Cultures of purified human natural killer cells: Growth in the presence of interleukin 2

We have isolated highly enriched populations of LGL, which are virtually devoid of mature typical lymphocytes (as enumerated by morphological and surface antigen analysis using monoclonal antibodies e.g., OKT3) in comparison to T cells which contain greater than 95% sheep erythrocyte-forming cells and are devoid of LGL and $\text{NK}\text{K}$ activities. Both types of cells grew in the presence of crude or partially purified IL-2. Cultures of LGL could be initiated consistently even in the absence of lectins and the cultured LGL retained their characteristic morphology and cytotoxic activity. However, within 7–10 days after initiation, the cultured LGL changed in surface phenotype to become antigenically indistinguishable from cultured T cells.     

Author index for volume 49

Using mouse thymocytes, mitogen-induced [³H]thymidine incorporation was compared with a recently developed flow-cytometric technique, based on acridine orange staining of cells, which differentiates the G₀ and G₁ phase of thymocytes. PHA induces a transient but considerable G₀-G₁ shift without any substantial proliferation. On the other hand, crude supernatants derived from Con A-stimulated human peripheral blood mononuclear cells induce only a minor G₀-G₁ shift and no proliferation. However, PHA in the presence of this supernatant induced an increased [³H]thymidine uptake in thymocytes and a shift from G₁ to S. These results support the current hypothesis that a factor present in Con A-activated supernatants in conjunction with PHA stimulation indeed facilitates the entrance of G₁ cells into the S phase. The flow-cytometric technique might be used in the study of the interaction of endogenous mediators with exogenous mitogenic agents in activating lymphocytes to proceed through the initial G₀-G₁ phases of the cell cycle.     

Structural features of proteins responsible for resistance of tryptophan residues to nitrosylation

It is known that potentially reactive groups of the protein molecule may be most efficiently nitros(yl)ated only when located within hydrophobic globules or built into the membrane. N1-nitrosotryptophan (NOW) is a stable product of nitrosation in vitro. However, the NOW fraction in proteins is small in ordinary proteins. It suggests the existence of unknown mechanisms preventing the accumulation of NOW. Here we show that these mechanisms are underlain by the protein structure. Analysis of protein structure databases to explore the atomic surroundings of tryptophan residues revealed preferential selection of certain surroundings. N(E) atoms of tryptophan residues, which are the targets for nitrosation, have usually polar and nucleophilic groups in their environment. Residues of Asp, Glu, Cys, His, and Met act as catalysts of denitrosation (internal denitrosilase). We found that short peptides with the same residues possessed denitrosilase activity even in solution. This selection might explain both the resistance of tryptophan residues in proteins to nitrosation and the mechanisms of chemical communication by means of reversible nitrosation of proteins.     

NO-dependent modifications of nucleic acids

This review is devoted to chemical transformations of nucleic acids and their components under the action of nitrogen oxide metabolites. The deamination reaction of bases is discussed in the context of possible competing transformations of its intermediates (nitrosamines, diazonium cations, diazotates, triazenes, and diazoanhydrides) and mechanisms of crosslink formation with proteins and nucleic acids. The oxidation and nitration of bases by NO2 is considered together with the possibility of radical transfer to domains from the base stacks in DNA. Reduction of redox potentials of bases as a result of stacking interactions explains the possibility of their reactions within nucleic acids with the oxidants whose redox potential is insufficient for the effective reactions with mononucleotides. Modifications of nucleic acids with peroxynitrite derivatives are discussed in the context of the effect of the DNA primary structure and the modification products formed on the reactivity of single bases. The possibility of reduction of nitro groups within modified bases to amino derivatives and their subsequent diazotation is considered. The substitution of oxoguanine for nitroguanine residues may result; the reductive diazotation can lead to undamaged guanine. The intermediate modified bases, e.g., 8-aminoguanine and 8-diazoguanine, were shown to participate in noncanonical base pairing, including the formation of more stable bonds with two bases, which is characteristic of the DNA Z-form. A higher sensitivity of RNA in comparison with DNA to NO-dependent modifications (NODMs) is predicted on the basis of the contribution of medium microheterogeneity and the known mechanisms of nitrosylation and nitration. The possible biological consequences of nucleic acids NODMs are briefly considered. It is shown that the NODMs under the action of nitrogen oxide metabolites generated by macrophages and similar cells in inflammations or infections should lead to a sharp increase in the number of mutations in the case of RNA-containing viruses. As a result, the defense mechanisms of the host organism may contribute to the appearance of new, including more dangerous, variants of infecting viruses.     

Inorganic nitric oxide metabolites participating in NO-dependent modifications of biopolymers

Biogenous nitric(II) oxide (NO), the higher nitrogen oxides (NO2, isomeric N2O3 and N2O4, ONOO-, etc.) that are NO-derived in vivo, and the products of their transformations are active compounds capable of reactions with biopolymers and low-molecular metabolites. The products of these reactions are often considered to be various NO-dependent modifications (NODMs). The nitrated, nitrosylated, nitrosated, and other NODMs play key roles in the regulation of the most important biochemical processes. In this review, we briefly discuss the metabolic reactions of nitrogen oxides that supply active intermediates for NODMs, the NODM reaction products, and some mechanisms of NODM reparation that allow the recovery of chemically intact biopolymer molecule from a modified (chemically damaged) NODM. For example, residues of 3-nitrotyrosine arising due to the NODM reactions of proteins can be reduced to unsubstituted Tyr residues as a result of alternative NODM reactions through intermediate diazotyrosine derivatives. The heterogeneity of a medium in vivo is an important factor controlling the proceeding of NODM reactions. We showed that many processes determining NODM efficiency proceed differently in the heterogeneous media of organisms and in homogeneous aqueous solutions.     

In Vivo Evaluation of Safety and Toxicity of a Lactobacillus jensenii Producing Modified Cyanovirin-N in a Rhesus Macaque Vaginal Challenge Model

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across the cervicovaginal mucosa in women is influenced by many factors including the microbiota and the presence of underlying inflammation. It is important that potential HIV preventative agents do not alter the mucosal environment in a way that enhances HIV acquisition. We examined the impact of a “live” microbicide on the vaginal mucosal environment in a rhesus macaque repeated vaginal simian-HIV (SHIV_SF162P3) challenge model. The microbicide contained a human vaginal Lactobacillus jensenii expressing the HIV-1 entry inhibitor, modified Cyanovirin-N (mCV-N), and henceforth called LB-mCV-N. Macaques were colonized vaginally each week with LB-mCV-N and sampled six days after colonization for culturable bacteria, pH and cervical-vaginal cytokines during the duration of the six-week study. We show that macaques that retained the engineered LB-mCV-N strain in their vaginal microbiota, during SHIV challenge, had lower pH, when colonization levels were higher, and had no evidence of inflammatory cytokines. Indeed, Interleukin-13, a mediator of inflammation, was detected less often in LB-mCV-N colonized macaques than in controls and we found higher levels of Interleukin 1 receptor antagonist (IL-1RA) in LB-mCV-N colonized macaques during the SHIV challenge period. We noted an inverse correlation between levels of mucosal IL-1RA and peak plasma viral load, thus higher IL-1RA correlated with lower viral load in LB-mCV-N treated macaques. These data support the use of LB-mCV-N as a safe “live” microbicide and suggest that lactobacilli themselves may positively impact the mucosal environment.