[Threshold rat neonatal cardiomyocyte response to gradual cryptosporidial infection severity increase].

Infectious gastroenteritis is one of the common causes of tachyarrythmia, malabsorbtion and growth retardation in children. Our recent studies have indicated that neonatal.cryptosporidial gastroenteritis is associated with long-term cardiomyocyte abnormalities. The aim of the present study was to find out how neonatal cryptosporidiosis of various severities affects cardiac anatomy and cardiomyocyte polyploidization, remodeling and HIF-1α expression. Using real-time PCR, cytometry, immunohistochemistry, image analysis and interatrial septum visual examination, we revealed that gradual increase in cryptosporidial invasion was associated with threshold changes. At weak parasitic infection, interatrial septum was entire and there was no statistically significant change in cardiomyocytes. At moderate and severe infection, all changes in cardiac anatomy and cardiomyocytes were statistically significant and demonstrated approximately similar degree. Compared to control, heart were atrophied and elongated, interatrial septum contained a small window (patentforamrn ovale), and cardiomyocytes lost protein, became elongated, thin and accumulated additional genomes. Also we found HIF-1α mRNA hyperexpression. Notable, the threshold response to gradual stimulus is an important criterion of development programming since such a response is commonly a consequence of abnormal anatomic structure formation and cell differentiation failure. Our results can be interesting for physicians because they indicate that even moderate cryptosporidiosis can be dangerous for neonatal heart and can trigger neonatal programming of cardiovascular pathology. Also, our results for the first time demonstrate the association between gastroenteritis, patent foramen ovale and cardiomyocyte malfunction.

[Changes in the heart of neonatal rats after cryptosporidial gastroenteritis of different degrees of severity].

Disturbances at the childhood age increase risk of the appearance of cardiovascular diseases decades later. The nature of this interconnection called ontogenetic programming is not completely understood. Valuable sources of knowledge about mechanisms of ontogenetic programming are data of interspecies study of biology of the body life cycles and of heart physiological capabilities. Taken into account the interspecies differences, these data allow finding the correct direction of experimental investigations. Results of studies of almost 100 homoiothermal species have shown the slow growth and a high loading on the heart at postnatal development to decrease its aerobic capability in adults. Basing on these data, we suggested that the neonatal gastroenteritis causing tachyarrhythmia, malabsorption, and the growth deceleration might lead to pathological changes in the heart. Our task was to evaluate the effect of cryptosporidial gastroenteritis of different degrees of severity on heart of the neonatal rats. By using methods of Real-Time PCR, immunocytochemistry, image analysis, and study of interatrial septum, we have established that a gradual increase of intensity of infestation with Cryptosporidium parvum oocytes causes sharp changes corresponding to the "all or nothing" response. At a weak infestation the interatrial septum was close (like in control), while significant changes in expression of isoforms of heavy chains of alpha- and beta-myosin were absent. At the intermediate and severe infestation, in the interatrial septum the foramen ovale was visualized and there were observed cardiac atrophy and a strong shift of ration of expression of myosin heavy chains toward the low-velocity beta chain. Thus, by disturbing the frequency-strength ratios and causing outflow of resources from the formed heart, the neonatal gastroenteritis produces pathological changes of the organ molecular and anatomical structures. Our results can be interest to evolutionary biologists and physicians, as they show importance of knowledge of evolutionary-comparative investigations for the search for novel risk factors of heart diseases and demonstrate interconnection between gastroenteritis, pathology of interatrial septum, and a change of composition of the main contractile proteins in cardiomyocytes.

[Rat cardiomyocyte remodeling after neonatal cryptosporidiosis. II. Elongation, excessive polyploidization and HIF-1alpha overexpression].

Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization.

[Cardiomyocyte myosin heavy chain composition change after cryptosporidial gastroenteritis].

