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The basis of interpersonal relationships is communication as a means of information exchange and an important factor in influence on the of personality`s development. There is a world perception, norms awareness and society values which are perceived through the prism of interpersonal relations. The young people are characterized by the lack of accumulated life experience; values are influenced from the outside, so that influence is the reason for their unstable behavior in the legal field. In a transitive society, all processes are exacerbated, including growing deviant behavior of adolescents. Qualitative work in the organization of legal support of interpersonal relations in the educational environment can help to keep students from committing negative manifestations in interpersonal communication. This form of activity of the teaching staff could be an integral part of the preventive impact on students.

Ixodid ticks (Acarina, Ixodidae) are vectors of dangerous human infections. The main tick species that determine the epidemiological situation for tick-borne diseases in northern Europe are Ixodes ricinus and Ixodes persulcatus. In recent years, significant changes in the number and distribution of these species have been observed, accompanied by an expansion of the sympatric range. This work summarizes the data of long-term studies carried out in Karelia since 2007 on the infection of I. persulcatus and I. ricinus ticks with various pathogens, including new viruses with unclear pathogenic potential. As a result, tick-borne encephalitis virus (TBEV, Siberian genotype), Alongshan virus, several representatives of the family Phenuiviridae, Borrelia afzelii, Borrelia garinii, Ehrlichia muris, Candidatus Rickettsia tarasevichiae and Candidatus Lariskella arthropodarum were identified. Data were obtained on the geographical and temporal variability of tick infection rates with these main pathogens. The average infection rates of I. persulcatus with TBEV and Borrelia burgdorferi sensu lato were 4.4% and 23.4% and those of I. ricinus were 1.1% and 11.9%, respectively. We did not find a correlation between the infection rate of ticks with TBEV, B. burgdorferi s.l. and Ehrlichia muris/chaffeensis with the sex of the vector. In general, the peculiarities of the epidemiological situation in Karelia are determined by the wide distribution and high abundance of I. persulcatus ticks and by their relatively high infection rate with TBEV and B. burgdorferi s.l. in most of the territory, including the periphery of the range., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022. Published by Elsevier GmbH.)

The inhibitory effect of p53 on homologous recombination (HR) is exerted through sequestration of replication protein A (RPA). Release of the p53/RPA complex in response to replication stress is crucially dependent on the phosphorylation status of both proteins and is required for efficient DNA repair by HR. Phosphorylation of RPA within its RPA2 subunit by cyclin-dependent kinases (CDK) is an early event in the replication stress response. Here we investigated the role of transcriptional activation of the p53 downstream target, p21 Waf1 , on RPA2 phosphorylation, the stability of the p53/RPA complex and HR in cells undergoing replication arrest induced by camptothecin (CPT). We show that in CPT-treated cells, activation of p53 and p21 Waf1 impedes RPA2 phosphorylation, while their depletion by siRNA stimulates it. The p53/RPA complex is more stable in wild-type cells than in cells depleted of p21 Waf1 . We used nocodazole-synchronized cells treated with CPT at the entrance to S phase to assess rates of HR. Regardless of their p53 or p21 Waf1 status, the cells proceed through S phase at a similar rate and enter G2. While HR is low in wild-type cells and high in p53-depleted cells, only partial inhibition of HR is observed in the p21 Waf1 -depleted cells. This correlates with the extent of RPA sequestration by p53. Thus, in CPT-treated cells, p53-induced transcriptional activation of p21 Waf1 regulates RPA2 phosphorylation, the stability of the p53/RPA complex and HR., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Coordinated changes of actin cytoskeleton and cell adhesion accompany maturation of lymphoid cells, their migration through lymphoid organs and to sites of inflammation, as well as metastasis of transformed cells. Here we discuss the central role of the actin-regulating adaptor protein, paxillin, during lymphocyte transition from a polarized, motile cell phenotype with partially active LFA-1 integrins to a round and immobile one with fully active LFA-1. In Baf3 murine pro-B lymphocytes, the former phenotype is induced by IL-3 that stimulates a FAK-mediated phosphorylation of paxillin at tyrosines (Y) 31 and 118 and a consequent Rac1 activation. Rearrangements of actin cytoskeleton that lead to the cell's acquisition of a spherical shape and LFA-1 activation are achieved upon activation of PKC-δ that binds and directly phosphorylates paxillin at threonine (T) 538 with consequent RhoA activation. This is accompanied by dephosphorylation of paxillin Y31/118 and by Rac1 inactivation. We propose a model of signaling cascades that reflects the interplay between the IL-3- and PKC-δ-mediated pathways.

