High frequency vibrational modulations in two-dimensional electronic spectra and their resemblance to electronic coherence signatures.

In this work we analyze how nuclear coherences modulate diagonal and off-diagonal peaks in two-dimensional electronic spectroscopy. 2D electronic spectra of pinacyanol chloride are measured with 8 fs pulses, which allows coherent excitation of the 1300 cm(-1) vibrational mode. The 2D spectrum reveals both diagonal and off-diagonal peaks related to the vibrational mode. On early time scales, up to 30 fs, coherent dynamics give rise to oscillations in the amplitudes, positions, and shapes of the peaks in the 2D spectrum. We find an anticorrelation between the amplitude and the diagonal width of the two diagonal peaks. The measured data are reproduced with a model incorporating a high frequency mode coupled to an electronic two-level-system. Our results show that these anticorrelated oscillations occur for vibrational wavepackets and not exclusively for electronic coherences as has been assumed previously.

Excitonic couplings and interband energy transfer in a double-wall molecular aggregate imaged by coherent two-dimensional electronic spectroscopy.

The early stage of molecular excitonics and its quantum-kinetic dynamics in the multiband, bitubular cyanine dye aggregate C(8)O(3) at room temperature are revealed by employing two-dimensional (2D) coherent electronic spectroscopy in the visible spectral region. The sub-20 fs measurements provide a direct look into the details of elementary electronic couplings by spreading spectroscopic transitions into two frequency axes. Correlation spectra of rephasing (k(I) = -k(1) + k(2) + k(3)) and nonrephasing (k(II) = +k(1) - k(2) + k(3)) data in emission (omega(3))-absorption (omega(1)) 2D-frequency space image interband excitons into cross-peak signals and unveil the quantum-dissipative regime of exciton relaxation. Spectral streaking of cross peaks directly reveals interband dephasing and exciton population relaxation on the road to tube-to-tube energy transfer without making recourse to an a priori model. Theory and simulations, based on an effective multilevel scheme and a quantum-dissipative model with experimental pulse envelopes, explain the origin of the cross peaks, reveal the underlying sequences of electronic transitions, recover the streaking patterns of relaxing cross peaks along omega(1), and reconstruct the space-energy pathways of electronic excitation flow.

A contribution to solve the arsenic problem in groundwater of Ganges Delta by in-situ treatment.

Arsenic in groundwater is a huge problem in numerous regions of the world. Many people are exposed to high arsenic concentrations and consequently risk getting ill or even die as a result of arsenic poisoning. There are several efficient technologies for the removal of arsenic but often these methods have disadvantages, e.g. high costs for installation and/or operation, the need for chemicals or the production of arsenic contaminated filter sludge. These disadvantages can make the application difficult, especially in poor regions. Under suitable ancillary conditions the subterranean (in-situ) treatment, which is often used for iron and manganese removal from groundwater, can also be applied for the removal of arsenic and can be a cost-effective treatment technology. A field trial was carried out with a low-cost in-situ treatment plant in West Bengal/India which is described in this paper, in order to investigate whether this treatment technology is also applicable under the boundary conditions there. As for the in-situ treatment technology besides oxygen no additives are required and no arsenic contaminated filter sludge is produced this technology could be a suitable method for arsenic removal especially in poor regions.

Acute and repeat-dose toxicity studies of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH), an albumin-binding prodrug of the anticancer agent doxorubicin.

