Phylogenetic and structural analysis of annexins in pea (Pisum sativum L.) and their role in legume-rhizobial symbiosis development.

Annexins as Ca2+/phospholipid-binding proteins are involved in the control of many biological processes essential for plant growth and development. In a previous study, we had shown, using a proteomic approach, that the synthesis of two annexins is induced in pea roots in response to rhizobial inoculation. In this study, phylogenetic analysis identif ied these annexins as PsAnn4 and PsAnn8 based on their homology with annexins from other legumes. The modeling approach allowed us to estimate the structural features of these annexins that might inf luence their functional activity. To verify the functions of these annexins, we performed comparative proteomic analysis, experiments with calcium inf lux inhibitors, and localization of labeled proteins. Essential down-regulation of PsAnn4 synthesis in a non-nodulating pea mutant P56 (sym10) suggests an involvement of this annexin in the rhizobial symbiosis. Quantitative RT-PCR analysis showed that PsAnn4 was upregulated at the early stages of symbiosis development, starting from 1-3 days after inoculation to up to 5 days after inoculation, while experiments with the Ca2+ channel blocker LaCl3 revealed its negative inf luence on this expression. To follow the PsAnn4 protein localization in plant cells, it was fused to the f luorophores such as red f luorescent protein (RFP) and yellow f luorescent protein (YFP) and expressed under the transcriptional regulation of the 35S promoter in Nicotiana benthamiana leaves by inf iltration with Agrobacterium tumefaciens. The localization of PsAnn4 in the cell wall or plasma membrane of plant cells may indicate its participation in membrane modif ication or ion transport. Our results suggest that PsAnn4 may play an important role during the early stages of pea-rhizobial symbiosis development.

[Immunolocalization of microsporidium paranosema locustae canning lSP70 family proteins in locust infected fat body].

Immunolabeling method of microsporidium Paranosema locustae proteins on cryosections of locust infected fat body was proposed. In contrast to single parasite cells and artificially infected host cell cultures, this method allows to study molecular mechanisms of host-parasite relationships and in particular the secretory microsporidial proteins either at cellular or tissue level. Immunolocalization of the EPR-specific and cytoplasmic forms of Hsp70 family of molecular chaperones on cryosections showed accumulation of these proteins in the respective compartments of intracellular developmental stages of P. locustae and their absence in host structures. This allows to use them in diagnostics of microsporidiosis lesions in infected tissues as well as in colocalization analysis with P. lociustatre secretory proteins as a marker of parasite. The cytoplasmic chaperone stains cytoplasmic compartment homogeneously, but in the infected host cell during sporogony it disappears partially from the intracellular stages of development which damaged by maturing spores. Thereby study of molecular mechanisms of host-parasite relationships is to be carried out at the earlier stages of infection before active sporogony.

[Peculiarities of the expression, structure, and localization of the subtilisin-like protease in the microsporidium Paranosema locustae].

Peculiarities of the expression, localization, and structure of the subtilisin-like protease from the microsporidium Paranosema locustae, a parasite of the migratory locust and other orthopteran species, are analyzed. Heterologous expression of the microsporidian ferment in the bacterium Escherichia coli allowed obtaining antibodies to the recombinant protein and to start its examination. In spite of the presence of the N-tail signal peptide in the ferment, potentially able to secret it into the cytoplasm of the infected cell, immunoblotting with obtained antibodies had demonstrated specific accumulation of the protease in the insoluble fraction of spore homogenate. At the same time, the ferment was absent in intracellular stages.of the parasite and also in the cytoplasm of infested host cells. Accumulation of mRNA, coding the studied protein in microsporidian spores was confirmed with the use of RT-PCR method. Heterologous expression of the protease in the methylotrophic yeast Pichiapastoris demonstrated the same result. The ferment of P. locustae was not secreted into a culture medium and was absent in the cytoplasm of yeast cells, accumulating in a dissoluble (membrane) fraction of the homogenate. On the whole, the obtained data testify to the fact that the subtilisin-like protease of P. locustae plays an important role in the physiology of spores rather than participates in host-parasite relations during intra-cellular development.

