Formation of a bioconjugate composed of hemin, smectite, and quaternary ammonium chloride that is soluble and active in hydrophobic media

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) intercalated with a quaternary alkenylammonium compound, dioleyldimethylammonium chloride (DOA), to form a hemin-smectite-DOA conjugate. The hemin-smectite-DOA conjugate was soluble in organic solvents such as benzene and toluene to form a transparent colloidal solution with a light yellow color. Its absorption spectrum in benzene showed two bands, 600 and 568 nm, in the visible region and a sharp Soret band at 400 nm with the molar extinction coefficient of 7.5 x 10(4) M(-1) cm(-1). The formation of the conjugate of smectite and DOA was confirmed by X-ray diffraction analysis: the basal spacing, d(001), of hemin-smectite-DOA conjugate was 19 A which is an expansion of the interlayer space by 5 A based upon the basal spacing of smectite of 14 A. Hemin-smectite-DOA conjugate catalyzed the peroxidase-like reaction in organic solvents using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The temperature-dependent peroxidase-like activity of the conjugate was compared with peroxidase activity of horseradish peroxidase. The hemin-smectite-DOA conjugate exhibited higher activity as the temperature was increased from 30 to 70 degrees C, while horseradish peroxidase activity was reduced as the temperature was increased.     

Improved functional properties of the ovoinhibitor by conjugating with galactomannan

The chicken egg white ovoinhibitor, a multi-type proteinase inhibitor, was conjugated with galactomannan through the Maillard reaction in a controlled dry heating state at 60 degrees C and 65% relative humidity. The formation of an ovoinhibitor-galactomannan conjugate during dry heating was confirmed by SDS-PAGE. The resulting ovoinhibitor-galactomannan conjugate showed almost the same inhibitory activity toward trypsin, chymotrypsin and elastase as that of the untreated ovoinhibitor, while the conjugate showed stronger heat stability and better emulsifying properties than the untreated ovoinhibitor. These results suggest that the ovoinhibitor-galactomannan conjugate can be used as a protease inhibitor having heat stability and outstanding emulsifying properties for industrial application.     

The antioxidant and antiaggregant effects of the covalent bienzyme superoxide dismutase — Chondroitin sulfate — Catalase conjugate on platelets

A covalent bienzyme superoxide dismutase-chondroitin sulfate-catalase conjugate (SOD-CHS-CAT) demonstrated the dose-dependent inhibitory action on induced platelet aggregation both in the presence and the absence of hydrogen peroxide. The antioxidant activity of SOD-CHS-CAT was noted at much lower doses than that of other CAT derivatives. The conjugate SOD-CHS-CAT inhibited platelet aggregation regardless of the nature of inducers; it also prevented platelet spreading on the glass surface. The latter suggests conjugate influence on adhesive properties of platelets. Novel properties of the SOD-CHS-CAT conjugate underlie prospects of its biopharmaceutical applicability as a tool for antioxidant therapy.     

Design and synthesis of novel antibacterial peptide-resin conjugates

We synthesized a novel peptide-resin conjugate by immobilizing beta-sheet antibacterial peptide on PEG-PS resin. The peptide-resin conjugate, similar to cationic antimicrobial peptides, demonstrated unique properties such as potent antibacterial activity, no hemolytic activity, lipid membrane perturbation activity, and potent synergism with vancomycin. Specially, the peptide-resin conjugate showed a more increased lipid membrane perturbation activity in comparison to unbound beta-sheet antibacterial peptide.     

