Specific interactions between lectins and red blood cells of Chornobyl cleanup workers as indicator of some late radiation effects.

AIM: Growing interest in lectins is based on their diagnostic and pharmacological potential, especially the ability to inhibit proliferation and initiate apoptosis of cancer cells. In our research microplate lectinoassay able to detect carbohydrate containing structures (receptors) on erythrocyte surface have been proposed for Chornobyl cleanup workers (1986) monitoring. It was expected to reveal specific abnormalities associated with pathological condition arising as a result of late radiation effects. MATERIALS AND METHODS: Red blood cell (RBC) specimens were taken from 171 persons distributed into the six cohorts: nonexposed donors (1); chronically exposed to the doses below (2) and over 50 cGy (3); exposed to acute radiation without (4) and with manifestation of acute radiation syndrome (5 and 6). Lectins from 24 species of medicinal plants were purified by ethanol fractionation and electrofocusing. Intensity of lectin-receptor interactions was determined in reaction of hemagglutination. Method of flow cytofluorometry was used to study B-cell counts. Hormone levels in blood serum were determined by radioimmunoassay. RESULTS: An elevated ability of RBC to interact with the panel of lectins was found in all cohorts of exposed persons versus nonexposed donors, moreover, changes in the intensity of lectin-receptor binding depended on the dose of irradiation. Diagnostic value of specific RBC reactions with some individual lectins has been elucidated. Elevated intensity of RBC reaction with Zea mays lectin was accompanied by a decrease in serum content of thyroid hormones T4 and T3, as well as reduction of B-cell counts. In the case of Rubus caesius lectin the more intensive reaction with RBC, the higher level of hormone cortisol was observed. CONCLUSIONS: Deviations from donor's norm in intensity of lectin - RBC interactions in radiation exposed men are supposed to carry information about negative changes in their health status following Chornobyl catastrophe and show the diagnostic potential. The most sensitive reactions have been associated primarily with shifts in endocrine and immune systems. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

[Extracellular lectins from saprophytic strains of bacteria of the genus Bacillus (review)].

The present review summarizes both the authors' own and other researchers' reports concerning the synthesis and properties of sialic acid-specific extracellular lectins from saprophytic bacteria of the genus Bacillus. Bacilli of this genus isolated from different ecological niches differ with regard to the ability to synthesize lectins. The biphasic temporal pattern of lectin synthesis in culture and the effect of cultivation conditions on lectin production have been demonstrated. The advantages of the technological procedure used for the isolation and purification of these biopolymers have been characterized. Specificity towards sialic acid, which is characteristic only of a small number of lectins from bacilli and underlies a wide range of biological effects of these proteins, receives particular attention. Adaptation mechanisms involving lectins--carbohydrate-recognizing proteins--are suggested to have developed in saprophytic bacilli to ensure the survival of these organisms in a constantly changing environment.

[Influence of chemically different antiviral substances on the expression of IFNα, PKR, OAS1a and RNAse L genes].

The aim of the study was to determine the ability of several chemically different antiviral substances to induce the expression of interferon α(IFNα), PKR, OAS1a and RNAse L genes in the rat liver. The investigated substances included Amizon, Altabor and Proteflasid, which are already used in practical medicine, and 3',7-dimethylquercetin extracted from Proteflasid, the mixture of synthesized trimethyl- and tetramethylquercetin and Sialospecific lectin from persimmon, which are at the stage of preclinical trial and experimental research respectively. The content of corresponding mRNAs in total RNA was detected with the help of reverse transcription and polymerase chain reaction in real time. The results have shown that all investigated substances induce the expression of genes α, PKR, OAS RNAse L in specific manner. The combination of 3',7-dimethylquercetin + lectin from persimmon had the highest stimulating effect exceeding the effect of each component of the mixture and the influence of Heberon (recombinant IFNα2b) and PolyI-polyC as the standard inducers of IFNα and its target genes. The ability of all substances to specifically induce the expression of IFNa and its target genes, the absence of correlation between the levels of IFNα and its target genes expression as well as between target genes themselves indicate that the mechanism of antiviral activity of the investigated substances is connected not only with up-regulation of IFNα and potential IFNα mediated effects.

