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.     

A comparison of the in vitro and in vivo activities of conjugates of anti-mouse lymphocyte globulin and abrin

Anti-mouse lymphocyte globulin and normal immunoglobulin have been conjugated to abrin using two procedures, one involving linkage through an amide bond and a piperazine ring and the other the introduction of two amide bonds flanking a disulphide bridge. The four conjugates produced were equipotent as inhibitors of protein synthesis in rabbit reticulocyte lysates. Each antibody-containing conjugate was a more effective inhibitor of protein synthesis in cultured cells than the equivalent normal immunoglobulin-containing conjugate. In addition the conjugates with disulphide linkage groups were ten times more potent than their counterparts. The disulphide conjugates were also twice as toxic to mice in an acute toxicity test but when used to suppress their immune responses to sheep red blood cells it was the non-disulphide-linked conjugates that were superior. In all instances antibody-containing conjugates were more powerful immunosuppressants than those containing normal IgG. The results are taken to indicate a relative lack of stability of the disulphide conjugates in the tissues.     

Evidence That IAA Conjugates Are Slow-Release Sources of Free IAA in Plant Tissues

Evidence that indoleacetic acid (IAA) conjugates are metabolized via enzyme-catalyzed hydrolysis to free IAA and that their biological activities are related to the rates at which they are hydrolyzed by the tissue is presented. These conclusions are based on the following observations. Slow but continuous decarboxylation of the IAA moiety of IAA-l-alanine and IAA-glycine occurs when these conjugates are applied to pea (Pisum sativum L. cv. Alaska) stem segments. Inasmuch as IAA conjugates are protected from peroxidase-catalyzed oxidative decarboxylation, the conjugates are probably hydrolyzed and the freed IAA then further metabolized. Free IAA and IAA-l-alanine are converted, by pea stem tissue, into the same metabolites. The metabolism is enzymic, since conjugates of IAA with the d-isomers of the amino acids are inactive. Ethylene production induced by IAA-l-alanine and by IAA-glycine is correlated with their hydrolysis, as indicated by their decarboxylation and with the appearance or nonappearance of IAA metabolites in the tissues.     

New Polyazamacrocycle-Nucleoside Conjugates: Synthesis,Anti-HIV Evaluation and Interaction with CXCR-4 Coreceptor

Abstract

We report the synthesis of new conjugates that incorporate in their structure bis-tetraazamacrocycle coupled with AZT via enzymolabile bond.

Two series of bis-polyazamacrocycles-AZT conjugates were designed, synthesized and evaluated for their antiviral effect in vitro as well as their capability to bind to CXCR-4 coreceptor.     

多糖-药物轭合物的研究与展望

多糖类物质作为赋形剂在药物制剂中已被广泛使用,多糖结构中包含了多种活性基团如羟基、羧基、氨基等,具有良好的亲水性、生物可降解性以及生物安全性,使其在聚合物-药物轭合物的构建中成为理想的载体材料.目前天然的多糖大分子及其衍生物作为药物载体的研究方兴未艾,以多糖为载体的聚合物-药物轭合物在定位或靶向给药、组织工程、生物黏附等领域也备受关注.本文以天然多糖-药物轭合物的研究现状为切入点,总结归纳了多糖-药物轭合物的设计与构建途径,介绍了其在药物传递中的应用,讨论并分析了多糖在轭合物体系中的角色和发挥的作用,对以多糖为载体的聚合物-药物轭合物发展的方向予以了讨论.     

RNA cleavage by 2,9-diamino-1,10-phenanthroline PNA conjugates

We report on the synthesis of 2,9-diamino-1,10-phenanthroline PNA conjugates as well as on their action in cleavage of a target RNA. Synthesis of the PNA conjugates are performed on solid support and the phenanthroline derivative is conjugated either to the amino-end or to a centrally positioned diaminopropionic acid in the PNA via a urea linker. Cleavage of the target RNA is achieved and compared to cleavage with the corresponding 2,9-dimethyl-1,10-phenanthroline and glycine conjugates.     

