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The results of structural and functional analyses of harpin-like peptides from the EcAMP group isolated from the barnyard grass (E. crusgalli) seeds are summarized from the viewpoint of their participation in plant innate immunity to environmental biotic stress factors.

Successful disease prevention and therapy critically depend on timely diagnosis of infections. Quantitative immuno-PCR (qiPCR) technology improves the sensitivity in the detection of antibodies to pathogens. A qiPCR-based assay was developed to determine IgG antibodies to Epstein-Barr virus (EBV) in the human blood serum. EBV nuclear protein 1 fragment (pEBV) was expressed in Escherichia coli. A synthetic single-stranded deoxyribonucleotide was conjugated to streptavidin, and the conjugate was used to detect рEBV-IgG1-biotin complexes by qiPCR. The IgG1 titers determined by qiPCR were compared to the results of enzyme-linked immunosorbent assay (ELISA). The sensitivity of qiPCR was one order of magnitude higher than that of ELISA. Thus, a highly sensitive qiPCR-based assay was developed to quantitate antibodies specific to the recombinant EBV antigen.

The class E immunoglobulins (IgE) is known to recognize conformational epitopes and therefore the native conformation of recombinant allergens is essential for their using in test-systems. Recombinant Dermatophagoides farinae house dust mite (HDM) allergens Der f1 and Der f2 were expressed in bacteria Escherichia coli and Nicotiana benthamiana plants. It has been shown that IgE in sera from children allergic to HDM recognizes Der f2 expressed both in E. coli and N. benthamiana. Mature form of Der f1 expressed in E. coli does not interact with IgE while the protein purified from N. benthamiana is able to recognize IgE as a native allergen.

The genome of viruses of the genus Carlavirus codes for a small cysteine-rich protein (CRP) with unknown functions. To study the role of CRP of the chrysanthemum virus B (CVB), a recombinant potato virus X (PVX) genome containing the CVB CRP gene was constructed. Expression of CVB CRP in the PVX genetic surrounding drastically changed the character of symptoms produced by PVX in Nicotiana benthamiana. The recombinant virus caused local necrotic lesions of inoculated leaves and necrosis of apical leaves rather than asymptomatic infection and mild mosaic, which are caused by PVX in this host plant. In N. tabacum, the infection pattern depended on the plant host genotype: recombinant PVX spread systemically only in plants carrying the N gene. Agroinfiltration-mediated transient expression assays showed that CRP neither acts as an avirulence factor in N. benthamiana nor suppresses posttranscriptional gene silencing. CVB CRP was identified as a virus pathogenicity determinant that controls the interaction of the virus with the host plant in a way that depends on plant defense, which is mediated by resistance genes such as the N gene.

A mutation resulting in substitution of positively charged Lys53 with negatively charged Glu in the coat protein was introduced in the infectious cDNA copy of the genome of wild-type tobacco mosaic virus strain U1. Kinetic analysis of long-distance virus transport in plants showed that systemic spread of the mutant virus was delayed by 5–6 days as compared with the wild-type one. On evidence of RNA sequencing in the mutant progeny, Glu50 of the coat protein was substituted with Lys after passage I to compensate for the loss of the positive charge at position 53. Electron microscopy revealed atypical inclusions (rodlike structures, multiple electron-dense globular particles) in the nuclear interchromatin space of leaf mesophyll cells infected with the mutant but not with the wild-type virus.

Flexible filamentous shallot virus X (ShVX) particles were detected in extracts of Beta vulgaris protoplasts inoculated with transcripts from a full-length ShVX cDNA. Extracts from ShVX-infected protoplast were infectious for ShVX-healthy shallot seedlings. Western blot analysis of inoculated plants revealed the accumulation of the ShVX coat protein, while electron microscopy confirmed the presence of ShVX virions. The results suggest that the in vitro RNA transcripts from full-length ShVX cDNA give rise to infectious viral particles.

A new RNA of about 900 nt was found in the virions of cocksfoot mottle virus (CfMV) and in infected plants by RNA hybridization and RT-PCR. Structural features suggested that this RNA is a defective interfering RNA (diRNA). The CfMV diRNA was shown to consist of a 35-nt 5′-terminal genomic region, which formed a hairpin, and a 3′-terminal genomic region, which included the coat protein (CP) gene lacking the first 120 nt.In vitro translation of the diRNA started at the third Met codon to produce truncated CP. The CfMV diRNA was assumed totrans-activate synthesis of the CP subgenomic RNA (sgRNA).

The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation.

Optimization problems involving an infinite number of constraints are considered. This paper presents a general stochastic outer approximations method which incorporates mechanisms for active search of relevant constraints and for dropping of irrelevant constraints. The method extracts the characteristic features of several stochastic outer approximations algorithms suggested by Wardi [J. Optim. Theory Appl., 56 (1988), pp. 285--311; J. Optim. Theory Appl., 64 (1990), pp. 615--640] and furthermore develops the approach to get advantages of the Eaves--Zangwill scheme. Similarly to Gonzaga and Polak [SIAM J. Control Optim., 17 (1979), pp. 477--493] the method is based on the use of quasi-optimality functions satisfying some general unrestricted assumptions. These functions are usually employed in the stopping criteria of numerical techniques for solving simpler problems. It is shown that the method's trajectories almost surely converge to the quasi-optimal set. Following the proposed approach a stochastic algorithm for solving the approximation problem is constructed and studied. The proposed general method can be considered as a developed Eaves--Zangwill method applying the multistart technique at each iteration for the search of relevant constraints' parameters.

