Activation of mercuric reductase by the substrate NADPH

Preincubation of the oxidized form of the flavoenzyme mercuric reductase with the reducing substrate, NADPH, or with a high concentration of cysteine (30 mM) results in a substantial increase of the catalytic activity as measured in a standard spectrophotometric assay. Also NADH has some activating effect but NADP+ or EDTA have no effect. In the presence of 1 mM cysteine only one equivalent of NADPH per FAD seems to be required for full activation which occurs after an incubation time of about 10 min. Activated mercuric reductase appears to be stable under anaerobic conditions but eventually returns to the original level of activity in the presence of oxygen. The activated state seems to be stabilized by 1 mM cysteine. Activation of mercuric reductase does not seem to be correlated with a change in the number of reactive thiol groups. The chemical nature of the activation process is not yet understood. Stopped-flow studies have shown that the nonactivated enzyme is practically inactive prior to contact with the substrates. The enzyme is gradually activated during the assay. The kinetics of activation of the 'native' enzyme is biphasic but 'clipped' enzyme, lacking an 85-residue N-terminal domain, is activated in a single first-order process. The progress curves obtained with preactivated enzyme are approximately exponential even at saturating concentrations of NADPH (Km = 0.4 microM at 25 degrees C, pH 7.3) and Hg2+ (Km = 3.2 microM in the presence of 1 mM cysteine). The initial rates yield kcat values of about 13 s-1 per FAD molecule (25 degrees C, pH 7.3). We find no evidence for a thiol-dependent change from a rapid to a slow kinetic phase. The shape of the progress curves presumably depends on product inhibition, but NADP+ is not a sufficiently effective inhibitor to explain the effect fully.     

Selenite-induced variation in glutathione peroxidase activity of three mammalian cell lines: no effect on radiation-induced cell killing or DNA strand breakage

The selenium-dependent glutathione peroxidase activities of three mammalian cell lines, HT29, P31, and N-18, cultured in medium with low serum content, increased about 2-, 5-, and 40-fold, respectively, after supplementation with 100 nM selenite. Catalase, CuZn superoxide dismutase, and Mn superoxide dismutase activities were not generally influenced by selenite supplementation, and there was only a minor nonselenium-dependent glutathione peroxidase activity in the investigated cell lines. Gamma-irradiated control and selenite-supplemented cells showed no changes in the surviving fractions, as estimated by clonogenic survival or [3H]-thymidine uptake, nor were there any significant differences between the two groups in the induction of DNA strand breaks after gamma irradiation under repairing (37 degrees C) or nonrepairing (0 degrees C) conditions. The results suggest that selenium-dependent glutathione peroxidase does not contribute significantly to the radiation resistance of cultured mammalian cells.     

Infections with Francisella tularensis biovar palaearctica in hares (Lepus timidus, Lepus europaeus) from Sweden

The occurrence of tularemia was studied in 1,500 hares submitted to the National Veterinary Institute, Uppsala, Sweden for postmortem examination during 1973 through 1985. A total of 109 tularemia cases was recorded based on the fluorescent antibody (FA) test for Francisella tularensis and on the gross and microscopic pathology. Tularemia was diagnosed only in the varying hare (Lepus timidus) and not in the European brown hare (Lepus europaeus). The geographical distribution of the 109 cases indicates that tularemia has not spread in Sweden during the last 45 yr, with the exception of an endemic occurrence of the disease on the island of Stora Karls? in the Baltic sea. The disease was most frequent in the autumn and only a few cases were recorded during winter. Cases were not seen in the spring. The annual prevalence varied, with several cases in 1974 and 1981, but there were no cases in 1976 and 1980. The postmortem findings in hares dying of tularemia in the autumn were characterized by focal coagulative necrosis in liver, spleen and bone marrow, with high numbers of bacteria FA-positive for F. tularensis. In hares dying during winter months, the most characteristic findings were hemorrhagic enteritis and typhlitis, although necrotic lesions could occur in liver, spleen and bone marrow. Diseased hares on the island of Stora Karls? were demonstrated to be infected with ticks, while hares on the mainland of Sweden generally were fed upon by mosquitoes. Twenty-six of the 109 hares with tularemia were examined bacteriologically and F. tularensis biovar palaearctica was isolated from eight. The lung extract antibody test for F. tularensis was performed in 18 of the 109 hares. All were negative. In addition to the field study, an experimental study with F. tularensis biovar palaearctica was performed. Four varying hares and three European brown hares were inoculated. None of the hares died from tularemia, and generalized infection was not demonstrated.     

