Ultrafast dynamics of resonance energy transfer in cryptochrome

In this communication, we report the ultrafast dynamics of resonance energy transfer in a blue-light photoreceptor, Vibrio cholerae cryptochrome. The transfer was observed to occur in 60 ps. We also studied the local rigidity and solvation around the binding site of the photoantenna molecule. The results for the first time show energy transfer in cryptochrome suggesting some mechanistic similarities between photolyase that repairs damaged DNA and cryptochrome that mediates blue-light signaling.     

Therapeutic Monitoring of Isoniazid, Pyrazinamide and Rifampicin in Tuberculosis Patients Using LC

The objective of this study was to measure plasma concentration of isoniazide (INH), pyrazinamide (PZA) and rifampisin (RIF) in tuberculosis patients by using HPLC. 100 μL of plasma was deproteinized by adding trichloroacetic acid and acetonitrile to yield INH, PZA and RIF respectively. They were analysed by HPLC using a reversed phase C₁₈ pre-column linked to a 4 μm C₁₈ analytical column with a gradient solvent programme, which delivered 3% to 40% (v/v) acetonitrile in phosphate buffer in 20 min at rate of 0.8 mL min^?1. Signals were monitored by diode-array detector. Acetanilide was used as internal standard. The method is reproducible and accurate with lower limits of quantification of 0.6 mg L^?1 for INH, 1.5 mg L^?1 for PZA and 0.7 mg L^?1 for RIF. The plasma of 25 patients receiving daily standard therapy were assayed for INH, PZA and RIF 3 h after administration. Plasma concentration were found between 0.98 and 6.27 mg L^?1 for INH, 11.05 and 47.26 mg L^?1 for PZA, 5.09 and 33.20 mg L^?1 for RIF respectively. Many of the plasma levels were found to be sub therapeutic. This practical method may be used for monitoring drug plasma levels of patients who fail to respond to treatment.     

A role for cryptochromes in sleep regulation

Background  

The cryptochrome 1 and 2 genes (cry1 and cry2) are necessary for the generation of circadian rhythms, as mice lacking both of these genes (cry1,2 ^-/-) lack circadian rhythms. We studied sleep in cry1,2 ^-/- mice under baseline conditions as well as under conditions of constant darkness and enforced wakefulness to determine whether cryptochromes influence sleep regulatory processes.     

The effect of topical ethanol extract of Cotinus coggygria Scop. on cutaneous wound healing in rats

The aim of this study is to determine the cutaneous wound healing effects of the ethanol extract of Cotinus coggygria leaves in rats by excision wound model to provide scientific evidence for the traditional use of C. coggygria Scop. The levels of malondialdehyde, catalase, superoxide dismutase, glutathione and hydroxyproline were investigated in wound tissues. Histopathological examination was also performed. The hydroxyproline content of the granulation tissue and the glutathione levels were both significantly higher in the treatment group than in the control group (p < 0.05 for both); while the malondialdehyde levels were significantly lower in the treatment group (p < 0.05). These results were supported with histological results. The ethanol extract of C. coggygria Scop could be considered as an effective agent in wound healing in accordance with its traditional use.     

Purification and Partial Characterization of (6-4) Photoproduct DNA Photolyase from Xenopus laevis

Abstract— The (6-4) photoproduct DNA photolyase was detected in two vertebrate animals Crotalus atrox (rattlesnake) and Xenopus laevis (South African clawed toad). The enzyme was extensively purified from X. laevis and characterized. The highly purified enzyme is fluorescent with an excitation maximum at 420-440 nm and emission maximum at 460-480 nm. The photorepair action spectrum matches the fluoresoence excitation spectrum with a 430 nm maximum.     

A third member of the photolyase/blue-light photoreceptor family in Drosophila: a putative circadian photoreceptor

Two photolyases, specific for cyclobutane pyrimidine dimers and (6-4) photoproducts, have been reported in Drosophila. These enzymes share extensive sequence homologies with the plant blue-light photoreceptor. We have now identified a third gene in Drosophila melanogaster with extensive sequence homology to the photolyase gene. The newly identified gene, which we named dCRY, was expressed as a recombinant protein and tested for photolyase activity. The recombinant protein exhibited photochemical properties similar to those of Drosophila pyrimidine dimer and (6-4) photolyases but lacked photolyase activity. In light of recent evidence that blue-light photoreceptors regulate the circadian clock in mammals, we propose that dCRY is the circadian photoreceptor in this organism.     

PHOTOCHEMISTRY, PHOTOPHYSICS, AND MECHANISM OF PYRIMIDINE DIMER REPAIR BY DNA PHOTOLYASE

Abstract— DNA photolyases photorepair pyrimidine dimers (PyroPyr) in DNA as well as RNA and thus reverse the harmful effects of UV-A (320–400 nm) and UV-B (280–320 nm) radiations. Photolyases from various organisms have been found to contain two noncovalently bound cofactors; one is a fully reduced flavin adenine dinucleotide (FADH^-) and the other, commonly known as second chromophore, is either methenyltetrahydrofolate (MTHF) or 8-hydroxydeazaflavin (8-HDF). The second chromophore in photolyase is a light-harvesting molecule that absorbs mostly in the near-UV and visible wavelengths (300–500 nm) with its high extinction coefficient. The second chromophore then transfers its excitation energy to the FADH^-. Subsequently, the photoexcited FADH^- transfers an electron to the Pyr<>Pyr generating a dimer radical anion (Pyr<>Pyr^-) and a neutral flavin radical (FADH^-). The Pyr<>Pyr^- is very unstable and undergoes spontaneous splitting followed by a back electron transfer to the FADH^-. In addition to the main catalytic cofactor FADH^-, a Trp (Trp277 in Escherichia coli ) in apophotolyase, independent of other chromophores, also functions as a sensitizer to repair Pyr <> Pyr by direct electron transfer.     

Femtosecond dynamics of flavin cofactor in DNA photolyase: radical reduction, local solvation, and charge recombination

We report here our femtosecond studies of the photoreduction dynamics of the neutral radical flavin (FADH) cofactor in E. coli photolyase, a process converting the inactive form to the biologically active one, a fully reduced deprotonated flavin FADH(-). The observed temporal absorption evolution revealed two initial electron-transfer reactions, occurring in 11 and 42 ps with the neighboring aromatic residues of W382 and F366, respectively. The new transient absorption, observed at 550 nm previously in photolyase, was found from the excited-state neutral radical and is probably caused by strong interactions with the adenine moiety through the flavin U-shaped configuration and the highly polar/charged surrounding residues. The solvation dynamics from the locally ordered water molecules in the active site was observed to occur in approximately 2 ps. These ultrafast ordered-water motions are critical to stabilizing the photoreduction product FADH(-) instantaneously to prevent fast charge recombination. The back electron-transfer reaction was found to occur in approximately 3 ns. This slow process, consistent with ultrafast stabilization of the catalytic cofactor, favors photoreduction in photolyase.