Testing for the ability to modify antibiotics of Panus tigrinus 8/18 Lentinus strigosus 1566 laccase

Abstract In advanced biotechnology, the utilization of enzymes to achieve new or modified compounds with antibacterial, fungicidal, and anti-cancer specifications is crucial. Mushroom lactases are a hopeful biocatalyst for the synthesis and modification of different compounds. They are an accessible and inexpensive enzyme for the preparation of reaction objects and have recently received attention. Laccase purification was performed from basidiomycete Lentinus strigosus (LS) in several stages: Stage 1. On ion-exchange chromatography on TEAE Servacell 23 (400 ml), two distinctly separated laccase activity peaks were observed, eluted from the carrier at 0.21 and 0.27 M NaCl. In order to reduce the loss of enzymes, all fractions with laccase activity were collected, concentrated, and desalted using an ultrafiltration cell (Amicon, United States) with a UM-10 membrane. Stage 2. The resulting preparation with laccase activity was applied to a Q-Sepharose column (60 ml). Two well-separated peaks with laccase activity were obtained during the elution: laccase I (0.12 M NaCl) and laccase II (0.2 M NaCl). Stage 3. In the course of further purification of both enzymes, carried out on anion-exchange carrier Resource Q (6 ml), a broken gradient was used: 0 - 10%, 10 - 20%, and 20 - 100% with 1M NaCl. Stage 4. Both laccase I and laccase II, obtained after Resource Q, were desalted, concentrated to 1 ml each, and applied to a Superdex 75 gel filtration column. As a result, two laccases were obtained in a homogeneous form.

Resumo Na biotecnologia moderna, o uso de enzimas para obter compostos novos ou modificados com propriedades antibacterianas, antifúngicas e anticancerígenas é crucial. Lactases de cogumelos são biocatalisadores promissores para síntese e modificação de diferentes compostos, por serem enzimas baratas e disponíveis para a preparação de componentes de reação, e vem recebendo a devida atenção recentemente. A purificação da lacase foi realizada a partir do basidiomiceto Lentinus strigosus em vários estágios: Etapa 1 - na cromatografia de troca iônica em TEAE Servacell 23 (400 ml), foram observados dois picos de atividade da lacase distintamente separados, com eluição do transportador a 0,21 e 0,27 M de NaCl. Para reduzir a perda de enzimas, todas as frações com atividade de lacase foram coletadas, concentradas e dessalinizadas em uma célula de ultrafiltração (Amicon, Estados Unidos) com membrana UM-10; Etapa 2 - a preparação resultante com atividade de lacase foi aplicada a uma coluna Q-Sepharose (60 ml). Durante a eluição, foram obtidos dois picos bem separados com atividade de lacase: lacase I (NaCl 0,12 M) e lacase II (NaCl 0,2 M); Etapa 3 - no decurso da purificação adicional de ambas as enzimas, realizada no Recurso Q de transportador de troca aniônica (6 ml), um gradiente quebrado foi usado: 0-10%, 10-20% e 20-100% com NaCl 1M; Etapa 4 - tanto a lacase I como a lacase II, obtidas após o Recurso Q, foram dessalinizadas e concentradas para 1 ml cada e aplicadas a uma coluna de filtração em gel Superdex 75. Como resultado, duas lacases foram obtidas de forma homogênea.

Testing for the ability to modify antibiotics of Panus tigrinus 8/18 Lentinus strigosus 1566 laccase / Teste de capacidade para modificar antibióticos a partir da lacase de Panus tigrinus 8/18 e Lentinus strigosus 1566

In advanced biotechnology, the utilization of enzymes to achieve new or modified compounds with antibacterial, fungicidal, and anti-cancer specifications is crucial. Mushroom lactases are a hopeful biocatalyst for the synthesis and modification of different compounds. They are an accessible and inexpensive enzyme for the preparation of reaction objects and have recently received attention. Laccase purification was performed from basidiomycete Lentinus strigosus (LS) in several stages: Stage 1. On ion-exchange chromatography on TEAE Servacell 23 (400 ml), two distinctly separated laccase activity peaks were observed, eluted from the carrier at 0.21 and 0.27 M NaCl. In order to reduce the loss of enzymes, all fractions with laccase activity were collected, concentrated, and desalted using an ultrafiltration cell (Amicon, United States) with a UM-10 membrane. Stage 2. The resulting preparation with laccase activity was applied to a Q-Sepharose column (60 ml). Two well-separated peaks with laccase activity were obtained during the elution: laccase I (0.12 M NaCl) and laccase II (0.2 M NaCl). Stage 3. In the course of further purification of both enzymes, carried out on anion-exchange carrier Resource Q (6 ml), a broken gradient was used: 0 - 10%, 10 - 20%, and 20 - 100% with 1M NaCl. Stage 4. Both laccase I and laccase II, obtained after Resource Q, were desalted, concentrated to 1 ml each, and applied to a Superdex 75 gel filtration column. As a result, two laccases were obtained in a homogeneous form.

