Inability of IL-2 and IL-10 to counteract B cell clonal deletion.

The B cell antigen receptor (BCR) delivers inhibitory signals in nascent B cells leading to the establishment of tolerance via clonal deletion or clonal anergy depending upon the type of antigen to which the B cells are exposed. In previous work, it has been demonstrated that activated Th2 cells, as well as some recombinant lymphokines, prevent the inhibition of growth and subsequent cell death induced through the BCR in model B cell lymphomas. Herein, we extend this work to another Th2 lymphokine, IL-10, that in contrast to IL-4 does not interfere with the deletion promoted by IgM crosslinking. The effect of individual lymphokines has also begun to be analyzed in a transgenic model of B cell clonal deletion. To this end, we have administered a recombinant vaccinia virus producing human IL-2 to mice expressing an autoreactive H-2Kk,b-specific transgenic IgMk and found that IL-2 does not abrogate B cell deletion in vivo.     

Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents.

Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+, ascorbate, and O2, but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+, and catalase have no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site for ATPA (ATPA yields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOH-terminal domains. The oxidative inactivation promoted by ATP is inhibited by Mg2+, Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337. Groups in the 60-kDa domain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPB and support the binding of ATPB in the 60-kDa domain. We also demonstrate that mercaptoethanol, reducing impurities in glycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in the oxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.     

Platinum complexes of p- and m-aminobenzamidine. Synthesis, characterization, and cytotoxic activity.

Four platinum(II) aminobenzamidine complexes have been prepared and characterized by IR and 1H and 13C NMR spectroscopy, and tested for their ability to interact with the nicked and closed circular forms of the pUC8 plasmid DNA. The results show that the complexes of formula [Pt(LH)2Cl2]2X have a cis- geometry with an amino-Pt bonding, where L is either p- or m-aminobenzamidine and where 2X is 2Cl- or PtCl4(2-). It was observed that these complexes significantly alter the electrophoretic mobility of nicked and closed circular forms of DNA and that the alteration in electrophoretic mobility due to Pt(II)-p-aminobenzamidine binding is higher than that due to Pt(II)-m-aminobenzamidine. No difference in mobility was observed whether the DNA interacted with complexes having as counteranion Cl- or PtCl4(2-). The synthesized compounds were, in addition, assayed for antitumor activity in vitro against colon (CX-1), lung (LX-1), and mammary (MX-1) human tumor cells. The results show that these complexes inhibited the multiplication of the tumor cells and that they show higher specificity for lung cells.     

Differentiation of EC cells in vitro by the fluorescent dye Hoechst 33342

The fluorescent dye Hoechst 33342 is able to differentiate F9 EC cells at low concentrations. This differentiation is accompanied by synthesis of large amounts of laminin, production of a well-developed cytoskeleton, disappearance of the SSEA-1 antigen, and synthesis of large amounts of fibronectin, all characteristics of the primitive endoderm. The dye immediately blocks the cells at the S/G2 phase of the cell cycle and produces a complete arrest in proliferation. This effect is not specific for the nullipotent F9 cell line, as multipotent EC cell lines like PCC3, P19, and PCC4 can also be easily differentiated into the same pathway by treatment with the Hoechst dye. In contrast, the dye has no remarkable effects on terminal differentiated, immortalized cells like NIH 3T3 or the parietal endoderm-like cell PYS-2.     

The lamellar to hexagonal phase transition in phosphatidylethanolamine liposomes: a fluorescence anisotropy study

The steady-state anisotropy of trimethylammonium diphenylhexatriene fluorescence has been used to monitor the thermotropic lamellar to HII hexagonal phase transition in an unsaturated phosphatidylethanolamine. The transition is observed in lipid aggregates when they are heated above the transition temperature Th, as well as in diluted liposomes after aggregation above Th. Changes in fluorescence anisotropy are not observed with Ca(++)-induced fusion of phosphatidylserine vesicles, a process not involving hexagonal phase formation.     

