PRODUCTION OF SINGLE CHAIN FRAGMENT VARIABLE (scFv) ANTIBODY AGAINST PARANOSEMA LOCUSTAE ALPHA/BETA-HYDROLASE AND IMMUNOLOCALIZATION OF MICROSPORIDIAN ENZYME IN THE INFECTED HOST CELL.

Microsporidia is a widespread group of fungi-related intracellular parasites. Direct contact of the most microsporidia species with host cytoplasm suggests that these parasites may control physiological processes of infected cells by secretion of various proteins. In previous experiments, secretion of significant amounts of microsporidia Paranosema locustae alpha/beta-hydrolase into infected cells of Locusta migratoria fat bodies was demonstrated using polyclonal antibodies against the enzyme. However, heterologous expression of microsporidian hydrolase in yeast Pichia pastoris cells was not accompanied by its secretion. In this study, we have constructed library of recombinant single chain antibodies (scFv-fragments) against proteins of fat bodies of infected locusts and isolated mini-antibody specifically recognizing the studied enzyme using phage display technology. Immunoblotting and immunofluorescent microscopy with selected scFv-fragment confirmed secretion of two different in size forms of P. locustae alpha/beta-hydrolase into infected host cell. Prospects of scFv-fragment use to explore the role of microsporidian hydrolase in host-parasite relations and mechanism of its secretion are discussed in the paper.

INTERACTIONS OF MICROSPORIDIA WITH INFECTED HOST CELL.

Microsporidia comprise a group of fungi-related obligate intracellular eukaryotic pathogens with extremely wide host range: from protists to mammals. Adaptation to intracellular parasitism drives these parasites towards significant reduction and modification of the genome and functional apparatus, which causes extreme dependence on the host cell, as well as sophisticated host-parasite relationships. In this review we summarize our results and recent literature data about microsporidian interactions with the host at the cellular level. The impacts of these pathogens to infected cells include induction of hypertrophy, restructuring and modification of the cytoskeleton and the vesicular transport system of the host cells. Microsporidians also able to stimulate the metabolic processes in the infected cells and inhibit their defensive reactions. The main tool of the direct regulatory impact of microsporidia on the host cell apparently is the secretion of the special protein effectors capable to interfere to regulatory and signaling pathways of the host cell.

[Current Concepts of Human Microsporidiosis].

Microsporidiosis is an ubiquitous opportunistic disease that usually appears in immunocompromised patients: AIDS patients or organ-transplant recipients. The infectious agents of disease are fungi-related obligate intracellular parasites - microsporidia. Alongside with Cryptosporidium and Cytomegalovirus, these parasites are the most common causative agents of diarrhea in HIV-infected patients. Intestinalform of microsporidiosis has been mostfrequently observed, but microsporidia can affect almost any organs of the human body, eyes, lungs, muscles, organs of the nervous system. The present paper overviews the current data on the etiology, pathogenesis, epidemiology, clinical presentation, diagnosis and treatment methods of microsporidiosis.

[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.

[Urinary calcium excretion in a renal form of primary hyperparathyroidism]. / Otsenka mochevoi ékskretsii kal'tsiia pri pochechnoi forme pervichnogo giperparatireoidizma.

The renal calcium transport was studied in 31 patients with the renal form of initial hyperparathyroidism. The amount of calcium excreted from 100 ml-glomerular filtrate was lowered in patients with hyperparathyroidism comparatively to normal (0.11 +/- 0.005 mg% against 0.19 +/- 0.005 mg%). Determination of filtration calcium is proposed to be used as a diagnostic test in this pathology. In spite of the fact that the increased percentage of calcium tubular reabsorption detected simultaneously is statistically significant, this test seems to be less reliable diagnostic criterion because of minute quantitative differences (99.0 +/- 0.054%, the normal being 98.1 +/- 0.31%).