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Biomedical subjects

E S Severin

Publications and source records attributed to E S Severin.

At least 19 recordsLinked to original sources
PubMed1992

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. The toxin's C-terminal fragment possesses an ability to activate calmodulin-dependent enzymes and is probably the toxicogenic part of the enterotoxin.

Amino Acid Sequence↗
PubMed1992

Nitroprusside stimulates the cysteine-specific mono(ADP-ribosylation) of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes.

In human erythrocyte membranes incubated with [adenylate-32P]NAD the 36 kDa protein is predominantly labeled. The labeling is greatly stimulated by nitroprusside in the presence of dithiothreitol. We have purified the 36 kDa protein and identified this modification as cysteine-specific mono(ADP-ribosylation) because: (i) labeling occurred only when [32P]NAD was replaced by adenine[U-14C]NAD, but not by [carbonyl-14C]NAD; (ii) treatment of the prelabeled protein with snake venom phosphodiesterase led to releasing 5'-[32P]AMP; (iii) the bond between the protein and the nucleotide was hydrolyzed by HgCl2, but was resistant to hydroxylamine. The 36 kDa protein reacted on Western blots with two different monoclonal antibodies (MAbs) against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and was immunoprecipitated by both MAbs.

Adenosine Diphosphate Ribose↗
PubMed1992

Internalizable insulin-BSA-chlorin E6 conjugate is a more effective photosensitizer than chlorin E6 alone.

Experiments with human hepatoma PLC/PRF/5 cells involving the use of two different tests (colony formation and Trypan blue exclusion) have demonstrated a significantly higher photosensitizing activity of chlorin e6 conjugates with bovine serum albumin (BSA) and internalizable ligand insulin as compared to that of chlorin e6 itself. Receptor-mediated internalization of insulin-BSA-chlorin e6 conjugates ensures greater photosensitization of cells than the binding of those conjugates to cell surface receptors. The suitability of such conjugates permitting the delivery of a photosensitizer to most sensitive cell structures is discussed.

Carcinoma, Hepatocellular↗
PubMed1992

Human lung adrenergic and muscarinic cholinergic receptors in cancer and previous airways diseases.

In the present study adrenergic and muscarinic cholinergic receptors have been investigated in human lung parenchyma in cancer and previous airways diseases (chronic pneumonia, tuberculosis). It has been found that the number of muscarinic sites significantly increases in cancer and does not change both in chronic pneumonia and tuberculosis lung parenchyma in comparison with the normal tissue; the number of beta-adrenergic sites decreases in cancer, chronic pneumonia, as well as severe tuberculosis lung parenchyma. The important role of beta-adrenergic and muscarinic receptors in the formation of human lung adenocarcinoma is discussed.

Adenocarcinoma↗
PubMed1991

DNA interpolyelectrolyte complexes as a tool for efficient cell transformation.

A tool was developed for enhancement of plasmid penetration into an intact cell, based on increasing DNA hydrophobicity via inclusion into a soluble interpolyelectrolyte complex (IPC) with polycations. The characteristics of formation of DNA IPC with synthetic polycations [poly(N-ethyl-4-vinylpyridinium)bromide (PVP) and PVP modified with 3% of N-cetyl-4-vinylpyridinium units (PVP-C)] were studied using ultracentrifugation and polyacrylamide gel electrophoresis methods. The conditions were established under which the mixing of DNA and polycation aqueous solutions results in the self-assembly of soluble IPC species. Incorporation of DNA into IPC results in the enhancement of DNA binding with isolated Bacillus subtilis membranes. A considerable increase in the efficiency of transformation of B. subtilis cells with pBC16 plasmid resulted from incorporation of the plasmid into the IPC with PVP and CVP.

Bacillus subtilis↗
PubMed1991

Platelet-activating factor stimulates receptor-mediated formation of reactive oxygen intermediates in human monocytes.

