Binding of the S7 Protein with Fragment 926–986/1219–1393 of the 16S rRNA As a Key Step in the Assembly of the Small Subunit of Prokaryotic Ribosomes

Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coliandThermus thermophilusS7 with a fragment of the 3" domain of the E. coli16S rRNA. Both proteins showed high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA–protein interactions.     

Activity of the nucleolar organizers in cultured cells from the bone marrow erythroblastic islets

The erythroblast islands of the bone marrow are the morphofunctional units of erythropoiesis. In this work, the functional state of erythroblast islands' cells of the bone marrow for the first time was defined by estimation of the activity of the nucleolar organizers of erythroid cells in the erythroblast islands cultivated during 24 and 48 hours in presence of various doses of erythropoietin. The findings indicated that an increase in doses of erythropoietin was accompanied by a corresponding increase of the activity of nucleolar organizers in erythrokaryocytes of erythroblast islands. The nucleolar organizers of erythroid cells in cultures of erythroblast islands responded with activation to very small doses of erythropoietin; besides, a proliferative response of erythrokaryocytes was observed after activation of the nucleolar organizers.     

Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon

In this work it is shown by deletion analysis that an intercistronic region (ICR) approximately 80 nucleotides in length is necessary for interaction with recombinant E. coli S7 protein (r6hEcoS7). A model is proposed for the interaction of S7 with two ICR sites-region of hairpin bifurcations and Shine-Dalgarno sequence of cistron S7. A de novo RNA binding site for heterologous S7 protein of Thermus thermophilus (r6hTthS7) was constructed by selection of a combinatorial RNA library based on E. coli ICR: it has only a single supposed protein recognition site in the region of bifurcation. The SERW technique was used for selection of two intercistronic RNA libraries in which five nucleotides of a double-stranded region, adjacent to the bifurcation, had the randomized sequence. One library contained an authentic AG (?82/?20) pair, while in the other this pair was replaced by AU. A serwamer capable of specific binding to r6hTthS7 was selected; it appeared to be the RNA68 mutant with eight nucleotide mutations. The serwamer binds to r6hTthS7 with the same affinity as homologous authentic ICR of str mRNA binds to r6hEcoS7; apparent dissociation constants are 89 ± 43 and 50 ± 24 nM, respectively.     

The number of the rat peritoneal cells in normal condition, blood loss, and under colchicine injection

The modification of peritoneal cells derivation method proposed for the peritoneum rat, and the absolute amount of peritoneal lymphocytes, monocytes, macrophages, stem and polymorphonuclear neutrophyles, mat cells was determined. The procedure of acute 2% blood loss was found to reduce the total amount of the peritoneal cells due to depletion of lymphocytes accompanied by an increase in amount and proliferative activity of monocyte peritoneal cells, stem neutrophiles appearance in the peritoneal cavity. The subcutaneous injection of colchicine solution reduces the number of the peritoneal macrophages in normal rats, decreases the number of the monocytes, polymorphonuclear neutrophiles and led to disappearance of the stem neutrophiles on the second day of blood loss.     

Electric Field Exposure Triggers and Guides Formation of Pseudopod-Like Blebs in U937 Monocytes

We describe a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells stimulated by nanosecond pulsed electric field (nsPEF). In contrast to “regular,” round-shaped blebs, which are often seen in response to cell damage, pseudopod-like blebs (PLBs) formed as longitudinal membrane protrusions toward anode. PLB length could exceed the cell diameter in 2 min of exposure to 60-ns, 10-kV/cm pulses delivered at 10–20 Hz. Both PLBs and round-shaped nsPEF-induced blebs could be efficiently inhibited by partial isosmotic replacement of bath NaCl for a larger solute (sucrose), thereby pointing to the colloid-osmotic water uptake as the principal driving force for bleb formation. In contrast to round-shaped blebs, PLBs retracted within several minutes after exposure. Cells treated with 1 nM of the actin polymerization blocker cytochalasin D were unable to form PLBs and instead produced stationary, spherical blebs with no elongation or retraction capacity. Live cell fluorescent actin tagging showed that during elongation actin promptly entered the PLB interior, forming bleb cortex and scaffold, which was not seen in stationary blebs. Overall, PLB formation was governed by both passive (physicochemical) effects of membrane permeabilization and active cytoskeleton assembly in the living cell. To a certain extent, PLB mimics the membrane extension in the process of cell migration and can be employed as a nonchemical model for studies of cytomechanics, membrane–cytoskeleton interaction and cell motility.     

Selection of random RNA fragments as method for searching for a site of regulation of translation of <Emphasis Type="Italic">E. coli</Emphasis> streptomycin mRNA by ribosomal protein S7

In E. coli cells ribosomal small subunit biogenesis is regulated by RNA-protein interactions involving protein S7. S7 initiates the subunit assembly interacting with 16S rRNA. During shift-down of rRNA synthesis level, free S7 inhibits self-translation by interacting with 96 nucleotides long specific region of streptomycin (str) mRNA between cistrons S12 and S7 (intercistron). Many bacteria do not have the extended intercistron challenging development of specific approaches for searching putative mRNA regulatory regions, which are able to interact with proteins. The paper describes application of SERF approach (Selection of Random RNA Fragments) to reveal regulatory regions of str mRNA. Set of random DNA fragments has been generated from str operon by random hydrolysis and then transcribed into RNA; the fragments being able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to single serfamer, 109 nucleotide long (RNA109), derived from the intercistron. After multiple copying and selection, the intercistronic mutant (RNA109) has been isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than authentic intercistronic str mRNA; apparent dissociation constants are 26 +/- 5 and 60 +/- 8 nM, respectively. Location of S7 binding site on the mRNA, as well as putative mode of regulation of coupled translation of S12 and S7 cistrons have been hypothesized.     

the effect of colonystimulating macrophage factor on activity of the nuclear organisers in central macrophages of the cultures of the bone marrow erythroblastic islands

Erythroblastic islands of the bone marrow are morpho-functional units of erythropoiesis. The functional state of erythroblastic islands' cells of the bone marrow was for the first time defined by estimation of activity of the nuclear organisers of the central macrophages in the erythroblastic islands cultivated for 24 hrs in presence of various doses of the colony-stimulating macrophage factor. The findings indicate that increased doses of the colonystimulating macrophage factor was accompanied by a respective enhancement of the activity of nucleolar organisers in central macrophages of erythroblastic islands.