Na biotecnologia moderna, o uso de enzimas para obter compostos novos ou modificados com propriedades antibacterianas, antifúngicas e anticancerígenas é crucial. Lactases de cogumelos são biocatalisadores promissores para síntese e modificação de diferentes compostos, por serem enzimas baratas e disponíveis para a preparação de componentes de reação, e vem recebendo a devida atenção recentemente. A purificação da lacase foi realizada a partir do basidiomiceto Lentinus strigosus em vários estágios: Etapa 1 - na cromatografia de troca iônica em TEAE Servacell 23 (400 ml), foram observados dois picos de atividade da lacase distintamente separados, com eluição do transportador a 0,21 e 0,27 M de NaCl. Para reduzir a perda de enzimas, todas as frações com atividade de lacase foram coletadas, concentradas e dessalinizadas em uma célula de ultrafiltração (Amicon, Estados Unidos) com membrana UM-10; Etapa 2 - a preparação resultante com atividade de lacase foi aplicada a uma coluna Q-Sepharose (60 ml). Durante a eluição, foram obtidos dois picos bem separados com atividade de lacase: lacase I (NaCl 0,12 M) e lacase II (NaCl 0,2 M); Etapa 3 - no decurso da purificação adicional de ambas as enzimas, realizada no Recurso Q de transportador de troca aniônica (6 ml), um gradiente quebrado foi usado: 0-10%, 10-20% e 20-100% com NaCl 1M; Etapa 4 - tanto a lacase I como a lacase II, obtidas após o Recurso Q, foram dessalinizadas e concentradas para 1 ml cada e aplicadas a uma coluna de filtração em gel Superdex 75. Como resultado, duas lacases foram obtidas de forma homogênea.

[APPswe/PS1dE9/Blg Transgenic Mouse Line for Modeling Cerebral Amyloid Angiopathy Associated with Alzheimer's Disease].

Alzheimer's disease (AD) is the most common proteinopathy, which is accompanied by a steady decrease in the patient's cognitive functions with a simultaneous accumulation of amyloid plaques in brain tissues. Amyloid plaques are extracellular aggregates of amyloid ß (Aß) and are associated with neuroinflammation and neurodegeneration. Unlike humans and all other mammals, rats and mice do not reproduce AD-like pathology because there are three amino acid substitutions in their Aß. Amyloid plaques form in the brains of transgenic mice with overexpression of human Aß, and such mice are therefore possible to use in biomedicine to model the key features of AD. The transgenic mouse line APPswe/PS1dE9 is widely used as an animal model to study the molecular mechanisms of AD. A study was made to characterize the APPswe/PS1dE9/Blg subline, which was obtained by crossing APPswe/PS1dE9 mice on a CH3 genetic background with C57Bl6/Chg mice. No difference in offspring's survival and fertility was observed in the subline compared to wild-type control mice. Histological analysis of the brain in the APPswe/PS1dE9/Blg line confirmed the main neuromorphological features of AD and showed that amyloid plaques progressively increase in number and size during aging. The APPswe/PS1dE9/Blg line was assumed to provide a convenient model for developing therapeutic strategies to slow down AD progression.

Testing for the ability to modify antibiotics of Panus tigrinus 8/18 Lentinus strigosus 1566 laccase.

