[Allogeneic umbilical cord blood cell therapy for children with autism: safety and efficacy of the method]. / Primenenie kontsentrata yadrosoderzhashchikh kletok pupovinnoi krovi u detei s autizmom: bezopasnost' i effektivnost' metoda.

OBJECTIVE: To evaluate effectiveness and safety of umbilical cord blood cells (UCBC) in children with ASD. MATERIAL AND METHODS: The study comprised 13 boys and 2 girls, mean age 7.0±0.5 years (test group), and 9 boys and 1 girl, mean age 6.0±1.3 years (control group) diagnosed with autism or autistic syndrome. UCBC were infused intravenously in a single dose of 250±20 million cells, four times at 14±3-day intervals. Dynamics of cognitive functions were assessed with WISC subscales, questionnaire Skvortsov Developmental Profile Survey and brainstem auditory evoked potentials (BAEPs) conducted before therapy and six months after the first injection. The dynamic of autistic symptoms was explored with CASD and ATEC questionnaires, fulfilled at the start of the study, 3 and 6 months later (test group) or at the start of the study, and 6 months later (control group). RESULTS: UCBC was well tolerated and caused no appreciable adverse effects. Observation revealed improvement in cognitive functioning and alleviation of autistic symptoms in patients of the test group six months after the first UCBC injection. Positive dynamics were more noticeable in the test group than in the control group receiving standard therapy. CONCLUSION: The use of UCBC is safe and might be effective in the complex therapy of autism.

[Endothelial microtubules as integral part of the mechanism controlling the level of stimulated secretion of van Willebrand factor]. / Sistema mikrotrubochek v kletkakh sosudistogo endoteliia kak vozmognyi reguliator urovnia seketsii faktora Villebranda.

We used double immunofluorescence and electron microscopy to study the spatial relationships between Weibel--Palade bodies (WPBs) and cytoskeletal elements in endothelial cells treated with thrombin or cytoskeleton-damaging agents. We have found that some WPBs undergo translocation towards the centrosome in 5 min in the cells treated with thrombin, cytochalasin B or calyculin A. The cells treated with thrombin or cytochalasin exhibit depletion of WPBs, whereas WPBs found at the cell periphery were colocalized with intermediate filaments. There was a precise colocalization observed between the WPBs and microtubules in the calyculin-treated cells in which all WPBs undergo centrosome-directed translocation within 15 min after the agent addition. When vimentin filaments were induced to collapse by demecolcine, intermediate filaments and WPBs both translocated to the perinuclear region. The data provide the first direct evidence that secretory granules utilize microtubules to move in retrograde direction, i.e., away from the plasma membrane, towards the centrosome. We suggest that anterograde movement of WPBs is dependent on their interaction with vimentin filaments.

Translation termination factors function outside of translation: yeast eRF1 interacts with myosin light chain, Mlc1p, to effect cytokinesis.

The translation termination factor eRF1 recognizes stop codons at the A site of the ribosome and induces peptidyl-tRNA hydrolysis at the peptidyl transferase centre. Recent data show that, besides translation, yeast eRF1 is also involved in cell cycle regulation. To clarify the mechanisms of non-translational functions of eRF1, we performed a genetic screen for its novel partner proteins. This screen revealed the gene for myosin light chain, Mlc1p, acting as a dosage suppressor of a temperature-sensitive mutation in the SUP45 gene encoding eRF1. eRF1 and Mlc1p are able to interact with each other and, similarly to depletion of Mlc1p, mutations in the SUP45 gene may affect cytokinesis. Immunofluorescent staining performed to determine localization of Mlc1p has shown that the sup45 mutation, which arrests cytokinesis, redistributed Mlc1p, causing its disappearance from the bud tip and the bud neck. The data obtained demonstrate that yeast eRF1 has an important non-translational function effecting cytokinesis via interaction with Mlc1p.

[A new concept of development of neointimal hyperplasia]. / Novaia kontseptsiia razvitiia giperplazii intimy sosudov.

The intima hyperplasia is a major morphological feature of various arterial pathologies such as atherosclerosis, postangioplasty restenosis and transplantation arteriopathy. It is commonly assumed that smooth muscle cells (SMC) comprising loci of the intima hyperplasia originate from arterial media. However, recent studies suggest that the bone marrow could also supply circulating vascular progenitor of SMCs and endothelial cells (EC). Such bone marrow progenitors participate in the formation of a cellular mass of neointima after experimental allotransplantation, mechanical vessel injury or hyperlipidemia induced experimental atherosclerosis. Circulating SMC and EC progenitors are also likely to be involved in the transplantation arteriopathy development in humans but their roles in the atherosclerosis and restenosis remain to be determined. Stages of the mobilization, defferentiation and proliferation of SMC progenitors could provide point of attack for new therapeutic strategies for the treatment of proliferative vascular diseases. The precise understanding of the neointima cells origin could provide a key for development of the optimal therapeutic strategy of treatnent of such disorders. This review is focused on the pathological significance of circulating progenitors of the bone marrow origin, particularly on the SMC progenitors, for development of vascular wall disorders.