Antioxidative and Prooxidative Activity of Caffeic Acid toward H2O2-induced DNA Strand Breakage Dependent on the State of the Fe Ion in the Medium

Hen egg lysozyme–carboxymethyl dextran (HEL–CMD) conjugate was prepared by using water-soluble carbodiimide as a model protein-acidic polysaccharide conjugate for improving the protein function. An acid-amide bond between HEL and CMD was confirmed by SDS-PAGE, isoelectric focusing and IR spectra. The molar ratio of CMD to HEL in the conjugate was 1:1. The isoelectric point of the conjugate was 5.5–6.0, which is much lower than that of HEL. Spectroscopic studies suggested that the conformation around the Trp residue had not changed but the α-helix content had decreased to about 1/3 that for native HEL. The conjugate maintained about 60% of the enzymatic activity of native HEL at 40–60°C, while it was about 1.4 times as active as native HEL at 4°C and 80°C. The conjugate was more stable to proteolysis than native HEL. The denaturation temperature of the conjugate was about 73°C, which is almost the same as that of native HEL. However, the enthalpy for denaturation of the conjugate was about 1/3 that of native HEL, which corresponds to the decrease in α-helix content.     

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen.

The biochemical and biologic properties of a purified disulfide conjugate of diphtheria toxin fragment A and human placental lactogen (toxin A-hPL) have been studied by (a) assaying the ADP-ribosyltransferase activity of the intact conjugate, (b) assaying the binding of the intact conjugate to mammary gland plasma membrane lactogenic receptors, and (c) assaying the effect of the conjugate on the rate of protein synthesis in rabbit mammary gland explants maintained in organ culture. The toxin A-hPL conjugate retains one-third of the NAD+:EF-2 ADP-ribosyltransferase activity of toxin A, and 26% of the hPL-binding activity to lactogenic receptors. Binding activity was demonstrated by radioreceptor assay and by assaying toxin A activity bound to membranes which was competitively displaced by excess hPL. Since the toxin A-hPL conjugate retained activities of its separate subunits, it could be regarded as a structural analogue of nicked diphtheria toxin with replacement of the original membrane-binding chain by another binding chain that is specific for lactogenic receptor. However, the conjugate failed to inhibit protein synthesis in organ-cultured mammary gland explants, although these were sensitive to native diphtheria toxin and could bind hPL. It is concluded from these results that the toxin A-hPL conjugate does not act as a functional analogue of diphtheria toxin with altered receptor specificity, and that the hPL receptor cannot mediate the entry of toxin A or toxin A-hPL from membrane-bound conjugate into the cytosol site of action of toxin A.     

The biological and structural similarity between lunularic acid and abscisic acid

Lunularic acid (LA) inhibited not only the germination and the growth of cress and lettuce at 1 mM but also the gibberellic acid (GA3)-induced alpha-amylase induction in embryoless barley seeds at 120 microM, which was recognized as a specific activity of abscisic acid (ABA). Moreover LA and ABA equally inhibited the growth of Lunularia cruciata A18 strain callus at 40 and 120 microM. A computational analysis revealed that the stable conformers of LA could be superimposed on the stable ABA conformers. In addition, the antibody raised against the conjugate of C1-ABA-bovine serum albumin (ABA-BSA) reacted with LA-horse-radish peroxidase (LA-HRP) conjugate as well as ABA-HRP conjugate, apparently. These results can explain why LA has ABA-like activity in higher plants. Moreover the results suggest that LA and ABA bind to the same receptor in higher plants.     

In vivo andin vitro antitumor activity of mitomycin C conjugates at 7-N position through a linker containing thiocarbamate bond with CD10 monoclonal antibody

Through a linker containing thiocarbomate bound to the 7-N position of mitomycin C (MMC), conjugates with a monoclonal antibody to CD10 (NL-1) were prepared, and their antitumor activities were examined. All five conjugates, except one, showedin vitro cytotoxity to two CD10⁺ lymphoid cell lines superior to MMC. The conjugate displaying the highest cytotoxicity was selected and further tested against three CD10⁺ and two CD10 lymphoid cell linesin vitro. The conjugate with NL-1 antibody demonstrated higher cytotoxic activity against CD10+ tumor cells than the control conjugate with normal immunoglobulin, while there was no significant difference, when tested against CD10^– tumors. The cytotoxic activity of the NL-1 conjugate to CD10+ tumors was significantly blocked by NL-1 antibody. In vivo antitumor activity of the NL-1 conjugate was then tested against a CD10⁺ tumor transplanted to nude mice, and side effects were recorded. The NL-1 conjugate (4 mg/kg) showed anin vivo antitumor effect similar to MMC (2 mg/kg), which is at nearly maximal tolerable dose; the latter induced decreases in numbers of leukocytes and platelets, while the former did not, suggesting less side effect by the NL-1 conjugate. Since MMC demonstrates a broad spectrum of antitumor activity, the conjugate, as such, may be applicable for the treatment of cancer patients.     