[Using of antibodies against Microsporia Hsp70 family proteins for analysis of secretome of intracellular parasites].

Microsporidia is a large group of fungi-related unicellular parasites with obligate intracellular lifestyle. Unlike other protozoan intracellular parasites (Kinetoplastida and Apicomplexa), most microsporidian species develop in direct contact with the host cell cytoplasm. This fact, acquisition of unique transporters to exploit host metabolic system (alongside the strong minimization of own machinery) and predicted repertoire of microsporidia secretome altogether suggest an active role of parasite proteins in the control of infected cell. Lack of information about secretome of microsporidia intracellular stages is largely due to the methodological difficulties of working with the obligate intracellular parasites. An important problem of such study is the contamination of preparations of host cell cytoplasm by inner (nonsecreted) parasite proteins. Even the homogenization of infected tissue in mild conditions and removal of parasite cells by low-speed centrifugation may result in their partial disruption. We expressed the fragments of three Hsp70 family chaperones from the microsporidium Paranosema (Antonospora) locustae in bacteria Escherichia coli. Immunoblotting with proteins of microsporidia intracellular stages and infected host tissue (locust fat bodies) demonstrated that antibodies against recombinant polypeptides may be used to monitor the integrity of parasite cells during homogenization of infected host tissue and subsequent removal of parasites by centrifugation.

[Unique characteristics of the energy metabolism in Microsporidia as a result of durational adaptation to the intracellular development].

Microsporidia is a large group of fungi-related unicellular eukaryotes with obligate intracellular lifestyle infecting a wide range of invertebrate and vertebrate hosts. Long adaptation of the parasites to intracellular development resulted in extraordinary minimization of their metabolic system. The paper summarizes the original results and literature data on the study of microsporidian carbohydrate and energy metabolism. On the basis of the material, it is concluded that minimization of microsporidian cell machinery was accompanied by the acquisition of a number of unique characteristics, which were not found in other eukaryotes.

[Analysis of expression of vesicular transport genes in avesicular cells of the microsporidium Paranosema (Antonospora) locustae].

Long adaptation of microsporidia, a large group fungi-related protozoa, to intracellular lifestyle has resulted in a drastic minimization of parasite cell. Ultrastructural analysis has shown that the Golgi complex of the microsporidia Paranosema (Antonospora) grylli and P. locustae appears as branching or varicose networks of thin tubules. These tubular networks are connected to endoplasmic reticulum, plasma membrane and forming polar tube but have no vesicles. Vesicles were not found even if ultra-fast cryofixation and membrane fusion/uncoating inhibition were used. However, a limited number of genes involved in vesicular transport were found in microsporidia genomes. In this study we used RT-PCR to analyze the content of mRNA transcripts encoding beta and beta' subunits COPI coatomer complex, Sec13 and Sec31 subunits COPII, SNARE-proteins synaptobrevin and syntaxin-like member of SFT family in P. locustae intracellular stages. The level of expression of studied genes was comparable with that of gene encoding alternative oxidase, enzyme envolved in microsporidia core metabolism. Moreover, polyclonal antibodies raised against recombinant Sec13 subunit COPII, expressed in B Escherichia coli, has shown accumulation of the protein is spores and stages of intracellular development as well as its association with membranes. The presence of components of vesicular transport machinery in avesicular microsporidia cells requires their functional analysis.

[Posttranslationally modified microcins].

Microcins are antibacterial compounds that are encoded in the bacterial genome and synthesized via ribosomal translation. Microcins play an important role in microbial ecology and are promising as antibiotics. To exert their effect, most microcins are incorporated in the membrane of sensitive cells to increase its permeability. The review considers the known classes of posttranslationally modified microcins. These microcins are unusual in structure and inhibit the grown of sensitive cells by entering their cytoplasm and affecting intracellular targets, such as DNA gyrase, DNA-dependent RNA polymerase, and aspartyl-tRNA synthase.

