[Replacement of Hydroxylated His39 in Ribosomal Protein uL15 with Ala or Thr Impairs the Translational Activity of Human Ribosomes].

Post-translational hydroxylation occurs in three mammalian ribosomal proteins, uS12, uL2, and uL15, which are located in the small (S) and large (L) subunits of the ribosome near the most important decoding and peptidyltransferase functional centers. We have used cell cultures, which produce protein uL15 labeled with the 3xFLAG epitope at the C-terminus (uL15^(3xFLAG)) or mutant forms of uL15^(3xFLAG) that contain His39Ala, His39Thr, or His40Ala substitutions, to examine the role of hydroxylated His39 of uL15 in maintaining the translational activity of ribosomes. It has been found that exogenous uL15^(3xFLAG) is able to functionally replace endogenous uL15 in HEK293 cells transfected with an appropriate DNA construct. However, the translational activity of ribosomes decreases by about 35% in cells that produce the above mutant forms of uL15^(3xFLAG) compared with that in cells that produce nonmutated uL15^(3xFLAG). Analysis of the structural model of the human ribosome has allowed us to assume that the hydroxyl group in His39 is involved in the local stabilization of the ribosome structure through the formation of a hydrogen bond between this group and the nitrogen atom of the His40 imidazole ring. Given that His39 is located near the E site of the ribosome, we believe that this stabilization of the ribosome structure ensures the maintenance of its translational activity.

Reproducible Peak Clusters on Differential Mouse Mortality Curves and Their Relation to the Gompertz Model.

It is shown that differentiation of mouse mortality curves (number of animals that died at a certain age plotted versus their lifespan) results in the appearance of eight clearly distinguished clusters of peaks corresponding to increased mortality rates. Smoothing of the original mortality curves and subsequent transformation of the differential mortality curves according to the Gompertz model makes the peaks and the corresponding clusters less pronounced and drives the logarithm of the force mortality curve toward a straight line. The positions of the clusters on the lifespan axis (expressed in days) were calculated as weighted means by dividing the sum of the products of multiplication of the peak heights and their position on the lifespan axis by the sum of the peak heights within a cluster. To prove that the peaks and their clusters are not random, we have demonstrated that the positions of the clusters on the lifespan axis do not depend on the extent of mortality curve smoothing or the group of mice analyzed.

Very late biolimus-eluting coronary stent thrombosis: case report.

The case of very late everolimus-eluting stent thrombosis in left arteria descendant (LAD) was presented. Risk factors and possible ways of this complication prevention are discussed.

New Data on Programmed Risks of Death in Normal Mice and Mutants with Growth Delay.

Study of the lifespans of normal (non mutant) mice and growth delay mutants has shown that mortality rates for both kinds of animals exhibit reproducible fluctuations. In the case of the mutant mice, the positions of peaks on the differential mortality curves (mortality rate plotted against lifespan) coincided in different-sex groups of animals and in same-sex subgroups of animals. Differential mortality curves of the mutant mice also had a peak at 1 month of age that was absent from the differential mortality curves of the normal mice. In the case of normal animals, positions of most peaks were the same in the studied independent subgroups of males, and to a lesser extent - independent subgroups of females, which might be explained by a shift in mortality peak positions due to the reproductive activity of females. Similar positions of mortality rate peaks in the differential mortality curves for animals from independent groups and subgroups indicate the existence of increased risks of death at specific ages. The observed pattern could be due to the programming in the genome of both the periods of increased risk of death and the intermitting intervals of stable development.

[CONTRIBUTION OF LIPOPROTEIN(A) TO CARDIOVASCULAR RISK IN PATIENTS UNDER 40 YEARS OF AGE AFTER ACUTE MYOCARDIAL INFARCTION OR ACUTE CEREBRAL CIRCULATION DISORDER].

