The heterocytotoxicity of human serum. II. Role of natural antibody and of the classical and alternative complement pathways.

Mouse lymphoid cells and tumor cell lines were employed as target cells for the investigation of the mechanism of heterocytotoxicity in human serum samples. It was shown that the heterocytotoxic effects were due to two differing mechanisms. Cytotoxicity was mediated in part, by activation of the alternative complement pathway on target cell membrane, a process which was antibody-independent. A second mechanism of cytotoxicity was induced by natural antibodies to a target cell, which probably mediated activation of the classical complement pathway. These data may shed light on the frequently observed cytotoxicity in mammalian sera for various target cells.     

In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.     

New structure–activity relationship studies in a series of N,N-bis(cyclohexanol)amine aryl esters as potent reversers of P-glycoprotein-mediated multidrug resistance (MDR)

As a continuation of previous research on a new series of potent and efficacious P-gp-dependent multidrug resistant (MDR) reversers with a N,N-bis(cyclohexanol)amine scaffold, we have designed and synthesized several analogs by modulation of the two aromatic moieties linked through ester functions to the N,N-bis(cyclohexanol)amine, aiming to optimize activity and to extend structure–activity relationships (SAR) within the series. This scaffold, when esterified with two different aromatic carboxylic acids, gives origin to four geometric isomers (cis/trans, trans/trans, cis/cis and trans/cis).The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Most of them resulted in being potent modulators of the extrusion pump P-gp, showing potency values ([I]_0.5) in the submicromolar and nanomolar range. Of these, compounds 2b, 2c, 3d, 5a–d and 6d, showed excellent efficacy with a α_max close to 1. Selected compounds (2d, 3a, 3b, 5a–d) were further studied to evaluate their doxorubicin cytotoxicity potentiation (RF) on doxorubicin-resistant erythroleukemia K562 cells and were found able to enhance significantly doxorubicin cytotoxicity on K562/DOX cells.The results of both pirarubicin uptake and the cytotoxicity assay, indicate that the new compounds of the series are potent P-gp-mediated MDR reversers. They present a structure with a mix of flexible and rigid moieties, a property that seems critical to allow the molecules to choose the most productive of the several binding modes possible in the transporter recognition site.In particular, compounds 5c and 5d, similar to the already reported analogous isomers 1c and 1d,²⁹ are potent and efficacious modulators of P-gp-dependent MDR and may be promising leads for the development of MDR-reversal drugs.     

Integrin alpha3 subunit participates in myoblast adhesion and fusion in vitro

Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin alpha3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin alpha3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin alpha3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin alpha3 subunit co-localizes with actin, and binds the integrin beta1 subunit and ADAM12, suggesting that the complex alpha3beta1/ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin alpha3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin alpha3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.     

The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS

Remyelination of primary demyelinated lesions is a common feature of experimental models of multiple sclerosis (MS) and is also suggested to be the normal response to demyelination during the early stages of MS itself. Many lines of evidence have shown that remyelination is preceded by the division of endogenous oligodendrocyte precursor cells (OPCs) in the lesion and its borders. It is suggested that this rapid response of OPCs to repopulate the lesion site and their subsequent differentiation into new oligodendrocytes is the key to the rapid remyelination. Antibodies to the NG2 chondroitin sulphate proteoglycan have proved exceedingly useful in following and quantitating the response of endogenous OPCs to demyelination. Here we review the literature on the response of NG2-expressing OPCs to demyelination and provide some new evidence on their response to the chronic inflammatory demyelinating environment seen in recombinant myelin oligodendrocyte glycoprotein (MOG) induced experimental allergic encephalomyelitis (EAE) in the DA rat. NG2-expressing OPCs responded to the inflammatory demyelination in this model by becoming reactive and increasing in number in a very focal manner. Evidence of NG2⁺OPCs in lesioned areas beginning to express the oligodendrocyte marker CNP was also seen. The response of OPCs appeared to occur following successive relapses but did not always lead to remyelination, with areas of chronic demyelination observed in the spinal cord. The presence of OPCs in the adult human CNS is clearly of vital importance for repair in multiple sclerosis (MS). As in rat tissue, the antibody labels an evenly distributed cell population present in both white and grey matter, distinct from HLA-DR⁺microglia. NG2⁺cells are sparsely distributed in the centre of chronic MS lesions. These cells apparently survive demyelination and exhibit a multi-processed or bipolar morphology in the very hypocellular environment of the lesion.     

