DNA unwinding induced by photoaddition of psoralen derivatives and determination of dark-binding equilibrium constants by gel electrophoresis.

Derivatives of furo[3,2-g]coumarin (psoralen) can bind to the DNA double helix and, in the presence of long-wavelength UV light, the bound psoralen may react covalently with pyrimidine residues on one or both strands of the helix. By using agarose gel electrophoresis, we have determined the unwinding angle associated with each of four different psoralen derivatives to be 28 degrees +/- 4 degrees . For 4,5',8-trimethylpsoralen (trioxsalen) the unwinding angle was found to be independent of the initial DNA superhelix density in the range that is accessible to agarose gel electrophoresis. Also by using agarose gel electrophoresis, we have determined the unwinding angle for ethidium intercalation. This was done by the total relaxation of supercoiled DNA in the presence of a series of ethidium concentrations. By using published values for the association constant for ethidium binding to DNA and evaluating the final superhelix density (after removal of ethidium) of the DNA on gels, we calculated an unwinding angle of 29 degrees +/- 3 degrees . Assuming an unwinding angle of 28 degrees for the noncovalent intercalation of psoralen derivatives, we used the same procedure to determine intercalation binding constants. The association constants for 4'-aminomethyltrioxsalen were 300-1400 M(-1) in NaCl at 0.2-0.05 M and 300-2500 M(-1) in Mg(2+) at 4-0.5 mM. The association constant for 4'-hydroxymethyltrioxsalen in 0.5 mM Mg(2+) was determined to be 70 M(-1).

Psoralen-DNA photoreaction: controlled production of mono- and diadducts with nanosecond ultraviolet laser pulses.

Samples of DNA which contain adducts of a new psoralen derivative, but no cross-links, have been prepared by irradiating mixtures of DNA and the derivative with single, 15-nanosecond pulses of laser light. Succeeding pulses introduce cross-links. The ability to rapidly and selectively create monoadducts and corss-links may allow the use of psoralens as probes of dynamic processes in living cells.

The photoinactivation of an RNA animal virus, vesicular stomatitis virus, with the aid of newly synthesized psoralen derivatives.

The newly synthesized psoralen derivatives, 4' hydroxymethyl 4,5',8 trimethylpsoralen, 4' methoxymethyl 4,5',8 trimethylpsoralen, and 4' aminomethyl 4,5',8 trimethylpsoralen hydrochloride photoreact with the single-stranded RNA animal virus, Vesicular Stomatitis virus, VSV. This virus is inactivated 10(3) times more effectively by photoreaction with these compounds than when photoreacted with 4,5',8 trimethylpsoralen. Under these conditions the RNA virus remains more than 10(3) times less sensitive to inactivation by these new photoreagents than were two double-stranded DNA viruses, Herpes Simplex type 2 (HSV-2) and Vaccinia. Preliminary evidence for the generality of this result is discussed.

Photochemotherapy (PUVA) and psoriasis: comparison of 8-MOP and 8-MOP/5-MOP.

Twenty-eight psoriatic patients received PUVA treatment (psoralen and long ultraviolet irradiations. Two preparations were used; 8-methoxypsoralen and a mixture of 5-methoxypsoralen and 8-methoxypsoralen. Both gave considerable improvement, but in 6 cases the lesions reappeared after 2 to 8 weeks, in spite of maintenance treatment. In this report, photochemotherapy of psoriasis was compared, using a UVA emitting lamp, 8-MOP, and a mixture of 8-MOP and 5-methoxypsoralen (5-MOP).

Electron microscopy of DNA crosslinked with trimethylpsoralen: test of the secondary structure of eukaryotic inverted repeat sequences.

