Rapid backward movement of anaphase chromosome whose kinetochore fibers were cut by ultraviolet microbeam irradiation

Summary— kinetochore spindle fibers in meiosis I and II grasshopper spermatocytes were cut with a heterochromatic ultraviolet (UV) microbeam converging on the specimen to form a slit-shaped microspot 1.5 × 8 μm or 3 × 8 μm. A total exposure of 3 × 10^?8 joules per μm² was administered within 0.8–2.4 s, which was sufficient for severing. The cells were observed with a high extinction polarizing microscope or phase contrast optics and a record made by time-lapse video microscopy, continuously before, during and after the irradiation. When kinetochore fibers were irradiated i anaphase with UV, an area of reduced birefringence (ARB) was produced at the exposed site. The newly created + ends of the microtubules rapidly disassembled poleward, at a constant speed of 17 μm/min. The — ends at the edge of ARB also depolymerized at a slower rate. When a kinetochore fiber was cut with UV in early anaphase at which time its associated chromosome had not disjoined from the partner chromosome, the chromosome of the irradiated kinetochore fiber moved rapidly back to its partner. The speed during this movement was faster than the normal poleward chromosome movement in anaphase by an order to magnitude or more. When a kinetochore and its associated kinetochore fiber were included in the irradiation are, the effects were more pronounced than the effects of irradiation on a kinetochore fiber alone; the direction of the line connecting the irradiated half-bivalent with the partner half-bivalent deviated so much from the longitudinal axis of the original spindle with time that the division assumed a tripolar figure.     

Inhibition of ATP-regulated K+-channels by a photoactivatable ATP-analogue in mouse pancreatic β-cells

The effects of a photoactivatable (DMNPE-caged) ATP-analogue on ATP-regulated K⁺-channels (K_ATP-channel) in mouse pancreatic β-cells were investigated using the inside-out patch configuration of the patch-clamp technique. The caged precursor caused a concentration-dependent reduction of channel activity with a K_i of 17 μM; similar to the 11 μM obtained for standard Mg-ATP. The small difference in the blocking capacity between the precursor and ATP is probably the reason why no change in channel activity was observed upon photolysis of the caged molecule and liberation of ATP. It is suggested that the part of the ATP molecule involved in the blocking reaction of the K_ATP-channel is not sufficiently protected in DMNPE-caged ATP making this compound unsuitable for studying the rapid kinetics of ATP-induced K_ATP-channel inhibition.     

Repair mechanisms of UV-induced DNA damage in soybean chloroplasts

In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage rapir in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.     

Radiative properties of hardwood leaves to ultraviolet irradiation

Spectral reflectance and transmittance of leaves to ultraviolet irradiation were determined under laboratory conditions for seven species of hardwood trees, namely red oak (Quercus rubra, L), black oak (Q. velutina, Lamarch), white oak (Q. alba, L.), sugar maple (Acer saccharum), Norway maple (A. plantanoides), hickory (Carya tomemtosa), sweetgum (Liquidambar styraciflua), and black oak litter. The experimental system consisted of a solar simulator, an integrating sphere, and a spectroradiometer. Experiments were repeated three to five times for both adaxial and abaxial surfaces of fresh leaves chosen at randomly. The spectral distributions and simple averages of the radiative properties in the wavelength ranges of ultraviolet-B (UV-B, 280–320 nm) and ultraviolet-A (UV-A, 320–400 nm) were determined. The spectral distributions of reflectance were similar between adaxial and abaxial surfaces, although the magnitude varied among tree species. Leaf reflectance was very low for the ultraviolet spectrum in general and varied among species and between adaxial and abaxial surfaces. It was generally higher over the UV-A waveband compared to UV-B, and higher on the abaxial than adaxial surface. The broadband reflectance in the UV-A range (over all species) was 5.0 and 3.9% for abaxial and adaxial surface, respectively, compared to 3.5 and 2.8% in UV-B. The transmittance through leaves was extremely small in the UV-B (<0.1%) and nearly zero in the UV-A spectral range. Consequently, the absorptance of ultraviolet radiation by leaves, as determined from the measured reflectance and transmittance, was quite high, being more than 90% for all the combinations of species and wavebands examined. The reported results are useful for studies requiring spectral radiative properties of the examined leaves with respect to ultraviolet irradiation.     

Cell stress and implications of the heat-shock response in skin

Heat-shock proteins (HSPs), or so-called stress proteins may play an important role in cutaneous pathophysiology. HSPs are a group of highly conserved molecules that are expressed by all cells when subjected to heat or other forms of physical or chemical stress. The physiological roles of stress proteins are varied and are important in stress and nonstress conditions. They bind to other cellular proteins and participate in protein folding pathways during stress and also during the synthesis of new polypeptides. HSPs are also essential for thermotolerance and for prevention and repair of damage caused in DNA after ultraviolet exposure. Although HSPs are expressed in the skin in both epidermis and dermis, HSPs may influence many other cellular processes in the inflammatory and immune skin response. Many authors have speculated on a link between HSPs and human skin disease characterized by inflammation and proliferation.Abbreviations HSP heat-shock protein - IL-1 interleukin-1     

Design of new inhibitors specific to the adenosine triphosphate binding site of DNA topoisomerases II

Summary DNA topoisomerases II are involved in the segregation of chromosomes which occurs after DNA replication. These enzymes proceed by nicking and resealing of a phosphodiester bond of the DNA double helix and require the hydrolysis of ATP into ADP and inorganic phosphate. Studies of ATP hydrolysis showed specific properties according to the source of isolation of the enzymes, suggesting the existence of an evolution of the ATP binding site of DNA topoisomerases II. In order to study this evolution, two experimental strategies were followed, first of all an analysis of the topography of the ATP binding site by forming UV crosslinks between ATP and the enzymes, and second the effects of new inhibitors.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.