[Stability of microbial association in the process of industrial biofiltration cleaning of gaseous discharges]. / Issledovanie ustoichivosti mikrobnoi assotsiatsii v protsesse promyshlennoi biofil'tratsionnoi ochestki gazovozdushnykh vybrosov.

Results of industrial exploitation of a biofiltration plant tailored for purifying gaseous discharges of hazardous organic components such as toluene, cyclohexane, and xylene, are examined. Both numerical and compositional variations were monitored for a long-term (more than 1.5 years) utilization process in an association of microorganisms decomposing organic pollutants. A population of microbial association composed by one yeast and two bacterial strains in the biofilm on the surface of filtering sheets was abundant (10(8)-10(9) yeast cells/cm2 and 10(10)-10(11) bacterial cells/cm2) and stable during the whole period of monitoring. A microbial association in the culture medium averaging 10(6) yeast cells/l and 10(8) bacterial cells/l is more susceptible to technogenic impacts and seasonal fluctuations. Overall, the biofilter as an open and autonomic system maintained its microbial association, thereby providing a high-degree (93-98%) purification of industrial gaseous discharges from organic pollutants.

Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae.

BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl - dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough. RESULTS: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p. CONCLUSIONS: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.

[Psi(+)] prion generation in yeast: characterization of the 'strain' difference.

The yeast cytoplasmically-inherited nonsense suppressor [PSI(+)] determinant is presumed to be a manifestation of the aggregated prion-like state of the Sup35 protein. Overexpression of the Sup35 protein induces generation of [PSI(+)] determinants with various suppressor efficiency and mitotic stabilities. Here, we demonstrate that the relative frequency of appearance of [PSI(+)] with different properties depends on the SUP35 allele used to induce their generation. The difference in properties of [PSI(+)] determinants was preserved after their transmission from one yeast strain to another. This difference correlated with variation in properties of the Sup35 protein. A novel type of prion instability was observed: some [PSI(+)] with weak suppressor efficiency could convert spontaneously into strong suppressor determinants.

Chaperones that cure yeast artificial [PSI+] and their prion-specific effects.

The [PSI(+)] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI(+)] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI(+)] against Hsp104 [3,4]. We used an artificial [PSI(+)(PS)] based on the Sup35 prion-forming domain from yeast Pichia methanolica [5] to find other prion-curing factors. Both [PSI(+)(PS)] and [PSI(+)] have prion 'strains', differing in their suppressor efficiency and mitotic stability. We show that [PSI(+)(PS)] and a 'weak' strain of [PSI(+)] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI(+)(PS)] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.

Centrosome-directed translocation of Weibel-Palade bodies is rapidly induced by thrombin, calyculin A, or cytochalasin B in human aortic endothelial cells.

To examine the possible role of the cytoskeleton in exocytosis of Weibel-Palade bodies (WPBs), we used double immunofluorescence and electron microscopy to study the spatial relationships between WPBs and main cytoskeletal elements in endothelial cells treated with secretagogue, such as thrombin, or cytoskeleton-damaging agents. Unexpectedly, we have found that WPBs undergo rapid translocation towards the centrosome both in cells treated with thrombin and in those treated with cytochalasin B or calyculin A. Typically, 3 or 5 min after agent addition compact cluster of WPBs became visible near the microtubule-organizing center (MTOC) in most endothelial cells in which a fivefold increase in WPBs localized in close proximity to the mother centriole had been detected. In both thrombin- and cytochalasin-treated cells that exhibit a noticeable depletion in WPBs compared to control cells, WPBs located at the cell periphery were found to colocalize with vimentin intermediate filaments, but not with microtubules. In contrast, there was precise colocalization observed between WPBs and microtubules in calyculin-treated cells in which all WPBs undergo centrosome-directed translocation within 15 min after the agent addition. When vimentin filaments were induced to collapse to a perinuclear location by the microtubule-disrupting agent demecolcine, WPBs also translocated to the perinuclear region, where numerous WPBs were found to be localized within the bundles of intermediate-sized filaments. The data provide the first direct evidence that secretory granules utilize microtubule-based transport system to move in retrograde direction, i.e., away from the plasma membrane, towards the centrosome. We suggest that anterograde movement of WPBs is primarily dependent on their interaction with vimentin intermediate filaments.

Cytomegalovirus genome and the immediate-early antigen in cells of different layers of human aorta.