The (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH) is an albumin-binding prodrug of doxorubicin with acid-sensitive properties that demonstrates superior antitumor efficacy in murine tumor models, and has been evaluated in a phase I study. In order to establish the toxicity profile of this prodrug, acute and repeat-dose toxicity studies were performed with DOXO-EMCH in CD1-mice, Sprague-Dawley rats and Beagle dogs. Although the objective of the acute toxicity studies was not the determination of LD50 values, the LD50 of DOXO-EMCH was >60 mg/kg doxorubicin equivalents in both male and female mice (the LD50 of doxorubicin in CD-1 mice is -12 mg/kg). In Sprague-Dawley rats, the LD50 was 23.4 and 45.9 mg/kg doxorubicin equivalents for males and females, respectively. For comparison, the LD50 of doxorubicin in Sprague-Dawley rats is -10.5 mg/kg. The major clinical sign noted following intravenous administration of DOXO-EMCH in mice and rats was a dose-dependent peripheral neuropathy which, in general, developed as a delayed toxicity 1-3 weeks after application. The observed neurotoxicity has been well documented for Sprague-Dawley rats treated with doxorubicin at a dose of 5 and 10 mg/kg. In Beagle dogs, LD10 was not reached for DOXO-EMCH at 4.5 mg/kg doxorubicin equivalents. A four-cycle intravenous study with DOXO-EMCH at dose levels of 4 x 2.5, 5.0 or 7.5 mg/kg doxorubicin equivalents in rats revealed approximately three-fold less side effects on the hemolymphoreticular system when compared to 4 x 2.5 mg/kg doxorubicin dose, whereas effects on the testes/oligospermia seem to be comparable between both drugs at equitoxic dose. A No Observable Adverse Effect Level (NOAEL) for DOXO-EMCH of 4 x 2.5 mg/kg doxorubicin equivalents was established in this study. This dose is equivalent to the maximum tolerated dose (MTD) of doxorubicin in rats. In a two-cycle study over a period of 6 weeks in Beagle dogs (intravenous administration of DOXO-EMCH at dose levels of 1.5, 3.0 or 4.5 mg/kg doxorubicin equivalents), dose-related systemic histamine-like reactions within the first 3 hours after injection were noted in all treated groups. Only transient and temporary effects on hematology, urinary function, as well as on histopathology in mid- and/or high-dose animals, were observed. The low dose of 2 x 1.5 mg/kg was considered to be the NOAEL in this study, which is equivalent to twice the MTD o f doxorubicin i nBeagle dogs. In summary, the toxicity studies with DOXO-EMCH in mice, rats or dogs have not identified any other special toxicity when compared to the toxicity data for doxorubicin. Preclinical tolerance of DOXO-EMCH was higher in mice, rats and dogs compared to doxorubicin. A dose of 20 mg/m2 doxorubicin equivalents was recommended as the starting dose for a phase I study with DOXO-EMCH.

Two-dimensional electronic spectra of symmetric dimers: Intermolecular coupling and conformational states.

We study the information content of two-dimensional (2D) electronic photon-echo (PE) spectra, with special emphasis on their potential to distinguish, for waiting times T=0, between different conformations of electronically coupled symmetric dimers. The analysis is performed on the basis of an analytical formula for the frequency-domain 2D PE signal. The symmetric dimers are modeled in terms of two identical, energy-degenerate, excitonically coupled pairs of electronic states in the site representation. The spectra of conformationally weighted ensembles, composed of either two or four dimers, are compared with their one-dimensional linear absorption counterparts. In order to provide a realistic coupling pattern for the ensemble consisting of four dimers, excitonic couplings are estimated on the basis of optimized geometries and site-transition dipole moments, calculated by standard semiempirical methods for the bridged bithiophene structure 1,2-bithiophene-2-yl-ethane-1,2-dion (T2[CO]2). In the framework of our model, the highly readable 2D PE spectra can unambiguously identify spectral doublets, by relating peak heights and positions with mutual orientations of site-localized transition dipoles.

Correlation of femtosecond wave packets and fluorescence interference in a conjugated polymer: Towards the measurement of site homogeneous dephasing.

