[Changes in the chromatin structure of lymphoid cells under the influence of low-intensity extremely high-frequency electromagnetic radiation against the background of inflammatory process].

Using the alkaline single cell gel electrophoresis technique (comet assay), changes in chromatin structure of peripheral blood leukocytes and peritoneal neutrophils have been studied in mice exposed to low-intensity extremely high-frequency electromagnetic radiation (42.2 GHz, 0.1 mW/cm2, 20 min at 1 h after induction of inflammation) against the background of the systemic inflammatory process. It was revealed that the exposure of mice with the developing inflammation leads to a pronounced decrease in the level of DNA damage to peripheral blood leukocytes and peritoneal neutrophils. It is supposed that the changes in the chromatin structure of lymphoid cells have a genoprotective character in the inflammatory process and can underlie the mechanisms of realization of antiinflammatory effects of the electromagnetic radiation.

Role of efflux pumps in adaptation and resistance of Listeria monocytogenes to benzalkonium chloride.

In this study, potential mechanisms underlying resistance and adaptation to benzalkonium chloride (BC) in Listeria monocytogenes were investigated. Two groups of strains were studied. The first group consisted of strains naturally sensitive to BC which could be adapted to BC. The second group consisted of naturally resistant strains. For all adapted isolates, there was a correlation between the resistance to BC and ethidium bromide, but this was not the case for the naturally resistant isolates. To investigate the role of efflux pumps in adaptation or resistance, reserpine, an efflux pump inhibitor, was added to the strains. Addition of reserpine to the sensitive and adapted strains resulted in a decrease in the MIC for BC, whereas no such decrease was observed for the resistant strains, indicating that efflux pumps played no role in the innate resistance of certain strains of L. monocytogenes to this compound. Two efflux pumps (MdrL and Lde) have been described in L. monocytogenes. Studies showed low and intermediate levels of expression of the genes encoding the efflux pumps for two selected resistant strains, H7764 and H7962, respectively. Adaptation to BC of sensitive isolates of L. monocytogenes resulted in significant increases in expression of mdrl (P < 0.05), but no such increase was observed for lde for two adapted strains of L. monocytogenes, LJH 381 (P = 0.91) and C719 (P = 0.11). This indicates that the efflux pump Mdrl is at least partly responsible for the adaptation to BC.

Assessment of photodynamic destruction of Escherichia coli O157:H7 and Listeria monocytogenes by using ATP bioluminescence.

Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.

Regulation of NO-producing function of the lungs with salmeterol.

The state of epithelial nitric oxide synthase, bronchomotor reaction, and concentration of NO metabolites (NO(2)(-) and NO(3)(-)) in bronchoalveolar lavage fluid were studied in healthy rats and rats with experimental bronchial asthma induced by inhalations of long-acting beta(2)-agonist salmeterol. The effects of salmeterol on NO-producing function of the lung in healthy animals and in animals with bronchial asthma were studied.

Bioluminescence of free and poly(vinyl alcohol) cryogel-entrapped recombinant Escherichia coli cells expressing firefly luciferase.

Bioluminescence of free and poly(vinyl alcohol) cryogel-entrapped recombinant E. coli cells expressing firefly luciferase was investigated. It was shown that bioluminescence intensity and time-course of the bioluminescent signal changed upon immobilization and depended on intracellular ATP concentration and permeability of the cell membrane.

[Comparative evaluation of methods of intracellular ATP extraction from different types of microorganisms for bioluminescent determinationof microbial cells]. / Sravnenie metodov ekstraktsii vnutrikletochnogo ATF mikroorganizmov razlichnogo tipa dlia bioliuminestsentnogo opredeleniia kletok mikro organizma.

The efficiency of dimethyl sulfoxide (DMSO), trichloroacetic acid (TCA), and cetyltrimethylammonium bromide (CTAB) as extractants of intracellular ATP from various microorganisms was compared in bioluminescent measurements of microbial cell concentrations. Extraction with CTAB was found to provide an approximately ten times higher sensitivity of the bioluninescent assay of microbial cells than extraction with DMSO or TCA. In Gram-positive bacteria and yeasts, the ATP concentration in the extract was a linear function of the microbial suspension density only within a cell concentration range of D600 0.02-3.5 in the three types of tested extracts. In Gram-negative bacteria, a significant deviation from the linear dependence between ATP concentration and microbial suspension density was observed in CTAB extracts at D600 > 1.

[The ATP content of the neutrophils and whole blood in the inhabitants of regions in the Altai Territory subjected to nuclear testing exposure]. / Soderzhanie ATF v neitrofilakh i tsel'noi krovi u zhitelei raionov Altaiskogo kraia, podvergshikhsia vozdeistviiu iadernykh ispytanii.

