MONITORING ULTRAVIOLET-B-INDUCED DNA DAMAGE IN INDIVIDUAL DIATOM CELLS BY IMMUNOFLUORESCENT THYMINE DIMER DETECTION1

We developed a method to investigate the effect of ultraviolet-B radiation (UVBR) on the formation of thy-mine dimers in microalgal DNA that can be used for both laboratory and in situ research. Antibody labeling of dimers was followed by a secondary antibody (fluorescein isothiocyanate) staining to allow visualization of DNA damage with flow cytometry or fluorescence microscopy. Thymine dimer-specific fluorescence in nuclear DNA of the marine diatom Cyclotella sp. was linearly related to the UVBR dose. Simultaneous measurements of cellular DNA content showed that the vulnerability of G2 cells to DNA damage did not differ significantly from the vulnerability of G1 cells. The formation and removal of thymine dimers in Cyclotella sp. cells was monitored for 3 consecutive days at two realistic UVBR irradiance levels. Thy-mine dimers were removed within 24 h when exposed to a saturating photosynthetically active radiation intensity following the UVBR treatment. This new method allows the study of UVBR-induced DNA damage on a cell-to-cell basis. It is also feasible for field studies because cells remain intact and can be recognized readily after antibody treatment.     

Enzymatic Elimination of Collagen in the Alcian Blue Staining of Connective Tissue Ground Substance

Collagenase pretreatment of frozen-dried sections permits Alcian blue staining of mucopolysaccharides of connective tissue ground substance without the interference of collagen staining. Hyaluronidase elimination of Alcian blue staining confirms mucopolysaccharide as a substrate of the staining reaction.     

Planktonic copepods of the Bras d'Or Lakes system,Nova Scotia,Canada

Fifteen species of planktonic copepods are recorded from Bras d'Or Lakes, Nova Scotia, Canada. Pseudocalanus minutus, Oithona similis, Temora longiremis, and Tortanus discaudatus are the dominant species. The distribution of planktonic copepods in Bras d'Or Lakes as well as in the adjacent Gulf of St. Lawrence is discussed.     

Interaction of the B subunit of cholera toxin with endogenous ganglioside GM1 causes changes in membrane potential of rat thymocytes

Summary The fluorescent anionic dye, bisoxonol, and flow cytometry have been used to monitor changes in the membrane potential of rat thymocytes exposed to the B subunit of cholera toxin. The B subunit induced a rapid hyperpolarization, which was due to activation of a Ca²⁺-sensitive K⁺ channel. Reduction of extracellular Ca²⁺ to <1 m by the addition of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid immediately abolished the hyperpolarization caused by the B subunit. Cells treated with quinine and tetraethylammonium lost their ability to respond to the B subunit, whereas 4-aminopyridine did not have any effect. Thus, calcium-sensitive and not voltage-gated K⁺ channels appeared to be responsible for the hyperpolarization. The results of ion substitution experiments indicated that extracellular Na⁺ was not essential for changes in membrane potential. Further studies with ouabain, amiloride and furosemide demonstrated that electrogenic Na⁺/K⁺ ATPase, Na⁺/H⁺ antiporter and Na⁺/K⁺/Cl^– cotransporter, respectively, were not involved in the hyperpolarization process induced by the B subunit. Thus, crosslinking of several molecules of ganglioside GM1 on the cell surface of rat thymocytes by the pentavalent B subunit of cholera toxin modulated plasma membrane permeability to K⁺ by triggering the opening of Ca²⁺-sensitive K⁺ channels. A role for gangliosides in regulating ion permeability would have important implications for the function of gangliosides in various cellular phenomena.     

Fractal analysis on root systems of rice plants in response to drought stress

A fractal analytical method was used to examine the developmental responses of root systems in upland rice genotype CT9993-5-10-1-M (japonica) and lowland genotype IR62266-42-6-2 (indica) (abbreviated as CT9993 and IR62266, respectively) to soil water stress. The root systems were grown for one month in root boxes with 25 cm in length, 2 cm in width and 40 cm in depth, which were filled with soil. The root systems were sampled by following the needle-pinboard method, and then spread on the transparent plastic films with nets after carefully washing out the soils. The two-dimensional images of root systems were digitized by using a scanner. The digitized images were used for analysis based on fractal geometry with the box-counting method. The reductions in shoot dry weight, photosynthesis rate and transpiration rate of IR62266 by soil drought were greater than those of CT9993. The change of fractal parameters in response to soil moisture conditions differed between the two rice genotypes. The values of fractal abundance (FA) and fractal dimension (FD) in well-watered IR62266 plants were larger than in CT9993. The value of FA of IR62266 was decreased more by drought stress than that of CT9993, indicating that the volume of soils explored by the whole root systems of CT9993 was maintained or less decreased under drought stress in comparison to IR62266. Moreover, the values of FD tended to increase in CT9993 while it tended to decrease in IR62266 in response to drought. These root responses detected by the fractal analysis in CT9993 may be advantageous for its extracting more water from drying soils, which explains its better growth under drought-stressed condition.