In advanced biotechnology, the utilization of enzymes to achieve new or modified compounds with antibacterial, fungicidal, and anti-cancer specifications is crucial. Mushroom lactases are a hopeful biocatalyst for the synthesis and modification of different compounds. They are an accessible and inexpensive enzyme for the preparation of reaction objects and have recently received attention. Laccase purification was performed from basidiomycete Lentinus strigosus (LS) in several stages: Stage 1. On ion-exchange chromatography on TEAE Servacell 23 (400 ml), two distinctly separated laccase activity peaks were observed, eluted from the carrier at 0.21 and 0.27 M NaCl. In order to reduce the loss of enzymes, all fractions with laccase activity were collected, concentrated, and desalted using an ultrafiltration cell (Amicon, United States) with a UM-10 membrane. Stage 2. The resulting preparation with laccase activity was applied to a Q-Sepharose column (60 ml). Two well-separated peaks with laccase activity were obtained during the elution: laccase I (0.12 M NaCl) and laccase II (0.2 M NaCl). Stage 3. In the course of further purification of both enzymes, carried out on anion-exchange carrier Resource Q (6 ml), a broken gradient was used: 0 - 10%, 10 - 20%, and 20 - 100% with 1M NaCl. Stage 4. Both laccase I and laccase II, obtained after Resource Q, were desalted, concentrated to 1 ml each, and applied to a Superdex 75 gel filtration column. As a result, two laccases were obtained in a homogeneous form.

[The ultrastructural changes of Sarcocystis ovifelis infested sheep tongue myofibrils].

Structural changes were observed in filaments of Sarcocystis ovifelis infected sheep tongue myofibrils. In sarcocysts containing myofibrils, actin filaments and Z-disks, myosin filaments and M-line were seen destroyed. Protein bridges, uniting actin and myosin filaments into a joint complex (net), eventually become not visible, and as a result separate Z-disks and free filaments appear. Fibrils, referred to as leptomeric, have been first revealed between protrusions of the sarcocyst surface apparatus. These are striated filaments with periodic 100 nm striation of dark and light bands, made of thin and short 120-200 nm long filaments 5 nm in diameter. The genesis of leptomeric fibrils still remains obscure. In sarcocysts infected myofibrils these may be involved in metabolite transportation to the intercellular space and back.

[The structural and functional analysis of the surface apparatus in four species of Sarcocystis (Sporozoa, Apicomplexa)].

The comparable ultrastructural analysis of the sarcocyst surface apparatus (SSA) was made for four species of Sarcocystis: Sarcocystis muris, S. fusiformis, S. medusiformis, and Sarcocystis sp. from buffalo heart muscles. In all these species, SSA contains a surface membrane, overmembrane complex with glycocalyx, and submembrane complex made of two glycoprotein SSA primembrane layers. SSA makes numerous primary vesicle-like protrusions and pits in between. Some vesicles containing two layers, PM1 and PM2, are pinching off from the totally formed protrusions. Then these vesicles are directed into infected host cell to participate in its degradation. In the SSA pits neither over-, nor submembrane complex is present, the pits being made of the surface membrane only. It is important that fibrillar structures penetrate through the SSA membrane into pits from the host cell. Besides, SSA forms secondary protrusions with different structures in various species of Sarcocystis. They increase the sarcocyst surface and transport different substances along intermediate filaments from the SSA pits membrane to the sarcocyst body. At the same time, deep invaginations are found in the SSA of old sarcocysts. We thought that these structures increased the sarcocyst surface and thus promote to intensify metabolism. This study-defined presence of membranous vesicles in secondary protrusions. According to their structure and localization, the membranous vesicles may be involved in the building of the sarcocyst surface membrane.

[Structural and functional characterization of cyst cells in Sarcocystis sp. (Sporozoa, Apicomplexa)]. / Osobennosti stroeniia i funktsional'nye kharakteristiki kletok v tsistakh sarkosporidii.

By means of light and electron microscopy, the structural pattern of muscle cysts (sarcocysts) was examined for the four species of the genus Sarcocystis: S. muris (from murine skeletal muscles), Sarcocystis sp. and S. fusiformis (from, respectively, heart and skeletal muscles of buffalo), and S. ovifelis (from ovine tong muscles). The orderly fashion of the interior of the cyst is attained by partitition of its space into numerous compartments with the involvement of the intermediate filaments. These, in their turn, are bound to each other by thin filaments to make eventually a common filamentous net. The net limits separate groups of cells referred to as cyst zoites. The common net of filaments and microtubules (when present) may be regarded not only as the organizer of the cyst interior cytoskeleton, but also as the main mechanism of substance transportation in various directions: from the host cell to the sarcocyst, and within or outside the cyst. The role of dedifferentiation, proliferation and differentiation processes is suggested in the establishment of the fixed sequence of events throughout the unidirectional development of cyst cells and their interaction, from precystic meronts to cyst merozoites (gamonts). Special attention is paid to metrocyte morphogenesis and functioning. In the present work, metrocytes subjected to apoptosis were recognized. It is suggested that phenomenon of programmed cell death in metrocytes may be associated with the control of cell number in mature and ageing sarcocysts.

