[Light- and electron-microscopic study of pelobiont Pelomyxa secunda (Gruber, 1884) comb. nov. (Archamoebae, Pelobiontida)].

Morphology of a pelobiont Pelomyxa secunda (Gruber, 1884) comb. nov. was investigated at light- and electron-microscopical levels. Locomotive forms are elongated or cigar-shaped. The size of active forms varies from 200 to 300 µm. Larger individuals (up to 400 µm) are not able to directed movement. Organism can produce short, usually finger-shaped hyaline pseudopodia at the frontal side or laterally. The cell coat is represented by amorphous glycocalix, up to 300 nm in thickness. A thin periphery cytoplasmic zone is deprived of any organelles, vacuoles, endocytobionts and other inclusions and separated from main cytoplasm by a layer of arranged microfilaments. P. secunda is multinucleate organism; nuclei are of granular type. The nucleolar material is represented by two forms of discrete structures differing in size and electron density. Two or three layers of short microtubules organized in the parallel arrangement are associated with outer side of the nuclear envelop. P. secunda possess two types of obligate prokaryotic endocytobionts lying in individual symbiontophoric vacuoles. Undulipodia, kinetosomes and root microtubular derivatives are not observed in P. secunda cells as well as any developed cytoplasmic microtubular cytoskeleton.

[Stress proteins in the cells of Porphyra purpurea (Rhodophyta) thallus].

Heat shock proteins have been revealed for the first time by the methods of Western blotting using alkaline phosphatase and ECL in the cells of Porphyra purpurea from Kattegat area of the Baltic Sea in normal and experimental stress conditions. It was demonstrated with application of monoclonal anti-Hsp70 antibodies that a slight band about 70 kDa is present constitutively at the film; additionally the polypeptide of about 40 kDa ("Hsp40") has been detected. After heat shock at 28 degrees C during 1 hr significant "expenditure" of Hsp70 was observed, as well as the pronounced induction of "Hsp40"; the induction was expressed especially strongly in 24 hr after the stress application.

[Reisolation and redescription of pelobiont Pelomyxa paradoxa Penard, 1902 (Archamoebae, Pelobiontida)].

Morphology of a pelobiont Pelomyxa paradoxa Penard, 1902 was investigated at light- and electron-microscopical levels. Locomoting cells are cigar-shaped. The cells produce many hyaline pseudopodia of digital and conical form at lateral sides of the body. The organism has a pronounced hyaline bulbous uroid with broad peripheral zone of hyaloplasm and many conical hyaline villi. There is a thin layer of amorphous glycocalix at the cell surface. "Structure" and food vacuoles of different size are very abundant in the endoplasm. Two different species of prokaryote endocytobionts are peculiar for P. paradoxa. Uninucleate stage dominates in the life cycle of P. paradoxa. Usually there are no more than 10-12 nuclei in multinucleate forms of P. paradoxa. Pelomyxae nuclei are closely surrounded by thick multilaminar layer and additionally by one more layer, which is formed by small vesicles with electron-dense content. Several irregular-shaped nucleoli are situated at the nucleus periphery. Inside the nucleoli, and sometimes directly in nucleoplasm the small round bodies are revealed, these bodies being formed by tightly packed electron-dense fibrils. Many non-motile flagellae are located mainly in the uroidal zone of the cell. Pronounced lateral root and 50-60 radial microtubules originate from the electrone-dense muft around the kinetosome. All elements of the rootlet system of flagella are limited by peripheral layers of cytoplasm. P. paradoxa occupy an intermediate position between two groups of species of Pelomyxa genus--P. gruberi + P. prima and P. palustris + P. stagnalis + P. belewski, which differ greatly by the organization of their flagella basal apparatus.

[A contact electroimpulse impact on the ureteral and bladder wall of mature dogs: a 1-year results of a morphological prospective study].

The aim of the study was investigation of morphological changes in the urinary bladder and ureteral wall of dogs in response to contact electroimpulse impact (CEII); to develop recommendations for utilization of contact electroimpulse lithotripsy in humans. Effects of single-impulse CEII on the bladder and ureteral mucosa produced by an electroimpulse lithotripter were studied in 23 mature mongrel dogs. The morphological material was studied immediately after the impact, 1, 6, 14 days and 1, 3, 6 and 12 months after it. The 0.1-0.7 J electroimpulse impact resulted in fragmentary epithelial necrosis and aceptic inflammation within a muscular layer. An enhanced to 0.8 J impact caused local damage to all layers of the wall including adventitium while a 0.9-1 J impact caused ureteral perforation in 3 cases. Duration of morphological rehabilitation depended on the impact power and was optimal for 0.1-0.5 J impulses with complete mucosal repair to day 14 and complete morphological rehabilitation to month 6 after the procedure. In power 0.6-1 J mucosal recovery increased to 1 month while complete formation of the connective tissue finished after 1 year. None cases of development of ureteral strictures after CEII was observed in follow-up for 1 year.