We have developed phosphate permease gene sequence-based PCR detection system of Fusarium cerealis phytopathogenic fungus. Sequencing and analysis revealed that the gene displayed unique polymorphism and could serve to establish phylogenetic relations as well as a marker to design specific primers. The specificity assay has confirmed the absence of cross reactions with DNAs of closely related Fusarium species. The qPCR assay demonstrated the 10 pg detection limit of specific DNA per reaction.

The steepest-descent technique dealing with the perturbed values of the objective function and its gradients and with nonexact line searches is considered. Attention is given to the case where the perturbations do not decrease on the algorithm trajectories; the aim is to investigate how perturbations at every iteration of the algorithm perturb its original attractor set. Based on the Liapunov direct method for attraction analysis of discrete-time processes, a sharp estimation of the attractor set generated by a perturbed steepest-descent technique with respect to the perturbation magnitudes is obtained. Some global optimization properties of finite-difference analogues of the gradient method are discovered. These properties are not inherent in methods which use exact gradients.

RAPD analysis for ten F. sporotrichioides strains of different geographical origin was done for DNA loci, potentially suitable as a new markers for taxonomic characterization and identification of toxigenic Fusarium fungi. Three selected monomorphic fragments--products of amplification with one of standard RAPD primers were sequenced that allowed creating SCAR markers for identification of Fusarium fungi on the species group level with similar profiles of produced mycotoxins.

Serological analysis (ELISA, immunoblotting, and immunoelectron microscopy) of the partially purified virus preparations obtained from shallot plants (selection sample no. 83) inoculated with the Mongolian isolate of onion yellow dwarf potyvirus (OYDV) revealed neither an OYDV coat protein antigen nor virions decorated by the corresponding antiserum. At the same time, these preparations contained many flexuous filamentous virus particles (FFVP) with a modal length of 780 nm. It is suggested that shallot no. 83 is resistant at least to the Mongolian isolate of OYDV, while the discovered FFVPs are the virions of some other viral agent. The nature and origin of a new virus are now under study in this laboratory

Potato, one of the most widespread agricultural plants in Russia, is strongly affected by various pathogens of viral, bacterial, and fungal origin as well as by pests. Their simple and accurate diagnostics and identification sound rather important both for production of virus free planting material and to perform monitoring of the phytosanitary state of planting areas. Based on qualitative Fluorescent Amplification--based Specific Hybridization Polymerase Chain Reaction (FLASH-PCR) we have developed the diagnostic systems, which provided fast, careful, and with the minimum risk of contamination in the working zone by amplification products, detection of the major potato pathogens, i. e. A, Y, X, M, S potato viruses, potato leafroll virus, potato mop top virus, as well as potato spindle tuber viroids.

The minimization of the maximum function of a set-valued map is considered. The problem has a number of applications in game theory, in dynamic system design models, etc.

Two-stage stochastic optimization of dynamic system parameters is considered. It is shown that the original optimization problem can be approximated with any degree of accuracy by a finite-dimensional minimax stochastic problem. An Arrow—Hurwicz type stochastic quasigradient algorithm is proposed for solving such problems.

Viruses of genus Carlavirus encode a small cysteine-rich protein (CRP) of unknown function. To investigate the role of CRP of carlavirus chrysanthemum virus B (CVB), a recombinant potato virus X (PVX) genome was constructed, which carried the CVB CRP gene. Expression of CVB CRP in the PVX genetic background drastically changed the PVX symptom phenotype in N. benthamiana. Instead of symptomless infection and mild mosaic, which are characteristic of PVX in this plant host, the recombinant virus expressing CVB CRP induced formation of necrotic local lesions on inoculated leaves and necrosis of the apical leaves. In N. tabacum, the infection pattern depended on the host genotype: the recombinant PVX was able to spread systemically only in N gene-carrying plants. In agroinfiltration-mediated transient expression assay, CVB CRP did not exhibit the properties of avirulence factor in N. benthamiana and was unable to suppress post-transcriptional gene silencing. Thus, CVB CRP is the viral pathogenicity determinant controlling the virus interaction with plant hosts in a manner which depends on plant defense mediated by resistance genes such as the N gene.

The new plant virus family Flexiviridae is described. The family is named because its members have flexuous virions and it includes the existing genera Allexivirus, Capillovirus, Carlavirus, Foveavirus, Potexvirus, Trichovirus and Vitivirus, plus the new genus Mandarivirus together with some related viruses not assigned to any genus. The family is justified from phylogenetic analyses of the polymerase and coat protein (CP) sequences. To help to define suitable molecular criteria for demarcation of species, a complete set of pairwise comparisons was made using the nucleotide (nt) and amino acid (aa) sequences of each fully-sequenced gene from every available accession in the family. Based on the distributions and on inspection of the data, it was concluded that, as a general rule, distinct species have less than ca. 72% identical nt or 80% identical aa between their entire CP or replication protein genes.

