Variations of the effect of insulin on neutrophil respiratory burst. The role of tyrosine kinases and phosphatases.

The priming effect of insulin on the fMLP-induced respiratory burst of mouse neutrophils as well as the involvement of tyrosine protein kinases and phosphatases in this process have been studied. Peritoneal evoked neutrophils of NMRI strain mice were incubated with 0.01-100 nM insulin for 1-60 min at 22, 30, or 37 degrees C and activated by 0.1-50 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP). The production of reactive oxygen species (ROS) by neutrophils was monitored by luminol-dependent chemiluminescence. We found that 125I-labeled insulin binding by mouse neutrophils occurred with saturation and high affinity. Insulin itself did not change the basal level of the ROS production but could modulate fMLP-induced respiratory burst. The effect of insulin depended on temperature and duration of pretreatment of the neutrophils with insulin and the concentration combination of the insulin and fMLP. The tyrosine kinase inhibitor tyrphostin 51 decreased the fMLP-induced respiratory burst significantly. Insulin did not change the fMLP response of neutrophils pretreated with tyrphostin. However, the effect of tyrphostin on the response to 50 microM fMLP was considerably decreased in neutrophils treated with insulin. There was no such effect during activation by 5 microM fMLP, for which the priming effect of insulin was not observed. Insulin did not increase the fMLP-induced respiratory burst in neutrophils treated with the protein phosphatase inhibitors orthovanadate and pyrophosphate. If the inhibitors were added after insulin, the combined effect was nearly additive. It is possible that priming by insulin of the fMLP-induced respiratory burst is triggered by tyrosine phosphorylation, realized with its participation, and involves the signaling pathways initiated by tyrosine phosphorylation but subsequently is not dependent on the latter. The role of protein phosphatases in priming by insulin is of little importance. The data indirectly confirm the idea that priming of the neutrophil respiratory burst is a result of crosstalk of signaling pathways of the insulin and fMLP receptors with the participation of tyrosine phosphorylation.

Structural and functional similarity between Yersinia pestis capsular protein Caf1 and human interleukin-1 beta.

A comparative study of the structural and functional properties of recombinant Yersinia pestis Caf1 and human IL-1beta was performed. According to Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) data, IL-1beta and Caf1 are typical beta-structural proteins. Neither protein interacts with the hydrophobic probe ANS (8-anilino-1-naphthalenesulfonate) under physiological conditions. Specific binding of Caf1 [K(d) = (5.4 +/- 0.1) x 10(-10) M] to interleukin-1 receptors (IL-1Rs) on the surface of finite mouse fibroblasts (line NIH 3T3) was observed. Caf1 is able to inhibit high-affinity binding of (125)I-labeled IL-1beta to NIH 3T3 cells, and in the presence of Caf1, the binding of [(125)I]IL-1beta is characterized by a K(d) of (2.0 +/- 0.3) x 10(-9) M. Caf1 binding to IL-1R could reflect adhesive properties of the capsular subunits responsible for the contact of bacteria with the host immunocompetent cells. In its turn, this may represent a signal for the initiation of the expression and secretion of the proteins of Y. pestis Yop virulon. Thus, these results help to explain the importance of Caf1 in the interaction of Y. pestis with the host immune system.

Zn(2+)-mediated structure formation and compaction of the "natively unfolded" human prothymosin alpha.

Human recombinant prothymosin alpha (ProTalpha) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProTalpha conformation is unaffected by large excess of Ca(2+), Mg(2+), Mn(2+), Cu(2+), and Ni(2+). However, Zn(2+) induces compaction and considerable rearrangement of the protein structure. This means that ProTalpha can specifically interact with Zn(2+) (K(D) approximately 10(-3) M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein.

[Production of recombinant hIL-4delta2-a native isoform of human interleukin-4 in Escherichia coli cells]. / Produktsiia rekombinantnogo hIL-4delta2-prirodnoi izoformy interleikina-4 cheloveka, v kletkakh Esherichia coli.

