Development of a quantitative PCR variant, and use of it for assessing gene expression

The data on the development of a quantitative PCR technique are presented, including the synthesis of modified primers, the choice of conditions for the removal of reaction-mixture components from the amplified DNA, and the colorimetric detection of the PCR product. The application of this technique to the assessment of the level of gene expression in nonculturable bacterial forms characterized by a drastically reduced metabolism is described.     

Study of the core gaps formed accidentally during wire explosion

During wire explosion, along with striations (a regular structure with alternating lower and higher density bands), low-density regions the characteristic axial size of which differs substantially from that of striations and can reach 1–2 mm are also observed in the discharge channel. Such irregular structures came to be known as “gaps” (D. B. Sinars et al., Phys. Plasmas 8, 216 (2001)). In the present study, the mechanism of the formation of core gaps during explosions of 25- and 50-μm-diameter copper and nickel wires in air is investigated. It is shown that the specific energy deposited in the gap region substantially exceeds the average specific energy deposited in the wire material.     

Changes in the chromatin structure of lymphoid cells under the influence of low-intensity extremely high-frequency electromagnetic radiation against the background of inflammatory process

Using the alkaline single cell gel electrophoresis technique (comet assay), changes in chromatin structure of peripheral blood leukocytes and peritoneal neutrophils have been studied in mice exposed to low-intensity extremely high-frequency electromagnetic radiation (42.2 GHz, 0.1 mW/cm², 20 min at 1 h after induction of inflammation) against the background of the systemic inflammatory process. It was revealed that the exposure of mice with the developing inflammation leads to a pronounced decrease in the level of DNA damage to peripheral blood leukocytes and peritoneal neutrophils. It is supposed that the changes in the chromatin structure of lymphoid cells have a genoprotective character in the inflammatory process and can underlie the mechanisms of realization of antiinflammatory effects of the electromagnetic radiation.     

Cytoarchitectonics of the cerebral cortex in mice selected for high and low brain weight

The cortex cytoarchitectonics was studied in mice, which had been genetically selected for high (H-line) and low (L-line) brain weight and manifested some differences in their behaviour. Microscopic brain sections were analysed to estimate the areas of the neocortex, archicortex and paleocortex and to investigate quantitatively the microstructure in different zones of the neocortex. In the H-line, absolute value of total cortical area is, on the average, by 18% higher than that in the L-line--mainly owing to the larger area of neocortex, while the mean values of neurone volume fraction and profile field reveal no significant difference between the H- and L-line. Hence, the total number of neurones in the neocortex of mice from H-line should be greater, as compared with the L-line. Besides, within the framework of the same cerebral organization in general, the obtained data are indicative of a somewhat higher spatial differentiation of the neocortex in mice from the H-line.     

Sodium oxybutyrate and piracetam acceleration of the compensatory-reparative processes after damage to the cerebral cortex in rats

The effects of sodium hydroxybutyrate and piracetam on compensatory-reparative processes in the central nervous system have been investigated in rats after extirpation of the frontal cortex. The animals were pretrained to conditioned reflex of active avoidance. Extirpation of the frontal cortex has been shown to disturb the conditioned reflex. Sodium hydroxybutyrate (50 mg/kg) and piracetam (200 mg/kg) were found to precipitate the recovery of the damaged reflexes.     

Microbial associations in chronic respiratory tract diseases

In the peripheral blood of patients with chronic bronchitis the presence of Mycobacterium pneumoniae antigens has been registered in 20.0% of cases and the presence of group A Streptococcus haemolyticus antigens, in 24.0% of cases, the transformation of streptococci into the L-form being observed in 12.0% of cases. The presence of M. pneumoniae, streptococci and their L-forms, as well as associations of these microorganisms, is characteristic of patients with chronic obstructive bronchitis, which is, probably, one of the reasons for the maintenance of bronchial obstruction. In patients with chronic pneumonia and fibrosing alveolitis the antigens of the bacterial form of streptococcus and its L-forms have been detected only in a few cases.     

Inhibitors of Antioxidant Enzymes Systemically Protect Cucumber Plants from Scab

Russian Journal of Plant Physiology - The study is aimed at the induction of systemic disease resistance by a local oxidative burst caused by inhibition of plant antioxidant enzymes. A possible...     

