Analysis of non-linearities in frequency curves for UV-induced mitotic recombination in wild-type and excision-repair-deficient strains of yeast

Frequency curves for UV-induced mitotic recombination often are linear at low doses. As dose increases, these curves either increase at higher powers of dose and/or reach a maximum induced frequency and then decline. Similar dose-response patterns have been observed previously for mutation. The non-linearities can arise from higher order effects inherent in the molecular mechanisms of mutagenesis and/or from 'delta-effects' (Eckardt and Haynes, 1977a), i.e., differential probabilities of clone formation for mutant and non-mutant cells. Previously, we have shown that one can distinguish between these two possibilities by plotting the ratio of the induced mutant yield to the linear component of frequency as a function of dose (Haynes et al., 1985). In this study, we have used this ratio, a quantity we call 'apparent survival', to analyse the non-linear regions of the dose-response curves for UV-induced mitotic crossing-over and gene conversion in wild-type (RAD) and excision-repair-deficient (rad3) strains of yeast. Plots of apparent survival versus dose reveal the existence of a positive, non-linear component associated with UV-induced gene conversion in RAD, but not rad3, cells. A high dose decline in frequency, which is observed for UV-induced recombination in both strains, can be attributed to delta-effects.     

Induction of somatic and male crossing-over by bleomycin in Drosophila melanogaster

Bleomycin (BLM) is well known as an antibiotic as well as for its antineoplastic activity. A clinical preparation of BLM was tested for its recombinogenicity in a higher eukaryotic organism, Drosophila melanogaster. Feeding of the F1 larvae on a medium with BLM increased somatic crossing-over spots on female tergites and induced recombination in male germ cells. However, nonlinear dose-response curves were obtained. Malformed tergites were also observed in females treated with BLM.     

Homeologous recombination in Solanum lycopersicoides introgression lines of cultivated tomato

A library of "introgression lines" containing Solanum lycopersicoides chromosome segments in the genetic background of cultivated tomato (Lycopersicon esculentum) was used to study factors affecting homeologous recombination. Recombination rates were estimated in progeny of 43 heterozygous introgressions and whole-chromosome substitution lines, together representing 11 of the 12 tomato chromosomes. Recombination within homeologous segments was reduced to as little as 0-10% of expected frequencies. Relative recombination rates were positively correlated with the length of introgressed segments on the tomato map. The highest recombination (up to 40-50% of normal) was observed in long introgressions or substitution lines. Double-introgression lines containing two homeologous segments on opposite chromosome arms were synthesized to increase their combined length. Recombination was higher in the double than in the single segment lines, despite a preference for crossovers in the region of homology between segments. A greater increase in homeologous recombination was obtained by crossing the S. lycopersicoides introgression lines to L. pennellii--a phylogenetically intermediate species--or to L. esculentum lines containing single L. pennellii segments on the same chromosome. Recombination rates were highest in regions of overlap between S. lycopersicoides and L. pennellii segments. The potential application of these results to breeding with introgression lines is discussed.     

Tachyphylaxis to inhaled aerosolized histamine in anesthetized dogs

Three consecutive dose-response curves to inhaled aerosolized histamine, separated by 1-h intervals, were obtained in 20 anesthetized mongrel dogs. In general, successive histamine dose-response curves shifted progressively rightward. Changes in pulmonary resistance (RL) and dynamic compliance (Cdyn) in response to low concentrations of histamine were reproducible, but responses to high concentrations (sufficient to at least double RL or decrease Cdyn by at least 30%) decreased on successive dose-response curves. The concentration of histamine required to double RL increased significantly (P less than 0.05) from 1.01 mg/ml on the first to 1.62 and 2.02 mg/ml on the second and third dose-response curves. In contrast, consecutive methacholine dose-response curves were not significantly different. Indomethacin pretreatment (5 mg/kg iv) prevented histamine tachyphylaxis, whereas atropine (4 mg iv) did not. However, indomethacin did not alter base-line pulmonary mechanics or histamine responsiveness as measured on the first dose-response curve. We conclude that tachyphylaxis to inhaled aerosolized histamine occurs in anesthetized dogs. Our results are consistent with an important role for endogenous prostaglandins in modulating the airway responses to repeated histamine exposures.     