It has been suggested that inverted repeat (palindrome) sequences, which are widespread in eukaryotic genomes, exist in two alternate configurations, a linear form and a cruciform. To investigate the relative frequency of these forms, the DNA of intact mouse tissue culture cells was covalently crosslinked with 4,5',8-trimethylpsoralen (me3-psoralen) in order to prevent rearrangement of the DNA secondary structure during DNA isolation. The distribution of me3-psoralen crosslinks was determined by electron microscopy after denaturation of the DNA in the presence of glyoxal. Because of the high frequency and the relatively uniform distribution of the me3-psoralen crosslinks, it could be concluded that almost all of the inverted repeat sequences had been crosslinked. In spite of this, no significant number of cruciforms was detected by electron microscopy. To determine whether the me3-psoralen might itself be disrupting cruciform structures, cruciforms were first produced in isolated Tetrahymena rDNA by heat treatment and then crosslinked in vitro. The crosslinking was found to stabilize rather than disrupt these cruciforms. We conclude that the inverted repeat sequences of the mouse tissue culture cells we tested are predominantly in linear forms rather than in cruciform structures inside the cell.

Psoralen-crosslinked secondary structure map of single-stranded virus DNA.

The photochemical crosslinking of DNA by 4,5',8-trimethylpsoralen (trioxsalen) has been used to freeze the secondary structures of single-stranded DNA molecules of bacteriophage fd at different ionic strengths. These secondary structures (hairpins or looped hairpins) have been visualized in the electron microscope. Most of the single-stranded circular fd DNA molecules show only one hairpin after irradiation at 15 degrees in 20 mM NaCl in the presence of trioxsalen. As the ionic strength is increased, more hairpins appear on the DNA molecules. To map these secondary structures, double-stranded supercoiled fd DNA (RFI) was cleaved with the restriction enzyme HindII, which makes only one cut on each RFI molecule. After denaturation and crosslinking of the linear single-stranded fd DNA (a mixture of viral and complementary strands), all the hairpins have been mapped on the DNA molecule with respect to this HindII site. The results show that these hairpins occur at specific sites. The most stable hairpin has been assigned to the position where the initiation site for the conversion of single-stranded fd DNA to the double-stranded covalently closed form has been mapped. The remaining hairpins map in or near regions corresponding to in vitro promoter sites on the fd DNA.

[Treatment of psoriasis (author's transl)]. / Therapie der psoriasis

From an examination of all possible methods for the treatment of psoriasis and the therapeutic results obtained with them, the author emphasizes the outstanding efficacy of a method for the treatment of psoriasis only recent described in the medical literature, which consists of a combination of methoxsalen by mouth and longwave ultraviolet light. In almost all cases treated with this "photochemotherapy" a rapid and complete regression of the extensive psoriasis lesions could, in fact, be observed without being able to detect toxic systemic side effects or secondary alterations of the skin. As a result of a recent trial of his own, the author points out that the results of photochemotherapy of psoriasis can equally well be obtained with "Trisoralen" by mouth and sunlight.

Studies on the photosensitizing properties of angelicin, an angular furocoumarin forming only monofunctional adducts with the pyrimidine bases of DNA.

The bioligical photosensitizing properties of furocoumarins are due to the formation of adducts with the pyrimidine bases of DNA under irradiation with long wavelength ultraviolet light. The greatest importance is attributed to the difunctional adducts, which form cross-linkings between the 2 strands of DNA. As angelicin, photoreacting with DNA, forms only monofunctional adducts, and therefore no cross-linkings, its photosensitizing properties have been studied in order to evaluate the ability of monofunctional adducts to produce biological effects. The results obtained studying the inhibition of DNA, RNA and protein synthesis in Ehrlich ascite tumor cells after irradiation in the presence of angelicin and psoralen (for a comparison), and the inhibition of the ability of identically treated cells to transmit the tumor showed a remarkable ability of monofunctional adducts to produce biological effects.

[Case contribution on a group affliction with photodermatitis bullosa striata pratensis (Oppenheim)]. / Kasuistischer Beitrag über eine Gruppenerkrankung mit Photodermatitis bullosa striata pratensis (Oppenheim)

After duty in the open field a group of 58 soldiers were simultaneously affected by photodermatitis bullosa striata pratensis Oppenheim. Among others Heracleum sphondylium was found in the botanical inventory of the lower vegetation of the terrain in question. This plant is in our latitudes the most important trigger of phototoxic reactions of the skin.