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1.
Int J Biol Macromol ; 177: 455-462, 2021 Apr 30.
Article in English | MEDLINE | ID: mdl-33636261

ABSTRACT

Anionic liposomes were electrostatically adsorbed onto the surface of cationic chitosan particles cross-linked by sulfate anions, forming multi-liposomal containers (MLCs) for encapsulation and delivery of bioactive substances. An increase in molecular mass of chitosan from 30 to 300 kDa results in a size increase of chitosan particles, from 200 to 400 nm. Being saturated by liposomes, chitosan particles give MLCs of 320-540 nm. Each chitosan particle carries between 60 and 200 liposomes. The proteolytic complex Morikrase, a mixture of enzymes with various specificities, induces degradation of MLCs down to particles of size 10-15 nm; the higher the molecular mass of chitosan, the slower the enzyme-induced MLCs' degradation. pH variation within 5.5-7 and cholesterol incorporation into the liposomal membrane both have a minor effect on the rate of MLCs' biodegradation. Both the MLCs and the products of their biodegradation show low cytotoxicity. These results are of interest for constructing biodegradable capacious carriers of bioactive substances.


Subject(s)
Chitosan/chemistry , Cytotoxins/chemistry , Endopeptidases/chemistry , Multienzyme Complexes/chemistry , Chitosan/pharmacology , Cytotoxins/pharmacology , Humans , Liposomes , MCF-7 Cells , Particle Size
2.
Environ Res ; 191: 110049, 2020 12.
Article in English | MEDLINE | ID: mdl-32926891

ABSTRACT

Many natural substances exhibit anti-inflammatory activity and considerable potential in prophylaxis and treatment of allergies. Knowing exact molecular targets, which is required for developing these as medicinal products, is often challenging for multicomponent compositions. In the present study we examined novel polyphenolic substance, a water-soluble fraction of wood lignin (laboratory code BP-Cx-1). In our previous study, a number of polyphenolic components of BP-Cx-1 (flavonoids, sapogenins, phenanthrenes etc.) were identified as the major carriers of biological activity of BP-Cx drug family, and several molecular targets involved in cancer and/or inflammation signaling pathways were proposed based on the results of the in vitro and in silico screening studies. In the present study, half maximal inhibitory concentration (IC50) of BP-Cx-1 was established with a radioligand method and a range of IC50 values between 22.8 and 40.3 µg/ml were obtained for adenosine receptors A1, A2A and prostaglandin receptors EP2, IP (PGI2). IC50 for serotonin 5-HT1 and for glucocorticoid GR receptors were 3.0 µg/ml and 12.6 µg/ml, respectively, both being within the range of BP-Cx-1 concentrations achievable in in vivo models. Further, distribution of [3H] labelled BP-Cx-1 in NIH3T3 murine fibroblasts and MCF7/R carcinoma cells was studied with autoradiography. [3H]-BP-Cx-1 (visualized as silver grains produced by tritium beta particles) was mainly localized along the cell membrane, in the perinuclear region and in the nucleus, suggesting ability of BP-Cx-1 to enter cells and bind to membrane or cytosol receptors. In our experiment, we observed the effect of BP-Cx-1 on maturation of dendritic cells (DCs): downregulation of expression of the lipid-presentation molecule CD1a, co-stimulatory molecules CD80, CD83 and CD 40, decreased production of pro-inflammatory cytokines IL-4 and TNF-α and increased production of anti-inflammatory cytokine IL-10. It is hypothesized that [3H]-BP-Cx-1 detectable in the nucleus is part of the activated GR complex, known to be involved in regulation of transcription of genes responsible for the anti-inflammatory response. Based on IC50, cell distribution data and results of the experiment with DCs it is suggested that the in vivo effects of BP-Cx-1 are mediated via GR and 5-HT1 receptors thus promoting development of tolerogenic effector function in dendritic cells.


Subject(s)
Dendritic Cells , Lignin , Animals , Cytokines , Mice , NIH 3T3 Cells , Water
3.
Prikl Biokhim Mikrobiol ; 53(2): 164-72, 2017.
Article in Russian | MEDLINE | ID: mdl-29508977

ABSTRACT

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.


