Comparative study of calcification in human choroid plexus, pineal gland, and habenula.

Choroid plexus, pineal gland, and habenula tend to accumulate physiologic calcifications (concrements) over a lifetime. However, until now the composition and causes of the intracranial calcifications remain unclear. The detailed analysis of concrements has been done by us using X-ray diffraction analysis (XRD), X-ray diffraction topography (XRDT), micro-CT, X-ray phase-contrast tomography (XPCT), as well as histology and immunohistochemistry (IHC). By combining physical (XRD) and biochemical (IHC) methods, we identified inorganic (hydroxyapatite) and organic (vimentin) components of the concrements. Via XPCT, XRDT, histological, and IHC methods, we assessed the structure of concrements within their appropriate tissue environment in both two and three dimensions. The study found that hydroxyapatite was a major component of all calcified depositions. It should be noted, however, that the concrements displayed distinctive characteristics corresponding to each specific structure of the brain. As a result, our study provides a basis for assessing the pathological and physiological changes that occur in brain structure containing calcifications.

Immunohistochemical Study of Dark and Progenitor Carotid Body Cells: Artefacts or Real Subtypes?

Postmortem changes occurring in human carotid body were simulated on the Wistar rat model. It was shown that light, dark, and pyknotic (progenitor) subtypes of human carotid body cells are an artifact and cannot be used in clinical practice to study the characteristics of various human diseases. The differences between the control group of healthy individuals and individuals with the various pathologies are most likely due to the different levels of premortal hypoxia that the tissue had been exposed to. Moreover, widespread antigens used in practice were divided into 2 groups by their tolerance to autolysis: stable and unstable ones. This can be useful for the development of immunohistochemical test algorithms for the diagnostics on autopsy material.

Individual features of play behavior in thick-toed geckos in weightlessness and normal gravity conditions.

The object play behavior in thick-toed geckos (Chondrodactylus turneri GRAY 1864) was studied during a 30-day orbital experiment onboard the Bion-M1 biosatellite. The play object for five geckos was a marking collar that one of the geckos in the flight group removed immediately before the launch. The play behavior started when either the gecko observed the approaching floating collar or when the gecko independently approached the stationary collar, followed by manipulation of the collar and subsequent observation of its moving away. While playing with the collar, the individuality of geckos' behavior was manifested in the frequency and number of play episodes, the nature of manipulations, and the duration of interest in play during the flight. We found that thick-toed geckos could play not only with an unknown object (marking collar) but also with familiar molting skins. In weightlessness, the play behavior of geckos with molting skin fragments was similar to the play behavior with the collar and also varied between individuals. It was established that geckos maintained a similar individual level of play activity with different objects (collar and molting skins). It was found that geckos also played with fragments of molting skin under normal gravity conditions. In contrast to weightlessness, play behavior at normal gravity was rare and limited to short durations of object manipulation.

Attachment of Turner's thick-toed geckos (Chondrodactylus turneri GRAY 1864) during weightlessness and their responses to flotation.

We investigated the behavior of 15 female Turner's thick-toed geckos (Chondrodactylus turneri GRAY 1864) during a 30-day orbital experiment on the unmanned spacecraft "BION-M" No. 1. During weightlessness, the geckos maintained their ability to attach to the surfaces using the subdigital pads on their toes. On average, the geckos spent 99.9% of the time adhering to surfaces during the flight and only 0.1% floating freely. The active geckos, when starting to float, immediately restored attachment by a number of behavioral responses. The floating quiescent geckos, when resuming their active condition, responded in the same manner. The responses during flotation are similar to the behavioral reflexes triggered by a fall under normal gravity; i.e.: 1) the ventral extension of the limbs, 2) a skydiving posture, and 3) postural righting reflexes. Ventral extension of limbs was described for the first time in weightlessness. Individual variability in the frequency of flotations was found for both active and quiescent geckos during the flight. The findings show that the ability to attach to surfaces is an important factor in the geckos' adaptation to weightlessness. The behavioral responses that originated during freefall in conditions on Earth (one-G) appear as adaptations to weightlessness and remain partially effective.

[The profile of plasma non-esterified fatty acids in children with different terms of type 1 diabetes mellitus].

