Binding Of Conjugate Research Articles

Characterization of self-assembled redox polymer and antibody molecules on thiolated gold electrodes

Multilayer immobilization of antibody and redox polymer molecules on a gold electrode was achieved, as a strategy for the potential development of an amperometric immunosensor. The step-by-step assembly of antibiotin IgG on Os(bpy) 2ClPyCH 2NH poly(allylamine) redox polymer (PAH-Os) adsorbed on thiolated gold electrodes was proved by quartz crystal microbalance (QCM) and atomic force microscopy (AFM) experiments, confirming the electrochemical evidence. The increase of redox charge during the layer-by-layer deposition demonstrated that charge propagation within the layers is feasible. The multilayer structure proved to be effective for the molecular recognition of horseradish peroxidase–biotin conjugate (HRP–biotin), as confirmed by the QCM measurements and the electrocatalytic reduction current obtained upon H 2O 2 addition. The catalytic current resulting from PAH-Os mediation was shown to increase with the number of assembled layers. Furthermore, the inventory of IgG molecules on the supramolecular self-assembled structure and the specific and non-specific binding of HRP–biotin conjugate were confirmed by the QCM transient studies, giving information on the kinetics of IgG deposition and HRP–biotin conjugate binding to the IgG.

Highly sensitive amperometric detection of genomic DNA in animal tissues.

A simple and highly sensitive method for the detection of genomic DNA in tissue samples is described. It is based on amperometric detection of target DNA by forming an analyte/polymeric activator bilayer on a gold electrode. The biotinylated target DNA is hybridized to oligonucleotide capture probes immobilized on the gold electrode, forming the first layer. A subsequent binding of glucose oxidase-avidin conjugate to the target DNA and the introduction of a second layer of a redox polymer to the electrode, via layer-by-layer electrostatic self-assembly, allow for electrochemical detection of the catalytic oxidation current of glucose in a PBS solution. Less than 2.0 fg of rat genomic DNA, for both regulated and house-keeping genes, can be easily detected in 2.5 microl droplets. The proposed procedure shows very high specificity for genomic DNA in a RT-PCR mixture.

SELECTIVE INTRACELLULAR DELIVERY OF DEXAMETHASONE INTO ACTIVATED ENDOTHELIAL CELLS USING AN E-SELECTIN-DIRECTED IMMUNOCONJUGATE

Everts M, Kok RJ, Ásgeirsdóttir SA, et al. J Immunol. 2002;168:883–889To determine if a dexamethasone-anti-E-selectin conjugate can be specifically delivered to activated endothelial cells and exert its biological effects in a cell-specific manner.Experiments were performed using human and mouse endothelial cell cultures.Dexamethasone was conjugated to anti-human E-selectin and the resulting immunoconjugate was characterized in terms of its ability to bind to E-selectin and endothelial cells, to be internalized, and to exert biological activity on the endothelial cells. Binding of the dexamethasone-anti-E-selectin conjugate to endothelial cells was analyzed by immunohistochemistry and flow cytometry. Internalization and localization of the dexamethasone conjugate was studied with confocal laser scanning microscopy (CLSM) and immuno-transmission electron microscopy (Immuno-TEM). Activity of the drug was analyzed using a mouse endothelial cell line transfected with a glucocorticoid-responsive reporter gene construct and the drug’s effect on interleukin (IL)-8 expression was assayed using Northern blotting.The dexamethasone conjugate bound to activated, but not resting, human endothelial cells when the drug-treated cell cultures were stained with antibodies directed against anti-human E-selectin or dexamethasone. Flow cytometry confirmed these results. CLSM and Immuno-TEM demonstrated binding of the dexamethasone conjugate to activated, but not resting, cell surfaces 10 and 20 minutes after incubation and after 1 hour, cell surface staining began to decrease and intracellular staining increased. The dexamethasone conjugate was able to activate a glucocorticoid-responsive reporter gene to a similar degree to that seen with free dexamethasone. Northern blotting demonstrated downregulation of IL-8 expression in activated human endothelial cells treated with the dexamethasone conjugate. Again, this effect was similar to that seen with free dexamethasone.Conjugated dexamethasone-anti-E-selectin is specifically targeted to active endothelial cells and internalized by these cells. The results also suggest that the conjugate drug retains the ability to regulate transcription.Although targeted drug therapy is not a novel concept, these experiments suggest that a noncytotoxic drug can be targeted to a specific cell type and retain biological activity. Unlike a drug targeted to kill cancer cells, this approach utilizes an antiinflammatory drug to downregulate proinflammatory cytokines and cell surface receptors with the idea that the toxicity associated with immune suppression would be minimized.