Subject(s)
Antineoplastic Agents, Phytogenic/chemistry , Asparaginase/chemistry , Bacterial Proteins/chemistry , Pectobacterium carotovorum/chemistry , Polyethylene Glycols/chemistry , Antineoplastic Agents, Phytogenic/biosynthesis , Antineoplastic Agents, Phytogenic/pharmacology , Asparaginase/biosynthesis , Asparaginase/genetics , Asparaginase/pharmacology , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Cell Survival/drug effects , Chromatography, Gel , Cloning, Molecular , Cross-Linking Reagents/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , HL-60 Cells , Humans , Jurkat Cells , K562 Cells , Pectobacterium carotovorum/enzymology , Polyethylene Glycols/pharmacology , Protein Engineering , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Succinimides/chemistry
4.
Biomed Khim ; 61(3): 312-24, 2015.
Article in Russian | MEDLINE | ID: mdl-26215408

ABSTRACT

For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemia in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases as Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA) and Rhodospirillum rubrum (RrA); special attention is paid to isolation of purified enzymes and their crystallization, modification by chitosan/polyethylene, physicochemical, kinetic and structural properties characterization, and the study of the cytotoxic or anti-proliferative activity of new recombinant L-asparaginases on cell cultures in vitro. The resultant recombinant L-asparaginases (EwA, YpA, HpA и RrA) exhibit reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available L-asparaginase EcA and represent practical interest in respect to creation, on their basis, new effective antineoplastic remedies. Further prospects of researches on bacterial L-asparaginases are associated with development of analogs of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes of the protein structure using genetic engineering, development of chito-PEGylation for receiving L-asparaginase preparations with improved pharmacokinetic characteristics.


Subject(s)
Antineoplastic Agents/pharmacology , Asparaginase/chemistry , Asparaginase/pharmacology , Bacterial Proteins/pharmacology , Amino Acid Sequence , Antineoplastic Agents/chemistry , Asparaginase/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cell Line, Tumor/drug effects , Helicobacter pylori/enzymology , Humans , Leukemia/drug therapy , Leukemia/pathology , Molecular Sequence Data , Pectobacterium carotovorum/enzymology , Protein Engineering/methods , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Rhodospirillum rubrum/enzymology , Yersinia pseudotuberculosis/enzymology
5.
Klin Lab Diagn ; (12): 19-24, 2009 Dec.
Article in Russian | MEDLINE | ID: mdl-20140999

ABSTRACT

A number of interleukins and iron metabolic parameters were studied in acute leukemia over time. Regulation of hemopoiesis and the cytokine network has been found to be impaired, which appears as the increased synthesis of ferritin and hepsidin by macrophages that are activated by imbalance in the cytokine network. A severe impairment of the parameters responsible for a regulatory process is shown to occur in leukemia. There is no complete normalization of these parameters during hematological remission, which is likely to lead to a further relapse of leukemic process.


Subject(s)
Cytokines/blood , Leukemia/blood , Leukocytes/metabolism , Acute Disease , Agranulocytosis/blood , Agranulocytosis/chemically induced , Antimicrobial Cationic Peptides/blood , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Carrier Proteins/blood , Ferritins/blood , Folate Receptors, GPI-Anchored , Hepcidins , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/blood , Interleukins/blood , Leukemia/drug therapy , Leukemia/immunology , Receptors, Cell Surface/blood , Serum , Tumor Necrosis Factor-alpha/blood
6.
Biochim Biophys Acta ; 1758(10): 1685-95, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16901462

