scholarly journals Biotin-conjugated tumour-targeting photocytotoxic iron(III) complexes

2013 ◽  
Vol 371 (1995) ◽  
pp. 20120190 ◽  
Author(s):  
Sounik Saha ◽  
Ritankar Majumdar ◽  
Akhtar Hussain ◽  
Rajan R. Dighe ◽  
Akhil R. Chakravarty
Keyword(s):  
Hepg2 Cells ◽  
Hek293 Cell ◽  
Saturated Calomel Electrode ◽  
Specific Interaction ◽  
Fluorescein Isothiocyanate ◽  
Binding Studies ◽  
Sodium Dependent ◽  
Microscopic Studies ◽  
Biotin Conjugation ◽  
High Spin Iron

Iron(III) complexes [FeL(B)] ( 1 – 4 ) of a tetradentate phenolate-based ligand (H 3 L) and biotin-conjugated dipyridophenazine bases (B), viz. 7-aminodipyrido [3,2- a :2′,3′- c ]-phenazine (dppza in 1 ), ( N -dipyrido[3,2- a :2′,3′- c ]-phenazino)amidobiotin (dppzNB in 2 ), dipyrido [3,2- a :2′,3′- c ]-phenazine-11-carboxylic acid (dppzc in 3 ) and 2-((2-biotinamido)ethyl) amido-dipyrido[3,2- a :2′,3′- c ]-phenazine (dppzCB in 4 ) are prepared, characterized and their interaction with streptavidin and DNA and their photocytotoxicity and cellular uptake in various cells studied. The high-spin iron(III) complexes display Fe(III)/Fe(II) redox couple near −0.7 V versus saturated calomel electrode in dimethyl sulfoxide–0.1 M tetrabutylammonium perchlorate. The complexes show non-specific interaction with DNA as determined from the binding studies. Complexes with appended biotin moiety show similar binding to streptavidin as that of free biotin, suggesting biotin conjugation to dppz does not cause any loss in its binding affinity to streptavidin. The photocytotoxicity of the complexes is tested in HepG2, HeLa and HEK293 cell lines. Complex 2 shows higher photocytotoxicity in HepG2 cells than in HeLa or HEK293, forming reactive oxygen species. This effect is attributed to the presence of overexpressed sodium-dependent multi-vitamin transporters in HepG2 cells. Microscopic studies in HepG2 cells show internalization of the biotin complexes 2 and 4 essentially occurring by receptor-mediated endocytosis, which is similar to that of native biotin and biotin fluorescein isothiocyanate conjugate.

Lectin Binding Studies on RNA Tumor Virus-Infected Cells

1975 ◽  
Vol 33 ◽  
pp. 326-327
Author(s):  
D. C. Hixson
Keyword(s):  
Binding Sites ◽  
Lectin Binding ◽  
Culture Conditions ◽  
3T3 Cells ◽  
Binding Studies ◽  
Con A ◽  
Microscopic Studies ◽  
Tumor Virus ◽  
Infected Cells ◽  
Cell Culture Conditions

The abilities of plant lectins to preferentially agglutinate malignant cells and to bind to specific monosaccharide or oligosaccharide sequences of glycoproteins and glycolipids make them a new and important biochemical probe for investigating alterations in plasma membrane structure which may result from malignant transformation. Electron and light microscopic studies have demonstrated clustered binding sites on surfaces of SV40-infected or tryp- sinized 3T3 cells when labeled with concanavalin A (con A). No clustering of con A binding sites was observed in normal 3T3 cells. It has been proposed that topological rearrangement of lectin binding sites into clusters enables con A to agglutinate SV40-infected or trypsinized 3T3 cells (1). However, observations by other investigators have not been consistent with this proposal (2) perhaps due to differences in reagents used, cell culture conditions, or labeling techniques. The present work was undertaken to study the lectin binding properties of normal and RNA tumor virus-infected cells and their associated viruses using lectins and ferritin-conjugated lectins of five different specificities.

scholarly journals Binding of perlecan to transthyretin qin vitro

1997 ◽  
Vol 326 (3) ◽  
pp. 829-836 ◽  
Author(s):  
Sigbjørn SMELAND ◽  
Svein Olav KOLSET ◽  
Malcolm LYON ◽  
Kaare R. NORUM ◽  
Rune BLOMHOFF
Keyword(s):  
Hepg2 Cells ◽  
Hydrophobic Interactions ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Binding Protein ◽  
Heparan Sulphate ◽  
Retinol Binding Protein ◽  
Affinity Column ◽  
Serum Retinol ◽  
Binding Studies

Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein–glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions.

scholarly journals Dibutylchloromethyltin chloride, a covalent inhibitor of the adenosine triphosphate synthase complex

1977 ◽  
Vol 166 (3) ◽  
pp. 593-602 ◽  
Author(s):  
Kelvin Cain ◽  
Michael D. Partis ◽  
David E. Griffiths
Keyword(s):  
Oxidative Phosphorylation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Interaction ◽  
Sodium Dodecyl ◽  
Mitochondrial Atpase ◽  
Protein Subunit ◽  
Binding Studies ◽  
Mitochondrial Atp Synthase ◽  
Chloroform Methanol ◽  
Covalent Inhibitor

1. The synthesis of dibutylchloromethyltin chloride, a new covalent inhibitor of the mitochondrial ATP synthase [oligomycin-sensitive ATPase (adenosine triphosphatase)] complex is described, together with a method for preparing dibutylchloro[3H]methyltin chloride. 2. Studies with the yeast mitochondrial oligomycin-sensitive ATPase complex show that dibutylchloromethyltin chloride inhibits both the membrane-bound enzyme and also the purified Triton X-100-dispersed preparation. 3. F1-ATPase is not inhibited even at 500nmol of dibutylchloromethyltin chloride/mg of protein, and the general inhibitory properties are similar to those of triethyltin, oligomycin and dicyclohexylcarbodi-imide, known energy-transfer inhibitors of oxidative phosphorylation. 4. Binding studies with yeast submitochondrial particles show that dibutylchloromethyltin chloride antagonizes the binding of triethyl[113Sn]tin, indicating that there is an interaction between the two inhibitor-binding sites. 5. Unlike triethyltin, inhibition by dibutylchloromethyltin chloride is due to a covalent interaction which titrates a component of the inner mitochondrial membrane present at a concentration of 8–9nmol/mg of protein. 6. All of the labelled component can be extracted with chloroform/methanol (2:1, v/v), and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chloroform/methanol extract indicates that the labelled component has an apparent mol.wt. of 6000–8000. However, t.l.c. reveals the presence of only one labelled component which is lipophilic and non-protein and is distinct from the free inhibitor, mitochondrial phospholipids and the dicyclohexylcarbodi-imide-binding protein (subunit 9). 7. Inhibition of mitochondrial ATPase and oxidative phosphorylation is correlated with specific interaction with a non-protein lipophilic component of the mitochondrial inner membrane which is proposed to be a co-factor or intermediate of oxidative phosphorylation.

Imprinting Effects of Proline Containing Dipeptides (Proline-Glycine, Proline-Leucine, Proline-Valine and Their Retro Variants) in Tetrahymena. Evolutionary Conclusions

1997 ◽  
Vol 17 (6) ◽  
pp. 537-542 ◽  
Author(s):  
G. Csaba ◽  
P. Kovács
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Peptide Binding ◽  
Fluorescein Isothiocyanate ◽  
Physiological Effects ◽  
Small Peptides ◽  
Binding Studies ◽  
Hormone Formation ◽  
Selection Of

Proline-glycine, proline-leucine and proline-valine dipeptides and their retro variants were used in the experiments to study the effects of pretreatment (imprinting) in Tetrahymena, by investigating fluorescein isothiocyanate (FITC)-conjugated peptide binding. The protozoan organism could differentiate between the proline-dipeptides containing different partner amino-acids and between the dipeptides having the amino acids in reversed positions. The effect of imprinting was positive or negative and this was dependent on the type of the partner amino acid and on its position. Pro-Gly and Pro-Leu induced positive imprinting (elevated FITC-dipeptide binding) and Pro-Val induced negative imprinting (decrease of FITC-peptide binding). There was positive imprinting induction in two cases for the retro FITC-peptide and in one case for the FITC-conjugate of the imprinter peptide itself. The highest positive imprinting (almost 60% increase) was induced by Pro-Gly for FITC-Gly-Pro. Considering earlier—chemotaxis—experiments, the results of the present—binding—studies run parallel with the physiological effects. The experiments call attention to the sharp differentiating ability of small peptides at a unicellular level, that could have some role in the selection of molecules for hormone formation, during evolution.

