Effects of gold nanoparticles on erythrocyte hemolysis.

We studied hemolytic activity of gold nanoparticles added to the whole blood (ex vivo) and of nanoparticles coated and not coated with plasma components on erythrocytes in hypotonic medium (osmotic hemolysis) in vitro. Gold nanoparticles did not stimulate erythrocyte hemolysis after 4-h incubation with the whole blood ex vivo. Hemolysis tended to increase in the presence of small gold nanoparticles (5, 10, 20 nm) at the maximum concentration of 20 µM (by gold content) used in our study in comparison with the control. This tendency was detected during the 1st hour of the nanoparticles incubation with blood. Gold nanoparticles in the used concentrations (up to 20 µM of gold) coated with plasma components after preincubation with autologous plasma and nanoparticles without coating caused no osmotic hemolysis of erythrocytes in vitro.

Studies of oxidant-induced changes in albumin transport function with a fluorescent probe K-35. Metal-catalyzed oxidation.

The dynamics of albumin transport function was studied during metal-catalyzed oxidation of albumin in diluted blood plasma from healthy donors and in the solution of purified albumin using fluorescent probe K-35. The changes were compared with the dynamics of free radical oxidation markers. For oxidation, different concentrations of Cu(2+), Fe(2+), Fe(3+) ions as well as EDTA and H(2)O(2) were used. Oxidative modification of proteins was assessed by carbonyl and bityrosine fluorescent products. Oxidation of plasma lipids was assessed by the levels of TBA-reactive products. It was found that oxidation markedly decreased effective concentration of albumin characterizing albumin binding capacity, and leads to accumulation of carbonyl products of protein oxidation, bityrosine fluorescent products in proteins, and TBA-active products of lipid oxidation. It was hypothesized that reduced effective concentration of albumin is related to impairment of its binding sites and/or accumulation of free-radical oxidation products filling the binding sites of albumin.

Studies of oxidant-induced changes in albumin transport function with a fluorescent probe k-35. Effect of hypochlorite.

The dynamics of changes in albumin transport function during hypochlorite-induced oxidation of isolated albumin in blood plasma and serum was studied with a fluorescent probe K-35. Binding of the probe K-35 to albumin was characterized by effective concentration of albumin. Oxidative modification of proteins was evaluated by the content of carbonyl products of protein oxidation and bityrosine fluorescent products. Oxidation with hypochlorite was accompanied by a decrease in the effective concentration of albumin in albumin, diluted plasma, and serum and accumulation of carbonyl products of protein oxidation and bityrosine fluorescent products. The decrease in the effective concentration of albumin during oxidation with hypochlorite can be explained by oxidative damage to albumin binding sites. Oxidative modification of probe K-35 binding sites with hypochlorite contributes to a decrease in effective concentration of albumin under pathological conditions.

Detection of mutations in Mycobacterium tuberculosis genome determining resistance to fluoroquinolones by hybridization on biological microchips.

We developed a method of identification of Mycobacterium tuberculosis with simultaneous evaluation of the sensitivity to fluoroquinolones on a biological microchip array. The method of multiplex two-staged PCR followed by hybridization of a biochip makes it possible to detect 8 mutant variants of gyrA gene occurring in fluoroquinolone-resistant strains (approximately 85% all resistant forms) within 1 day. Using this method we analyzed 107 cultures isolated from patients with tuberculosis and 78 sputum samples. Mutations in gyrA gene were detected in 48 (92%) resistant strains. Natural S95T polymorphism in gyrA gene was detected in all resistant and in 76% sensitive strains. The sensitivity and specificity of the proposed method calculated on the basis of the analysis of sputum samples (n=78) were 94 and 100%, respectively.

[Use of molecular-biological microchip TB-BIOCHIP-2 for detecting of Mycobacterium tuberculosis with multidrug resistance to fluoroquinolones in patients with new detected and chronic tuberculosis].

At present the left-handed "respiratory" quinolones such as moxifloxacin and levofloxacin are the most promising drugs for therapy of multidrug resistant tuberculosis (MDR). Fast and specific diagnostics of sensitivity of M. tuberculosis (MBT) with MDR to this group of drugs is required for timely prescription of adequate chemotherapy and its correction in case of MBT resistance to fluoroquinolones. A new generation of biological microchips - TB-BIOCHIP-2 makes possible to detect 9 mutation types in quinolones resistant determination region (QRDR) of gene. About 800 samples from 169 patients in Antituberculosis center were studied. In patients with new detected tuberculosis 23.5% MBT resistant to isoniazid and rifampicin (MDR) and sensitive to fluoroquinolones were revealed. In patients with chronic tuberculosis 65.5% MBT-MDR were revealed. Our results were confirmed with detecting ofloxacin resistance on Lowenstein - Jensen. In addition efficiency of TB-BIOCHIP-2 to control drug testing sensitivity of MBT-MDR on fluoroquinolones was confirmed.