Structural dynamics of individual Holliday junctions

The four-way DNA (Holliday) junction is the central intermediate of genetic recombination, but the dynamic aspects of this important structure are presently unclear. Although transitions between alternative stacking conformers have been predicted, conventional kinetic studies are precluded by the inability to synchronize the junction in a single conformer in bulk solution. Using single-molecule fluorescence methodology we have been able to detect these transitions. The sequence dependence, the influence of counterions and measured energetic barriers indicate that the conformer transition and branch migration processes share the unstacked, open structure as the common intermediate but have different rate-limiting steps. Relative rates indicate that multiple conformer transitions occur at each intermediate step of branch migration, allowing the junction to reach conformational equilibrium. This provides a mechanism whereby the sequence-dependent conformational bias could determine the extent of genetic exchange upon junction resolution.     

The inhibition of food intake in the dog by LDS, mescaline, psilocin, d-amphetamine and phenylisopropylamine derivatives.

Both LSD and $\text{d}$-amphetamine decreased food intake in the dog; however, the dose-response curve for amphetamine had a significantly steeper slope than that for LSD. Psilocin and mescaline also suppressed food intake with dose-response curves parallel to that of LSD. Seven ring-substituted phenylisopropylamine derivatives, that have been abused for their supposed hallucinogenic properties, also decreased food intake and produced dose-response curves parallel to that of $\text{d}$-amphetamine. It is concluded that the appetite suppressant effects of amphetamine and LSD can be distinguished by differences in slopes of the dose response curves. However, this difference does not pertain to all LSD-like drugs since some substituted amphetamines with LSD-like neuropharmacologic and behavioral profiles can show slopes for appetite suppression as steep as that of amphetamine.     

Photodynamic toxicity of porphyrins and chlorins for a human tumor cell line: combined light and concentration dose responses for the retained fraction

In recent years porphyrins and related materials have been tested as antitumor agents. A technique was devised to obtain dose-response curves for the sensitizer fraction that resists one day of elution by tissue culture medium--the retained fraction. We found a steep "threshold" dose response relationship that helps to explain tumor destruction without damage to normal tissues. The family of dose-response curves produced by a wide range of light exposures suggests that chlorins and porphyrins do not act by identical mechanisms. Moreover, they suggest that chlorins will prove superior in practical use.     

Kinetics of indole acetic acid-induced growth in hypocotyl sections of Vigna mdiata

The kinetics of auxin-induced elongation of segments from Vigna radiata (L.) Wilczek hypocotyls have been investigated using auxanometer measurements. Doseresponse curves were established for several well-defined parameters of the growth response. The experimental data revealed that different kinetic parameters were affected differently by increasing auxin concentrations. The dose-response curves are slightly sigmoid for fresh weight, nearly bell-shaped for total elongation or maximal elongation rate, and nearly linear for maximal growth acceleration. The effects of auxin concentration on regulation of growth orientation are discussed.
A biphasic response is observed mainly with segments taken from the middle of the hypocotyl ('C') which exhibit maximal growth rates. Segments from other levels, with lesser growth potentials, exhibit a very weak response. The two successive phases may then require different maturation states. Kinetics of the acceleration curves are very similar all along the hypocotyl and remain very homogeneous with increasing IAA concentrations.     

Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells

The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to gamma-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of approximately 2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the alpha parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.     

Steady-state enzyme kinetics with high-affinity substrates or inhibitors. A statistical treatment of dose–response curves

A statistical treatment of steady-state enzyme kinetic measurements is described that allows for depletion of free substrate or free inhibitor concentrations owing to significant binding to the enzyme. V(max.), K(m) or K(i), enzyme concentration, the concentration of substrate or inhibitor required for a half-maximal effect and standard errors of these parameters can be calculated from dose-response measurements; the concentration of each component of the system may be estimated also. The statistically best values of the parameters are used to convert dose-response curves into convenient linear forms. The method is applied to dose-response measurements of hydroxyquinoline N-oxide inhibition of bacterial respiration and aminopterin inhibition of dihydrofolate reductase. Two FORTRAN programs for this method have been deposited as Supplementary Publication no. SUP 50019 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973) 131, 5.     

On the evolution of mutation in changing environments: recombination and phenotypic switching

Phenotypic switching has been observed in laboratory studies of yeast and bacteria, in which the rate of such switching appears to adjust to match the frequency of environmental changes. Among possible mechanisms of switching are epigenetic influences on gene expression and variation in levels of methylation; thus environmental and/or genetic factors may contribute to the rate of switching. Most previous analyses of the evolution of phenotypic switching have compared exponential growth rates of noninteracting populations, and recombination has been ignored. Our genetic model of the evolution of switching rates is framed in terms of a mutation-modifying gene, environments that cause periodic changes in fitness, and recombination between the mutation modifier and the gene under selection. Exact results are obtained for all recombination rates and symmetric fitnesses that strongly generalize earlier results obtained under complete linkage and strong constraints on the relation between fitness and period of switching. Our analytical and numerical results suggest a general principle that recombination reduces the stable rate of switching in symmetric and asymmetric fitness regimes and when the period of switching is random. As the recombination rate increases, it becomes less likely that there is a stable nonzero rate of switching.     

