The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.     

GABA(A) receptor function is regulated by lipid bilayer elasticity

Docosahexaenoic acid (DHA) and other polyunsaturated fatty acids (PUFAs) promote GABA(A) receptor [(3)H]-muscimol binding, and DHA increases the rate of GABA(A) receptor desensitization. Triton X-100, a structurally unrelated amphiphile, similarly promotes [(3)H]-muscimol binding. The mechanism(s) underlying these effects are poorly understood. DHA and Triton X-100, at concentrations that affect GABA(A) receptor function, increase the elasticity of lipid bilayers measured as decreased bilayer stiffness using gramicidin channels as molecular force transducers. We have previously shown that membrane protein function can be regulated by amphiphile-induced changes in bilayer elasticity and hypothesized that GABA(A) receptors could be similarly regulated. We therefore studied the effects of four structurally unrelated amphiphiles that decrease bilayer stiffness (Triton X-100, octyl-beta-glucoside, capsaicin, and DHA) on GABA(A) receptor function in mammalian cells. All the compounds promoted GABA(A) receptor [(3)H]-muscimol binding by increasing the binding capacity of high-affinity binding without affecting the associated equilibrium binding constant. A semiquantitative analysis found a similar quantitative relation between the effects on bilayer stiffness and [(3)H]-muscimol binding. Membrane cholesterol depletion, which also decreases bilayer stiffness, similarly promoted [(3)H]-muscimol binding. In whole-cell voltage-clamp experiments, Triton X-100, octyl-beta-glucoside, capsaicin, and DHA all reduced the peak amplitude of the GABA-induced currents and increased the rate of receptor desensitization. The effects of the amphiphiles did not correlate with the expected changes in monolayer spontaneous curvature. We conclude that GABA(A) receptor function is regulated by lipid bilayer elasticity. PUFAs may generally regulate membrane protein function by affecting the elasticity of the host lipid bilayer.     

Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections

Summary The mechanism of intracellular maturation and sorting of herpes simplex virus type I glycoproteins is not known in details. To elucidate the intracellular sorting of viral glycoproteins and their possible interaction with the cytoskeleton, a method for simultaneous immunogold staining of three antigens in ultrathin cryosections is described. Each antigen is stained by an indirect technique using mouse monoclonal IgG as first layer, rabbit antimouse IgG as second and gold-conjugated goat anti-rabbit IgG as third layer antibody. After each staining cycle the paraformaldehyde vapour at 80° C for 30 min. This destroys the free antigen combining sites of the second and the third layer IgG and abolish contaminating staining. Simultaneous triple-staining is documented with three mouse monoclonal antisera specific for 1) herpes simplex virus type 1 glycoprotein C, 2) glycoprotein D and 3) - and -tubulin as primary antibodies. Labelling for virus glycoproteins was found in some Golgi vesicles and close to the cytoplasmic microtubules as well as on the cell surface and on intracytoplasmic and extracellular virus particles.Presented in part at the 9th European Congress on Electron Microscopy, York, England, September 4–9, 1988     

Negative relationships between species richness and evenness render common diversity indices inadequate for assessing long-term trends in butterfly diversity

Species richness and evenness, the two principle components of species diversity, are frequently used to describe variation in species assemblages in space and time. Compound indices, including variations of both the Shannon–Wiener index and Simpson’s index, are assumed to intelligibly integrate species richness and evenness into all-encompassing measures. However, the efficacy of compound indices is disputed by the possibility of inverse relationships between species richness and evenness. Past studies have assessed relationships between various diversity measures across survey locations for a variety of taxa, often finding species richness and evenness to be inversely related. Butterflies are one of the most intensively monitored taxa worldwide, but have been largely neglected in such studies. Long-term butterfly monitoring programs provide a unique opportunity for analyzing how trends in species diversity relate to habitat and environmental conditions. However, analyzing trends in butterfly diversity first requires an assessment of the applicability of common diversity measures to butterfly assemblages. To accomplish this, we quantified relationships between butterfly diversity measures estimated from 10 years of butterfly population data collected in the North Saskatchewan River Valley in Edmonton, Alberta, Canada. Species richness and evenness were inversely related within the butterfly assemblage. We conclude that species evenness may be used in conjunction with richness to deepen our understandings of assemblage organization, but combining these two components within compound indices does not produce measures that consistently align with our intuitive sense of species diversity.     

