Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.     

Interposition of different chemical linkages between antibody and 111In-DTPA to accelerate clearance from non-target organs and blood

Two chemically labile linkages, disulfide and diester, and two stable linkages, thioether and hydrocarbon, were introduced between antibody and ¹¹¹In-DTPA in order to modify their biodistributions. The biodistributions of the new linkages were evaluated in rats with target antigens localized in lungs. For a comparison purpose, the antibody-DTPA conjugate with a peptide linkage was used as a control conjugate. The antibody conjugates with the stable linkages produced the biodistributions similar to that of the peptide linked conjugate during a 48 h period. The disulfide and diester conjugates, however, cleared from blood much faster and are retained in normal organs much lower than the peptide conjugate. The disulfide and the diester conjugate amplified the lung (target) to blood ratio by 15 and 6 times, respectively at 48 h, as compared to the corresponding target to blood ratio of the control conjugate. Compared to the control conjugate, a 3 times higher target to liver ratio was also obtained by the disulfide conjugate and a 4 times higher target to kidney ratio was obtained by the diester conjugate at 48 h.     

Preparation of an insulin conjugate with beta-galactosidase from E. coli for immunoenzyme assay

A modified procedure has been worked out for preparing a conjugate of porcine insulin with E. coli beta-galactosidase employing a heterobifunctional reagent, N-hydroxysuccinimidyl m-maleimidobenzoate. Optimal conditions for insulin acylation and subsequent coupling with beta-galactosidase were selected that afforded the conjugate in a high yield. The ability of the modified antigen to react with antibody was evaluated in the reaction of conjugate binding with immobilized monoclonal antibody to insulin. The conjugate almost completely retained the enzymatic activity and reacted with high specificity with the antibody to insulin. The conjugate can be used in competitive ELISA of insulin.     

Sequence-specific affinity precipitation of oligonucleotide using poly(N-isopropylacrylamide)-oligonucleotide conjugate

In this study we develop a sequence-specific precipitation separation system of oligonucleotide (ODN) using a conjugate between poly(N-isopropylacrylamide) (PNIPAM) and ODN. PNIPAM is known as a thermoresponsive polymer and dehydrates to precipitate above its phase transition temperature in an aqueous milieu. The principal advantage of this separation system using the conjugate is that the hybridization reaction between the conjugate and oligonucleotide is conducted in homogeneous solution. The conjugate was prepared by copolymerization between N-isopropylacrylamide and a vinyl-derivatized (dT)(8). The obtained conjugate efficiently precipitated (dA)(8) from solution when the solution contained more than 1.5 M NaCl. The conjugate containing 3 nmol of (dT)(8) residue was able to precipitate 1.4 nmol of (dA)(8), suggesting that the (dT)(8) residue of the conjugate formed a triple helix with (dA)(8). From an equimolar mixture of (dA)(8) and its one point mutant, the conjugate selectively precipitated (dA)(8): the highest selectivity was obtained for the isolation of (dA)(8) from the mixture consisting of (dA)(4)dT(dA)(3) and (dA)(8). When the conjugate was applied for the precipitation of five oligo(dA)s having different chain lengths, the longer oligo(dA)s tended to be precipitated by the conjugate more efficiently than the shorter ones. The conjugate could be used repeatedly for precipitation of (dA)(8) without showing any loss in precipitation efficiency.     

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.     

Antitumor activity of alpha-fetoprotein conjugate with doxorubicin in vitro and in vivo

alpha-Fetoprotein (AFP) was conjugated with doxorubicin (DR) using glutaraldehyde as a cross-linking agent. The protein/DR molar ratio in the conjugate is 1 : 2. Cytotoxic activities (CTA) of the AFP-DR conjugate and of the free DR were compared using human mammary gland carcinoma cells, both DR-sensitive (MCF-7Wt) and DR-resistant (MCF-7AdrR). The CTA of the AFP-DR conjugate was fivefold higher than the CTA of the free DR for sensitive cells of the MCF-7Wt line and sevenfold higher for resistant cells of the MCF-7AdrR line. The CTA of the AFP-DR conjugate was also studied in vitro using the proliferating endothelium taken for a model of endothelial cell lining of blood vessels that supply the tumor. The AFP-DR conjugate was shown to have a high CTA for the endothelial cells (IC50 = 2.5 nM); thus, the conjugate is suggested to manifest an anti-angiogenic effect in vivo. The antitumor activity of the AFP-DR conjugate was studied using mice with inoculated melanoma B16 tumors. The treatment of animals significantly inhibited the tumor growth (>97%) and increased by 60% the mean life span of the animals compared to the control. The high antitumor efficiency of the AFP-DR conjugate and the possibility to significantly decrease the tumor cell resistance to DR make this conjugate a promising chemotherapeutic agent.     

