Generation of reactive oxygen species from the photolysis of histidine by near-ultraviolet light: effects on T7 as a model biological system.

Near-ultraviolet (NUV) light (280-400 nm) has a variety of effects on biological systems; these effects are increased, often synergistically, in the presence of sensitizers. A variety of both man-made and naturally occurring sensitizers have been identified, but their precise roles and relative contributions to cellular damage are not yet fully established. DNA seems to be a major target and a variety of types of damage have been observed. In this report we present evidence that histidine can also act as a sensitizer of NUV. Upon NUV photolysis a variety of reactive oxygen species, including superoxide anions, hydroxyl radicals and hydrogen peroxide, are produced as determined by the effects of various scavengers. pH influences the reaction, alkaline media being most effective, as has previously been reported for the photolysis of H2O2, tyrosine, phenylalanine and tryptophan. Exposure of phage T7 to a combination of histidine and NUV leads to synergistic inactivation and scavengers of O2.-, .OH and H2O2 reduce this effect. These results point to a possible involvement of sunlight-induced histidine photolysis in cellular damage. The fact that photolysis is maximal at high pH indicates that biological effects are likely to be highly localized, e.g., at enzyme active sites.

Detection of Borrelia burgdorferi-specific DNA in urine specimens from patients with erythema migrans before and after antibiotic therapy.

A nested PCR was developed for the detection of Borrelia burgdorferi-specific DNA in the urine of patients with erythema migrans. The target for the nested PCR was a specific region of the flagellin gene; the detection limit was less than five organisms of B. burgdorferi including all three species B. burgdorferi sensu stricto, B. afzelii, and B. garinii. A prospective, controlled, blinded study was performed with 26 patients with erythema migrans to evaluate the nested PCR method with clinical samples. B. burgdorferi-specific DNA could be detected in urine specimens from 22 of 24 patients with erythema migrans (sensitivity, 91.61%). Immediately after therapy, 11 of 19 patients still yielded positive results (58%). Eight weeks after therapy, 2 of 16 patients (13%) were positive by PCR of urine, and 20 weeks after treatment none of seven investigated urine samples was reactive. Essential for the sensitivity that was obtained was the development of a simple DNA extraction procedure. The results of the study indicate that the described method is highly sensitive and allows for the effective control of the efficacy of antibiotic therapy in patients with early Lyme borreliosis.

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in the urine and breast milk of patients with Lyme borreliosis.

Current laboratory diagnosis of Lyme borreliosis relies on tests for the detection of antibodies to Borrelia burgdorferi with known limitations. By using a simple extraction procedure for urine samples, B. burgdorferi DNA was amplified by a nested PCR with primers that target the specific part of the flagellin gene. To control possible inhibition of the enzyme (polymerase), a special assay using the same primers was developed. We examined 403 urine samples from 185 patients with skin manifestations of Lyme borreliosis. Before treatment, B. burgdorferi DNA was detected in 88 of 97 patients with Lyme borreliosis. After treatment, all but seven patients became nonreactive. Six of these seven persons suffered from intermittent migratory arthralgias or myalgias, and one from acrodermatitis chronica atrophicans. Two of 49 control patients with various dermatologic disorders and none out of 22 presumably healthy persons were reactive in the PCR. In addition to urine, breast milk from two lactating women with erythema migrans was tested and also found reactive. Borrelia burgdorferi DNA can be detected with high sensitivity (91%) by a nested PCR in urine of patients with Lyme borreliosis. In addition, this test can be a reliable marker for the efficacy of treatment.