Biomedical subjects

M J Kurth

Publications and source records attributed to M J Kurth.

25 records · Page 2Linked to original sources

PubMed1988

Production of antibodies and development of an immunoassay for the anticoagulant, diphacinone.

Diphacinone, a commonly used anticoagulant rodenticide, was coupled to protein via an (O-carboxymethyl) oxime bridge. Immunization in rabbits produced antibodies with good ability to recognize the hapten as demonstrated by indirect EIA and affinity column adsorption. A competitive EIA was developed which clearly measured 10 micrograms/L diphacinone concentrations showing the sensitivity of the assay. Cross-reactivity study with chlorophacinone showed that the antisera possessed a high degree of diphacinone specificity.

Animals↗

PubMed1988

Polypeptide fluoroacetate derivatives. Synthesis and 19F n.m.r.

The synthesis and characterization of monofluoroacetyl (MFAc) functionalized haptens are described. These were covalently bound to polypeptide carriers (bovine serum albumin and poly-D-lysine) by primary amine/succinimide ester or primary amine/acid chloride coupling. Epitopic densities of the resulting antigens were determined by both 19F n.m.r. and picryl sulfonic acid assays. 19F n.m.r. experiments defining the stability of MFAc ester and amide linkages as a function of media (including in vitro), time, and temperature are presented. These results indicate that MFAc functionalized antigens are well suited for further immunologic studies.

Fluorine↗

PubMedDate not supplied

Cationic lipid polymerization as a novel approach for constructing new DNA delivery agents.

In vivo gene delivery mediated by cationic lipids is often compromised by aggregation due to complexation with proteins in the blood. To improve the stability of cationic lipid-DNA complexes, the present study aimed to develop a novel approach in which a poly(cationic lipid) (PCL) is utilized to form stable cationic polyplexes for gene transfection. Hydrogenation of the acrylamide analogue of betaAE-DMRI, the polymerizable precursor of PCL, provided a monomeric lipid derivative (MHL) which was used for direct comparison of corresponding lipoplex stability, toxicity, and transfection activity. Various formulations of cationic liposomes, such as MHL, MHL-cholesterol (Chol), PCL, PCL-Chol, DOTAP-Chol, and commercially available lipofectamine were generated and examined in this study. The new poly(cationic lipid) did not display any significant toxicity to rat hepatocytes or Hep G2 cells as indicated by an LDH leakage assay. Furthermore, PCL was significantly less toxic than MHL, DOTAP-Chol or lipofectamine. Suspensions of PCL were resistant to aggregation even after 24 h of exposure to solutions containing 50 and 100% fetal bovine serum (FBS). In contrast, suspensions of lipofectamine extensively aggregated after 24 h of exposure to 50% FBS. To examine the influence of lipid polymerization on gene transfer activity, liposome-mediated transfections of a luciferase vector (pGL3) were performed in Hep G2 and Alexander cell lines. The luciferase activity of the PCL formulations in Hep G2 cells were similar to those of the MHL, DOTAP-Chol and lipofectamine formulations, demonstrating that lipid polymerization does not compromise transfection activity. In comparison to the monomeric precursor MHL and to the industry transfection standards DOTAP and lipofectamine, the novel poly(cationic lipid) exhibited the lowest cytotoxicity, was the most resistant to serum-induced aggregation and had comparable transfection activity when coformulated with cholesterol. This novel polymerization approach for the development of stable and active polyplexes may prove a valuable alternative for in vivo gene delivery.

Animals↗

PubMedDate not supplied

Enzyme-linked immunosorbent assay detection of pyrrolizidine alkaloids: immunogens based on quaternary pyrrolizidinium salts.

Polyclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of retrorsine (1, 351 g/mol), monocrotaline (2, 325 g/mol), and retronecine (3, 155 g/mol) in the parts per billion (ppb) range. A set of three bifunctional linking arms (6-8) was synthesized. By N-alkylation of pyrrolizidine alkaloids (PAs) retrorsine, monocrotaline, and retronecine acetonide (9), six haptens (6.1, 6.2, 7.1, 7.2, 7.9, and 8.9) were synthesized and used to generate rabbit antisera. The resulting anti-retrorsine antiserum gave a 50% inhibition (I50) value of 0.9 +/- 0.2 ppb for retrorsine with detection limits of 0.5-10 ppb. The same ELISA system also detected isatidine (4, retrorsine N-oxide) dihydrate (403 g/mol) with an I50 of 1 ppb and senecionine (5,352 g/mol) with an I50 of 100 ppb. A second monocrotaline-based ELISA detected monocrotaline with an I50 of 36 +/- 9 ppb 2 with detection limits of 5-500 ppb and shows no cross-reactivity with 1 or 5; this ELISA demonstrates the potential for the substrate-specific detection method. A third retronecine-based ELISA detects 3 with an I50 of 3000 +/- 600 ppb (3 +/- 0.6 ppm) and detection limits of 600-10,000 ppb. None of these ELISAs cross-react with the structurally similar swainsonine (10) or lupinine (11) alkaloids. PAs were detected in extracts of Senecio vulgaris and Crotalaria retusa, but not in Lupinus spp., as a demonstration of the ELISA's usefulness.