Stimulation of production of reactive oxygen intermediates (ROI) was examined in human peripheral blood monocytes by luminol-dependent chemiluminescence. The dose-response curve characterizing the dependence of ROI production on the concentration of platelet-activating factor (PAF) showed that stimulation occurred within a concentration range of 2 x 10(-9)M to 5 x 10(-6)M. Transformation of the dose-response curve to an Eadie-Hofstee plot indicated that the process is characterized by two Km values. The Km value corresponding to the high-affinity branch of the curve is 1.3 +/- 0.14 nM. In the same cells, the dissociation constant for the [3H]PAF/receptor complex was determined. The Kd value was 0.8 +/- 0.1 nM, which agreed quite well with the high-affinity Km value obtained in the Eadie-Hofstee plot. The data indicate that stimulation of ROI generation is mediated through PAF binding at specific receptor sites at nanomolar PAF concentrations. Along with the specific receptor-mediated ROI generation, a nonspecific effect of PAF at a high concentration was demonstrated.

Cells, Cultured↗
PubMed1991

Hydrophobized antiviral antibodies and antisense oligonucleotides.

A method of suppressing virus reproduction in cells has been proposed. The approach consists of affecting the cells with antiviral antibodies artificially hydrophobized with fatty acid residues. Reproduction of influenza viruses in MDCK cells and respiratory-synticial virus in HeLa cells was used as a model to demonstrate that poly- and monoclonal antibodies, modified by 1 or 2 stearic acid residues, are potent, unlike the non-modified antibodies, at inhibiting viral reproduction. The observed phenomenon is apparently due to penetration of hydrophobized antibodies into the cells. Thus, in particular, considerable antiviral activity is exhibited by monoclonal antibodies against NP-protein of influenza virus, which is an antigen accessible to antibodies only inside the infected cells. Hydrophobized antibodies do not affect the kinetics of viral protein synthesis; they block the virus withdrawal from the cells, probably by interfering with the assembling and budding of virus particles. To enhance penetration of oligonucleotides ("oligos") into cells, chemical modification of the former at the 5'-end phosphate group by fatty radicals has been suggested. The undecanol-modified oligo namely an oligo complementary to the protein binding sites located at the influenza virus polymerases encoding RNA, was synthesized using a DNA-synthesator. The above modified oligo effectively suppressed the influenza A/PR8/34 virus reproduction and inhibited synthesis of the virus-specific proteins in MDCK cells. The non-modified antisense oligo and the modified nonsense oligo did not affect the virus development under the same conditions.

Animals↗
PubMed1991

[cGMP-activated phosphodiesterase from human brain: kinetic and regulatory properties].

The kinetic and regulatory properties of cGMP-activated phosphodiesterase (PDE) from human brain were studied. In double reciprocal plots the enzyme activity is characterized by a linear dependence of cAMP and a nonlinear one for cGMP. Micromolar concentrations of cGMP accelerate cAMP hydrolysis (7-14-fold) with Ka for cGMP of 0.36 microM. Stimulation of cAMP hydrolysis is accompanied by a decrease of Km with no changes in Vmax. With a rise in the cGMP concentration above 5 microM PDE activation is changed by its inhibition. Both substrates act as competitive inhibitors towards each other. The Ki value for both cGMP and cAMP is 30 microM. After the increase in the cAMP (Bt)2 concentration the activation of 5 microM cAMP hydrolysis is accompanied by the enzyme inhibition. Both analogs competitively inhibit cGMP hydrolysis with Ki of 10 and 1500 microM for cGMP(Bt)2 and cAMP(Bt)2, respectively. The data obtained point to the existence of two binding sites for cyclic nucleotides, namely, a regulatory site which is highly specific for cGMP and a catalytic site responsible for the hydrolysis of the both substrates which displays no apparent specificity either for cAMP or for cGMP. The different affinity of natural and synthetic cyclic nucleotides for these sites is determined, to a large extent, by the amino groups in the 2nd and 6th positions of the purine ring.

Binding Sites↗
PubMed1991

Chemical modification enhances the inhibitory effect of regulatory subunit antisense oligodeoxynucleotide of cAMP-dependent protein kinase type I on cell proliferation.

