Inhibition of ammonia monooxygenase in Nitrosomonas europaea by carbon disulfide.

Carbon disulfide has long been recognized as a potent inhibitor of nitrification, and it is the likely active component in several nitrification inhibitors suitable for field use. The effects of this compound on Nitrosomonas europaea have been investigated, and the site of action has been determined. Low concentrations of CS2 (less than 400 microM) produced a time-dependent inhibition of ammonia-dependent O2 uptake but did not inhibit hydrazine-oxidizing activity. CS2 also produced distinct changes in difference spectra of whole cells. These results suggest that ammonia monooxygenase (AMO) is the site of action of CS2. Unlike the case for thiourea and acetylene, saturating concentrations of CS2 did not fully inhibit AMO, and the inhibition resulted in a low but significant rate of ammonia-dependent O2 uptake. The effects of CS2 were not competitive with respect to ammonia concentration, and the inhibition by CS2 did not require the turnover of AMO to take effect. The ability of CS2-treated cells to incorporate [14C]acetylene into the 28-kilodalton polypeptide of AMO was used to demonstrate that the effects of CS2 are compatible with a mode of action which involves a reduction of the rate of turnover of AMO without effects on the catalytic mechanism. It is proposed that CS2 may act on AMO by reversibly reacting with a suitable nucleophilic amino acid in close proximity to the active site copper.     

14C2H2- and 14CO2-labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase.

Incubation of cells of the nitrifying bacterium Nitrosomonas europaea with 14C2H2 results in the covalent attachment of 14C label to a membrane-bound polypeptide of an approximate Mr of 28,000 (Hyman, M.R., and Wood, P.M. (1985) Biochem. J. 227, 719-725). A labeling procedure using 14C2H2 generated from Ba14CO3 has been used to investigate the correlation between the extent of covalent modification of this polypeptide by 14C from 14C2H2 and the level of ammonia oxidizing activity in whole cells. The time-dependent inactivation of ammonia monooxygenase by 14C2H2 resulted in a progressive and saturable incorporation of 14C into a 27-kDa polypeptide. In contrast, the specific, time-dependent and complete inactivation of ammonia monooxygenase by light resulted in concomitant decrease in the ability of cells to incorporate 14C from 14C2H2 into this polypeptide. The 14C2H2 labeling procedure was also used to investigate the recovery of ammonia monooxygenase activity after complete inactivation of pre-existing ammonia monooxygenase by either C2H2 or light. The recovery of ammonia monooxygenase activity was closely correlated with a recovery of ability of cells to incorporate 14C label from 14C2H2 into the 27-kDa polypeptide. This recovery process was energy (NH4+)-dependent and was inhibited by chloramphenicol and rifampicin, implying that de novo protein synthesis was required. Additional polypeptides labeled with 14C from 14CO2 were also identified during recovery from C2H2 or light inactivation of ammonia monooxygenase.     

Investigating the healing mechanisms of an angiogenesis‐promoting topical treatment for diabetic wounds using multimodal microscopy

Impaired skin wound healing is a significant comorbid condition of diabetes that is caused by poor microcirculation, among other factors. Studies have shown that angiogenesis, a critical step in the wound healing process in diabetic wounds, can be promoted under hypoxia. In this study, an angiogenesis‐promoting topical treatment for diabetic wounds, which promotes angiogenesis by mimicking a hypoxic environment via inhibition of prolyl hydroxylase resulting in elevation or maintenance of hypoxia‐inducible factor, was investigated utilizing a custom‐built multimodal microscopy system equipped with phase‐variance optical coherence tomography (PV‐OCT) and fluorescence lifetime imaging microscopy (FLIM). PV‐OCT was used to track the regeneration of the microvasculature network, and FLIM was used to assess the in vivo metabolic response of mouse epidermal keratinocytes to the treatment during healing. Results show a significant decrease in the fluorescence lifetime of intracellular reduced nicotinamide adenine dinucleotide, suggesting a hypoxic‐like environment in the wounded skin, followed by a quantitative increase in blood vessel density assessed by PV‐OCT. Insights gained in these studies could lead to new endpoints for evaluation of the efficacy and healing mechanisms of wound‐healing drugs in a setting where delayed healing does not permit available methods for evaluation to take place.      

