HspBP1 in Complex with the Peptide of the Innate Immunity Protein Tag7 is Able to Lyse Tumor Cells Carrying TNFR1 Receptor.

The search for new cytotoxic agents capable of lysing tumor cells is an important task in the fight against cancer. Here we have shown that the HspBP1 protein, the chaperone of the heat shock protein Hsp70, is able to form a complex with the previously discovered peptide (17.1) of the innate immunity protein Tag7. Experiments using thermophoresis demonstrated that the affinity of the Tag7 protein peptide 17.1 to the HspBP1 molecule is 100 times higher than that of the full-sized Tag7 molecule. The addition of the 17.1-HspBP1 complex to tumor cells induces apoptosis and necroptosis in them. The results obtained in this work can be used to develop promising antitumor drugs.

[Isolated splenic lymphangioma]. / Laparoskopicheskaya rezektsiya pri izolirovannoi limfangiome selezenki.

Splenic lymphangioma is a rare malformation of splenic lymphatic channels characterized by cysts resulting from increased number of enlarged thin-walled lymphatic vessels. In our case, there were no clinical manifestations. Lymphangioma was congenital and diagnosed by ultrasound as an accidental finding. Surgery is the only method of radical treatment of splenic lymphangioma. We describe an extremely rare case of pediatric isolated splenic lymphangioma and laparoscopic resection of spleen as the most advantageous variant of surgical treatment.

Incubation of Lymphocytes with IL-2 Causes the Appearance of Antitumor T Cells Carrying FC, CD25, and LFA-1 on the Surface.

To carry out antitumor activity against cells that have lost surface antigens, human lymphocytes must have a certain repertoire of surface proteins capable of contacting a tumor cell and inducing programmed cell death in it. In this work, we showed that activation of healthy donor cells by IL-2 cytokine within 6 days causes the appearance of FasL, CD25, and LFA-1 proteins on CD8+CD25+ T lymphocytes, and also converts the LFA-1 protein into an active form having a high affinity for its target, ICAM-1 integrin. The appearance of these proteins on the surface of this subpopulation of lymphocytes allows them to induce programmed cell death in HLA-negative tumor cells.

Tag7-Mts1 Complex Activates Chemotaxis of Regulatory T Cells.

One of the basic features of immune system is the ability to sustain balance between activation and suppression of effector lymphocytes. In this process a key role belongs to the subpopulation of cells called regulatory T cells (Treg). Many cancer and autoimmune diseases are caused by malfunctions of Treg, and investigation of this subpopulation is important for development of new therapeutic approaches. In this study, we demonstrate that regulatory T cells can migrate along the concentration gradient of Tag7-Mts1 complex, and also they produce agents that induce blood cells migration.

[MutL Protein from the Neisseria gonorrhoeae Mismatch Repair System: Interaction with ATP and DNA].

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18-50 Šfrom the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

[The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA].

Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only one strand. The mechanism of their action is important to study because NEs with new specificities are necessary to design to solve the practical tasks of biotechnology. One of the modern approaches for investigation of protein-nucleic acid interactions is fluorescence spectroscopy, which involves the introduction of fluorophores into proteins, mainly through Cys residues due to the high reactivity of their thiol group. To implement this approach, it is necessary to clarify the role of Cys residues in the functioning of the native protein and the possible consequences of their modification. Crosslinking was used to study whether Cys residues are close to DNA in the complex with NE BspD6I. Reactions were carried out using the wild-type enzyme, its mutant form NE BspD6I(C11S/C160S), and modified DNA duplexes containing the 2-pyridyldisulfide group at the C2' atom of the sugar-phosphate moiety in different positions of the oligonucleotide strand. The Cys residues of NE BspD6I were for the first time shown to be in close proximity to DNA during the binding process, including the step of a nonspecific complex formation. The substitutions C11S and C160S in the N-terminal domain of the enzyme slightly decreased the efficiency of substrate hydrolysis. Construction of a cysteine-free NE BspD6I variant and examination of its properties will provide additional information about the functional significance of the Cys residues for this unique enzyme.

Innate Immune Protein Tag7 Stimulates the Appearance of Cytotoxic NK Cells after Incubation with Lymphocytes.

Tag7 (PGRP-S) is an innate immune protein that is involved in the antibacterial and antitumor defense and stimulates the maturation of cytotoxic lymphocyte subpopulations. It was found that the incubation of lymphocytes with Tag7 for 3 days promotes the appearance of cytotoxic NK cells that are active against a number of tumor cell lines.