Small-molecule RETRA suppresses mutant p53-bearing cancer cells through a p73-dependent salvage pathway.

Identification of unique features of cancer cells is important for defining specific and efficient therapeutic targets. Mutant p53 is present in nearly half of all cancer cases, forming a promising target for pharmacological reactivation. In addition to being defective for the tumor-suppressor function, mutant p53 contributes to malignancy by blocking a p53 family member p73. Here, we describe a small-molecule RETRA that activates a set of p53-regulated genes and specifically suppresses mutant p53-bearing tumor cells in vitro and in mouse xenografts. Although the effect is strictly limited to the cells expressing mutant p53, it is abrogated by inhibition with RNAi to p73. Treatment of mutant p53-expressing cancer cells with RETRA results in a substantial increase in the expression level of p73, and a release of p73 from the blocking complex with mutant p53, which produces tumor-suppressor effects similar to the functional reactivation of p53. RETRA is active against tumor cells expressing a variety of p53 mutants and does not affect normal cells. The results validate the mutant p53-p73 complex as a promising and highly specific potential target for cancer therapy.

Toll-like receptors and their adapter molecules.

Toll-like receptors (TLR) are among key receptors of the innate mammalian immune system. Receptors of this family are able to recognize specific highly conserved molecular regions (patterns) in pathogen structures, thus initiating reactions of both innate and acquired immune response finally resulting in the elimination of the pathogen. In this case every individual TLR type is able to bind a broad spectrum of molecules of microbial origin characterized by different chemical properties and structures. Recent data demonstrate the existence of a multistep mechanism of the TLR recognition of the pathogen in which, in addition to receptors proper, the involvement of different adapter molecules is necessary. However, functions of separate adapter molecules as well as the principles of formation of a multicomponent system of ligand-specific recognition are still not quite understandable. We describe all identified as well as possible (candidate) adapter TLR molecules by giving their brief characteristics, and we also propose generalized possible variants of the TLR ligand-specific recognition with involvement of adapter molecules.

[Correlations between salinity-persistence of ciliate species and their constitutive heat shock protein of 70 kDa contents].

Our own studies of alterations in the level of constitutive heat shock protein of 70 kDa family (Hsp70) in freshwater (Paramecium jenningsi), meta-freshwater (Tetrahymena pyriformis) and curyhaline (P. nephridiatum) ciliates acclimated to salt-water and fresh-water medium were reviewed. It has been shown that the level of constitutive Hsp70 content correlates with the salinity-resistance of ciliate species: in P. jenning i it was lower in freshwater, than in marine water, in euryhaline P. nephridiatum it was higher in freshwater, than in marine water, and was more or less stable in T. pyriformis, the ciliate that occupies intermediate position between two mentioned above species according its salinity-resistance. The ecological importance of constitutive heat shock protein level is discussed in the context of salinity adaptations studies.

[Identification of Mycoplasma in patients with suspected prostate cancer].

AIM: Tests for Mycoplasma hominis, M. genitalium, Ureaplasma urealyticum in males with suspected prostate cancer. MATERIALS AND METHODS: Identification of mycoplasms was performed in prostate tissue samples using universal PCR as well as in serum samples of patients with suspected prostate cancer using ELISA for detection of IgG to M. hominis. Two hundred and fifty samples from each lobe of prostate were obtained from 125 patients with suspected prostate cancer by transrectal polyfocal biopsy. Blood samples were drawn from the same patients for ELISA. RESULTS: Out of 125 patients with suspected prostate cancer, 20.5% were positive for Mycoplasma by PCR. Between studied species, only M. hominis was found in big proportion of analyzed samples. Out of 118 serum samples, 30.5% were positive for IgG to M. hominis in ELISA. CONCLUSION: Fact of presence of Mycoplasma species in tissue of prostate was established in 20.5% pf patients with suspected prostate cancer. Obtained results show that M. hominis is frequently infects prostate tissue and that this infection was more common in patients with high grade prostatic interstitial neoplasia and prostate cancer than in patients with benign changes of prostate tissue or in persons without prostate disease. This allows to suggest that infection with M. hominis could play an important role in development of cancer.