Alkylation↗

PubMedDate not supplied

Preparation of a 990-member chemical compound library of hydantoin- and isoxazoline-containing heterocycles using multipin technology.

The development of a useful chemistry for the construction of polyfunctional heterocycles--first through solution and solid phase (resins) and then library production via SynPhase crowns--is reported. Bead-based synthetic work was done on Merrifield resin where treatment with benzylamine in the presence of DBU followed by reaction with 4-chloromethylbenzoyl chloride afforded amide-linked resin 9. Finally, TFA.NH2-polystyrene macro crowns were derivatized with 4-(hydroxymethyl)benzoic acid to afford pin 14 which was coupled with Boc-protected amino acid 2 in the presence of DIC to deliver pin 15. Deprotection and reaction with phenyl isocyanate afforded urea functionalized pin 17 which underwent 1,3-dipolar cycloaddition reaction to give pin 19. Finally, compound 20 was obtained with moderate diastereoselectivity (20:21::8:1) by the reaction of pin 19 with a catalytic amount of Et3N.

Alkylation↗

PubMedDate not supplied

Application of ELISA techniques to metabolic disposition studies for 1,3-dinitrobenzene: comparison with HPLC and radiochemical methods.

An enzyme-linked immunosorbent assay (ELISA) is reported for the detection and quantitation of 1,3-dinitrobenzene, a widely used chemical intermediate in industrial syntheses and potent testicular toxicant in rats. Radiolabeled [14C]-1,3-dinitrobenzene (25 mg/kg, ip) was administered to rats, and the blood disappearance curve generated with the ELISA assay was compared with HPLC separation and quantitation by using both UV and radiochemical methods. An excellent correlation was found between the two methods. Matrix effects from the blood samples were minimal. Cross reactivity with metabolites of 1,3-dinitrobenzene occurred only at levels that were 3 orders of magnitude greater than for the parent compound. The specificity of the ELISA for 1,3-dinitrobenzene was further demonstrated with a variety of other nitroaromatic compounds, of which only 2,4-dinitrotoluene showed significant cross reactivity. The sensitivity and specificity of the 1,3-dinitrobenzene ELISA demonstrate the usefulness of an approach which, if verified with conventional analytical methods, allows rapid and inexpensive measurement of xenobiotic levels in biological samples.

Animals↗

PubMedDate not supplied

Enzyme-linked immunosorbent assay for the specific detection of the mercapturic acid metabolites of naphthalene.

The measurement of metabolites constitutes a useful tool for detection of exposure and in pharmacokinetic studies. Epoxidation with subsequent glutathione conjugation and mercapturic acid formation is an important deactivation pathway for naphthalene, a toxin which presumably causes lung disease. The mercapturic acid conjugates of naphthalene [NaphMA (1), N-acetyl-S-(1,2-dihydro-1-hydroxy-2-naphthyl)cysteine (1a), and N-acetyl-S-(1,2-dihydro-2-hydroxy-1-naphthyl)cysteine (1b)], its most important urinary metabolites, and other structurally related derivatives, such as N-acetyl-S-(1,2,3,4-tetrahydro-2-hydroxy-1-naphthyl) cysteine (2), N-acetyl-S-(3-hydroxy-1,2,3,4-tetrahydro-2-naphthyl)cysteine (3), and N-acetyl-S-(2-hydroxy-1-phenylethyl)cysteine (4a) and N-acetyl-S-(2-hydroxy-2-phenylethyl)cysteine (4b) as an isomeric mixture, were synthesized to develop an ELISA (enzyme-linked immunosorbent assay) for the specific detection of NaphMA (1). Compound 1, as an isomeric mixture, was used to raise antibodies by immunizing six rabbits with the corresponding KLH (keyhole limpet hemocyanin) and BSA (bovine serum albumin) derivatives (1KLH and 1BSA). The remaining compounds were covalently attached to BSA, conalbumin, and ovalbumin to be used as coating antigens. The best assay was obtained in a homologous system combining serum Ab2357 (1KLH) and 1BSA as coating antigen. The immunoassay has an I50 of 4-6 ng/mL and a detection limit of 1-2 ng/mL. Because of the known instability of the mercapturic acid conjugate of naphthalene 1, leading to the fully aromatic compound 20, a system involving HPLC is described to check the stability of the NaphMA stock solutions used in the assay. Cross-reactivity studies show high specificity toward the NaphMA. Other related compounds as well as the dehydrated derivative 20 are not recognized by the antibody in this ELISA system.

Acetylcysteine↗