Effects of structure of polyamidoamine dendrimer on gene transfer efficiency of the dendrimer conjugate with alpha-cyclodextrin

To improve gene transfer activity of a new nonviral vector, a polyamidoamine dendrimer (G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugates with dendrimer having different generations (G3 and G4), and their gene transfer activities were compared with those of alpha-CDE conjugate (G2) and TransFast, a novel transfection reagent. alpha-CDE conjugates (G2, G3, and G4) formed the complexes with pDNA, changing the zeta-potential and particle size of pDNA complexes and the protection of pDNA from DNase I in a charge ratio-dependent manner, although their differences at higher charge ratios (vector/pDNA) were small. The gene transfer activity of alpha-CDE conjugates (G2, G3, and G4) was higher than that of the corresponding dendrimer alone in NIH3T3 and RAW264.7 cells. Of these CDE conjugates, alpha-CDE conjugate (G3) had a superior gene transfer activity which was comparable to that of TransFast in NIH3T3 cells. The intracellular distribution of pDNA after application of the pDNA complex with alpha-CDE conjugate (G3) to NIH3T3 cells was different from that with dendrimer alone (G3), although the cellular association of pDNA was almost comparable among all vectors. alpha-CDE conjugate (G3) strongly interacted with a fluorescence probe, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS), suggesting that the conjugate possesses the inclusion ability with biomembrane constituents such as phospholipids after transfection. These results suggest that alpha-CDE conjugates, particularly the G3 conjugate, could be novel nonviral gene transfer agents.     

An antisense oligodeoxynucleotide-doxorubicin conjugate: preparation and its reversal multidrug resistance of human carcinoma cell line in vitro

An antisense oligodeoxynucleotide-doxorubicin conjugate was synthesized by an aminocaproic acid linker. The synthetic conjugate was identified by HPLC analysis and UV-vis spectra. Properties of the conjugate in vitro conditions were investigated. The results demonstrated that the conjugate was remarkably stabilized by doxorubicin. When incubated in Dulbecco Phosphate-Buflered Saline (pH 7.4) at 37 degrees C, the conjugate was more stable than doxorubicin or the mixture of doxorubicin and antisense oligodeoxynucleotide. When incubated in 10% fetal serum at 37 degrees C, the conjugate showed a remarkable stabilization as compared to the unmodified oligodeoxynucleotide. Melting experiments demonstrated that the covalent attachment of doxorubicin strongly stabilized the binding of the oligodeoxynucleotide to its complementary sequence. In addition, in vitro reversion of multidrug resistance by the conjugate was assayed in a human carcinoma cell line (KB-A-1) resisting to doxorubicin. The result showed that the conjugate displayed very high reversal multdrug resistance activity in KB-A-1 cells in vitro. The conjugate lowered the IC50 value from 21.5 microM to 2.2 microM with a fold-reversal factor of 10. In contrast, a slight decrease of the IC50 value was observed when they combined with the "free" antisense oligodeoxynucleotide: the IC50 value was down from 21.5 microM to 16.8 microM. This study suggested that antisense oligodeoxynucleotide-doxorubicin conjugate might be helpful in multidrug resistance reversal.     