Heavy Meromyosin Binding Studies of Myocardial Cells in Cardiac Lethal Mutant Salamanders (Ambystoma Meximanum

1975 ◽  
Vol 33 ◽  
pp. 540-541
Author(s):  
Larry F. Lemanski
Keyword(s):  
Heart Development ◽  
Recessive Gene ◽  
Genetic Mutation ◽  
Heart Beat ◽  
Myocardial Cells ◽  
Binding Studies ◽  
Electron Microscopic ◽  
Lethal Mutant ◽  
Microscopic Studies ◽  
Gross Examination

A naturally-occurring genetic mutation, designated c for “cardiac lethal”, was discovered in Ambystoma meximanum. The effect of homozygosity for recessive gene c is the absence of a heart beat, even though initially heart development appears normal. Mutant embryos are first distinguishable form their normal siblings at Harrison stage 34, when the normals develop contracting hearts. The mutant hearts at this stage, upon gross examination appear structurally normal but fail to beat. Nevertheless, the mutants survive through stage 41, which is about 20 days beyond the heart-beat stage and exhibit normal swimming movements indicating that gene c does not affect skeletal muscle. Electron microscopic studies of normal hearts at stage 34 reveal that the myocardial cells contain organized myofibrils; these myofibrils first form directly beneath the plasma membrane. By stage 41, the normal myocardial cells contain numerous well-organized myofibrils and thus have now become highly differentiated muscle cells.

scholarly journals Specific interaction of ivermectin with retinol-binding protein from filarial parasites

1988 ◽  
Vol 249 (3) ◽  
pp. 929-932 ◽  
Author(s):  
B P Sani ◽  
A Vaid
Keyword(s):  
Retinoic Acid ◽  
Binding Proteins ◽  
Binding Protein ◽  
Specific Interaction ◽  
Retinol Binding Protein ◽  
Binding Affinities ◽  
Binding Studies ◽  
Retinoic Acid Binding Proteins ◽  
Parasitic Worms ◽  
Host Tissues

Specific cellular binding proteins for retinol and retinoic acid from mammalian and avian species may mediate the action of retinoids in the control of epithelial differentiation, growth and tumorigenesis. Parasite retinol-binding protein (PRBP) and parasite retinoic acid-binding protein (PRABP) isolated and characterized from parasitic worms of the family Filarioidea might be involved in some possible action of vitamin A compounds in these parasites. Ivermectin, a potent and widely used anti-parasitic drug, competes efficiently with retinol for retinol-binding sites on PRBP, but not for the host-tissue retinol-binding-protein sites. The drug has no affinity for retinoic acid-binding proteins from either parasite or host tissues. Binding studies using radiolabelled ivermectin and retinol reveal that ivermectin has a higher affinity than retinol for PRBP. A correlation exists between the binding affinities of ivermectin analogues and their anti-parasitic activities. A binding-protein-mediated interrelationship may exist between the actions of retinol and ivermectin in the parasites, but not in the host tissues.

The cell surface ofPlasmodium gallinaceum sporozoites: microelectrophoretic and lectin-binding characteristics

Parasitology ◽  
1982 ◽  
Vol 84 (2) ◽  
pp. 227-238 ◽  
Author(s):  
D. P. Turner ◽  
N. A. Gregson
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Lectin Binding ◽  
Surface Membrane ◽  
Significant Proportion ◽  
Fluorescein Isothiocyanate ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Cell Surface Membrane ◽  
Nitrobenzoic Acid

SUMMARYThe cell surface properties ofPlasmodium gallinaceumsporozoites have been investigated by means of microelectrophoretic and lectin-binding studies. Their electrophoretic mobility has been measured as a function of pH, the results suggesting qualitative and quantitative differences in the surface ionogenic groups between sporozoites from mature oocysts and those from salivary glands. Reaction of sporozoites with citraconic anhydride produced a small but significant increase in mobility, whereas 5,5-dithio-bis-(2-nitrobenzoic) acid had no effect on mobility; thus there appear to be amino groups but not –SH groups at the surface of sporozoites. Treatment of sporozoites with trypsin considerably reduced their mobility and suggests that a significant proportion of the cell surface charge is associated with protein. Incubation with neuraminidase, however, had no effect on sporozoite mobility and indicates that sialic acid residues, responsible for much of the negative charge associated with mammalian cells, are probably not present on the cell surface of sporozoites. Evidence for the presence of carbohydrates on the cell surface membrane of sporozoites was sought using fluorescein isothiocyanate–Concanavalin A. Results demonstrated that ligands similar to α-D-glucose and α-D-mannose are not present in an exposed or reactive form on the cell surface membrane ofP. gallinaceumsporozoites.