[Formation and diversity of parasitophorous vacuoles in parasitic protozoa. The Coccidia (Sporozoa, Apicomplexa)]. / Puti formirovaniia parazitofornykh vakuolei i ikh raznoobrazie u paraziticheskikh prosteishikh. Koktsidii (Sporozoa, Apicomplexa).

Data on parasitophorous vacuole (PV) formation in host cells (HC) harbouring different intracellular protozoan parasites have been reviewed and critically analysed, with special reference to the main representatives of the Coccidia. The vacuole membrane (PVM) is the interface between host and parasite, playing a role in nutrient acquisition by the parasite from the HC. The PV phenomenon is regarded as a generalized HC response to the introduction of alien bodies (microorganisms), which eventually reflects the evolutionary established host-parasite relationships at cellular, subcellular and molecular levels. Special attention has been paid to the existing morpho-functional diversity of the PVs within the same genera and species of parasites, and even at different stages of the parasite life cycle. The PVM is generally considered to derive from the HC plasmalemma, whose biochemical composition undergoes significant changes as the intravacuolar parasite grows. The original HC proteins are selectively excluded from the PVM, while those of the parasite are incorporated. As the result, the changed PVM becomes not fusigenic for HC lysosomes. For Toxoplasma gondii and other cyst-forming coccidia (Isospora, Sarcocystis), a definite correlation has been noticed between the extent of rhoptry and dense granule secrets released by a zoite during HC internalization, on the one hand, and the pattern of the PV that forms, on the other one. In T. gondii, tachyzoites, known to discharge abundant secrets, commonly force the development of PVs limited with a single unit membrane and equipped with a tubulovesicular network in the lumen. Unlike, bradyzoites known to be deficient in secretory materials trigger the formation of PVs with a three-membrane lining composed of the changed invaginated plasmalemma in addition to two membranes of endoplasmic reticulum. The two different types of PV harbour, respectively, exoenteric and enteric stages of T. gondii, the latter being confined to the cat intestine only. Unlike, all endogenous stages of the classic intestinal coccidia (Eimeria spp.) develop within PVs limited with a single membrane, with some invaginations extending into the PV lumen. Unusual PV patterns are characteristic of the extracytoplasmic eimerian coccidia (Cryptosporidium, Epieimeria) and adeleid haemogreagarines (Karyolysus). In cyst-forming coccidia, the PVM is actively involved in tissue cyst wall formation, thus protecting the encysted parasites from recognition by the host immune system. All this strongly suggests that the PV is far from being an indifferent membraneous vesicle containing a parasite, but represents a metabolically active compartment in infected cells. Since all the coccidia are obligate intracellular parasites, the mode of their intimate interaction with the HC, largely accomplished via the PV and its membrane, is vital for their survival as biological species.

[The role of the sarcocyst surface apparatus in utilizing the host cell muscle structures]. / Rol' poverkhnostnogo apparata myshechnykh tsist sarkosporidii v protsesse utilizatsii struktur myshechnoi kletki khoziaina.

The participation of the sarcocyst surface apparatus (SSA) of two sarcosporidian species, Sarcocystis muris and S. ovifelis (Coccidia, Sporozoa, Apicomplexa), in degradation of disrupted host cell substances was investigated. After degradation, these substances are transported through the membrane of the SSA to the sarcocyst ground substance (GS), but this process cannot be regarded as endocytosis. At first, the transported substances were found in SSA pits in the form of fibrillar structures. Later on, these were seen as twisted up granules. In some cases, such granules restore their fibrillar shape, penetrate through the SSA membrane and appear in the sarcocyst GS. In other cases, the small granules may be released from SSA pits directly to the sarcocyst GS. Besides, two SSA primembrane layers were seen to disappear during the transportation of host cell substances. In addition, multimembrane structures (membranous whorls) were first demonstrated between the plasmalemma and inner membrane complex of the zoite pellicle. Multimembrane structures were found, in addition, in the zoite cytoplasm in connection with micronemes. These structures resembling chloroplast granae of thylakoids may presumably fill the gap in membrane pool of the SSA contributing to its renewal.

