Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry

Procedures are described for the treatment of phase and modulation lifetime data in fluorescent systems having multiexponential decay. All computer procedures (called FIT programs) arise from the lifetime resolution theory for phase-modulation measurements (Weber, G (1981) J. Phys. Chem. 85, 949–953). The programs most successful in resolving heterogeneous lifetimes use a Monte Carlo approach in which phase and modulation lifetime data at three modulation frequencies are simultaneously utilized. These programs are shown to have more utility than the final closed form procedure presented by Weber (1981). The FIT routines are simple and require little computer time while yielding excellent results. To illustrate the applicability of these programs, defined binary (carbazole and pyrene) and ternary systems (carbazole, pyrene and POPOD) were examined. In most cases, the resolved lifetimes were within 5% of the independently measured value and the fractional fluorescence contributions were within 10% of that expected. These results demonstrate that phase-modulation measurements analyzed by appropriate computer programs are capable of solving for lifetimes in both binary and, in selected cases, ternary systems. An example is given from the recent literature (Dalbey, R., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696–4706) in which the above programs allowed the resolution of both binary and ternary lifetimes of a dansyl label on myosin, where Förster energy transfer was occuring. These lifetimes] were used to quantify changes in distances between two activity-related thiols on myosin upon the addition of Mg-ATP or its analogs.     

Albumin extinction followed by de novo methylation of its gene in somatic hybrids of a rat hepatoma

We have previously identified an Msp I site at the 5′ end of the rat albumin gene whose undermethylation is necessary but not sufficient for stable albumin expression in rat hepatoma cells [1]. We have also shown that the extinction of albumin expression in somatic hybrids is not the result of methylation at this site, since for two different crosses, rapid extinction was found to occur in the absence of any de novo methylation of the previously active gene[2]. In the present study, we examine albumin expression and albumin gene methylation for independent hybrid clones isolated from crosses between albumin expressing rat hepatoma cells and cells of two different non-expressing lines. The cells from hybrid clones of both crosses are characterized by stable extinction of albumin expression. Moreover, we find that de novo methylation of the “extinguished” albumin gene can occur in somatic hybrids, but only some weeks after the gene has ceased to be expressed.     

A method facilitating the demonstration of synergism in the tissue-responses to mixed peptide stimuli

A methodology for the pharmacodynamical assay of mixtures of 2 spasmogenic peptides is described, illustrated and discussed, with reference to a method intended for the chemists (P. Job, 1927). This allows a quick appreciation of the existence of synergism, of its own order of magnitude and of that of the doses for which it is significant. Also given is a fast way of curve-fitting the dose response to individual peptides, according to Clark's approximation.     

Prolactin increases the density of striatal dopamine receptors in normal and hypophysectomized male rats

Administration of prolactin to adult male rats, by s.c. injection, significantly increases the density of the striatal dopamine (DA) receptors, without altering the apparent affinity of the receptors for [³H]spiroperidol. Larger doses of prolactin are required to increase the density of the striatal DA receptors in hypophysectomized rates compared to normal rats. These results suggest that prolactin might be the common mediator of the increase in striatal DA receptor density produced by either estrogen or haloperidol administration. Monitoring and/or altering prolactin levels might be informative in neurologic or psychiatric disorders involving striatal DA neurotransmission.     

Five 3β, 6α-dihydroxysterols in organ-pipe cactus

The sterol diol fraction from the lipids of organ-pipe cactus, Stenocereus thurberi, was separated into five compounds: macdougallin, peniocerol, cyclostenol, stenocereol and thurberol. The last three compounds have not been described before. All compounds were characterized by physical and spectroscopic properties.     

The structures of garcinones a,b and c: Three new xanthones from Garcinia mangostana

Three new tetraoxygenated xanthones (garcinones A, B and C), each disubstituted with C₅-units, have been isolated from the chloroform extract of the fruit-hulls of Garcinia mangostana. Their structures were established by a combination of spectral interpretation and chemical correlation.     

A rapid and specific fluorescent activity staining procedure for transamidating enzymes.

A rapid, sensitive, and specific procedure has been developed for detection of transsamidatingenzymes (transglutaminase, e.g., factor XIII) after agarose gel electrophoresis. The technique is based on the transamidase-catalyzed incorporation of the fluorescent monodansylthiacadaverine into casein. The high sensitivity enables detection and characterization of transamidases in blood plasma, platelet, and red blood cell lysate and tissue extracts. The technique can also be combined with crossed immunoelectrophoresis.     

Frequency changes with time in vivo of radiation-induced chromosomal aberrations in skin fibroblasts

5 Syrian hamster litter mates were each irradiated with X-rays on one flank to 300 rad. Skin biopsies were taken from both the irradiated and unirradiated (control) flanks of each animal at one day and at about 6 months after irradiation. The cells cultured from these biopsies were used to determine the frequencies of chromosomal aberrations. During the 6-month period there were significant reductions in the frequencies of both reciprocal translocations and terminal deletions.

Translocations involving the short arm of the Y-chromosome, however, showed a significant increase during this period.

It is possible that while the latter phenomenon was due to cell selection in vivo the general fall off in translocations and deletions was the result of a long term in vivo repair mechanism or perhaps the results of certain aberrations proving to be lethal with prolonged expression times.     

Effect of N2 on the mutagenic and lethal activities of ICR-170 in Neurospora crassa

The nature of the N₂ effect for ICR-170, i.e., the greater mutagenic and lethal activities of this agent in the presence of N₂ than O₂, has been studied at the ad-3 region of Neurospora crassa. The characteristics of the N₂ effect for ICR-170 were that (1) the N₂ effect with ICR-170 was displayed in conidia when N₂ was administered during, but not before or after, ICR-170 treatment, (2) the highly increased mutagenic and lethal activities of ICR-170 in the presence of N₂ were due to an anoxic condition rather than to the presence of N₂ per se, (3) the high killing activity of ICR-170 under N₂ was due largely to increased cytoplasmic inactivation, (4) the N₂ effect was a general phenomenon at the ad-3 region of N. crassa, and (5) the highly ICR-170-induced mutation in conidia under N₂ was attributable to an actual enhancement in the mutagenic activity of ICR-170 rather than to selective killing. With regard to the mechanisms of the N₂ effect with ICR-170, results indicate that this effect (1) was not due to more extracellular oxidative degradation of ICR-170 molecules in the presence of O₂, or to a greater uptake of ICR-170 by conidia under N₂, but (2) was due to the inhibition of conidial respiration under an anoxic environment.