Antiproliferative and Pro-Apoptotic Effects of Thiazolo[3,2–b][1,2,4]triazoles in Breast and Cervical Cancer Cells

2021 ◽  
Vol 21 ◽  
Author(s):  
Imtiaz Khan ◽  
Sumera Zaib ◽  
Mohsin Javed ◽  
Faisal Rashid ◽  
Jamshed Iqbal ◽  
...  
Keyword(s):  
Dna Binding ◽  
Hela Cells ◽  
Docking Studies ◽  
Binding Studies ◽  
Caspase 9 ◽  
Flow Cytometric ◽  
Ic50 Values ◽  
Microscopic Studies ◽  
Dna Binding Studies ◽  
Mcf 7

Background and Objectives: Cancer is one of the leading causes of death in the world affecting millions of people. The commercially available anticancer drugs lack the selectivity and show several undue side effects during the biologically targeted therapy, thus calling for the exploration of wider chemical space to furnish new structural leads with promising anticancer potential. In this endeavor, we synthesized a series of coumarinyl thiazolotriazoles with diverse functional group tolerance and will be tested for their anticancer properties against cancer cell lines (HeLa and MCF-7) and a normal cell line (BHK-21). Materials and Methods: To overcome such complications, in the current study, we evaluated the cytotoxic effects of coumarinyl thiazolotriazoles hybrids on human breast adenocarcinoma (MCF-7), cervical adenocarcinoma (HeLa) cells and normal cells i.e., baby hamster kidney cells (BHK-21) using MTT (dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide) assay. DNA binding studies of compound 6c was performed on Herring-sperm DNA (HS-DNA) and docking studies were also carried out. The mechanistic studies were performed on potent compounds by fluorescent microscopic studies, release of lactate dehydrogenase (LDH) and mitochondrial membrane potential, activation of caspase-9 and -3 and flow cytometric analysis. Results: As revealed by MTT assay, compound 6m and 6c were identified as the most potent derivative among the tested series with IC50 values of 5.64 and 29.1 μM against HeLa and MCF cells, respectively as compared to cisplatin which gave IC50 values of 11.3 and 6.20 μM, respectively. DNA binding studies of compound 6c showed the binding of compound in DNA with Gibbs free energy of ‒17 KJ/mol and docking studies validated the DNA binding studies. Fluorescent microscopic studies using 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) staining confirmed the occurrence of apoptosis in HeLa cells treated with the most active compound 6m. Moreover, compound 6m and 6c also triggered the release of lactate dehydrogenase (LDH) in treated HeLa and MCF-7 cells while a luminescence assay displayed a remarkable increase in the activity of caspase-9 and -3. Moreover, flow cytometric results revealed that compound 6m caused G0/G1 arrest in the treated HeLa cells. Conclusion: Our results suggested that the compound possesses chemotherapeutic properties against breast cancer and cervical adenocarcinoma cells, thus warranting further research to test the anticancer efficacy of this compound at clinical level.

scholarly journals Stereoselectivity of Ins(1,3,4,5)P4 recognition sites: implications for the mechanism of the Ins(1,3,4,5)P4-induced Ca2+ mobilization

1993 ◽  
Vol 294 (1) ◽  
pp. 191-194 ◽  
Author(s):  
R A Wilcox ◽  
R A Challiss ◽  
G Baudin ◽  
A Vasella ◽  
B V Potter ◽  
...  
Keyword(s):  
Binding Site ◽  
Specific Binding ◽  
Specific Interaction ◽  
Neuroblastoma Cells ◽  
Dependent Manner ◽  
Binding Studies ◽  
Concentration Dependent Manner ◽  
Human Neuroblastoma ◽  
Specific Binding Sites ◽  
Ic50 Values