[The structure of the surface apparatus in sarcocysts of Sarcosporidia in the persistence process]. / Strukturnaia organizatsiia poverkhnostnogo apparata sarkosporidii v protsesse persistirovaniia.

The structure of the sarcocyst surface apparatus (SSA) was investigated for two sarcosporidian species: Sarcocystis muris (non-pathogenic) and S. fusiformis (pathogenic). The surface membrane, being the main SSA subsystem, makes numerous vesicle-like protrusions with different ultrastructural patterns. This made it possible to distinguish between four and three types of these protrusions in S. fusiformis and S. muris, respectively. Vesicles of similar structure, pinched off from the fully formed protrusions, were classified, correspondingly, in the same four and three different types. A presumable functional role of both protrusions and membrane-coated vesicles in pathogenicity of different sarcosporidian species is proposed. The vesicles pinched off from corresponding protrusions may be involved in transporting certain substance complexes from the sarcocyst to the harbouring host cell. In addition, another way of substance transporting was observed, when the cystic substances, not surrounded with any membrane coating, are thrown from open protrusions directly into the immediate cytoplasm of the host cell.

[Effect of developing Sarcocystis muris tissue cysts on ultrastructural change of murine muscle fibers]. / Vliianie razvivaiushchikhsia tkanevykh tsist Sarcocystis muris na izmenenie ul'trastruktury myshechnogo volokna myshi.

A study was made of the influence exerted by developing sarcocysts of Sarcocystis muris on the ultrastructural organization of muscle fibres, both harbouring the sarcocysts (HSM) and sarcocyst-free (SFM), from skeletal muscles of experimentally infected mice. Muscle fibres of non-infected mice of the same age served as a control. Mice were sacrificed 6 months following feeding S. muris oocysts (or sporocysts). The developing sarcocysts seriously destroyed HSM: their myofilaments were no hold in register, cross-bridges almost entirely disappeared, M-lines and Z-disks looked as broken structures. The majority of actin myofilaments were arranged along myosin myofilaments as discrete units. The host cell sarcoplasm was packed with numerous vacuoles of different form and size. Compared to muscle fibres in the control, SFM of infected mice also displayed an obvious ultrastructural alteration. On the periphery of SFM, some destroyed sarcomeres with swollen myofilaments were noticed whose cross-bridges were totally lacking. In other extreme areas myosin and actin myofilaments were disintegrated into thin straightened filaments 2.0-2.5 nm in diameter. It is supposed that HSM and SFM of the infected mice may experience different kinds of influence on the part of the developing intracellular parasite (sarcocyst). And it dos not seem unlikely that various biologically active substances, produced by the parasite, may be vesicle transported to SFN through the endomysium space.

[Intracellular parasitism and sarcocystosis]. / Vnutrikletochnyi parazitizm i problema sarkotsistoza.

The obligatory heterogenous tissue cyst-forming coccidia of the genus Sarcystosis are regarded as an excellent example of specific coexistence of two organisms, i.e., the host and parasite. These parasitic protozoans are known as causative agents of the chronic, often life-threatening disease, sarcocystosis, which still cannot be effectively controlled. In Sarcocystis, the entire phase of asexual multiplication was transferred to the intermediate host. Of special interest is the parasite's ability to persist in this host at the stage of tissue cyst or sarcocyst. This is a giant meront, in which unidirectional development proceeds starting from a little differentiated metrocyte, through intermediate cells, and towards highly differentiated cyst merozoites (gamonts) unable to further divide. The life span of the sarcocyst depends, to a great extent, on self-regulation within the cyst itself and on relations between the cyst and its immediate environment. A totally new field of research into Sarcocystis was initiated by the discovery that the intracellular parasite damages both cyst harboring and intact muscle cells, apart from the adjacent connective and nervous tissue. The previously unknown cytopathological effects of sarcocysts have been described and characterized. The changes observed within and outside the sarcocysts have been analyzed in terms of general biological processes: proliferation, differentiation, and programmed cell death.

[Current views on the life cycle of cyst-forming Coccidia (Eucoccidia, Sporozoa, Apicomplexa)]. / Sovremennoe predstavlenie o zhiznennykh tsiklakh tsistoobrazuiushchikh koktsidii (Eucoccidia, Sporozoa, Apicomplexa).