[The influence of lectin isoforms of Bacillus subtilis saprophytic strain IMV B-7014 on viability of normal and cancer cells in vitro].

Structural and functional polymorphism of saprophytic bacterium lectin was demonstrated to be due to subunit organization of the molecule as it was shown for many lectins of plant and animal origin. Three isoforms of extracellular sialic acid-specific lectin produced by Bacillus subtilis saprophytic strain IMV B-7014 were discovered that differed for physicochemical and biological properties. The influence of the lectin isoforms on mammalian cells proliferation and morphology in vitro depends both on the subunit organization of the protein molecule and the type of cells under study.

[Molecular genetic study of acute intermittent porphyria in Russia: mutation analysis and functional polymorphism search in porphobilinogen deaminase gene].

Acute intermittent porphyria (AIP) is an autosomal dominant hereditary disease, caused by partial deficiency of porphobilinogen deaminase (PBGD), one of the key enzymes ofheme biosynthesis. This study describes molecular genetics of AIP in Russia. Mutation analysis of PBGD gene in 70 unrelated patients revealed 47 various genetic defects, 28 of which had not been described previously. Mutations 53delT and Argl 73 Trp (recorded 8 times, in total 23%) proved to be the most common in Russia. Microdeletion 53delThas monophyletic origin and was found only in Russia. Molecular genetic examination of 132 relatives of AIP patients from 40 families revealed 52 latent carriers of the disease. Low (about 10%) AIP penetrance indicates that a mutation in the PBGD gene is an important but not sufficient prerequisite for clinical manifestation of the disease. Modulation of penetrance in erythropoietic protoporphyria by coinheritance of a mutant allele and a functionally defective wild type allele of ferrochetalase gene has been shown previously. We hypothesized that similar mechanism works in AIP. Sequencing of the full length PBGD genes from unrelated AIP patients as well as SN P analysis, and the analysis of abnormal PBGD mRNA splicing showed that in case ofAIP, this hypothesis is not true and some other factors are responsible for the penetrance of this disease.

[Isoforms of Bacillus subtilis IMV B-7014 lectin].

The subunit organization and regulatory features of extracellular sialic acid specific lectin produced by saprophytic strains Bacillus subtilis IMV B-7014 have been investigated. Autofocusing method was used to identify three lectin isoforms that were distinguished by physicochemical and biological properties. Using the transcription system in vitro it was established that one of the targets of bacterial lectin action was DNA-dependent RNA-synthesis. Lectin isoforms affect differently the synthesis: from its full inhibition to the absence of the effect on the yield of RNA-transcription product.

[Lectins from Sambucus nigra L inflorescences: isolation and investigation of biological activity using procaryotic test-systems].

Isolation of lectins from extracts of the Sambucus nigra inflorescences and of pollen material have been performed using isoelectric focusing without carrier ampholytes (autofocusing). Fractions active in agglutination tests with different carbohydrate specificity were subjected to SDS-PAGE. The major lectin found in whole inflores-cences was GalNAc specific and is proposed to be a heterotetramer with subunits of about 30 and 33 kDa. It was called SNAflu-I. At least two other lectins were present in the pollen material and supposed to consist of identical subunits. Major positively charged lectin was Glc/Man specific with subunit of 26 kDa and called SNApol-I. Other pollen component (SNApol-II) was Gal specific with subunit of about 20 kDa. In order to elucidate cell targets sensitive for the S. nigra lectin's activity the combined effects of the lectins and transcriptional of phenazine origin on B. subtilis cells growth have been studied. Only SNApol-I demonstrated the antagonistic activity against these inhibitors in vivo. This lectin but not the SNAflu-I can also inhibit transcription in vitro. It is supposed that lectins from the same source may act in different directions on cell metabolism. Particularly one of the common targets may be the DNA-dependent synthesis of RNA.

