Anticardiolipin antibodies in healthy and diseased dogs.

The concentrations of anticardiolipin immunoglobulin G (IgG) were measured in 134 healthy dogs and 63 diseased dogs by an elisa. The mean (sd) concentration in the healthy dogs was 5.40 (2.60) IgG phospholipid (gpl) units, and concentrations greater than 11 gpl were considered as above the normal range. On this basis, 30 (47.6 per cent) of the diseased dogs were within the normal range, with a mean of 5.45 (3.07) gpl and the other 33 had levels above the normal range (P<0.001); 19 of them had a mean level of 22.2 (5.66) gpl, 10 had a mean level of 49.1 (11.2) gpl, and four had a mean level of 85.8 (9.64) gpl. Levels above the normal range were more frequent in females (59.4 per cent) than in males (45.1 per cent), but were higher in males (45.5 [34.71] v 42.91 [22.0] gpl). In addition, they were more frequent and higher in older dogs (66.7 per cent, 40.4 [24.0] gpl) than in younger dogs (33.3 per cent, 33.5 [21.4] gpl).

Overproduction and improved strategies to purify the threenative forms of nuclease-free HU protein.

The heterodimeric HU protein was isolated from Escherichia coli as one of the most abundant DNA binding proteins associated with the bacterial nucleoid. HUalphabeta is composed of two very homologous subunits, but HU can also be present in E. coli under its two homodimeric forms, HUalpha(2) and HUbeta(2). This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts and in some viruses. HU can participate, like the histones, in the maintenance of DNA supercoiling and in DNA condensation. This protein which does not recognize any specific sequence on double-stranded DNA, has been shown to bind specifically to cruciform DNA as does the eukaryotic HMG1 protein and to a series of structures which are found as intermediates of DNA repair, e.g., nick, gap, 3'overhang, etc. The strong binding of HU to these diverse DNA structures could explain, in part at least, its pleiotropic role in the bacterial cell. To understand all the facets of its interactions with nucleic acids, it was necessary to develop a procedure which allowed the purification of the three forms of HU under their native form and without the nuclease activity strongly associated with the protein. We describe here such a procedure as well as demonstrating that the three histidine-tagged HUs we have produced, have conserved the binding characteristics of native HUs. Interestingly, by two complementation tests, we show that the histidine-tagged HUs are fully active in vivo.

The Escherichia coli ribosomal protein S16 is an endonuclease.

The histone-like protein HU isolated from Escherichia coli exhibited, after several purification steps, a Mg(2+)-dependent nuclease activity. We show here that this activity can be dissociated from HU by a denaturation-renaturation step, and is due to a small fraction of ribosomal protein S16 co-purifying with HU. S16 is an essential component of the 30S ribosomal particles. We have cloned, overproduced, and purified a histidine-tagged S16 and shown that this protein is a DNA-binding protein carrying a Mg(2+)-Mn(2+)-dependent endonuclease activity. This is an unexpected property for a ribosomal protein.

Modulation of stress proteins by Cd2+ in a human T cell line.

We previously showed in a human T cell line (CEM-C12 cells) that Cd2+ induced gene expression of stress proteins, metallothionein-IIA and heat shock protein 70 in a time- and dose-dependent manner. In the present study, CEM-C12 cells were pretreated for 24 h with 1 microM Cd2+ and then challenged with toxic concentrations of this metal. We found that maximal expression of the metallothionein-IIA and heat shock protein 70 genes was increased and this maximal level occurred at higher Cd2+ toxic concentrations. Actinomycin D chase experiments indicated that Cd2+ pretreatment did not modify metallothionein-IIA mRNA stability. The modulatory effect of Cd2+ pretreatment was dose-dependent from 100 pM to 1 microM. Such pretreatment also enhanced resistance to Cd2+ toxicity. Finally, verapamil, a calcium/potassium channel blocker displaced the dose-response curve for Cd2+ toxicity as well as metallothionein-IIA and heat shock protein 70 gene expression to higher Cd2+ concentrations.

Cadmium induces apoptosis in a human T cell line.