A mutation resulting in substitution of positively charged Lys53 with negatively charged Glu in the coat protein was introduced in the infectious cDNA copy of the genome of wild-type tobacco mosaic virus strain U1. Kinetic analysis of long-distance virus transport in plants showed that systemic distribution of the mutant virus was delayed by 5-6 days as compared with the wild-type one. On evidence of RNA sequencing in the mutant progeny, Glu50 of the coat protein was substituted with Lys after passage I to compensate for the loss of the positive charge at position 53. Electron microscopy revealed atypical inclusions (rodlike structures, multiple electron-dense globular particles) in the nuclear interchromatin space of leaf mesophyll cells infected with the mutant but not with the wild-type virus.

Flexible filamentous shallot virus X (ShVX) particles were detected in extracts of Beta vulgaris protoplasts inoculated with transcripts from a full-length ShVX cDNA. Extracts from ShVX-infected protoplast were infectious for ShVX-healthy shallot seedlings. Western blot analysis of inoculated plants revealed the accumulation of the ShVX coat protein, while electron microscopy confirmed the presence of ShVX virions. The results suggest that the in vitro RNA transcripts from full-length ShVX cDNA give rise to infectious viral particles.

The main difference between genome structures of shallot virus X (ShVX) and related potex- and carlaviruses is the unique gene of ShVX coding for a 42K protein. The amino acid sequence of this protein was analyzed and compared with those of similar proteins from several newly characterised viruses of garlic. Using antibodies against the recombinant 42K protein, expression of the 42K protein of ShVX was detected in most of plants where the ShVX coat protein is present.

The complete nucleotide sequence of potato virus M genomic RNA has been determined to be 8534 nucleotides (with the exception of the poly(A) tail at the 3' end). The sequence contains six large open reading frames coding for proteins of mol. wt. 223206, 25438, 11893, 6793, 33906, and 12183 (in 5'----3' direction). According to its primary sequence analysis the 223K protein ORF codes for a virus RNA replicase. The in vitro translation product of 34K protein gene precipitates by the antisera against the RVM indicating that the 34K protein is the virus coat protein. The general aspects of carla- and potexvirus gene organization are discussed.

The sequence of 2630 3'-terminal nucleotides has been determined for the genomic RNA of potato virus M (PVM), a type member of the carlavirus group. Analysis of this nucleotide sequence revealed five open reading frames coding for proteins of mol. wt. 25, 12, 7, 34 and 11 kDa (in 5'----3' direction). The PVM genome organization has been shown to be basically analogous to that of potexviruses, except that the latter at their 3' end lack the gene encoding 11K protein. Amino acid sequence comparison between the PVM 34K protein and the coat proteins of potexviruses has shown a high extent of homology in the C-terminal amino acids. Homology has also been found between the 25, 12 and 7K proteins encoded by PVM RNA and corresponding potexvirus proteins. All this gave grounds for suggestion that carlaviruses and potexviruses belong to the same subgroup of phytoviruses.

The circular dichroism (CD) spectra of flow-oriented complexes of DNA with proflavine (PF), ethidium (ET) and distamycine (DS) have been studied in the ultraviolet region. The CD spectra with light propagating in parallel to the flow direction were measured by the method of Chung and Holzwarth [1]. Delta epsilon and delta epsilon values have been obtained by this method. It was shown that all the complexes studied exhibit a strong CD anisotropy so that "isotropic" CD spectra measured with a conventional procedure can be attributed to the mutual compensation of the two components of opposite signs.

A membrane filter assay has been devised to study the influence of ionic strength (0--150 mM NaCl) and actinomycin D on the kinetics of the open promoter complex formation between E. coli RNA-polymerase and [3H]DNA of T7 phage. The dependence of the complex formation rate upon the ionic strength is non-monotoneus with a maximum at 75--100 mM. The addition of one actinomycin molecule per 200 base pairs decreases the open promoter complex formation rate at ionic strength less than 100 mM. A promoter site selection model including liner diffusional selection is proposed which is in a good agreement with the experimental results obtained.

The role of ATP and membrane potential in phage T7 DNA injection into E. coli during infection has been studied. Entrance of phage T7 genes of class II and III was shown to be prevented by arsenate, indicating the requirement for phosphorylated macroergs in the phage DNA injection. The injection process was also inhibited by exposition of the cells to the uncoupler of oxidative phosphorylation. Dependence of the injection efficiency on the membrane-potential value has been shown to be sigmoidal, which suggests a regulatory role of the membrane potential in phage T7 DNA injection from the virion into the host cell.

The mechanism by which bacteriophage T3 DNA is transported into the E. coli cell during infection was studied. The data obtained testify that bacteriophage T3, similarly to what we have earlier found for bacteriophage T7, introduces its DNA into the infected cell is a transcription-dependent way. A detailed discussion is presented on the occurrence of the transcription-coupled transport of viral DNA into the infected cell and on a number of general issues concerning the transport functions of template-directed enzymes.