[Regulatory properties of Bacillus subtilis isolectins].

The ability of natural and mutant Bacillus subtilis cultures with imperfect reparation/recombination system to synthesis of extracellular and surface lectins was investigated, and dependence of lectin production process on cultures' genotype was proved. Mutant B. subtilis recP has practically lost its ability to produce the extracellular lectins as a result of mutation of a gene of the reparation/recombination system. The application of the method "autofocusing" allowed to investigate all the spectrum oflectin molecular forms of natural B. subtilis culture and to reveal isoforms distinguished by physico-chemical and hemagglutination properties. It was shown that lectin cathode forms inhibit the transcription process from plasmid promnoter completely, and anodic forms activate the transcript formation slightly in the transcription in vitro with T7 bacteriophage DNA-dependent RNA-polymerase.

[Investigation of Bacillus subtilis exolectin ability to modulate the antibiotics effect on growth of Bacillus subtilis mutants].

The ability of Bacillus subtilis exolectin to modulate the effect of antibiotics acting as metabolic inhibitors, which can suppress the biosynthesis of cell wall glycans (ampycillin), replication (mitomycin C) and transcription (rifampycin), have been investigated on mutants of B. subtilis. Extracellular lectin was produced by B. subtilis strains B-7014 isolated from new-born calve's intestines. It was shown that the exolectin displays affinity for to sialic and uronic acids in the decreasing order: mucin, glycuronic acid, N-acetylneuraminic acid, galacturonic acid. It was established by the diffusion method and by determination of minimum inhibitory concentrations (MIC) that B. subtilis exolectin had selective activity as to bacteriostatic effect of antibiotics under study. The activity depended on the antibiotic structure and on mutant genotype defective as to the state of its replication and repair system. The lectin under study had no modulating effect on ampycillin action. There was a tendency to lower the bacteriostatic effect of rifampycin on the growth of strain BD170 (rec+) with the help of exolectin. Only in the case of mitomycin C the significant modulating effect of the bacterial lectin was manifested and its dependence on the mutant genotype was shown. The mutants sensitivity to exolectin effect decreased in the order: BD293 (polC), SB25 (recP), BD224 (recE), BD170 (rec+). Revealed ability of B. subtilis exolectin to protect the action of mitomycin C on growth of mutant BD293 (polC) with defect in the enzyme-DNA polymerase III permits supposing that the process of DNA replication is the most sensitive target for the lectin. The found dependence of modulation of the mitomycin C effect by the bacterial lectin on the genotype of mutants (rec-) demonstrated that the lectin acted following a complex mechanism mediated by a replicative and reparative complex.

[Lectins modulate the cytostatic action of the composite bioregulators-conjugates of azaanalogues of pyrimidine and phenazine-1-carboxilic acid in Bacillus subtilis test-system].

The idea of the work was to study a combined effect of some lectins (Con A, PHA, STA, WGA, SNA, VAA) and new composite bioregulators (PhCA-1 and azapyrimidine derivatives) on growth of Bacillus subtilis cells in order to elucidate cell targets sensitive to lectin's activity. Study of combined effects of high- and low-molecular bioregulators may also be the subject of practical interest with a prospect of obtaining new bacterio-and carcinostatic drugs. Using B. subtilis mutants it was shown that lectins can modulate the cytostatic effect of initial substanses and their pyrimidine derivatives in the range: phenazine < PhCA-1 < 6-azacytosine derivative of PhCA-1 < 6-azauracyl derivative of PhCA-1. This modulating effect was absent in recP mutant, which has lost the ability to produce its own bacterial lectin, demonstrating its possible role as a mediator. The antagonistic effect of all plant lectins under study on cytostatic action of <6-azauracyl derivative of PhCA-1 in rec+ culture was observed as the result of possible competition for some common target. As both the B. subtilis lectin and the low-molecular bioregulator can inhibit transcription in vitro, it is supposed that their common target may be the DNA-dependent synthesis of RNA.