A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy.

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects are in part determined by p53-mediated apoptosis, temporary suppression of p53 has been suggested as a therapeutic strategy to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility, a small molecule was isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound, pifithrin-alpha, protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors. Thus, inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated with p53 induction.

[Lipid-associated membrane lipopeptides of M. arginini activate NF-kB by interacting with TLR2/1, TLR2/6, and TLR2/CD14].

Various strains of mycoplasmas cause activation of transcriptional factor NF-kB as a result of interaction with different combinations of Toll-like receptors (TLR). It is well known that the MALP-2 protein of M. fermentans activates the NF-kB through interaction with the TLR2/6, lipid-associated membrane lipopeptides (LAMPs) of M. penetrans through the TLR1/2, LAMPs of M. pneumoniae through combinations of Toll-like receptors (TLR2/6 and TLR1/2), and superantigene of M. arthritidis through the TLR2 and TLR4-dependent pathways. In this study, we defined specific Toll-like receptors for LAMPs of M. arginini. For carrying out the research we used cell lines 293-null, 293-hTLR2, 293-hTLR1/2, 293-hTLR2/CD14, 293-hTLR2/6, 293-hTLR4/ CD14-MD2 expressing certain combinations of TLR and their coreceptors. It was shown that LAMPs of M. arginini cause activation of NF-kB interacting with TLR2/1, TLR2/6 and TLR2/ CD14, but not with TLR2 alone or TLR4.

Experience of long-term afala treatment in benign prostatic hyperplasia.

The use of ultralow doses of antibodies to prostate-specific antigen (afala) for long-term treatment of benign prostatic hyperplasia in patients with moderate symptoms rapidly and effectively reduces irritative and obstructive symptoms, significantly decreases residual urine volume, and increases the rate of urination. Afala therapy is indicated for patients with stage I-II benign prostatic hyperplasia of moderately pronounced symptoms.

[Heat shock proteins of 70 kDa family in the cells of free living and amphizoic amoeboid organisms].

The content of constitutive from of 70 kDa family heat shock pritein (Hsp70) was determined by the method of immunoblotting. 9 strains of representatives of the genus Acanthamoeba including 8 amphizoic (facultative parasitic) strains and one free-living (isolated from upper horizons of Arctic soils) were studied. We also examined 15 strains of free-living freshwater amoebae of various geographic origin, age and species. 14 of them belonging to the genus Amoeba and one to the genus Trichamoeba. The presence of Hsp70 was demonstrated in the cells of all 25 freshwater amoeba strains, whereas it was shown only for 2 of amphizoic acanthamoebae strains. In all these cases, the position of zone at the blot, revealed by monoclonal anti-HSP70 antibodies, corresponded to polypeptide with molecular mass about 70 kDa. We also found rather high level of constitutive Hsp70 in the cells of contemporary free-living tundra soil representative. However, in this case, the stained zone occupied the position corresponding to MW about 60 kDa which was just the same as earlier obtained for the ancient tundra acanthamoebae strain from permafrost.

[Contact electroimpulse lithotripsy].

Efficacy and safety of endoscopic contact electroimpulse lithotripsy (EILT) were studied in 146 patients with urolithiasis (mean age 48+/-16 years). Of them, 10 (7%) had ureteropelvic (UP) concrements, 124 (85%) had ureteroliths and 12 (8%) had bladder stones. The impulses were generated by the electroimpulse lithotripter Urolit-105M (Lithotech Medical, Israel; MedLine, Russia). EILT produced a complete destruction of UP concrements, ureteroliths and bladder stones in 96% cases. Complications occurred in 8.2% cases. The risk of intraoperative EILT complications was higher in destruction of stones more than 8 mm in the largest parameter of size, long-standing ureteral concrements complicated by ureteritis. EILT of UP concrements must be conducted by means of single impulses (impulse energy 0.45 J), of ureteroliths--by single or paired impulses (impulse energy 0.45-0.6 J), of bladder stones--by paired or serial impulses (impulse energy 0.6-0.7 J).

[Mycoplasma M. arginini infection induces constitutive activation of NF-kappaB and inhibits apoptosis in cells expressing toll-like receptors TLR2/6].