Probing electronic femtosecond (fs) coherence among segmental sites that are congested by static and dynamic site disorder and subject to structural relaxation is a big, experimental challenge in the study of photophysics of poly(p-phenylenevinylene). In this work, fs-wave-packet fluorescence interferometry experiments are presented that measure macroscopic coherent kernels and their phase-relaxation in the low-temperature, bottom-state regime of the density-of-states below the migrational threshold energy where downhill site-to-site transfer is marginal. By using freely propagating and tunable 70 fs excitation/probing pulses and employing narrow-band spectral filtering of wave packets, fluorescence interferograms with strongly damped beatings can be observed. The coherences formally follow the in-phase superpositions of two site-optical free-induction-decays and originate from distinct pairs of coherent doorway-states, different in energy and space, each of them being targeted, by two discrete quantum-arrival-states 1(alpha) and 1(beta), via independent, isoenergetic 0-->1 fluorescence transitions. The coherent transients are explained as site-to-site polarization beatings, caused by the interference of two fluorescence correlation signals. The numerical analysis of the damping regime, based upon second-order perturbational solutions, reveals the lower limit value of homogeneous dephasing in the range from T(2) approximately 100 fs to T(2) approximately 200 fs depending on the site-excitation energy of the bottom-states. The experiments enable to look into the formation of the relaxed state as a special molecular process of electron-phonon coupling and hence open-up a quite new perspective in the puzzle of multichromophore optical dynamics and structural relaxation in conjugated polymers.

Identification of human multidrug resistance protein 1 (MRP1) mutations and characterization of a G671V substitution.

The multidrug resistance protein 1 (MRP1) belonging to the ATP-binding cassette (ABC) superfamily of transport proteins can confer resistance to multiple natural product drugs and methotrexate in human tumor cells. In addition, MRP1 is expressed in normal tissues acting as an efflux pump for glutathione, glucuronate, and sulfate conjugates and may thus influence the pharmacokinetic properties of many drugs. Using polymerase chain reaction-single-strand conformation polymorphism analysis, we screened 36 Caucasian volunteers for mutations in the coding exons of the MRP1 gene, including the adjacent intron sequences. Among several mutations found, two are expected to cause amino acid substitutions. One of these mutations (G671V) was of special interest because it is located near the first nucleotide binding domain. To determine whether this mutation caused a change in the MRP1 phenotype, a mutant MRP1 expression vector was constructed and transfected into SV40-transformed human embryonic kidney cells (HEKSV293T) and the transport properties of the mutant protein were examined. Transport of the MRP1 substrates leukotriene C4, 17beta-estradiol 17beta-(D)-glucuronide, and estrone sulfate by membrane vesicles prepared from transiently transfected HEKSV293T cells was comparable to that of wild-type MRP1.

Basal expression of the rat, but not of the human, multidrug resistance protein 2 (MRP2) gene is mediated by CBF/NF-Y and Sp1 promoter-binding sites.

The most important biliary efflux transporter known so far is the multidrug resistance protein 2 (MRP2). Previously, we isolated and characterized the 5'-flanking region of the rat mrp2 gene. In the present study, we performed site-directed mutagenesis experiments indicating that both a Y-Box and a GC-Box in the rat mrp2 promoter are essential for the full basal expression of the gene, but have no significant relevance for its inducibility by the chemical carcinogen 2-acetylaminofluorene. Gel mobility shift experiments demonstrated the binding of the transcription factor CBF/NF-Y, but not of EFIA/YB-1, to the Y-Box. Site-directed mutations in the Y-Box decreasing reporter gene activity of a promoter construct prevented the binding of NF-Y. Consequently, NF-Y contributes substantially to the basal expression of the gene. A site-directed mutation in the GC-Box also reduced basal expression and resulted in a reduced complex formation with the transcription factor Sp1. The corresponding region of the human MRP2 promoter comprises no Sp1 site, but a Y-Box-like element binding YB-1 but not NF-Y, which, however, does not contribute to basal expression. In conclusion, NF-Y and Sp1 binding sites play a decisive role in the basal expression of the rat mrp2 gene, while the human MRP2 gene is regulated differently.