Tag7 (PGLYRP1) Can Induce an Emergence of the CD3+CD4+CD25+CD127+ Cells with Antitumor Activity.

We have shown that in the human peripheral blood cells, the innate immunity protein Tag7 can activate a subpopulation of CD3+CD4+CD25+ cells, which have antitumor activity. These cells can induce lysis of HLA-negative tumor cell lines. The Hsp70 stress molecule on the surface of the tumor cells is used as a recognition target, while the Tag7 protein on the lymphocyte membrane acts as a receptor for Hsp70. We have also demonstrated that this subpopulation of the CD4+CD25+ cells is CD127 positive and hence is not the Treg cells. Our data suggest that this subpopulation of cells is identical to the CD4+CD25+ lymphocytes, which are activated in the leukocyte pool by the IL-2 cytokine.

Lymphocytes incubated in the presence of IL-2 lose the capacity for chemotaxis but acquire antitumor activity.

Naïve non-activated lymphocytes are capable of releasing the chemoattractant complex Tag7-Mts1 and can migrate along the gradient of its concentration. After activation of these cells by IL-2, they acquire the abilities to kill tumor cells and to release the cytotoxic Tag7-Hsp70 complex, which is accompanied by a loss of both the Tag7-Mts1-mediated lymphocyte chemotaxis and the ability to release this chemoattractant into the conditioned medium.

Apoptotic tumor cell death under the influence of the cytotoxic complex Tag7-Hsp70 is induced by interaction with the TNFR1 receptor.

The results of comparative analysis of interaction between the protein cytotoxic complex Tag 7-Hsp70 and the Tag 7 component of this complex with TNFR1 receptor in solution and in tumor cells are presented.

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1.

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7-Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4(+) and CD8(+) lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.

Cell death of L-929 cells induced by cytotoxic complex Tag7-Hsp70 is analogous to the death of the same cells induced by TNF-α.

The identification and studying the molecular bases of functioning of new cytotoxic agents finds an important implication in developing drugs for fighting with tumors. While investigating the cytotoxic action of protein complex Tag7-Hsp70 which was opened in our laboratory previously we found that Tag7-Hsp70 demonstrated the same specificity in regard to different tumor target cells as it was for classical cytokine TNF-α. L-929 cells and Jurkat cells appeared to be good targets representing up to 30% of dead cells within a population and HeLa cells--bad targets representing less than 5% of dead cells after 20 h of incubation with either of the cytotoxic agents. While investigating the action of either TNF-α or Tag7-Hsp70 on L-929 cells we detected two peaks of death: after 3 h and after 20 h. For both cytotoxic agents we observed the first, smaller (13-15%), peak to be eliminated after the addition of caspase inhibitor YVAD-CHO and the second, greater (25-30%), peak to become even bigger in presence of caspase inhibitor. Probably, protein complex Tag7-Hsp70 interacts like TNF-α with a receptor on the surface of tumor cells that results in triggering two alternative mechanisms of programmed cell death: apoptosis and necroptosis.

[Determinants for iron overload in patients with acute leukemias and aplastic anemia].

AIM: to reveal the determinants of the development of iron overload in patients with acute leukemias (AL) and aplastic anemia (AA). SUBJECTS AND METHODS: The investigation included 104 patients, including 64 with various types of AL, 31 with AA, and 9 with myelodysplastic syndromes (MDS). A group affiliation and an erythrocyte phenotype were determined from rhesus system antigens in all the patients and the HFE gene was studied to identify mutations. For control of siderosis, the authors determined serum iron (SI) by a colorimetric technique, by applying the kits of the AGAT firm (Russia), serum ferritin (SF) by an immunoradiometric method, by using the kits of Immunotech (Czechia). The volume of transfusion was estimated in the period of June 2007 to November 2009. RESULTS: There is evidence for a relationship between the higher level of SF and the number of transfusions. SF was 1046.1 microg/l in patients, H63D heterozygous carriers who had received less than 10 packed red blood cell transfusions and 2856 microg/l in those who had 20 transfusions (p < 0.005). HFE gene mutation carriage accelerates iron accumulation and is an additional risk factor for siderosis. In patients with transfusion chimeras and a rare phenotype in terms of rhesus antigens, packed red blood cell transfusion results in a much more increase in iron stores. CONCLUSION: The most important factor of iron overload acceleration is no specific choice of packed red blood cells for patients with rare combinations of red blood cell antigens and for those with artificially induced chimeras.