NF-kappaB is one of the main transcriptional factors that is responsible for cell survival under stresses. It was shown that various species of mycoplasma and their structural components were able to stimulate NF-kappaB activation as a result of their interaction with specific toll-like receptors on eukaryotic cell surface. Based on these studies, we suggested that activation of NF-kappaB in response to mycoplasmal infection could enhance the resistance of infected cells in response to proapoptotic stimuli. In this study we showed that infection of cells expressing toll-like receptors TLR2/6 with mycoplasma M. arginini leaded to suppression of apoptosis induced by chemotherapeutic agents (cisplatin, 5-fluorouracil, taxol).

[Alterations of the level of 70 kDa family heat shock protein in the ciliate Tetrahymena pyriformis in the process of the cells adaptation to the medium salinity changes].

Alterations of Hsp70 level were studied in the cells of freshwater ciliate Tetrahymena pyriformis after medium salinity changes. It is shown that ciliates, acclimated to fresh water (0 per thousand) and to salt water of 2 and 10 per thousand have similar constitutive levels of Hsp70 in their cells. Neither pronounced induction of Hsp70, was not decrease of its level, revealed in ciliates after salinity stresses. These data differ from the results obtained while studying euryhaline ciliate Paramecium nephridiatum and strongly freshwater one Paramecium jenningsi. We presume that the differences in the mode of chaperone system reaction of these ciliates species might be connected with different extents of salinity persistence - the least in P. jenningsi, intermediate in T. pyriformis and the most pronounced in P. nephridiatum.

Mobilan: a recombinant adenovirus carrying Toll-like receptor 5 self-activating cassette for cancer immunotherapy.

Toll-like receptor 5 (TLR5) is considered an attractive target for anticancer immunotherapy. TLR5 agonists, bacterial flagellin and engineered flagellin derivatives, have been shown to have potent antitumor and metastasis-suppressive effects in multiple animal models and to be safe in both animals and humans. Anticancer efficacy of TLR5 agonists stems from TLR5-dependent activation of nuclear factor-κB (NF-κB) that mediates innate and adaptive antitumor immune responses. To extend application of TLR5-targeted anticancer immunotherapy to tumors that do not naturally express TLR5, we created an adenovirus-based vector for intratumor delivery, named Mobilan that drives expression of self-activating TLR5 signaling cassette comprising of human TLR5 and a secreted derivative of Salmonella flagellin structurally analogous to a clinical stage TLR5 agonist, entolimod. Co-expression of TLR5 receptor and agonist in Mobilan-infected cells established an autocrine/paracrine TLR5 signaling loop resulting in constitutive activation of NF-κB both in vitro and in vivo. Injection of Mobilan into primary tumors of the prostate cancer-prone transgenic adenocarcinoma of the mouse prostate (TRAMP) mice resulted in a strong induction of multiple genes involved in inflammatory responses and mobilization of innate immune cells into the tumors including neutrophils and NK cells and suppressed tumor progression. Intratumoral injection of Mobilan into subcutaneously growing syngeneic prostate tumors in immunocompetent hosts improved animal survival after surgical resection of the tumors, by suppression of tumor metastasis. In addition, vaccination of mice with irradiated Mobilan-transduced prostate tumor cells protected mice against subsequent tumor challenge. These results provide proof-of-concept for Mobilan as a tool for antitumor vaccination that directs TLR5-mediated immune response toward cancer cells and does not require identification of tumor antigens.

Targeted disruption of the mouse ing1 locus results in reduced body size, hypersensitivity to radiation and elevated incidence of lymphomas.

Ing1 belongs to the family of evolutionary conserved genes encoding nuclear PHD finger-containing proteins implicated in a variety of processes, including tumorigenesis, replicative senescence, excision repair and response to genotoxic stress. We have generated mice deficient in all the isoforms of Ing1 by targeted disruption of the exon that is common for all ing1 transcripts. Embryonic fibroblasts from ing1-knockout mice were similar to the wild-type cells in their growth characteristics, replicative lifespan in culture, p53 induction and sensitivity to various cytotoxic treatments with minor alterations in cell cycle distribution in response to genotoxic stress. ing1-deficient animals are characterized by reduced size with no obvious morphological, physiological or behavioral abnormalities, indicating that ing1 function is dispensable for the viability of mice under normal physiological conditions. Loss of ing1 was associated with earlier onset and higher incidence of lymphomas. Consistent with the possible involvement of Ing1 in DNA repair, ing1-deficient mice were more sensitive to total body gamma radiation. Our observations are well in line with the suggested role of ing1 as a candidate tumor suppressor gene involved in control of DNA damage response.