An oestrogen membrane receptor participates in estradiol actions for the prevention of amyloid-beta peptide1-40-induced toxicity in septal-derived cholinergic SN56 cells.

Although oestrogen [17 beta-estradiol (E2)]-related neuroprotection has been demonstrated in different models, the involvement of non-classical oestrogen receptors (ERs) remains unexplored. Using the SN56 cholinergic cell line, we present evidence indicating that an ER associated with the plasma membrane participates in oestrogen-dependent inhibition of cell death induced by amyloid-beta peptide (A beta) toxicity. Similarly to E2 alone, a 15-min exposure to estradiol-horseradish peroxidase (E-HRP) significantly reduced A beta-induced cell death. This effect was decreased by the ER antagonist ICI 182,780 as well as by MC-20 antibody directed to a region neighbouring the ligand-binding domain of ER alpha. Using confocal microscopy on unpermeabilized SN56 cells exposed to MC-20 antibody, we identified a protein at the plasma membrane level. Western blot analysis of purified SN56 cell membrane fractions using MC-20 antibody revealed the presence of one band with the same electrophoretic mobility as intracellular ER alpha. Using conjugated forms of the steroid, E-HRP and E2 conjugated to bovine serum albumin-FITC, we demonstrated by confocal microscopy that SN56 cells contain surface binding sites for E2. Binding of both conjugates was blocked by pre-incubation with E2 and decreased by either ICI 182,780 or MC-20 antibody in a concentration-dependent manner. Thus, a membrane-related ER that shares some structural homologies with ER alpha may participate in oestrogen-mediated neuroprotection.

Analysis of Protein Adsorption and Binding at Biosensor Polymer Interfaces Using X-ray Photon Spectroscopy and Scanning Electrochemical Microscopy

We describe a method, based on X-ray photoelectron spectroscopy (XPS) measurements, to assess the extent of protein adsorption or binding on a variety of different muTAS and biosensor interfaces. Underpinning this method is the labeling of protein molecules with either iodine- or bromine-containing motifs by using protocols previously developed for radiotracer studies. Using this method, we have examined the adsorption and binding properties of a variety of modified electrodeposited polymer interfaces as well as other materials used in muTAS device fabrication. Using polymer interfaces modified with poly(propylene glycol) (PPG) chains, our results indicate that a chain of at least approximately 30 monomer units is required to inhibit nonspecific adsorption from concentrated protein solutions. The XPS methodology was also used to probe specific binding of avidins and enzyme conjugates thereof to biotinylated and mixed biotin/PPG-modified polymer interfaces. In one example, using competitive binding, it was established that the mode of binding of a peroxidase-streptavidin conjugate to a biotinylated modified polymer interface was primarily via the streptavidin moiety (as opposed to nonspecific binding via the enzyme conjugate). XPS evaluation of nonspecific and specific peroxidase-streptavidin immobilization on various functionalized polymers has guided the design and fabrication of functionalized interdigitated electrodes in a biosensing muTAS device. Subsequent characterization of this device using scanning electrochemical microscopy (SECM) corroborated the adsorption and binding previously inferred from XPS measurements on macroscale electrodes.

Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues.

Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.

Lymphocyte-facilitated tumour cell adhesion to endothelial cells: the role of high affinity leucocyte integrins

