[Integration of DNA oscillatory vector pSV neo to genome of NIH3T3: importance in investigations of transformation with c-Ha-ras oncogene]. / Integracja DNA wektora wahadlowego pSVneo do genomu komórek NIH3T3: znaczenie w badaniach transformacji onkogenem c-Ha-ras.

Main aim of this study was evaluation of application of stable clones of transforming cells, containing DNA of plasmid vector for the investigation of oncogenes. Plasmid vector was constructed basing on pSV2neo vector, containing activated oncogene c-Ha-ras-1, derived from pT 24-C3 which was followed by evaluation of phenotypic and genetic changes in standard line of NIH3T3 mouse fibroblast line after transfection with constructed plasmid. After two weeks of culture in selective conditions, transformants resistant to geneticin were obtained and analysis of clones was performed after transfection with constructed vector containing ras oncogene and after transfection pSV2neo. Analysis of efficiency of cloning and transformation basing on growth independent from placement and morphology and investigation of karyotypes demonstrated similar irregularities in both investigated groups and subchromosomal aberrations of NIH3T3 cells were even more frequent than in initial lines of NIH3T3 cells. Southern hybridization with pSV2neo-ras probe demonstrated that only restrictive DNA fragments, obtained by Pst1 enzyme contain copies of neo in cell genomes. Integration of gene cf geneticin-resistance increases thus normally unstable genetically NIH3T3 cells.

Instability of hybrid plasmids in Bacillus subtilis.

We studied the mechanisms responsible for instability of hybrid derivatives of staphylococcal R plasmids in B. subtilis cells. Four pBCH plasmids were constructed from staphylococcal chloramphenicol-resistance plasmid pC756 ligated with pBR322 or pUC8 vectors, and two pAEC plasmids contained pC756 plasmid, erm gene from S. aureus pE3692 and pBR322 plasmid vector. The level of instability of these recombinant plasmids was clearly related to their sizes, the larger derivatives were segregationally highly unstable. The four hybrid pBE plasmids constructed from erythromycin-resistant pE3692 plasmid or its deletion derivative and pBR322 vector were poorly maintained in B. subtilis rec+ strain in comparison with the B. subtilis (recE4) strain. Moreover, the absence in pBE-hybrids of a part of palA sequence led to considerable reduction of plasmid stability in B. subtilis cells. However, the clones of B. subtilis harbouring pBE plasmids integrated into the bacterial chromosome presented much higher stability of the plasmid erm gene than the clones maintaining autonomously replicating plasmids.

[Modulation of lymphocytes and immunoglobulins by serine proteinase of Staphylococcus aureus]. / Modulacje limfocytów i immunoglobulin pod wplywem proteinazy serynowej Staphylococcus aureus.

Serine proteinase of S. aureus binds with surface of peripheral blood lymphocytes and by proteolysis of polypeptides on the membrane and transduction of signals, changes in polyclonal activation of T and B lymphocytes occur. Cleavage of immunoglobulins tested in three classes and evaluated by electrophoresis in polyacrylamide gel (SDS-Page) of Ig fragments, was differing. IgG and IgG fragments were degraded in higher degree than IgM, H and L chains or i Fc and i Fab fragments. Influence of proteolytic modulation of IgG particles on fixation antigenic fractions homogenized by ultrasound cells of S. aureus was evaluated in ELISA test. Basing on detailed results postulated mechanism of modification effect of immunoglobulins and lymphocyte reaction under influence of serine proteinase of S. aureus in microenvironment of after-inflammatory reaction in staphylococcal infection, has been accepted.

Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus.

The serine proteinase (SP) released into the environment by most strains of S. aureus cleaves human IgG, IgM and IgA of both subclasses--IgA 1 and IgA 2. SP cleaves H chains of all immunoglobulin classes and the SC of S-IgA, the L chains are degraded partially. The SP-induced cleavage results in a large spectrum of fragments under reducing conditions within a broad range of Mr (approx. 41,000 to less than 12,400). This indicates that the enzyme does not affect the Ig molecule in the hinge region only. The degree of cleavage depends on the enzyme:substrate ratio and on the duration of incubation. The generation of small fragments is associated with the loss of antigenic determinants that results from the decreased binding of the cleaved material in the ELISA method. Partial cleavage of L chains suggests that the enzyme alters part of the molecule that is involved in antigen binding. Even if the ability of antigen binding remains preserved after cleaving Ig with SP, the antibody function is disturbed by splitting off the Fc region or by its degradation into small fragments. SP has to be considered as one of the virulence factors of S. aureus that may protect bacteria against the defence mechanisms of the host.

Genetic changes in Morris hepatoma 7777 cells.

Analysis of genetic changes in Morris hepatoma 7777 is described. Three different approaches were applied: DNA and chromosome transfection, karyotype analysis, and Southern hybridization modified by use of PFGE. Changes in the genome of tested cells were identified by PFGE and chromosome transfection. This supports the statement that PFGE is a useful method in transformed cell genome analysis.

