Tad H. Koch

Photocross-Linking of Nucleic Acids to Associated Proteins

Photocross-linking is a useful technique for the partial definition of the nucleic acid-protein interface of nucleoprotein complexes. It can be accomplished by one or two photon excitations of wild-type nucleoprotein complexes or by one photon excitation of nucleoprotein complexes bearing one or more substitutions with photoreactive chromophores. Chromophores that have been incorporated into nucleic acids for this purpose include aryl azides, 5-azidouracil, 8-azidoadenine, 8-azidoguanine, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-iodocytosine. The various techniques and chromophores are described and compared, with attention to the photochemical mechanism.

Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers

High sensitivity and specificity of two modified ssDNA aptamers capable of photocross-linking recombinant human basic fibroblast growth factor (bFGF((155))) were demonstrated. The aptamers were identified through a novel, covalent, in vitro selection methodology called photochemical systematic evolution of ligands by exponential enrichment (PhotoSELEX). The aptamers exhibited high sensitivity for bFGF((155)) comparable with commercially available ELISA monoclonal antibodies with an absolute sensitivity of at least 0.058 ppt bFGF((155)) under prevailing test conditions. The aptamers exquisitely distinguished bFGF((155)) from consanguine proteins, vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). A commercially viable diagnostic system incorporating PhotoSELEX-evolved aptamers capable of simultaneous quantification of a large number of analyte molecules is also described. Such a system benefits from covalent bonding of aptamer to target protein allowing vigorous washing with denaturants to improve signal to noise.

Cardiovascular implant calcification: a survey and update.

Calcification of cardiovascular prosthetic implants is a common and important problem. This review provides an update based upon the Conference on Cardiovascular Implant Calcification held as part of the 13th World Congress of the International Society for Heart Research, 1989. A variety of cardiovascular prostheses are affected clinically by calcification, including bioprosthetic heart valves, aortic homografts and trileaflet polymeric valve prostheses. In addition, experimental studies have demonstrated calcification of artificial heart devices in ventricular assist systems in long-term calf studies. The pathophysiology of this disease process is incompletely understood. A common element between the various types of cardiovascular implant calcification is the localization of calcific deposits to devitalized cells and membranous debris. Prevention of cardiovascular implant calcification by either biomaterial modifications or regional drug therapy (controlled release) is being investigated.

Sensitivity and Specificity of Photoaptamer Probes

The potential of photoaptamers as proteomic probes was investigated. Photoaptamers are defined as aptamers that bear photocross-linking functionality, in this report, 5-bromo-2′-deoxyuridine. A key question regarding the use of photoaptamer probes is the specificity of the cross-linking reaction. The specificity of three photoaptamers was explored by comparing their reactions with target proteins and non-target proteins. The range of target/non-target specificity varies from 100- to >106-fold with most values >104-fold. The contributions of the initial binding step and the photocross-linking step were evaluated for each reaction. Photocross-linking never degraded specificity and significantly increased aptamer specificity in some cases. The application of photoaptamer technology to proteomics was investigated in microarray format. Immobilized anti-human immunodeficiency virus-gp120 aptamer was able to detect subnanomolar concentrations of target protein in 5% human serum. The levels of sensitivity and specificity displayed by photoaptamers, combined with other advantageous properties of aptamers, should facilitate development of protein chip technology.

Photochemical coupling of 5-bromouracil to tryptophan, tyrosine and histidine, peptide-like derivatives in aqueous fluid solution.

Direct irradiation of 5‐bromouracil (BU) in aqueous fluid solution in the presence of tryptophan (trp), tyrosine (tyr) or histidine (his) derivatives using a XeCl excimer laser at 308 nm yielded photocoupling of BU to the aromatic ring of each amino acid. Irradiation of BU at 308 nm most likely results in excitation of the n‐φ* transition, intersystem crossing to the triplet manifold, and coupling via electron transfer from the aromatic amino acid. The coupling observed was regiospecific between the 5‐position of uracil (U) and the 2‐position of the indole and phenol rings and the 5‐position of the imidazole ring of the respective amino acids. Quantum yields of photocoupling to BU ranged from 1 × 10‐3 to 7 × 10‐3 and paralleled known rates of electron transfer and ionization potentials of the aromatic rings. The photocoupling between BU and some of the aromatic amino acid peptide‐like derivatives possibly mimics photocrosslinking of BU‐DNA to associated proteins, a potentially useful photoreaction for studying nucleic acid‐protein interactions. Formation of crosslinks of the type proposed here might be detected by the characteristic fluorescence emission of the uracil amino acid adducts.

Nuclear targeting and retention of anthracycline antitumor drugs in sensitive and resistant tumor cells

Recent and new results which support a drug-DNA covalent bonding mechanism for cell toxicity of the clinical antitumor drugs, daunorubicin, doxorubicin, and epidoxorubicin, are summarized. The mechanism involves the iron complex of the drugs inducing oxidative stress to yield formaldehyde, which then mediates covalent attachment to G-bases of DNA. At NGC sites the combination of covalent and non-covalent drug interactions serve to virtually crosslink the DNA. Structural data for virtual crosslinks are compared as a function of drug structure. Elucidation of the mechanism led to the synthesis and evaluation of drug formaldehyde conjugates, Daunoform, Doxoform, and Epidoxoform, as improved chemotherapeutics. Drug uptake, nuclear targeting, drug release, and cytotoxicity of the clinical drugs by sensitive and resistant breast and prostate cancer cells are contrasted with those of the corresponding formaldehyde conjugates. Conjugates are taken up better, retained longer, and are more toxic to a wide variety of tumor cells. The kinetics of drug release from Doxoform and Epidoxoform treated MCF-7/Adr cells are biexponential and correlate with the biexponential kinetics of drug release from extracellular DNA. The results of the lead conjugate, Epidoxoform, in the National Cancer Institute 60 human tumor cell screen are presented and discussed in terms of some resistance mechanisms. Epidoxoform shows increased toxicity to all panels relative to doxorubicin and epidoxorubicin, and this enhanced toxicity is especially evident with the more resistant cell lines.