Lymphocytes transiently express an active form of the beta_2 integrin LFA-1 (LFA-1Af) which has conformational changes in extracellular domains enabling higher affinity binding to the ligand ICAM-1. In this study, we investigated the role of lymphocytes bearing LFA-1Af as potential mediators of binding of ICAM-1-positive tumour cells to endothelium. LFA-1 expression on 51Cr-PBLs was modulated in order to express high affinity LFA-1Af and conjugates were formed with 35S-labelled COLO526. The binding of the conjugates to resting or IL-1beta-stimulated human umbilical vein endothelial cells (HUVECs) was then assessed via a modified radioactive HUVEC binding assay. In addition, the binding of PBL-COLO526 conjugates to HUVECs was demonstrated by confocal microscopy. The binding of COLO526 to endothelial cells did not change significantly between unstimulated and stimulated HUVECs. In addition, pre-incubating the COLO526 with fresh PBLs did not significantly alter the binding of COLO526 to resting or activated HUVECs; whereas, in the presence of PBLs with LFA-1Af, the COLO526 conjugate binding dramatically increased from basal levels to 41% on resting HUVECs and 81% on stimulated HUVECs. COLO526-PBL(LFA-1Af) conjugate adhesion to stimulated HUVECs was inhibited by blocking antibody to LFA-1 (50%), VLA-4 (32%) or L-selectin (40%). Antibodies to the HUVEC adhesion molecules ICAM-1, VCAM-1 and E-selectin also inhibited COLO526-PBL(LFA-1Af) conjugate binding to activated HUVECs by 79, 60 and 73%, respectively. PBLs bearing LFA-1Af can enhance COLO526 adhesion to both resting and activated HUVECs. Furthermore, blocking studies demonstrate that a range of pathways are involved in this phenomenon (LFA-1/ICAM-1, VLA4/VCAM-1, L-selectin/E-selectin). These studies have identified a novel alternative pathway for lymphocyte-facilitated tumour cell adhesion to endothelial cells.

Targeting of Human Squamous Carcinomas by SPA470-doxorubicin Immunoconjugates

In a quest to identify a favorable target for head and neck cancers and squamous cell carcinoma, we sought to determine if Hsp47/CBP2 could be used as a target and whether the expression of this target was influenced by hypoxia. Moreover, we determined if doxorubicin (DOX) immunoconjugates directed against Hsp47/CBP2 that linked monoclonal antibodies (MAbs) to the 13-keto position of the drug possessed high cytotoxic drug activity and antibody-directed killing of antigen bearing tumor target cells. Experiments were performed using established cell lines of human oral squamous carcinoma cells (SCCs) (SCC-4, -9, -15 and -25) obtained from American Type Culture Collection (ATCC) (Manassas, VA). In addition, the UMB2 cell line is a spontaneous mutant of SCC-9 that does not express Hsp47/CBP2 was also used. Synthesis of the immunoconjugates was accomplished by thiolating the MAbs with 2 IT and reacting the MAbs with the DOX-hydrazone. The binding of MAb-DOX conjugates to SCC cells was determined by indirect immunofluorescence and analyzed using a Becton Dickinson FACS scan with Cell Quest software. Comparison of the cytotoxicity of DOX, MAb-DOX conjugates and MAb+DOX were determined using a limited dilution assay and colony survival assays during normoxia and hypoxia. These studies revealed that SCC cells treated with the SPA470-DOX conjugate for 2 h retained the original binding activity for targeted SCC cells and was significantly more potent that unconjugated DOX, DOX-hydrazone or equivalent MAb protein+DOX. Also, SPA47-DOX produced equal to and at lower concentrations greater cell killing than equivalent dose of free DOX. During hypoxia cells treated with SPA470-DOX demonstrated a small increase in colony survival and a diminishment in cytotoxicity. SPA470-DOX conjugates target SCC cells that express Hsp47/CBP2. The demonstration that SPA470-DOX is effective during hypoxia or conditions that mimic hypoxia presumes the further utility of SPA470-DOX in treating head and neck cancers.

Development of generic continuous-flow enzyme immunoassay system for analysis of aminoglycosides in serum

