β1 Integrin triggering affects leukemic cell line sensitivity to natural killer cells

The role of beta1 (CD29) integrins in natural killer (NK) cell-target cell conjugation and cytotoxicity has not been clearly established. Ligation of beta1 integrins in NK cells can modulate the lytic capacity in both a positive and a negative manner; however, the contribution of the beta1 integrins present on target cells remains to be evaluated. Here, we analyzed the effect of beta1 integrins expressed by potential tumor target cells on conjugation and cytotoxicity. Using normalized flow cytometry binding assays, we demonstrated that the pretreatment of MOLT-4, K562, U-937 and HL-60 human leukemia target cell lines with selected anti-beta1 monoclonal antibodies (mAb) increased conjugation to human NK cell line NKL as well as to purified NK cells. Only mAb recognizing residues 207-218 of the beta1 subunit and functionally involved in the induction of homotypic adhesion (functional epitope A1) increased conjugation of all the target cells. Moreover, mAb to adhesion molecules different from beta1 but also inducers of homotypic adhesion of the target cells, i.e. CD43 and CD50 (ICAM-3), failed to increase conjugation to NKL cells. Cytotoxicity assays demonstrated that lysis of NK-sensitive target cells (MOLT-4) also increased after pretreatment with anti-beta1 epitope A1 mAb. Importantly, pretreatment of NK-resistant target cells (U-937 and HL-60) with anti-beta1 mAb was not able to outweigh the cytotoxic inhibitory mechanisms controlled by HLA class I molecules. However, simultaneous masking of HLA class I molecules with mAb and pretreatment with anti-beta1 mAb rendered NK-resistant cells susceptible to lysis, as predicted by the missing self hypothesis. Triggering of tumor target cells through beta1 integrins may thus play a role in conjugation to NK cells as well as in co-stimulation of cell-mediated cytotoxicity.     

The role of mitochondrial nitric oxide synthase in inflammation and septic shock

Nitric oxide and cytokines constitute the molecular markers and the intercellular messengers of inflammation and septic shock. Septic shock occurs with an exacerbated inflammatory response that damages tissue mitochondria. Skeletal muscle appears as one of the main target organs in septic shock, showing an increased nitric oxide (NO) production, an early oxidative stress, and contractile failure. Mitochondria isolated from rat and human skeletal muscle in septic shock show a markedly increased NO generation and a decreased state 3 respiration, more marked with nicotinamide adenine dinucleotide (NAD)-linked substrates than with succinate, without uncoupling or impairment of phosphorylation. One of the current hypothesis for the molecular mechanisms of septic shock is that the enhanced NO production by mitochondrial nitric oxide synthase (mtNOS) leads to excessive peroxynitrite (ONOO(-)) production and protein nitration in the mitochondrial matrix, to mitochondrial dysfunction and to contractile failure. Surface chemiluminescence is a useful assay to assess inflammation and oxidative stress in in situ liver and skeletal muscle. Liver chemiluminescence in inflammatory processes and phagocyte chemiluminescence have been found spectrally different from spontaneous liver chemiluminescence with increased 440-600 nm emission, likely due to NO and ONOO(-) participation in the reactions leading to the formation of excited species.     

Direct evidence for membrane pore formation by the apoptotic protein Bax

Direct imaging of the interaction of the apoptotic protein, Bax, with membrane bilayers shows the presence of toroidal-shaped pores using atomic force microscopy. These pores are sufficiently large to allow passage of proteins from the intermitochondrial space. Both the perturbation of the membrane and the amount of protein bound to the bilayer are increased in the presence of calcium. The results from the imaging are consistent with leakage studies from liposomes of the same composition. The work shows that Bax by itself can form pores in membrane bilayers.     

Decidual lymphocytes of human spontaneous abortions induce apoptosis but not necrosis in JEG-3 extravillous trophoblast cells

The human decidua contains an unusually high proportion of lymphocytes, mainly NK and T cells, which are potentially cytotoxic to the trophoblast when they are stimulated with certain cytokines. Given the high incidence of spontaneous abortion in humans and other species, our working hypothesis is that decidual lymphocytes are involved in immunological mechanisms that attack the trophoblast and induce abortion when any gestational problem arises. To test this hypothesis, flow cytometry was used to compare decidual lymphocyte populations in first-trimester spontaneous abortions and elective terminations of first-trimester pregnancy. We found significantly higher proportions of decidual lymphocytes that expressed activation markers, and of T cells (mainly T helper cells) in spontaneous abortions than in elective terminations of pregnancy. Decidual lymphocytes from spontaneous abortion, like decidual lymphocytes from elective termination of pregnancy and peripheral blood lymphocytes, were however, unable to lyse the JEG-3 extravillous cytotrophoblast cell line in a (51)Cr-release assay. Nevertheless, decidual lymphocytes from spontaneous abortion, unlike decidual lymphocytes from elective termination of pregnancy and peripheral blood lymphocytes, induced apoptosis in JEG-3 cells as determined by DNA fragment-release assay. Hematoxylin and eosin staining showed a significantly higher proportion of apoptotic JEG-3 cells when these cells were treated with decidual lymphocytes from spontaneous abortion than when JEG-3 cells were cultured with decidual lymphocytes from elective termination of pregnancy. The ultrastructural signs of apoptosis were confirmed by electron microscopy. These data support the hypothesis that activated decidual lymphocytes participate in human spontaneous abortion by inducing apoptosis but not necrosis of the trophoblast.     