Cardiovascular diseases are the most common case of human death in developed countries. Thus, the discovering of their new risk factors is of primary importance. Based on epidemiology studies, vertebrate life-history traits comparison and cross-species cardiomyocyte transcriptome analysis, we suggest that one of these factors could be infectious gastroenteritis. This disease outflows recourses from cardio-vascular system and triggers pathological stimuli, like tachyarrhythmia, inflammation, malapsorption and energy depletion thereby disturbing cardiomyocyte metabolism and function. To test this hypothesis, we challenged gastroenteritis in neonatal rats with widespread human parasite Cryptosporidium parvum (Apicomplexa, Sporozoa). The results obtained by the methods of immunocytochemistry, quantitative morphometry and real-time PCR, indicate that moderate cryptosporidiosis lasting four days induces dramatic shift in myosin isoform expression ration toward isoform beta (with low ATPase activity) both at mRNA (by 1.7-4.5 folds) and protein (by 2.5-6 folds) levels. Antithetical manner of this shift and coherence between changes in mRNA and protein suggest that cryptosporidiosis affects all main steps of a complex myosin heavy chain regulatory network. Since the overexpression of myosin heavy chain beta (showing several times lower ATPase activity than myosin heavy chain alfa) is a generally accepted marker of human cardiac failure, we can consider cryptosporidial gastroenteritis as a new risk factor of cardiac contractile ability impairment. Our data can be interesting for clinicians.

[The plate in the zone of oocyte and germinal epithelium contact in scyphomedusa Aurelia aurita binds antibodies to ZP-domain-containing protein mesoglein].

Cnidaria are lower multicellular animals with the body consisting of two epithelial layers. An extracellular substance--mesoglea--is situated between epidermal and gastrodermal layers of these animals. Mesoglein is one of the major mesogleal proteins of adult medusa of Scyphozoan jellyfish Aurelia aurita. Search for the known domains in mesoglein amino acid sequence reveals prominent zona pellucida (ZP) domain (which was found at first in the mammal oocyte zona pellucida proteins), so the protein belongs to ZP family of extracellular matrix proteins and it is an early metazoan member of ZP-domain-containing protein family. However, nothing is known about oogenesis related ZP-domain proteins in the lower multicellular animals. Oogenesis in Scyphozoa is described poorly. In this work morphological features of the zone in contact area between the oocyte and the germinal epithelium were investigated in semi-fine sections: To make it more convenient we identified seven stages according to the oocyte size and the structure found in this area was named the plate. It was shown that the components of the plate bound specifically the antibodies against mesoglein. So it seems the plate material contains ZP-domain proteins. Electrophoresis and immunoblot results give evidence that the proteins immunologically related to mesoglein have a higher molecular mass. It might be due to either the posttranslational modifications of the precursors or that they represent other proteins of ZP-domain family in Cnidaria.

[Heterochromatin and centromere structure paradox].

Centromere (CEN) is the structure responsible for the chromatid association, chromosome attachment to the spindle, and correct position in the plate. The only DNA found in the mammalian CEN belongs to the satellite DNA--high repeated tandem repeats. Mounting evidence indicates that both types of chromatin (CEN and peri-CEN) are required for proper centromere function. CEN, peri-CEN and peritelomeric regions remain white spots at the chromosome maps appeared after reading genomes of human, mouse, and rat. SatDNA is considered to be species-specific. Library hypothesis regards heterochromatin as the library of different satDNA one fragments of which became spread and fixed in species fixation. We have analyzed database Chromosome Unknown (ChrUn) and found several new classes of mouse tandem repeats. The features of these classes are similar with the ones from rat ChrUn, as well as their distributions according to GC-richness. We believe that similar fragments' structure, i. e. intermingling of fragments with different curvature rather than their primary sequence will help to solve the paradox, when CEN or peri-CEN fragments from different animals have nothing in common, but bind the same sets of proteins.

A novel Aurelia aurita protein mesoglein contains DSL and ZP domains.