[Investigation of Mycobacterium tuberculosis sensitivity to fluoroquinolone, by revealing gyrA gene mutations].

The resistance of Mycobacterium tuberculosis (MBT) to fluoroquinolones is associated with the mutations concentrated in the gyrA gene that is a structural gene of a gyrase A subunit. Detection of mutations in this portion of the gene allows the sensitivity of MBT to this group of drugs to be rapidly determined.

[Use of molecular genetic studies to determine the species of the mycobacteria mais and M. tuberculosis complex].

The genus Mycobacterium currently comprises more than 90 species of Mycobacterium, of which a third is able to induce human diseases. With a rise in the incidence of diseases induced by non-tuberculosis mycobacteria, tuberculosis caused by M. bovis that is characterized by a severe cause and a high frequency of poor outcomes cannot be remembered. The species of mycobacteria should be identified to establish a diagnosis and to prescribe adequate chemotherapy. For this purpose, cultural, biochemical, chromatographic, and molecular genetic studies are conducted. The present study using the hsp65 gene restriction fragment length polymorphism test on museum mycobacterial strains and strains isolated from the diagnostic material of patients with suspected tuberculosis by means of Hind61 restrictase has provided a clear differentiation of the restriction profiles of MAIS complex mycobacteria and some other species of non-tuberculosis mycobacteria. To determine the species of representatives of M. tuberculosis complex (M. tuberculosis, M. bovis, BCG M. bovis), the authors have successfully used the test system "TUB-dif" developed at the Institute of Molecular Genetics, Russian Academy of Sciences, by applying the chain polymerase reaction of the senX3-regX3 region.

[Determination of multidrug resistance of M. tuberculosis by different methods].

Testing the direct nitrate reductase technique versus the absolute concentration test has indicated that the former may be successfully used for rapid determination of the sensitivity of Mycobacterium tuberculosis (MBT) to isoniazid and rifampicin and it can reduce the time of obtaining a result by 4-5 times in the cases that sputum bacterioscopy yielded a positive result that allows the modified method to be applied. The advantages of the TB-Biochip technique are the time of detection multidrug-resistant MBT (24 hours), a possibility of obtaining these data just when analyzing sputum-isolated MBT DNA, and characterization of the MBT genomic elements that are responsible for drug sensitivity to antituberculous agents, by determining mutations in the examined genes and this all by using one chip. The agreement of results of microbiological and molecular genetic studies study of drug MBT sensitivity was 98%. There were no differences in the results of those using isoniazid. As for rifampicin, there was a difference in two samples (3.8%). Analysis of a combination of mutations forming multidrug resistance indicated that 74.3% of multidrug-resistant MBT isolates had mutations in the codon Ser531 > Lue of the rpoB gene and in the codon Ser315 > Thr of the katG gene. 97.4% of strains with signs of multidrug resistance had mutations in the codon 315 of the katG gene. 20.5% of isoniazid-resistant strains were observed to have mutations in two genes (katG and inhA) and 28.2% of the strains exhibited double mutation in the katG gene - Ser315Thr and Ile335 > Val.

Identification of mycobacteria of the MAIS Complex and M. tuberculosis by restriction fragment length polymorphism analysis of hsp65 gene.

Restriction fragment length polymorphism analysis of hsp65 gene was performed on museum strains of mycobacteria using Hin6I restrictase. Study of restriction profiles allowed us to distinguish mycobacterial species of the MAIS complex and several strains of nontuberculous mycobacteria.

[The composition and drug sensitivity of a mycobacterial population in patients with suspected tuberculosis].

By using the diagnostic material (175 sputum samples and 103 bronchoalveolar lavage fluid samples) taken from 39 patients with suspected tuberculous infection during a 2.5-month follow-up, the authors traced the time course of changes in the composition and drug sensitivity of a mycobacterial population to rifampicin. Along with the traditional microbiological studies, the latest molecular biological studies, a TB-BIOCHIP test system (enzyme immunoassay) in particular, were employed to detect the bacterial and L-transformed forms of the causative agent. A molecular biological assay was first developed to detect the drug sensitivity of L-forms of Mycobacterium tuberculosis.

[The characteristics of the sensitivity of Mycobacterium tuberculosis to rifampicin and isoniazid through determination of mutations in the genes rpoB, katG, inhA, oxyR, and kasA by different molecular biological assays].