Up-regulation of transporters of the MRP family by drugs and toxins.

Expression of a variety of ABC efflux pumps including certain conjugate transporters of the multidrug resistance protein (MRP) subfamily is inducible in primate and rodent tissues, and in a variety of cell lines and primary cells in culture. In human cell lines (HepG2, MCF-7), we studied the inducibility of MRPs 1-5. Similar to the rat mrp2 gene, human mrp2 is inducible by the chemical carcinogen 2-AAF, the chemotherapeutic drug cisplatin and the barbiturate phenobarbital, as demonstrated in Northern and Western Blots. Furthermore, the antibiotic rifampicin was identified as MRP2 inducer in HepG2 cells. MRP1 and 4 mRNAs being expressed in human liver at a very low level could not be detected in HepG2 cells after treatment with various agents. However, MRP3 and 5 mRNAs were detected in addition to MRP2 and their expression was found to be increased by 2-AAF, cisplatin and rifampicin. MRP1 expression was studied in MCF-7 cells where the chemotherapeutic drug vinblastine and tert-butyl hydroquinone but not the MRP2 inducing agents described above acted as inducers.

Sequencing and tissue distribution of the canine MRP2 gene compared with MRP1 and MDR1.

The effects of xenobiotic drugs and toxic compounds depend largely on their kinetic properties, which can be influenced by transmembrane drug transporters like MDR1/P-glycoprotein and the drug-conjugate transporters multidrug resistance protein (MRP) 1 and 2. As the dog is a preferential species used in the pharmacological and toxicological evaluation of new drugs, we sequenced the canine MRP2 cDNA and investigated its expression in various canine tissues compared with the related transporters MRP1 and P-glycoprotein. The tissue distribution pattern of these ABC-transporters differs partially from the distribution described in humans. So we found relatively high renal and low hepatic canine MRP2 expression levels, relatively high hepatic canine MRP1 expression levels, and quite high levels of MRP1 and P-glycoprotein in the dog brain. The knowledge of the tissue distribution pattern of these transporters will aid to interpret pharmacokinetic and toxicokinetic data gained from dog studies and to extrapolate them to humans.

Evaluation of a vincristine resistant Caco-2 cell line for use in a calcein AM extrusion screening assay for P-glycoprotein interaction.

AIM: To develop a fast fluorometric screening assay based on vincristine resistant Caco-2 cells (Caco-2VCR) in order to elucidate potential P-glycoprotein (Pgp) interactions of compounds, and to characterise Caco-2VCR cells with regard to their expression of the efflux transporters Pgp, MRP1 and MRP2. METHODS: We applied the Caco-2VCR cells to a 96-well plate-based calcein AM extrusion assay. The Caco-2VCR cells were cultured as monolayers and incubated with calcein AM with/without addition of Pgp modulators. Fourteen known Pgp modulators were tested in the assay (chloropromazine, cyclosporin A, domperidone, digoxin, ivermectin, ketoconazole, loperamide, metoprolol, propranolol, progesterone, quinidine, quinine, verapamil and vincristine). For each compound an EC50 value was calculated. Protein and mRNA levels of the efflux transporters were analysed by Western blot and polymerase chain reaction techniques. RESULTS: All compounds with the exception of digoxin displayed increased calcein levels. Protein and mRNA analysis showed increased levels of Pgp after vincristine exposure, while expression of the efflux transporters MRP1 and MRP2 remained unchanged. CONCLUSIONS: The calcein AM extrusion assay applied to Caco-2VCR cells can be a valuable tool as a screening assay for new compounds and their potential interaction with P-glycoprotein.

The effect of rifampin treatment on intestinal expression of human MRP transporters.