Expression plasmids containing the synthetic gene hil-4 delta 2 was constructed to produce human interleukin-4 in Escherichia coli cells. Strains TG1 (pBTIL-4 delta 2) and BL21 (DE3) (pETIL-4 delta 2) produced the recombinant protein as inclusion bodies, and its production level was up to 30% of the total cell protein. The renatured hIL-4 delta 2 inhibited IL-4-stimulated T cell proliferation, and this effect was enhanced by cyclosporin A.

Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH.

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.

The sequence 130-137 of human interferon-alpha 2 is involved in the competition of interferon, prothymosin alpha and cholera toxin B subunit for common receptors on human fibroblasts.

125I-labelled recombinant human interferon alpha 2 (rHuIFN-alpha 2) capable of high-affinity binding (Kd = 2.46 +/- 0.18 x 10(-10) M) with receptors expressed on mouse thymocytes was obtained. Prothymosin alpha (proTM-alpha) but not cholera toxin was found to compete with radiolabelled IFN-alpha 2 for binding to the same receptor (Ki = 3.68 +/- 0.21 x 10(-11) M). The synthetic peptide covering the sequence 130-137 of IFN-alpha 2 (authors' definition: alpha-peptoferon) was shown to have the capacity to displace the labelled IFN-alpha 2 from the IFN-alpha 2/receptor complex (Ki = 7.19 +/- 0.12 x 10(-11) M). It was shown that receptors of this type are localized in plasmatic membrane fraction. Using [125I]-alpha-peptoferon, specific and saturable binding was detected on human fibroblasts and the data fitted a single binding site. Scatchard analysis yielded a Kd of 9.63 +/- 0.17 x 10(-8) M. The binding was competitively inhibited by IFN-alpha 2 (the Ki value in competition assays was 1.37 +/- 0.12 x 10(-8) M), proTM-alpha(Ki = 2.2 +/- 0.2 x 10(-7) M) and cholera toxin B subunit (Ki = 5.5 +/- 0.2 x 10(-7)). The present study has demonstrated that the sequence 130-137 of HuIFN-alpha 2 is involved in the competition of HuIFN-alpha 2, proTM-alpha and cholera toxin B subunit for common receptors on human fibroblasts.

[The effect of antigenic fractions of the outer membrane in Francisella tularensis on the functional activity of macrophages]. / Vliianie antigennykh fraktsii vneshnei membrany Francisella tularensis na funktsional'nuiu aktivnost' makrofagov.

The influence of different gel-chromatographic antigenic fractions (GAF) of the membrane of F. tularensis, strain A'Cole, on different forms of reactivity of mouse peritoneal macrophages, such as the adhesion, ingestion and presentation of antigen on the cell surface, has been immunologically evaluated. GAF isolated from F. tularensis have been shown to produce a pronounced modulating effect on all forms of macrophagal functional activity under study. Thus, GAF II with a molecular weight of 85-200 kD inhibits the adhesion, ingestion and presentation of antigens and, on the contrary, GAF IV with a molecular weight of 15-35 kD stimulates these functions.

Synthesis and properties of the peptide corresponding to the ACTH-like sequence of human immunoglobulin G1.

The decapeptide Val-Lys-Lys-Pro-Gly--Ser-Ser-Val-Lys-Val (termed immunocorticotropin) corresponding to the sequence 11-20 of the variable part of the human immunoglobulin G1 heavy chain was synthesized. The ACTH-like peptide was found to have an ability to increase body temperature. Intracerebroventricular injection of 5-10 micrograms of the peptide to rabbits induced an elevation of body temperature by 0.7-1.3 degrees C. The ACTH-like peptide was shown to compete with [125I]-ACTH (13-24) for binding to receptors on murine brain synaptic membranes and to activate adenylate cyclase of these membranes.