AIM: To evaluate the importance of lipoprotein(a) for the evaluation of cardiovascular risk in patient under 40 years of age after acute myocardial infarction or acute cerebral circulation disorder. MATERIALS AND METHODS: We analysed the data from two departments of the Regional Vascular Centre for 2013-2015 including 90 case histories of patients of different age (mean 57.8 ± 3.4 yr) and studied standard risk factors, such as age, sex, smoking habits, dyslipidemia, aggravated heredity, arterial hypertension (AH), obesity. Standard examination of 7 patients under 40 years of age was supplemented by measuring lipoprotein(a) by the immunoturbodimetric method regarding the levels over 0.3 g/l as abnormally elevated. RESULTS: The study group was dominated by young and middle-aged men (85.2 and 84% respectively). The key risk factors were increased LDLP level (88%) and smoking (70%) in patients under the age of 40 and AH in middle-aged men (100%, p < 0.004). Arterial hypertension was also diagnosed in 59% of the younger subjects. Increased LDLP levels most frequently occurred in senior patients (90%). The group of patients under 40 yr included 15% of those having a single risk factor. In this group, 22% of the patients were at high risk calculated prior to the development of vascular events, 58% at moderate and 20% at low risk. 42.8% of the patients had elevated lipoprotein(a) levels. CONCLUSION: Based on the relative risk scoring scale, 22% of the patients under 40 years of age were at risk of myocardial infarction or cerebral circulation disorders prior to the development of vascular events. However, these patients like those of other age groups frequently had traditional risk factors, such as smoking (67.5%), AH and dyslipidemia (66.6% each). Total cholesterol was elevated only in 47.6% of the patients while LDLP and LP(a) in 92 and 42.8% respectively.

[Common features in arrangements of ribosomal protein S26e binding sites on its own pre-mRNA and the 18S rRNA].

It is known that human ribosomal protein (rp) S26e can bind to the first intron of its own pre-mRNA and thereby inhibit its splicing. In this work, hydroxyl radical footprinting was applied for detailed mapping of the rpS26e binding site on an RNA transcript corresponding to the rpS26e pre-mRNA fragment containing the first intron flanked by the first exon and a part of the second exon sequences. Nucleotides of this RNA protected from hydroxyl radical attack in the presence of rpS26e were identified. Most of them are found in the region of the 3'-splice site of the first intron within a purine-rich sequence, which forms a loop connecting two helices in the predicted secondary structure of the rpS26e pre-mRNA fragment, and the remaining nucleotides are located near the 5'-splice site. Comparison of arrangements of rpS26e binding sites on the pre-mRNA and 18S rRNA secondary structures reveals similar elements in the organization of these sites. It was found that both sites contain a structural motif, represented by an extended purine-rich loop between two helices, which could be recognized by rpS26e upon binding to these RNAs. The data obtained shed light on the structural aspects of RNA-protein interactions underlying autoregulation of human RPS26e gene expression at the splicing step.

Age fluctuations in mortality of mice with mutation causing growth retardation.

Lifespan of mice over a number of consecutive generations of descendants of a male with a mutation causing growth retardation was studied. The mutant and normally developing (normal) mice were obtained by crossbreeding of mutant males with normal females from the same brood. The mutant females were infertile. Mortality of the mutant and normal mice was shown to fluctuate depending on age. The curve of dependence of lifespan on their serial number in a series of lifespan increase (mortality rank curve) had the form of evident steps for the mutant mice, while in normal mice this feature was less pronounced. These steps indicate that in the course of development of mice stages with low mortality are alternately replaced by stages with increased mortality. One month after birth, the first stage of stable development of mutant males and females is replaced by a stage with abnormally high mortality, which coincides with the period of their maximal backlog in weight compared to the normal animals. Within two months, surviving mutants catch up in weight with normally developing mice and externally become indistinguishable from them. The steps are reproduced on mortality rank curves in mutant and normal mice, both in groups of mice of different sexes and in parallel same-sex groups. The observed phenomenon is interpreted within the hypothesis of a genetic aging program in mice that provides periodic changes when stages of great viability are followed by stages of increased sensitivity to the external risk factors causing death. Less-expressed steps on mortality rank curves of normal females were shown to be enhanced by the removal from the sample of parous females and animals with tumors. Results of the study indicate the possibility of detecting in humans of ontogenesis-programmed stages of high and low sensitivity to external influences and the prospect of the development of effective measures to prevent risks of premature death.

[Graduated change of life expectancy in mice in ontogenesis].