Penicillium verrucosum in feed of ochratoxin A positive swine herds

Ochratoxin A contamination of cereal feed grain was monitored during October 1989–September 1990 by analysis of blood samples from slaughter swine in Sweden. The detection of ochratoxin A in swine blood was used as a method to identify swine herds fed ochratoxin A contaminated feed. The contamination level of ochratoxin A in the blood of the positive herds was in the range 2–45 ng/ml with the mean concentration 5.2 ng/ml. Feed samples for mycological analysis were collected from both ochratoxin A positive herds (2 ng/ml blood) and ochratoxin A negative herds (<2 ng/ml blood). From the ochratoxin A positive herds and the ochratoxin A negative herds 22 and 21 feed samples were collected, respectively. No quantitative differences in mould content, as determined by colony forming units, were observed between the two groups. However, there were differences in the mycoflora. The incidence of storage fungi (Penicillium and Aspergillus spp.) was significantly higher (p < 0.05) in feed from ochratoxin A positive herds. Particularly, Penicillium verrucosum was found to be significantly more common (p < 0.001). Altogether 274 isolates were screened for their ability to produce ochratoxin A. Ochratoxin A producers were found only within P. verrucosum; 38% of the 63 isolates produced detectable amounts of ochratoxin A. Ochratoxin A producing isolates of P. verrucosum were found in 60% of the feed samples collected from ochratoxin A positive swine herds and in one sample (5% ) of the feed samples collected from the ochratoxin A negative herds.     

Stomatal closure by ultraviolet radiation

The effect of ultraviolet radiation (UV) (255–325 nm) on stomatal closure was investigated on tef [ Eragrostis tef (Zucc) Trotter] in the presence of white light (ca 50 ·mol m⁻² s⁻¹). The action spectrum showed that UV (ca 2 ·mol m⁻² s⁻¹, half band width about 10 nm) of 285 nm or shorter wavelengths was very efficient in causing stomatal closure. The effectiveness decreased sharply towards longer wavelengths. Radiation of 313 nm or longer wavelengths was practically without effect. Increasing UV intensity increased stomatal resistance. When stronger white light (5 to 9 times stronger than the one used during irradiation) was administered, stomates re-opened rapidly irrespective of whether the UV was on or off, although a subsequent gradual closing tendency was observed when the UV was on.     

Light-induced linear dichroism in photoreversibly photochromic sensor pigments. – V. Reinterpretation of the experiments on in vivo action dichroism of phytochrome

Experiments by several authors on the effects of polarized light on phytochromemediated responses in fern gametophytes and in the green alga Mougeotia have earlier been interpreted as showing that the transition moment of phytochrome in the P_r form is parallel to the plasmalemma, but perpendicular to the plasmalemma for the P_fr form of phytochrome. It is now shown that the experimental results can be interpreted differently, and that they are also consistent with a chromophore rotation of about 30° (instead of 90°), as found for immobilized phytochrome molecules in vitro. Thus there is no evidence for a rotation of the whole phytochrome protein. For the gametophyte of Adiantum it is calculated that the P_r transition moment is inclined 17° to the plasmalemma, and the P_fr transition moment ca 50°, corresponding to an in vivo chromophore rotation of ca 33°; however, these values are very approximate.     

Light-induced linear dichroism in photoreversibly photochromic sensor pigments. – VI. Relation between the two pigments of the mycochrome system in Alternaria cichorii

Conidiation in Alternaria cichorii Nattras is reversibly stimulated by near ultraviolet radiation (NUV, ca 313 nm) and inhibited by blue light (ca 450 nm) and seems to be a mycochrome-mediated process. After induction with plane-polarized NUV, blue light polarized perpendicularly to the NUV was more effective in counteracting the induction than was blue light polarized parallel to the NUV. From this the conclusions are drawn that (a) both the blue-absorbing component (presumably a flavo-protein) and the P_NUV of the mycochrome system are membrane-bound and that (b) the transition moment associated with blue light absorption in the presumed flavoprotein forms an angle of at least 53° with the transition moment associated with NUV absorption in P_NUV.     

A histochemically demonstrable thiaminepyrophosphatase activity in the sinusoidal endothelium of the liver

Summary A new histochemically demonstrable thiaminepyrophosphatase activity in liver tissue is described. It appears to be localized solely to the cytosomes of the sinusoidal endothelium. The activity, which requires high concentration of lead ion for its demonstration, has a high degree of substrate specificity. It resists formaldehyde fixation, ethanol and many inhibitors. The optimum pH is at pH 6.This study was supported in part by a grant from the Swedish Medical Research Council.     

A comparison between histochemically demonstrable Golgi-associated and bile canalicular TDP-ase activity in liver

Summary Histochemically demonstrable Golgi-associated TDP-ase activity in liver cells from cat, chicken, rat and frog has been investigated.This activity is highly substrate-specific, insensitive to aldehyde fixation, ethanol, acetate, lead and most enzyme inhibitors. It is stimulated by divalent manganese, calcium, magnesium and cobalt and optimum pH is at pH 6 to 7.The characteristics are identical for all four species but significant differences exist at a comparison with bile canalicular activity, Golgi-associated activity in other cells and biochemical findings.This study was supported in part by a grant from the Swedish Medical Research Council.