The bioluminescent method was used in the studies of the influence of ionizing irradiation and/or xenobiotics on the content of ATP in RBC and neutrophils of rats, and in whole blood and neutrophils of 80 examined women of Altai Region exposed to ionizing radiation during a series of nuclear tests in Semipalatinsk in 1949-1965. Deviations from the normal ATP content were measured with due to account of the natural variability of a given metabolite. For rats, deviations from the content of ATP in erythrocytes were short-term, those in neutrophils were long-term. For people, a statistically significant increase in the content of ATP in neutrophils as compared to the control was observed. A non-linear correlation between the content of ATP in neutrophils and the calculated dose of radiation was observed. An increase in the ATP content in whole blood, with regard to the control, was not statistically significant for all groups of examined persons.

Bioluminescent assay for human lymphocyte blast transformation.

One of the basic tests of in vitro evaluation of immune cell functional activity is a proliferative response of lymphocytes on the action of external stimuli such as mitogenic lectines, antigens, etc. We compared two methods used to assess the lymphocyte functional status. (1) [3H]thymidine incorporation and (2) bioluminescence for determination of intracellular ATP in blast cells. Comparison has been done for healthy donors and patients with proven low immunological status. The proposed bioluminescent method for evaluation of the proliferative response was shown to be sensitive enough for diagnostic purposes. This method allows one to process a large number of samples at the same time and correlates highly with the radionuclide test use hazardous radioactive materials.

Bioluminescent assay of bacterial intracellular AMP, ADP, and ATP with the use of a coimmobilized three-enzyme reagent (adenylate kinase, pyruvate kinase, and firefly luciferase).

A three-enzyme coimmobilized system (firefly luciferase, pyruvate kinase, and adenylate kinase) was constructed for the bioluminescent assay of ATP, ADP, and AMP in bacterial cell extracts. Data for the reproducibility and sensitivity of the proposed method are presented. Detection limits were 1.5 pmol of ADP and 15 pmol of AMP in the sample. With this system, changes in adenine nucleotide concentrations in bacterial cells were measured during the actions exerted by external chemical and physical sources, such as additives to nutrient media and low-power He-Ne laser irradiation.

[Change in adenine nucleotide pool in E. coli 1257 bacterial cells under the action of a low intensity He-Ne laser]. / Izmenenie pula adeninovykh nukleotidov v kletkakh bakterii E. coli 1257 pri vozdeistvii nizkointensivnogo He-Ne lazera.

Evidence is presented of co-immobilized bioluminescent reagents which include firefly luciferase, pyruvate kinase and adenylate kinase used for an adenylate assay in bacterial cells. The changes in the ATP, ADP and AMP content induced by irradiation with a low-power He-Ne-laser depend on the laser power and irradiation dose. The data obtained suggest that laser irradiation causes the activation of adenine nucleotide synthesis de novo.

[The radiobiological characteristics of the P+ variant of Bacillus brevis var. G.--B. in relation to gramicidin S synthesis]. / Radiobiologicheskie osobennosti P+-varianta Bacillus brevis var. G.--B.v sviazi s sintezom gramitsidina S.

The radiosensitivity of P(+) variant Bacillus brevis var. G.-B. cells cultured under condition of normal and inhibited gramicidin S synthesis, antibiotically high-active strain and high radioresistant cells has been studied. It has been shown that the radioresistance of bacterial cells correlates, in general, with their antibiotic activity: the antibiotic superproduced is more radioresistant than P(+) variant, the inhibition of antibiotic synthesis by beta-phenil-beta-alanin rises a little the sensitivity of P(+) variant cells. But the radioresistant fraction of P(+) variant contains the lower antibiotic amount than the whole population. It has been concluded that the radioprotective action of gramicidin S can not be the only reason of the above-mentioned differences in radiosensitivity.

[A bioluminescent method of determining the sensitivity of microflora to antibiotics]. / Bioliuminestsentnyi metod opredeleniia chuvstvitel'nosti mikroflory k antibiotikam.

A bioluminescent rapid method (a three-hour test) has been developed to assess the susceptibility of microflora to antibiotics. It is based on comparison of intracellular ATP content in a sample (pus) after 3 h incubation at 37 degrees C in liquid culture medium (dilution: sample/culture medium--1:9) on a shaker in the presence and absence (control) of a single antibiotic concentration. A criterion has been offered for quantitative estimation of antibiotic susceptibility of microorganisms. The authors have optimized the measurements of intracellular ATP by the rapid bioluminescent method using immobilized firefly luciferase-based ATP reagent. The results of the method are in good correlation with those of the standard agar diffusion technique.

Interaction of purified rat liver glucocorticoid-receptor complexes with polynucleotides: strong base composition dependence.

The binding of 10(3)-fold purified rat liver glucocorticoid receptors to natural and synthetic polynucleotides has been studied. The receptors may bind to RNA as well as to DNA. The binding effectiveness of nucleic acids is strongly base composition dependent, with natural DNAs (double-stranded or denatured) greater than E. coli rRNA greater than poly(dA).poly(dT) greater than or equal to poly d(A,T,G)7 greater than or equal to poly(U) greater than poly(A).poly(U) greater than poly(A) approximately poly(C). The equilibrium (apparent) constants of receptor binding to a number of polynucleotides were calculated by a method proposed.