Binding of different antigen-enzyme and antibody-enzyme conjugates by intracellular antibodies in cytoplasm and Golgi complex of plasma cells

Summary 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 antibody producing plasma cells can be demonstrated both by TNP-enzyme conjugates and by anti-immunoglobulin ( or chain specific) antibody-enzyme conjugates. Once arrived in the Golgi complex (GC) detection with TNP-enzyme conjugates remains possible, but anti-immunoglobulin antibody-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.     

Peptide-and Biotin-Oligonucleotide-Pyrrole Conjugates for Electrochemical Addressing on Silicon Chip

Abstract

The syntheses of pyrrole-oligonucleotide-peptide conjugates and pyrrole-oligonucleotide-biotin conjugates were described. The oligonucleotide moiety acted as an ?active linker? which allowed the easy purification and quantitation of the conjugates and in turn controlled the grafting. The peptide conjugates were immobilised on silicon array and their immunoreactivity was tested using biotinylated antibodies and a phycoerythrin-streptavidin staining. The biotin conjugate provided a fluorescence scale.     

Acetals as pH-sensitive linkages for drug delivery

pH-Sensitive linkages designed to undergo hydrolysis at mildly acidic pH can trigger the release of therapeutics selectively at targets such as tumor and inflammatory tissues and in the endosomes and lysosomes of cells. Acetals have the potential to be used as linkages for a range of alcohol functionalities, and, by altering their chemical structure, it is possible to tune their hydrolysis rate. The syntheses of four conjugates of model drug molecules with PEO using acetals of varying chemical structure are described herein. Primary and secondary alcohols, as well as syn-1,2-diols, were incorporated in the conjugates. The hydrolysis kinetics were investigated by HPLC, and the conjugates had half-lives ranging from less than 1 min to several days at pH 5.0, with slower hydrolysis at pH 7.4 in all cases. These acetal linkages are therefore promising for use in a variety of drug delivery applications ranging from polymer-drug conjugates to pH-sensitive micelles and nanoparticulate systems.     

Specific and nonspecific macromolecule-drug conjugates for the improvement of cancer chemotherapy

Antineoplastic drugs such as daunomycin, adriamycin, methotrexate, 5-fluorouridine, cytosine arabinoside, and platinate were bound to antibodies directly or via a polymeric bridge. The drug antibody conjugates retained most of their drug and antibody activities when tested in vitro. Daunomycin–antibody conjugates were shown to penetrate tumor cells in the conjugated form. In animals, daunomycin–antibody conjugates were at least as effective chemotherapeutically as the corresponding free drugs and considerably less toxic. In some tumor systems, the daunomycin–antibody conjugates represented an improvement over the free drug. This improvement was restricted in some tumors to a particular injection route of the tumor and the treatment.     

In vitro and in vivo gene transfer by an optimized alpha-cyclodextrin conjugate with polyamidoamine dendrimer

The purpose of the present study is to optimize the structure of the polyamidoamine starburst dendrimer (dendrimer) conjugate with alpha-cyclodextrin (alpha-CDE conjugate) as a nonviral vector. alpha-CDE conjugates of dendrimer (generation 3, G3) with various average degrees of substitution (DS) of alpha-CyD of 1.1, 2.4, and 5.4 were prepared. alpha-CDE conjugates formed the complexes with pDNA, resulting in a change of the particle sizes of pDNA complexes, but the distinction of physicochemical properties among their vector/pDNA complexes was only very slight. The membrane-disruptive ability of alpha-CDE conjugates on liposomes encapsulating calcein and their cytotoxicity to NIH3T3 and HepG2 increased with an increase in the DS value of alpha-CyD. In vitro gene transfer activity of alpha-CDE conjugates in both NIH3T3 and HepG2 cells augmented as the charge ratio (vector/pDNA) increased, and the activity of alpha-CDE conjugate (DS 2.4) was the highest at higher charge ratios among dendrimer (G3), the three alpha-CDE conjugates, and TransFast. After intravenous administration of pDNA complexes in mice, alpha-CDE conjugate (DS 2.4) delivered pDNA more efficiently in spleen, liver, and kidney, compared with dendrimer and other alpha-CDE conjugates (DS 1.1 and 5.4). The potential use of alpha-CDE conjugate (G3, DS 2.4) could be expected as a nonviral vector in vitro and in vivo, and these data may be useful for design of alpha-CyD conjugates with other nonviral vectors.     

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.     