Life expectancy of descendants of a normal female mouse and a male with an inherited growth inhibition mutation discovered in a laboratory population was investigated. The hereditability of the characteristic allows us to consider it a result of mutation. It was shown that, in mice, the curve of dependence of life expectancy on their serial number in a row of increase in life expectancy (curve of rank distribution) has step-like shape for mutant males and females, as well as for males with normal development. The first grade of mice death on the curve of rank distribution was observed at one month after their birth and was characteristic only of males and females with a mutation during the period of maximum lag in weight as compared with their normal relatives. The surviving mutants catch up to the normally developing individuals within two months and externally become indistinguishable from them. The subsequent grades of death in mutants and normal males coincide on the time axis. The steps are absent on the rank curves of life expectancy of normally developing females. The time intervals between the steps are reproduced in parallel groups of mice and, hence, are not casual deviations from theoretical curves and are of a regular nature. The discovered phenomenon is interpreted within the scope of a hypothesis about the realization of the genetic program of ontogenesis, which provides periodic change of vitality stages with stages of sensitivity to external risk factors, which increase the probability of death, by mice. Absence of such stages in the group of normally developing females can be explained by shifts in development, which are produced by the irregular performance of reproductive functions.

[Mg2+ ions affect the structure of the central domain of the 18S rRNA in the vicinity of the ribosomal protein S13 binding site].

It is known that Mg2+ ions at high concentrations stabilize the structure of the 16S rRNA in a conformation favorable for binding to the ribosomal proteins in the course of the eubacterial 30S ribosomal subunits assembly in vitro. Effect of Mg2+ on the formation of the 18S rRNA structure at the 40S subunit assembly remains poorly explored. In this paper, we show that the sequentional increase of the Mg2+ concentration from 0.5 mM to 20 mM leads to a significant decrease of the affinity of recombinant human ribosomal protein S13 (rpS13e) to a RNA transcript corresponding to the central domain fragment of the 18S rRNA (18SCD). The regions near the rpS13e binding site in 18SCD (including the nucleotides of helices H20 and H22), whose availabilities to hydroxyl radicals were dependent on the Mg2+ concentration, were determined. It was found that increase of the concentrations of Mg2+ results in the enhanced accessibilities of nucleotides G933-C937 and C1006-A1009 in helix H22 and reduces those of nucleotides A1023, A1024, and A1028-S1026 in the helix H20. Comparison of the results obtained with the crystallographic data on the structure of the central domain of 18S rRNA in the 40S ribosomal subunit led to conclusion that increase of Mg2+ concentrations results in the reorientation of helices H20 and H24 relatively helices H22 and H23 to form a structure, in which these helices are positioned the same way as in 40S subunits. Hence, saturation of the central domain of 18S rRNA with coordinated Mg2+ ions causes the same changes in its structure as rpS13e binding does, and leads to decreasing of this domain affinity to the protein.

Proteins of the human 40S ribosomal subunit involved in hepatitis C IRES binding as revealed from fluorescent labeling.

Initiation of translation of genomic RNA (gRNA) of hepatitis C virus (HCV) is provided by a highly structured fragment in its 5'-untranslated region, the so-called Internal Ribosome Entry Site (IRES). In this work, the exposed NH2-groups of proteins in the 40S subunit of the human ribosome and in its binary complexes with RNA transcripts corresponding to the full-size HCV IRES or its fragments were probed using the N-hydroxysuccinimide derivative of the fluorescent dye Cy3. Comparison of efficiencies of modification of ribosomal proteins in free subunits and in their binary complexes with the RNA transcripts revealed ribosomal proteins involved in the HCV IRES binding. It was found that binding of the 40S subunits with the RNA transcript corresponding to full-size HCV IRES results in a decrease in modification levels of ribosomal protein (rp) S27 and, to a lesser extent of rpS10; also, a noticeable decrease in the efficiency of labeling of proteins RACK1/S2/S3a was observed. When a fragment of HCV IRES containing the initial part of the open reading frame (ORF) of the viral gRNA was deleted, the level of rpS10 modification became the same as in free subunits, whereas the levels of modification of rpS27 and the RACK1/S2/S3a group remained virtually unchanged compared to those observed in the complex of 40S subunit with the full-size HCV IRES. Binding of 40S subunits to a fragment of the HCV IRES lacking an ORF and domain II increased the modification level of the RACK1/S2/S3a proteins, while the efficiencies of labeling of rpS10 and rpS27 remained the same as upon the deletion of the ORF fragment. Comparison of these results with known structural and biochemical data on the organization of 40S subunit and the location of the HCV IRES on it revealed structural elements of the IRES contacting exposed lysine residues of the above-mentioned ribosomal proteins. Thus, it was found that the majority of exposed lysine residues of rpS27 are involved in the binding of the HCV IRES region formed by the junction of subdomains IIIa, IIIb, and IIIc with the central stalk of domain III, and that several lysine residues of rpS10 participate in the binding of the HCV IRES region corresponding to the initial part of the ORF of the viral gRNA. In addition, we concluded that lysine residues of rpS3a are involved in the binding of domains II and III of HCV IRES.