[The efficacy of high-dose chemotherapy and the transplantation of autologous hemopoietic cells in lymphogranulomatosis]. / Effektivnost' vysokodoznoi khimioterapii i transplantatsii autologichnykh gemopoéticheskikh kletok pri limfogranulematoze.

AIM: To determine clinical effectiveness of high-dose polychemotherapy (PCT) and transplantation of autologous hemopoietic cells (TAHC) in patients with lymphogranulomatosis (LGM). MATERIAL AND METHODS: 27 LGM patients aged 16-42 years who have undergone TAHC after high-dose PCT (BEAM--17 patients or CBV--10 patients). 4 patients given high-dose PCT were in the first-second complete remission (CR), 7 patients--in the first partial remission (PR). Prior to TAHC, 8 patients had one, two and more relapses of LGM, and 8 patients had no remission at all. Bone marrow, hemopoietic blood cells and both were transplanted to 17, 2 and 8 patients, respectively. Mobilization of hemopoietic blood cells and stimulation of hemopoiesis after TAHC were achieved using colony-stimulating factors. RESULTS: The treatment resulted in CR or PR (from 6 to 95 months) in 70.4% of patients. The remission duration varied depending on the disease phase at transplantation. Four patients who underwent TAHC in PR maintained it for 13-95 months (median 47.5 months). Lasting remissions (29-59 months) were achieved in 42.9 and 37.5% of patients who underwent TAHC in the first PR or in recurrent LGM. None of the patients was in remission longer than 2 years after TAHC if high-dose PCT was conducted in advanced tumor process due to resistant LGM or inadequate previous treatment. Infectious complications lethality early after the transplantation reached 7.4%(2 patients). CONCLUSION: High-dose PCT followed by TAHC is effective in LGM if the tumor is chemosensitive.

[Specific aspects of thrombocyte system of serotonin in patients with different manifestations of schizoaffective psychosis]. / Osobennosti serotoninovoi sistemy trombotsitov bol'nykh s razlichnymi klinicheskimi proiavleniiami shizofaffektivnogo psikhoza.

45 women with different manifestations of schizoaffective psychosis (SAP) were examined. The diagnosis corresponded to ICD-10 (F25). According to the classification elaborated in Mental Health Research Centre of Russian Academy of Medical Sciences, groups of patients were identified with different variants of the psychoses course: a nuclear SAP type; a borderline SAP variation with phasic-recurrent course; SAP with progredient variation (schizoaffective variation of schizophrenia). The patients were examined both during the attack and remission. A rate of serotonine uptake (Vmax) in blood platelets, a specific imipramine binding (Bmax) and the level of serotonin in blood platelets were evaluated. It was found that dynamics of both Vmax and the level of serotonin in different SAP types were different, that was related to clinical and biological SAP heterogeneity. A tendency to decreasing of serotonin system functional activity was found in progredient SAP variations, especially during the remission, which was of low quality in these cases. On the contrary, in the borderline variations the indices of the decreased function of serotonin system corresponded well to those of acute psychosis. In nuclear type--a type with the most favourable course of psychosis--any significant changes weren't revealed as compared with the normal parameters.

[Analyzing correlation between neurophysiological parameters and level of stress hormone cortisol in aging]. / Analiz korreliatsii mezhdu neirofiziologicheskimi pokazateliami i urovnem gormona stressa kortizola pri starenii.

A correlation between the serum levels of the stress hormone cortisole, neurophysiological parameters, and age was examined in 23 elderly healthy examinees (whose mean age was 65.7 +/- 2.1 years). The latency of the components of visual evoked potentials (VEP) was found to progressively increase with age. The individuals having high cortisole levels showed an increase in the latency of late VEP components associated with accelerated ageing and/or with the degenerative changes in the brain limbicoreticulocortical pathways. The DC potential level (DCPL) reflecting the intensity of brain energy expenditures correlated with the content of cortisole. Increased DCPL and longer latency of late VEP components were interrelated. It is suggested that high cortisole level produces a neurotoxic action and promotes accelerated ageing of brain regions having glucocorticoid receptors. Imbalance between the significant intensification of catabolic processes, the rise of energy expenditures and the limited increase in anabolism in elderly age is a course of this process.