Critical Role of Nucleostemin in Pre-rRNA Processing

Nucleostemin is a nucleolar protein widely expressed in proliferating cells. Nucleostemin is involved in the regulation of cell proliferation, and both depletion and overexpression of nucleostemin induce cell cycle arrest through the p53 signaling pathway. Although the presence of p53-independent functions of nucleostemin has been previously suggested, the identities of these additional functions remained to be investigated. Here, we show that nucleostemin has a novel role as an integrated component of ribosome biogenesis, particularly pre-rRNA processing. Nucleostemin forms a large protein complex (>700 kDa) that co-fractionates with the pre-60 S ribosomal subunit in a sucrose gradient. This complex contains proteins related to pre-rRNA processing, such as Pes1, DDX21, and EBP2, in addition to several ribosomal proteins. We show that the nucleolar retention of DDX21 and EBP2 is dependent on the presence of nucleostemin in the nucleolus. Furthermore, the knockdown of nucleostemin delays the processing of 32 S pre-rRNA into 28 S rRNA. This is accompanied by a substantial decrease of protein synthesis as well as the levels of rRNAs and some mRNAs. In addition, overexpressed nucleostemin significantly promotes the processing of 32 S pre-rRNA. Collectively, these biochemical and functional studies demonstrate a novel role of nucleostemin in ribosome biogenesis. This is a key aspect of the role of nucleostemin in regulating cell proliferation.Nucleostemin (NS)² is a nucleolar protein preferentially expressed in actively proliferating cells. The structure of NS is characterized by two GTP-binding domains, which are involved in the regulation of its dynamic shuttling between the nucleolus and nucleoplasm (1). NS was originally identified as a nucleolar protein prominently expressed in rat neural stem cells and down-regulated during differentiation of these cells in vitro (2). The same authors also found that NS is widely expressed in neural precursor cells in early mouse embryos as well as in a variety of cancer cells and stem cells, including embryonic stem cells and a hematopoietic stem cell-enriched fraction. NS is generally down-regulated in the early stage of differentiation before exit from the cell cycle. In addition, knockdown of NS significantly inhibits proliferation of cortical stem cells and cancer cells. These initial observations led to suggestions that NS is involved in multipotency in stem cells as well as in the regulation of cancer and stem cell proliferation (2).Recent work, however, has demonstrated that NS is in fact widely expressed in many types of normal proliferating cells at levels similar to those in malignant cells. For instance, NS is expressed in normal kidney cells and renal carcinoma cells at comparable levels as detected in histological sections (3). The expression of NS is significantly up-regulated when normal T lymphocytes are activated by concanavalin A (3) and when bone marrow stem cells are stimulated by fibroblast growth factor 2 (4). Cells in NS-null mouse embryos fail to enter the S phase, resulting in embryonic death at the blastocyst stage (5, 6). In early Xenopus embryos NS is also expressed in the sites of active cell proliferation and local depletion of NS results in a decrease in proliferating neural progenitor cells (6). Based on these observations, it was proposed that expression of NS is more closely linked with cell proliferation than with the malignant state or differentiation status of a cell.Several studies have provided evidence that the p53 signaling pathway is involved in the G₁ arrest of the cell cycle induced by the down-regulation of NS. Physical interaction between NS and p53 was initially reported by Tsai and McKay (2). Later, it was shown that the G₁ arrest requires the presence of p53 (7). In the most recent study Dai et al. (8) showed that knockdown of NS enhances the interaction between the p53-binding protein MDM2 and the ribosomal protein L5 or L11, preventing MDM2 from inducing ubiquitylation-based p53 degradation. However, other studies have also suggested that NS may have a p53-independent role in the regulation of cell proliferation. For instance, the depletion of p53 from NS-null blastocysts did not rescue them from the embryonic lethality (6). In addition, NS partial loss-of-function in mouse fibroblasts did not result in any change in the p53 level (5). Furthermore, knockdown of L5 and L11 only partially rescued the G₁ arrest in NS knockdown cells (8). Finally, the fact that NS is primarily localized in the nucleolus, whereas the p53-mediated mechanism occurs in the nucleoplasm, suggests that NS might have an additional role more directly relevant to nucleolar functions.To identify novel functions of NS, we purified an endogenous NS complex from HeLa cell extract and investigated whether NS interacts with other proteins not described previously. Identification of the components of this complex and the alterations of the expression level of NS in HeLa cells led us to uncover a novel role of NS in the processing of rRNA. Our findings not only provide supporting evidence for the hypothesis that NS has a p53-independent function but also demonstrate that NS is critical for ribosome biogenesis, one of the most fundamental processes common for all cell types.