The importance of the ATP-dependent transporter P-glycoprotein, which is expressed in the brush border membrane of enterocytes and in other tissues with excretory function, for overall drug disposition is well recognized. For example, induction of intestinal P-glycoprotein by rifampin appears to be the underlying mechanism of decreased plasma concentrations of P-glycoprotein substrates such as digoxin with concomitant rifampin therapy. The contribution of transporter proteins other than P-glycoprotein to drug interactions in humans has not been elucidated. Therefore, we tested in this study the hypothesis whether the conjugate export pump MRP2 (cMOAT), which is another member of the ABC transporter family, is inducible by rifampin in humans. Duodenal biopsies were obtained from 16 healthy subjects before and after nine days of oral treatment with 600 mg rifampin/day. MRP2 mRNA and protein were determined by reverse transcription-polymerase chain reaction and immunohistochemistry. Rifampin induced duodenal MRP2 mRNA in 14 out of 16 individuals. Moreover, MRP2 protein, which was expressed in the apical membrane of enterocytes, was significantly induced by rifampin in 10 out of 16 subjects. In summary, rifampin induces MRP2 mRNA and protein in human duodenum. Increased elimination of MRP2 substrates (eg, drug conjugates) into the lumen of the gastrointestinal tract during treatment with rifampin could be a new mechanism of drug interactions.

Induction of P-glycoprotein by rifampin increases intestinal secretion of talinolol in human beings: a new type of drug/drug interaction.

BACKGROUND: P-Glycoprotein is an efflux pump in many epithelial cells with excretory function. It has been demonstrated that rifampin (INN, rifampicin) induces P-glycoprotein, particularly in the gut wall. We therefore hypothesized that rifampin affects pharmacokinetics of the P-glycoprotein substrate talinolol, a beta1-blocker without appreciable metabolic disposition but intense intestinal secretion in human beings. METHODS: Pharmacokinetics of talinolol (a single dose of 30 mg administered intravenously or 100 mg administered orally for 7 days) and duodenal expression of the MDR1 gene product P-glycoprotein as assessed by reverse transcriptase-polymerase chain reaction of the MDR1-messenger ribonucleic acid, by immunohistochemistry and Western blot analysis were analyzed before and after coadministration of rifampin (600 mg per day for 9 days) in 8 male healthy volunteers (age 22 to 26 years). RESULTS: During rifampin treatment, the areas under the curve of intravenous and oral talinolol were significantly lower (21% and 35%; P < .05). Treatment with rifampin resulted in a significantly increased expression of duodenal P-glycoprotein content 4.2-fold (2.9, 6.51) (Western blot) and messenger RNA was increased in six of the eight volunteers. P-Glycoprotein expression in biopsy specimens of gut mucosa correlated significantly with the systemic clearance of intravenous talinolol (rs = 0.74; P < .001). CONCLUSIONS: Rifampin induces P-glycoprotein-mediated excretion of talinolol predominantly in the gut wall. Moreover, clearance of talinolol from the blood into the lumen of the gastrointestinal tract may be predicted by the individual intestinal P-glycoprotein expression. Thus we describe a new type of steady-state drug interaction affecting compounds that are subject to transport rather than metabolism.

Sequence analysis and functional characterization of the 5'-flanking region of the rat multidrug resistance protein 2 (mrp2) gene.

Gene expression of the canalicular conjugate transporter mrp2 is inducible by treatment with the DNA-damaging agents 2-acetylaminofluorene (50 and 100 microM), and cisplatin (20 microM) in primary rat hepatocytes as well as in the rat hepatoma cell line H4IIE. Furthermore, phenobarbital (1 and 2 mM) induces mrp2 gene expression, probably explaining the increase in bile-salt-independent bile flow caused by phenobarbital, while the cholestatic drug ethinyl estradiol (10(-6) M) leads to an increase in mrp2 mRNA but decreases Mrp2 protein level probably via a posttranscriptional mechanism. The 5'-flanking region of the rat mrp2 gene was sequenced and cloned into a luciferase reporter vector. Transient transfection assays with reporter vectors containing unidirectionally deleted 5'-flanking regions using H4IIE cells indicate that two different sequences of 17 and 37 bases comprising a Y-Box and a GC-Box are required for mrp2 gene basal expression. Sequences mediating 2-AAF induction are located within a region 250 bases upstream of the translation start site while the inducing effect of phenobarbital seems to be mediated by another domain located further upstream.