The analysis of the action of antisense oligodeoxynucleotide complementary to mRNA of the type I cAMP-dependent protein kinase regulatory subunit has revealed a stable inhibitory effect of the substance on Molt-4 leukemic cell proliferation. To enhance the efficiency of the oligodeoxynucleotide action, its molecule was subjected to terminal modifications. The hydrophobic radical-effected modification at the 5'-end was carried out at the last stage of automated synthesis using undecanol. At the 3'-end, the molecule was modified with an acridine residue. The substances synthesized were significantly more potent in inhibiting thymidine incorporation in the acid-insoluble fraction, compared to the nonmodified nucleotide. Examination of the cell cycle progression of Molt-4 cells has demonstrated a considerable shift of cells from G2/M stage to GI, the process being more effective in the case of modified oligonucleotides.

Base Sequence↗
PubMed1991

[Tyrosine protein kinase from cattle cerebral cortex: purification, characteristics, protein substrates for phosphorylation and inhibitors of activity].

Tyrosine protein kinase present in the membrane fraction of bovine cerebral cortex were extracted and chromatographically fractionated. The activity associated with tyrosine protein kinases was fully extracted from the membranes by 1% sodium cholate and eluted in two peaks (I and II) during chromatography of protein extracts on DEAE-Toyopearl in the presence of sodium cholate. The predominant in cerebral cortex membrane tyrosine protein kinase of peak I (about 75% of the total activity) was purified 1930-fold by gel filtration on Sephacryl S-300, chromatography on hexyl- and phenyl-Sepharose and by rechromatography on DEAE-Toyopearl. The amount of the enzyme prepared from 250 g of bovine brain was 20 micrograms, the enzyme yield and specific activity being 3.8% and 3.9 nmol/mg protein/min, respectively. The purified protein kinase of peak I represents a protein with Mr of 62-63,000 (p62) capable of being autophosphorylated in the presence of [gamma-32P]. Protein kinase p62 phosphorylates enolase, tubulin and calpactin I as well as model substrates in the series: histone H5 greater than poly(G, T)n greater than or equal to histone H2A greater than poly(G, A, T)n, histone H4 greater than caseins, histones H1 and H2B, poly(G, A, L, T)n. The enzyme is specific for Mn2+ at the optimal concentration about 1 mM. The KmMn-ATP is 0.3 microM; Km for histone H5 and poly(G, T)n are 0.45 mg/ml and 0.06 mg/ml, respectively. The protein kinase p62 activity is inhibited by NaCl (IC50 approximately 75-100 mM) as well as by quercetin, adriamycin and lasalocid (IC50 approximately 14-34, 23 and 90 microM, respectively). It is concluded that protein kinase p62 is analogous to the c-src gene protein kinase.

Animals↗
PubMed1990

Nerve growth factor stimulates protein kinase C translocation in PC12 cells.

The effect of nerve growth factor (NGF) on the activity and intracellular localization of protein kinase C (PKC) in pheochromocytoma PC12 cells was studied. By using immunoblotting, immunofluorescence method and phorbol ester binding, NGF was found to induce PKC translocation from the cytoplasm into the cell membrane. This process was accompanied by an increase in the protein kinase activity in the membrane fraction. Translocation was completely blocked by H-7, a protein kinase inhibitor potentiating the neurite-stimulating activity of NGF.

Adrenal Gland Neoplasms↗
PubMed1990

Immunofluorescence localization of the regulatory subunit type II of cAMP-dependent protein kinase in PC12 and 3T3 cells in different proliferative states.

Localization of the regulatory subunit of cAMP-dependent protein kinase type II was studied in proliferating and quiescent fibroblasts 3T3 and in a cell line of neural origin pheochromocytoma PC12. In actively proliferating PC12 cells the regulatory subunit was found to be localized in the nucleus. Transition of these cells into a quiescent state was accompanied by a regulatory subunit translocation to the cytoplasm. In 3T3 cells the regulatory subunit was localized in the cytoplasm both in the quiescent and proliferating (though less actively than PC12 cells) states. Similar results were obtained both with monoclonal antibodies and with rabbit monospecific antiserum raised against the regulatory subunit type II from pig brain.

Adrenal Gland Neoplasms↗
PubMed1990

A new class of antivirals: antisense oligonucleotides combined with a hydrophobic substituent effectively inhibit influenza virus reproduction and synthesis of virus-specific proteins in MDCK cells.