Effect of metalloproteinase from Staphylococcus aureus on in vitro stimulation of human lymphocytes.

Metalloproteinase (MP) produced by the majority of Staphylococcus aureus strains exerts, in a wide concentration range (0.1-100 micrograms/ml), no cytotoxic action on mononuclear leukocytes of human peripheral blood. The enzyme itself does not appreciably stimulate proliferation and differentiation of lymphocytes in culture, but affects the stimulation of both T and B lymphocytes by polyclonal activators. The action is dose-dependent. High doses of MP (100 micrograms/ml) lower the blastic transformation after stimulation with Con A, SpA, NDCM, S. aureus strain Wood 46 and with suboptimal doses of PWM. Optimal concentrations of the enzyme potentiate the stimulation of lymphocytes by PWM, PHA, S. aureus strains Cowan 1 and Wood 46, and by NDCM. The same potentiation effect was achieved whether the enzyme was added concurrently with the mitogen or 18 h later. This implies that the beginning of cell activation is not affected. A high MP concentration decreases the production of Ig in culture after stimulation with PWM whereas lower concentrations of MP enhance this production. Production of Ig after stimulation with NDCM and Wood 46 is decreased by MP. The possible action of exoproteinase from S. Aureus on the immune response during infection is discussed.

Anti-staphylococcal serine proteinase and other serum factors in phagocytosis.

The interactions between polymorphonuclear cells (PMN), Staphylococcus saprophyticus cells and rabbit antibodies against Staphylococcus aureus V8 serine proteinase or normal rabbit serum proteins were investigated. The effect of opsonization on phagocytosis due to human peripheral polymorphonuclear cells was measured. The results were as follows: phagocytosis index values were relatively increased after the incubation of PMN cells with anti-serine proteinase gamma-globulin serum fraction, anti-serine proteinase IgG, non-immunized rabbit serum or with complement.

Stabilization vs. degradation of Staphylococcus aureus metalloproteinase.

Purified Staphylococcus aureus metalloproteinase contains trace amounts of a serine proteinase which rapidly degrades the metalloproteinase when EDTA is present. However, no degradation occurs when Ca2+ is added or if the serine proteinase is removed by immunoaffinity chromatography. Selective chelation of Zn2+ by o-phenanthroline, which reversibly inactivates the metalloproteinase, does not result in the degradation of the apometalloproteinase, even with excess of serine proteinase. These data are interpreted as follows: EDTA chelates enzyme-bound Ca2+ and Zn2+, causing irreversible inactivation as well as a conformational change in the metal-free protein. This allows proteolysis by the contaminating serine proteinase and explains why the metalloproteinase purified from serine proteinase-deficient strains of S. aureus was previously thought to be stable to autolysis.

Chemoattractant activity of Staphylococcus aureus serine proteinase modified human plasma alpha-1-proteinase inhibitor.

S. aureus serine proteinase inactivates human alpha-1-proteinase inhibitor (alpha-1-PI) by attacking a single peptide bond between Glu354 and Ala355 giving a modified inhibitor which is a tight complex of Mr = 4,000 and 48,000 fragments. In the present paper we show that this proteolytically inactivated alpha-1-PI is a potent chemotactic factor for human neutrophiles at a nanomolar concentration, and we discuss its potential involvement in the inflammatory reaction due to S. aureus infections.

Mechanism of staphylococcal serine proteinase inactivation by lymphocytes and granulocytes.

Stricking differences were observed in the mechanism of interaction between staphylococcal serine proteinase and surface of human granulocytes or lymphocytes despite the fact that incubation of this enzyme with both types of cells leads to analogical decrease of proteinase activity. Interaction of proteinase with lymphocytes releases peptides smaller than these released spontaneously by non-treated lymphocytes or lymphocytes treated with DFP-proteinase. However, in supernatants of lymphocytes neither complex of proteinase with cell derived molecules nor changes of electrophoretic mobility of proteinase was found. Products of proteinase-lymphocyte reaction have a proliferative effect on intact lymphocytes, which is greater that the one of active proteinase. On the other hand granulocytes are resistant to proteinase and bind active proteinase as well as the DFP-proteinase in the receptor mediated way, followed by endocytosis with the affinity similar to the one in monocytes.

Effects of serine proteinase from Staphylococcus aureus V8 cells on Con A stimulation of human lymphocytes.

The combined effect of serum and Staphylococcus aureus serine proteinase on human lymphocyte Con A stimulation was assayed. Serum was found to protect the proteinase-treated lymphocytes. It is suggested that not only the immunogenicity of proteinase itself, but also proteinase modified serum and lymphocyte-derived components affect lymphoproliferative response.

Effect of serine proteinase from Staphylococcus aureus on in vitro stimulation of human lymphocytes.

