DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety.

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eukaryotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNA-binding domains. Successful transferrin-receptor-mediated delivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (greater than 100 mg) of conjugates.     

Multidrug resistance-associated protein2 (MRP2) plays an important role in the biliary excretion of glutathione conjugates of 4-hydroxynonenal

Glutathione (GSH) conjugates of 4-hydroxy-trans-2,3-nonenal (HNE) are the final products of lipid peroxidation. In the present study, the role of multidrug resistance-associated protein 2 (MRP2) in biliary excretion of GSH conjugates of HNE (HNE-SG) was studied in vitro by using Madin-Darby canine kidney II (MDCK II) cells expressing human MRP2 and in vivo using a mutant rat strain whose MRP2 expression is defective (Eisai-hyperbilirubinemic rats [EHBR]). A high-performance liquid chromatography method was developed to assay HNE-SG conjugates. Four diastereomeric HNE-SG conjugates could be separated with this method. Three of four HNE-SG conjugates were detectable after incubation of the cell monolayers with HNE. Expression of human MRP2 resulted in a 10-fold increase in HNE-SG conjugates excretion across the apical membrane of MDCK II cells. The four HNE-SG conjugates appeared swiftly in bile from Sprague Dawley rats after intravenous administration of HNE, whereas no detectable HNE-SG conjugates were observed in the bile of EHBR. These results demonstrate the role of MRP2 in the biliary excretion of HNE-SG conjugates.     

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated frrm normal human urine was acheived by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urin.     

Aptamer–Au NPs conjugates-enhanced SPR sensing for the ultrasensitive sandwich immunoassay

The goal of this work is to explore the amplification effect of aptamer–gold nanoparticles (Au NPs) conjugates for ultrasensitive detection of large biomolecules by surface plasmon resonance (SPR). A novel sandwich immunoassay is designed to demonstrate the amplification effect of aptamer–Au NPs conjugates by using human immunoglobulin E (IgE) as model analyte. Human IgE, captured by immobilized goat anti-human IgE on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 1 ng/ml after anti-human IgE aptamer–Au NPs conjugates is used as amplification reagent. Meanwhile, the non-specific adsorption of aptamer–Au NPs conjugates on goat anti-human IgE is confirmed by SPR spectroscopy and then it is minimized by treating aptamer–Au NPs conjugates with 6-mercaptohexan-1-ol (MCH). These results confirm that aptamer–Au NPs conjugates is a powerful sandwich element and an excellent amplification reagent for SPR-based sandwich immunoassay.     

Gold labeling of thrombin and ultrastructural studies of thrombin-gold conjugate binding by fibrin

Monodispersed thrombin-gold (T-Au) conjugates were prepared by the absorption of a monolayer (3.8 nm thick) of human alpha-thrombin around individual monodispersed colloidal gold particles (16.5 +/- 1.8 nm). Like free molecular thrombin, T-Au conjugates can cause platelet aggregation, plasma clotting, and the release of fibrinopeptides A and B from fibrinogen. At the same thrombin concentration, T-Au conjugates have only one-tenth the fibrinogen-clotting activity of free thrombin and one-third the amidolytic activity of free thrombin. Hirudin can completely inhibit the fibrinogen-clotting activity of both T-Au conjugates and free thrombin, but can inhibit only half of the amidolytic activity of the conjugates. Diisopropyl fluorophosphonate can completely inhibit the fibrinogen-clotting activity and the amidolytic activity of both T-Au conjugates and free thrombin. T-Au conjugates were further characterized by studying the mechanism of their binding to fibrin and the location of the binding site on fibrin. The results of electron microscopic studies showed that T-Au conjugates, but not albumin-Au conjugates, are bound by fibrin. Increasing T-Au conjugate concentrations are associated with an increase in the number of T-Au conjugates binding to fibrin. At 0.1 microM thrombin, 73% of the T-Au conjugates are bound to branch points of the fibrin network with 27% of the T-Au conjugates present in the fibrin strands. At higher thrombin concentration (e.g., 0.5 microM) the percentage of T-Au conjugates bound to locations other than branch points increases to 62%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of different antigen-enzyme and antibody-enzyme conjugates by intracellular antibodies in cytoplasm and Golgi complex of plasma cells. A double immunocytochemical study

