Simulation of the effect of population size on the evolution of the recombination fraction

A study, by means of computer simulation, has been performed on the evolution of recombination rate modifier genes in a system with three diallelic loci (A, B and C). The locus C, selectively neutral, is responsible for the modification of the recombination fraction between the major loci (A and B) which are subjected to selection. Two models have been analysed, the modifier allele being recessive in one of them, and codominant in the other, with infinite and finite populations. Distinct initial genic frequencies of the major loci and different selection coefficients have been utilised. We have found that the frequency of the allele which favours recombination increases in finite populations, and decreases slightly in infinite populations. These results are consistent with previous theory; presumably, selection favours alleles reducing recombination between epistatically interacting loci in a infinite population, since this reduces the breakup of advantageous combinations of alleles. However, in finite populations, selection favours the breakup of the random linkage disequilibria which are produced by random drift.     

The effect of propylbenzilylcholine mustard on contraction and radioligand binding parameters of muscarinic receptors in guinea pig ileum

The receptor occupancy-biological effect relationship for muscarinic receptors in guinea pig ileal smooth muscle has been studied by comparison of radioligand binding and contractile response. Muscarinic receptors in homogenates of ileal smooth muscle were labeled with [3H]-1-Quinuclidinyl benzilate. Treatment with propylbenzilylcholine mustard (PrBCM), to inactivate irreversibly muscarinic receptors, caused a large dose dependent rightward shift of the dose-response curve to three agonistic furtrethonium derivatives with a concomitant decrease in maximal response. Using those data, the fraction of receptors remaining unoccupied (q-values) and "true affinity constants" (-log KA-values) were calculated. Exposure to 20 or 60 nM PrBCM for 15 minutes resulted in a 39% and a 61% reduction in specific [3H]-1-Quinuclidinyl benzilate binding sites respectively to be compared with a 62% and a 85% decrease expected from calculated q-values. KA-values for the methyl and ethyl derivative agreed well with the dissociation constants for the high affinity agonist sites determined from displacement of [3H-]-1-Quinuclidinyl benzilate. The KA-value for the propylfurtrethonium corresponds to the low affinity agonist dissociation constant. The fraction of receptors in the high affinity agonist state differs considerably for the three furtrethonium derivatives investigated. Neither the fraction of receptors in the high affinity agonist state, nor the ratio of dissociation constants for these states is affected by the alkylation of 85% of the functional muscarinic receptors. The inactivation of components of the effector system by PrBCM seems unlikely.     

β-adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation

Specific binding of [¹²⁵I]-(−)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (K_d=5.3±0.9 pmol/l and R_T=78±7fmol/g tissue). The β₁-selective antagonists atenolol and LK 203-030 inhibited specific [¹²⁵I]-(−)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous β₂-adrenoceptor population. In one subject using LK 203-030 a small β-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pK_H- and pK_L- values for the high and low affinity sites were 8.0±0.2 and 5.9±0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD₂-value of 6.63±0.19.     

Modulation of the immune response by emotional stress

The influence of mild, emotional stress was investigated for its effect on the immune system by subjecting rats to the one-trial-learning passive avoidance test. The reactivity of the immune system was tested by determining the proliferative response after mitogenic stimulation in vitro as well as the capacity to generate a primary antibody response in vivo after immunization with sheep red blood cells. Our results demonstrate that exposure of rats to a single electric footshock (learning trial) or habituation to the passive avoidance apparatus, induces an increase of the immune response in vitro and in vivo. Thus, emotional stimuli seem to facilitate immunological responsiveness. However, when the animal is confronted with a conflict situation, as tested by the retention of the avoidance response after a single learning trial, the initially enhanced reactivity of the immune system decreases. It is concluded that the immune system is capable of reacting specifically and immediately to distinct psychological stimuli.     

Immunological discrimination between self and non-self by precursor depletion and memory accumulation

We study processes by which T-lymphocytes "learn" to discriminate "self" from "non-self". We show that intrinsic features of the T cell activation and proliferation process are sufficient to tolerize (self) reactive T-lymphocyte clones. Self vs non-self discrimination therefore develops without any down-regulatory (e.g. suppressive) interactions. T-lymphocyte clones will expand by proliferation only if the IL2 concentration is high enough to induce a proliferation rate larger than the rate of cell decay. This concentration is the proliferation threshold. Because effector T cells are short-lived the proliferation threshold must be quite high. Such high numbers of cells producing IL2 are achieved only when sufficient (memory) precursors are activated. Self and non-self antigens differ with respect the number of (memory) precursor cells they accumulate, as a result of two processes, i.e. precursor depletion and memory accumulation, and can thus be discriminated. Precursor depletion: the dynamics of long-lived precursors can cause tolerization. In neonatal circumstances precursor influx is still low, newborn cells reacting with self antigens are immediately activated, generating (few), i.e. fewer than the proliferation threshold, effectors that decay rapidly. Thus total lymphocyte numbers remain low, yielding self tolerance. Conversely, large doses of similar antigens introduced in mature systems push "their" lymphocyte clone over the proliferation threshold because a large (accumulated) precursor population is rapidly activated. Small doses are however low zone tolerized. Memory accumulation: peripheral T-lymphocyte populations in fact consist of a mixture of virgin precursors and memory cells. If the formation process of (long-lived) memory cells is taken into account and virgin precursors are made short-lived, the proliferation threshold again accounts for self non-self discrimination. Memory cells accumulate when antigenic restimulation is low; it is low when the antigen concentration and/or the antigen affinity is low. Therefore self antigens, which are present in relatively high concentrations, fail to accumulate high affinity memory cells, and are hence tolerated. Memory cells crossreacting to self antigens with low affinity, however accumulate neonatally, pushing those clones over the proliferation threshold whenever "their" high affinity antigen enters the immune system. Thus the model generates differences in the antigenicity (i.e. memory precursor frequency) of self and non-self.(ABSTRACT TRUNCATED AT 400 WORDS)     

