Whole -root iron(III)-reductase activity throughout the life cycle of iron-grown Pisum sativum L. (Fabaceae): relevance to the iron nutrition of developing seeds

To understand the whole-plant processes which influence the Fe nutrition of developing seeds, we have characterized root Fe(III)-reductase activity and quantified whole-plant Fe balance throughout the complete 10-week (10-wk) life cycle of pea (Pisum sativum L., cv. Sparkle). Plants were grown hydroponically in complete nutrient solution with a continuous supply of chelated Fe; all side shoots were removed at first appearance to yield plants with one main shoot. Root Fe(III)-reductase activity was assayed with Fe(III)-EDTA. Flowering of the experimental plants began on wk 4 and continued until wk 6; seed growth and active seed import occurred during wks 5–10. Vegetative growth terminated at wk 6. Iron(III) reduction in whole-root systems was found to be dynamically modulated throughout the plant's life cycle, even though the plants were maintained on an Fe source. Iron(III)-reductase activity ranged from 1–3 mol Fe reduced · g ^–1 DW · h^–1 at early and late stages of the life cycle to 9.5 mol Fe reduced · ^g–1 DW · h^–1 at wk 6. Visual assays demonstrated that Fe(III)-reductase activity was localized to extensive regions of secondary and tertiary lateral roots during this peak activity. At midstages of growth (wks 6–7), root Fe(III)-reductase activity could be altered by changes in internal shoot Fe demand or external root Fe supply: removal of all pods or interruption of phloem transport from the reproductive portion of the shoot (to the roots) resulted in lowered root Fe(III)-reductase activity, while removal of Fe from the nutrient solution resulted in a stimulation of this activity. Total shoot Fe content increased throughout the 10-wk growth period, with Fe content in the non-seed tissues of the shoot declining by 50% of their maximal level and accounting for 35% of final seed Fe content. At maturity, total seed Fe represented 74% of total shoot Fe; total Fe in the roots (apoplasmic and symplasmic Fe combined) was minimal. These studies demonstrate that the root Fe(III)-reductase system responds to Fe status and/or Fe requirements of the shoot, apparently through shoot-to-root communication involving a phloem-mobile signal. During active seed-fill, enhanced root Fe(III)-reductase activity is necessary to generate sufficient Fe²⁺ for continued root Fe acquisition. This continuing Fe supply to the shoot is essential for the developing seeds to attain their Fe-content potential. Increased rates of root Fe(III) reduction would be necessary for seed Fe content to be enhanced in Pisum sativum.Abbreviations BPDS bathophenanthrolinedisulfonic acid - DAF days after flowering - DW dry weight - EDDHA N,N-ethylenebis[2-(2-hydroxyphenyl)-glycine] - wk week This project has been funded in part with federal funds from the U.S. Department of Agriculture, Agricultural Research Service under Cooperative Agreement number 58-6250-1-003. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The author wishes to acknowledge S. Pezeshgi and K. Koch for their excellent technical assistance, L. Loddeke for editorial comments, and A. Gillum for assistance with the figures.     

The lipidosterolic extract fromSerenoa repens interferes with prolactin receptor signal transduction

The lipidosterolic extract from the saw palmettoSerenoa repens (LSESr) is commonly used for medical treatment of benign prostatic hypertrophia due to its ability to inhibit 5-reductase which permits the conversion of testosterone to dihydrotestosterone, the active androgen on prostate cell proliferation. However, the complete action mechanism of LSESr is still unknown. Several lines of evidence suggest that, in addition to inhibition of 5-reductase, it may interfere with the action of prolactin (PRL). We therefore investigated a possible interference of this plant extract with another hormone that controls prostate gland growth, PRL. As the action mechanism of PRL is now fully documented in Chinese hamster ovary cells expressing the PRL receptor, we have conducted our experiments on these cells. In this study, using electrophysiological (whole-cell recording and single-channel recording), microspectrofluorimetric and biochemical techniques, we show that LSESr (1–30 µg/ml) reduced the basal activity of a K⁺ channel and of protein kinase C (PKC) in CHO cells. In addition, pretreatment of the cells with 1–10 µg/ml LSESr for 6–36 h abolished the effects of PRL on [Ca²⁺]_i, K⁺ conductance and PKC. LSESr may block PRL-induced prostate growth by inhibiting several steps of PRL receptor signal transduction. LSESr may also be useful for diseases implicating PRL.     