We investigated the PKCdelta-mediated phosphorylation of paxillin within its LIM4 domain and the involvement of this phosphorylation in activation of LFA-1 integrins of the Baf3 pro-B lymphocytic cell line. Using phosphorylated-threonine-specific antibodies, phosphorylated amino acid analysis and paxillin phosphorylation mutants, we demonstrated that TPA, the pharmacological analog of the endogenous second messenger diacyl glycerol, stimulates paxillin phosphorylation at threonine 538 (T538). The TPA-responsive PKC isoform PKCdelta directly binds paxillin in a yeast two-hybrid assay and phosphorylates paxillin at T538 in vitro and also co-immunoprecipitates with paxillin and mediates phosphorylation of this residue in vivo. Recombinant wild-type paxillin, its phospho-inhibitory T538A or phospho-mimetic T538E mutants were expressed in the cells simultaneously with siRNA silencing of the endogenous paxillin. These experiments suggest that phosphorylation of paxillin T538 contributes to dissolution of the actin cytoskeleton, redistribution of LFA-1 integrins and an increase in their affinity. We also show that phosphorylation of T538 is involved in the activation of LFA-1 integrins by TPA.

Mutant p53 is a cancer-specific target for pharmacologic intervention. We show that histone deacetylase inhibitors such as FR901228 and trichostatin A completely depleted mutant p53 in cancer cell lines. This depletion was preceded by induction of p53-regulated transcription. In cells with mutant p53 pretreated with histone deacetylase inhibitors, DNA damage further enhanced the p53 trans-function. Furthermore, histone deacetylase inhibitors were preferentially cytotoxic to cells with mutant p53 rather than to cells lacking wild-type p53. We suggest that, by either restoring or mimicking p53 trans-functions, histone deacetylase inhibitors initiate degradation of mutant p53. Because mutant p53 is highly expressed, a sudden restoration of p53-like functions is highly cytotoxic to cells with mutant p53. In a broader perspective, this shows how selectivity may be achieved by targeting a non-cancer-specific target, such as histone deacetylases, in the presence of a cancer-specific alteration, such as mutant p53.

By activating anti-apoptotic factors (e.g., Hsp70, Raf-1, Bcl-xL), Bcr-Abl blocks apoptotic pathways at multiple levels, thus rendering leukemia cells resistant to chemotherapeutic agents such as doxorubicin (DOX). In Bcr-Abl-transfected HL60 (HL/Bcr-Abl) cells, procaspase-9 was increased and partially processed. The Bcr-Abl inhibitor imatinib (Gleevec, STI-571) released the apoptotic stream. Also, HL/Bcr-Abl cells were hyper-sensitive to geldanamycin (GA), which depletes Bcr-Abl and Raf-1. Raf-1 and Bcr-Abl-transfected FDC-P1 hematopoietic cells were selectively sensitive to GA and imatinib, respectively. Remarkably, cell clones with high levels of Bcr-Abl that could not be depleted by GA were relatively resistant to both GA and imatinib. GA and flavopiridol sensitized such resistant cells to imatinib. These data suggest bi-phasic sensitivity to mechanism-based therapeutic agents. Although Bcr-Abl renders cells hyper-sensitive, an excess of Bcr-Abl results in resistance (due to the remaining activity). We discuss therapeutic approaches to overcome bi-phasic resistance to mechanisms-based agents.

