Rapid detection of isoniazid resistance in Mycobacterium tuberculosis isolates by use of real-time-PCR-based melting curve analysis.

The MeltPro TB/INH assay, recently approved by the Chinese Food and Drug Administration, is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test specially designed to detect 30 isoniazid (INH) resistance mutations in katG position 315 (katG 315), the inhA promoter (positions -17 to -8), inhA position 94, and the ahpC promoter (positions -44 to -30 and -15 to 3) of Mycobacterium tuberculosis. Here we evaluated both the analytical performance and clinical performance of this assay. Analytical studies with corresponding panels demonstrated that the accuracy for detection of different mutation types (10 wild-type samples and 12 mutant type samples), the limit of detection (2×10(3) to 2×10(4) bacilli/ml), reproducibility (standard deviation [SD], <0.

Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.

Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA.

Modern Approaches to Cancer Therapy: Immunotherapy and Targeted Drug Delivery.

This article reviews recent achievements in molecular immunology that have given a new insight into the mechanisms of tumor escape from the control of immunocompetent cells and opened the way to the development of radically new immunotherapeutic procedures and further improvement of existing chemotherapeutic techniques. The application of new updated techniques in molecular biology, biochemistry and cellular biotechnology has made it possible to detect molecular changes in tumor cells which help to escape them from immunological control and develop on their basis some novel approaches to immunotherapy of malignant tumors. One of the main challenges to immunotherapy, which is related to recognition of tumor antigens by immunocompetent cells, has been solved through generation of antitumor vaccines based on dendritic cells.

Structural and functional properties of Langmuir films of antibodies based on amphiphilic polyelectrolytes.

We optimized the procedure for the formation of Langmuir films of antibodies based on amphiphilic polyelectrolytes and studied the physicochemical and immunochemical properties of the films obtained. Their immunochemical properties were compared with the immunochemical activity of antibodies in Langmuir films without amphiphilic polyelectrolytes and with antibodies adsorbed on the surface of polystyrene and graphite. The efficiency of immune adsorption by the films based on amphiphilic polyelectrolytes was shown to be greater; the affinity of antibodies and surface concentration of their active conformation depended on the type of amphiphilic polyelectrolytes used to obtain the films.

STM studies of LB films of amphiphilic polyelectrolytes with antibodies and enzymes.

The structure of LB films of protein-polyelectrolyte complexes transferred onto the pyrographite surface was studied by STM. The images of the films obtained at various protein concentrations in the water subphase and different values of surface pressure were captured. The topology of the surface covered by one or three layers of the films studied was investigated.

Langmuir films from antibodies based on amphiphilic polyelectrolytes.

A technique for forming Langmuir films from antibodies based on an amphiphilic polyelectrolyte was developed. The physicochemical and immunochemical properties of the Langmuir films obtained were studied. The interaction of HBsAg with the films was found to be described by a model with one binding site, whereas that of HBsAg with antibodies adsorbed on a polystyrene plate, by a model with a positive cooperativity.

Properties of the regulatory subunit of cAMP-dependent protein kinase type II from human brain.

The regulatory subunit type II (RII) of cAMP-dependent protein kinase purified from human brain was represented by two proteins with apparent molecular masses of 51-52 kD and 54 kD. Dephosphorylation of human RII containing 3 mol phosphate/mol protein did not change the electrophoretic pattern. One-dimensional peptide mapping of 51-52 kD and 54 kD proteins after digestion with St.

Anthracycline antibiotics non-covalently incorporated into the block copolymer micelles: in vivo evaluation of anti-cancer activity.

The chemosensitising effects of poly(ethylene oxide)-poly(propylene oxide)-poly-(ethylene oxide) (PEO-PPO-PEO) block copolymers (Pluronic) in multidrug-resistant cancer cells has been described recently (Alakhov VY, Moskaleva EY, Batrakova EV, Kabanov AV 1996, Biocon. Chem., 7, 209).