Composition and quantitative content of non-esterified fatty acids (NEFA) were investigated in plasma samples of healthy children (12) and children with type 1 diabetes mellitus (DM1) (31) by gas chromatography (GC) after preliminary NEFA solid-phase extraction from plasma lipids. There was a significant (p<0.001) 1.6-fold increase in the total level of NEFA regardless of the disease duration. In the group of DM1 children with the disease period less than 1 year there was an increase in the arachidonic acid (20:4) content (30%) and the oleic acid trans-isomer (18:1) content (82%), and also a decrease in the docosahexaenoic acid (22:6 n3) content (26% ) and the docosapentaenoic acids (22:5 n-6) content (60%). In the group of DM1 children with prolonged course of this disease the altered NEFA levels returned to the normal level.

[Development of the Human Olfactory Bulbs in the Prenatal Ontogenesis: an Immunochistochemical Study with Markers of Presynaptic Terminals (anti-SNAP-25, -Synapsin-I, -Synaptophysin)].

We provide the data of the olfactory bulbs (OB) development in the human fetuses on the stages from 8 week to birth. Immunochistochemical markers of presynaptic terminals (anti-SNAP-25, -synapsin-I, -synaptophysin) were used to evaluate the maturation of the OB. Differentiation of the OB layers begins from periphery, which implicitly evidences that growth of the olfactory nerves fibers induses not only anatomical differentiation of the OB, but also differentiation of its functional layers. The sites of the developing glomerulus are revealed using the immunochistochemical prosedure on the stage before distinct glomerulus can be identified with common histological procedure. OB conductive system demonstrates immunoreactivity with the antibodies to the presynaptic proteins on the all stages from 10-11 weeks of fetus development. Four stages of the OB development are described. All functional layers of the OB are mature at the 22-weeks stage. Further differentiation of the OB neuroblasts, including lamina formation of the internal granular leyer, glomerular layer development, OB growth continue after 20-22 weeks stage until 38-40 weeks of the fetus develoment. Patterns of the immunoreactivity with antibodies to SNAP-25, synapsin-I and synaptophysin are completely appropriate to those of adult's OB on the 38-40 weeks of the prenatal development. Complete maturity of the human OB is achived at 38-40 weeks of the prenatal development.

Soft X-ray imaging of thick carbon-based materials using the normal incidence multilayer optics.

The high transparency of carbon-containing materials in the spectral region of "carbon window" (lambda approximately 4.5-5nm) introduces new opportunities for various soft X-ray microscopy applications. The development of efficient multilayer coated X-ray optics operating at the wavelengths of about 4.5nm has stimulated a series of our imaging experiments to study thick biological and synthetic objects. Our experimental set-up consisted of a laser plasma X-ray source generated with the 2nd harmonics of Nd-glass laser, scandium-based thin-film filters, Co/C multilayer mirror and X-ray film UF-4. All soft X-ray images were produced with a single nanosecond exposure and demonstrated appropriate absorption contrast and detector-limited spatial resolution. A special attention was paid to the 3D imaging of thick low-density foam materials to be used in design of laser fusion targets.

Comparative analysis of the skeletal changes in tetrapods after brief influence of microgravity.

Experiments involving lower tetrapods demonstrate that the degree of skeletal demineralization in spaceflights is related to the type of environmental behaviour of the animal. Probably the sensing of support reaction decreases the negative effect of spaceflight upon the bone tissue.

Quantitative analysis of bone mineral content by x-ray microtomography.

A new non-destructive method based on x-ray microtomography (micro-CT) was developed to measure calcium density in bone. X-ray micro-CT was used as a quantitative approach to acquire and reconstruct virtual cross-sections through the sample. Accurate beam-hardening correction was implemented. Grey values in the virtual cross-sections were calibrated as calcium mineral density in bone. From these cross-sections, three-dimensional models were created. Calcium content was calculated directly from images and expressed as percentage per volume and per weight. Calcium mineral density was studied by this method in a unique set of bones isolated from newts (Pleurodeles waltlii Michah) that had travelled into space. A demineralization of 10% was shown as a consequence of sustained micro-gravity.

Product analysis illuminates the final steps of IES deletion in Tetrahymena thermophila.