Lack of glutathione conjugation to adriamycin in human breast cancer MCF-7/DOX cells. Inhibition of glutathione S-transferase p1-1 by glutathione conjugates from anthracyclines.

One of the proposed mechanisms for multidrug resistance relies on the ability of resistant tumor cells to efficiently promote glutathione S-transferase (GST)-catalyzed GSH conjugation of the antitumor drug. This type of conjugation, observed in several families of drugs, has never been documented satisfactorily for anthracyclines. Adriamycin-resistant human breast cancer MCF-7/DOX cells, presenting a comparable GSH concentration, but a 14-fold increase of the GST P1-1 activity relative to the sensitive MCF-7 cells, have been treated with adriamycin in the presence of verapamil, an inhibitor of the 170 P-glycoprotein (P-gp) drug transport protein, and scrutinized for any production of GSH-adriamycin conjugates. HPLC analysis of cell content and culture broths have shown unequivocally that no GSH conjugates are present either inside the cell or in the culture broth. The only anthracycline present inside the cells after 24 hr of incubation was > 98% pure adriamycin. Confocal laser scanning microscopic observation showed that in MCF-7/DOX cells adriamycin was localized mostly in the Golgi apparatus rather than in the nucleus, the preferred site of accumulation for sensitive MCF-7 cells. These findings rule out GSH conjugation or any other significant biochemical transformation as the basis for resistance to adriamycin and as a ground for the anomalous localization of the drug in the cell. Adriamycin, daunomycin, and menogaril did not undergo meaningful conjugation to GSH in the presence of GST P1-1 at pH 7.2. Indeed, their synthetic C(7)-aglycon-GSH conjugates exerted a strong inhibitory effect on GST P1-1, with K(i) at 25 degrees in the 1-2 microM range, scarcely dependent on their stereochemistry at C(7).

PHOTOCHEMICAL REDUCTION OF 5‐BROMOURACIL BY CYSTEINE DERIVATIVES AND COUPLING OF 5‐BROMOURACIL TO CYSTINE DERIVATIVES

Abstract Irradiation of pH 7, aqueous solutions of 5‐bromouracil (BU) in the presence of cysteine peptide‐like derivatives at 308 nm using a XeCl excimer laser yielded initial formation of only uracil (U) and the corresponding cystine derivative. Continued irradiation yielded an S‐uracilylcysteinyl adduct as well as additional U and cystine derivative. Similar irradiation of a solution of BU and a cystine derivative yielded initial formation of U and the S‐uracilylcysteinyl adduct. Formation of these products as well as secondary products of uracil photochemistry was observed upon irradiation of the respective solutions with 254 nm light. With 308 nm laser excitation, U‐Cys adduct formation and reduction of BU to U are proposed to occur via initial electron transfer from the disulfide of the cystine derivative to triplet BU. The quantum yield of BU destruction with 308 nm excitation in the presence of cystine derivative is 1.1 × 10‐3. Reaction of triplet BU with the cysteine derivative does not yield U‐Cys adduct but U and cystine derivative. A possible byproduct of reduction of triplet BU to U by a cysteinyl residue in a protein BU‐DNA complex is a sulphenyl bromide which might yield a protein‐DNA crosslink via nucleophilic substitution on sulfur by a nucleophilic site in DNA.

Dabco stabilization of coumarin dye lasers

Abstract 1.4-diazabicyclo [2.2.2] octane (DABCO) has been shown to extend the lifetime of several coumarin dyes in nitrogen-laser-pumped and flash-lamp-pumped dye lasers. With 0.010 M DABCO average power output remains at better than 907 of initial power at least three times longer than without DABCO.DABCO is effective in stabilizing dye solutions which are not oxygen degassed and to a lesser extent, in stabilizing oxygen degassed dye solutions. Average power output, pulse duration, and spectral linewidth are not significantly affected. Stabilization is proposed to occur through a combination of dye triplet excited state quenching and quenching of singlet oxygen which results from oxygen quenching of dye triplet states.

Detection and possible origins of aminomalonic acid in protein hydrolysates

Aminomalonic acid (Ama) was first detected in alkaline hydrolysates of proteins in 1984. In this work we describe our search for the origin of aminomalonic acid in alkaline hydrolysates of proteins. We have developed a technique for quantitation of aminomalonic acid based upon gas chromatography/mass spectrometry. Using this technique, we find approximately 0.3 Ama/1000 amino acids in hydrolysates of Escherichia coli protein. We have demonstrated that Ama is not formed from any of the 20 major amino acids during the hydrolysis procedure. Furthermore, the amount of Ama found does not depend on the presence of small amounts of O2 during the hydrolysis. Thus far, we have not been able to demonstrate an artifactual origin for Ama. The results described above suggest that Ama may indeed be a constituent of proteins before the hydrolysis procedure. Possible origins of Ama include errors in protein synthesis and oxidative damage to amino acid residues in proteins.