and on the protein undergoing the separate effects of metal ligation and temperature. The chaperone-like activity of alphaB-crystallin increased in the presence of zinc and when temperature was increased. By using fluorescent probes to monitor hydrophobic surfaces on alphaB-crystallin, it was found that exposed hydrophobic patches on the protein surface increased significantly both in the presence of zinc and when the temperature was raised from 25 to 37 degrees C. The zinc-induced increased exposure of lipophilic residues is in agreement with theoretical calculations performed on 3D-models of monomeric alphaB-crystallin, and may be significant to its increased biological activity.     

Half-sandwich Ru II[9]aneS3 complexes structurally similar to antitumor-active organometallic piano-stool compounds: preparation, structural characterization and in vitro cytotoxic activity

The preparation, structural characterization, and chemical behavior in aqueous solution of a series of new Ru[9]aneS3 half-sandwich complexes of the type [Ru([9]aneS3)Cl(NN)][CF3SO3] and [Ru([9]aneS3)(dmso-S)(N-N)][CF3SO3]2 (5-15, NN=substituted bpy or 2x1-methylimidazole) are described. The X-ray structures of [Ru([9]aneS3)Cl(3,3'-H2dcbpy)][CF3SO3] (9) (3,3'-H2dcbpy=3,3'-dicarboxy-2,2'-bipyridine), [Ru([9]aneS3)Cl(4,4'-dmobpy)][CF3SO3] (13) (4,4'-dmobpy=4,4'-dimethoxy-2,2'-bipyridine), and [Ru([9]aneS3)Cl(1-MeIm)2][CF3SO3] (15) (1-MeIm=1-methylimidazole) were also determined. The new compounds are structurally similar to anticancer-active organometallic half-sandwich complexes of formula [Ru(eta6-arene)Cl(NN)][PF6]. Three chloro compounds (5, 9, 15) were tested in vitro for cytotoxic activity against two human cancer cell lines in comparison with the previously described [Ru([9]aneS3)Cl(en)][CF3SO3] (1, en=ethylenediamine), [Ru([9]aneS3)Cl(bpy)][CF3SO3] (2), and with their common dmso precursor [Ru([9]aneS3)Cl(dmso-S)2][CF3SO3] (3). Only the ethylenediamine complex 1 showed some antiproliferative activity, ca. one order of magnitude lower than the reference organometallic half-sandwich compound RM175 that contains biphenyl instead of [9]aneS3. This compound was further tested against a panel of human cancer cell lines (including one resistant to cisplatin).     

Studies on the anti-proliferative effects of novel DNA-intercalating bipyridyl-thiourea-Pt(II) complexes against cisplatin-sensitive and -resistant human ovarian cancer cells

Six bipyridyl complexes of platinum(II) with thiourea, with different substituents on thiourea moiety [Pt(bipy)(R,R'NCSNR',R')(2)]Cl(2) (bipy=2,2'-bipyridine: R=R'=R'=R' =H; R=Me, R'=R'=R'=H; R=n-Bu, R'=R'=R'=H; R=Et, R'=H, R'=Et, R'=H; R=p-tolyl, R'=R'=R'=H; R=phenyl, R'=H, R'=phenyl, R'=H), rationally designed to intercalate into DNA, have been tested against a cisplatin (cDDP)-sensitive human ovarian carcinoma cell line (2008) and its -resistant variant (C13( *)). We show here that the anti-proliferative efficacy of these drugs was dependent on molecular structure, since it increased with ancillary ligand bulkiness and hydrophobicity of substituents on thiourea moiety. In particular, the presence of two phenyl groups on thiourea moiety confers an outstanding cytotoxicity. The increasing cell growth inhibition along the series of complexes partially paralleled with drug accumulation, particularly in resistant cells, but not with drug intercalation into DNA since all compounds exerted comparable ethidium bromide displacement ability. The cDDP-resistant phenotype seems, at least in part, to be involved in the action of these compounds, since the level of cross-resistance established for most complexes appeared to be in agreement with the observed impairment of drug accumulation in the resistant subline. These findings indicate that resistance to alkylating agents such as cDDP confers low level of cross-resistance to this class of DNA intercalators, which, however, depending on substituents on thiourea moiety may present remarkable cell growth inhibition even of resistant cells.     

Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity

Proteinase-activated receptor 2 (PAR(2)), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR(2), often measured by Ca(2+) release. PAR(2) contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR(2) in HEK293T cells resulted in concentration-dependent, PAR(2) AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple G(i) proteins or sequester intracellular Ca(2+). Because previous studies described synergistic PAR(2)- and TLR4-mediated cytokine production, we hypothesized that PAR(2) and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR(2)-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR(2) in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR(2) and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR (-/-)(2) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.     

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr(78)) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.     

Modified T-RFLP methods for taxonomic interpretation of T-RF

Terminal restriction fragment length polymorphism (TRFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct TRFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.