[Influence of carbon dioxide on the impregnation of the nervous tissue by silver].

It has been shown that 4% carbon dioxide (CO2) in the air above reaction mixture inhibits the initiation of the formation of silver nanoparticles from complexes with biogenic amines (noradrenaline and serotonin). At the same concentration of CO2 in the air above solution of AgNO3, which is used for staining nerve tissues by the method of Golgi, neurons are preferentially stained, whereas at a concentration of 0.06%, vessels and poor neurons are stained. It is suggested that the entry of free silver ions to neurons is due to the inhibition of sites of initiation of silver nanoparticles in vessels at high CO2 concentrations, while the lack of inhibition leads to silver precipitation in vessels at low CO2 concentrations. It can be assumed that, for stable silver impregnation, the concentration of CO2 must be controlled.

[The different effects of carbon dioxide on the toxicity of silver ions for prokaryotic and eukaryotic microorganisms].

The effect of carbon dioxide on survivability of bacteria Escherichia coli and the germination ability ofconidia of the fungus Neurospora crassa in the presence of silver nitrate was studied. It was shown that carbon dioxide increased the toxic effect of silver ions on prokaryotic cells of E. coli but did not change the survivability of spores of the eukaryote N. crassa.

[Binding of human ribosomal protein S13 to the central domain of 18S rRNA].

Human ribosomal protein S13 is a structural element of the small subunit of ribosome. It is a homologue of eubacterial ribosomal protein S15, and, besides, it possesses an extended N-terminal region, characteristic of the S15p family in eukaryotes and archaea. In the present study, we investigated binding of recombinant ribosomal protein S13 and its mutants containing deletions or substitutions of amino acid residues in different regions with an RNA transcript corresponding to a fragment of the central domain of 18S rRNA. We found that replacement of ultra-conservative residues H101 and D108 as well as deletions of either 29 C-terminal or 27 N-terminal residues substantially reduced affinity of the protein to the RNA transcript. Deletion of 54 C-terminal or 80 N-terminal residues completely deprived the protein of binding capacity. Using a footprinting assay, we identified sites in the RNA transcript changing their accessibilities to action of hydroxyl radicals under binding of either full-length protein S13 or its mutant lacking 27 N-terminal residues. It is shown that these sites are located mainly in helix H22 of the 18S rRNA and in the region of its junction with helix H20 and are consistent predominantly with contacts of the rRNA with the conserved part of the protein. We concluded that binding of ribosomal protein S13 to 18S rRNA is provided mainly by conserved motifs of the protein corresponding to those motifs in its eubacterial homologue that are involved in the interaction with 16S rRNA in the 30S subunit. Role of the N-terminal region of the protein in its binding to the central domain of 18S rRNA is discussed.

[Surgical treatment of vesicovaginal fistulas].

To improve surgical outcomes in patients with complicated recurrent, radiation-induced, giant and multiple vesicovaginal fistulas, we have developed a new combined method of fistuloplasty (patent 21350999). The method was used in 12 of 32 operations made in 1997-2007 in the urological clinic of the Kazan Medical University for vesicovaginal fistula in patients aged 19 to 72 years. The technique was applied in women with vesicovaginal fistula located close to the ureteral orifice. Good results of the operation were achieved due to leak-proof sutures, accurate dissection of the bladder from the vagina and intact blood supply of the tissues. Two surgical approaches were used: transvesical and vaginal. Neither complications nor relapses occurred in all 12 patients operated by the proposed technique of combined fistuloplasty which proved to be effective in recurrent, complicated, combined fistulas and is a method of choice in complicated, recurrent, radiation-induced, giant and multiple fistulas.