To enhance the penetration of oligonucleotide ('oligo') into cells, the oligo was combined with the hydrophobic undecyl residue. Using the 'DNA-synthesator', we synthesized oligo, complementary to the loop-forming site of the RNA, encoding polymerase 3 of the influenza virus (type A), and combined it with the undecyl residue added to the 5' terminal phosphate group. It was found that the modified oligo effectively suppresses the influenza A/PR8/34 (H1N1) virus reproduction and inhibits the synthesis of virus-specific proteins in MDCK cells. Under the same conditions, the non-modified antisense oligo and modified nonsense oligo did not affect the virus development.

Animals↗
PubMed1990

Calmodulin-dependent enzymes as a target of staphylococcal enterotoxin A.

The response of lymphoid and nerve cells to the action of SEA has been investigated. It has been established that the toxin acts as a mitogen with respect to resting cells and suppresses the DNA biosynthesis in proliferating cells. Interaction of SEA with the systems of second messengers in lymphoblastoid cells has been studied. The results obtained suggest a mechanism of the antiproliferative action of SEA on these cells. Studies on the structural organization of the toxin molecule have revealed that the latter contains a polypeptide (BacM) capable of activating calmodulin-dependent enzymes both in the presence and absence of Ca2+. These findings permit us to assume that the cytostatic effect of SEA is conditioned by the formation of BacM and phosphorylation of elongation factor 2.

Adenylyl Cyclases↗
PubMed1990

Amplification of DNA sequences of Epstein-Barr and human immunodeficiency viruses using DNA-polymerase from Thermus thermophilus.

Using thermophilic DNA-polymerase from Thermus thermophilus we have amplified by polymerase chain reaction (PCR) specific DNA sequences of Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV). DNA-polymerase from Thermus thermophilus (molecular mass of 80-86 kDa) differs in its physico-chemical properties from DNA-polymerase from Thermus aquaticus (molecular mass of 62-68 kDa). To amplify the specific EBV DNA sequence, oligonucleotide primers for the virus replicon region (oriP region) were used. As a result of amplification, a specific 405-bp DNA fragment was produced.

Base Sequence↗
PubMed1990

[Localization of antigenic sites for monoclonal antibodies against alpha-subunit of Go-protein from the bovine brain].

The properties of monoclonal antibodies (MA) specifically raised against the alpha-subunit of the GTP-binding protein from bovine brain, G0, were studied. The hybridoma clones were found to secrete MA capable to interact with different antigenic sites of G0 alpha. Clone 1D2 MA interacted with the N-terminal domain of G0 alpha. The antigenic sites for clones 3DE. 1H6 and 2E3 MA were localized in the C-terminal domain of the protein molecule. Using clone 1H6 MA, the site of G0 alpha involved in the interaction with the beta gamma complex located in the C-terminal domain of the alpha-subunit, was revealed. It was found that the interaction of the alpha-subunit with the beta gamma complex changed the conformation of the C-terminal fragment of G0 alpha (Mr5000) together with an increase in the alpha-subunit affinity for clone 2E3 MA. It was concluded that the observed conformational changes may be the reason for the increased affinity of the alpha-subunit for the receptor.

Animals↗
PubMed1990

[Interaction of alpha-subunit of the GTP-binding protein Go with cytoskeleton].

It was found that about 30% of the alpha-subunit of the bovine brain GTP-binding protein, Go, is bound to the cytoskeleton. The efficiency of Go alpha binding to the cytoskeleton components depends on the nature of the guanyl nucleotides [GDP-beta-S, Gpp (NH) p] present in the incubation medium. It was shown that the alpha-subunit interaction with cytoskeleton components is controlled by ATP-dependent reactions. ATP diminishes the degree Go alpha adsorption. The non-hydrolysable ATP analog, App (NH) p, has no effect on the Go alpha interaction with the cytoskeleton. It was found that one of the cytoskeleton components capably of binding to Go alpha is tubulin. Similar interactions were detected in human neuroblastoma N2A cells.

Adenosine Triphosphate↗