Characterization of a secreted cholesterol oxidase from Rhodococcus sp.GKI (CIP 105335)

Extracellular cholesterol oxidase (COX) (EC 1.1.3.6) was produced by Rhodococcus sp. GK1 cells grown in a defined mineral salt medium containing a mixture of phytosterols (sitosterol, campesterol, stigmasterol) as the sole source of carbon and energy. In the same time, the sterols acted as enzyme inducers. The medium was enriched with yeast extract in order to stimulate enzyme secretion. COX was purified from the culture supernatants by affinity-like chromatography on a column packed with kieselguhr and cholesterol. Enzyme bound onto the column was eluted with 0.05 M phosphate buffer pH 7.0 containing Triton X-100 at 0.1% (w/v). Some properties of the purified COX were determined. Its specific activity at pH 7.0 and 30 °C, was around 5.5 units mg^–1. The molecular mass of the enzyme, as estimated by SDS-PAGE, was 59 kDa. Its isoelectrofocusing point was around pH 8.9. The C-5 double bond and the alkyl chain moiety in sterol molecules were necessary for an adequate oxidation of the sterol 3-ol. Enzyme inhibition by the ions (0.1 mM): AsO₂ ^–, Ba²⁺, Co²⁺, Cd²⁺, Cu²⁺, N₃ ^–, Ni²⁺, and Pb²⁺ was negligible (around 10%). However, COX inhibition by 0.1 mM of either Zn²⁺, 2-[(ethylmercurio)-thio]benzoic acid, or Hg²⁺ was 18%, 22% and 93% respectively. Inhibition of activity by Hg²⁺ was significant, even at 1 M. The purified COX (0.1–0.15 mg ml^–1 in 0.05 M phosphate pH 7.0) was relatively heat-stable at temperatures up to 50 °C. At this temperature, the half-life of its activity was around 70 min. However, 90% of the enzyme initial activity was lost by 20 min incubation at 60 °C. The aminoacid sequence of the COX N-terminal segment was: H2N–Ala–Pro–Pro–Val–Ala–Ser–X–Arg–Tyr–X–(Phe)– (X might be 2 Cys residues).     

The Enzymes of Carbon Metabolism in the Thermotolerant Bacillar Strain K1

To determine enzymatic activities in the thermotolerant strain K1 (formerly Sulfobacillus thermosulfidooxidans subsp. thermotolerans), it was grown in a mineral medium with (1) thiosulfate and Fe²⁺ or pyrite (autotrophic conditions), (2) Fe²⁺, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. The increased content of carbon dioxide (up to 5 vol %) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide is fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO₂ in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phospho-enolpyruvate carboxytransphosphorylase decreased with increasing content of CO₂ in the medium.     

Quantitative and qualitative changes in the endoplasmic reticulum of barley aleurone layers

Changes in the level of the endoplasmicreticulum (ER) marker enzyme cytochrome-c reductase (EC 1.6.2.1) were followed with time of imbibition of de-embryonated half-seeds of barley (Hordeum vulgare L.) and the subsequent incubation of their aleurone layers in gibberellic acid (GA₃) and H₂O. During imbibition there is an increase in the level of cytochrome-c-reductase activity and in the amount of 280-nm absorbance associated with this enzyme. When aleurone layers are incubated for a further 42 h in water, there is a doubling of the cytochrome-c-reductase activity. In GA₃, the activity of cytochrome-c reductase reaches a maximum at 24 h of incubation and thereafter falls to below 70% of its level at the beginning of the incubation period. Changes in the cytochrome-c-reductase activity correlate with changes in the fine structure of the aleurone cell. The ER isolated in low Mg²⁺ from aleurone layers incubated in buffer for up to 18 h has buoyant density of 1.13–1.14 g cc^-1 while that from layers incubated in GA₃ for 7.5–18 h has a density of 1.11–1.12 g cc^-1. The -amylase (EC3.2.1.1) isolated with the organelle fraction by Sepharose gel filtration is associated with the ER on isopycnic and rate-zonal density gradients, and its activity can be enhanced by Triton X-100. The soluble -amylase fraction from Separose-4B columns, on the other hand, is not Triton-activated but is acid-labile. Acid phosphatase (EC3.1.3.2) is distributed in at least three peaks on isopycnic gradients. In low Mg²⁺ the second peak of activity has a density of 1.12 g cc^-1 in GA₃-treated tissue and 1.13–1.14 g cc^-1 in H₂O-treated tissue. With high-Mg²⁺ buffers, this peak of phosphatase activity disappears. Acid-phosphatase activity is not enhanced by Triton X-100 nor is it acid-labile.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Regucalcin modulates hormonal effect on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes

The interaction of various hormones and regucalcin on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10^–6–10^–4 M), and insulin (10^–8–10^– M) in the reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10^–8–3×10^–6 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10^–4 M) which can inhibit the Ca²⁺-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 M), isolated from rat liver cytosol, elevated significantly (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10^–4 M). the epinephrine (10^–5 M) or phenylephrine (10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10^–6 M) was not weakened by the presence of regucalcin (0.5 M). Moreover, GTP (10^–5 and 10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was not seen in the presence of regucalcin (0.25 M). The present finding suggests that the activating mechanism of regucalcin on (Ca²⁺–Mg²⁺)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.     

Suitability of the enzyme treatment and ammonium sulfate precipitation method for detection of staphylococcal enterotoxin from different foods

Summary An enzyme treatment and ammonium sulfate precipitation procedure was adapted to the detection of staphylococcal enterotoxin from high-protein foods. The enterotoxin is extracted from food with distilled water, after which soluble proteins are acid-precipitated (pH 4.5) and the supernatant washed with chloroform (pH 7.5). The extract is then treated with trypsin andPseudomonas peptidase for 2 h at +37°C. Residual unhydrolyzed material is precipitated with 60% ammonium sulfate for 15 min at +4°C. The precipitate is redissolved in phosphate buffer and concentrated by dialysis against polyethyleneglycol. The concentrate is washed with chloroform and lyophilized. The dry material is dissolved in 0.2 ml distilled water and enterotoxin detected by the micro-slide method with 24 h incubation at +37°C. Using this method, it has been possible within three days to detect 0.2–1.0 g staphylococcal enterotoxin A added to minced meat, dry sausage, smoked fish, cheese and milk.     

Assessment of Al3+ availability in callus culture media for screening tolerant genotypes of Cynodon dactylon

Aluminium-tolerant genotypes of Cynodon dactylon are potential candidates for the vegetation of gold mine tailings in South Africa. As a prerequisite to in vitro selection of tolerant genotypes, this work aimed at assessing and adapting micropropagation media to ensure Al³⁺ activity and toxicity. This was investigated using MINTEQA2, a chemical equilibrium speciation model. The maximum AlAl³⁺ activity achieved in any medium was 7.5 M. Of the seven published media investigated, four never achieved an activity greater than 4 M at 3–4 mM aluminium. The most appropriate medium was that of Yamamoto et al. (1996) (modified MS without KH₂PO₄ and EDTA), as it showed an increasing range of AlAl³⁺ activities from 2 to 7.5 M at aluminium concentrations from 0.25–2.5 mM. An improved modified MS formulation retaining phosphate was investigated because phosphate is an important component of our medium for callus induction in C. dactylon. Using MINTEQA2, no reduction in AlAl³⁺ activity by phosphate was detected in standard MS medium at pH 4. Through further simulations a new modified MS medium was derived with 1 mM SO ₄ ^2– and no EDTA at pH 4, which gave the maximum AlAl³⁺ activity (7.5 M) at 2 mM aluminium. This medium gave the highest AlAl³⁺ activities for the 0.25–2 mM concentration range of all the tested formulations, including the seven published media. It also resulted in significantly higher callus growth rates than standard MS media and other tested media. This new medium is currently being used to screen C. dactylon for aluminium tolerance at pH 4.     

Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts

An alkaline 5-phosphodiesterase (5-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5-PDE was purified 40-fold to a specific activity of 30 U mg^–1 protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 °C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg²⁺ up to 168% of the original activity, while Zn²⁺, Mn²⁺ and Cu²⁺ ions, chelating agent (EDTA) and NaN₃ (1–10 mM), and 5-ribonucleotides (1–5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 °C), at 70 °C for up to 120 min and without loss of activity over 90 d at –18 °C.     

Reduction of Mo6+ with elemental sulfur by Thiobacillus ferrooxidans.

In the presence of phosphate ions, molybdic ions (Mo6+) were reduced enzymatically with elemental sulfur by washed intact cells of Thiobacillus ferrooxidans to give molybdenum blue. The whole-cell activity that reduced Mo6+ was totally due to cellular sulfur:ferric ion oxidoreductase (SFORase) (T. Sugio, W. Mizunashi, K. Inagaki, and T. Tano, J. Bacteriol. 169:4916-4922, 1987). The activity of M06+ reduction with elemental sulfur was competitively inhibited by Fe3+, Cu2+, and Co2+. The Michaelis constant of SFORase for Mo6+ was 7.6 mM, and the inhibition constants for Fe3+, Cu2+, and Co2+ were 0.084, 0.015, and 0.17 mM, respectively, suggesting that SFORase can reduce not only Fe3+ and Mo6+ but also Cu2+ and Co2+ with elemental sulfur.     