An enzyme-linked immunosorbent assay (ELISA) for human sex hormone-binding globulin

An enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for sex hormone binding globulin (SHBG) has been developed. A rabbit anti-SHBG antibody (RAb) is immobilized to the microtitre plate. After incubation with standards and samples a second monospecific rabbit anti-SHBG antibody, labelled with alkaline phosphatase is added (RAb). Following further washing substrate is added, colour developed and the plate read at 405 nm wavelength on a standard ELISA plate reader. The assay is not influenced by the presence of steroids at the binding site, and shows good agreement to SHBG binding capacity assay and commercially available IRMA. Its sensitivity, specificity and precision allows its use in the routine laboratory. The SHBG ELISA has been used to measure SHBG concentrations in sera of normal men, women, pregnant women, and women receiving high-dose medroxyprogesterone acetate as a treatment of metastatic breast cancer.     

Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied. pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation. After transformation into E. coli cells with different repair capacities (uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed. Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria. Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway (uvrA-dependent). Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain. In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed. Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery. Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells. Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts. The greatest increase in plasmid recovery was found in the wild-type strain. This likely involved the repair of monoadducts and some fraction of the crosslinks. We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.     

The use of bromodeoxyuridine labeling in the human lymphocyte HGPRT somatic mutation assay

The autoradiographic assay developed by Strauss and Albertini (1979) to quantitate human in vivo somatic mutation at the hypoxanthine guanine phosphoribosyl-transferase locus uses tritiated thymidine to identify mutant cells by their ability to pass through 'S' phase in the presence of 6-thioguanine. An alternative method, based on the incorporation of bromodeoxyuridine (BrdUrd) into the DNA of proliferative cells, followed by differential staining with the fluorescence-plus-Giemsa method, was used to identify 3 classes of lymphocyte nuclei: (a) small darkly stained nuclei, (b) large, reddish-colored nuclei with an apparent nucleolus, and (c) large, bluish-colored nuclei. By double labeling with BrdUrd and tritiated thymidine, it was determined that only the nuclei of the third class had incorporated BrdUrd. These results demonstrate that the technique used for sister-chromatid differentiation can be used to detect putative HGPRT mutants and to determine variant frequencies at the HGPRT locus.     

Induction of SOS and adaptive responses by alkylating agents in Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase activities

The induction of SOS and adaptive responses by alkylating agents was studied in Escherichia coli mutants tagA and alkA deficient in 3-methyladenine-DNA glycosylase activities. The SOS response was measured using an sfiA::lacZ operon fusion. The sfiA operon, in the double mutant tagA alkA, is induced at 5-50-fold lower concentrations of all tested methylating and ethylating compounds, as compared to the wild-type strain. In all cases, the tagA mutation, which inactivates the constitutive and specific 3-alkyladenine-DNA glycosylase I (TagI), sensitizes the strain to the SOS response. The sensitization effect of alkA mutation, which inactivates the inducible 3-alkyladenine-DNA glycosylase II (TagII), is observed under conditions which allow the induction of the adaptive response. We conclude that the persistence of 3-methyladenine and 3-ethyladenine residues in DNA most likely leads to the induction of the SOS functions. In contrast, the adaptive response, evaluated by O6-methylguanine-DNA methyltransferase activity in cell extracts, was not affected by either tagA or alkA mutations. The results suggest that the SOS and adaptive responses use different alkylation products as an inducing "signal". However, adaptation protein TagII inhibits the induction of the SOS response to some extent, due to its action at the level of signal production. Finally, we provide conditions to improve short-term bacterial tests for the detection of genotoxic alkylating agents.     

Release of dormancy during after-ripening of Agrostemma githago seeds

Freshly harvested seeds of Agrostemma githago L. do not germinate when they are imbibed at 20°C. The block is located in the embryo and is relased by dry storage at 20°C (after-ripening). Freshly harvested seeds complete only a small part of the processes that occur in after-ripened seeds during the lag phase prior to germination (radicle protrusion). After-ripening removed the block on lag phase processes much faster than the block on germination. This was shown both by direct determinations of the completion of lag phase processes and by measurements of the rate of axial protein synthesis, which approximately doubles when seeds are progressing through the lag phase. It is concluded that the percentage germination does not adequately reflect the extent to which the dormancy mechanism has been overcome.