Syntheses and DNA binding of new cationic porphyrin-tetrapeptide conjugates

Recently cationic porphyrin-peptide conjugates were synthesized to enhance the cellular uptake of porphyrins or deliver the peptide moiety to the close vicinity of nucleic acids. DNA binding of such compounds was not systematically studied yet.We synthesized two new porphyrin-tetrapeptide conjugates which can be considered as a typical monomer unit corresponding to the branches of porphyrin-polymeric branched chain polypeptide conjugates. Tetra-peptides were linked to the tri-cationic meso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin. DNA binding of porphyrin derivatives, and their peptide conjugates was investigated with comprehensive spectroscopic methods. Titration of porphyrin conjugates with DNA showed changes in Soret bands with bathocromic shifts and hypochromicities. Decomposition of absorption spectra suggested the formation of two populations of bound porphyrins.Evidence provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and induced CD signals reveals that peptide conjugates of di- and tricationic porphyrins bind to DNA by two distinct binding modes which can be identified as intercalation and external binding. Tri-cationic structure and elimination of negative charges in the peptide conjugates are preferable for the binding. Our findings provide essential information for the design of DNA-targeted porphyrin-peptide conjugates.     

Metabolism of Indole-3-acetic Acid: IV. Biological Properties of Amino Acid Conjugates

The biological activity of 20 l-alpha-amino acid conjugates of indole-3-acetic acid (IAA) to stimulate cell elongation of Avena sativa coleoptile sections and to stimulate growth of soybean cotyledon tissue cultures has been examined at concentrations of 10(-4) to 10(-7)m. In the Avena coleoptile test, most of the amino acid conjugates stimulated elongation. Several of the conjugates stimulated as much elongation as IAA but their half-maximum concentrations tended to be higher. Some of the more active conjugates were alanine, glycine, lysine, serine, aspartic acid, cystine, cysteine, methionine, and glutamic acid.In the soybean cotyledon tissue culture test, all of the l-alpha-amino acid conjugates of IAA stimulated growth except for the phenylalanine, histidine, and arginine conjugates. Most of the conjugates produced responses at least as great as that caused by IAA. Conjugates with half-maximum concentrations lower than IAA included cysteine, cystine, methionine, and alanine. These conjugates exceed the IAA-induced callus growth at all tested concentrations. Other conjugates significantly better than IAA at 10(-6)m were serine, glycine, leucine, proline, and threonine.     

Selective growth promotion and growth inhibition of Gram-negative and Gram-positive bacteria by synthetic siderophore-β-lactam conjugates

Conjugates of a carbacephalosporin with hydroxamate, spermexatol, N,N-bis(2,3-dihydroxybenzoyl)-L-lysine, mixed catecholate/hydroxamate and cyanuric acid-based siderophores were investigated for their potential to promote growth of siderophore indicator strains of Gram-negative and Gram-positive bacteria under iron depleted conditions, for their antibacterial activity and for their ability to use iron transport path-ways to penetrate the Gram-negative bacterial outer membrane. The selective growth promotion of enter-obacterial and pseudomonas strains by hydroxamate, spermexatol and mixed catecholate-hydroxamate siderophore-based conjugates bearing a L- or D-amino acid spacer was correlated with TonB dependent uptake routes. The preferred outer membrane siderophore receptor used in Escherichia coli was found to be Fiu, followed by Cir. Antagonistic effects of siderophores administered with the conjugates to determine antibacterial activity confirmed the active transport of conjugates via siderophore receptors. All of the conjugates were still able to diffuse through the porin proteins OmpC and OmpF. Nevertheless, strong inhibition of E. coli and Pseudomones aeruginosa outer membrane mutants DC2 and K799/61 compared to the parent strains indicated inefficient penetrability of all types of conjugates tested. Mycobacterium smegmatis SG 987 was able to use all of the siderophore-cephalosporin conjugates as growth promotors. Consequently there was no growth inhibition of this strain. © Rapid Science 1998.     