ABSTRACT

Both natural and synthetic polycations can induce demixing of negatively charged components in artificial and possibly in natural membranes. This process can result in formation of clusters (binding of several components to a polycation chain) and/or domains (aggregation of clusters and formation of a separate phase enriched in some particular component). In order to distinguish between these two phenomena, a model lipid membrane system containing ion channels, formed by a negatively charged peptide, O-pyromellitylgramicidin, and polycations of different structures was used. Microelectrophoresis of liposomes, changes in boundary potential of planar bilayers, the shape of compression curves and potentials of lipid and lipid/peptide monolayers were used to monitor the electrostatic factors in polymer adsorption to the membrane and peptide-polymer interactions. The synthesized PEO-grafted polylysine, PLL-PEO20000, did not induce peptide demixing monitored by stabilization of the gramicidin channels, in contrast to parent polylysine (PLL). Both polymers were shown to bind effectively to negatively charged liposomes and lipid monolayers, suggesting that the ineffectiveness of PLL-PEO20000 was not due to reduction of its binding. It was hypothesized that PLL-PEO20000 could not induce domain formation due to steric hindrance of long PEO chains preventing lateral fusion of clusters. Another copolymer, PLL-PEO4000, having four PEO chains of 4000 Da, exhibited intermediate effect between PLL and PLL-PEO20000, which shows the importance of the copolymer architecture for the effect on the lateral distribution of OPg channels. The model system can be relevant to regulation of lateral organization of ion channels and other components in natural membrane systems.


Subject(s)
Gramicidin/analogs & derivatives , Lipid Bilayers/chemistry , Membrane Lipids/chemistry , Membranes, Artificial , Polyethylene Glycols/chemistry , Polylysine/chemistry , Gramicidin/chemistry , Liposomes/chemistry
7.
Langmuir ; 20(16): 6575-9, 2004 Aug 03.
Article in English | MEDLINE | ID: mdl-15274557

ABSTRACT

Complexation of antitumor drug, doxorubicin (DOX), with poly(acrylic acid) (PAA) in buffer solutions was examined. The DOX-to-PAA binding was governed by electrostatic and stacking interactions resulting in a complex of characteristic composition with a PAA/DOX = 1.6 molar ratio. Sizes of the complex particles were found to lie in 600-900-nm range. However, the particles were able to interact with small neutral egg yolk lecithin liposomes (80-100 nm in diameter), a ternary DOX/PAA/liposome complex being formed. The observations and conclusions we made may be useful for interpreting biological effects of polymer-based bioactive constructs.


Subject(s)
Acrylic Resins/chemistry , Doxorubicin/chemistry , Liposomes/chemistry , Egg Yolk/chemistry , Particle Size , Phosphatidylcholines/chemistry , Static Electricity , Surface Properties
8.
Langmuir ; 20(16): 6796-9, 2004 Aug 03.
Article in English | MEDLINE | ID: mdl-15274587

ABSTRACT

A model, based on fluorescence data, is developed for the poly(acrylic acid)-assisted transport of doxorubicin, a cationic antitumor drug, through a bilayer membrane. Accordingly, the doxorubicin binds to the poly(acrylic acid) via electrostatic polymer-drug interactions plus drug-drug stacking within the complex. This complex associates with neutral egg lecithin vesicles by means of hydrophobic attraction between the doxorubicin and the vesicle bilayers. In the process, the doxorubicin "destacks", providing a fluorescence change that can be monitored. Finally, the doxorubicin enters the vesicle interior which has been imparted with an acidic pH to protonate the doxorubicin and thus, in a second stage, yield an additional fluorescence change that can also be detected. A portion of the poly(acrylic acid), now devoid of doxorubicin, then leaves the outer vesicle surface and enters to external solution.


Subject(s)
Acrylic Resins/chemistry , Doxorubicin/chemistry , Membranes, Artificial , Egg Yolk/chemistry , Fluorescence , Hydrogen-Ion Concentration , Lipid Bilayers/chemistry , Phosphatidylcholines/chemistry , Static Electricity , Surface Properties , Time Factors
9.
Biochim Biophys Acta ; 1611(1-2): 44-54, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12659944

ABSTRACT

Small (40-60 nm in diameter) and large (300-350 nm) negative vesicles were complexed with a cationic polypeptide, poly-L-lysine (PL). Laser microelectrophoresis experiments showed that in small vesicles rendered anionic with the addition of cardiolipin (CL(2-)), only the CL(2-) in the outer leaflet is involved in the complexation with PL. Calorimetric and other data demonstrate that the binding of PL to the membrane surface causes domains ("rafts") of CL(2-) to form in the outer leaflet, and it is these domains that electrostatically bind the polymer. The kinetics of transmembrane permeation of doxorubicin (Dox, a fluorescent anti-tumor drug) was monitored with and without PL binding to the outer surface of the vesicles. It was found that PL mediates the permeation of Dox into the vesicle interior. In the absence of PL, the Dox molecule (possessing an amino group of pK(a)=8.6) binds to the anionic vesicles in the protonated form and, consequently, suffers an impaired mobility through the membrane. On the other hand, when the PL covers the vesicle surface, Dox passes though the membrane with greater ease. The effects of salt and polyanion on the stability of PL-vesicle complexes and the PL-mediated Dox permeation are also discussed.