[The indices of the thrombocyte serotonin system and of the nerve growth factor system in children with hereditary disorders of neuropsychic development]. / Pokazateli serotoninovoi sistemy trombotsitov i sistemy faktora rosta nervov u detei s nasledstvennymi narusheniiami nervno-psikhicheskogo razvitiia.

Indices of both platelet serotoninergic system and the system of nerve growth factor (NGF) were examined in children with neurofibromatosis (15 patients), polygenic oligophrenia (24 patients) and Rett's syndrome (14 ones). There was an increase of both the level of blood serum autoantibodies (aAB) to NGF and the value of specific binding of 3H-imipramine (Bmax) in platelets of patients with oligophrenia. For this group of patients a significant negative correlation exists between the rate of platelet uptake of serotonin (Vmax value) and the degree of mental retardation (r = -0.425, p < 0.03). Decrease of both Vmax and activity of platelet NGF receptors was revealed in patients with neurofibromatosis. In such patients there was positive correlation between sensitivity of platelet NGF receptors to NGF (during their stimulation by test dose of purified NGF) and the degree of mental retardation (r = 0.697, p < 0.04). In patients with Rett's syndrome a significant increase of activity of platelet NGF receptors to NGF was observed. The conclusion was made on the existence of some general mechanism of intellectual defect development. Autoimmune processes considered to be such mechanism.

[A clinico-biological study of the thrombocyte serotonin-transport complex in therapeutically resistant patients with endogenous depression undergoing combined treatment]. / Kliniko-biologicheskoe issledovanie serotonintransportnogo kompleksa trombotsitov u terapevticheski rezistentnykh bol'nykh éndogennoi depressiei pri kompleksnom lechenii.

The platelet parameters of the functional status of the serotonin-reuptake complex were studied in drug-resistant depressive patients given various regimens of complex therapy or selective serotonin re-uptake inhibitors (fluvoxamine and fluoxetine). The most persistent and marked clinical effect was seen in electroconvulsive therapy and it was due to the normalization of the serotonin-reuptake system.

[The thrombocytic serotoninergic markers of the efficacy of electroconvulsive therapy in endogenous depressions]. / Trombotsitarnye serotoninergicheskie markery éffektivnosti élektrosudorozhnoi terapii éndogennykh depressii.

The changes of main platelet serotonergic parameters (maximal velocity of 3H-serotonin uptake, Vmax; and density of (3)3H-imipramine binding sites, Bmax) were followed up in 25 endogenously depressed patients before and after electroconvulsive therapy (ECT). It was found a marked decrease in the velocity of 3H-serotonin uptake into platelets (Vmax) from patients before ECT (especially in non-responder group) and the significant increase of Vmax- and Bmax-values after the course of ECT in responders but not in resistant patients (non-responders). The increase of Vmax- and Bmax-values in responders was evidence of the rise of the functional activity serotonin system after treatment. The results evidence about the significance of serotonergic-mechanisms in the therapeutic efficacy of ECT.

[Thrombocytic serotoninergic markers in therapy-resistant patients with endogenous depression undergoing alpha-tocopherol treatment]. / Trombotsitarnye serotoninergicheskie markery u rezistentnykh k terapii bol'nykh éndogennymi depressiiami pri lechenii al'fa-tokoferolom.

The main platelet serotoninergic parameters (maximal velosity of 3H-serotonin uptake, Vmax and density of 3H-imipramine binding sites, Bmax) were measured in 17 therapy-resistant endogenously depressed patients before and after combined treatment (antidepressants and alpha-tocopherol). It was found a significant decrease of Vmax-, Vmax/Bmax- values and the increase of Bmax- values in patients before treatment. A beneficial effect of the combined therapy was found in 47% of patients. However, it was not followed by the changes of main platelet serotoninergic parameters in patients after treatment in spite of the obtained clinical improvement. There were not also observed the significant differences of measured biochemical parameters between responders and non-responders. These findings suggest that the beneficial clinical effect of the combined antidepressive therapy with alpha-tocopherol does not involve serotoninergic mechanisms.