[Glucocorticoid-receptor complexes of rat liver. II. Interaction with natural and synthetic polynucleotides]. / Gliukokortikoid-retseptornye kompleksy pecheni krys. II. Vzaimodeistvie s estestvennymi i sinteticheskimi polinukleotidami.

The relative affinities of [3H]dexamethasone-labelled glucocorticoid-receptor complexes (GRC) from rat liver to various natural and synthetic polynucleotides differing in base composition and secondary structure have been determined. The modified competition assay procedure with use of DNA-cellulose was employed. The interaction of 150-2000 fold purified GRC with polyribo- and polydeoxyribonucleotides greatly depends on their base composition and nucleotide sequences. This interaction is hardly affected by the structure (ribo- or deoxyribo-) of the sugar-phosphate backbone or its size and is in fact independent of the secondary structure (single- or double-stranded forms) of polynucleotides. Mixtures of mononucleotides or apurinic DNA do not compete with DNA-cellulose for GRC binding. In the presence of cytosol, GRC bind double-stranded rather than single-stranded DNA, the fact possibly reflecting different effects of cytoplasmic proteins on the accessibility to GRC of polynucleotides of dissimilar secondary structure. The apparent equilibrium dissociation constants (Kd) of the ternary complexes of GRC with some synthetic polynucleotides were calculated. In case of double-stranded polydeoxyribonucleotide poly(dA) . poly(dT) Kd is equal to (1.2 +/- 0.6) x 10(-4) M; this value may characterize a nonspecific GRC--DNA interaction. It is supposed that GRC interact with high affinity (Kd approximately 10(-7)--10(-8) M) with short (5-10 nucleotides long) DNA sequences in which certain purine and pyrimidine residues are interspersed. Such DNA sequences may represent sites of primary action of GRC on the cellular genome in vivo.

[Glucocorticoid-receptor complexes of rat liver. I. Kinetic and equilibrium parameters of their interaction with DNA: ionic strength and temperature dependence]. / Gliukokortikoid-retseptornye kompleksy pecheni krys. I. Kineticheskie i ravnovesnye parametry vzaimodeistviia s DNK: vliianie ionnoi sily i temperatury.

The interaction of about 2000-fold purified rat liver glucocorticoid-receptor complexes (RGC) with DNA immobilized on cellulose was investigated. The sedimentation coefficient of GRC before or after purification is about 4S in 0.3 M NaCl. The parameters of GRC-DNA interaction were quantitatively determined using arbitrary constants expressed in M of DNA nucleotide residues. The GRC oligomeric forms which are predominant in low ionic strength media are supposed to have a higher DNA-binding affinity as compared to the monomeric forms which are predominant in high salt solutions. The interaction of GRC with DNA-cellulose qualitatively differs from the interaction of GRC with phosphocellulose. This suggests that electrostatic forces do not determine the formation of GRC-DNA complexes. So, in addition to nonspecific interaction, some specific "recognition" in GRC-DNA complexes may occur. Calculations made on the basis of GRC-DNA binding parameters derived for the conditions close to physiological (0.15 M NaCl, 20-30 degrees C, pH 7.4) demonstrate that rapid and nearly complete in vivo translocation of GRC form cytoplasm to nuclei may be a consequence of direct interaction of GRC with cellular DNA.

[Various properties of partially purified dexamethasone receptor complexes of the rat liver]. / Nekotorye svoistva chastichno ochishchennykh deksametazon-retseptornykh kompleksov pecheni krys.

A 300-fold purification of glucocorticosteroid-receptor complexes (GRC) of the rat liver with the 10--25% yield was conducted by combining the methods of binding with DNA-cellulose and salting out with ammonium sulfate at 30% saturation. An important feature of this method was a preponderant removal of protein admixtures capable of dissociation with DNA. In comparison of column chromatography and batch-procedure the latter proved to possess a number of advantages. Some properties of partially purified GRC were also studied. As revealed, the purified GRC which rapidly decomposed at 2 degrees C, could be partially stabilized by addition of inert protein, glycerine, SH-reagents and an excess of dexamethazone. Under such conditions about 13% of the initial amount of purified GRC was preserved after a week of storage at 2 degrees C. Determination of purified GRC content in the preparation by sorption on dextran coated carbon gave sharply diminished results; as to the method of oxyapatite sorption--it proved to be adequate. In comparing the GRC properties without and after the purification demonstrated that sedimentation and other properties of these preparations largely coincided. GRC-acceptor capacity of one- and two-chain DNA depended to a different degree on cytoplasmic admixtures in the GRC preparation removed in its purification.