Subject(s)
Cardiolipins/chemistry , Membrane Lipids/chemistry , Polylysine/chemistry , 1,2-Dipalmitoylphosphatidylcholine , Adsorption , Anions , Antineoplastic Agents/chemistry , Conductometry , Doxorubicin/chemistry , Hydrogen-Ion Concentration , Permeability/drug effects , Phosphatidylcholines/chemistry , Polylysine/pharmacology , Transport Vesicles/chemistry , Transport Vesicles/drug effects
11.
Biochim Biophys Acta ; 1514(1): 139-51, 2001 Sep 03.
Article in English | MEDLINE | ID: mdl-11513811

ABSTRACT

Proteoliposomes were prepared by making bilayer vesicles from neutral egg yolk lecithin and negatively charged alpha-chymotrypsin that had been previously stearoylated. Interaction of these proteoliposomes with a cationic polymer, poly-(N-ethyl-4-vinylpryidinium bromide) (PEVP) was examined. For comparison purposes, interaction of PEVP with egg lecithin vesicles containing an anionic phospholipid, cardiolipin, was also examined. Binding of PEVP to both types of vesicles was electrostatic in nature with the polymer manifesting a higher affinity to the cardiolipin relative to the enzyme. PEVP had no effect on the permeability of the bilayer membranes to sodium chloride. On the other hand, PEVP increased the transmembrane permeability of the nonionic anti-tumor drug, doxorubicin. The greater the negatively charged component in the membrane, the greater the PEVP effect. Polycation binding to the vesicles was accompanied by clustering of the stearoylated chymotrypsin (sCT) molecules within the membrane. This protein clustering is most likely responsible for the increase in the doxorubicin permeation. Enzymatic activity of the membrane-associated sCT remained unchanged upon PEVP binding. These findings seem relevant to the effects of polyelectrolytes on cellular membranes.


Subject(s)
Liposomes/chemistry , Polymers/chemistry , Polyvinyls/chemistry , Proteolipids/chemistry , Cations , Chymotrypsin/chemistry , Phosphatidylcholines/chemistry , Static Electricity
12.
Chemistry ; 7(22): 4835-43, 2001 Nov 19.
Article in English | MEDLINE | ID: mdl-11763452

ABSTRACT

Laser microelectrophoresis (coupled with conductance, fluorescence, and dynamic light scattering) is shown to be a highly instructive tool in comparing the dynamics of conventional and gemini surfactants embedded within vesicle bilayers. The following can be listed among the more important observations and conclusions: a) Cationic conventional surfactant, added to a "solid" (gel) lipid vesicle containing an anionic phospholipid, charge-neutralizes only half the anionic charge. With a "liquid" (liquid crystalline) vesicle, however, the entire negative charge is neutralized. Thus, the cationic conventional surfactant can "flip-flop" readily only in the liquid membrane. b) A cationic gemini surfactant charge-neutralizes only the anionic lipid in the outer membrane leaflet of either solid or liquid membranes, thus indicating an inability to flip-flop regardless of the phase-state of the bilayer. c) Mixed population experiments show that surfactants can hop from one vesicle to another in liquid but not solid membranes. d) In liquid, but not solid, bilayers, a surface-adsorbed cationic polymer can electrostatically "drag" anionic surfactant from the inner leaflet to the outer leaflet where the polymer resides. e) Peripheral fluorescence quenching experiments show that a cationic polymer, adhered to anionic vesicles, can be forced to dissociate in the presence of high concentrations of salt or an anionic polymer. f) Adsorbed polymer, of opposite charge to that imparted to vesicles by a gemini surfactant, is unable to dislocate surfactant even in a liquid membrane. g) In our systems, ionic polymers will not bind to neutral vesicles made solely of zwitterionic phospholipid. On the other hand, ionic polymers bind to neutral vesicles if charge neutrality is obtained by virtue of the membrane containing equimolar amounts of cationic and anionic surfactant. This is attributable to surfactant segregation within the bilayer. h) Experiments prove that polymer migration can occur among a population of neutral ternary vesicles.


