Times 10-6M Research Articles

Effects of an antipsoriatic agent, dithranol, on mitochondrial and nuclear functions in yeast cells

Respiration-deficient (petite) cells are much more resistant than respiration-competent (grande) cells of Saccharomyces cerevisae to the killing effect of dithranol at concentrations of 2 times 10-6 — 10 times 10-6M. The high proportions of cytoplasmic petites found in cultures of yeast treated at these concentrations are mainly due to this selective killing. However, at lower concentrations with little killing effect on grandes a strong net increase of the number of cytoplasmic petites was found. Dithranol can act as a photosensitizer. Results from photodynamic treatment indicate that the resistance of petites depends on their inability to take up dithranol. Cells grown in media with glycerol as carbon source were more resistant to the petite-inducing effect of dithranol than cells grown with glucose. Kinetic studies showed that both killing and petites can be induced by very short treatments, the maximal effect being obtained within 2–5 min. Reconstruction experiments showed that the dithranol available in the treatment solution was quickly bound to the cells. Dithranol was found to be much more effective than ethidium bromide in inducing petites by treatments under no-growth conditions in phosphate buffer. Mitotic recombination and gene conversion were not increased by treatment with dithranol in the dark. Light + dithranol gave a small increase of the frequency of mitotic gene conversion.

The High-Velocity Clouds: Galactic or Intergalactic?

Nature and origin of the high-velocity clouds (HVCs) remain enigmatic after thirty years (Wakker & van Woerden 1997,ARA&A,35, 217), owing to lack of distance information. Hypotheses range from supernova shells at 100 pc to intergalactic clouds at 1 Mpc. On statistical grounds, Blitz et al. (1996,BAAS,28, 1349) claim that the HVCs are “remnants of Local Group formation, best explained as members of the Local Group of galaxies”. Reliable distances must come from the presence or absence of absorption at the HVC's velocity in spectra of stars at different distances. For Complex A, MgII absorption is seen inHSTspectra of the Seyfert galaxy Mrk 106, but not in the star PG0859+593 at 4 kpc (Wakker et al. 1996,ApJ,473, 834). La Palma spectra of the RR Lyr star AD UMa at 11 kpc distance show CaII absorption by Complex A at both K and H, which is lacking at 4 kpc. These absorptions are not confused with stellar metal lines. Our distance bracket 4 <d< 11 kpc places Complex A in the Galactic Halo, at 2.5 <z< 7.5 kpc above the plane; a distance similar to Local Group galaxies is excluded. The HI mass implied lies between 0.15 and 1.2 times 106M⊙. Our result precludes local origins for this HVC. It allows an origin in a Galactic Fountain, or in interaction of infalling intergalactic material (from the Magellanic System or the Local Group) with the Galactic Halo.

Beta2-adrenoceptors mediate melanosome dispersion in winter flounder (Pleuronectes americanus))

Although the melanosome-aggregating mediation of catecholamines through alpha -adrenoceptors is well established for teleost melanophores, the regulation of the dispersive process is not as clearly understood. The melanosome-aggregating effect of high concentrations of catecholamines in vitro is reversed at low concentrations with melanophores of the winter flounder, Pleuronectes americanus. In vitro incubation in low concentrations of isoproterenol ( <<= 10-7M) and noradrenaline ( <<= 10-8M) enhances Na+-induced melanosome dispersion in balanced salt solution, which can be depressed by propranolol (10-4M), indicating beta -adrenoceptor mediation in pigment dispersion. The subtype of this adrenoceptor appears to be the beta2conformation, since the beta2-adrenoceptor agonist terbutaline (>=>3.15 times 10-6M) reverses the melanosome-aggregating effect associated with higher concentrations of noradrenaline and with electrical stimulation. It is concluded that in this species there is adrenergic neuronal control of melanosome aggregation through alpha -adrenoceptors on release of noradrenaline and of dispersion through beta2-adrenoceptors during a subsequent decrease in concentration.