A simple generic continuous-flow enzyme immunoassay (CFEIA) for analysis of aminoglycosides in serum has been successfully developed. The developed assay employed a specific monoclonal antibody and β-galactosidase (β-GAL) enzyme as label. The assay involves an off-line competitive binding reaction between the analyte and free labelled analyte for the binding sites of the antibody. After equilibrium is reached, the sample was injected into the flow system. The bound antibody complexes with the analyte and the labelled analyte were trapped in a protein G column, while the unbound free labelled analyte was eluted and detected colorimetrically down-stream, after reaction with chlorophenolic red-β- d-galactopyranoside as a substrate for the β-GAL enzyme. The concentration of the analyte in a sample was quantified by its ability to inhibit the binding of the analyte-enzyme conjugate to the antibody, and the signal was directly proportional to the concentration of the analyte in the original sample. The optimum conditions for the developed CFEIA were investigated and applied to the analysis of tobramycin, as a representative example of the aminoglycosides, in serum samples. The detection limit of the assay was 0.06 μg ml −1. The assay showed good precision; the coefficients of variation were 2.49–4.33 and 3.30–6.82% for intra- and inter-assay precision, respectively. Serum matrix constituents and the endogenous compounds did not interfere with the assay. Analytical recovery of spiked tobramycin, in the concentration range between 0.5 and 8.0 μg ml −1, was 101.55±3.14. The assay results correlated well with those obtained by high-performance liquid chromatography ( r=0.991). All the obtained results strongly demonstrate that the developed CFEIA is a suitable method for a rapid and reliable analysis of aminoglycosides in serum.

Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4.

The multiple functions of the p97/Cdc48p ATPase can be explained largely by adaptors that link its activity to different cellular pathways, but how these adaptors recognize different substrates is unclear. Here we present evidence that the mammalian adaptors, p47 and Ufd1-Npl4, both bind ubiquitin conjugates directly and so link p97 to ubiquitylated substrates. In the case of Ufd1-Npl4, which is involved in endoplasmic reticulum (ER)-associated degradation and nuclear envelope reassembly, binding to ubiquitin is mediated through a putative zinc finger in Npl4. This novel domain (NZF) is conserved in metazoa and is both present and functional in other proteins. In the case of p47, which is involved in the reassembly of the ER, the nuclear envelope and the Golgi apparatus, binding is mediated by a UBA domain. Unlike Ufd1-Npl4, it binds ubiquitin only when complexed with p97, and binds mono- rather than polyubiquitin conjugates. The UBA domain is required for the function of p47 in mitotic Golgi reassembly. Together, these data suggest that ubiquitin recognition is a common feature of p97-mediated reactions.

Expression of membrane associated non-genomic progesterone receptor(s) in caprine spermatozoa

Mammalian spermatozoa have been used recently to model the study of rapid, non-genomic effects of progesterone on cell. Our study used progesterone–BSA–fluorescein isothiocyanate conjugate to indicate the presence of a progesterone receptor on the surface of >90% of a goat sperm population. The sperm possessed the receptor at 0 h and capacitation had no modulating effect on the number of sperm responsive to P–BSA–FITC. Although a decrease in receptor bearing cells was observed during the course of capacitation, the effect may have been due to the induction of acrosome reaction (AR) by the conjugate. This decrease was blocked by the pre-treatment of the spermatozoa with EGTA. Binding of conjugate occurred at the apical portion of the acrosome and at the post-acrosomal region in all the sperm, possibly mediating sperm functions other than the acrosome reaction. The P–BSA–FITC treated cells showed a single peak in a flow cytometer suggesting that the sperm population was homogeneous. Competition studies with free progesterone and GABA with P–BSA–FITC confirmed that the binding was specific and that progesterone mediated its action via a GABA A/Cl − channel complex akin to the one present in neuronal cells.

Supramolecular calsequestrin complex.

As recently demonstrated by overlay assays using calsequestrin-peroxidase conjugates, the major 63 kDa Ca(2+)-binding protein of the sarcoplasmic reticulum forms complexes with itself, and with junctin (26 kDa), triadin (94 kDa) and the ryanodine receptor (560 kDa) [Glover, L., Culligan, K., Cala, S., Mulvey, C. & Ohlendieck, K. (2001) Biochim. Biophys. Acta1515, 120-132]. Here, we show that variations in the relative abundance of these four central elements of excitation-contraction coupling in different fiber types, and during chronic electrostimulation-induced fiber type transitions, are reflected by distinct alterations in the calsequestrin overlay binding patterns. Comparative immunoblotting with antibodies to markers of the junctional sarcoplasmic reticulum, in combination with the calsequestrin overlay binding patterns, confirmed a lower ryanodine receptor expression in slow soleus muscle compared to fast fibers, and revealed a drastic reduction of the RyR1 isoform in chronic low-frequency stimulated tibialis anterior muscle. The fast-to-slow transition process included a distinct reduction in fast calsequestrin and triadin and a concomitant reduction in calsequestrin binding to these sarcoplasmic reticulum elements. The calsequestrin-binding protein junctin was not affected by the muscle transformation process. The increase in calsequestrin and decrease in junctin expression during postnatal development resulted in similar changes in the intensity of binding of the calsequestrin conjugate to these sarcoplasmic reticulum components. Aged skeletal muscle fibers tended towards reduced protein interactions within the calsequestrin complex. This agrees with the physiological concept that the key regulators of Ca(2+) homeostasis exist in a supramolecular membrane assembly and that protein-protein interactions are affected by isoform shifting underlying the finely tuned adaptation of muscle fibers to changed functional demands.