Downregulation of topoisomerase IIbeta in myeloid leukemia cell lines leads to activation of apoptosis following all-trans retinoic acid-induced differentiation/growth arrest.

Among the topoisomerase (topo) II isozymes (alpha and beta), topo IIbeta has been suggested to regulate differentiation. In this study, we examined the role of topo IIbeta in all-trans retinoic acid (ATRA)-induced differentiation of myeloid leukemia cell lines. Inhibition of topo IIbeta activity or downregulation of protein expression enhanced ATRA-induced differentiation/growth arrest and apoptosis. ATRA-induced apoptosis in topo IIbeta-deficient cells involved activation of the caspase cascade and was rescued by ectopic expression of topo IIbeta. Gene expression profiling led to the identification of peroxiredoxin 2 (PRDX2) as a candidate gene that was downregulated in topo IIbeta-deficient cells. Reduced expression of PRDX2 validated at the mRNA and protein level, in topo IIbeta-deficient cells correlated with increased accumulation of reactive oxygen species (ROS) following ATRA-induced differentiation. Overexpression of PRDX2 in topo IIbeta-deficient cells led to reduced accumulation of ROS and partially reversed ATRA-induced apoptosis. These results support a role for topo IIbeta in survival of ATRA-differentiated myeloid leukemia cells. Reduced expression of topo IIbeta induces apoptosis in part by impairing the anti-oxidant capacity of the cell owing to downregulation of PRDX2. Thus, suppression of topo IIbeta and/or PRDX2 levels in myeloid leukemia cells provides a novel approach for improving ATRA-based differentiation therapy.

[Heat shock protein of Hsp70 in Paramecium nephridiatum and its role in adaptation to environmental salinity changes].

The level of Hsp70 was studied in the cells of eurihaline ciliate Paramecium nephridiatum after the environmental salinity changes. Two types of treatment were applied. "Shock": ciliates were placed for 1 h to the medium with stress salinity, then transferred back to the medium, they were acclimated to, for 2 h; "adaptation": ciliates were placed for 3 h into stress salinity. It has been shown, that ciliates, acclimated to fresh water (0%) have the higher constitutive level of Hsp70, than those, acclimated to 10%. Transfer from fresh water to 10% does not cause the increase of Hsp70 synthesis in protists, whereas the reciprocal transfer results in induction of Hsp70 in the cells. "Adaptation" results in induction of Hsp70 in both "directions" of salinity changes. The results obtained allow to presume that the possibility to survive in the media of various salinity in eurihaline ciliates is somehow determined by the higher initial level of Hsp70 in their cells, than in stenohaline representatives of the same genus.

[The morphological study of the cysts Pelomyxa palustris Greeff, 1874].

Pelomyxa palustris Greeff, 1874, is the only species of pelomixoid amoebas with the rest cysts in its life cycle. The morphology of the P. palustris has been studied by the light and electronic microscopy. Encystation of P. palustris under climatic conditions of North-West of Russia occurs within August-September. Rest cysts have a complex, trilaminar wall. Two inner lamina are the dense endocyst and the laminated mesocyst, thickness of each layer runs up to 0.6-0.7 microm. Thickness of the electron-dense ectocyst usually does not exceed 0.1-0.2 microm. The encystated cell of P. palustris has the unique structure. About 60 % of the cell volume are occupied by a huge vacuole placed in the center and filled up with the prokaryotic cytobionts. Different vacuoles, small vesicles of various nature, autophagosomes and lipid drops could be found inside that huge vacuole. The amoebae cytoplasm occupies the space in between endocyst's inner surface and the central vacuole. No any inclusions, prokaryotic cytobionts and most of cell organelles are absent in the cytoplasm. There are 4 large nuclei filled with relatively homogeneous karyoplasm lying in the cytoplasm. Nuclear envelope forms a lot of long tubular channels, running through the cytoplasm and lining the membrane of the central vacuole. Encysted pelomixoid stay in this state up until the beginning of excystation. Excystation of P. palustris in the studied region occurs in spring, during the latter half of April and the beginning of May. Cysts undergo complex morphofunctional changes, related to the reorganization of the wall and formation of young multinucleate amoebas. Only one wall lamina of the 3 initial ones is left up to the moment of excystation. The central vacuole endures ruination and its content penetrates into the cytoplasm. Pelomixoid nuclei divide twice. Prokaryotic cytobionts are localized in cytoplasm and in the perinuclear area. Young multinuclear species of P. palustris coming out of the cysts do not differ in their structure from the adult forms.