Immunochemical studies on human, bovine and pig brain regulatory subunits of cAMP-dependent protein kinase type II.

Regulatory subunits type II (RII) purified from human, pig and bovine brains were compared using II monoclonal antibodies (MoAb) against bovine brain RII. Bovine RII has at least 5 antigenic sites located in the N-terminal 1-110 residues. Immunochemical difference detected between human and animal RII was more pronounced than between pig and bovine RII.

mRNA stabilization in continuous flow translation system.

In contrast with standard in vitro translation systems, where 1 to 2 copies of polypeptide per mRNA molecule are produced, the continuous flow cell-free translation system is able to synthesize hundreds of polypeptide molecules per one mRNA molecule. Our investigations have shown that the poor yield obtained in the standard analytical system is due to rapid mRNA decay as opposed feedback inhibition by low molecular weight translation by products. In contrast, continuous flow system was found to stabilize mRNA for up to two-three days.

Fatty acid acylated peroxidase as a model for the study of interactions of hydrophobically-modified proteins with mammalian cells.

Artificial fatty acylation of proteins has attracted significant attention during the last decade as a method for modification of protein specificity and efficacy of action on mammalian cells (A. V. Kabanov and V.

Modification of fluid lipid and mobile protein fractions of reticulocyte plasma membranes affects agonist-stimulated adenylate cyclase. Application of the percolation theory.

The technique of fluorescence recovery after photobleaching was used to measure the lateral mobility of membrane integral proteins in reticulocyte plasma membranes which were treated to modify the 'fluid' lipid or immobilized protein fractions, hence increasing the relative prevalence of obstacles to protein lateral motion. This was achieved by either: (1) treating the plasma membranes with phospholipase A2 followed by extraction of the hydrolysis products using fatty-acid-free bovine serum albumin, resulting in a decrease in the membrane 'fluid' lipid portion; or (2) preincubating the plasma membranes with polylysines, resulting in plasma membrane protein aggregation and immobilization. As the prevalence of obstacles to lateral motion increased in plasma membranes through the treatments described above, the mobility of the membrane integral proteins diminished.

Insulin-mediated intracellular targeting enhances the photodynamic activity of chlorin e6.

Photodynamic therapy has been applied quite extensively over the last few years, whereby the activation of photosensitizers by light causes the production of reactive oxygen species such as singlet oxygen, which is cytotoxic. The goal of this study was the enhancement of the photodynamic activity of photosensitizers through their delivery to specific, sensitive intracellular compartments of target cells. We synthesized a BSA-insulin-chlorin e6 conjugate that bound specifically to the insulin receptors (EC50, 1 nM) of the human hepatoma cell line PLC/PRF/5 and could be internalized by receptor-mediated endocytosis.

New catalytic properties of monoamine oxidase immobilized in Langmuir-blodgett films with amphiphilic polyelectrolytes.

Langmuir-Blodgett (LB) films of monoamine oxidase (MAO) have been formed on the surface f a polypropylene membrane using amphiphilic polyelectrolytes. The enzyme activity of such protein-polyelectrolyte films was measured by a Clark electrodes. It was shown that in LB films thus formed the use of amphiphilc polyelectrolytes, MAO activity was higher than in polyelectrolyte-free LB films.

Monoclonal antibodies directed against two different corticotropin-releasing factor determinants.

Two hybridomas secreting monoclonal antibodies (mAbs) against human/rat corticotropin-releasing factor (CRF) have been produced by the cell fusion technique. Isotyping of the mAbs revealed that both belong to the IgG1 subclass. Human serum containing CRF-binding protein inhibits the binding protein inhibits the binding of CRF to both mAbs.

The study of histamine H1- and H2-receptors in human lung cancer.

Data on human lung histamine H1- and H2-receptors in cancer and chronic inflammatory processes are reported. It has been found that the number of histamine H1-receptors significantly increases both in cancer and chronic pneumonia and does not practically change in tuberculosis lung parenchyma. The binding parameters of histamine H2-receptors both in cancer and inflammatory processes were similar to those obtained for the normal tissue.