The sequential action of a dipeptidase and a beta-lyase is required for the release of the human body odorant 3-methyl-3-sulfanylhexan-1-ol from a secreted Cys-Gly-(S) conjugate by Corynebacteria

Human axillary odor is formed by the action of Corynebacteria on odorless axilla secretions. Sulfanylalkanols, 3-methyl-3-sulfanylhexan-1-ol in particular, form one key class of the odoriferous compounds. A conjugate with the dipeptide Cys-Gly has been reported as the secreted precursor for 3-methyl-3-sulfanylhexan-1-ol. Here, we confirm the Cys-Gly-(S) conjugate as the major precursor of this odorant, with lower levels of the Cys-(S) conjugate being present in axilla secretions. The enzymatic release of 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate by the axilla isolate Corynebacterium Ax20 was thus investigated. Cellular extracts of Ax20 released 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate and from the Cys-(S) conjugate, whereas the previously isolated C-S lyase of this bacterial strain was only able to cleave the Cys-(S) conjugate. o-Phenanthroline blocked the release from the Cys-Gly-(S) conjugate but did not affect cleavage of the Cys-(S) conjugate, indicating that in a first step, a metal-dependent dipeptidase hydrolyzes the Cys-Gly bond. This enzyme was purified by four chromatographic steps and gel electrophoresis, and the partial amino acid sequence was determined. The corresponding gene was cloned and expressed in Escherichia coli. It codes for a novel dipeptidase with a high affinity toward the Cys-Gly-(S) conjugate of 3-methyl-3-sulfanylhexan-1-ol. Co-incubating either the synthetic Cys-Gly-(S) conjugate or fresh axilla secretions with both the C-S lyase and the novel dipeptidase did release 3-methyl-3-sulfanylhexan-1-ol, proving that the sequential action of these two enzymes from the skin bacterium Corynebacterium Ax20 does release the odorant from the key secreted precursor.     

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 conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate.

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.     

A daidzein-daunomycin conjugate improves the therapeutic response in an animal model of ovarian carcinoma

The use of daunomycin against neoplasms is limited due to its severe cardiotoxicity. The cytotoxicity of daunomycin can be minimized by linking it to an affinity tag. Since ovarian cancer cells are sensitive to isoflavone action, we synthesized a daidzein daunomycin conjugate. In MLS human ovarian cancer cells, the conjugate was shown to have a larger cytotoxic effect than daunomycin per se at a low concentration. The conjugate was then tested in vivo in mice carrying MLS xenografts. Tumour growth in the groups of conjugate and daunomycin was inhibited by >50% as compared to vehicle treated mice. In contrast to daunomycin treated mice, no weight reduction or death was seen in mice treated with the conjugate. In vivo imaging of the fluorescence signal generated by daunomycin indicated uptake of both conjugate and daunomycin by the tumour. Tumour fluorescence was, however, higher in the conjugate treated mice than in the daunomycin treated mice, thus suggesting specific delivery of the drug to the tumour. Histological examination of myocardial tissue indicated that only the daunomycin, but not conjugate treated mice showed cardiac damage. These results indicate that targeting of daunomycin via carboxymethyldaidzein retains daunomycin's cytotoxic effects while averting its toxicity in an ovarian xenograft.     

Lipid conjugates of antiretroviral agents. I. Azidothymidine-monophosphate-diglyceride: anti-HIV activity, physical properties, and interaction with plasma proteins

3'-Azido-3'-deoxythymidine-5'-phosphate diglyceride (16:0/18:1 omega 9), a phosphatic acid conjugate of AZT, is active against HIV replication in H9 cells and syncytia formation in MOLT-3 cells. The activities rank as AZT greater than pure conjugate greater than conjugate in mixed liposomes, with the pure conjugate having about one-third the activity of free AZT. The compound binds very rapidly to serum lipoproteins, but not to serum albumin, alpha and beta globulins, or red cells. Pancreatic phospholipase A2 hydrolyzes it to the lysophosphatidic acid conjugate.     