The tumor suppressor protein p53 is emerging as a central regulator of homologous recombination (HR) processes and DNA replication. P53 may downregulate HR through multiple mechanisms including the reported associations with the Rad51 and Rad54 recombinases, and the BLM and WRN helicases. Here, we investigated whether the interaction of p53 with human replication protein A (RPA) is necessary for the regulation of HR. By employing a plasmid-based HR assay in p53-null H1299 lung carcinoma cells, we studied the HR-suppressing properties of a panel of p53 mutants, which varied in their ability to interact with RPA. Both wild-type p53 and a transactivation-deficient p53 mutant (L22Q/W23S) suppressed HR and prevented RPA binding to ssDNA in vitro and in vivo. Conversely, p53 mutations that specifically disrupt the RPA-binding domain, while not compromising p53 transactivation function (D48H/D49H and W53S/F54S), did not affect HR. Suppression of HR was also not seen with missense mutations in the p53 core domain (His175 and His273), which retained the ability to interact with RPA, suggesting that the disruption of additional binding interactions of p53, for example, with Rad51 or recombination intermediates, also impacts on HR. We hypothesize that sequestration of RPA by p53 at the sites of recombination is one means by which p53 can inhibit HR processes. Our data support and extend the previously formulated 'dual model' of p53's role as guardian of the genome.

Tyrosine phosphorylation of paxillin regulates actin cytoskeleton-dependent changes in cell morphology and motility in adherent cells. In this report we investigated the involvement of paxillin tyrosine phosphorylation in the regulation of actin cytoskeleton-dependent polarization and motility of a non-adherent IL-3-dependent murine pre-B lymphocytic cell line Baf3. We also assessed the effect of phorbol myristate acetate (PMA), a phorbol ester analogous to those currently in clinical trials for the treatment of leukemia, on paxillin phosphorylation. Using tyrosine-to-phenylalanine phosphorylation mutants of paxillin and phosphospecific antibody we demonstrated that IL-3 stimulated phosphorylation of paxillin tyrosine residues 31 and 118, whereas the tyrosines 40 and 181 were constitutively phosphorylated. Phosphorylation of paxillin residues 31 and 118 was required for cell polarization and motility. In the presence of IL-3, PMA dramatically reduced the phosphorylation of residues 31 and 118, which was accompanied by inhibition of cell polarization and motility. This PMA effect was partially recapitulated by expression of exogenous tyrosine 31 and 118 mutants of paxillin. We also demonstrated that PMA inhibited the IL-3-induced and activation-dependent tyrosine phosphorylation of focal adhesion kinase. Thus, our results indicate that phosphorylation of paxillin tyrosine residues 31 and 118 regulates actin-dependent polarization and motility of pre-B Baf3 cells, both of which could be inhibited by PMA. They also suggest that inhibition of upstream signaling by PMA contributes to the decrease of paxillin phosphorylation and subsequent changes in cell morphology.

Overexpression of ectopic mutant p53 represses wild-type p53-stimulated transcription, known as a dominant negative effect. On the other hand, overexpression of wild-type p53 can repress transcription stimulated by several transcription factors, including hypoxia-inducible factor-1 (HIF-1). Using a panel of well-characterized Arg175 p53 mutants we found that only mutants (Tyr175, Trp175, Asp175 and Phe175) which have completely lost their ability to transactivate repress wild-type p53-stimulated Bax, p21 and PG13 promoter constructs. In contrast, Asn175, Gln175, Leu175 and Pro175 mutants which partially retained transactivating functions did not exert dominant negative effects against PG13 and p21 promoter constructs. However, these latter mutants failed to activate Bax and, instead, exerted a dominant negative effect on a Bax-Luc promoter construct. We conclude that a dominant negative effect is promoter selective as a consequence of selective loss of transactivating function. Albeit less potent than wild-type p53, all Arg175 p53 mutants retained partial ability to repress HIF-1-stimulated transcription. We propose that transrepression and the dominant negative effect have similar mechanisms and may involve competition with transcription factors (wild-type p53, HIF-1, etc.) for cofactors such as p300. Thus, a p53(22/23) mutant, which is deficient in p300 binding, did not exert dominant negative effects. Like transrepression, the dominant negative effect required overexpression of mutant p53 and, therefore, is not dominant. In the presence of a wild-type p53 allele, levels of endogenous mutant p53 protein were low in heterozygous cells. Endogenous mutant p53 became overexpressed only after loss of the second p53 allele. Therefore, endogenous mutant p53s are unable to display a dominant negative effect. This explains why loss of the second p53 allele is required to eliminate p53 functions in cancer cells.

