[Peripheral refraction and retinal contour in children with myopia by results of refractometry and partial coherence interferometry].

OBJECTIVE: To determine the posterior pole contour of the eye based on the relative peripheral refractive error and relative eye length. MATERIAL AND METHODS: A parallel study was performed, which enrolled 38 children (76 eyes) with myopia from -1.25 to -10.82 diopters. The patients underwent peripheral refraction assessment with WR-5100K Binocular Auto Refractometer ("Grand Seiko", Japan) and partial coherence tomography with IOLMaster ("Carl Zeiss", Germany) for the relative eye length in areas located 15 and 30 degrees nasal and temporal from the central fovea along the horizontal meridian. RESULTS: In general, refractometry and interferometry showed high coincidence of defocus signs and values for the areas located 15 and 30 degrees nasal as well as 15 degrees temporal from the fovea. However, in 41% of patients defocus signs determined by the two methods mismatched in one or more areas. Most of the mismatch cases were mild myopia. CONCLUSION: We suppose that such a mismatch is caused by optical peculiarities of the anterior eye segment that have an impact on refractometry results.

Correlates of measures of voluntary force with the functional state of the motor system.

Oscillation spectra were analyzed for prolonged isometric force recorded in healthy subjects of three age groups. Changes in the distributions of spectral components of the oscillations in force were noted, along with differences in the distributions of spectral density as exhaustion developed in the age groups. The amplitude-frequency ranges of changes in the spectral densities of oscillations in force characterized the activity at the suprasegmental and segmental levels of the motor system which support the voluntary control and automatic regulation of posture during the performance of movements. Correlates of the functional state of the motor system are discussed in terms of the voluntary and involuntary components of control. A significant increase in activity in the central structures of the movement control system was seen with the development of exhaustion, along with decreases in the frequency range of the activity of subcortical structures with age.

[Parameters of voluntary effort correlates with functional state of the motor control system].

The spectrum oscillations of a prolonged supported effort that was registered at isometric regimen in 3 age groups of healthy volunteers, were analyzed. Changes in distribution of spectral density of effort oscillations and distinction in distribution of spectral density as a reaction to fatigue in age groups, were shown. The revealed amplitude-frequency ranges of changes of spectral density of effort oscillations characterize activity of suprasegmental and segmental levels of motor control system fulfilling the voluntary movement control and the automatic regulation of posture. The correlates with functional state of motor control system are considered in the terms of voluntary and involuntary components of regulation. The substantial growth of activity in the central structures of motor control system in process of development of fatigue and the narrowing of the frequency range of activity of sub-cortical structures with age, were revealed.

[Characteristics of microsporogenesis in the Siberian larch growing under the conditions of technogenic load].

Studies of microsporogenesis in the Siberian larch growing in Krasnoyarsk and its suburbs have shown that meiosis starts in October. Microsporocytes winter at prophase I (leptoneme, diploneme). Reduction divisions in male generative buds are resumed and terminated in spring, in March. However, in the case of thaws during the autumn-winter period, meiotic division proceeds in the larch buds and this leads to the formation of degrading tetrads and pollen. Hence, the organic quiescence is absent in the larch in winter. It was shown that in the larch growing in the city, meiosis proceed more asynchronously than in the background tree stands. An increase of chromosomal aberrations during the reduction division was noted under the conditions of technogenic pollution.

Some problems of molecular biology of poliovirus infection relevant to pathogenesis, viral spread and evolution.

Molecular mechanisms of poliovirus reproduction in the human gut remain largely unexplored. Nevertheless, there are grounds to believe that the virus spreads from cell to cell, like that from person to person during natural circulation, and involves a relatively small proportion of the highly heterogeneous viral population generated by the previous host. This mechanism of random sampling is responsible for the majority of fixed mutations, and contributes to the maintenance of a certain level of viral fitness (virulence). In the long term, random sampling may lead to the decrease in fitness and even to extinction of some viral evolutionary branches, explaining cases of self-limiting poliovirus infection in immunodeficient patients. A low propensity of the Sabin viruses for natural circulation may also be a related phenomenon. The trend to decrease in fitness may be interrupted by the appearance of rare, fitter (more virulent) variants, which may be responsible for poliomyelitis outbreaks caused by wild type virus, and for the development of paralytic disease in chronic carriers of the Sabin vaccine. All these evolutionary events are largely stochastic and hence are unpredictable in principle.

Competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle.

Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first approximately 2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors.

Two types of death of poliovirus-infected cells: caspase involvement in the apoptosis but not cytopathic effect.

The death of poliovirus-infected cells may occur in two forms: canonical cytopathic effect (CPE) (on productive infections) or apoptosis (when the viral reproduction is hindered by certain drugs or some other restrictive conditions). Morphological manifestations of the CPE and apoptosis, being distinct, share some traits (e.g., chromatin condensation and nuclear deformation). It was shown here that a permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD.fmk), prevented the development of the poliovirus-induced apoptosis on abortive infection. The apoptotic pathway could be dissected by an inhibitor of chymotrypsin-like serine proteases, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), which prevented the cleavage of DNA to oligonucleosome-sized pieces and nuclear fragmentation but did not suppress cellular shrinkage, cytoplasmic blebbing, and partial chromatin condensation. These results demonstrate that caspase activation is involved in the execution phase of the viral apoptosis and suggest that a nuclear subset of the apoptotic program is under a separate control, involving a TPCK-sensitive event. Neither zVAD.fmk nor TPCK, at the concentrations affecting the apoptotic response, exerted appreciable influence on the virus growth or cellular pathological changes on productive infection, indicating that the pathways leading to the poliovirus-evoked CPE and apoptosis are different.

Final checkpoint in the drug-promoted and poliovirus-promoted apoptosis is under post-translational control by growth factors.

The treatment of HeLa subline (HeLa-B) cells with cycloheximide or Actinomycin D resulted in a rapid (approximately 1.5 h and approximately 2.5 h, respectively) development of morphological and biochemical signs of apoptosis. The addition of fetal bovine serum to the cycloheximide-treated or Actinomycin D-treated cells suppressed the apoptotic reaction, as evidenced by the postponement of the DNA fragmentation for at least 9 and 5 h, respectively. A similar suppressive effect was observed upon the serum addition to cells undergoing abortive infection with poliovirus, which died of apoptosis in the absence of the serum. The serum appeared to exert its anti-apoptotic effect without any appreciable lag and even immediately blocked further progress of ongoing DNA fragmentation. The epidermal growth factor also suppressed, although less efficiently and more transiently, the apoptotic reaction promoted by the metabolic inhibitors. It is concluded that growth factors may affect, without modulating either transcription or translation, the balance of pro-apoptotic and anti-apoptotic activities at a final checkpoint, just preceding the irreversible effector step of apoptosis.

Apoptosis-inducing and apoptosis-preventing functions of poliovirus.

Data showing that an apoptotic reaction (the exit into the cytoplasm and nucleolytic internucleosomal degradation of chromosomal DNA, compaction and fragmentation of chromatin, cellular shrinkage, and cytoplasmic blebbing) developed in a subline of HeLa-S3 cells upon nonpermissive poliovirus infection with either a guanidine-sensitive poliovirus in the presence of guanidine, a guanidine-dependent mutant in the absence of guanidine, or certain temperature-sensitive mutants at a restrictive temperature are presented. Essentially, no apoptotic reaction occurred upon permissive infection of these cells. Both permissive and nonpermissive infections resulted in the inhibition of host protein synthesis. Actinomycin D or cycloheximide also elicited a rapid apoptotic reaction in uninfected cells. However, preinfection or coinfection with poliovirus prevented the apoptotic response to the addition of actinomycin D, and preinfection blocked cycloheximide-induced apoptosis as well. These data fit a model in which the cells used are prepared to develop apoptosis, with their viability due to the presence of certain short-lived mRNA and protein species. Poliovirus infection turns on two oppositely directed sets of reactions. On the one hand, the balance is driven toward apoptosis, probably via the shutoff of host macromolecular synthesis. On the other hand, viral protein exhibits antiapoptotic activity, thereby preventing premature cell death. To our knowledge, this is the first description of an antiapoptotic function for an RNA virus.