Subject(s)
Lipid Bilayers , Membranes, Artificial , Surface-Active Agents/chemistry
13.
Biochim Biophys Acta ; 1468(1-2): 73-86, 2000 Sep 29.
Article in English | MEDLINE | ID: mdl-11018653

ABSTRACT

The effects of ethylene oxide and propylene oxide block copolymers (pluronics) on the permeability of several weak acids and bases through bilayer lipid membranes have been studied by the methods of monitoring (1) pH shifts near planar bilayers, (2) doxorubicin fluorescence quenching inside liposomes, and (3) current transients in the presence of hydrophobic anions. It has been shown that pluronics facilitate the permeation of comparatively large molecules (such as 2-n-undecylmalonic acid and doxorubicin) across lipid bilayers, while the permeation of small solutes (such as ammonium and acetic acid) remains unaffected. Pluronics also accelerate the translocation of large hydrophobic anions (tetraphenylborate). The effect of pluronics correlates with the content of propylene oxide units: it is enhanced when the portion of polypropylene oxide block in the copolymer is increased. The action of the pluronic on lipid membrane permeability differs from the effect of the conventional detergent Triton X-100, which does not affect doxorubicin transport if added at concentrations similar to those used for pluronics. It has been proposed that pluronics accelerate the processes of solute diffusion within lipid bilayers (in a structure-dependent manner) rather than influencing the rate of solute adsorption/desorption on the membrane surface. We suppose that the effect of pluronics on doxorubicin permeation across lipid bilayers along with the known effect on the multidrug resistance protein determines its influence on the therapeutic activity of anthracycline drugs.


Subject(s)
Doxorubicin/chemistry , Epoxy Compounds/pharmacology , Ethylene Oxide/pharmacology , Lipid Bilayers/chemistry , Biological Transport , Diffusion , Doxorubicin/metabolism , Epoxy Compounds/chemistry , Ethylene Oxide/chemistry , Fluorescence , Hydrogen-Ion Concentration , Kinetics , Liposomes/chemistry , Permeability/drug effects , Polymers , Structure-Activity Relationship , Tetraphenylborate/chemistry , Tetraphenylborate/metabolism
14.
FEBS Lett ; 461(3): 141-4, 1999 Nov 19.
Article in English | MEDLINE | ID: mdl-10567685

ABSTRACT

A set of artificially hydrophobized alpha-chymotrypsin derivatives, carrying 2-11 stearoyl residues per enzyme molecule, were synthesized and their catalytic parameters and conformation in water solution and in the liposome-bound state were investigated. Hydrophobization of alpha-chymotrypsin and its further incorporation into phosphatidylcholine (PC) liposomes have no effect on the rate constant of the N-acetyl-L-tyrosine ethyl ester (ATEE) ester bond hydrolysis (k(cat)). At the same time, an increase in the number of stearoyl residues attached to the enzyme results in a drastic decrease of ATEE binding to the active center (K(M) increase). Incorporation of the hydrophobized enzyme into the PC liposome membrane results in K(M) recovery to nearly that of native alpha-chymotrypsin. The above changes are accompanied by partial unfolding of the enzyme molecules observed by fluorescence measurements. The obtained results are of interest to mimic the contribution of surface hydrophobic sites in the functioning of membrane proteins.


Subject(s)
Chymotrypsin/chemistry , Lipid Bilayers/chemistry , Protein Conformation , Proteolipids/chemistry , Animals , Binding Sites , Catalysis , Chemical Phenomena , Chemistry, Physical , Chymotrypsin/metabolism , Hydrolysis , Liposomes , Phosphatidylcholines , Protein Binding , Stearic Acids/chemistry , Tyrosine/analogs & derivatives , Tyrosine/metabolism
15.
Anal Biochem ; 269(2): 393-8, 1999 May 01.
Article in English | MEDLINE | ID: mdl-10222015

ABSTRACT

A rapid magnetic beads-based immunoassay for the immunodepressant drug cyclosporin A (CsA) has been developed. The method allows CsA determination in medium with a higher content of ethanol compared to conventional immunochemical techniques due to increased antibody stability. Monitoring of the drug in ethanol extracts from patient's whole blood without many-fold dilution with aqueous buffer is possible. The assay has adequate specificity and sensitivity for CsA to be suitable for the routine monitoring of therapy.