The life cycles of cyst-forming coccidia of the genera Sarcocystis and Toxoplasma have been first analysed in terms of generally recognized biological phenomena, such as proliferation, differentiation, dedifferentiation, programmed cell death. The differences, existing between the respective obligatory heteroxenous (Sarcocystis) and facultatively heteroxenous (Toxoplasma) life cycles, have been found to involve the obvious differences in the degree of a zoite's differentiation, occurring in the course of asexual development in the intermediate host. In Sarcocystis spp., the degree of differentiation in merozoites, throughout their development, is much higher than in those of T. gondii. This level of merozoite differentiation in Sarcocystis is thought to determine the irreversible, one-directional way of both pre-cystic and cystic development of the pathogen, starting from the sporozoite stage and terminating in gamont formation within the tissue cyst. Unlike, in T. gondii, the level of merozoite differentiation is not so strong as in Sarcocystis spp., which may account for the reversible merozoite development in the former, which is clearly demonstrated by a ready conversion of pre-cystic tachyzoites into cystic bradyzoites, and the other way round. In the course of endogenous development, the pathogens adversely affect their environment (the infected cells and tissues), which, in its turn, may exert its influence on the particular parasites. Thus, both the parasite and the host represent a unique feed-bach regulatory system.

[One of the possible ways of propagation of the unicellular parasites Sarcocystis muris in the musculature of the intermediate host]. / Odin iz vozmozhnykh putei rasprostraneniia odnokletochnykh parazitov Sarcocystis muris v muskulature promezhutochnogo khozaina.

20 laboratory mice (Mus musculus) were fed each a single dose of 20,000 Sarcocystis muris sporocysts to be then sacrificed 1, 2.5, 4, 6 and 10 months following infection (p.i.). A visual infection of the murine corps demonstrated that the number of sarcocysts per animal increased regularly as the time of infection was progressing, being eventually higher after 6 and 10 months p.i. than within 1-4 months p.i. This phenomenon was poorly understood from the knowledge that the tissue cysts (sarcocysts) are able to increase in size, rather than in number, and that the original number of sarcocysts largely depends on the number of precystic merozoites available. In our experiments, each of 20 mice was fed an equal number of sporocysts, and thus the number of precystic merozoites ought to be expected also more or less equal. EM investigation of murine skeletal muscles 6 and 10 months p.i. revealed, along with numerous normal sarcocysts, the presence of some separate, individual zoites, both within and outside the muscle fibre, in the endomysium. Besides, a colony of zoites, living freely without any visible common wall, was detected within a muscle fibre adjacent to another one, containing a sarcocyst of normal structure. These zoites may have originated from one or more sarcocysts, whose cyst walls were spontaneously broken, time after another, and thus led the cyst cells go out. These discharged zoites could either perish, being enzymatically degraded, or penetrate the neighbouring muscle fibres to proceed their further development. The colony making zoites were confined to two cell types only: the intermediate cells and the merozoites (gamonts). No metrocytes were recognized due, presumably, to inability of these little differentiated cells, devoid of penetrative organelles, to invade the host muscle cell. The colony of zoites turned out to be a developing population of live cells, able to destroy progressively the harbouring muscle fibre, except its basal membrane and sarcolemma. It does not seem unlikely that the outer coverings of the infected cell could be transformed eventually into a cyst wall to make, thus, a new sarcocyst. The above phenomena have never been found in murine muscles earlier than 6 months p.i. Although these facts are few and far between, they may prompt a possible mechanism of sarcocyst increase in number, in the intermediate host with age, even without any additional sporocyst contamination.

[The localization and functional significance of arylsulfatases in parasitic sarcosporidian protozoa in the tissue-cyst life cycle phase]. / Lokalizatsiia i funktsional'noe znachenie arilsul'fataz u paraziticheskikh prosteishikh sarkosporidii v faze tkanevoi tsisty zhizhnennogo tsikla.