Synthesis, characterization, and thermodynamic study of a polyvalent lytic peptide-polymer conjugate as novel anticancer agent

We designed and synthesized a new polyvalent lytic peptide-polymer conjugate as a novel chemotherapeutic agent capable of overcoming multidrug resistance. A hexapeptide (KWKWKW or (KW)?) was designed and conjugated to dextran in multiple copies to afford a polyvalent conjugate. A robust synthesis procedure involving click chemistry and the detailed characterization of the conjugate were reported here. The conjugate Dex-(KW)? exhibited significantly enhanced anticancer potency in vitro by up to 500-fold compared to monomeric (KW)?. The LC?? value was comparable to that of conventional lytic peptides which have more than 20 residues. No hemolytic activity was shown by the conjugates up to 300 μM. Thermodynamic study indicated that the binding of conjugates was predominantly entropy-driven while the binding of free peptides was mainly enthalpy-driven, implying a deeper penetration of conjugate into the core of lipid bilayer. The binding affinity of conjugate to neutral membrane is much higher than that to free peptide (K(conj) ≈ 8822.9 M?1, K(pep) ≈ 1884.7 M?1). In binding to negatively charged membrane, the conjugate surpassed free peptides at high concentrations when the binding of free peptides became saturated. The higher binding capability, attributed to the high local concentration of peptides mounted on a polymer backbone, explains the superior anticancer activity of polyvalent Dex-(KW)?.     

Dendrimer-epidermal growth factor conjugate displays superagonist activity

Binding of ligands on to epidermal growth factor receptor (EGFR) can stimulate cell growth; therefore, any application employing EGF as a targeting ligand for a "drug carrier" must evaluate the effect of the conjugate on cell growth. We report the synthesis and in vitro biological activity of EGF molecules coupled to a fluorescein-labeled polyamidoamine dendrimer. The conjugate bound and internalized into several EGFR-expressing cell lines in a receptor-specific fashion. The conjugate effectively induced EGFR phosphorylation and acted as a superagonist by stimulating cell growth to a greater degree than free EGF. Concomitant administration of the chemotherapeutic drug methotrexate completely inhibited cell growth to a degree similar to its effect in the absence of the conjugate. Thus, dendrimer-EGF conjugates serve as EGFR superagonists, but this activity can be overcome by chemotherapeutic drugs. The agonist activity of these materials must be taken into consideration when using EGF conjugates for imaging applications.     

Assay of cysteine conjugate β-lyase activity with S-(2-benzothiazolyl)cysteine as the substrate

Cysteine conjugate β-lyases convert S-substituted cysteine conjugates to pyruvate, ammonia, and thiols. A simple assay for cysteine conjugate β-lyase activity was developed with S-(2-benzothiazolyl)cysteine as the substrate. The production of 2-mercaptobenzothiazole was measured by its intense absorbance at 321 nm in trichloroacetic acid-quenched reaction mixtures. The formation of 2-mercaptobenzothiazole was directly proportional to protein concentrations of 0.17 to 1.2 mg/ml with rat liver cytosol as the source of β-lyase activity. Production of 2-mercaptobenzothiazole was stoichiometric with pyruvate and was increased by addition of pyridoxal phosphate only at reaction times of 5 min or longer. The simplicity, sensitivity, and specificity of this procedure offer significant advantages over other methods for the assay of cysteine conjugate β-lyase activity.     

Isolation and characterization of a rat liver enzyme with both cysteine conjugate beta-lyase and kynureninase activity

Cysteine conjugate beta-lyase is a name applied to enzymes which cleave the S-cysteine conjugates of some xenobiotics to pyruvate, ammonia, and a thiol. Recently, several laboratories have characterized these enzymes from kidney, liver, and bacterial sources in an effort to understand their role in the genesis of novel sulfur-containing metabolites of xenobiotics and in the toxicity of some S-cysteine conjugates. Kynureninase is an enzyme which plays a key role in the biosynthesis of nicotinamide ribonucleotides. This investigation demonstrates that rat hepatic cysteine conjugate beta-lyase is the same enzyme as kynureninase. Both activities copurify on ion exchange, hydroxylapatite, and molecular exclusion chromatography. The subunit composition of enzyme prepared by two different methods is identical, Mr = 55,000. The Km values for 3-OH-kynurenine and kynurenine are 13 and 400 microM, respectively. Kynurenine and 3-hydroxykynurenine inhibit cysteine conjugate beta-lyase activity. Inactivation of the enzyme by substrates which undergo beta-elimination results in loss of kynureninase activity, but kynurenine does not inactivate the enzyme. Both enzyme activities react with anti-cysteine conjugate beta-lyase antibody. Product inhibitors of kynureninase, anthranilate, and 3-hydroxyanthranilate are also inhibitors of cysteine conjugate beta-lyase. Heat inactivation at 70 degrees C produced coincident loss of both activities. The enzyme has an absorption maximum at 432 nm suggesting a bound pyridoxal phosphate. These data show that at least one cysteine conjugate beta-lyase is a pyridoxal phosphate enzyme with a biological function other than xenobiotic metabolism. The enzyme can catalyze two distinct types of reactions, i.e. beta-elimination and the kynureninase reaction.     