Synthesis of an amphiphilic poly(gamma-glutamic acid)-cholesterol conjugate and its application as an artificial chaperone

A poly(gamma-glutamic acid) (gammaPGA)-cholesterol conjugate was synthesized and the properties of an aqueous solution were evaluated. The conjugate showed amphiphilic nature derived from the hydrophilic gammaPGA backbone and the hydrophobic cholesterol side chain. The conjugate spontaneously formed nanoparticles in the aqueous solution of the low concentration, and the high concentration resulted in the formation of the physical gel. By utilizing the self-aggregating properties of the conjugate in water, an artificial chaperone was developed. The complex of protein with the nanoparticles of the conjugate was formed and the protein was released upon the dissociation of the nanoparticles by the addition of beta-cyclodextrin. For denatured carbonic anhydrase, the activity was recovered in the artificial chaperone of the nanoparticle conjugate.     

Specific alkylation of human telomere repeats by hairpin pyrrole-imidazole polyamide

A novel hairpin polyamide-cyclopropapyrroloindole (CPI) conjugate PyImImIm-gamma-PyPyPyLDu86 (conjugate 11), which targets human telomere repeats d(TTAGGG)(n)/d(CCCTAA)(n), was synthesized. High resolution denaturing polyacrylamide gel electrophoresis using 44 bp DNA fragments and HPLC product analysis of a synthetic nonanucleotide demonstrated that conjugate 11 alkylates the target adenine in the telomere repeats, 5'-CCCTAA-3'. Examination of the antitumor activity of conjugate 11 using a panel of 39 cancer cell lines demonstrated that the average concentration of conjugate 11 required for 50% growth inhibition was 5.75 microM, which is superior to pepleomycin and bleomycin and comparable to cisplatin.     

A Novel Anti-HIV Dextrin–Zidovudine Conjugate Improving the Pharmacokinetics of Zidovudine in Rats

The aim of this study was to investigate a newly synthesized dextrin–zidovudine (AZT) conjugate designed as a sustained release prodrug of AZT for parenteral administration. AZT was first reacted with succinic anhydride to form a succinoylated AZT which was subsequently coupled with dextrin to yield the dextrin–AZT conjugate. The structure of the conjugate was characterized by FT-IR and ¹H-NMR spectroscopy. The drug content of the conjugate was 18.9 wt.%. The release in vitro of free AZT and succinoylated AZT was investigated in buffer solutions at pH 5.5 and 7.4 and in human plasma. AZT and succinoylated AZT release from the conjugate was 1.4% (pH 5.5), 41.7% (pH 7.4) and 78.4% in human plasma after 24 h. Release was complete in human plasma after 48 h. A pharmacokinetic study in rats following intravenous administration of the conjugate showed prolonged plasma levels of AZT compared to free AZT. The use of the conjugate extended the plasma half-life of AZT from 1.3 to 19.3 h and the mean residence time from 0.4 to 23.6 h. Furthermore, the conjugate provided a significant greater area under the plasma concentration-time curve and reduced the systemic clearance of AZT. This study suggested the potential of this novel dextrin–AZT conjugate as a new intravenous preparation of AZT.     

Optimization of the lowry method of protein precipitation from theH. influenzae type b conjugate vaccine using deoxycholic acid and hydrochloric acid

The Lowry method was used in this study to measure protein inHaemophilus influenzae type b (Hib) conjugate vaccines (polyribosylibitol phosphate-tetanus toxoid; PRP-TT) using deoxycholic acid (DOC) to induce protein precipitation. Trichloroacetic acid (TCA) did not induce precipitation adequately from the Hib conjugate bulk and the freeze-dried Hib conjugate product. Its yield was approximately 50%. The matrix structure of Hib conjugate inhibits precipitation by TCA. Although the Lowry method can be carried out without precipitation in Hib conjugate bulk when no residual impurities (adipic acid dihydrazide [ADH], 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCI [EDAC], phenol and cyanogens bromide [CNBr],etc.) are present, it cannot be used for Hib conjugate products that contain sucrose 8.5%, because 8.5% concentration of sucrose enhanced the protein concentration. DOC- and HCI-induced precipitation is an alternative method for evaluating the protein content of the Hib conjugate bulk and the Hib conjugate product. The precipitation was optimal with a final concentrate of 0.1% for DOC at 4°C and pH 2. This Lowry method, using DOC/HCI precipitation to induce protein precipitation, was confirmed a consistent, reproducible, and valid test for proteins in Hib conjugate bulk and its freeze-dried product.     