In a broad concentration range (0.1-100 micrograms/ml) the serine proteinase (SP) from Staphylococcus aureus has no cytotoxic effect on human peripheral blood lymphocytes and does not stimulate them in culture. However, it affects the action of a number of polyclonal activators. In a concentration of 100 micrograms/ml SP completely eliminates blastic transformation after stimulation with B cell mitogens (NDCM, S. aureus and Escherichia coli), lowers the blastic transformation after stimulation with PWM and SPA, and does not affect the blastic transformation after stimulation with PHA. SP (100 micrograms/ml) reduces the concentration of Ig in stimulated cultures (stimulation with PWM, NDCM, S. aureus and E. coli) far below the Ig level of unstimulated controls. This effect can be ascribed to an influence on cell stimulation, not to the proteolytic cleavage of secreted Ig, although SP can partially digest Ig. The effect on lymphocyte stimulation takes place when the SP is added to the culture together with the mitogen, or 18 h after the mitogen. This implies that SP does not affect the first stage of stimulation.

Preliminary estimation of chemoattractant activity of staphylococcal serine proteinase in vitro. Chemoattractant activity of staphylococcal serine proteinase.

Human polymorphonuclear leukocytes isolated from peripheral blood of healthy donors migrated toward the staphylococcal serine proteinase.

Surface interaction of human granulocytes and lymphocytes with staphylococcal extracellular serine proteinase.

Enzymatic activity of purified staphylococcal extracellular serine proteinase decreases as a result of incubation with granulocytes as well as with lymphocytes taken from peripheral blood of healthy donors. However, specific proteinase binding was observed only in the case of granulocytes but not in peripheral lymphocytes.

Evaluation of the effect of staphylococcal serine proteinase on phagocytosis.

Correlations between values of phagocytosis index and values of concentrations of staphylococcal serine proteinase were analysed. Preincubation of granulocytes with the proteinase stimulated phagocytosis of three bacterial strains: S. saprophyticus, S. aureus VS and S. aureus Smith diffuse. Significant correlations were also observed for S. saprophyticus strain in phagocytosis performed with or without bovine serum. Specific rabbit IgG anti-serine proteinase effected the increase of phagocytosis index only in the case of S. aureus Smith diffuse. Summary statistical analysis for all experimental conditions exhibits significant correlations also for S. aureus VS strain. No significant correlations were noted for the three remaining strains taken from patients.

Delayed hypersensitivity to polyoma and "self" antigens in syngeneic mice.

Using the tail swelling test, delayed-type hypersensitivity (DTH) to tumor and self antigens accompanying the development of syngeneic polyoma tumor in CBA mice was observed. The soluble polyoma cell-surface antigen contained both polyoma and H-2k specificities and induced weak proliferation response of spleen cells in the presence of Il-2 in vitro. Appearance of a measurable tail swelling reaction at the side of the midtail, subcutaneous injection of the eliciting antigen not earlier than at 16th hour, maximum of swelling after 24 hours and the results of histological examination proved that the swelling was caused by a typical DTH to the antigens. Maximum DTH to both antigens occurred in the mice 6 days after polyoma cell transplantation (about 3 days before the appearance of palpable tumor) and weakened as the tumor progressed. DTH activity was transferred by spleen T lymphocytes to naive recipients. Five-day restimulation of splenocytes from polyoma transplanted donors with antigen in the presence of Il-2 led to an increase of antigen-lymphocytes affinity but resulted in a decrease of DTH activity of these cells. The mechanisms of these processes are still discussed.

Influenza virus hemagglutinin-binding T cells in delayed hypersensitivity induced in mice.

The ability of spleen T cells from mice inoculated with influenza virus to transfer delayed hypersensitivity increases after 5 days culture in the presence of TCGF and influenza virus or its hemagglutinin. Enriched T cells have also a greater ability to bind viral hemagglutinin than cells which were not cultured and restimulated with either antigen. The results suggest the selection in vitro of specific antigen-binding T cells, able to transfer delayed hypersensitivity.

T-enriched spleen cells in delayed-type hypersensitivity to influenza virus in mice.

Restimulation in vitro of T-enriched spleen cells from CBA mice with influenza virus A/Bangkok 1/79/H3N2 or its hemagglutinin (HA) leads to enhancement of delayed-type hypersensitivity (DTH) to virus and HA in recipients of transfer. The enhancement of DTH measured by tail swelling is accompanied by 20-fold increase of binding affinity of transferring cells to HA measured by saturation analysis. DTH induced by HA in vivo is weaker than induced by virus in this system. However, when HA is used in vitro as a restimulating antigen of virus primed in vivo lymphocytes it leads to generation of such lymphocytes population which after transfer mediates DTH response to virus or HA to the same level as virus restimulated cells. The increase of binding affinity of restimulated T lymphocytes to HA accompanying the enhancement of DTH activity is considered in the relation to quantitative and qualitative changes of antigen binding cell populations and their role in antiviral response in this systems.