Isoform-selective interaction of cyclooxygenase-2 with indomethacin amides studied by real-time fluorescence, inhibition kinetics, and site-directed mutagenesis.

Conversion of carboxylate-containing nonsteroidal antiinflammatory drugs, such as indomethacin, to esters or amides provides potent and selective inhibitors of cyclooxygenase-2 (COX-2) [Kalgutkar et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 925-930]. Synthesis of cinnamyl- or coumarinyl-substituted ethanolamide derivatives of indomethacin produced fluorescent probes of inhibitor interaction with COX-2 and COX-1. Binding of either derivative to apoCOX-2 or apoCOX-1 resulted in a rapid, reversible enhancement of fluorescence. Following this rapid phase, a slow additional increase in fluorescence was observed with apoCOX-2 but not with apoCOX-1. The slow, COX-2-specific increase in fluorescence was prevented or reversed by addition of the nonfluorescent COX inhibitor (S)-flurbiprofen. Detailed kinetic studies of the interaction of the coumarinyl derivative with COX-2 showed that the observed changes in fluorescence could be described by two sequential equilibria, the first of which is rapid, reversible, and bimolecular in the forward direction. The second equilibrium is slower, reversible, and unimolecular in both directions. The forward rate constant for the slow equilibrium determined by fluorescence enhancement [(3.1 +/- 0.6) x 10(-3) s(-1)] corresponded closely to the forward rate constant for inhibition of COX-2 activity [(6.8 +/- 2.3) x 10(-3) s(-1)], suggesting that the slow fluorescence enhancement is associated with selective COX-2 inhibition. Site-directed mutagenesis indicated that residues in the carboxylate-binding region of the COX-2 active site (Arg-120, Tyr-355, and Glu-524) are critical for the binding of the indomethacin conjugates that leads to slow fluorescence enhancement and cyclooxygenase inhibition. The indomethacin conjugates described herein represent powerful tools for the investigation of a novel class of selective inhibitors of COX-2.

Analysis and quantitation of biotinylated apoB-containing lipoproteins with streptavidin-Cy3

Non denaturing gradient gel electrophoresis (GGE) is commonly used to analyze the size distribution of lipoprotein particles. Its relatively low sensitivity and linear dynamic range limit use of GGE to quantify protein content of lipoproteins. We demonstrate a new high sensitivity method for analysis and quantitation of biotinylated apolipoprotein B (apoB)-containing lipoproteins using a fluorescent streptavidin-Cy3 conjugate and non covalent preelectrophoretic binding. Forty-four lipoprotein subfractions spanning the VLDL and LDL particle spectrum subfractions (11 each from four human subjects) were prepared by density gradient ultracentrifugation. An aliquot of each sample was biotinylated and GGE was performed. Gels also were stained for lipid with Oil Red O (32 samples) and for protein with Coomassie Brilliant Blue (30 samples). There was a significant relationship between the Cy3 fluorescent label area under the curve and the mass of apoB (P < 0.02–0.004) and total cholesterol (P < 0.03–0.004). Particle diameters of each absorbence/fluorescent peak were comparable between Oil-Red O and streptavidin-Cy3 treated biotinylated lipoproteins (±3.54 Å, P = 0.3).Biotinylation and prestaining of lipoprotein particle with streptavidin-Cy3 provides a new fluorescence-based method for detection and quantitative analysis of lipoprotein subspecies by gradient gel electrophoresis.

Transport of phosphatidylserine via MDR1 (multidrug resistance 1)P-glycoprotein in a human gastric carcinoma cell line.