[Human ribosomal protein S16 inhibites excision of the first intron from its own].

Recombinant human ribosomal protein S16 (rpS16) is shown to bind specifically to a fragment of its own pre-mRNA that includes exons 1 and 2, intron 1, and part of intron 2, and to inhibit the splicing of the fragment in vitro. The weaker binding of other recombinant human ribosomal proteins, S10 and S13, to this pre-mRNA fragment indicated that the binding of rpS16 was specific. Besides, poly(AU) and rpS16 mRNA fragment affected poorly the binding of rpS16 to its pre-mRNA, providing another evidence that the interaction was specific. RpS16 specifically inhibited the pre-mRNA fragment splicing whereas recombinant rpS10 and rpS16 did not affect excision of intron from this pre-mRNA fragment in contrast to rpS16. Those positions in rpS16 pre-mRNA fragment that were protected by rpS16 against cleavage by RNases T1, T2 and V1 were found to be located closely to the branch point and 3' splice site in the pre-mRNA. Results obtained support the possibility of the autoregulation of rpS13 pre-mRNA splicing through feedback mechanism.

C-terminal fragment of human laminin-binding protein contains a receptor domain for venezuelan equine encephalitis and tick-borne encephalitis viruses.

Polyclonal and monoclonal antibodies (MABs) to human laminin-binding protein (LBP) can efficiently block the penetration of some alpha- and flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 protein and tick-borne encephalitis virus (TBEV) E protein. Mapping of binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187-210, and MAB 4F6 interacts with the fragment of residues 263-278 of LBP protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187-210 and 263-278) as well as the central beta-folded region turning into the alpha-helical site including residues 200-216 of the LBP molecule and providing for the interaction with the laminin-1 molecule has been proposed.

[Mutual effect of human ribosomal proteins S5 and S16 on their binding with 18S rRNA fragment 1203-1236/1521-1698].

Human ribosomal proteins S5 and S16 are homologues of prokaryotic ribosomal proteins S7p and S9p, respectively, that according to X-ray crystallography data on the Thermus thermophilus 30S ribosomal subunit contact the 3'-terminal 16S rRNA region formed by helices H28-H30 and H38-H43. In the present work we report studying mutual effect of human ribosomal proteins S5 and S16 on their binding with RNA transcript corresponding to the region 1203-1236/1521-1698 of the 18S rRNA (helices H28-30 and H41-43), which is homologous to thel6S rRNA region known to contain binding site of S7p and part of binding site of S9p. It was shown that simultaneous binding of ribosomal proteins S5 and S16 with this RNA transcript causes conformational changes in it stabilizing the complex by involvement of new parts of the RNA that interact with neither S5 nor S16 in the respective binary complexes.

[Molecular environment of the IIId subdomain of the IRES element of hepatitits C virus RNA on the human 40S ribosomal subunit].

The molecular environment of the key subdomain IIId of the internal ribosome entry site (IRES) element of hepatitis C virus (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, HCV IRES derivatives bearing perfluorophenylazido groups activatable by mild UV at nucleotide G263 or A275 in the subdomain IIId stem were used. They were prepared by the complementarily addressed modification of the corresponding RNA transcript with alkylating oligodeoxynucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA. It was found that the photoreactive groups of the IRES G263 and A275 nucleotides are crosslinked to ribosomal proteins S3a, S14, and S16. For the IRES derivative with the photoreactive group in nucleotide G263, the degree of modification of proteins S14 and S16 was greater than that of S3a, whereas the derivative containing the same photoreactive group in nucleotide A275 was mainly crosslinked to proteins S3a and S14. An analysis of the data led to the conclusion that, in the binary complex of HCV IRES elements with the small subunit of the 80S ribosome, its subdomain IIId stem is located on the outer subunit surface between the head and the body next to the "beak" near the exit of mRNA from the ribosome.