[Ability of lectins to model the action of antibiotics on growth of mutants of Bacillus subtilis].

Using the bacterium B. subtilis as a model and antibiotics as metabolic inhibitors which can suppress replication (mitomycin), transcription (rifampycin) or translation (streptomycin), it is shown that carbohydrate-binding proteins (lectins of plant origin) have different action on intracellular processes under study. The effect depends on lectin's structure and on the condition of bacterial reparation/recombination system. Lectins with chitin-binding domain (STA, WGA) is characterized by the most expressed effect on the repair-proficient strain when the inhibitor of template function of DNA (mitomycin C) was used. The absence of such effect on mutants recP and polC may prove that the corresponding bacterial proteins play the role of mediators in transmission of the signal influence of lectins. The representative of ribosome-inactivating lectins--SNA-I--could increase the streptomycin effect. It is proposed that intracellular effects of lectins have complex mechanisms which need participation of repair functions.

[The DNA of recombinant plasmids acquires mutagenic activity in a Bacillus subtilis culture after the insertion of the human genome Alu repeat]. / DNK rekombinantnykh plazmid priobretaiut mutagennuiu aktivnost' v kulture Bacills subtilis posle vvedeniia Alu-povtora genoma cheloveka.

In our previous work the mutagenic activity of recombinant plasmids (pBR322 carrying the human Alu repeat alone or in combination with preproinsulin or apo AI gene) in competent Bacillus subtilis culture was demonstrated. In present work it was shown that among seven tested plasmids only three revealed mutagenic activity (pBR322 and two Alu repeat-containing constructions). It seems that mutagenic activity is not inherent to any recombinant molecule in applied test system but depends on its structure. For example, Alu repeat of human genome may attach mutagenic properties to plasmids which either had no such properties, or lost them after gene engineering manipulations.

[The effect of the lectin from Sambucus nigra inflorescences on spontaneous and alkylating agent-induced mutagenesis in mammalian somatic cells]. / Vliianie lektina sotsvetii Sambucus nigra na spontannyi i indutisrovannyi alkiliruiushchim agentom mutagenez v somaticheskikh kletkakh mlekopitaiushchikh.

Data on separation of purified galactose-specific lectin as well as its two polypeptide chains from inflorescences of European elder Sambucus nigra are represented. Their molecule quaternary structure after the pattern a2 b2 is suggested. It was shown that purified protein in concentration 2.0 mg/ml had mutagenic activity and might enhance mutagenic effect of alkylating substance MNNG. Thus, preparations of lectin from inflorescences of European elder influenced on spontaneous and induced by the alkylating agent mutagenicity in mammalian somatic cells in vitro.

[The mutagenic activity of the DNA of recombinant plasmids in a competent Bacillus subtilis culture]. / Mutagennaia aktivnost' DNK rekombinantnykh plazmid v kompetentnoi kul'ture Bacillus subtilis.

The method for testing foreign plasmid DNA mutagenicity on the competent culture of B. subtilis has been developed. High mutagenic effect of DNA of recombinant plasmids carrying a single human Alu-repeat or the same repeat in combination with human apoAi gene or human insulin gene was demonstrated. The vector plasmid pUC18 had no mutagenic activity. According to the data of dot-blotting some fragments of recombinant plasmid DNA of human origin can integrate in B. subtilis chromosome by means of illegitimate recombination. It is concluded that B. subtilis test system is suitable for detection of potential mutagenic polynucleotide sequences in recombinant plasmid constructions produced for gene therapy purposes.

[The effect of pyridinolcarbamate (parmidine) on the microcirculation and immunity of patients with stable angina]. / Vliianie piridinolkarbomata (parmidina) na mikrotsirkuliatsiiu i immunitet bol'nykh so stabil'noi stenokardiei.