Subject(s)
Blood Chemical Analysis/methods , Cyclosporine/blood , Fluorescent Antibody Technique , Immunosuppressive Agents/blood , Antibody Affinity , Blood Chemical Analysis/statistics & numerical data , Cyclosporine/immunology , Ethanol , Fluorescent Antibody Technique/statistics & numerical data , Humans , Immunosuppressive Agents/immunology , Magnetics , Sensitivity and Specificity
16.
Biochemistry (Mosc) ; 64(4): 390-8, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10231591

ABSTRACT

A Triton X-100 extract from rat brain mitochondria was obtained using low detergent/protein ratio. From this extract a proteinaceous complex was purified; its molecular weight was as high as 880 kD. The complex contained both hexokinase and creatine kinase activity. When incorporated into phospholipid bilayer membranes, the complex formed a channel whose activity was different than the channel activity of purified porin isolated either by adsorption chromatography or by dissociation from protein complexes. A ligand of the mitochondrial benzodiazepine receptor (Ro5-4864) in submicromolar concentrations had an apparent influence on the kinetic behavior of enzymatic coupling of hexokinase and creatine kinase. It is suggested that the 880-kD complex is formed by mitochondrial contact sites. The role of the isolated protein complex in the formation of nonspecific permeability in mitochondria is discussed.


Subject(s)
Mitochondria/metabolism , Nerve Tissue Proteins/metabolism , Animals , Benzodiazepinones/metabolism , Brain/enzymology , Brain/metabolism , Brain/physiology , Creatine Kinase/metabolism , Female , Hexokinase/metabolism , Membrane Potentials , Mitochondria/enzymology , Mitochondria/physiology , Rats , Receptors, GABA-A/metabolism
17.
FEBS Lett ; 446(1): 194-8, 1999 Mar 05.
Article in English | MEDLINE | ID: mdl-10100641

ABSTRACT

Ethylene oxide and propylene oxide block copolymers (pluronics) are widely known as agents that promote drug penetration across biological barriers. We have studied the interaction of normal and malignant blood cells with pluronics L61 and P85 that have different hydrophobicity. SP2/0 myeloma cells accumulated pluronics while normal cells adsorb most of the polymer on the surface. Interaction of pluronics with cells resulted in drastic changes of membrane microviscosity. Tumor cell membrane microviscosity decreased after pluronics adsorption, in contrast to normal cells, whose membrane microviscosity was enhanced. We suppose that sensitivity of tumor cell membrane microviscosity to the pluronics action correlates with its permeability for molecular substances.


Subject(s)
Erythrocytes/drug effects , Lymphocytes/drug effects , Poloxamer/pharmacology , Surface-Active Agents/pharmacology , Animals , Erythrocyte Membrane/drug effects , Erythrocytes/chemistry , Erythrocytes/pathology , Erythrocytes/ultrastructure , Lymphocytes/chemistry , Lymphocytes/pathology , Lymphocytes/ultrastructure , Membrane Fluidity/drug effects , Mice , Mice, Inbred BALB C , Multiple Myeloma/blood , Poloxamer/metabolism , Surface-Active Agents/metabolism , Tumor Cells, Cultured
18.
Biochem Mol Biol Int ; 40(6): 1159-66, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8988327

ABSTRACT

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. aureus V8 protease evidenced to their being distinct proteins. The data obtained permit to assume that human RII of neural type is represented by two isoforms.