Kreuzungsversuche mit Triticale

Zusammenfassung In unseren Versuchen wurde, neben Kreuzungen zwischen Weizen und Roggen, insbesondere die Kreuzbarkeit zwischen hexaploidem und oktoploidem Triticale geprüft. Außerdem wurden sowohl die F₁-Hybriden als auch die späteren Generationen genauer untersucht.Die oktoploiden Triticale-F₁ hatten keinen höheren Kornansatz als der bessere Elter. Auch in den späteren Generationen gelang es nicht, daraus geeignete Typen für den praktischen Anbau zu selektieren.Obwohl die hexaploiden und oktoploiden F₁-Pflanzen einen beträchtlich schwächeren Kornansatz als beide Eltern hatten, ist es gelungen, aus späteren Generationen wertvolle hexaploide Individuen auszulesen. Während unser 1952 hergestellter hexaploider Triticale Nr. 1 in den vergangenen 14 Jahren nicht verbessert werden konnte, scheint unser aus der Kreuzung von Triticale verschiedener Polyploidiegrade hergestellter hexaploider Triticale Nr. 30 für den praktischen Anbau von Bedeutung zu sein. — In diesem Triticale gelang es, das für den hexaploiden Weizen F 481 charakteristische Merkmal, die rote Farbe der Auricula, zu fixieren.Im Jahre 1964 begannen wir mit größeren Anbauversuchen. Der erste Anbau erfolgte auf einer Fläche von 2,6 ha Sandboden in der LPG Aranykalász in Kecskemét (Kornertrag 21,1 dt/ha). — Tetraroggen und Wintergerste erbrachten unter ähnlichen Bedingungen geringere Erträge.Im Jahr 1965 wurde Triticale Nr. 30 von der gleichen LPG auf einer Fläche von rd. 17 ha (Durchschnittsertrag 23,0 dt/ha) und in unserem Institut auf rd. 3 ha (Durchschnittsertrag 24,6 dt/ha) geprüft. Im Herbst 1965 wurde dieser sekundäre hexaploide Triticale auf rd. 170 ha ausgesät.

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Crossing experiments with Triticale

Summary Besides crosses between wheat and rye, crossability between hexaploid and octoploid Triticale was tested. F₁ hybrids as well as subsequent generations were examined.Octoploid Triticale F₁ showed no higher seed productivity than did the better parent. Even in subsequent generations an attempt to select suitable types for practical cultivation failed.Though hexaploid and octoploid F₁ plants produced considerably less seed than both parents, we succeeded in selecting valuable hexaploid individuals from subsequent generations. While for the past 14 years we were unable to improve our hexaploid Triticale No. 1 produced in 1952, hexaploid Triticale No. 30 produced from crossing Triticale of different degrees of polyploidy appears to be important for practical breeding. In this Triticale we succeeded in fixing the red color of the auricola characteristic of hexaploid wheat F 481.In 1964 we started growth experiments on a larger scale. The first sowing was made on an area of 2.6 ha in sandy soil on the co-operative farm Aranykalász in Kecskemét (grain yield 21.1 quintals/ha). — Tetra rye and winter rye gave lower yield under similar conditions.In 1965 Triticale No. 30 was tested on an area of about 17 ha on the same farm, and in our institute on an area of about 3 ha. In autumn 1965 this secondary hexaploid Triticale was sown over about 170 ha.

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Für die Problemstellung sowie für seine zahlreichen Ratschläge spreche ich Herrn Prof. Barna Györffy, Budapest, für ihre gewissenhafte Arbeit in der Züchtung und Durchführung der Versuche meinen technischen Mitarbeitern den besten Dank aus.     

A multi‐level response to DNA damage induced by aluminium

Aluminium (Al) ions are one of the primary growth‐limiting factors for plants on acid soils, globally restricting agriculture. Despite its impact, little is known about Al action in planta. Earlier work has indicated that, among other effects, Al induces DNA damage. However, the loss of major DNA damage response regulators, such SOG1, partially suppressed the growth reduction in plants seen on Al‐containing media. This raised the question whether Al actually causes DNA damage and, if so, how. Here, we provide cytological and genetic data corroborating that exposure to Al leads to DNA double‐strand breaks. We find that the Al‐induced damage specifically involves homology‐dependent (HR) recombination repair. Using an Al toxicity assay that delivers higher Al concentrations than used in previous tests, we find that sog1 mutants become highly sensitive to Al. This indicates a multi‐level response to Al‐induced DNA damage in plants.     

