Some Properties of New DNA-Specific Bisbenzimidazole Fluorochromes without a Piperazine Ring

Three new bisbenzimidazole (BBI) compounds, which differ from Hoechst 33258 mainly by substitution of a N-dimethylaminopro-pylcarboxamide group in place of the N-methyl-piperazine ring, were studied for their DNA- and AT-base pair specificity as well as for their ability to be quenched by incorporated 5-bromodeoxy-uridine (BrdU). Each of them had DNA binding specificity comparable to or greater than that of Hoechst 33258 and each had a greater specificity for AT-rich regions than did Hoechst 33258. The dependence of fluorescence of new dyes on the BrdU-incorporation into DNA is different from that of Hoechst 33258 and related compounds with piperazine ring. The quenching effect is much weaker, and two of the new compounds (BBI-1 and BBI-2) even show somewhat enhanced binding (fluorescence) at lower concentrations. Certain BBI dyes without piperazine ring may have some advantage over Hoechst for accurate DNA [AT-specific] measurements. The piperazine ring appears to play an important role in the yet unknown mechanism of Hoechst quenching by incorporated BrdU.     

Bivariate flow cytometry of farm animal chromosomes: a potential tool for gene mapping

Bivariate flow karyotypes of chromosomes from sheep, cattle and pig lymphocytes and from a cattle-mouse somatic cell hybrid line were obtained using a dual laser fluorescence-activated cell sorter (FACS). Pig chromosomes were resolved into 19-20 peaks, indicating that most, if not all, pig chromosomes could be separated by this technique. Sheep chromosomes showed incomplete separation but three clear peaks, presumably representing the three large metacentric chromosomes, plus five other clusters were obtained. Cattle chromosomes showed poor separation but about four peaks could be distinguished, indicating that certain chromosomes could be sorted in this species. The use of cattle-mouse hybrids may enable other individual cattle chromosomes to be obtained. It is concluded that FACS separation will be a useful additional tool for gene mapping.     

Dipole source localization by the mottled sculpin II. The role of lateral line excitation patterns

Extracellular, single unit recording techniques were used to measure the responses of posterior lateral line nerve fibers to a 50-Hz dipole source that slowly changed its location along the length of the fish. The flow-field equations for a dipole source were used to model the pressure gradient pattern and thus, the expected excitation pattern along a linear array of lateral line receptor organs for different source locations. Finally, excitation patterns were similarly modeled along the left and right side of the fish's head for actual steps taken by sculpin in approach pathways to the 50-Hz dipole source. Spatial histograms of posterior lateral line nerve fiber responses to different locations of the dipole source could be predicted from pressure gradient patterns modeled from the flow-field equations, confirming that the modeling approach applied to behavioral results was a good predictor of excitation patterns likely to be encoded by the lateral line periphery. An examination of how modeled excitation patterns changed from one position to the next in typical approach pathways and how patterns differed between positions from which successful and unsuccessful strikes were launched suggests that approach and strike strategies can indeed be explained by the information available in excitation patterns. In particular, changes in the spatial distribution of pressure gradient directions (polarities), available only when the source is lateral (as opposed to directly in front of the fish), appear to enhance the ability of sculpin to determine source distance. Without such information, misses are more likely to occur and successful strikes are more likely to be launched from short distances only. Accepted: 23 October 1996     

Oligomerized backbone pilin helps piliated Lactococcus lactis to withstand shear flow

The present work focuses on the role of pili present at the cell surface of Lactococcus lactis in bacterial adhesion to abiotic (hydrophobic polystyrene) and biotic (mucin-coated polystyrene) surfaces. Native pili-displaying strains and isogenic derivatives in which pilins or sortase C structural genes had been modified were used. Surface physico-chemistry, morphology and shear-flow-induced detachment of lactococcal cells were evaluated. The involvement of pili in L. lactis adhesion was clearly demonstrated, irrespective of the surface characteristics (hydrophobic/hydrophilic, presence or not of specific binding sites). The accessory pilin, PilC, and the backbone pilin, PilB, were revealed to play a major role in adhesion, provided that the PilB was present in its polymerized form. Within the population fraction that remained attached to the surface under increasing shear flow, different association behaviors were observed, showing that pili could serve as anchoring sites thus hampering the effect of shear flow on cell orientation and detachment.     

In vitro dynamics of chromatin organization and migration

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.     