Deamination of 5-methylcytosine residues in Mammalian cells.

DNA demethylation in mammalia occurs after fertilization and during embryogenesis and accompanies cell aging and cancer transformation. With the help of the primer extension reaction, MALDI MS and DNA cleavage by thymine DNA glycosylase deamination of 5-methylcytosine residues has been shown to take place when the model methylated DNA duplexes are treated with nuclear extracts from the cell lines CHO, HeLa, and Skov3. The hypothesis that deamination of 5-methylcytosine is the first stage of demethylation in mammalia has been postulated.

Comparative analysis of secretion of S100A4 metastatic marker by immune and tumor cells.

S100A4 protein is present in low concentrations (2.1-15.7 ng/10(6) cells) in lymphocyte and neutrophil culture medium. Addition of stimulants to the cells did not lead to an appreciable increase in the content of this protein. The initial content of S100A4 is significantly higher (92-447 ng/10(6) cells) in culture media of highly metastatic KSML-100 adenocarcinoma and M3 and B16 melanoma cells. The release of S100A4 by these cells significantly increased after addition of lymphocytes and Tag7/Hsp70 cytotoxic complex. Repeated injection of antibodies to S100A4 to mice with transplanted M3 melanoma inhibited tumor growth.

[Hyperhomocysteinemia--one of the factors underlying thrombotic complications in patients with chronic myeloproliferative diseases].

AIM: To assess incidence of hyperhomocysteinemia (HHC) in patients with chronic myeloproliferative diseases (CMPD) and to analyse possible correlation between an elevated concentration of plasma homocystein (HC) and thrombotic complications. MATERIAL AND METHODS: The trial enrolled 61 patients: 39 CMPD patients with thrombotic complications and free of them, 22 nonhematological patients with thrombosis. The control group consisted of 40 healthy donors. The examination protocol included determination with standard methods of HC plasma concentration, platelet and plasma components of hemostasis, mutation of factor V Leiden gene, prothrombin and methylenetetrahydrofolate reductase (MTHFR). RESULTS: Mean HC concentration in the serum in CMPD patients was 19 +/- 1.7 mcmol/l which appeared higher than in healthy donors (12 +/- 1.3 mcmol/l). The highest HC was in patients with subleukemic myelosis (SLM)--23 +/- 2.3 mcmol). No difference in HC concentration in plasma was observed in CMPD carriers of homo- or heteroxygous mutation of C667T gene or CMPD patients without the mutation. In CMPD content of factor VIII was higher in HHC than in normal HC (222 +/- 26.5 and 116 +/- 20%, respectively, p = 0.002). For von Willebrand factor 202 +/- 15.6 and 120 +/- 14.6%, respectively (p < 0.003). HC reduction in response to vitamin therapy was the greater the higher its initial level was. CONCLUSION: There is correlation between HHC and thrombosis in CMPD patients. HC concentration may depend on the proliferative stage of CMPD. As HC is a significant independent factor of thrombotic complications risk, it is necessary to detect and treat HHC.

[Chronic myeloproliferative diseases and pregnancy].

AIM: To analyse the course of pregnancy in chronic myeloproliferative diseases (CMPD) with hyperthrombocytosis, primarily, essential thrombocytemia. MATERIAL AND METHODS: The analysis of thrombogenic risk factors covered literature data and 8 cases observed by the authors. RESULTS: Six pregnant women received long-term treatment with preparations of interferon-alpha in a dose 9-20 million IU a week (both before and during pregnancy). Rapid reduction of hyperthrombocytosis (1100-4000 x 10(9) l) and the absence of a negative effect on development of the fetus were seen in all the cases. Normal delivery on week 37-39 was in 4 patients, spontaneous abortion on week 24 was provoked by a car accident. Three gravidas (gestational week 28, 33 and 34) are still under observation. Lupus anticoagulant or elevation of anticardiolipin antibodies level was detected in 4 of 8 patients, 2 patients had heterozygous mutation of methylentetrahydrofolatereductase genes and factor V (Leiden). These patients were given lannacher, faxiparine, folic acid and discrete plasmapheresis (in 2 cases). CONCLUSION: Gravidas with hyperthrombocytosis, if not contraindicated, must be treated with aspirin and interferon-alpha preparations at any gestational term. Moreover, it is necessary to exclude additional most prevalent causes of thrombophilia for adequate prevention of thromboses.