Surface plasmon resonance (SPR) as a tool for antibody conjugate analysis

Surface plasmon resonance (SPR) analysis was used to assess the immunoreactivity of anti-biotin (4) and anti-fluorescein (5) monoclonal antibody after conjugation with the N-hydroxysuccinimide ester of acridinium-9-carboxamide 1. Only minor changes in the apparent equilibrium dissociation constants of the antibody conjugates for their ligands resulted from the conjugation process. However, comparison of the initial binding rate of the conjugates with their ligands with those of the unmodified antibodies over a range of concentrations showed that the antibody conjugates were partially inactivated. The anti-fluorescein conjugates retained at least 90% of their immunoreactivity over the range of modification tested, while anti-biotin conjugates showed a progressive loss of reactivity with increased substitution by the label.     

Synthesis and bioevaluation of aryl-guanidino polyamine conjugates targeting the polyamine transporter

Aryl-guanidino polyamine conjugates were prepared to evaluate their recognition for polyamine transporter (PAT) via a-difluoromethylornithine (DFMO) and spermidine (SPD)-treated B16 cell lines. The potent synergistic effects of DFMO on guanidino polyamine conjugates indicated that the presence of DFMO strongly facilitates the transport of conjugates into cells via PAT on cell membrane. The apoptotic mechanisms of triamine conjugates 10 and 1 (with and without guanidine groups) revealed that they induced apoptosis of Hela cells through the mitochondrial pathway associated with lysosomes, while DFMO strongly synergizes the function of 10 without changing the apoptotic route. Taken together, guanidino polyamine conjugates can target PAT for transport as normal polyamine ones, and the presence of guanidine in the polyamine vectors does not seem to alter the cellular targets of the conjugates, which may depend mainly on the pharmacophore.     

Lectin-bound alcohol and lactic dehydrogenases as a reagent for the visual detection of glycoproteins.

Alcohol and lactic dehydrogenases were covalently bound to concanavalin A. The conjugates obtained retained their ability to bind glycoprotein as well as to carry out NAD⁺-oxidoreduction in the presence of the appropriate substrates. The dual capacity renders these protein conjugates as a useful reagent for the location of glycoprotein bands on polyacrylamide gels. Visualization of the conjugates absorbed to the glycoprotein is made by precipitation of formazan dye produced as the result of dehydrogenase activity. It is suggested that the present method and its variations may provide a useful analytical tool for histochemical and other detection procedures for glycoconjugates.     

Auxin metabolism in representative land PLANTS

The plant hormone auxin (indole-3-acetic acid, IAA) appears to control many plant developmental processes, and studies performed in seed plants suggest that IAA conjugation is the critical mechanism to regulate free IAA concentration. The purpose of this investigation is to characterize the biochemical ability of one charophyte and 23 land plants ranging from liverworts to angiosperms to produce IAA conjugates, and to study the complexity of their conjugation patterns. Actively growing tissue was incubated with ¹⁴C-IAA, after which labeled IAA conjugates were separated using thin-layer chromatography. The conjugates were analyzed using radioimaging techniques and their tentative identity assigned by co-chromatography and/or by differential hydrolysis. The charophyte and the liverworts appear unable to conjugate IAA. The mosses and the hornwort are able to conjugate IAA into a few amide and ester conjugates. The tracheophytes examined synthesize several conjugates unique to the vascular plants, indole-3-acetyl-aspartic acid (-glutamic acid) and/or indole-3-acetyl-β-1-O-glucose, as well as a variety of other amide and ester conjugates. These three conjugation patterns are correlated to the type of conducting tissue characteristic of the plants analyzed. These biochemical differences may be indicative of significative differences in the hormonal regulation in these plant groups, thus suggesting that changes in IAA regulation accompanied the major evolutionary events in land plants.     

Cyanocobalamin (vitamin B12) conjugates with enhanced solubility

Cyanocobalamin (vitamin B12) is an essential nutrient as well as a very useful carrier in drug delivery. Conjugates of vitamin B12 are investigated due to their wide range of therapeutic applications. We report the synthesis of six vitamin B12 conjugates, and the effect of conjugation on their solubilities and stabilities in various media. We reveal here that vitamin B12 can be released readily if a 2'-hydroxyl group is conjugated rather than the 5'-hydroxyl group, and the solubility (thus the equivalents of vitamin B12) could be enhanced as much as 19-fold, by simple conjugates such as glycolates. Findings disclosed here provide insights into the reactivities of vitamin B12 conjugates, the design of future prodrugs and similar conjugated moieties using vitamin B12.