Growth hormone-releasing peptide-biotin conjugate stimulates myocytes differentiation through insulin-like growth factor-1 and collagen type I

Based on the potential beneficial effects of growth hormone releasing peptide (GHRP)-6 on muscle functions, a newly synthesized GHRP-6-biotin conjugate was tested on cultured myoblast cells. Increased expression of myogenic marker proteins was observed in GHRP-6-biotin conjugate-treated cells. Additionally, increased expression levels of insulin-like growth factor-1 and collagen type I were observed. Furthermore, GHRP-6-biotin conjugate-treated cells showed increased metabolic activity, as indicated by increased concentrations of energy metabolites, such as ATP and lactate, and increased enzymatic activity of lactate dehydrogenase and creatine kinase. Finally, binding protein analysis suggested few candidate proteins, including desmin, actin, and zinc finger protein 691 as potential targets for GHRP6-biotin conjugate action. These results suggest that the newly synthesized GHRP-6-biotin conjugate has myogenic stimulating activity through, at least in part, by stimulating collagen type I synthesis and several key proteins. Practical applications of the GHRP-6-biotin conjugate could include improving muscle condition. [BMB Reports 2015; 48(9): 501-506]     

Plasminogen activator-anti-human fibrinogen conjugate

A covalent conjugate between the plasminogen activator urokinase and polyclonal rabbit anti-human fibrinogen has been formed using the heterobifunctional coupling reagent N-succinimidyl 3-(2-pyridyldithio) propionate. The resultant urokinase-anti-human fibrinogen conjugate was separated from unreacted material by gel filtration. The conjugate exhibited amidase activity against the small chromogenic substrate pyroglutamyl-glycyl-arginine-p-nitroanilide as well as plasminogen activator activity in an assay employing plasminogen and the plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide. Retention of antibody specificity for fibrinogen was demonstrated using an enzyme linked immunoassay procedure. The conjugate was found to have greater stability in human plasma than unconjugated urokinase.     

Transferrin-adriamycin conjugates which inhibit tumor cell proliferation without interaction with DNA inhibit plasma membrane oxidoreductase and proton release in K562 cells.

Conjugates of adriamycin crosslinked to transferrin with glutaraldehyde inhibit proliferation of transformed cells. Conjugates of this type inhibit oxidoreductase activity in the plasma membrane of K562 cells, and the inhibition of electron transport is found at concentrations ten times lower than concentrations of free adriamycin which inhibit electron transport and cell growth. The transferrin-adriamycin conjugate inhibits ferricyanide reduction, diferric transferrin reduction and plasma membrane NADH oxidase activity stimulated by transferrin. Activation of proton release from the K562 cells by diferric transferrin also is inhibited by the conjugate, and conjugate kills cells more effectively than free adriamycin. Since the conjugate does not transfer adriamycin to the nucleus, the growth control may be based on inhibition of the transferrin regulated redox system and Na+/H+ antiport activity at the plasma membrane.     