In the murine pre-B lymphoid cell line Baf3, the presence of IL-3 is required for the formation of membrane ruffles that intensely stain for actin and are responsible for the elongated cell phenotype. Withdrawal of IL-3 dissolves ruffled protrusions and converts the cell phenotype to round. Flow cytometric analysis of the cell shape showed that an inactive analog of Rac1 but not inactive RhoA or inactive cdc42 rounds the cells in the presence of IL-3. Constitutively activated Rac1 restores the elongated cell phenotype to IL-3-starved cells. We conclude that the activity of Rac1 is necessary and sufficient for the IL-3-induced assembly of membrane ruffles. Similar to the IL-3 withdrawal, phorbol 12-myristate 13-acetate (PMA) dissolves actin-formed membrane ruffles and rounds the cells in the presence of IL-3. Flow cytometric analysis of the cell shape demonstrated that in the presence of IL-3 the PMA-induced cell rounding cannot be abolished by constitutively active Rac1 but can be imitated by inactive Rac1. These data indicate that PMA disrupts the IL-3 pathway downstream of Rac1. Cells rounded by PMA return to the elongated phenotype concomitantly with PKC depletion. PMA-induced cell rounding can be reversed by the PKC-specific inhibitor GF109203X. Experiments with overexpression in Baf3 of individual PKC isoforms and a dominant negative PKC-delta indicate that activation of PKC-delta but not other PKC isoforms is responsible for disruption of membrane ruffles.

We investigated the p53 status and the ability of exogenous wildtype (wt) p53 to affect chemosensitivity in three anaplastic thyroid carcinoma cell lines (BHT-101, SW-1736, and KAT-4). All three cell lines had nonfunctional p53. Treatment with mitomycin C or adriamycin did not result in accumulation of p53 or induction of p21WAF1/CIP1 or Mdm-2 and did not cause Rb dephosphorylation. BHT-101 and KAT-4 cells had mutant p53. SW-1736 cells were functionally mutant because of marked down-regulation of wt p53 messenger ribonucleic acid, representing a novel mechanism of p53 dysfunction. Infection with a p53-expressing adenovirus (Ad-p53) induced high levels of p21 and Mdm-2 proteins. In BHT-101 cells, induction of p21 and Mdm-2 was evident 10 h after infection. In KAT-4 cells, induction of p21 and Mdm-2 was observed 1 day after infection, and continued to increase over the ensuing 24 h. SW-1736 cells demonstrated intermediate kinetics. Sensitivity to the cytotoxic effect of Ad-p53 paralleled the kinetics of p21/Mdm-2 induction. BHT-101 cells were most sensitive to killing by Ad-p53, with an IC50 of less than 2 multiplicity of infection; SW-1736 cells were intermediate in sensitivity; KAT-4 cells were resistant. All three cell lines became more sensitive to adriamycin after wt p53 expression, with a 10-fold decrease in IC50 values. The latter observation may make a combination of wt p53 and chemotherapeutic drugs an attractive modality for treating anaplastic thyroid cancer.