Genetic studies on the poliovirus 2C protein, an NTPase. A plausible mechanism of guanidine effect on the 2C function and evidence for the importance of 2C oligomerization.

Poliovirus RNA replication is known to be inhibited by millimolar concentrations of guanidine. A variety of guanidine-resistant (gr) and guanidine-dependent (gd) poliovirus strains were selected, and mutations responsible for the phenotypic alterations were mapped to distinct loci of the viral NTP-binding pattern containing protein 2C. Together with already published results, our data have demonstrated that the overwhelming majority of guanidine mutants of poliovirus 2C can be assigned to one of the two classes, N (with a change in Asn179) or M (with a change in Met187). As inferred from the structure/function relations in other NTP-binding proteins, both these "main" mutations should reside in a loop adjoining the so-called B motif known to interact with the Mg2+ involved in the NTP splitting. In classes M (always) and N (not infrequently), these B motif mutations were combined with mutations in, or close to, motif A (involved in binding of the NTP phosphate moieties) and/or motif C (another conserved element of a subset of NTP-binding proteins). These data strongly support the notion that the region of polypeptide 2C involved in the NTP utilization is affected by the guanidine mutations and by the presence of the drug itself. The mutations, however, never altered highly conserved amino acid residues assumed to be essential for the NTP binding or splitting. These facts and some other considerations led us to propose that guanidine affects coupling between the NTP binding and/or splitting, on the one hand, and the 2C function (related to conformational changes), on the other. Both N and M classes of mutants contain gr and gd variants, and the gr/gd interconversion as well as modulations of the guanidine phenotype can be caused by additional mutations within each class; sometimes, these additional substitutions are located far away from the "main" mutations. It is suggested that the target for guanidine action involves long-range tertiary interactions. Under conditions restrictive for the individual growth of each parent, efficient reciprocal intra-allelic complementation between guanidine-sensitive (gs) and gd strains (of M or N classes) was observed. The complementation occurred at the level of viral RNA synthesis. These data allowed us to propose that oligomerization of polypeptide 2C is an essential step in the replication of viral genome.

Insertion of short hepatitis virus A amino acid sequences into poliovirus antigenic determinants results in viable progeny.

In an infectious poliovirus cDNA construct, the determinant encoding antigenic epitope N-Ag1 (in a loop located between two beta-strands in poly-peptide VP1) was altered by site-directed mutagenesis, to be partially similar with the determinants for presumptive epitopes in polypeptides VP1 or VP3 of hepatitis A virus (HAV). The modified constructs proved to be infectious. However, another construct, in which the same locus encoded a 'nonsense' and a relatively hydrophobic amino acid sequence, exhibited no infectivity. These data showed the feasibility of the insertion of foreign sequences in a specific antigenically active locus of the poliovirus icosahedron, and suggest some limitations with respect to the sequences to be 'transplanted'.

Conserved structural domains in the 5'-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence.

A model of secondary structure common for the central part (ca. 400 nucleotides) of the 5'-untranslated regions (5'-UTR) of all the so far sequenced genomes of polioviruses, coxsackieviruses, and rhinoviruses was derived on the basis of evolutionary and thermodynamic considerations. According to the model, this part of the genome comprises three domains, which appear to be involved, at least in the poliovirus genome, in the control of viral neurovirulence and in vitro translation. Some salient features of this model were supported by investigating RNAs of five poliovirus and one coxsackievirus strains with respect to their accessibility to modifications with dimethyl sulfate and sensitivity to single-strand- and double-strand-specific nucleases. In contrast to the previous suggestion, no major changes in the conformation of the Sabin vaccine poliovirus type 3 5'-UTR due to the transition in position 472 were observed. The biological relevance of the conserved primary and secondary structure elements in the picornaviral 5'-UTRs is discussed.

Studies on the recombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants.