The hydrolytic enzymes arylsulphatases (AS) were detected in developing tissue cysts of Sarcocystis ovifelis, using two methods: the Goldfischer lead technique, with two different pH values-5.5 (AS-A) and 4.2 (AS-B), and the Hopsu-Havu barium technique (Gayer, 1974). The enzymatic activity was identified by the presence of an electron dense finely granulated precipitation. In cyst cells, lead sulphate precipitation was spotted only in the inner membrane complex (IC) of the pellicle, whereas barium sulphate marked, in addition, the plasma membrane. Besides, AS activity was detected in the endoplasmic reticulum, Golgi complex, lysosomes and micronemes of cyst cells. Of interest is the finding of AS in the outer membrane of IC and matrix of pellicular evaginations. In the cyst ground substance (CGS) of S. ovifelis AS activity is confined to the membrane and matrix of transport vesicles, originating from cyst cell pellicle evaginations. These cystic vesicles carry enzymes from the places of their synthesis, in the cyst cells, to the tissue cyst periphery near the cyst wall. In the CGS, the obvious precipitations of lead and barium sulphate, respectively, are seen around some cyst cells being in the state of destroying due to natural death, and around so-called apoptotic-like bodies made from the destroyed cells. AS activity is seen both in the cyst wall and in vesicles separating from the wall ("wall vesicles") that find eventually their way in the cytoplasm of infected muscle cells, the granulation being observed around destroyed organelles of such cells. The investigated dynamics of AS movement, by means of the transport cystic and wall vesicles, extends general knowledge of the distant metabolic interaction between cells of the host and the parasite in tissue cysts of Sarcocystis spp.

[The effect of coccidia of the genus Sarcocystis on the morphofunctional organization of the skeletal musculature in experimentally infected mice. I. The muscle fiber]. / Vliianie koktsidii roda Sarcocystis na morfofunktsional'nuiu organizatsiiu skeletnoi muskulatury éksperimental'no zarazhennykh myshei. I. Myshechnoe volokno.

At the ultrastructural level, the cellular response of skeletal muscles on developing Sarcocystis muris sarcocysts has been followed in mice at different times after sporocyst feeding, i.e. in 1, 2.5, 6 and 10 months, resp. The developing cyst creates a progressive degeneration of the infected muscle cell that involves organelle disorganization and formation of numerous vacuoles in the cytoplasm as a consequence of cell edema. Products of the host cell degradation, shaped as fibrillar-granular structures, are seen to find their way to the cyst wall outgrowings, where they become denser and on being covered with membranes appear eventually in the sarcocyst ground substance. Later on, the membranes around the granules disappear. In the course of its development, the sarcocyst totally destroys not only the harbouring muscle cell and the nearest connective tissue elements of the endomysium, but also the previously intact neighbouring cells. The involvement of some proteolytic enzymes in this process is suggested.

[The effect of coccidia of the genus Sarcocystis on the morphofunctional organization of the skeletal musculature in experimentally infected mice. II. The connective tissue elements of the endomysium]. / Vliianie koktsidii roda Sarcocystis na morfofunktsional'nuiu organizatsiiu skeletnoi muskulatury éksperimental'no zarazhennykh myshei. II. Soedinitel'notkannye élementy éndomiziia.

As an extension of the previous communication (Radchenko, et al., 1996), a study was made of the response of connective tissue elements, surrounding the Sarcocystis muris infected muscle fibers. As earlier, the S. muris sarcocysts were examined in mice 1, 2.5, 6, and 10 months after sporocyst feeding. Within the first 2.5 months after infection, marked accumulations of lymphocyte-like cells and collagen fibres are observed in the endomysium, and simultaneously the activity of capillary endothelial cells is seen to enhance due to the appearance of much more micropinocytotic vesicles, compared to the uninfected control. All this may be qualified as the host organism protective reaction to the parasitic infection. 6 and 10 months after infection, not only collagen fibres, but also some other fibrillar structures of the endomysium undergo degradation, the damage of capillary endothelial cells starting from breaking the outer membrane (in 6 months) and terminating in lysing the whole cell (in 10 months). Besides, structural abnormalities were noticed in the axon endings.

[An electron microscopic study of the natural death of cyst cells in Sarcosporidia. I. Ultrastructural changes in the cytoplasm of the cyst cells of Sarcocystis muris and S. bovifelis]. / Elektronno-mikroskopicheskoe issledovanie estestvennoi gibeli tsistnykh kletok u sarkosporidii. I. Izmenenie ul'trastruktury tsitoplazmy tsistnykh kletok Sarcocystis muris i S. bovifelis.