Separation of 1,4-benzodiazepines by micellar elektrokinetic capillary chromatography

In this work the applicability of micellar elektrokinetic capillary chromatography (MECC) for the determination of benzodiazepines (BZD) has been studied. The applied method was used for the simultaneous separation of 8 BZDs (alprazolam, bromazepam, chlordiazepoxide, diazepam, flunitrazepam, medazepam, oxazepam, nitrazepam), and also for the study of stability in acidic medium. A fast and reliable method has been developed; using a separation buffer composed of sodium tetraborate 25 mM (pH 9.5), SDS (50 mM) and methanol (at least 12%) as an organic modifier.     

Thiosulfate elimination and permeability in a sulfide-adapted marine invertebrate.

Oxidation of hydrogen sulfide to thiosulfate is one of the best-characterized mechanisms by which animals adapted to sulfide minimize its toxicity, but the mechanism of thiosulfate elimination in these animals has remained unclear. In this study, we examined the accumulation and elimination of thiosulfate in the sulfide-adapted marine worm Urechis caupo. The coelomic fluid of U. caupo exposed to 50-100 micromol L-1 sulfide in hypoxic seawater (Po2 ca. 10 kPa) accumulated (mean+/-SD) 132+/-41 micromol L-1 thiosulfate after 2 h, reaching 227+/-113 micromol L-1 after an additional 4 h in aerated, sulfide-free seawater. In whole-animal thiosulfate clearance studies, the rate of thiosulfate elimination from the coelomic fluid followed a single exponential time course with a half-life of 6 h. The thiosulfate permeability coefficient of isolated preparations mounted in diffusion chambers was 7.6x10-5+/-7. 7x10-5 cm s-1 for the hindgut and 5.5x10-7+/-2.7x10-7 cm s-1 for the body wall. These rates were independent of the direction of net efflux (mucosal-to-serosal or serosal-to-mucosal). Using a simple mathematical model of U. caupo that incorporates the thiosulfate permeability coefficients, the thiosulfate half-life was calculated to be 23 h without hindgut ventilation but less than 1 h with normal hindgut ventilation. Based on this information, we propose that passive thiosulfate diffusion across the hindgut is adequate to explain the observed rates of thiosulfate elimination.     

Morphological adaptations of the respiratory hindgut of a marine echiuran worm

The echiuran worm Urechis caupo lives in U-shaped burrows in marine mudflats where levels of toxic hydrogen sulfide increase and water becomes hypoxic during low tide. Even in this low oxygen and high sulfide environment, the animal is capable of maintaining aerobic respiration. Gas exchange occures across both the body wall and hindgut. The hindgut functions as a type of water lung and is a thin walled, highly convoluted structure capable of considerable dilatation. It is rhythmically ventilated with water and its role as a respiratory organ becomes increasingly important as ambient pO₂ drops. In the deflated hindgut light microscopy reveals a pseudostratified appearing innermost mucosal epithelium composed of columnar cells with nuclei at different levels. When the hindgut is fully inflated, ultrastructural studies show a simple columnar epithelium with the nuclei at the same level. Ultrastructurally, the free surface of the hindgut cells bears numerous microvilli and a few cilia. The lateral cell membranes are highly folded in the deflated hindgut, but these folds are not visible in the fully inflated hindgut. The cytoplasm contains osmiophilic bodies which show a partially lamellated pattern which may be sulfide oxidizing bodies involved in sulfide detoxification. In the fully inflated hindgut, the entire perimeter of the lumenal mucosa is covered by electron dense inclusions, whose exact fuction is unknown. The lack of structural information on the respiratory organ of this echiuran worm renders the interpretation of its morphological and histological features at the ultrastructural level difficult, although the present study has broadened our understanding of the structural adaptations of the hindgut as a respiratory organ. © 1992 Wiley-Liss, Inc.