Refinements of the single photon technique are reported, which are concerned for the most part with the photon timing photomultiplier, its associated circuitry, and the signal processing equipment between this multiplier and the time to amplitude convertor. A photomultiplier voltage distribution circuit is described which permits one to optimize the performance of the photon counting photomultiplier either for sensitivity, time resolution, or both in order to assure the validity of the convolution integral as the representation of the instrument output. An investigation of the variation of the instrumental response with stop discriminator level is reported, and the origin and elimination of wavelength effects and photocathode area effects associated with the timing photomultiplier are also described.
Energy transfer from hen lysozyme to a complexed eosin molecule has been investigated with single photon counting and conventional spectrofluorimetry. Transfer takes place with approximately unit efficiency from one of the two strongly emitting tryptophyl residues, leading to partial quenching of the U.V. protein fluorescence and enhanced visible emission from the dye. In conjunction with reported work on fluorescence and photodynamic inactivation of lysozyme by singlet oxygen, the results indicate that the donor is tryptophyl residue 108 and that the dye acceptor is located in a nearby hydrophobic environment.The transfer of energy from a protein molecule to a complexed or conjugated dye has been studied extensively in connection with " fluorescent probes " of protein structure and function (e.g. the reviews of Pesce et al. ' and Brand and Gohlke 2). Much of this work has been done with sulphonated naphthalene derivatives such as ANS and DNS, in which the emission spectra are strongly dependent on the polarity of the environment. The present investigation was undertaken from the viewpoint of photodynamic properties of the dye, i.e. the effect of binding on the mechanism by which visible light excitation of the dye alters the protein substrate. A previous study on inactivation of hen lysozyme sensitized by eosin has shown that a non-inhibitory 1 : 1 complex is formed, leading to partial quenching of the native lysozyme U.V. fluorescence. It was demonstrated that excitation of the bound dye generates singlet oxygen which attacks the enzyme and destroys the lytic activity. Based on the fluorescence changes prior to and after irradiation and the photochemical results, it was proposed that the eosin molecule is located in a " hydrophobic box " in the vicinity of tryptophyl site 108 near the inner wall of the active site cleft. The fluorescence properties of the eosin-lysozyme complex have been investigated in greater detail in this work with the time-correlated single photon counting technique as well as conventional spectrofluorimetry.
EXPERIMENTALThe measurements were made with 6X crystallized, hen egg white lysozyme from Seikagaku Kogyo Co. (used as received), chromatographically purified eosin Y (cmax = 9.7 x lo4 dm3 mo1-I cm-I), and AnalaR grade L-tryptophan and indole-3-carboxylic acid. The fluorescence measurements were carried out with a Perkin-Elmer MPF-3 fluorescence spectrophotometer and the absorption measurements with a Perkin-Elmer model 124 spectrophotometer. The single photon counting apparatus employed for the measurements of lifetimes and time-resolved spectra is similar to that described by Lewis et aL4 The samples were excited at 337 nm using an air-filled lamp operated at 20 kHz providing 2.5 ns pulses and the spectra obtained with the method of Ware et aL5 t present address :
We evaluated the efficacy of palliative radiation therapy using 5 × 4 Gy given daily in 18 dogs with nasal tumors. Dogs with malignant nasal tumors were evaluated for response rate, response duration, and survival. Seventy-eight percent of the dogs achieved complete resolution of clinical signs, and 16.5% had partial resolution of their signs. Overall median response duration for all dogs was 178 days after one course of radiation therapy. Six dogs received a second course of therapy when their disease progressed using the same daily 5 × 4 Gy scheme, and all six responded for a median time of 129.5 days for an overall median survival time in these six dogs of 309 days. Based on these results, a radiation prescription of 5 × 4 Gy appears to be useful palliatively in dogs with a malignant nasal tumor.
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