Intracellular immunoglobulins in plasma cells were characterized by antigen-enzyme conjugates and anti-immunoglobulin antibody-enzyme conjugates applied in a double immunocytochemical approach. After their assemblage, immunoglobulins in the cytoplasm of anti-TNP anti-body producing plasma cells can be demonstrated both by TNP-enzyme conjugates and by anti-immunoglobulin (mu or gamma chain specific) antibody-enzyme conjugates. Once arrived in the Golgi complex (GC) detection with TNP-enzyme conjugates remains possible, but anti-immunoglobulin anti-body-enzyme conjugates did not bind to a detectable degree. Similar results were obtained in experiments where immunoglobulin-enzyme conjugates were used both as an antigen-enzyme conjugate and as an antibody-enzyme conjugate.     

Fixation of various aldehydic dextrans onto human hemoglobin: Study of conjugate stability

Formation and stability of different aldehydic dextran-hemoglobin conjugates were studied. Two types of polymers were used: sulfated or unsulfated oxidized dextrans and 4-carboxamidobenzaldehyde dextran. Periodate-oxidized dextran forms imine and ketoamine linkages by reaction with hemoglobin and the obtained conjugates are not completely stable, as their molecular size increases with time or decreases after incubation with lysine. The sulfated conjugates are more sensitive to lysine action than the unsulfated ones, which is consistent with the decreased possibilities of Amadori rearrangement. Therefore, this proves the importance of ketoamines for ensuring the cohesion of oxidized dextran-based conjugates. Carboxamidobenzaldehyde dextran forms only imine linkages with hemoglobin and the corresponding conjugates possess a marked instability in the absence of reductive treatment. The different types of conjugates could be stabilized by a sodium borohydride treatment in a satisfying manner.     

Conjugation of [125I]ubiquitin to cellular proteins in permeabilized mammalian cells: comparison of mitotic and interphase cells.

[125I]Ubiquitin introduced into permeabilized hepatoma tissue culture (HTC) cells rapidly forms conjugates with endogenous proteins. A characteristic pattern of low mol. wt conjugates is obtained which includes the ubiquitinated histone, uH2A, and unknown molecular species with MrS of 14, 23, 26 (two bands) and 29 kd. A broad spectrum of higher mol. wt conjugates is also produced. The formation of all conjugates is absolutely dependent on ATP, and upon depletion of ATP they are rapidly broken down. The 14, 23 and 29 kd species are found in all subcellular fractions examined. uH2A is located exclusively in the nuclear fraction. The pair of 26 kd bands is specifically associated with the ribosome fraction. A considerable percentage of the higher mol. wt conjugates sediments with the small particle (100,000 g) fraction in the ultracentrifuge but is solubilized with deoxycholate, indicating that there are many membrane-associated conjugates. The pattern of ubiquitin conjugation in interphase and metaphase cells was compared. The incorporation of ubiquitin into uH2A was markedly reduced in metaphase cells whereas its incorporation into other low mol. wt conjugates and into high mol. wt conjugates was affected slightly, if at all. This shows that the known decrease of uH2A levels in metaphase is due to a specific effect on histone ubiquitination and not to a general decrease in ubiquitination activity or increase of isopeptidase activity. Changes in the levels of uH2A during mitosis measured by immunoblotting were similar to those estimated in permeabilized cells. These experiments indicate that permeabilized cells provide a useful approach to the study of rapidly turning over ubiquitin conjugates in mammalian cells.     