Topographical localization of the receptors for luteinizing hormone- releasing hormone on the surface of dissociated pituitary cells

A derivative of the hypothalamic peptide luteinizing hormone-releasing hormone (LHRH) has been coupled to ferritin and the conjugate purified by gel chromatography. In its ability to stimulate the secretion of luteinizing hormone from pituitary cells in vitro, the conjugate has the same potency and specificity as the native peptide. When dissociated pituitary cells maintained in short-term culture are lightly fixed with formaldehyde and then incubated with the conjugate, examination in the electron microscope shows an even distribution of ferritin particles over the free cell surface of the gonadotrophin cells. This binding appears to be specific for the LHRH receptor since it is prevented by a 10-fold excess of native peptide. In addition to the gonadotrophin cells, some somatotrophin and thyrotrophin cells bind conjugate on their free surfaces under similar conditions. If living cells are incubated with the conjugate for 15 min, the bound conjugate becomes aggregated and then concentrated in one localized area of the cell surface. In this area, which lies immediately above the juxtanuclear Golgi complex, the plasma membrane is frequently invaginated in a manner which suggests that the bound, aggregated conjugate is internalized by endocytosis.     

Differences between the endocytosis of horseradish peroxidase and its conjugate with wheat germ agglutinin by cultured fibroblasts

A covalent conjugate of wheat germ agglutinin (WGA) with horseradish peroxidase (HRP) was used for a morphologic study of its adsorptive endocytosis by cultured human fibroblasts. Initial binding at 4°C of the conjugate was observed over the entire plasma membrane, including “coated” and smooth pits. Endocytosis of HRP and the WGA-HRP conjugate was observed in lysosomes, but only the conjugate was seen in a cisterna of the Golgi apparatus (GERL), and in adjacent coated vesicles.     

Production of antisera specific to aldosterone: effect of hapten density and of carrier proteins

In order to investigate the influence of hapten density and of carrier proteins on the immunological characteristics of antisera, 4 groups of rabbits were injected with different aldosterone-carboxymethoxime protein conjugates. Six animals immunized with an aldosterone rabbit serum albumin (RSA) conjugate carrying 15 steroid molecules (RSA-2 conjugate) showed markedly higher antibody titers than rabbits injected with a RSA conjugate carrying 8 aldosterone molecules (RSA-1 conjugate). Low antibody titers were found in 8 animals immunized with an aldosterone bovine gamma globulin (BGG) conjugate showing a molar incorporation of 15. In a group of rabbits which was first injected with the RSA-1 conjugate and re-immunized with the RSA-2 conjugate the magnitude of antibody production was not enhanced. No differences in antibody sensitivity or specificity were observed between the 4 groups. It was concluded from these experiments a) that the density of haptenic groups depending on the molar incorporation of haptens and on the molecular weight of the carrier protein had influenced the magnitude of antibody production, b) that hapten density or carrier proteins had no effect on antibody sensitivity or specificity, c) that the magnitude of antibody production cannot be altered by re-immunizing with a more potent antigen.     

液相色谱-ELISA测定结合疫苗中游离蛋白含量可行性的初步探讨

用适宜分离范围的色谱柱,预先确定细菌多糖蛋白结合疫苗载体蛋白的洗脱峰出峰时间;然后将细菌多糖-蛋白结合疫苗用相同条件进行分离,分部收集各洗脱峰并直接包被酶标板,与适宜浓度的载体蛋白特异抗体反应后,加入酶标二抗测定各洗脱峰样品中载体蛋白的吸光值;把载体蛋白出峰后的吸光值总和计为游离载体蛋白质吸光值;以游离载体蛋白吸光值占所有洗脱峰吸光值的比例计算游离载体蛋白含量。以伤寒Vi结合疫苗(载体蛋白rEPA)和甲型副伤寒结合疫苗(载体蛋白为TT)作为实验材料探讨方法的可行性。结果说明,该方法具有较好的特异性和敏感度。     

Bile acid metabolism in mammals. VIII. Biliary secretion of cholylarginine by the isolated perfused rat liver.

In studies of cholic acid metabolism using the isolated perfused rat liver system, an unknown conjugate of cholic acid was observed. This conjugate comprised 15-27% of the biliary bile acids in these experiments, was less polar than cholylglycine on thin-layer chromatography using butanol, acetic acid, and water, and had an apparent molecular weight greater than that of cholyltaurine on gas-liquid chromatography. Amino acid analysis of the hydrolyzed conjugate demonstrated the presence of arginine. Perfusion studies with radioactive arginine, and mass spectrometric analysis proved that the conjugate was cholylarginine. Secretion of this conjugate does not represent a deficiency of available glycine and taurine.