A complicated development of Sarcocystis muris and S.bovifelis, within tissue cysts (sarcocysts), was examined in terms of a phenomenon of programmed cell death well-known in Metazoa. It looks likely that this phenomenon of versatile significance in living organisms has not yet been followed in parasitic protozoa. This is the first attempt to find out a general picture of morphological changes, occurring in the course of natural death in parasitic protozoa. With electron microscope, a study was made of sarcocysts of S. bovifelis isolated from oesophagus muscles of a naturally infected cow. In 6-month old sarcocysts of S. muris, three morphofunctional cell types are commonly distinguished: little differentiated metrocytes, intermediate cells, and highly differentiated <>, homologous to gamonts of other Sporozoa. Among numerous cyst cells, looking overtly normal, some <> cells tend to be encountered, which increase in number in 10 month old cysts, i.e. as sarcocysts are getting older. At least three stages of morphological degradation are to be distinguished in the cyst cells, with special attention being paid to changes in their cytoplasm. The first stage degradation involves ultrastructural changes in cell organelles, primarily in rough endoplasmic reticulum, whose membranes form vacuoles, with various kinds of membranous and non-membranous materials inside. At the second stage, apical organelles of cyst cells are involved: rhoptries are seen to lose their contrast, suggesting that their proteinaceous content may be discharged into the cell cytoplasm; eventually, membranes of rhoptries disappear. Micronemes seem to break into composing channels of endoplasmic reticulum. Ultimately, in the cells committed to death, the main body of cell organelles looks indecipherable, and the whole cells become filled with numerous vacuoles of different sizes and configuration. At the third statge of morphologoical degradation, cell trans

[An electron microscopic study of the natural death of cyst cells in Sarcosporidia. II. Ultrastructural changes in the nuclei of cyst cells of Sarcocystis muris]. / Elektronno-mikroskopicheskoe issledovanie estestvennoi gibeli tsistnykh kletok u sarkosporidii. II. Izmenenie ul'trastruktury iader tsistnykh kletok Sarcocystis muris.

Nuclear changes in cyst cells, developing within 6 and 10 month old sarcocysts of Sarcocystis muris, were followed in terms of the programmed cell death phenomenon. This communication extends our previous studies in the cytoplasm of S. muris cyst cells (Radchenko et al., 1995) to include particular nuclear changes in different involve changes in nuclear configuration: the original spherical from is progressively substituted for irregular or lobulated shapes. This may suggest some corresponding changes in cytoskeleton, involved in formation and maintaining of some definite nuclear shape. In normal cyst cells, the nuclear chromatin appears as a filiform and reticulate structure with a few lumps made of granules and filaments. In the cyst stages subject to natural cell death, structural changes in nuclei involve disassembly of lumps into separate granules. Some spherical structures are seen outbudding from the nucleolus. These structures are presumably made of RNA-containing granules. The pattern of nucleolar segregation in S. muris cells resembles somewhat the changes in nucleoli reported for metazoan cells. However, the general picture of morphological evolution in the nuclei of S. muris cells, in the course of natural dying, differs from that in metazoan cell nuclei. No condensation of nuclear chromatin at the nuclear periphery, or blebbing of the nuclear and cytoplasmic membranes, so characteristic of the latter, was followed in the former. The peculiarities noticed in the sarcosporidia may reflect biological peculiarities of these specialized parasitic protozoa.

[The role of the endoplasmic reticulum in the formation of specialized cellular organelles in parasitic Sarcosporidia]. / Rol' éndoplazmaticheskoi seti v obrazovanii spetsializirovannykh kletochnykh organell u paraziticheskikh prosteishikh sarkosporidii.

A cytological study was made of participation of the endoplasmic reticulum in the formation of specific cell organelles in Sarcocystis muris zoites. The data presented in this paper confirm our earlier assumption (Radchenko, 1987) that the Golgi adjunct is a structure made of four membranes of the smooth endoplasmic reticulum (ER) channels. This organelle appeared to be available in all the three cell types recognized within the sarcocyst: metrocytes, intermediate cells and merozoites. In the dividing metrocytes and intermediate cells the Golgi adjunct is turning progressively in a polar ring, whose membranes are covered with a fibrillar material from which subpellicular microtubules are formed. It has been first shown that ER channels are involved in the formation of amylopectin granules and immediate glycoprotein regions around them. Large spiral structures, composed of the ER channels, were seen in the zoite cytoplasm. Besides, micronemes were also made of closely packed ER channels. It looks likely that individual micronemes may be connected to each other through these channels.