p53 is required for hypoxia-induced apoptosis in vivo, although the mechanism by which this occurs is not known. Conversely, induction of the hypoxia-inducible factor-1 (HIF-1) transactivator stimulates transcription of a number of genes crucial to survival of the hypoxic state. Here we demonstrate that p53 represses HIF-1-stimulated transcription. Although higher levels of p53 are required to inhibit HIF than are necessary to transcriptionally activate p53 target genes, these levels of p53 are similar to those that stimulate cleavage of poly(ADP-ribose) polymerase, an early event in apoptosis. Transfection of full-length p300 stimulates both p53-dependent and HIF-dependent transcription but does not relieve p53-mediated inhibition of HIF. In contrast, a p300 fragment, which binds to p53 but not to HIF-1, prevents p53-dependent repression of HIF activity. Transcriptionally inactive p53, mutated in its DNA binding domain, retains the ability to block HIF transactivating activity, whereas a transcriptionally inactive double point mutant defective for p300 binding does not inhibit HIF. Finally, depletion of doxorubicin-induced endogenous p53 by E6 protein attenuates doxorubicin-stimulated inhibition of HIF, suggesting that a p53 level sufficient for HIF inhibition can be achieved in vivo. These data support a model in which stoichiometric binding of p53 to a HIF/p300 transcriptional complex mediates inhibition of HIF activity.

Studies utilizing the overexpression of individual isoforms indicated that both PKC-alpha and -delta promote a number of biological effects, including inhibition of DNA synthesis associated with rearrangements of the actin cytoskeleton in the murine B-cell lymphoma (Baf3), differentiation of the murine promyelocyte line 32D, and activation of MAP kinase in CHO fibroblasts. We postulated that these results reflect some form of cross-regulation between PKC-alpha and -delta rather than their functional redundancy. In this report, we show that overexpression of PKC-alpha in Baf3 and 32D leads to an elevation of the endogenous PKC-delta mRNA and protein levels. The elevated steady-state PKC-delta mRNA level results from a combination of increased PKC-delta transcription and mRNA stability. Upregulation of PKC-delta mRNA by PKC-alpha occurs even after a selective depletion of the PKC-delta protein. In addition, phorbol ester-induced elevation of PKC-delta mRNA and protein levels can be prevented by the PKC inhibitor GF109203X, an indication of the requirement for PKC kinase activity. Inhibition of new protein synthesis by cycloheximide showed that upregulation of PKC-delta mRNA, as opposed to delayed downregulation of the PKC-delta protein, is primarily responsible for the accumulation of this isoform by PKC-alpha. In parental Baf3 and 32D cells and PKC-alpha overexpressers, PKC-alpha and PKC-delta are uniquely involved in cross-regulation, while PKC-epsilon, PKC-eta, and PKC-mu are not.

Conservation of DNA segments performing sequence-related functions is a landmark of selection and functional significance. Phylogenetic variability of alpha satellite and apparent absence of conserved regions calls its functional significance into question, even though sequence-specific alpha satellite-binding proteins pJ alpha and CENP-B have been discovered. Moreover, the function of pJ alpha is obscure and CENP-B binding satellite DNA, which is thought to participate in centromere formation, is found only in few species and not necessarily in all chromosomes. Analysis of alpha satellite evolution allows us to recognize the order in this variability. Here we report a new alpha satellite suprachromosomal family, which together with the four defined earlier, covers all known alpha satellite sequences. Although each family has its characteristic types of monomers, they all descend from two prototypes, A and B. We show that most differences between prototypes are concentrated in a short region (positions 35 to 51), which exists in two alternative states: it matches a binding site for pJ alpha in type A and the one for CENP-B in type B. Lower primates have only type A monomers whereas great apes have both A and B. The new family is formed by monomeric types almost identical to A and B prototypes, thus representing a living relic of alpha satellite. Analysis of these data shows that selection-driven evolution, rather than random fixation of mutations, formed the distinction between A and B types. To our knowledge, this is the first evidence for selection in any of the known satellite DNAs.