A LASER FLASH SPECTROSCOPY STUDY OF THE KINETICS OF ELECTRON TRANSFER FROM SPINACH PHOTOSYSTEM I TO SPINACH AND ALGAL FERREDOXINS

Abstract—Laser flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from P700 in Photosystem I (PSI)‐enriched particles from spinach to the ferredoxins from spinach and the green alga Monoraphidium braunii. Very similar behavior for the interaction of both ferredoxins with the PSI complex was observed, although the algal ferredoxin appears to be slightly more effective as an electron acceptor. For both proteins, a non‐linear protein concentration dependence of the rate constant for reduction was obtained, indicating complex formation preceding electron transfer. Estimates of 3 times 107M−1 s−1 and 140–180 s−l were obtained from these data for the second order rate constants for complex formation, and the limiting first order rate constants for electron transfer, respectively. At neutral pH, a biphasic dependence of the rate constant for ferredoxin reduction on the concentration of NaCl or MgCl2 was observed. This was interpreted in terms of the electrostatic interactions which occur between ferredoxin and the PSI membrane. In addition, magnesium cations appear to play a specific role in the interaction between PSI and ferredoxin. Thus, the addition of these ions under optimal conditions induces a 6‐f‐old increase in the electron transfer reaction rate constant, compared with a 2‐f‐old increase in the presence of an optimal amount of NaCI. This cannot be explained as arising from ionic strength effects. To our knowledge, this is the first time that a direct measurement of the rate constant for the reduction of ferredoxin by the PSI complex has been reported.

PHOTOCHEMISTRY OF 1,2,3‐INDANETRIONE*

AbstractThe photochemistry of 1,2,3‐indanetrione (1) has been examined in solution at room temperature by steady state and laser flash photolysis. The triplet state of 1 (T = 6.5 μs, δmux, = 360 and 570 nm, in dry acetonitrile) reacts preferentially via an a‐cleavage process followed by a considerably slower loss of carbon monoxide. Triplet 1 shows a remarkably fast hydrogen abstraction rate constant when in the presence of 1,4‐cyclohexadiene (kr= 1.4 times 106M−1s−1) in spite of its low excitation energy (ET= 42 kcal/mol). This behavior can be explained by assuming that the vicinal carbonyls coplanar to the ketyl radical play an important role in its stabilization.

The Action of the Prostaglandins on Isolated Human Ureteric Smooth Muscle

Summary— A study has been carried out on the actions of the prostaglandins E2 and F2α and their synthesis inhibitors, indomethacin and diclofenac sodium, upon isolated human ureteric smooth muscle, using the technique of microsuperfusion designed to ensure good tissue viability. Indomethacin and diclofenac sodium were shown to abolish almost completely the contractile response of ureteric muscle to electrical field stimulation. Contractile activity, in the presence of the inhibitors, could be restored by prostaglandin E2 or F2α or by increasing the external potassium concentration, [K+]o, of the superfusate. Prostaglandin E2 or F2α alone were shown to increase dramatically both the phasic and tonic component of the electrically stimulated contractions, on occasions inducing spontaneous activity. A possible mechanism of action was elucidated with an electrophysiological technique using intracellular microelectrodes. The mean membrane potential recorded was 54.7 mV (SD ± 10 mV, n=15). The depolarising action of raising [K+]o was demonstrated and prostaglandin F2α (3 times 10-6M) was shown to produce a small depolarisation of the ureteric muscle cell membrane.

The action of the prostaglandins on isolated human ureteric smooth muscle.

A study has been carried out on the actions of the prostaglandins E2 and F2 alpha and their synthesis inhibitors, indomethacin and diclofenac sodium, upon isolated human ureteric smooth muscle, using the technique of microsuperfusion designed to ensure good tissue viability. Indomethacin and diclofenac sodium were shown to abolish almost completely the contractile response of ureteric muscle to electrical field stimulation. Contractile activity, in the presence of the inhibitors, could be restored by prostaglandin E2 or F2 alpha or by increasing the external potassium concentration, [K+]O, of the superfusate. Prostaglandin E2 or F2 alpha alone were shown to increase dramatically both the phasic and tonic component of the electrically stimulated contractions, on occasions inducing spontaneous activity. A possible mechanism of action was elucidated with an electrophysiological technique using intracellular microelectrodes. The mean membrane potential recorded was 54.7 mV (SD +/- 10 mV, n = 15). The depolarising action of raising [K+]O was demonstrated and prostaglandin F2 alpha (3 x 10(-6) M) was shown to produce a small depolarisation of the ureteric muscle cell membrane.