Induction of hepatic mrp2 (cmrp/cmoat) gene expression in nonhuman primates treated with rifampicin or tamoxifen.

The multidrug resistance protein 2 (Mrp2) also called canalicular multidrug resistance protein (cMrp) or canalicular multispecific organic anion transporter (cMoat) is a transmembrane export pump located at the canalicular domain of hepatocytes. Mrp2 transports a broad spectrum of organic anions including glucuronides, glutathione conjugates, and organic sulphates into bile. Based on previous observations in rat hepatocytes, the inducibility of mrp2 gene expression in primate liver was investigated in rhesus monkeys treated with tamoxifen or rifampicin. It was found that treatment with tamoxifen (25 mg/kg per day; over 7 days) or rifampicin (15 mg/kg per day; over 7 days) leading to an induction of cytochrome P450 3A4, resulted in a strong increase in mrp2 mRNA in the liver of male and female rhesus monkeys. On the protein level, tamoxifen also was a highly effective inducer, while rifampicin showed some inducing effect in a female and was inactive in a male monkey. In sections of paraffin-embedded liver tissue, immunofluorescence staining confirmed the results of Western blot analysis. Induced Mrp2 was localized to the canalicular domain of the hepatocytes. In conclusion, our data show inducibility of mrp2 gene expression in the liver of primates which may represent an adaptive response aimed at the enhanced biliary elimination of the inducing drugs and/or their metabolites.

Induction of cMrp/cMoat gene expression by cisplatin, 2-acetylaminofluorene, or cycloheximide in rat hepatocytes.

The human multidrug-resistance-associated protein (MRP), a member of the adenosine triphosphate (ATP)-binding cassette transporter superfamily, is frequently overexpressed in tumor cells resistant to antineoplastic drugs. In the rat, two Mrp isoforms have been identified, Mrp and cMrp. cMrp, also called Mrp2 or cMoat (canalicular multispecific organic anion transporter), is expressed in the canalicular membrane of rat hepatocytes and mediates the excretion of glucuronate, sulfate, and glutathione conjugates into bile. We investigated the expression of cMrp and Mrp in rat hepatocytes in primary culture. Treatment with the chemical carcinogen 2-acetylaminofluorene (2-AAF), the antineoplastic drug cisplatin, and the protein-synthesis inhibitor cycloheximide led to a dose-dependent and time-dependent increase in cmrp gene expression. A 347-base pair cmrp complementary DNA (cDNA) probe served to demonstrate the induction of cmrp messenger RNA (mRNA) with 40 micromol/L 2-AAF, 5 micromol/L cisplatin, or 5 micromol/L cycloheximide. An analogous response was obtained for the increase in cMrp protein. Mrp mRNA was below the detection limit in Northern blots of RNA from liver and hepatocyte cultures, in contrast to rat testis mRNA which served as a positive control. Immunofluorescence microscopy of cultured hepatocytes was used to visualize cMrp in the plasma membrane. Treatment with 2-AAF led to a marked increase in the immunofluorescence signal confirming the cMrp-inducing potency of 2-AAF. In conclusion, the inducing effect of the compounds studied may reflect a general inducibility of hepatic cMrp by a variety of cytotoxic, carcinogenic, and chemotherapeutic agents which is likely to be of relevance for the acquisition of multidrug resistance during chemotherapy and in the process of chemical carcinogenesis in the liver.

Intensity-independent fluorometric detection of cellular nitric oxide release.