The rates of dissociation and reassociation of the subunits of human chorionic gonadotropin

A large change in quantum yield of the fluorescent probe 1,8-anilinonaphthalene sulfonate is produced when it combines with the glycoprotein hormone, human chorionic gonadotropin. A method of analyzing for the hormone in the presence of its subunits has been developed based on the finding that the subunits have no effect on 1,8-anilinonaphthalene sulfonate fluorescence. Quantitative rates of dissociation and recombination can be obtained with very small concentrations of hormone since fluorescence measurements are fast and sensitive. The effects of temperature, pH, and urea concentration on the rate of human chorionic gonadotropin dissociation have been measured. The rates of recombination of subunits have been studied as a function of temperature, pH, and KCl concentration. Human chorionic gonadotropin is stable in water to pH 12 and pH 4.5 at 37 °C.     

Analysis of dose-dependent antibody titration curves

Several types of dose-response titration curves were considered. It was demonstrated that the use of the so-called coordinates of dilution suggested earlier by us allows one to analyze the titration curves, obtained either by ELISA, or by agglutination. Theoretical curves, obtained by the developed theory are very similar to those obtained in experiments. It was shown, that the analysis of the titration curves could give important information concerning antibody-blocking factors in titration sera or other samples of studied antibodies.     

Role of DNA Sequences in Genetic Recombination in the Iso-1-Cytochrome c Gene of Yeast. I. Discrepancies between Physical Distances and Genetic Distances Determined by Five Mapping Procedures

Recombination rates have been examined in two-point crosses of various defined cyc1 mutants using five mapping methods. Nucleotide sequences of mutant codons were identified in previous studies from alterations in functional iso-1-cytochromes c produced by intragenic revertants. Heteroallelic diploids were analyzed for rates of mitotic recombination that occurred spontaneously and that were induced with x-rays, ultraviolet light and the near-ultraviolet light emitted by sunlamps, as well as rates of meiotic recombination that occur after sporulation. Frequencies of both mitotic and meiotic recombination do not necessarily correspond with physical distances separating altered nucleotides. The most extreme discrepancy involved two adjacent intervals of thirteen basepairs which differed approximately thirty-fold in their spontaneous and X-ray-induced recombination rates. Marked disproportions between genetic and physical distances appear to be due to the interaction of the two nucleotide sequences in the heteroallelic combination and not to the sequences of the mutant codons alone. Recombination values that were obtained by all five methods could not be used to establish to correct order of mutant sitesmrelationships of the recombination rates for the various pairwise crosses are different after mitosis from those after meiosis, suggesting that these two recombinational processes are to some extent different in their dependence on particular nucleotide configurations. On the other hand, the relationships of the rates induced by UV-, sunlamp- and X-irradiation were identical or very similar. In addition to the intrinsic properties of the alleles affecting frequencies of mitotic and meiotic recombination rates, two- to threefold variations in recombination rates could be attributed to genetic backgrounds.     

Toll-Like Receptor Induced CD11b and L-Selectin Response in Patients with Coronary Artery Disease

Toll-Like Receptor (TLR) -2 and -4 expression and TLR-induced cytokine response of inflammatory cells are related to atherogenesis and atherosclerotic plaque progression. We examined whether immediate TLR induced changes in CD11b and L-selectin (CD62L) expression are able to discriminate the presence and severity of atherosclerotic disease by exploring single dose whole blood TLR stimulation and detailed dose-response curves. Blood samples were obtained from 125 coronary artery disease (CAD) patients and 28 controls. CD11b and L-selectin expression on CD14+ monocytes was measured after whole blood stimulation with multiple concentrations of the TLR4 ligand LPS (0.01–10 ng/ml) and the TLR2 ligand P3C (0.5–500 ng/ml). Subsequently, dose-response curves were created and the following parameters were calculated: hillslope, EC50, area under the curve (AUC) and delta. These parameters provide information about the maximum response following activation, as well as the minimum trigger required to induce activation and the intensity of the response. CAD patients showed a significantly higher L-selectin, but not CD11b response to TLR ligation than controls after single dose stimulations as well as significant differences in the hillslope and EC50 of the dose-response curves. Within the CAD patient group, dose-response curves of L-selectin showed significant differences in the presence of hypertension, dyslipidemia, coronary occlusion and degree of stenosis, whereas CD11b expression had the strongest discriminating power after single dose stimulation. In conclusion, single dose stimulations and dose-response curves of CD11b and L-selectin expression after TLR stimulation provide diverse but limited information about atherosclerotic disease severity in stable angina patients. However, both single dose stimulation and dose-response curves of LPS-induced L-selectin expression can discriminate between controls and CAD patients.     