Body of the scyphoid jellyfish Aurelia aurita consists of 2 epithelia -- epidermis and gastroderm. The layers are separated by a thick layer of extracellular matrix -- mesoglea. A. aurita has a lot of cells in the mesoglea unlike many other Cnidarians. The major protein of the mesoglea with apparent molecular mass of 47 kDa was detected by SDS-PAGE. A partial mRNA of the protein 1421 bp long was cloned and sequenced. The search for homologous nucleotide and protein sequences shows that the mRNA sequence is novel. Deduced amino acid sequence of 416 aa contains zona pellucida (ZP) domain and Delta/Serrate/Lag-2 (DSL) domain. The protein was named mesoglein. According to reverse transcription PCR analysis it is expressed in the mature medusa exclusively in the mesogleal cells. Mesoglein belongs to the lowest phyla among ZP domain-containing proteins. The protein is supposed to be a structural element of the mesoglea extracellular matrix.

[Differential gene expression in the jellyfish Aurelia aurita].

The body of Aurelia aurita, as well as other diploblasts, consists of two epithelial layers: ectodermal and gastral epithelium. These two tissues are separated by mesoglea, or extracellular matrix. In most coelenterates mesoglea is acellular. In A. aurita mesogleal cells are scattered in mesoglea. Differential display PCR was used to compare mRNA pools from ectodermal epithelium, gastral epithelium and mesoglea. 4 novel gene fragments were cloned and sequenced. According to RTPCR results, one of these fragments is differentially expressed in the ectodermal epithelium.

Invasive characteristics of apathogenic Shigella flexneri 5a2c mutant obtained under the effect of furazolidone.

Apathogenic Shigella flexneri 5a2c mutant treated with furazolidone can infect eucaryotic cells. These bacteria contain no virulence genes responsible for Sh. flexneri invasion, which seems to be the cause of their apathogenicity. The capacity of bacteria to penetrate into eucaryotic cells correlates with the appearance of ECP 32 protease specifically cleaving actin.

Structure-specific DNA-binding proteins as the foundation for three-dimensional chromatin organization.

Any functions of tandem repetitive sequences need proteins that specifically bind to them. Telomere-binding TRF2/MTBP attaches telomeres to the nuclear envelope in interphase due to its rod-domain-like motif. Interphase nuclei organized as a number of sponge-like ruffly round chromosome territories that could be rotated from outside. SAF-A/hnRNP-U and p68-helicase are proteins suitable to do that. Their location in the interchromosome territory space, ATPase domains, and the ability to be bound by satellite DNAs (satDNA) make them part of the wires used to help chromosome territory rotates. In case of active transcription p68-helicase can be involved in the formation of local "gene expression matrices" and due to its satDNA-binding specificity cause the rearrangement of the local chromosome territory. The marks of chromatin rearrangement, which have to be heritable, could be provided by SAF-A/hnRNP-U. During telophase unfolding the proper chromatin arrangement is restored according to these marks. The structural specificity of both proteins to the satDNAs provides a regulative but relatively stable mode of binding. The structural specificity of protein binding could help to find the "magic" centromeric sequence. With future investigations of proteins with the structural specificity of binding during early embryogenesis, when heterochromatin formation goes on, the molecular mechanisms of the "gene gating" hypothesis (Blobel, 1985) will be confirmed.

[Position of the satellite DNA relative to heterochromatin in the interphase nuclei in cultured cells]. / Polozhenie satellitnykh DNK po otnosheniiu k geterokhromatinu v interfaznykh iadrakh kletok v kul'ture.

It is considered that centromeric (CEN) regions play the leading role in the formation of chromocentres predominantly consisting of satellite DNA. Cloned mouse and human satellite DNA sequences from CEN and periGEN regions were used in order to trace their positions relative to chromocentres. Methods of fluorescent in situ hybridization (FISH) and immunoFISH with antibodies against CENP-B, known is a marker of prekinetochore, were employed. Peripheral position of the signals was observed at the chromocentres, but a combined signal of GEN and periGEN satellite DNAs never covered the whole brightly DAPI stained regions. A reasonable amount of the chromocentre body is not a satellite DNA. We suppose that the matrix-associated regions (MAR) of structural genes and, probably, the heavy methylated coding sequences of the genes, which are not expressed in the given cell type, are included in the chromocentres in addition to satellite DNAs.