Analogs of phalloidin

The analogs [D-Abu2-Lys7]-phalloin (1) and [D-Ala2-Leu7]-phalloin (2) are obtained by cyclization of the monocyclic thioether peptides 5 and 7 with DCCI and diphenylphosphoro-azidate respectively. For the synthesis of 5 and 7 an intramolecular Savige-Fontana reaction of the linear heptapeptide tert.-butylesters 4 and 6 is applied. On treatment in dilute solution with TFA the classically synthesized peptides 4 and 6 lose their N-terminal Boc groups thus giving rise to the reaction of the Hpi residues with the released cysteine SH-groups. The lysine analog 1 binds to F-actin with an association constant of 1.3 X 10(-6) M whereas analog 2 exhibits practically no affinity. By reaction of 1 with tetramethyl-rhodaminyl-isothiocyanate a fluorescent derivative, rhodaminyl-lysine-phallotoxin (RLP), is obtained as a novel fluorescent probe for the visualization of F-actin in cell preparations.

Cyclic 3′, 5′-Adenosine Monophosphate Levels in Separated Bone Cells

It has been shown that both parathyroid hormone (PTH) and calcitonin (CT) increase the concentration of cyclic 3',5'-adenosine monophosphate (cyclic AMP) in skeletal tissue. Since these two hormones have opposing actions on bone resorption, it has been postulated that the cell types upon which the hormones act in skeletal tissue may be different. We have previously shown that osteoblasts and osteocytes contain adenylate cyclase responsive to both PTH and CT whereas the enzyme prepared from periosteum and marrow cells did not respond to either. To further examine the postulate, the effect of these hormones on the concentration of cyclic AMP was determined. Bovine PTH (5U/ml equal to 4.2 times 10-7M) significantly increased the concentration of cyclic AMP/mug DNA in periosteum, osteoblasts, and osteocytes but not in marrow cells. Porcine CT (0.5 U/ml equal to 8.0 mug/ml approximately equal to 3.0 times 10-6M) significantly increased the concentration of cyclic AMP/mug DNA in periosteum, osteoblasts, osteocytes, and marrow cells. Adenylate cyclase activity prepared from periosteum and marrow cells was re-examined using EGTA and DMSO to improve enzyme stability and activity. Wth these additions PTH (5 U/70 mul) increased activity of the preparation from periosteum, and CT (0.5 U/70 mul) increased activity from marrow cells and periosteum. These studies provide evidence that: 1) periosteum, osteoblasts, and osteocytes respond directly to both PTH and CT and 2) marrow cells respond to CT and not PTH.

Purification of Tubulin from Bovine Brain and Its Interaction with Guanine Nucleotides

A rapid and sensitive assay for [3H]GTP binding activity of tubulin has been developed. This assay method is based on the quantitative retention of [3H]GTP. Tubulin complex on a nitrocellulose membrane filter. It was also found that bovine brain tubulin is markedly stablized by glycerol and GTP against denaturation. A large-scale purification of bovine brain tubulin was achieved using the new assay procedure and by the inclusion of glycerol and GTP in a buffer solution used for column chromatograph. The purified tubulin could be stored at -80degrees in the presence of glycerol and GTP for at least a year without any apprecialbe loss of [3H]GTP- and [3H]colchicine binding activities. The interaction of tubulin with guanine nucleotides was also studied using the nitorcellulose membrane filter procedure. It was found that the binding of [3H]GTP to tubulin with an empty exchangeable site proceeded promptly within k sec while the exchange of [3H]GTP- with a GTP-tubulin complex in which the exchangeable site had been occupied with unlabeled GTP occured more slowly. The dissociation constants for GTP and GDP at the exchangeable site of tubulin were determined as 0.5 times 10-6M and 1.9 times 10-6M, respectively. 5'-Guanylylimidodiphosphate could interact, although less strongly, with tubulin at this site, whereas the interaction of other nucleoside triphosphates includint ATP, CTP, UTP, and 5'-guanylyl methylenediphosphonate was very weak, if it occured at all. The presence of Mg2+ and a free sulfhydryl group was found to be essential for binding of [3H]GTP to tubulin. Ca2+ was found to replace Mg2+ in this binding reaction.