Host plant discrimination within Cruciferae: Feeding responses of four leaf beetles (Coleoptera: Chrysomelidae) to glucosinolates, cucurbitacins and cardenolides

Feeding responses of four Chrysomelidae to six less acceptable plants and to compounds from them were investigated by means of leaf disc tests. Significant differences were found between responses of different species, and plants containing potent feeding inhibitors were always rejected. Cucurbitacins are potent feeding inhibitors to Phyllotreta nemorum, and this species does not eat Iberis species containing these compounds. Cardenolides are potent feeding inhibitors to P. undulata, P. tetrastigma and Phaedon cochleariae, and these three species do not eat the cardenolide containing Cheiranthus and Erysimum.Six different glucosinolates all proved to be stimulatory when applied to pea leaf discs. Although the glucosinolates differed somewhat in their ability to stimulate feeding, no correlation is found between content of glucosinolates and acceptability of the investigated plants. Application of sinigrin to Iberis and Cheiranthus did not improve their acceptability. The presence of glucosinolates is necessary for feeding to occur, but it is less important which glucosinolates are present.Cardenolides and cucurbitacins are suggested to be a second generation of protective compounds in Cruciferae, glucosinolates being the first.

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Zusammenfassung Der Einfluss einiger sekundärer Pflanzenstoffe aus Cruciferen auf die Futteraufnahme von vier Chrysomeliden, die auf dieser Pflanzenfamilie vorkommen, wurde mittels Blattscheiben-Tests untersucht. Cucurbitacine sind starke Frasshemmstoffe für Phyllotreta nemorum, weniger starke Hemmstoffe für P. undulata und schwache Hemmstoffe für P. tetrastigma und Phaedon cochleariae. Iberis-Arten, die Cucurbitacine enthalten, werden von P. nemorum und P. undulata abgelehnt, von den beiden anderen Arten aber akzeptiert. Cardenolid-Glykoside vom Strophanthidin-Typ sind starke Frasshemmstoffe für P. undulata, P. tetrastigma und Phaedon cochleariae. Diese Arten lehnen Cheiranthus-und Erysimum-Arten, die solche Stoffe enthalten, ab. Die Futteraufnahme von P. nemorum wird von diesen Stoffen nicht beeinflusst; P. nemorum akzeptiert Cheiranthus- und Erysimum-Arten.Futteraufnahme fand bei Abwesenheit von Senfölglukosiden nicht statt. Sechs verschiedene Senfölglukoside waren alle imstande, das Aufnehmen von Erbsen-Blattscheiben zu stimulieren. Gewisse Unterschiede in der stimulierenden Wirkung der einzelnen Glukoside wurden gefunden. Das Vorkommen bestimmter Glukoside und die Akzeptabilität der Pflanzen zeigten aber keine Korrelation. Anwesenheit oder Abwesenheit von Frasshemmstoffen beeinflusst die Akzeptabilität der Pflanzenarten mehr als die Anwesenheit bestimmter Senfölglukoside.Wenn Senfölglukoside als eine erste Generation von Abwehrstoffen in Cruciferen aufgefasst werden, können Cucurbitacine in Iberis und Cardenolid-Glykoside in Cheiranthus und Erysimum als eine zweite betrachtet werden.

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The Danish Natural Science Research Council supported the research.     