Glutathione conjugation as a bioactivation reaction

In general, glutathione conjugation is regarded as a detoxication reaction. However, depending on the properties of the substrate, bioactivation is also possible. Four types of activation reaction have been recognized: direct-acting compounds, conjugates that are activated through cysteine conjugate beta-lyase, conjugates that are activated through redox cycling and lastly conjugates that release the original reactive parent compound. The glutathione S-transferases have three connections with the formation of biactivated conjugates: they catalyze their formation in a number of cases, they are the earliest available target for covalent binding by these conjugates and lastly, the parent alkylating agents are regularly involved in the induction of the enzymes. Individual susceptibility for each of these agents is determined by individual transferase subunit composition and methods are becoming available to assess this susceptibility.     

Protein-polymer conjugates: synthetic approaches by controlled radical polymerizations and interesting applications

Protein-polymer conjugates are of interest to researchers in diverse fields. Attachment of polymers to proteins results in improved pharmacokinetics, which is important in medicine. From an engineering standpoint, conjugates are exciting because they exhibit properties of both the biomolecules and synthetic polymers. This allows the activity of the protein to be altered or tuned, anchoring to surfaces, and supramolecular self-assembly. Thus, there is broad interest in straightforward synthetic methods to prepare protein-polymer conjugates. Controlled radical polymerization (CRP) techniques have emerged as excellent strategies to make conjugates because the resulting polymers have narrow molecular weight distributions, targeted molecular weights, and attach to specific sites on proteins. Herein, recent advances in the synthesis and application of protein-polymer conjugates by CRP are highlighted.     

Preparation of protein conjugates via intermolecular hydrazone linkage

Proteins can be modified at their amino groups under gentle conditions to contain an average of three to six aryl aldehyde or acyl hydrazide groups. These two types of modified proteins at about 10 microM concentration condense with each other at pH approximately 5 to form conjugates linked by hydrazone bonds. Under proper conditions conjugates mainly of dimers and trimers in size or, if desired, higher oligomers can be obtained. The conjugates can be dissociated to their individual protein components by an exchange reaction with an excess of acetyl hydrazide. The reversible hydrazone bonds of conjugates can be reduced with NaCNBH3 to give stable hydrazide bonds. The stability of protein-hydrazone conjugates was found to be significantly greater than that of the model compound, the N-acetylhydrazone of p-carboxybenzaldehyde. This difference is believed to result from the presence of multiple hydrazone linkages in protein conjugates.     

Hapten-IgG-induced suppression of the in vitro antibody response: role of hapten density and of antigenic challenge.

We have studied the suppression of the in vitro antibody response of mouse spleen cells to the trinitrophenyl (TNP) hapten by conjugates of TNP-human IgG (TNP-HGG). Normal mouse spleen cells were incubated with conjugates of HGG and of HGG fragments, with different hapten densities. They were then challenged with either a T-dependent or a T-independent antigen. Highly substituted (12 mol of TNP/mol of HGG) conjugates induced a dose-dependent suppression, apparent after short-term incubation at 4 °C, of both T-dependent and T-independent responses. Conjugates of Fab′₂ and Fab′ were as suppressive as conjugates of IgG, whereas conjugates of albumin, aggregated IgG, and β2 microglobulin lacked suppressive activity. In contrast, the lightly substituted conjugates TNP₈-HGG and TNP₂-Fab′₂ induced a time-dependent suppression, affecting only the T-dependent response. This suggests that the suppressive effect of hapten-IgG conjugates is mediated by two different mechanisms according to the density of hapten on the IgG carrier. When these conjugates are used as tolerogens in the in vivo situation, both mechanisms would operate to a variable extent, and this could account for the remarkable tolerogenic properties of hapten-IgG conjugates.     

Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption

Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery.     

The design,synthesis and study of siderophore-antibiotic conjugates Siderophore mediated drug transport

Summary The use of conjugates of microbial iron chelators (siderophores) and antibiotics for illicit transport of antibiotics into cells is a potentially powerful method for the rational design of therapeutic agents. The structural complexity of most natural siderophores has impeded progress in this area. Described here are the design, syntheses and preliminary biological studies of several siderophore--lactam antibiotic conjugates. Both hydroxamic-acid-based and catechol-based conjugates with and without amino acid spacers to carbacephalosporins were synthesized and demonstrated to be effective inhibitors ofEscherichia coli X580. Mutant selection was noted for each class of conjugates. Mutants selected from exposure of theE. coli to the hydroxamate conjugates were susceptible to the catechol conjugates and vice versa. Combinations of hydroxamate-and catechol-carbacephalosporin conjugates were most effective inhibitors ofE. coli X580.     