DNA sequences (IES elements) eliminated from the developing macronucleus in the ciliate Tetrahymena thermophila are released as linear fragments, which have now been detected and isolated. A PCR-mediated examination of fragment end structures reveals three types of strand scission events, reflecting three steps in the deletion process. New evidence is provided for two steps proposed previously: an initiating double-stranded cleavage, and strand transfer to create a branched deletion intermediate. The fragment ends provide evidence for a previously uncharacterized third step: the branched DNA strand is cleaved at one of several defined sites located within 15-16 nucleotides of the IES boundary, liberating the deleted DNA in a linear form.

Chimeric brain: theoretical and clinical aspects.

Using xeno-transplantation, interactions of neural tissues of vertebrates and insects were studied. Ventral neurogenic primordium of Notch Drosophila melanogaster embryos was transplanted into neural tube of amphibian and mammalian embryos with the aid of microhydrofeeding. Embryos of four different amphibian species, random bred mice and rats were used as graft recipients. It was concluded that there is a possibility to incorporate nerve cells of insects into the brain of vertebrates. Morphological and functional contacts can be established between the transplanted cells and host brain tissues. Transplanted Drosophila cells preserve their viability for a long time, so that a prolonged influence of the transplant upon the recipient can be predicted, which may be used in medical practice. A mixture of human fetal brain and Notch Drosophila melanogaster neural embryonic tissues were transplanted into the ventro-lateral nucleus of the thalamus of the patients of Parkinson' disease. As a result, tremor and constrained movements disappeared. Post-operation patients have been observed within 13-38 months. No side effects were noted during this time.

Developmentally programmed DNA deletion in Tetrahymena thermophila by a transposition-like reaction pathway.

We provide a molecular description of key intermediates in the deletion of two internal eliminated sequences (IES elements), the M and R regions, during macronuclear development in Tetrahymena thermophila. Using a variety of PCR-based methods in vivo, double-strand breaks are detected that are generated by hydrolytic cleavage and correspond closely to the observed chromosomal junctions left behind in the macronuclei. The breaks exhibit a temporal and structural relationship to the deletion reaction that provides strong evidence that they are intermediates in the deletion pathway. Breaks in the individual strands are staggered by 4 bp, producing a four nucleotide 5' extension. Evidence is presented that breaks do not occur simultaneously at both ends. The results are most consistent with a deletion mechanism featuring initiation by double-strand cleavage at one end of the deleted element, followed by transesterification to generate the macronuclear junction on one DNA strand. An adenosine residue is found at all the nucleophilic 3' ends used in the postulated transesterification step. Evidence for the transesterification step is provided by detection of a 3' hydroxyl that would be liberated by such a step at a deletion boundary where no other DNA strand ends are detected.

Segment selection during 'phase variation'-type regulation of gene replacement mediated by FLP recombinase in the yeast Saccharomyces cerevisiae.

FLP recombinase has recently been used as a tool to direct the exchange between invertible DNA segments, called 'Phase variation'-type regulation of gene replacement in eukaryotic cells. Using an appropriate selective medium, positive segment selection was shown to be efficient during the regulation of gene replacement. The efficiency was determined from the copy number ratio of invertible segments with the use of the neomycinphosphotransferase II (NPTII) gene bearing invertible segments located on the episomal yeast plasmid, and the resident 2-microns circle. Without the selection the segments copy number ratio was retained in growing cells. The results obtained are an evidence for the efficiency of positive segment selection during the 'Phase variation'-type regulation of gene replacement in eukaryotic cells.

Transient DNA breaks associated with programmed genomic deletion events in conjugating cells of Tetrahymena thermophila.

Thousands of programmed genomic deletion events occur during macronuclear development in Tetrahymena thermophila. Two of the deleted segments, called M and R, have been particularly well-characterized. Using ligation-mediated PCR, we have detected DNA strand breaks that correlate temporally and structurally with the deletion events in the M and R regions. The ends appear at positions that correspond precisely to boundaries of deleted sequences, as defined by observed chromosomal junctions found after deletion is complete. They occur exclusively during the known DNA rearrangement period in macronuclear development. The breaks are staggered by 4 bp in the complementary strands. Several alternative breaks were found at the end of one deleted region, consistent with multiple alternative chromosomal junctions detected previously. The free 5' ends generated at the breaks are phosphorylated. A purine residue always occurs at the free 3' ends, with an adenosine appearing in 11 of 12 cases. Patterns found in the detected break sites suggest rules that define the ends of the deleted segments within a transposon-like deletion mechanism.