A series of intertypic (type 3/type 1) poliovirus recombinants was obtained whose crossover sites were expected to be located in the middle of the viral genome, between the loci encoding type-specific antigenic properties, on the 5' side, and an altered sensitivity to guanidine, on the 3' side. The primary structures of the crossover regions in the genomes of these recombinants were determined by the primer extension method. The length of the crossover sites (the uninterrupted sequences shared by the recombinant and both parental genomes that are flanked, in the recombinant RNAs, by two heterotypic segments) varied between 2 and 32 nucleotides, but the majority of the sites were 5 nucleotides long or shorter. The crossover sites were nonrandomly distributed over the presumably available genome region: only a single such site was found within the gene for polypeptide 2A, whereas an apparent clustering of the crossover sites was encountered in other genomic segments. When the crossover sites were superimposed on a model of the secondary structure of the relevant region of the viral RNA molecule, a pattern consistent with the previously proposed mechanism of poliovirus recombination (L.I. Romanova, V.M. Blinov, E.A. Tolskaya, E.G. Viktorova, M.S. Kolesnikova, E.I. Guseva, and V.I. Agol (1986) Virology 155, 202-213) was observed. It is suggested that the nonrandom distribution of the crossover sites in the genomes of intertypic poliovirus recombinants was due to two factors: the existence of preferred sites for recombination, and selection against recombinants with a lowered level of viability.

The primary structure of crossover regions of intertypic poliovirus recombinants: a model of recombination between RNA genomes.

The nucleotide sequence of crossover sites in the genome of four intertypic (type 3/type 1) poliovirus recombinants has been determined. The approximate boundaries of the crossover regions were first estimated by RNase T1 oligonucleotide mapping of the recombinant genomes; then appropriate regions were sequenced by the chain termination method using oligonucleotide-primed reverse transcription of the recombinant RNAs. The crossover sites (defined as the contiguous sequences shared by the recombinant and both parental genomes flanked, in the recombinant genome, by heterotypic RNA segments) are 5, 5, 7, and 11 nucleotides long, respectively. The recombination was precise and was not accompanied by any other genetic alterations. The recombination sites were found to be located within genome segments having a potential to form secondary structure elements. Based on this observation, a model of recombination between picornaviral RNA genomes has been proposed. The essence of this model consists in bringing together homologous regions of two recombining RNA genomes via formation of intermolecular duplexes, detachment of the nascent 3' end of the newly synthesized complementary RNA from a "parting" site on the first template and its subsequent "jumping" to the identical (or closely related) "anchoring" site on the other template. Features of this model are discussed in some detail.

[Primary structure of the crossover region in the genome of two intertype poliovirus recombinant]. / Pervichnaia struktura oblasti perekresta v genome dvukh mezhtipovykh rekombinantov poliovirusa.

The nucleotide sequence of the crossover region on genomes of two intertypic (type 3/type 1) poliovirus recombinant, has been determined by the primer extension method. No deletions, insertions or rearrangements have been observed. Identical contiguous sequences, 7 or 11 nucleotides in length, respectively, have been found in two regions of the parental genomes, involved in the recombination.

Recombinants between attenuated and virulent strains of poliovirus type 1: derivation and characterization of recombinants with centrally located crossover points.

Recombinants with a centrally located crossover point were selected from crosses between poliovirus type 1 strains and intertypic (type 3/type 1) recombinants. Two such recombinants were characterized in some detail. In one of them (v1/a1-6), the 5' half of the genome was derived from a virulent type 1 strain, while the 3' half came from an attenuated type 1 strain. The genome of the other recombinant (a1/v1-7) had the reverse organization, with the 5' and 3' halves being derived from the type 1 attenuated and virulent strains, respectively. As deduced from the RNase T1 oligonucleotide maps, the a1/v1-7 genome also had a relatively short centrally located insert of the poliovirus type 3 origin. Both recombinants exhibited ts phenotypes. The RNA phenotypes of the recombinants corresponded to that of the parent donating the 3' half of the genome, v1/a1-6 and a1/v1-7 expressing RNA- and RNA +/- characters, respectively. Despite being a ts RNA- virus, v1/a1-6 proved to be neurovirulent when injected intracerebrally into Cercopithecus aethiops monkeys, although it exhibited a somewhat diminished level of pathogenicity as compared to its virulent type 1 parent. Recombinant a1/v1-7 behaved as an attenuated strain. These data supported our previous conclusion drawn from the experiments with intertypic poliovirus recombinants that the attenuated phenotype of poliovirus depends largely on the structure of the 5' half of its genome, although mutations of the 3' half may alleviate the virulence of the virus to a degree.