The ATP-binding cassette transporter multidrug resistance 1 P-glycoprotein (MDR1 Pgp) has been implicated with the transport of lipids from the inner to the outer leaflet of the plasma membrane. While this has been unambigously shown for the fluorescent lipid analogues [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (C6-NBD)-phosphatidylcholine, -phosphatidylethanolamine, -sphingomyelin and -glucosylceramide, by using a novel approach we have now found significantly increased outward transport also for C6-NBD-phosphatidylserine (C6-NBD-PS) in EPG85-257 human gastric carcinoma cells overexpressing MDR1 (coding for MDR1 Pgp). The increased transport of C6-NBD-PS is mediated by MDR1 Pgp, shown by transport reduction nearly to the level of controls in the presence of MDR1 Pgp inhibitors [PSC 833, cyclosporin A and dexniguldipine hydrochloride (Dex)]. Addition of MK 571, a specific inhibitor of the MDR protein MRP1, does not decrease transport in either of the two cell lines. The plasma-membrane association of FITC-annexin V, a fluorescent protein conjugate binding PS, is significantly increased in MDR1-overexpressing cells as compared with controls, and can be reduced by an MDR1 Pgp inhibitor. This suggests that MDR1 Pgp transports endogenous PS, the lipid exhibiting the most pronounced transverse asymmetry in the plasma membrane.

Sequence selective recognition of DNA by hairpin conjugates of a racemic seco-cyclopropaneindoline-2-benzofurancarboxamide and polyamides

Conjugates of racemic seco-cyclopropaneindoline-2-benzofurancarboxamide (CI-Bf) and four diamides (ImIm 1, ImPy 2, PyIm 3, and PyPy 4, where Py is pyrrole, and Im is imidazole), linked by a γ-aminobutyrate group were synthesized. In addition to alkylating at adenine-N3 positions within an A 5 sequence, the imidazole-containing compounds 1 and 2 were found to also alkylate purine-N3 positions within a sequence 3′-GGGGG GA (888)CTGCTC(894)-5′. A model for the binding of hairpin conjugates 1 and 2 with the 3′-G A CT-5′ sequence is proposed.

Synthesis, in vitro receptor binding, and in vivo evaluation of fluorescein and carbocyanine peptide-based optical contrast agents.

Site-specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effects of drugs and enhances the contrast between normal and pathologic tissues. One approach to achieve selectivity is to target overexpressed receptors on the membranes of tumor cells and to visualize the tumors by a noninvasive optical imaging method. Accordingly, we conjugated fluorescein and carbocyanine dyes to somatostatin and bombesin receptor-avid peptides and examined their receptor binding affinities. We also prepared potential dual imaging probes consisting of a bioactive peptide for tumor targeting, a biocompatible dye for optical imaging, and a radioactive or paramagnetic metal chelator for scintigraphic or magnetic resonance imaging of tumors. Using these approaches, the resulting carbocyanine derivatives of somatostatin and bombesin analogues retained high binding for their respective receptors. Further evaluation of representative molecules in rats bearing somatostatin- and bombesin-positive tumors showed selective uptake of the agents by the tumor cells. Unlike carbocyanine derivatives, the receptor binding of fluorescein-somatostatin peptide conjugates was highly sensitive to the type of linker and the site of fluorescein attachment on the nonreceptor binding region of the peptide. In general, the presence of flexible linkers disrupted binding affinity, possibly due to the interaction of the linker's thiourea group with the peptide's cyclic disulfide bond. While the receptor binding affinity of the dual probes was not dependent on the type of chelating group examined, it was affected by the relative positions of fluorescein and chelator on the lysine linker. For somatostatin compounds, best results were obtained when the chelator was on the alpha-amino lysine linker and fluorescein was on the epsilon-amino group. In contrast, conjugation of the chelator to epsilon- and fluorescein to the alpha-amino lysine linker of bombesin peptides resulted in high receptor binding. These findings indicate that despite their small size, conjugation of dyes to truncated somatostatin and bombesin peptide analogues results in promising diagnostic agents that retain high receptor binding activity in vitro. The results further show that these contrast agents can selectively and specifically localize in receptor-positive tumors in rat models.

Complications in cell-surface labelling by biotinylation of Candida albicans due to avidin conjugate binding to cell-wall proteins.