Seventy years of changes in the abundance of Danish charophytes

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The influence of crop rotation on the onset of early blight (Alternaria solani)

Field experiments were carried out in 2016 and 2017 to determine the effect of crop rotation on the onset of early blight in three potato varieties in Denmark. Six and seven fields with different histories with potato (rotation‐fields) were used for the experiments in 2016 and 2017, respectively. The results showed that variety and the interaction between variety and rotation‐field had no significant effect on the onset of early blight. Rotation‐field, on the other hand, had a significant effect on the onset of early blight in both years of the study. Early blight occurred earlier in fields with <2 years between subsequent potato crops than in fields where potatoes have not been grown for at least 2 years. The onset of early blight was not different between the fields where there had not been potatoes for at least 2 years. For fields that were preceded by potatoes, early blight occurred earlier in fields in which the severity of early blight in the previous years was higher than that in the fields in which the severity of early blight in the previous years was low or moderate. In conclusion, the results of this study suggest that at least a 2‐year interval between subsequent potatoes in a rotation cycle is necessary to delay the onset of early blight. Moreover, the severity of early blight in the previous years can affect the onset of early blight.     

Presence of antibodies against Coxiella burnetii and risk of spontaneous abortion: a nested case-control study

Background and Aims

Q fever is a bacterial zoonosis caused by infection with Coxiella burnetii. It is well established that Q fever causes fetal loss in small ruminants. The suspicion has been raised that pregnant women may also experience adverse pregnancy outcome when the infection is acquired or reactivated during pregnancy. The purpose of this study was to assess the potential association between serologic markers of infection with C.burnetii and spontaneous abortion.

Methods

A nested case-control study within the Danish National Birth Cohort, a cohort of 100,418 pregnancies recruited from 1996–2002. Women were recruited in first trimester of pregnancy and followed prospectively. Median gestational age at enrolment was 8 weeks (25 and 75 percentiles: 7 weeks; 10 weeks). During pregnancy, a blood sample was collected at gestational week 6–12 and stored in a bio bank. For this study, a case sample of 218 pregnancies was drawn randomly among the pregnancies in the cohort which ended with a miscarriage before 22 gestational weeks, and a reference group of 482 pregnancies was selected in a random fashion among all pregnancies in the cohort. From these pregnancies, serum samples were screened for antibodies against C. burnetii in a commercial enzyme-linked immunosorbent assay (ELISA). Samples that proved IgG or IgM antibody positive were subsequently confirmatory tested by an immunofluorescence (IFA) test.

Results

Among cases, 11 (5%) were C. burnetii positive in ELISA of which one was confirmed in the IFA assay compared to 29 (6%) ELISA positive and 3 IFA confirmed in the random sample.

Conclusions

We found no evidence of a higher prevalence of C.burnetii antibodies in serum samples from women who later miscarried and the present study does not indicate a major association between Q fever infection and spontaneous abortion in humans. Very early first trimester abortions were, however, not included in the study.     

Simultaneous demonstration of two antigens in ultrathin cryosections by a novel application of an immunogold staining method using primary antibodies from the same species

A recently developed immunocytochemical double-staining method for ultrathin Epon and Lowicryl K4M sections has been adopted for use on ultrathin cryosections. The essential features of the method include: staining for the first antigen by the indirect method using sufficient concentrations of second antibodies conjugated to colloidal gold particles to saturate available epitopes on the primary antibodies; supporting the cryosections by methyl cellulose followed by paraformaldehyde vapour treatment (30-60 min at 80 degrees C); removal of the methyl cellulose followed by staining for the second antigen using primary antiserum from the same species and another size class of colloidal gold particles conjugated to second antibodies. Contaminating staining does not occur if the paraformaldehyde vapour treatment exceeds 30 min, as this treatment destroys the combining sites on the second antibodies applied in the first staining cycle. Successful double-staining was documented using primary rabbit antibodies to growth hormone and corticotropin and anti-rabbit IgG conjugated to 5 and 15 nm colloidal gold particles. Following double-staining, the ultrathin cryosections may be silver-enhanced to improve detectability of the markers at low magnification.