Structural and functional properties of apolipoprotein A-I mutants containing disulfide-linked cysteines at positions 124 or 232

Recombinant Cys mutants of apolipoprotein A-I (apoA-I) (A124C and A232C) have been prepared in disulfide-linked forms in order to assess the effects of unnatural covalent constraints on the folding of apoA-I in solution, its ability to bind lipids, form HDL-like particles, activate LCAT, and undergo structural adaptations to changing lipid contents. Both mutants, in dimer form, were shown to fold similarly to plasma apoA-I in solution, but had a slightly decreased alpha-helix content and no evidence of intermonomer interactions. All forms of the mutants bound to and disrupted dimyristoylphosphatidylcholine (DMPC) liposomes with similar kinetics and efficiency to plasma apoA-I, and formed reconstituted HDL (rHDL) particles with palmitoyloleoylphosphatidylcholine (POPC) in high yields at three different ratios of lipid/protein. While the monomeric mutants produced identical rHDL to plasma apoA-I, the disulfide-linked dimers had distinct particle distributions from each other and from native apoA-I. The A124C-dimer formed rHDL with diameters of 86 and 78 A, while the A232C-dimer predominantly formed 96 A rHDL. These particles, and particles containing plasma apoA-I (96 and 78 A), were purified prior to structural and functional analyses. The structural properties of particles with similar diameters were comparable, as were their reactivities with LCAT; however, their ability to undergo structural rearrangements differed. The larger rHDL particles (96 and 86 A) containing native apoA-I or A124C-dimer, rearranged into smaller 78 A particles, while the 96 A particles containing A232C-dimer were resistant to rearrangement and did not form 78 A particles. From the results, it is concluded that synthetic, random disulfide-linked dimers of apoA-I have many properties analogous to those of the naturally occurring Cys mutants, apoA-I-Milano and apoA-I-Paris, which are thought to have antiatherogenic effects in vivo. Also, the results have implications for current models of rHDL structure.     

First comparative phenetic studies of Argentinean species of Acacia (Fabaceae), using morphometric, isozymal, and RAPD approaches

Morphological and genetic diversity among Acacia aroma, A. macracantha, A. caven, and A. furcatispina were studied with morphometric, isozymal, and RAPD approaches. The analysis of seven isozyme systems revealed 21 loci, and RAPD analysis showed 34 loci. Most of these loci allowed us to differentiate the species, with the exception of A. aroma and A. macracantha, the two most similar species. The levels of genetic variability estimated by isozymes were higher than those obtained from RAPD analyses. Morphometric characters showed highly significant differences among the species, although A. aroma and A. macracantha are differentiated only by thorn length. The phenogram obtained from isozyme data is consistent with morphological data. The RAPD phenogram based on allelic frequencies showed agreement with morphological and isozymal approaches only at the intraspecific levels, while the RAPD phenogram based on Nei and Li's similarity measures agreed with the phenograms constructed from isozyme and morphological data. High similarities and high indirect gene flow were found between A. aroma and A. macracantha, results that call the relationship between them into question.     

Cell-cycle-dependent regulation of CNT1, a concentrative nucleoside transporter involved in the uptake of cell-cycle-dependent nucleoside-derived anticancer drugs

Most nucleoside-derived anticancer drugs are taken up by the high-affinity Na-dependent nucleoside transporter CNT1. Since such drugs are to some extent cell-cycle-dependent in their cytotoxic action, we examined the relationship between CNT1 expression and cell-cycle progression in the rat hepatoma cell line FAO. Cell cultures were synchronized either at late G1 or early S stages by combining mimosin treatment with either previous synchronization or not by serum starvation. Cell-cycle progression was then assessed by measuring [methyl-3H]thymidine incorporation into DNA and monitoring cyclin E and A protein levels. In these conditions, CNT1 protein amounts increase at the G1-S transition. When cells were synchronized using hydroxyurea (HU), which directly interacts with nucleotide metabolism by inhibiting ribonucleotide reductase, CNT1 protein amounts increased in synchronized cells and remained high during cell-cycle progression. These data indicate that CNT1 adapts to cell-cycle progression and responds to nucleos(t)ide metabolism status, a feature that might contribute to the cytotoxic action of cell-cycle-dependent anticancer drugs.     

Identification and characterization of the<Emphasis Type="Italic"> carAB</Emphasis> genes responsible for encoding carbamoylphosphate synthetase in<Emphasis Type="Italic"> Halomonas eurihalina</Emphasis>

Halomonas eurihalina is a moderately halophilic bacterium which produces exopolysaccharides potentially of great use in many fields of industry and ecology. Strain F2-7 of H. eurihalina synthesizes an anionic exopolysaccharide known as polymer V2-7, which not only has emulsifying activity but also becomes viscous under acidic conditions, and therefore we consider it worthwhile making a detailed study of the genetics of this strain. By insertional mutagenesis using the mini-Tn 5 Km2 transposon we isolated and characterized a mutant strain, S36 K, which requires both arginine and uracil for growth and does not excrete EPS. S36 K carries a mutation within the carB gene that encodes the synthesis of the large subunit of the carbamoylphosphate synthetase enzyme, which in turn catalyzes the synthesis of carbamoylphosphate, an important precursor of arginine and pyrimidines. We describe here the cloning and characterization of the carAB genes, which encode carbamoylphosphate synthetase in Halomonas eurihalina, and discuss this enzyme's possible role in the pathways for the synthesis of exopolysaccharides in strain F2-7.