On the Roles of Urocanic Acid in Photoimmunosuppression: Attempted Photorepair of Urocanic Acid-DNA Cyclobutane Adducts with DNA Photolyase

It has been reported that UV-induced immunosuppres-sion can be reversed by photoreactivation or exposure to T4 endonuclease V, two treatments that can repair cyclobutane pyrimidine dimers. These observations, together with the known role of urocanic acid (UA) in UV-induced immune suppression, prompted us to study the ability of DNA photolyase to repair UA-DNA cyclobutane photoadducts in single-stranded calf thymus DNA. We did not detect any release of UA, with a sensitivity implying that photolyase is at least 2900 times less active toward UA-DNA adducts than toward cis-syn thyminethymine dimers. This indicates that any reversal of photoimmunosuppression by photoreactivation cannot significantly involve cleavage of UA-DNA cyclobutane adducts.     

PHOTOSENSITIZED INACTTVATION OF INFECTIOUS DNA BY UROCANIC ACID, INDOLEACRYLIC ACID AND RHODIUM COMPLEXES

Naked, infectious single-stranded (ss) and double-stranded (ds) DNA from phages SI3 and G4 were irradiated with 308 nm UV radiation in the absence and presence of several photobiologically active compounds: E - and Z -urocanic acid ( E - and Z -UA), their methyl esters ( E - and Z -MU), E - and Z-indoleacrylic acid ( E - and Z -IA), cis -dichlorobis(1,10-phenanthroline)rhodium(III) chloride (cDCBPR) and tris(1,10-phenanthroline)rhodium (III) perchlorate (TPR). E -urocanic acid protects against cyclobutane pyrimidine dimer formation in ssDNA but concomitantly photosensitizes the formation of other lesions that inactivate ssDNA. Z -urocanic acid also protects ssDNA against such dimerization but without the associated sensitized damage. The methyl ester isomers behave similarly. There is no such differential activity observed for the IA isomers, both of which sensitize the inactivation of ssDNA. Photostationary state mixtures of both UA and IA efficiently sensitize the inactivation of dsDNA, and cDCBPR strongly protects ssDNA from UV damage, while TPR is a significant sensitizer. Both of these metal complexes sensitize the inactivation of dsDNA slightly. For all compounds, cyclobutane pyrimidine dimers were the predominant lethal lesions produced by sensitization of the dsDNA, but they were not the major lethal lesions created by sensitization of the ssDNA. In the case of dsDNA, both UA and IA created pyrimidine dimers with a high degree of potential for mutagenesis, as determined by an assay that monitors the frequency of mutations following the spontaneous deamination of cytosine in photodimers.     

THE SITE-SPECIFIC INHIBITION OF Bgl I CLEAVAGE BY PSORALEN PHOTOADDUCTS

Abstract— We have investigated the site specificity of furocoumarins by using fluorescent densitometry to examine the frequency of cleavage by the restriction enzyme Bgl I. This enzyme has an 11 base pair (bp) recognition sequence which varies slightly from site to site because it includes a 5 base pair neutral region. Cleavage at all three Bgl I recognition sites in pBR322 was inhibited by the photoaddition of the psoralen derivative 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) which forms both crosslinks and monoad-ducts in a dose-dependent manner. One site, which contains two thymidines in a crosslinkable configuration, was observed to be markedly more sensitive to HMT photoadducts. In contrast Bgl I cleavage at all sites was relatively resistant to the derivative 5-methylisopsoralen (5-MIP), which forms only monoadducts. When HMT-reacted DNA was generated with widely different ratios of monoad-ducts to crosslinks (3% and 40% crosslinks), essentially the same level and pattern of inhibition was observed in both cases. Taken together, the data imply that differences in inhibition seen at the three cutting sites of Bgl I with HMT are attributable to DNA sequence and the role it plays in adduct positioning.     

Computer Calculations of Acid–Base Equilibria in Aqueous Solutions Using the Acid–Base Calculator Program

The Acid–Base Calculator program is proposed for calculating pH and ionic strength in aqueous solutions of mixtures of strong and weak acids or bases and salts. The program works under the widespread Windows 95/98/NT/2000 operating system, has a simple user interface, and permits editing the input data and saving the calculated results. Examples of applications of the Acid–Base Calculator program in routine calculations and in calculations of pH gradients in ion-exchange chromatography are given.     

Photochemistry and Photobiology of lmidazole-4(5)-Methylidene Malonic Acid: An Analog of Both E- and Z-Urocanic Acid*

Imidazole-4(5)-methylidene malonic acid (IMMA) may be thought of as having its imidazole and carboxyl functionalities permanently fixed in a configuration that is simultaneously analogous to both E- (trans) and Z- (cis) urocanic acid (UA). Because the UA isomers affect the photoinactivation of bacteriophage single-stranded DNA differently (E-UA increases and Z-UA decreases viral DNA inactivation), IMMA was similarly tested and was found to change the inactivation rate by a factor of 0.43, which is intermediate between the values for E- and Z-UA (1.6 and 0.014, respectively). The IMMA likewise sensitizes double-stranded DNA by a factor of 10.3 compared to a value of 13 for UA. In several ways the effects of IMMA parallel the distinctive effects of UA on the UV inactivation of single- and double-stranded DNA, including the ability to prevent the formation of cyclobutane pyrimidine dimers in irradiated single-stranded DNA and to sensitize a large increase in the formation of those dimers in irradiated double-stranded DNA. The IMMA photodecarboxylates to UA with a low quantum efficiency (ca 1 × 10^?3) and photochemically binds to calf-thymus DNA.     

Information Database on Internal pH Gradients in Chromatofocusing

An information database on internal pH gradients in chromatofocusing was developed. The Windows 95/98/NT–based database contains information on model and experimental pH gradients and has a user-friendly interface for information retrieval and new data input. The database also contains two embedded programs: CF for simulating pH gradients by experimental conditions and ABC for rapidly calculating particular sections in gradient profiles. Examples of the use of embedded programs are given, and experimental and model pH gradients are compared.