A number of data suggest that reactivation of cytomegalovirus (CMV) latent in arterial wall cells may contribute to atherogenesis; however, there is no direct evidence available. To address this issue, we have examined, using in situ hybridization or immunohistochemical staining, the frequency of occurrence of cells containing viral genome and of those expressing the IE 70 viral antigen in the endothelial layer and in deeper layers of human aortas with or without visible atherosclerotic lesions. Using endothelial cell cultures or tissue endothelial preparations, we found CMV-hybridizing endothelial cells in 6 of 8 grossly normal aortas and in 16 of 18 lesioned aortas. Antigen-positive endothelial cells were detected in 1 of 5 grossly normal vessels and in 6 of 7 lesioned vessels. Infected endothelial cells were abundant in areas adjacent to orifices of intercostal arteries of grossly normal aortas and in fatty spots of lesioned aortas, but no infected endothelial cells were observed in most plaques examined. In paraffin sections of grossly normal vessels, we detected CMV genome in cells adjacent to lumen and in cells randomly scattered through subendothelial intima and the media; however, no immunoreactive viral protein was found in the same tissue samples. In sections of lesioned vessels, clusters of CMV-hybridizing cells were found in the media in addition to infected cells randomly scattered through the intima and the media. In these samples of lesioned vessels, viral antigen was detected in cells adjacent to lumen and in cells clustered at the intima/media border. We found antigen-positive cells in grossly normal areas of lesioned aortas and in fatty lesions, but not in plaques of the same vessels. The data suggest that accumulation of the immediate-early CMV antigen in cells of endothelial layer and development of antigen-positive cell clusters in deeper layers of vascular wall accompany early atherogenic events in human aorta.

A novel vacuolar protein encoded by SSU21 / MCD4 is involved in cell wall integrity in yeast.

Using a screening procedure for obtaining yeast strains with enhanced ability to secrete heterologous protein, we have isolated a mutant with alteration of the cell wall structure. This mutant displayed strong decrease in cell wall mannoprotein content, which was not accompanied by decreased glycosylation of secreted proteins. The mutation defines a gene, designated SSU21(identical to previously characterized MCD4), which encodes a novel vacuolar protein. SSU21 is probably connected to the cell integrity protein kinase C-mediated pathway, since ssu21 and pkc1Delta double mutant is synthetic lethal. To our knowledge, this is the first example of a yeast vacuolar protein whose alteration results in a cell wall defect.

[Prion phenomenon in medicine and biology]. / Fenomen prionov v meditsine i biologii.

The data obtained suggest that the fatal changes in brain tissue associated with the prion diseases, are initiated by a conformational rearrangement of constitutively expressed cellular protein PrP. Possible mechanisms of such a conversion of this protein are discussed. Existence of the proteins with the prion properties in low eukaryotes may determine the unusual mechanisms of the "protein" inheritance. A new experimental model for studying the proteins with the prion properties in the yeast Saccharomyces cerevisiae, is described.

C-terminal truncation of the Sup35 protein increases the frequency of de novo generation of a prion-based [PSI+] determinant in Saccharomyces cerevisiae.

The yeast non-Mendelian [PSI+] determinant is presumed to be the manifestation of the aggregated prion-like state of the Sup35 protein. Plasmid-mediated amplification of the SUP35 gene greatly increases the frequency of Sup35p transition to this prion-like state. Here we show that the 3'-deletions of plasmid SUP35, leading to the C-terminal truncation of Sup35p, further increase the frequency of [PSI+] induction despite a marked decrease in Sup35p expression levels. The data suggest that the presence of Sup35p N-terminal proteolytic fragments can cause [PSI+] appearance in wild-type yeast cells.

Centrosome injury in cells infected with human cytomegalovirus.

The organization of the mitotic apparatus was studied in human embryo lung fibroblasts (HEL) and Vero cells at 4 days postinfection with human cytomegalovirus (HCMV) strain AD 169. The bipolar spindle was detected by immunofluorescence in p72-positive mitotic cells exhibiting a regular or C-metaphase-like chromosome configuration. Electron-microscopic study of C-metaphase-like cells revealed alteration of the centrosome structure which is characterized by the following features: (1) breakdown of the diplosome, (2) separation of the fibrillar material from centrioles, and (3) disruption of the centriolar cylinder. The spindle pole in the aberrant mitotic cells consisted of one or several foci of microtubules converging on the fibrillar aggregates. There are not any signs of the nuclear envelope reconstruction found in mitotic cells with highly condensed scattered chromosomes. Unlike in HEL cells, viral particles were not detected in Vero cells. A question arises as to whether centrosome injury is an integral part of the events leading to cell death unrelated to the reproduction of HCMV.

In vitro propagation of the prion-like state of yeast Sup35 protein.