Regulation of enzyme-substrate complexation by a substrate conjugated with a phospholipid polymer

To recognize and control ligand-receptor interactions at the interface between cells and polymer materials, we investigated a model system with an enzyme and a substrate conjugated with a biocompatible phospholipid polymer in an aqueous medium. We explored the regulation of enzyme-substrate (ES) complexation using horseradish peroxidase (HRP) as the enzyme and 4-aminoantipyrine (AAP) and 3-(p-hydroxyphenyl) propionic acid (HPPA) as substrates. The phospholipid polymer (PMBN), composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl poly(oxyethylene)methacrylate, was prepared and conjugated with AAP (PMBN-AAP conjugate). The formation and dissociation of the ES complex were investigated using capillary electrophoresis and fluorescence spectroscopy. In the chart of the capillary electrophoresis, a much longer retention time of HRP was observed in the PMBN-AAP conjugate-coated capillary compared with that in a nontreated capillary. The retention time was significantly longer in comparison with the case of a mixed solution of HRP and AAP. This result clearly shows that HRP forms an ES complex with the immobilized PMBN-AAP conjugate and that the addition of AAP to the medium inhibits the interactions between HRP and the PMBN-AAP conjugate. Though HRP forms an ES complex with both AAP and the PMBN-AAP conjugate, the ES complex with the PMBN-AAP conjugate was easily dissociated by addition of HPPA as an alternative substrate because HRP started to react with the HPPA immediately. However, the HRP that formed an ES complex with AAP fell behind in reacting with the HPPA. The activity of HRP was maintained at the initial level in the presence of the PMBN-AAP conjugate at 25 degrees C for 1 week. Additionally, even under H(2)O(2) conditions, HRP stored with the PMBN-AAP conjugate maintained 40% of the initial activity whereas HRP was deactivated within 6 h. This result indicates that the PMBN-AAP conjugate could block the active sites by formation of an ES complex. This is due to the formation of the ES complex, which retained the structure of HRP by blocking the active sites. On the basis of these results, we considered that the reversible attachment and detachment by PMBN conjugated with specific ligands from cellular receptors will be realized.     

Development of target delivery system based on actinomycin class drugs and recombinant alpha-fetoprotein

A recombinant alpha-fetoprotein (rAFP) was obtained in the yeast P. pastoris system, and its functional activity was confirmed. A method for producing polymer particles loaded with dactinomycin was developed, and a conjugate of these nanoparticles with rAFP was synthesized. The efficiency of the obtained conjugate on the HeLa, SKOV3, and MG-63 tumor cells and the absence of toxicity on the normal cells was shown. Experiments in vivo demonstrated a significant increase in the antitumor efficacy of the conjugate at a lower general toxicity as compared to the commercially available dactinomycin.     

Design of novel lipidated peptidomimetic conjugates for targeting EGFR heterodimerization in HER2 + cancer

The human epidermal growth factor receptor (EGFR) family is known to be involved in cell signaling pathways. The extracellular domain of EGFR consists of four domains, of which domain II and domain IV are known to be involved in the dimerization process. Overexpression of these receptors is known to play a significant role in heterodimerization of these receptors leading to the development of cancer. We have designed peptidomimetic molecules to inhibit the EGFR heterodimerization interaction that have shown antiproliferative activity and specificity for HER2-positive cancer cell lines. Among these, a peptidomimetic, compound 5, exhibited antiproliferative activity at low nanomolar concentrations in HER2-overexpressing cancer cell lines. To improve the stability of this peptidomimetic, we have designed and synthesized a novel conjugate of peptidomimetic compound 5 with a lipid, stearic acid. The antiproliferative activity of this conjugate was evaluated in HER2-positive cancer cell lines. Results suggested that the conjugate exhibited selective antiproliferative activity in HER2-overexpressing breast and lung cancer cell lines and was able to block HER2:HER3 heterodimerization. Also, the conjugate showed improved stability with a half-life of 5?h in human serum compared to the half-life of 2?h for parent compound 5. The binding affinity of the conjugate to HER2 protein was evaluated by SPR analysis, and the mode of binding of the lipid conjugate to domain IV of HER2 protein was demonstrated by docking analysis. Thus, this novel lipid conjugate can be used to target HER2-overexpressing cancers.