The histochemical pattern of mechanically or chemically injured rabbit cornea after aprotinin treatment: relationships with the plasmin concentration of the tear fluid

Summary Plasmin, a serine protease, was recently found to be involved in corneal ulcerative processes in humans and rabbits. In our experiments, plasmin activity was found in the tear fluid after mechanical and chemical damage of the rabbit cornea, such as de-epithelization and burning with alkali. The plasmin concentrations in the tear fluid were dependent on the severity of injury. The highest plasmin activity (2.0–3.0 g ml^–1) occurred after severe alkali damage to large areas of the cornea, and the lowest activity (0.4–1.0 g ml^–1) after mechanical injury (de-epithelization).Plasmin concentrations up to 1.0 ml^–1 were associated with increased activities of lysosomal hydrolases in epithelial cells and keratocytes beneath the epithelium. Plasmin activities increased as the inflammatory reaction developed. When plasmin activity in the tear fluid was higher than 1.0 g ml^–1, inflammatory cells were found in the corneal stroma. Levels of 1.5–2.0 g ml^–1 were connected with higher numbers of inflammatory cells (particularly polymorphonuclear leukocytes) with increased activities of lysosomal hydrolases. Very high plasmin activities (2.5–3.0 g ml^–1) accompanied corneal ulcerative processes.The local application of aprotinin (Trasylol, Bayer), an inhibitor of plasmin, and also of some other proteases, was found to be necessary for the healing of severe corneal injuries in which highly elevated plasmin activity in the tear fluid and inflammatory cellulization of the cornea occurred (severe damage). It was beneficial in cases in which medium plasmin activity occurred in the tear fluid and inflammatory changes in the cornea were not too extensive. If used very early after injury, aprotinin prevents the appearance of high plasmin activity in the tear fluid, reduces the invasion of inflammatory cells into the corneal stroma, and accelerates the healing. Even the corneal transparency is restored in many cases.     

Enhanced germination of artificial seeds by marine cynobacterial extract

Summary We have developed an improved artificial seed system by using a hot-water extract from a marine cyanobacterium, Synechococcus sp. NKBG 042902. Carrot somatic embryos (Daucus carota L.) were divided into two size categories (> 800 m and 425–800 m). High frequency germination (91%) was obtained using the large somatic embryos encapsulated in calcium alginate gel containing 400 mg 1^–1 of extract. This compares to 35% without addition of the extract. A non-dialysate fraction of the extract showed strong germination-promoting activity compared with a dialysate fraction. The germination frequency of artificial seeds containing 100 mg 1^–1 of non-dialysate fraction was more than 90%. Almost all germinating artificial seeds developed into plantlets within 4 days. We also achieved high frequency germination (60%) of artificial seeds encapsulating small somatic embryos (425–800 m) that contained 100 mg 1^–1 of non-dialysate (control 9%). Although the small somatic embryos showed a lower germination frequency than the large embryos, the plantlet development process in these seeds was far more vigorous. Such a high germination frequency has not previously been reported for a carrot artificial seed system.     

The Effect of Tetrahydrocortisol–Apolipoprotein A-I Complex on the RNA Polymerase Interaction with Eukaryotic DNA and the Rate of Protein Biosynthesis in Hepatocytes

A mechanism of activation of protein biosynthesis in hepatocytes was proposed as effected by the conditioned medium of nonparenchymal liver cells incubated in the presence of high density lipoproteins, cortisol, and lipopolysaccharides. It was found that the increase in the biosynthesis rate was associated with the formation of the tetrahydrocortisol–apolipoprotein A-I (THC–apoA-I) complex in macrophages, which display 5- and 5-reductase activity and are constituents of nonparenchymal liver cell. Using the small-angle X-ray scattering technique, it was shown that the THC–apoA-I–eukaryotic DNA interaction may break hydrogen bonds between pairs of complementary nucleic bases and cause the formation of single-stranded DNA fragments capable of binding to DNA-dependent RNA polymerase. The interaction is highly cooperative and has a saturating mode, up to six enzyme molecules being bound per DNA molecule.     

Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum

The production of a highly thermostable mannanase by Rhodothermus marinus was increased 16.5-fold by optimising the concentrations of locust bean gum and yeast extract using central composite designs. The optimised medium and culture conditions yielded mannanase activity at 495 nkat ml–1 (248 nkat mg–1 protein). In addition, -L-arabinofuranosidase, -xylanase, -xylosidase, -glucosidase, -mannosidase, -galactosidase, -galactosidase and endoglucanase activities were detected at 32 nkat ml–1, 30 nkat ml–1, 16 nkat ml–1, 15 nkat ml–1, 0.1 nkat ml–1, 1 nkat ml–1, 0.5 nkat ml–1 and 8 nkat ml–1, respectively. No filter paper cellulase activity could be detected. The optimum pH of the mannanase was 5.0–6.5 and it showed high stability from pH 5 to 10 after 16 h incubation at 50 °C. The enzyme activity was maximum at 85 °C, with half lives of 45.3 h at 85 °C and 4.2 h at 90 °C. This is the first report on the production of such a high activity of extremely thermostable mannanase by an extreme thermophilic bacterium. © Rapid Science Ltd. 1998     

Isolation of a highly copper-tolerant yeast, Cryptococcus sp., from the Japan Trench and the induction of superoxide dismutase activity by Cu2+

Thirteen yeast strains were isolated from deep-sea sediment samples collected at a depth of 4500 m to 6500 m in the Japan Trench. Amongst them, strain N6 possessed high tolerance against Cu²⁺ and could grow on yeast extract/peptone/dextrose/agar containing 50 mM CuSO₄. Analysis of the 18S rDNA sequence indicates strain N6 belongs to the genus Cryptococcus. In contrast, the type strain of C. albidus, a typical marine yeast Rhodotorula ingeniosa and Saccharomyces cerevisiae did not grow at high concentrations of CuSO₄. Superoxide dismutase (SOD) catalyzes the scavenging of superoxide radicals. The activity of SOD in cell extract of strain N6 was very weak (<1 mU g^–1 total protein) when the strain was grown in the absence of CuSO₄. However, the activity was stimulated (25.8 mU g^–1 total protein) when cells were grown with 1 mM CuSO₄ and further enhanced to 110 mU g^–1 total protein with 10 mM CuSO₄. Catalase activity was increased only 1.4 or 1.1-fold with 1 mM or 10 mM CuSO₄ in the growth medium, respectively. These results suggest that SOD may have a role in the defensive mechanisms against high concentrations of CuSO₄ in strain N6.     

Effect of calcium-binding protein regucalcin on hepatic protein synthesis: Inhibition of aminoacyl-tRNA synthetase activity

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, onin vitro protein synthesis in the 5500g supernatant fraction of rat liver homogenate was investigated. Addition of Ca²⁺ up to 5.0 M in the reaction mixture caused a significant decrease in protein synthesis. This decrease was saturated at 10 M Ca²⁺. The Ca²⁺ effect was not reversed by the presence of regucalcin (2.0 M); the protein caused a remarkable decrease in hepatic protein synthesis, and it enhanced significantly the Ca^2– effect. Meanwhile, calmodulin (2.5-20 g/ml), a calcium-binding protein, did not have an appreciable effect on the Ca²⁺ (10 M)-induced decrease in hepatic protein synthesis. [³H]Leucyl-tRNA synthetase activity in the 105000g supernatant fraction (cytosol) of liver homogenate was markedly decreased by addition of Ca²⁺ (1.0–50 M). This decrease was not reversed by the presence of regucalcin (2.0 M); the protein (1.0–2.0 M) caused a remarkable decrease in the enzyme activity. The present results suggest that regucalcin can regulate protein synthesis in liver cells.     

Effect of Naloxone on the Activity of Cl–-Activated Mg2+-ATPase from Plasma Membrane Fraction of Bream Brain (Abramis brama L.) in the Presence of GABAa-ergic Substances

We studied the effect of naloxone—an antagonist of the opioid receptors—on sensitivity of Cl^–-activated Mg²⁺-ATPase from the plasma membrane fraction of bream brain (Abramis brama L.) to GABA_a-ergic substances. Preincubation of the plasma membranes with 1–100 M naloxone increased the basal Mg²⁺-ATPase activity and suppressed its activation by chloride ions. The same effects were observed in the presence of the agonists of GABA_a/benzodiazepine receptors: 0.1–100 M GABA, 1–500 M pentobarbital, and 0.1–100 M phenazepam. Naloxone (10 M) inhibited activation of the basal Mg²⁺-ATPase by the studied ligands and restored the enzyme sensitivity to Cl^–. However, the effect of naloxone was not observed in the presence of high concentrations of pentobarbital (500 M) and phenazepam (100 M). The obtained data show that naloxone modulates the activity of Cl^–-activated Mg²⁺-ATPase from the plasma membranes of bream brain and antagonizes the GABA_a receptor ligands.     