Effect of two pyrimidine analogs on accumulation of tubulin in NHIK 3025 cells

Summary Accumulation of tubulin as compared with the accumulation of total cellular protein in human NHIK 3025 cells treated with the sulfone 2-(2-thenyl)sulfonyl-5-bromopyrimidine (NY 4137) and the sulfoxide 2-(2thenyl)sulfinyl-5-bromopyrimidine (NY 4138), two mitotic inhibitors, were investigated by two-parametric flow cytometry. Following a 4 h treatment with NY 4137 tubulin accumulation is inhibited while total protein continues to accumulate. After treatment for 4h with NY 4138 the accumulation of total protein is approximately constant, while the accumulation of tubulin is reduced although not to the same degree as that found for NY 4137-treated cells. In addition, the percentage tubulin SH-groups (6.89 ± 0.14) remaining after treatment of purified rat brain tubulin with NY 4137 or NY 4138 was determined. Treatment with 0.0125 mM NY 4137 reduced the number of tubulin SH-groups detectable with dithiobis benzoate or from 6.89 ± 0.14 before treatment to about 4 after treatment. However, practically all SH-groups of tubulin remain detectable following treatment with the same concentration of NY 4138. From the results described in this report we infer that NY 4137 binds to tubulin SH-groups and that inhibition of tubulin accumulation follows as a secondary effect.     

Is embryo abortion a post‐zygotic barrier to gene flow between Littorina ecotypes?

Genetic incompatibilities contribute to reproductive isolation between many diverging populations, but it is still unclear to what extent they play a role if divergence happens with gene flow. In contact zones between the "Crab" and "Wave" ecotypes of the snail Littorina saxatilis, divergent selection forms strong barriers to gene flow, while the role of post‐zygotic barriers due to selection against hybrids remains unclear. High embryo abortion rates in this species could indicate the presence of such barriers. Post‐zygotic barriers might include genetic incompatibilities (e.g. Dobzhansky–Muller incompatibilities) but also maladaptation, both expected to be most pronounced in contact zones. In addition, embryo abortion might reflect physiological stress on females and embryos independent of any genetic stress. We examined all embryos of >500 females sampled outside and inside contact zones of three populations in Sweden. Females' clutch size ranged from 0 to 1,011 embryos (mean 130 ± 123), and abortion rates varied between 0% and 100% (mean 12%). We described female genotypes by using a hybrid index based on hundreds of SNPs differentiated between ecotypes with which we characterized female genotypes. We also calculated female SNP heterozygosity and inversion karyotype. Clutch size did not vary with female hybrid index, and abortion rates were only weakly related to hybrid index in two sites but not at all in a third site. No additional variation in abortion rate was explained by female SNP heterozygosity, but increased female inversion heterozygosity added slightly to increased abortion. Our results show only weak and probably biologically insignificant post‐zygotic barriers contributing to ecotype divergence, and the high and variable abortion rates were marginally, if at all, explained by hybrid index of females.     

模拟穿透雨减少对锐齿栎(Quercus aliena var. acuteserrata)树干液流密度的影响

降水格局变化是全球气候变化的重要特征之一,未来气候变化下,较为频繁和严峻的干旱将威胁地球中纬度部分地区的森林,但森林植被如何响应季节性干旱胁迫及其机制尚不清楚。北亚热带-暖温带过渡区分布着以锐齿栎(Quercus aliena var.acuteserrata)为优势树种的落叶阔叶林,研究其水分蒸腾代谢过程对干旱的响应是评估气候变化对过渡区天然落叶阔叶林生态系统水碳影响的关键科学问题。在典型的锐齿栎天然林中通过开展模拟穿透雨减少大型野外实验,采用Granier热扩散式探针技术监测锐齿栎树干液流密度的动态变化,研究了不同径级锐齿栎树干液流密度对模拟干旱的响应规律。结果表明:(1)穿透雨减少对树干液流密度的影响呈现季节变异。在7月份,林内穿透雨减少显著降低了锐齿栎的树干液流密度,但生长季后期的10月份林内穿透雨减少反而使锐齿栎树干液流密度显著升高。(2)不同径级的锐齿栎树干液流密度在生长季内对干旱有不同的响应,特别是小径级的树干液流密度与其他径级有较多的不同。小径级的锐齿栎树干液流密度在5、7月份表现为减雨样地显著小于对照样地,在9、10月份则表现为减雨样地显著大于对照样地。中径级的锐齿栎树干液流密度在5、10月份表现为减雨样地显著大于对照样地,在7月份则表现为减雨样地极显著小于对照样地。大径级的锐齿栎树干液流密度在6、7月份表现为减雨样地显著小于对照样地,在10月份则表现为减雨样地显著大于对照样地。