A new fluorescence method is introduced in which nitric oxide (NO)-derived higher-order oxygen complexes (NO(x)) are quantified at physiological pH. Detecting the fluorescence lifetime shift between 2,3-diaminonaphthalene and the NO(x)-derived protonated 2,3-naphthotriazole allows an intensity independent determination of the NO(x) concentration. The NO release from LPS and IFNgamma-stimulated murine macrophages and iNOS transfected hamster cells was quantified. The lower detection limit for NO2- was found to be 800 pmol/ml. Since the influence of static fluorescence quenching due to cellular components can be neglected, the method is applicable for clear cellular supernatants as well as turbid cellular suspensions.

Molecular dynamics simulation of the rare amino acid LL-dityrosine and a dityrosine-containing peptide: comparison with time-resolved fluorescence.

The fluorescence of the rare amino acid LL-dityrosine, which is found in insoluble biological materials with structural features, was recently shown to decay non-exponentially (Kungl et al. (1992) J. Fluorescence 2, 63-74). Here we investigated the time-resolved fluorescence of a dityrosine-containing peptide (DCP) to study the influence of side chains on the fluorescence decay of the chromophore. The fluorescence decay of DCP was best fitted by three exponential terms including a sub-nanosecond rise term, the values of which are quite similar to the parameters obtained for the decay of free dityrosine. They were found to depend on the pH of the aqueous solution but not on the temperature. Analysis by an exponential series method revealed broad fluorescence lifetime distributions for DCP. Compared to the corresponding analysis of dityrosine transients, similar lifetime centers were found whereas the widths of the distributions were found broader for DCP. Molecular dyamics (MD) simulations of dityrosine at 300 K show that chi 1 and chi 2 side chain conformers (rotamers) of both tyrosine subunits interconvert on a picosecond timescale. The rates of interconversion were shown to depend critically upon the MD technique applied: in vacuo simulations yielded lower interconversion rates compared to stochastic dynamics (SD) and full MD (water explicitly included). However, MD simulations of the dityrosine-containing peptide revealed no interconversions of the chi 1 and chi 2 side chain rotamers of both tyrosine subunits within a 400 ps trajectory. Interconversions could be induced by raising the temperature of the system (DCP plus solvent) to 340 K. Side chain rotamers of dityrosine are not stable on a fluorescence time scale but are stable when a dityrosine-containing peptide is regarded. Nevertheless both molecules yield similar fluorescence decay patterns. We therefore conclude that the rotamer model proposed for the fluorescence decay of tyrosine and tryptophan cannot be applied to the fluorescence decay of dityrosine and peptides containing this chromophore. This should be of future interest when dityrosine is used as an intrinsic sensor to study complex dityrosine-containing macromolecules by fluorescence spectroscopy.

Time-resolved fluorescence studies of dityrosine in the outer layer of intact yeast ascospores.

The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.

Characterization of human immunodeficiency virus-1 (HIV-1) rev by (time-resolved) fluorescence spectroscopy.

Fluorescence spectroscopy has been applied to the single tryptophan-containing regulatory protein Rev of human immunodeficiency virus (HIV-1). The fluorescence emission was found to have a maximum at 336 nm which refers to a surrounding of the chromophore of intermediate polarity. Fluorescence transients recorded at the maximum of fluorescence were found to decay nonexponentially. A bimodal lifetime distribution is obtained from exponential series analysis (ESM) with centers at 1.7 and 4.5 ns. Two microenvironments for tryptophan are suggested to be responsible for the two lifetime distributions. No innerfilter effect occurred in a Rev solution up to a concentration of 40 µM. A data quality study of ESM analysis as function of collected counts in the peak channel maximum (CIM) showed that, for reliable reconvolution, at least 15,000 CIM are necessary. The widths of the two distributions are shown to be temperature dependent. The broadening of the lifetime distributions when the temperature is raised to 50°C is interpreted as extension of the number of conformational substates which do not interconvert on the fluorescence time scale. The thermal deactivation (temperature quenching) is reflected in a constant decrease in the center of the short-lived lifetime distribution.