The yeast cytoplasmically inherited genetic determinant [PSI+] is presumed to be a manifestation of the prion-like properties of the Sup35 protein (Sup35p). Here, cell-free conversion of Sup35p from [psi-] cells (Sup35ppsi-) to the prion-like [PSI+]-specific form (Sup35pPSI+) was observed. The conversion reaction could be repeated for several consecutive cycles, thus modeling in vitro continuous [PSI+] propagation. Size fractionation of lysates of [PSI+] cells demonstrated that the converting activity was associated solely with Sup35pPSI+ aggregates, which agrees with the nucleation model for [PSI+] propagation. Sup35pPSI+ was purified and showed high conversion activity, thus confirming the prion hypothesis for Sup35p.

[Regularities in the spread of hepatitis C virus and its genotypes in Russian and countries within the former USSR]. / Zakonomernosti rasprostraneniia virusa gepatita C i ego genotipov v Rossii i stranakh SNG.

The incidence of markers of hepatitis C virus (HCV in the blood of 4216 normal subjects living in the European Russia (Northern, North-Western, Central, Central Chernozem, Volga-Vyatka, Volga, and North Caucasian regions), in the Urals, in Siberia (Eastern Siberian region), in the Far East, and in Monogolia is assessed. The incidence of antibodies to HCV varied from 0.7% in the Central region to 3.8% in the Central Chernozem and 10.7% in Mongolia. HCV genotyping (identification of 1a, 1b, 2a, 2b, and 3a genotypes) was performed using 469 RNA of HCV-positive sera of donors and patients collected in Russia, Moldova, Turkmenistan, and Mongolia. The 1b genotype predominated everywhere (68.9%), its incidence being the highest in Moldova (96%). Unclassifiable variants of HCV were found in 28 (6%) of sera. The regularities of HCV genotypes circulation in the European Russia were the same as in other European countries, whereas their prevalence in Eastern Russia was rather like that in China or Japan. The prevalence of genotypes did not depend on the clinical manifestations of diseases caused by HCV.

Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for prion-dependent regulation.

The SUP45 and SUP35 genes of Saccharomyces cerevisiae encode polypeptide chain release factors eRF1 and eRF3, respectively. It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian [PSI+] nonsense suppressor determinant. In a [PSI+] state, Sup35p forms high-molecular-weight aggregates which may inhibit Sup35p activity, leading to the [PSI+] phenotype. Sup35p is composed of the N-terminal domain (N) required for [PSI+] maintenance, the presumably nonfunctional middle region (M), and the C-terminal domain (C) essential for translation termination. In this study, we observed that the N domain, alone or as a part of larger fragments, can form aggregates in [PSI+] cells. Two sites for Sup45p binding were found within Sup35p: one is formed by the N and M domains, and the other is located within the C domain. Similarly to Sup35p, in [PSI+] cells Sup45p was found in aggregates. The aggregation of Sup45p is caused by its binding to Sup35p and was not observed when the aggregated Sup35p fragments did not contain sites for Sup45p binding. The incorporation of Sup45p into the aggregates should inhibit its activity. The N domain of Sup35p, responsible for its aggregation in [PSI+] cells, may thus act as a repressor of another polypeptide chain release factor, Sup45p. This phenomenon represents a novel mechanism of regulation of gene expression at the posttranslational level.

Propagation of the yeast prion-like [psi+] determinant is mediated by oligomerization of the SUP35-encoded polypeptide chain release factor.

The Sup35p protein of yeast Saccharomyces cerevisiae is a homologue of the polypeptide chain release factor 3 (eRF3) of higher eukaryotes. It has been suggested that this protein may adopt a specific self-propagating conformation, similar to mammalian prions, giving rise to the [psi+] nonsense suppressor determinant, inherited in a non-Mendelian fashion. Here we present data confirming the prion-like nature of [psi+]. We show that Sup35p molecules interact with each other through their N-terminal domains in [psi+], but not [psi-] cells. This interaction is critical for [psi+] propagation, since its disruption leads to a loss of [psi+]. Similarly to mammalian prions, in [psi+] cells Sup35p forms high molecular weight aggregates, accumulating most of this protein. The aggregation inhibits Sup35p activity leading to a [psi+] nonsense-suppressor phenotype. N-terminally altered Sup35p molecules are unable to interact with the [psi+] Sup35p isoform, remain soluble and improve the translation termination in [psi+] strains, thus causing an antisuppressor phenotype. The overexpression of Hsp104p chaperone protein partially solubilizes Sup35P aggregates in the [psi+] strain, also causing an antisuppressor phenotype. We propose that Hsp104p plays a role in establishing stable [psi+] inheritance by splitting up Sup35p aggregates and thus ensuring equidistribution of the prion-like Sup35p isoform to daughter cells at cell divisions.