Ins(1,3,4,5)P4 was able to mobilize the entire Ins(1,4,5)P3-sensitive intracellular Ca2+ store in saponin-permeabilized SH-SY5Y human neuroblastoma cells in a concentration-dependent manner, yielding an EC50 value of 2.05 +/- 0.45 microM, compared with 0.14 +/- 0.03 microM for Ins(1,4,5)P3. However, L-Ins(1,3,4,5)P4 [= D-Ins(1,3,5,6)P4] failed to cause mobilization of intracellular Ca2+ at concentrations up to 100 microM. Binding studies using pig cerebellar membranes as a source of both Ins(1,4,5)P3/Ins(1,3,4,5)P4-specific binding sites have revealed a marked contrast in their stereospecificity requirements. Ins(1,4,5)P3-receptors from pig cerebella exhibited stringent stereospecificity, L-Ins(1,4,5)P3 and L-Ins(1,3,4,5)P4 were > 1000-fold weaker, whereas Ins(1,3,4,5)P4 (IC50 762 +/- 15 nM) was only about 40-fold weaker than D-Ins(1,4,5)P3 (IC50 20.7 +/- 9.7 nM) at displacing specific [3H]Ins(1,4,5)P3 binding from an apparently homogeneous Ins(1,4,5)P3 receptor population. In contrast, the Ins(1,3,4,5)P4-binding site exhibited poor stereoselectivity. Ins(1,3,4,5)P4 produced a biphasic displacement of specific [32P]Ins(1,3,4,5)P4 binding, with two-site analysis revealing KD values for high- and low-affinity sites of 2.1 +/- 0.5 nM and 918 +/- 161 nM respectively. L-Ins(1,3,4,5)P4 also produced a biphasic displacement of specific [32P]Ins(1,3,4,5)P4 binding which was less than 10-fold weaker than with D-Ins(1,3,4,5)P4 (IC50 values for the high- and low-affinity sites of 17.2 +/- 3.7 nM and 3010 +/- 542 nM respectively). Therefore, although L-Ins(1,3,4,5)P4 appears to be a high-affinity Ins(1,3,4,5)P4-binding-site ligand in pig cerebellum, it is a very weak agonist at the Ca(2+)-mobilizing receptors of permeabilized SH-SY5Y cells. We suggest that the ability of D-Ins(1,3,4,5)P4 to access intracellular Ca2+ stores may derive from specific interaction with the Ins(1,4,5)P3- and not the Ins(1,3,4,5)P4-receptor population.

scholarly journals N-formylpeptide and complement C5a receptors are expressed in liver cells and mediate hepatic acute phase gene regulation.

1995 ◽  
Vol 182 (1) ◽  
pp. 207-217 ◽  
Author(s):  
R McCoy ◽  
D L Haviland ◽  
E P Molmenti ◽  
T Ziambaras ◽  
R A Wetsel ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Acute Phase ◽  
Hepg2 Cells ◽  
Human Liver ◽  
Acute Phase Proteins ◽  
Mononuclear Phagocytes ◽  
Complement C3 ◽  
Binding Studies ◽  
C5a Receptor ◽  
Receptor Family

Although the classical chemotactic receptor for complement anaphylatoxin C5a has been associated with polymorphonuclear and mononuclear phagocytes, several recent studies have indicated that this receptor is expressed on nonmyeloid cells including human endothelial cells, vascular smooth muscle cells, bronchial and alveolar epithelial cells, hepatocytes, and in the human hepatoma cell line HepG2. In this study, we examined the possibility that other members of the chemotactic receptor family are expressed in HepG2 cells and human liver, and the possibility that such receptors mediate changes in acute phase gene expression in HepG2 cells. Using polymerase chain reaction (PCR) amplification of HepG2 mRNA with primers based on highly conserved regions of the chemotactic subgroup of the G protein-coupled receptor family, we identified a PCR fragment from the formyl-methionyl-leucyl-phenylalanine (FMLP) receptor, as well as one from the C5a receptor. Immunostaining with antipeptide antisera to FMLPR confirmed the presence of this receptor in HepG2 cells. Receptor binding studies showed specific saturable binding of a radioiodinated FMLP analogue to HepG2 cells (Kd approximately 2.47 nM; R approximately 6 x 10(3) plasma membrane receptors per cell). In situ hybridization analysis showed the presence of FMLPR mRNA in parenchymal cells of the human liver in vivo. Both C5a and FMLP mediated concentration- and time-dependent changes in synthesis of acute phase proteins in HepG2 cells including increases in complement C3, factor B, and alpha 1-antichymotrypsin, as well as concomitant decreases in albumin and transferrin synthesis. The effects of C5a and FMLP on the synthesis of these acute phase proteins was evident at concentrations as low as 1 nM, and they were specifically blocked by antipeptide antisera for the corresponding receptor. In contrast to the effect of other mediators of hepatic acute phase gene regulation, such as interleukin 6, the effects of C5a and FMLP were reversed by increased concentrations well above the saturation point of the respective receptor. These results suggest that acute phase gene regulation by C5a and FMLP is desensitized at high concentrations, a property that is unique among the several known mechanisms for hepatic acute phase gene regulation.

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