Subject(s)
Brain/enzymology , Carrier Proteins/chemistry , Cyclic AMP-Dependent Protein Kinases/chemistry , Intracellular Signaling Peptides and Proteins , Animals , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cattle , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase Type II , Cyclic AMP-Dependent Protein Kinases/isolation & purification , Cyclic AMP-Dependent Protein Kinases/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Immunoblotting , Isoenzymes/chemistry , Molecular Weight , Peptide Mapping , Phosphates/analysis , Phosphorylation , Swine
19.
Anal Biochem ; 234(1): 13-8, 1996 Feb 01.
Article in English | MEDLINE | ID: mdl-8742075

ABSTRACT

This work presents a new method for performing homogeneous fluoroimmunoassay in apolar organic media, quenching fluoroimmunoassay (QFIA). This method is based on utilization of the reverse micellar system of Aerosol OT (AOT) in n-octane as a medium for the analysis of compounds with low water solubility. It is shown using the system for determination of a hydrophobic pesticide atrazine as an example. The conjugate of atrazine with fluorescein (FA) serves as a label for fluorescence detection of antigen-antibody interaction in the reverse micellar system. The fluorescence quantum yield of this compound drastically depends on the micro-environment of the label in the reverse micelle system. Specifically, the binding of this conjugate with the antibodies solubilized in the reverse micelles results in fluorescence quenching. We found that quenching efficiency depends on the properties of the reverse micellar system (surfactant concentration, hydration degree w0, w0 = [water]/[surfactant], etc.). The optimal conditions for quenching of FA fluorescence by antibodies in reverse micelles of AOT in n-octane are low surfactant concentration and hydration degree, allowing one to get large reversed micelles (w0 = 15-20) capable of retaining solubilized antibodies. Addition of free atrazine results in displacement of the conjugate and restoration of its fluorescence. The sensitivity of the analysis to atrazine is only 10 times less than that of the commonly used method of homogeneous immunoassay, polarization fluoroimmunoassay, in aqueous solution using the same antibodies and conjugate. The advantage of QFIA in reverse micelles is that the analyte can be added when dissolved in nonpolar organic solvent.


Subject(s)
Antibodies , Atrazine/analysis , Atrazine/immunology , Animals , Antibodies/isolation & purification , Antigen-Antibody Reactions , Chromatography, Affinity/methods , Fluoresceins , Fluorescent Antibody Technique , Fluorescent Dyes , Indicators and Reagents , Kinetics , Micelles , Rabbits , Sensitivity and Specificity , Solvents , Spectrometry, Fluorescence
20.
Bioconjug Chem ; 6(5): 608-15, 1995.
Article in English | MEDLINE | ID: mdl-8974461

ABSTRACT

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. Yu. Alakhov (1994) J. Contr. Release 28, 15-35). Horse radish peroxidase (HRP) is used in this work to study the interaction of a fatty acylated protein with various mammalian cells. The HRP is modified with the chloranhydride of the stearic acid in the reversed micelles of sodium bis-(2-ethylhexyl)sulfosuccinate (Aerosol OT) in octane, a convenient protocol allowing production of protein molecules with a controlled, low modification degree (A.V. Kabanov et al. (1987) Ann. N. Y. Acad. Sci. 501, 63-66). The influence of the hydrophobic group on the binding and internalization of HRP in MDCK, P3-X63-Ag8, CHO, and HepG2 cells is examined. The major results are as follows: (i) the fatty acylation of a protein significantly enhances its binding to all tested mammalian cell lines, with a line-specific efficiency; (ii) the binding efficiency can be modified by changing growth conditions in a defined medium; (iii) along with the enhancement of protein adsorption on the plasma membrane, fatty acylation increases internalization of the protein during incubations at 37 degrees C; (iv) internalized protein was observed in endocytic vesicles; no evidence was obtained for a cytoplasmic distribution. These results are discussed in connection with previously observed effects of the fatty acylated proteins on cell activity.


Subject(s)
Proteins/chemistry , Proteins/metabolism , Acylation , Adsorption , Animals , Biological Transport, Active , CHO Cells , Cell Line , Cell Membrane/metabolism , Cricetinae , Dogs , Fatty Acids/chemistry , Horseradish Peroxidase/chemistry , Horseradish Peroxidase/metabolism , Humans , Mice , Micelles , Models, Biological , Protein Processing, Post-Translational , Subcellular Fractions/metabolism
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