Microinjection of ubiquitin: changes in protein degradation in HeLa cells subjected to heat-shock

Ubiquitin was radiolabeled by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using erythrocyte-mediated microinjection. The injected cells were then incubated at 45 degrees C for 5 min (reversible heat-shock) or for 30 min (lethal heat-shock). After either treatment, there were dramatic changes in the levels of ubiquitin conjugates. Under normal culture conditions, approximately 10% of the injected ubiquitin is linked to histones, 40% is found in conjugates with molecular weights greater than 25,000, and the rest is unconjugated. After heat-shock, the free ubiquitin pool and the level of histone-ubiquitin conjugates decreased rapidly, and high molecular weight conjugates predominated. Formation of large conjugates did not require protein synthesis; when analyzed by two-dimensional electrophoresis, the major conjugates did not co-migrate with heat-shock proteins before or after thermal stress. Concomitant with the loss of free ubiquitin, the degradation of endogenous proteins, injected hemoglobin, BSA, and ubiquitin was reduced in heat-shocked HeLa cells. After reversible heat-shock, the decrease in proteolysis was small, and both the rate of proteolysis and the size of the free ubiquitin pool returned to control levels upon incubation at 37 degrees C. In contrast, neither proteolysis nor free ubiquitin pools returned to control levels after lethal heat-shock. However, lethally heat-shocked cells degraded denatured hemoglobin more rapidly than native hemoglobin and ubiquitin-globin conjugates formed within them. Therefore, stabilization of proteins after heat-shock cannot be due to the loss of ubiquitin conjugation or inability to degrade proteins that form conjugates with ubiquitin.     

The effect of condition of oxidation of the carbohydrate component of peroxidase on the composition and properties of insulin-peroxidase conjugate

The influence of sodium metaperiodate concentration on kinetics and conversion degree of peroxidase carbohydrate moiety as well as the effect of the oxidation degree of the carbohydrate moiety on the composition, structure and properties of insulin-peroxidase conjugates were studied. The initial rate of peroxidase's oxidation is directly proportional to the periodate concentration; the oxidation rate constant of peroxidase carbohydrate moiety is 1.23 x 10(-3) M-1 min-1. At the molar ratio of metaperiodate to peroxidase 150:1 or higher, the maximal quantity of aldehyde groups (62 +/- 2) in the peroxidase molecule is formed and the oxidation of each carbohydrate chain leads to the formation of eight aldehyde groups. The molecular mass composition of the insulin-peroxidase conjugates was studied by HPLC. The conjugates proved to be multicomponent mixtures of oligomers (53, 83, 128, 174, 268, 440 kD and higher). The insulin-peroxidase molar ratio in the fractions of the conjugates with molecular masses higher than 83 kD is 8:1. It was shown that the affinity of insulin-peroxidase conjugates to antibodies depends on the oxidation degree of peroxidase used for production of conjugates.     

Effects of CKS-17, a synthetic retroviral envelope peptide,on cell-mediated immunity in vivo: Immunosuppression,immunogenicity, and relation to immunosuppressive tumor products