Two hundred and two patients with different forms of pulmonary tuberculosis were examined to study the characteristics of sensitivity with the signs of multidrug resistance to rifampicin and isoniazid, by using a microbiological assay of the absolute concentrations and determining mutations in the genes rpoB, katG, inhA, oxyR, and kasA, by employing different molecular biological assays. Mycobacterium tuberculosis (MBT) DNA was isolated from both a diagnostic material (such as sputum, bronchial secretion), and clinical MBT isolates. By showing a higher sensitivity and a higher specificity, as cultural techniques, molecular biological assays of MBT drug sensitivity in patients with tuberculosis were ascertained to accelerate its diagnosis until the patient was admitted to a clinic.

[New technologies in the determination of drug susceptibility in Mycobacterium tuberculosis]. / Novye tekhnologii opredeleniia lekarstvennoi chuvstvitel'nosti Mycobacterium tuberculosis.

A variety of mutations in the genes rpoB, katG, inhA, ahpC, kasA was studied by using different molecular biological methods (conformational polymorphism of single-chain fragments, heteroduplex analysis, biochips) in rifampicin- and isoniazid-resistant Mycobacterium tuberculosis (MBT) strains isolated from patients with pulmonary tuberculosis. Twenty-nine mutation combinations were identified in the MBT strains. The use of biochips is the most promising method for identifying the type of mutations responsible for the simultaneous resistance to rifampicin and isoniazid. Detection of several MBT strains in one patient requires the use a combination of molecular biological and microbiological studies.

Typing of Mycobacterium tuberculosis strains resistant to rifampicin and isoniazid by molecular biological methods.

Mutations in the rpoB, katG, inhA, oxyR/ahpC genes in rifampicin- and isoniazid-resistant M. tuberculosis strains isolated from residents of Moscow, Astrakhan', and Moldova Republic were studied by molecular biological methods (heteroduplex analysis, single strand conformational polymorphism, biochips). Twenty-five combinations of mutations were detected. Some differences in the type distribution of detected mutations were found. The use of biochips is the most perspective method for determining the type of mutation.

[Molecular genetic methods for the detection of rifampicin-resistant Mycobacterium tuberculosis strains]. / Molekuliarno-geneticheskie metody vyiavleniia rifampitsinrezistentnykh shtammov Mycobacterium tuberculosis.

RCR-heteroduplex (GDA) and chip methods were used to detect rifampricin-resistant (RR) and rifampicin-sensitive (RS) Mycobacterium tuberculosis (MTB) in the samples from patients (sputum) and in the clinical isolates of MTB from these patients (MB/BacT liquid medium and Lowenstein Jensen's (LJ) solid medium. The efficiency of detecting RR and RS of MTB (from the sputum) is 100 and 92.3% in the chip and GDA tests, respectively. Correlations between GDA (sputum) and drug test (LJ) were 91.7%, that of chip (sputum) and drug test LJ, 88.5%, chip (sputum) and chip clinical isolates (LJ), 100%. The efficacy of GDA and chip in the detection of RR of MTB strains is under discussion.

Detection of rifampicin-resistant Mycobacterium tuberculosis strains by hybridization and polymerase chain reaction on a specialized TB-microchip.

Two alternative methods for identification of rifampicin-resistant strains of Mycobacterium tuberculosis on biological microchips are developed. The methods are based on detection of point mutations and other rearrangements in the rpoB gene region determining rifampicin resistance. Hybridization on TB-microchip detects 30 mutant variants of DNA in rifampicin-resistant strains (about 95% of all resistant forms). Allele-specific microchip PCR shortens the duration of analysis to 1.5 h. These methods can be used in clinical diagnostic laboratories for evaluating drug resistance/sensitivity of tuberculosis agent and for monitoring of the efficiency of antibiotic therapy.

Application of nested-PCR technique for the diagnosis of tuberculosis.

A test-system based on amplification of IS 986 fragment (nested-PCR) was developed for the detection of Mycobacterium tuberculosis and M. bovis in different biological samples. We constructed external primers and selected appropriate amplifications parameters (annealing temperatures for states I and II, the number of cycles for each amplification stage, components of the amplification mixture, and pretreatment conditions for different biological samples). The developed parameters make the detection of mycobacteria more efficient and less expensive compared to commercial Cobas Amplicor system.

[The prediction of the individual chemosensitivity of human malignant tumors: the biological patterns and clinical results]. / Prognozirovanie individual'noi khimiochuvstvitel'nosti zlokachestvennykh opukholei cheloveka: biologicheskie zakonomernosti i klinicheskie rezul'taty.

Individual laboratory prediction of the sensitivity of human neoplasms to chemicals is justified on the basis of the authors' own data and those available in the literature. The potentialities and limitations of methods for individual prediction, as well as clinical results are discussed. Data are given on some biological mechanisms responsible for tumor growth which have been obtained in in vitro and in vivo tests with human tumor tissue and carcinoma cells. Further perspectives of this line of studies are discussed.