Cadmium, a potent toxic metal, poses a serious environmental threat but the mechanisms of its toxicity remain unclear. In the present study, we investigated the nature of cadmium-induced cell death in the human T cell line CEM-C12. Cadmium was time- and dose-dependently toxic for CEM-C12 cells, cell death being preceded by chromatin condensation and DNA fragmentation. Quantification of the latter indicated an increase above 4 microM cadmium, with maximal fragmentation at 8 to 10 microM. By contrast, when CEM-C12 cells were exposed to higher cadmium concentrations (50 microM), cell death increased without concomitant chromatin condensation or DNA fragmentation. Thus, cadmium at low and high concentration kills CEM-C12 cells by apoptosis and necrosis, respectively. Addition of cycloheximide reduced the apoptotic effect of cadmium, suggesting that cadmium-induced apoptosis is an process depending on protein synthesis. Verapamil, a calcium/potassium channel blocker, markedly increased the viability of CEM-C12 cells treated by low cadmium concentrations and prevented DNA fragmentation. The apoptotic effect of cadmium suggests a possible mechanism for lymphocyte damage occurring after in vivo exposure to cadmium.

Regulation of paf-acether receptor expression in human B cells.

Paf-acether (paf) is a phospholipid cytokine alloted with potent inflammatory and immunoregulatory properties. Recent reports indicated that in human B cell lines, paf modulated both early and late activation events. In our study, we showed that four of six human B cell lines specifically bound [3H]paf irrespective of the stage of differentiation, the presence of EBV genome or cell surface phenotype. Binding was saturated and fit a one receptor model with a dissociation constant ranging from 1 to 6 nM and a number of sites per cell ranging from approximately equal to 4000 in Rjc13 to approximately equal to 30,000 in Raji or IM9. In addition, our data indicate that 1) maximal expression occurred during the log phase growth; 2) paf itself (10-100 nM) or rIL-4 (100 U/ml) up-regulated by two- to threefold the number of paf binding sites without affecting the affinity. Finally, we found that activated normal B lymphocytes exhibited a higher capacity than resting B cells to incorporate and metabolize [3H]paf at 37 degrees C. Resting B lymphocytes lacked specific binding capacity for paf, yet specific paf receptors were induced upon stimulation via Staphylococcus aureus Cowan I or phorbol 12,13 dibutyrate plus ionomycin. These results suggest that B cell activation is a critical event for paf receptor expression and modulation.

Induction of high-affinity paf receptor expression during T cell activation.

Activated human T cells via the CD2 or the CD3 pathways exhibited a higher capacity than resting T lymphocytes to incorporate and metabolize [3H]pafacether (paf) at 37 degrees C. Resting T lymphocytes lacked specific binding capacity for paf, yet high-affinity paf receptors (paf-R) were induced on CD3- or CD2-dependent activation. This up-regulation in the number of paf-R became apparent by day 1 of culture, reached a maximum of about 25,000 sites cell by days 4 to 6 and subsequently declined. Interestingly, human recombinant interleukin-2 in a dose-dependent manner prevented the decrease of high-affinity paf-R expression on T cells. By contrast, the receptor affinity was constant throughout the culture period. Thus, paf-R at different stages of T cell activation were indistinguishable with respect to receptor-ligand interaction, and differed only in their number. Together, these data demonstrate that after activation human T cells develop membrane high-affinity paf-binding sites. They also suggest for the first time that expression of the paf-R are coupled to T cell activation and/or differentiation.

Metallothionein and tissue damage.

Metallothioneins (MTs) are a class of small cysteine-rich heavy metal binding proteins produced in response to a variety of stresses, inflammation, and as components of the acute-phase response. Although the exact role of MT in this phase is not known, a number of reports have shown that expression of hepatic MT is markedly increased in response to bacterial infection, an effect mediated by lipopolysaccharides. More recently, it has been suggested that MT may play a major role in the prevention of tissue damage. Thus MT has been shown to be an efficient free radical scavenger. Indirect activation of macrophages and neutrophils during the acute phase of inflammation result in a massive release of various species of oxygen metabolites which may be indirectly responsible for the initiation of apoptosis. In addition, expression of MT also increases cell resistance to radiation damage. Together, these results suggest that MT could be part of a generalized protective system in mammalian cells.