Nonapeptide corresponding to the sequence 27-35 of the mature human IL-2 efficiently competes with rIL-2 for binding to thymocyte receptors [corrected].

Previously it was shown [1] that amino acid substitutions at the region of the first alpha-helix of IL-2 specifically inactivate its reactivity with the intermediate-affinity receptor p70, and mutations in the fifth alpha-helix specifically inactivate the binding to the low-affinity receptor p55. We have synthesized the peptides corresponding to the putative binding site of IL-2 with the intermediate-affinity receptor p70 and found that the nonapeptide corresponding to the sequence 27-35 of the mature IL-2 [2] effectively competes with human rIL-2 for binding to thymocyte receptors. Two types of nonapeptide receptors were revealed: those with Kd1 = 1.84 x 10(-8) M and Kd2 = 1.6 x 10(-7) M. The rIL-2 provides a 100% inhibitory effect on the binding of the 125I-labeled nonapeptide to thymocyte receptors, Ki = 3.5 x 10(-8) M. Low immunoproliferative activity of the peptide allows one to recommend it as a specific antiproliferation drug, IL-2 inhibitor [corrected].

[The dependence of the functional activity of "immune" macrophages on T-cells]. / Zavisimost' funktsional'noi aktivnosti "immunnykh" makrofagov ot T-kletok.

The dependence of the functional activity of the peritoneal macrophages of mice immunized with Francisella tularensis vaccine strain on the presence of T-cells in the culture has been studied. The elimination of "immune" macrophages and sensitized T-lymphocytes by means of anti-Thy-1-2-serum has been shown to lead to a sharp decrease in both ingestive and digestive functions of the phagocytic mononuclears of peritoneal exudate to the level of the activity of macrophages isolated from intact animals.

The octapeptide corresponding to the region of the highest homology between alpha-interferon and thymosin-alpha 1 effectively competes with both cytokines for common high-affinity receptors on murine thymocytes.

The octapeptide corresponding to human interferon-alpha 2 (Hu IFN-alpha 2) sequence 131-138 has high affinity to murine thymocyte receptors (Kd = 4.2 x 10(-12) M, about 700 receptors per cell). The peptide receptor binding is inhibited by both Hu rIFN-alpha 2 (Ki = 8.6 x 10(-10) M) and thymosin-alpha 1 (TM-alpha 1) (Ki = 3 x 10(-7) M) as well as by the octapeptide homologous to the TM-alpha 1 sequence 16-23 (Ki = 4.5 x 10(-7) M). The peptide from IFN-alpha 2 (131-138) activates murine thymocyte blast transformation at a concentration of 10(-11) M in the presence of 2.5 micrograms/ml of concanavalin A.

[The immunoregulatory properties of reaferon]. / Izuchenie immunoreguliatornykh svoistv reaferona.

The influence of human recombinant alpha-interferon (reaferon) on cell-mediated and humoral immune response has been studied. Experimental facts on the blast transformation of lymphocytes, humoral immune response and the reaction of delayed hypersensitivity are presented. The study has shown that reaferon possesses the main immunoregulatory properties, characteristic of natural human leukocytic alpha-interferon. Manifestation of these properties depends on the dose of preparation and the time of its use.

[Effect of recombinant human alpha-interferon (reaferon) on macrophage functional activity in mice]. / Vliianie rekombinantnogo al'fa-interferona cheloveka (reaferona) na funktsional'nuiu aktivnost' makrofagov myshei.

The influence of human recombinant alpha 2-interferon (reaferon) on the parameters of the phagocytic activity of mouse peritoneal macrophages (migration, spreading, adhesion and absorption of corpuscular antigen) has been studied. Reaferon in doses of 5-5 X 10(2) I. U./ml has been found to produce a stimulating effect on all parameters under study. The data obtained in this study suggest that a stimulating effect on the functional activity of macrophages is the same for recombinant (alpha 2) interferon and natural alpha-interferon.