We show here that the mode of cell death in IL-6-starved T1165 and T1198 plasmacytoma cell lines is apoptosis, and that it can be suppressed by phorbol ester (PMA) treatment in a protein kinase C (PKC)-mediated process that involves alpha and/or delta isozymes. PMA-induced PKC activation, but not the depletion that follows it, participates in the suppression of apoptosis. Extended PKC activation is necessary but not sufficient for the apoptosis suppression. In addition, the cells must be in a "competent" state, which appears not to be determined by PKC. We observed two points of "competence" during the time between withdrawal of IL-6 and the start of massive cell death: one, immediately after withdrawal, and another, just before onset of apoptosis, at the time corresponding to maximal accumulation of cells in a G0/G1 block imposed by IL-6 withdrawal. Treatment with PMA and other PKC activators resulted in a shift of the cell population to S phase, lifting the G0/G1 block. We propose a model in which cells are rescued in a certain stage of the G1 phase of cell cycle. Death suppression occurs when a transient PMA-induced PKC activation occurs when a significant number of cells are in this part of G1, allowing them to pass the restriction point safely without initiating the cell death program.

Two alpha-satellite fragments specific for human chromosome 4 have been cloned and characterized. Under stringent annealing conditions, they hybridized in situ only to the pericentromeric region of chromosome 4, but under non-stringent conditions they hybridized to all chromosomes containing the sequences of alpha-satellite suprachromosomal family 2 (viz., chromosomes 2, 4, 8, 9, 13, 14, 15, 18, 20, 21 and 22). Southern blot analysis reveals the 3.2-kb higher-order repeated unit which exists in two forms: as a single MspI fragment or a combination of the 2.6-kb and 0.6-kb MspI fragments. The two chromosome-4-specific cloned sequences appear to be different parts of this repeated unit. Taken together they constitute about 60% of its length. The primary structure of the higher-order repeated unit is characterized by a dimeric periodicity of the D1-D2 type which is usual to suprachromosomal family 2. At least in one site this regularity is disrupted by monomer deletion leading to the D2-D2 monomeric order. The most likely mechanism of this monomer excision is homologous unequal crossing-over. These sequences may serve as both cytogenetic and restriction-fragment length polymorphism (RFLP) markers for the pericentromeric region of chromosome 4.

We have sequenced the full-length copy of the alpha satellite higher-order repeated unit characteristic of human chromosome 3. Its internal structure, the regular alterations of J1 and J2 type monomers, is typical of the alphoid suprachromosomal family 1. This dimeric order is disrupted by the substitution of one J1 unit by an alien dimer which is not clearly related to any of the established monomeric types. We have also observed some other similar cases of segment substitutions in alpha satellite DNA. They probably represent a special type of molecular event which could be generated by gene conversion. Segment substitutions may be one of the important factors responsible for the extreme variability of localization patterns and actual sequences of alpha satellite DNA that should be taken into account in reconstructions of alpha satellite evolution.

We have analyzed more than 500 alphoid monomers either sequenced in our laboratory or available in the literature. Most of them belonged to the well studied suprachromosomal families 1, 2 and 3 characterized by dimeric (1 and 2) and pentameric (3) ancestral periodicities. The sequences that did not belong to the previously known families were subjected to further analysis. About a half of them formed a relatively homogenous family. Its members were on average 80.5% identical and 89.5% homologous to the M1 consensus sequence derived from this group (39 monomers). In the genome they do not form any ancestral periodicities other than a monomeric one, and are found at least in chromosomes 13, 14, 15, 21, 22 and Y. The newly defined family was termed suprachromosomal family 4. Comparison of all 10 alphoid monomeric groups identified so far showed that the M1 sequence is closely related to the J1-D2-W4-W5 homology grouping. Notably the African Green Monkey alpha satellite, also characterized by monomeric construction, appears to be a member of the same group.