Summary CKS-17 is a heptadecapeptide corresponding to a region highly conserved in retroviral transmembrane proteins such as p15E. Because a relationship had previously been determined between p15E and immunosuppressive tumor cell products, we examined the effect of CKS-17, control peptides and conjugates thereof on the expression of cell-mediated immunity (delayed-type hypersensitivity, DTH) in mice. Conjugates of CKS-17 inhibited DTH reactions to sheep erythrocytes in the feet of mice. The degree of inhibition was dose-dependent. Unconjugated CKS-17 had almost no effect, and control peptide conjugates had no inhibitory effect. Immunization of mice with CKS-17 conjugates, but not with control conjugates, rendered them resistant to the depression of DTH reactions, not only by CKS-17 conjugates, but also by products of cultured tumor cells. CKS-17 conjugates, but not control conjugates, also depressed the cellular inflammatory reactions induced in mouse footpads by concanavalin A (ConA) and immunized mice against the depression of ConA reactions by products of cultured tumor cells. Injections of globulin from sera of mice immunized with CKS-17 conjugates conferred upon normal recipients resistance to the depression of footpad reactions to ConA by products of cultured tumor cells. Globulin from sera of normal mice or control immunized mice did not confer such resistance. Thus conjugates of a synthetic peptide not only mimic the immunosuppressive effects of tumor products in vivo, but can also immunize mice against those effects.     

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity

Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC) that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (ODN). We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL) responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.     

In vitro biological properties of flavonoid conjugates found in vivo

For some flavonoids such as quercetin, isoflavones and catechins, the pathways of absorption and metabolism are now reasonably well characterised and understood. By definition, for biological activity of flavonoids to be manifest, the target tissue, which includes the blood and vascular system, must respond to the form(s) of flavonoid that it encounters. Bioavailability studies have shown that the circulating form of most flavonoids is as conjugates, with a few notable exceptions. There have been several recent papers on the in vitro biological properties of conjugates that have been found in vivo. This paper reviews the properties of these conjugates. Most of the information currently available is on quercetin glucuronides, but also on isoflavone and catechin conjugates. In addition to the biological properties of the conjugates, the partition coefficients and methods of synthesis are also presented.     

Enhancement of immunotoxin efficacy by acid-cleavable cross-linking agents utilizing diphtheria toxin and toxin mutants

We have utilized a new class of acid-cleavable protein cross-linking reagents in the construction of antibody-diphtheria toxin conjugates (Srinivaschar, K., and Neville, D. M., Jr. (1989) Biochemistry 28, 2501-2509). The potency of anti-CD5 conjugates assayed by inhibition of protein synthesis on CD5 bearing cells (Jurkat) is correlated with cross-linker hydrolytic rates. The maximum increase in potency of the cleavable conjugates over non-cleavable conventional conjugates is 50-fold and is specific for the CD5 uptake route as judged by competition with excess anti-CD5. The potency of conjugates made from diphtheria toxin and the anti-high molecular weight melanoma-associated antigen (HMW-MAA) is enhanced 3-10-fold by a cleavable cross-linker. However the potency of transferrin or anti-CD3 diphtheria toxin conjugates is only minimally enhanced (2-3-fold). Mutant diphtheria toxins, CRM103 and CRM9, previously shown to express less than 1/100 of the wild type in binding affinity were substituted into these conjugates as probes for possible intracellular toxin receptor interactions. Both mutants were equally as toxic to Jurkat target cells exhibiting 1/700 the wild-type potency. CRM9 non-cleavable conjugates were equally as potent as wild-type conjugates for transferrin and anti-CD3-mediated uptake but not for anti-CD5-mediated uptake where toxicity was reduced 60-fold over the wild-type analog. The cleavable cross-linker enhanced the toxicity of anti-CD5-CRM103 and anti-CD5-CRM9 conjugates, but potency was only 1/10 that of the analogous wild-type cleavable conjugate. These data are consistent with a model in which potentiation of toxicity of the anti-CD5 and anti-high molecular weight melanoma-associated antigen conjugates by the cleavable cross-linker occurs from an enhanced intracellular toxin-toxin receptor interaction that ultimately results in increased toxin translocation to the cytosol compartment. In contrast, these data indicate that the anti-CD3 and transferrin uptake systems do not require this interaction in agreement with previous work (Johnson, V.G., Wilson, D., Greenfield, L., and Youle, R. J. (1988) J. Biol. Chem. 263, 1295-1300).