The use of internalizable derivatives of chlorin E6 for increasing its photosensitizing activity.

Experiments with human hepatoma PLC/PRF/5 cells and human embryo skin fibroblasts involving the use of three different tests (colony formation, Trypan blue exclusion, labeled thymidine incorporation) have demonstrated a significantly higher photosensitizing activity of chlorin e6 conjugates with internalizable ligands as compared to that of chlorin e6 itself. Receptor-mediated internalization of chlorin e6 conjugates ensures a greater photosensitization of cells than binding of those conjugates to cell surface receptors. The suitability of such conjugates that permit the delivery of a photosensitizer to sensitive intracellular targets is discussed.

alpha-Latrotoxin receptor. Implications in nerve terminal function.

alpha-Latrotoxin is a potent stimulator of neurotransmitter release from nerve terminals. High affinity membrane alpha-latrotoxin receptor was purified in an active binding form. It is a membrane glycoprotein (M(r) 160,000-220,000) which may be complexed to a smaller polypeptide (M(r) 29,000).

Interaction of hydrophobized antiviral antibodies with influenza virus infected MDCK cells.

The ability of artificially stearoylated antibodies to influenza virus hemagglutinin and M1 proteins to interfere with influenza infection in MDCK cells has been studied. Both the modified anti-hemagglutinin (polyclonal) and anti-M1 (monoclonal) antibodies neutralize the virus when added before the infection. The effect can be attributed to the interaction of stearoylated antibodies with the virus surface, which is enhanced by fatty acylation (anti-hemagglutinin), or to the antibody uptake in the cell endocytic compartments simultaneously with the virion which permits antibodies to interact with the virus envelope internal antigen (anti-M1).

Alterations in human lung adrenergic receptors in cancer.

Data on human lung alpha 1- and beta-adrenoceptors and the alpha 1/beta adrenergic ratio in cancer and chronic inflammatory processes are reported. The number of alpha 1-adrenergic sites markedly increased in lung cancer parenchyma, giving a ratio of alpha 1/beta binding sites of 12/1 in cancer but almost 1:1 in control specimens. The alpha 1/beta adrenergic ratio obtained both for mild and severe chronic pneumonia and tuberculosis lung parenchyma was similar to that demonstrated for normal tissue.

Identification of functionally active fragments of staphylococcal enterotoxin B.

It has been found that staphylococcal enterotoxin B contains a proteolysis-sensitive sequence in the cysteine loop formed by two half-cystines located in the middle of the toxin polypeptide chain. Fragments of the enterotoxin formed as a result of its digestion in this region have been isolated, their N-terminal sequences have been determined and sites of proteolysis have been identified. It has been demonstrated that the N-terminal fragment of staphylococcal enterotoxin B is capable of activating T cell proliferation in the culture of human mononuclear cells practically to the same degree as the intact enterotoxin.

Preparative in vitro synthesis of bioactive human interleukin-2 in a continuous flow translation system.

Recombinant human interleukin-2 has been synthesized in vitro in a continuous flow translation system based on the wheat germ extract. In the course of translation of mRNA the interleukin-2 becomes aggregated due to the adsorption of this protein onto the ribonucleoprotein complex. This process correlates with the cessation of translation that is usually observed in 25-30 min.

Antibodies to the regulatory subunit of cAMP-dependent protein kinase type II from bovine brain inhibit the holoenzyme formation.

Mouse monoclonal antibodies to the bovine brain regulatory subunit type II (RII) were produced and antibodies of 11 clones were characterized. We determined their subclass, affinity constants, competivity and influence on two RII functions, namely cAMP binding and inhibition of the catalytic subunit (C) activity. 4 clones produced antibodies that increased RII-cAMP binding 1,5-2-fold.