The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila.

Thousands of DNA deletion events occur during macronuclear development in the ciliate Tetrahymena thermophila. In two deleted genomic regions, designated M and R, the eliminated sequences form circles that can be detected by PCR. However, the circles are not normal products of the reaction pathway. The circular forms occur at very low levels in conjugating cells, but are stable. Sequencing analysis showed that many of the circles (as many as 50% of those examined) reflected a precise deletion in the M and R regions. The remaining circles were either smaller or larger and contained varying lengths of sequences derived from the chromosomal DNA surrounding the eliminated region. The chromosomal junctions left behind after deletion were more precise, although deletions in either the M or R regions can generate any of several alternative junctions (1). Some new chromosomal junctions were detected in the present study. The results suggest that the deleted segment is released as a linear DNA species that is degraded rapidly. The species is only rarely converted to the stable circles we detect. The deletion mechanism is different from those proposed for deletion events in hypotrichous ciliates (2-4), and does not reflect a conservative site-specific recombination process such as that promoted by the bacteriophage lambda integrase (5).

Results of transcatheter closure of the patent ductus arteriosus with the Botallooccluder.

OBJECTIVES: Evaluation of the immediate and long-term efficacy of a novel method of transvenous closure of the patent ductus arteriosus was the purpose of this study. BACKGROUND: In 1981 at the First Moscow Hospital, a new method of transvenous patent ductus arteriosus closure with the Botallooccluder was developed. METHODS: Transvenous closure of the patent ductus arteriosus with the Botallooccluder was carried out in 273 patients. All patients were regularly followed up as outpatients at intervals of 3, 6 and 12 months after intervention. A total of 171 patients were examined at a mean of 3.2 +/- 1.1 years (range 1 to 8) after the procedure. Late evaluation with cardiac catheterization was performed in 62 patients (36%); 50 (29%) of these 62 also underwent right heart catheterization. RESULTS: Successful implantation was achieved in 266 patients (97%). The late follow-up findings testify to the favorable clinical course, with disappearance of the continuous murmur and regression of the electrocardiographic signs of ventricular overloading in the vast majority of patients. In no case after complete patent ductus arteriosus closure was a late ductus recanalization observed. No patient had even a minor degree of postprocedural lower extremity venous damage. In 29 patients (24%), the femoral artery was imaged by ultrasonography at late follow-up, without evidence of any vascular abnormality. CONCLUSIONS: The clinical data suggest high long-term efficacy and reliability of this technique. Introducer sheaths (12F to 16F outer diameter) did not cause any long-term entry site sequelae.

'Phase variation'-type regulation of gene expression and gene replacement mediated by FLP recombinase in the yeast Saccharomyces cerevisiae.

Expression of a neomycin phosphotransferase II (NPTII) gene has been designed to be regulated by an FLP-mediated switching of the orientation of the NPTII coding region located on the invertible DNA segment in episomal yeast plasmids. Inversion of the segment from inverted to direct orientation with respect to the promoter resulted in a dramatic increase in G418 resistance. FLP also promoted a double reciprocal exchange between the transforming and the resident 2-microns plasmid, leading to insertion of the FLP and REP2 genes into the transforming plasmid. The results demonstrate a possible use of FLP recombinase for 'phase variation'-type regulation of gene expression and gene replacement in eukaryotic cells.

Patent ductus arteriosus: transcatheter closure with a transvenous technique.

Transvenous closure is described of the patent ductus arteriosus with a conical device that consists of polyurethane foam mounted on a stainless steel frame. Since 1981, the procedure has been performed in 273 patients in Russia. Permanent complete ductus closure was achieved in 258 (95%) patients. In eight cases (3%), the occlusion was incomplete. Complications occurred in 10 patients (4%), seven of whom required emergency surgery. The new technique combines certain advantages of both alternative methods. The conical shape of the occlusion device is well suited for the anatomic structure of the ductus. The possibility of selecting the size of the device permits reduction in the rate of incomplete closure while still taking advantage of transvenous delivery to avoid entry-site problems. In the initial 10 years of experience, the described technique proved effective, safe, and applicable in the vast majority of patients older than 2 years.