Direct demonstration of an acid-labile phosphoenzyme in the cycle of the sarcoplasmic reticulum Ca2(+)-dependent adenosinetriphosphatase

The Ca2(+)-dependent adenosinetriphosphatase (Ca2(+)-ATPase) from the sarcoplasmic reticulum (SR) of rat skeletal muscles is phosphorylated by inorganic phosphate (Pi) in the absence of Ca2+. The reaction can be described by the following simplified scheme: [formula: see text] where E-P is a covalent, acid-stable and ADP-insensitive phosphoenzyme, and E.Pi is a noncovalent and acid-labile complex. The reaction is Mg2(+)-dependent. Membrane fragments deposited on Millipore filters were successively perfused with two solutions, at constant flow. The effluent samples were analyzed. The perfused solutions were Ca2+ free and always contained 40% dimethylsulfoxide (DMSO), plus other reactants. Following the successive perfusion of solutions without and with [32P]Pi, 32P binding is only detected in the presence of Mg2+, indicating the formation of the phosphoenzymes (E.Pi and E-P). Following perfusions of the phosphoenzymes with 5% trichloroacetic acid, 32P release indicates the amount of the acid-labile moiety (E.Pi). After phosphorylations, the filters were washed with acid and unlabeled Pi, and the remaining radioactivity was measured to evaluate the acid-stable phosphoenzyme (E-P). The acid-labile and acid-stable phosphoenzymes amounted, respectively, 0.72 +/- 0.12, and 1.48 +/- 0.10 nmol of Pi/mg of protein ( +/- S.E., n = 5), after phosphorylations with 20 microM Pi. The results indicate: (1) The method allowed the evaluation of the acid-labile intermediate of the SR Ca2(+)-ATPase cycle. Keq = k2/k-2), in the above scheme, approaches 2.0. (2) The substrate of the phosphorylation reaction, in the presence of DMSO, is likely to be the Mg.Pi complex, since Mg2+ is necessary for step 1 in the above scheme.     

GC subtyping and HIV infection in a Spanish population: no evidence of an association between GC subtypes and AIDS

Group-specific component (GC) subtyping was performed by isoelectric focusing in 318 Spanish drug users at risk for infection or infected by HIV (85 HIV seronegatives, 111 HIV seropositives without symptoms, 89 seropositives with symptoms, 33 AIDS patients) and 187 healthy individuals. There was no significant association between GC subtypes and susceptibility to HIV infection and/or progression to AIDS.     

Phospholipid turnover during phagocytosis in human polymorphonuclear leucocytes

We have previously observed that the phagocytosis of zymosan particles coated with complement by human polymorphonuclear leucocytes is accompanied by a time- and dose-dependent inhibition of phosphatidylcholine synthesis by transmethylation [García Gil, Alonso, Sánchez Crespo & Mato (1981) Biochem. Biophys. Res. Commun. 101, 740–748]. The present studies show that phosphatidylcholine synthesis by a cholinephosphotransferase reaction is enhanced, up to 3-fold, during phagocytosis by polymorphonuclear cells. This effect was tested by both measuring the incorporation of radioactivity into phosphatidylcholine in cells labelled with [Me-¹⁴C]choline, and by assaying the activity of CDP-choline:diacylglycerol cholinephosphotransferase. The time course of CDP-choline:diacylglycerol cholinephosphotransferase activation by zymosan mirrors the inhibition of phospholipid methyltransferase activity previously reported. The extent of incorporation of radioactivity into phosphatidylcholine induced by various doses of zymosan correlates with the physiological response of the cells to this stimulus. This effect was specific for phosphatidylcholine, and phosphatidyl-ethanolamine turnover was not affected by zymosan. The purpose of this enhanced phosphatidylcholine synthesis is not to provide phospholipid molecules rich in arachidonic acid. The present studies show that about 80% of the arachidonic acid generated in response to zymosan derives from phosphatidylinositol. A transient accumulation of arachidonoyldiacylglycerol has also been observed, which indicates that a phospholipase C is responsible, at least in part, for the generation of arachidonic acid. Finally, isobutylmethylxanthine and quinacrine, inhibitors of phosphatidylinositol turnover, inhibit both arachidonic acid generation and phagocytosis, indicating a function for this pathway during this process.     

Methionine degradation inCandida utilis

Incubation of cell-free extracts ofC. utilis and its ethionine-resistant mutants with methionine, aspartate and alanine, and with various acceptor oxo-acids showed the presence of several transamination activities (Glu-Asp, Ala-Glu, Met-Glu, Met-Ala, Ala-Asp, but not Met-Asp). The lack of utilization of methionine by mec mutants appears to be due to a transport lesion.     

Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.