The characteristics of the protective and antigenic activities of purified staphylococcal anatoxin in trials with mice of different genotypes

The protective potency of purified staphylococcal toxoid by the survival rate of immunized mice challenged with the culture of Staphylococcus aureus strain L-1726 and the antigenic properties of the toxoid, determined by the level of antitoxin in the blood of mice and by the intensity of cell-mediated immunity in the spleen-cell migration inhibition test, were studied. The experiments were made on CBA and C57BL/6 mice. Purified staphylococcal toxoid was shown to possess antigenic and protective properties in a wide range of doses between 0.15 and 15 binding units per mouse. The protective effect of the toxoid in CBA mice was manifested in the presence of circulating antibodies and cell-mediated reaction or only in the presence of the toxoid. In C57BL/6 mice the protective effect of the toxoid was less pronounced and appeared in combination with the induction of cell-mediated immunity in the presence of an extremely low antibody level (0.062 I.U.).     

The role of cellular and humoral factors of body resistance in the development of experimental influenza-staphylococcal infections in mice

The clinico-experimental studies of mixed influenza-staphylococcal infection constantly point to the development of the aggravation of the infectious process due to the synergic action of the bacterial and viral infective agents. But, as shown by the authors of the present work, in those cases when the experimental infection with the virus was preceded by staphylococcal infection by 72 hours no synergism was observed. In cases of infection with adaptogenic virus the mortality rate of mice resulting from meningococcal infection was twice as low. The possible explanation of this fact is discussed.     

A novel method for preparation of HAMLET-like protein complexes

Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of α-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting α-LA - OA samples. In this study we present a novel method for preparation of α-LA - OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLA-OA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLA-OA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLA-OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca²⁺ transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active α-LA - OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties.     

The intracellular pH and cell proliferation of the LS cell line and its derivative LSM adapted to monolayer growth]

The dynamics of intracellular pH (pHi) during proliferation of cells of LS line in bicarbonate-containing media and of its derivative LSM line adapted to grow in a monolayer has been studied. The contact of LS cells with a solid substrate was not accompanied by their spreading and by an increase in pHi. The pHi values of growing and resting LS cells were practically equal (7.03 and 6.97, respectively). The adhesion and spreading of LSM cells were accompanied by an increase in pHi. The proliferation of LSM cells occurred at different pHi values: at 7.32 on solid substrate with serum, at 7.18 on substrate without serum, at 7.13 in a serum-containing suspension, at 6.97 in a suspension without serum. The highest growth rate was observed at the increased pHi value. Cell proliferation on the substrate stopped at pHi values within 7.10 and 7.13 which were equal to or exceeded the pHi of growing cells in suspension. No difference was observed between LS and LSM cells in the activities of Na+/H+ exchange and transport of Cl- into cells that are involved in pHi regulation. Transport of HCO3- into the cytoplasm of LSM cells was more active than that of LS cells. The role of pHi in the anchorage dependence of cell proliferation is discussed.     

Prooxidant and cytotoxic action of N-acetylcysteine and glutathione in combinations with vitamin B12b

Prooxidant and cytotoxic effects of thiols N-acetylcysteine (NAC) and glutathione (GSH) were studied in combinations with vitamin B_12b. Both GSH and NAC at physiological doses when combined with B_12b were shown to cause initiation of apoptosis. It was established that the prooxidant action of NAC (or GSH) combined with B_12b, i.e., generation and accumulation of hydrogen peroxide in culture medium, led to intractellular oxidative stress and cell redox imbalance. These effects are completely prevented by nonthiol antioxidants catalase and pyruvate. The chelators of iron phenanthroline and deferoxamine do not suppress the H₂O₂ accumulation in culture medium, but inhibit cell death induced by NAC combined with B_12b or by GSH combined with B_12b. Therefore, the thiols GSH or NAC in combination with vitamin B_12b reveal prooxidant properties and induce, with participation of intracellular iron, apoptotic HEp-2 cell death.