A minimal model of non-hyperbolic enzyme and receptor kinetics

A simplified version of P.W. Kühl's Recovery Model [Biochem. J. 298 (1994) 171-180] has been developed in which the duration of the recovery phase of receptor or enzyme (macro)molecule was assumed to be a random value distributed exponentially like other model parameters. The model has been shown to retain all the properties of the original Recovery Model except for its ability to yield asymmetric dose-response curves (if plotted in semi-logarithmic scale). Due to its simplicity, the present model is applicable for routine fitting to experimental data. In enzyme kinetics, the model yields a diversity of non-hyperbolic dose-response curves both with higher and lower steepness than that of Henri-type ones. In receptor kinetics, the diversity of dose-response curves is further increased due to virtually no restraints being imposed on the efficacies of any state of the macromolecule.     

Photoreceptor sensitivity and the shot noise of chemical processes.

The general modeling of dose-response curves to very low stimuli in a photosensory-effector system is critically reshaped starting from basic assumptions on the fluctuations of chemical signals inside the receptor cell, which add to those of the stimulus itself, both arising from their granular (or quantal) structure. We have shown, both through the analytical treatment of a simple kinetic scheme and by means of Monte Carlo simulations of the same, that shot noise arising from chemical transduction ("chemical shot noise") contributes considerably to the output noise of the receptor-effector system, thus affecting both the shape and the abscissa shift of dose-response curves under these conditions; the latter phenomenon has indeed been reported in Halobacterium halobium. After evaluating the general properties of a single-step amplifying mechanism, the effects of introducing several low-amplifying steps in cascade were investigated briefly. The results obtained were qualitatively and quantitatively at variance from those of earlier models on the same phenomenon, and the discrepancies are discussed in order to highlight the fundamental contribution of chemical shot noise to the response of any kind of sensory system to very low stimuli.     

Growth control variant cell line having increased serum requirement and decreased response to platelet-derived growth factor: reversion by 5-azacytidine

Variants of the mouse embryo fibroblast X melanoma hybrid clone 100A have been isolated by a procedure that selects against cells that are able to grow in medium containing low concentrations of serum plus insulin. Three variant clones derived from this selection were found to have a much higher serum requirement than the parental clone 100A cells, as evidenced by a very low rate of DNA synthesis and growth in medium containing low concentrations of serum. Two of the variants had approximately double the number of chromosomes as the parental cell line, while one had approximately the same number of chromosomes as the parental cells. One of the variants was very strongly reverted by 5- azacytidine but not by ethyl methanesulfonate, suggesting that it reverted by a nonmutational mechanism such as a stable change in DNA methylation. Analysis of the growth requirements in hormone- supplemented serum-free media of the 100A parent, the INS 471 variant, and revertants of the variant indicated that the variant had a specific deficiency in its growth response to platelet-derived growth factor (PDGF). PDGF dose-response curves obtained with the variant cells were shifted approximately an order of magnitude toward higher PDGF concentrations relative to PDGF dose-response curves obtained with the parental 100A cells. This quantitative increase in PDGF requirement of the INS 471 variant appears to explain the increased serum requirement of this variant. Equilibrium binding experiments performed with 125I- PDGF suggest that the variant does not have a decreased number of PDGF receptors.     

Induced structural changes in chromosomes of the Syrian hamster after X-irradiation of spermatogonia: comparison of dose-response curves derived from synaptonemal complexes and from air-dried preparations of metaphase I

The dose-response relationships of the induction of structural change in chromosomes after X-irradiation of spermatogonia have been determined from analyses of synaptonemal complexes in pachytene spermatocytes and from air-dried preparations of metaphase I. The dose-response curves were superficially the same shape, with a peak yield of cells containing a multivalent at 4 Gy, although only in the pachytene data was there any statistically significant hump. The pachytene preparations were much more sensitive, revealing nearly twice the proportion of cells containing multivalents than found at metaphase.