Chlorophyll breakdown in Chlorella protothecoides: characterization of degreening and cloning of degreening-related genes

Chlorella protothecoides cultures grown in a nitrogen-free bleaching medium (BM–N) in the dark rapidly degraded chlorophyll (Chl) to red catabolites. This degreening process was investigated under different growth conditions. Supply of nitrogen to the culture medium (BM+N) inhibited bleaching and the synthesis of catabolites as did the addition to BM–N of cycloheximide or a chelator, 2,2-bipyridyl. In contrast, chloramphenicol or the protease inhibitor E64 had no effect. During bleaching, Chl breakdown was accompanied by the degradation of cellular proteins such as light-harvesting complex II, cytochrome f and protochlorophyllide oxido-reductase. During growth in BM–N, protease activity increased and proteins immunologically detectable with an antibody against a senescence-enhanced cysteine protease accumulated. cDNAs from BM–N and BM+N cells were used for differential and subtractive screening to isolate cDNAs representing genes with degreening-enhanced expression (dee) in C. protothecoides. Several different dees were identified with different patterns of expression during Chlorella growth but which were all expressed at higher levels during bleaching. Among these, dee4 was most abundant and its expression was exclusive in BM–N cultures. Analysis of the dee sequences showed that they encode different proteins including a novel amino acid carrier (dee4), ferritin, ATP-dependent citrate lyase, a Ca²⁺-binding protein, MO25, ubiquinone-cytochrome c-reductase and several new proteins.     

Impact of Modified 2013 ASCO/CAP Guidelines on HER2 Testing in Breast Cancer. One Year Experience

Introduction

The latest guidelines of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) to test Human Epidermal Growth Factor Receptor 2 (HER2) in breast cancer after being revised in 2008 underwent a second modification in October 2013. The modification includes changes in cut-offs: 10% strong membranous staining for score 3+ on immunohistochemistry (IHC) (previously 30%) and using the ratio of >2 or absolute gene-copy-number (6 or more) alone or in combination with each other by in-situ-hybridisation technology (previously >2.2 and average copy-number of 6 or more). Hereby we addressed the question, which impact the modified cut-offs had on overall HER2-positivity in a single institution.

Methods

We prospectively analysed 617 consecutive diagnostic breast-cancer cases which underwent double HER2 testing by immunohistochemistry and fluorescent in-situ hybridisation (FISH), using the modified 2013 ASCO/CAP-guidelines for one year (October 2013–October 2014). Results were compared with HER2-test results on 1,528 consecutive diagnostic breast-cancer cases from two previous years (2011–2012), using the 2008 ASCO/CAP guidelines, also tested with IHC and FISH in each case.

Results

Between October 2013 and October 2014, overall HER2-positivity was 15.8% (98 of 617 cases were either IHC 3+ or FISH amplified). 79 of 617 cases (13%) were IHC 3+, 96 of 617 cases (15.5%) were FISH amplified. Equivocal cases were seen in 25 of 617 cases (4.1%). 22 of 25 equivocal cases (88%) in 2013–2014 were IHC 1+ or 2+. In 13 equivocal cases, there was a repeated IHC/FISH testing: 2 of 13 cases (15%) became FISH amplified, 1 of 13 cases (7.5%) became IHC 3+. In 2011–2012, overall HER2-positivity (IHC/FISH) was 13.8% (211 of 1,528 cases). 185 of 1,528 cases (12%) were 3+ on IHC, 181 of 1,522 cases (12%) were amplified by FISH. Six of 1,528 cases were equivocal by FISH, and interpreted as non-amplified (0.3%).

Conclusions

Applying the modified ASCO/CAP guidelines from 2013 resulted in an increase (2%) in overall HER2-positivity rate compared to overall-HER2-positivity rate using the 2008 ASCO/CAP guidelines. The increased positivity rate was mainly due to more FISH-positive cases (3.5% more than until 2013). The high rate of equivocal cases (4.1%) did not contribute to increase in overall HER2-positivity, but resulted in delay in definitive HER2-status.