A comparison of the efficacy of treating two patient group (Group 1, patients receiving nitrates in combination with parmidin and Group 2, those taking nitrates) revealed that the two groups did not differ greatly in the clinical effects. In heart failure patients with high capillary permeability, parmidin substantially decreased the permeability to protein, which was associated with decreases in the baseline high activity of primary lipid peroxidation product in plasma red blood cells, which was not observed in the controls. At the same time parmidin was demonstrated to exert an unfavourable effect on microhemocirculation and to increase the patient's autosensibilisation.

[Effect of hemosorption on microcirculation, humoral autoimmunity and lipid peroxidation in patients with stable angina pectoris]. / Vliianie gemosorbtsii na sostoianie mikrotsirkuliatsii, gumoral'nogo autoimmuniteta i perekisnoe okislenie lipidov u bol'nykh so stabil'noi stenokardiei.

Microhemo- and lymphocirculation, capillary permeability, humoral autoimmunity and lipid peroxidation were determined in 46 patients with Functional Classes III-IV stable angina concurrent with multiple atherosclerotic coronary lesions 24 hours following the second hemosorption session. The clinical efficiency of hemosorption was observed in 47.8% of patients with stenotic coronary atherosclerosis refractory to antianginal therapy. In this group of patients, hemosorption led to accelerated microcirculation, increased microcirculatory reserve potentials and decreased microvascular resistance at rest and in reactive postischemic hyperemia. In patients with abnormal humoral autoimmunity and lipid peroxidation, hemosorption resulted in their hormalization.

[Differential selection of anti-anginal drugs in stable stenocardia in relation to the data of central and intracardiac hemodynamics]. / Differentsirovannyi podbor antianginal'nykh preparatov pri stabil'noistenokardii s uchetom dannykh tsentral'noi i vnutriserdechnoi gemodinamiki.

Rheography and echocardiography were used to study central and intracardiac hemodynamic parameters in 196 patients with Functional Classes I-II from the screening of the main antianginal drugs during an acute pharmacological test under bicycle ergometric monitoring. It was established that the hemodynamic type of circulation and left ventricular end-diastolic and end-systolic volumes may be used as criteria for the differential choice of antianginal agents. The values of these parameters are given for sustac, propranolol, verapamil, nifedipine and sustac plus propranolol.

[Effect of xanthinol niacinate on the autoimmunity and capillary permeability in patients with stable stenocardia]. / Vliianie ksantinol-nikotinata na sostoianie autoimmuniteta i pronitsaemost' kapilliarov u bol'nykh so stabil'noi stenokardiei.

A three-month antianginal therapy with long-acting nitrates and xanthinol nicotinate given in various daily doses have shown that there is a positive clinical effect, a reduction in the level of immune complexes and IgG in most patients, which is followed by a decrease in capillary permeability and dienic conjugate levels both in erythrocytes and plasma.

[Bioenergetic processes in erythrocytes from patients with stable stenocardia]. / Bioénergeticheskie protsessy v éritrotsitakh bol'nykh stabil'noi stenokardiei.

Distinct impairments in energy metabolism were detected in erythrocytes of patients with persistent stenocardia. These impairments occurred both in the rate of high-energy substances synthesis and in their utilization. Possible mechanisms for regulation of bioenergetic processes in red blood cells under conditions of ischemic heart disease are discussed.

[Manifestation of instability in the leucine-dependent Bacillus subtilis auxotroph induced by herring DNA]. / Proiavlenie nestabil'nosti u leitsinzavisimogo auksotrofa Bacillus subtilis, poluchennogo s pomoshch'iu DNK sel'di.

Unstable leucine-depending Bacillus subtilis auxotroph previously induced by the herring DNA was studied. Instability was manifested as revertability in the leucine-free medium. Average frequency of revertants amounted to 50% of the number of viable cells. A clone analysis has shown that most of such colonies are mosaics consisting of leucine-independent and leucine-sensitive cells. Unstable mutation was cotransformed with leuA marker. Heating followed by material storage in demineralized water stimulated transposition of unstable mutation under study to the tryptophan operon region.