Initial contact between the opportunistic fungal pathogen Candida albicans and host tissue occurs at the cell surface. Biotin derivatives have been used to label the cell-surface proteins of yeasts, with labelled proteins subsequently detected by avidin-reporter conjugates. Previous work has indicated that avidin can bind to C. albicans proteins in the absence of biotin, suggesting a possible host-cell-recognition mechanism by fungal cell-surface proteins. To investigate this mechanism, Western blots of proteins extracted from biotinylated and mock-treated cells were probed with avidin or modified-avidin reagents. Each avidin reagent bound to cell-wall proteins extracted from non-biotinylated cells. Binding did not appear to be due to the lectin-like activity of the cell-wall proteins of C. albicans or to the presence of biotin in the sample itself. Binding was inhibited by added biotin, by the chaotrope KSCN and by NaCl in a concentration-dependent manner, although inhibition varied among the avidin conjugates tested. Thus, the non-specific binding of avidin to the cell-wall proteins of C. albicans appears to involve hydrophobic and electrostatic interactions, depending on the particular avidin species. These observations demonstrate potential pitfalls in the use of avidin-biotin complexes to identify cell-surface molecules and could provide insights into protein-protein interactions at the C. albicans cell wall.

Lectin-mediated drug delivery: binding and uptake of BSA-WGA conjugates using the Caco-2 model

To examine whether the dietary lectin wheat germ agglutinin (WGA) can facilitate binding and uptake of protein drugs due to its cytoadhesive and cytoinvasive properties, conjugates were prepared by covalent coupling of fluorescein-labeled bovine serum albumin (F-BSA) to WGA using divinylsulfone for crosslinking. Increasing the molar ratio of F-BSA/WGA resulted in 2.6–8.7 times higher Caco-2 binding as compared with glycyl-F-BSA. About 75% of F-BSA-WGA were bound specifically to Caco-2 cells according to inhibition studies in presence of the complementary carbohydrate. The Caco-2 association of F-BSA-WGA was temperature-dependent indicating active uptake of membrane bound conjugate, which was confirmed by confocal microscopy. The conjugate accumulated within lysosomal compartments followed by proteolytic degradation of F-BSA-WGA 1–4 h after conjugate loading as observed by equilibrating the intracellular pH with monensin. Finally low molecular weight degradation products of the proteinaceous prodrug appear in the extracellular medium. Contrary to Caco-2 single cells, a minor part of the conjugate is degraded by brush border proteases already 30 min after exposure to Caco-2 monolayers. But most of the conjugate is taken up into differentiated cells and processed as in single cells. Though the enzymic barrier remains to be surmounted, WGA-mediated drug delivery is a promising strategy for peroral delivery of even high molecular weight drugs to overcome the mucosal barrier.

Glutathione and mercapturic acid conjugates of sulofenur and their activity against a human colon cancer cell line.

Sulofenur is one of the diarylsulfonylureas developed as an anticancer agent. Sulofenur possesses a broad spectrum of activity in several solid tumor models and has undergone extensive clinical trials based on its impressive preclinical activity. However, the clinical response of sulofenur has been disappointing because of the side effect of anemia. Furthermore, the anticancer mechanism of sulofenur and its diarylsulfonylurea analogs still remains unknown. Elucidation of the metabolic fates of sulofenur may help to delineate the mechanism and provide information to guide the structural modification for more potent anticancer agents with less side effects. We have identified a glutathione conjugate and a mercapturic acid conjugate from sulofenur-dosed rats with the aid of liquid chromatography/mass spectrometry. The fraction of the dose of sulofenur as the glutathione conjugate in the dosed-rat bile over 5 h was 0.12 +/- 0.03%, and the mercapturic acid conjugate in urine over 24 h was 1.4 +/- 0.7%. Protein binding of the glutathione conjugate and mercapturic acid conjugate was determined to be 20 +/- 3 and 84 +/- 2%, respectively, as opposed to >99% of sulofenur. The high protein binding of sulofenur requires a higher than in vitro dose, which is believed to cause the side effect of anemia. The significance of this metabolic pathway is that both conjugates were found to be glutathione reductase inhibitors and to possess anticancer activity comparable to sulofenur against human colon adenocarcinoma GC(3)/c1 cells, a sulofenur-sensitive cell line. These conjugates may serve as new leads for the development of novel anticancer agents.