A block to efficient replication of simian immunodeficiency virus in C8166 cells can be overcome by duplication of the NF-κB binding site

Sequence analysis of the acutely lethal pbj14 strain of simian immunodeficiency virus (SIVpbj14) clone revealed among other differences from its less pathogenic counterparts a duplication of its binding site for nuclear factor kappa B (NF-B) in its long terminal repeats (LTR). We have investigated whether introducing a similar duplication into the pathogenic molecular clone SIVmac239 would alter its biological properties. We compared an SIV which possessed 2 NF-B sites to the wild type, a single NF-B site virus, with respect to its ability to replicate in vitro in established CD4+ T cell lines, primary peripheral blood mononuclear cells (PBMCs), and primary alveolar macrophages. The virus containing 2 NF-B sites exhibited no apparent difference from wild type in established cell lines 174×CEM, MT-2 and MT-4, or in primary PBMC or tissue macrophage cultures. However, the 2 B virus replicated well in the established cell line C8166, while the wild type, 1 B virus replicated very poorly in this cell type, suggesting that duplication of the NF-B site is capable of overcoming a block to efficient replication of SIVmac239 in C8166 cells. Interestingly, Em*, a macrophage tropic SIVmac that differs from SIVmac239 by 9 amino acids in the envelope region yet possesses only one NF-B binding site, also replicates well in C8166. The data suggest that the replication of wild type SIVmac239 is restricted in C8166 cells, but that this restriction can be overcome either by changes in the LTR or by changes in the envelope region.     

Characterization of phospholipase A2 activation by plasmin in cultured bovine endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 0.1 µM human plasmin has been reported to induce a receptor-mediated short burst of arachidonate release, which is a pertussis toxin-sensitive and extracellular calcium-dependent reaction. Plasmin-induced calcium influx in cells was significantly inhibited by pretreatment with pertussis toxin, indicating that the former was coupled with a pertussis toxin-sensitive guanosine 5-triphosphate (GTP)-binding protein. Plasmin significantly induced the formation of lysophosphatidylcholine but not lysophosphatidylethanolamine. A cellular phospholipase A₂ with an arachidonyl specificity at the sn-2 position of phosphatidylcholine, which required submicromolar calcium, was identified as a cytosolic phospholipase A₂ by immunoblot analysis. By a cell-free enzyme activity assay and immunoblot analysis, plasmin was found to induce a translocation of the cytosolic phospholipase A₂ from the cytosol to the membrane. Taken together, the results suggest that plasmin bound to its putative receptor and activated a GTP-binding protein coupled to calcium influx channel, followed by translocation and activation of cytosolic phospholipase A₂ in endothelial cells.     

Inhibition ofras oncogene: A novel approach to antineoplastic therapy

The most frequently detected oncogene alterations, both in animal and human cancers, are the mutations in the ras oncogene family. These oncogenes are mutated or overexpressed in many human tumors, with a high incidence in tumors of the pancreas, thyroid, colon, lung and certain types of leukemia. Ras is a small guanine nucleotide binding protein that transduces biological information from the cell surface to cytoplasmic components within cells. The signal is transduced to the cell nucleus through second messengers, and it ultimately induces cell division. Oncogenic forms of p21(ras) lead to unregulated, sustained signaling through downstream effectors. The ras family of oncogenes is involved in the development of both primary tumors and metastases making it a good therapeutic target. Several therapeutic approaches to cancer have been developed pointing to reducing the altered gene product or to eliminating its biological function: (1) gene therapy with ribozymes, which are able to break down specific RNA sequences, or with antisense oligonucleotides, (2) immunotherapy through passive or active immunization protocols, and (3) inhibition of p21(ras) farnesylation either by inhibition of farnesyl transferase or synthesis inhibition of farnesyl moieties.