Neurovirulence of the intertypic poliovirus recombinant v3/a1-25: characterization of strains isolated from the spinal cord of diseased monkeys and evaluation of the contribution of the 3' half of the genome.

A tsRNA- intertypic recombinant, v3/a1-25, which has the 5' and 3' halves of the genome derived from the neurovirulent type 3 poliovirus strain 452/62 3D and the attenuated type 1 poliovirus strain LSc-gr3, respectively, was previously shown to cause severe paralytic poliomyelitis after intracerebral inoculation of monkeys. To ascertain whether the illness was caused by the recombinant itself or by temperature-resistant trRNA+ mutants that might have arisen in the inoculated monkeys, five independent virus strains have been isolated from the spinal cord of the diseased animals. While two of these isolates exhibited RNA+ and RNA +/- phenotypes, respectively, the other three strains retained the parental RNA- character. Except for the RNA+ strain, the RNase T1 oligonucleotide maps of the genomes of all the isolates revealed only a minimal deviation from the parental pattern. These results were interpreted to mean that v3/a1-25 is intrinsically neurovirulent despite the presence of a tsRNA- mutation(s) in the 3' half of its genome. Nevertheless, this mutation, or other peculiarities of the 3' half of the recombinant genome, may somewhat alleviate the pathogenicity of the virus. This notion was inferred from the fact that, when used in a relatively small dose (about 10(3) p.f.u.), v3/a1-25 appeared to exhibit a lower level of neurovirulence compared to either the wild-type parent 452/62 3D, or a closely related intertypic recombinant having the genome 3' half derived from a neurovirulent trRNA +/- type 1 poliovirus strain. The problem of genetic determination of poliovirus neurovirulence and attenuation is briefly discussed.

Construction and properties of intertypic poliovirus recombinants: first approximation mapping of the major determinants of neurovirulence.

An attempt was made to map, in a general way, the region of the poliovirus genome that is responsible for the neurovirulent and attenuated phenotypes of different virus strains. A set of four recombinants was investigated, one described previously (E. A. Tolskaya, L. I. Romanova, M. S. Kolesnikova, and V. I. Agol, 1983, Virology 124, 121-132) and three obtained in the present work with the following genetic structure: a 5' end-adjacent segment of the genome derived from either a virulent strain (452/62 3D), or from an attenuated strain (Leon-2) of poliovirus type 3, the remaining RNA sequences being derived from either a virulent strain (Mgr), or an attenuated strain (LSc-gr3) of poliovirus type 1. The crossover points in the recombinant genomes were centrally located, somewhere between the gene(s) that determines antigenic specificity of the virus and the locus that determines resistance of virus multiplication to low doses of guanidine. The recombinant nature of the newly selected clones was definitively established by mapping RNase T1 oligonucleotides of their genome. The recombinants were characterized with respect to their ability to produce infectious progeny and synthesize viral RNA at an elevated temperature. Neurovirulence of the recombinants was assayed by intracerebral inoculation of monkeys. Irrespective of the origin of the 3' end-adjacent segment of the genome, the recombinants that inherited the 5' end-adjacent segment from the neurovirulent parent were neurovirulent, whereas the recombinants with the 5' end-adjacent segment derived from the attenuated parent were not. The results suggest that the major determinants of neurovirulence of these recombinants (and by inference, of their parental viruses) reside in the 5' end-adjacent segment of poliovirus genome, known to code for capsid proteins.