The nitrite reductase gene of Pseudomonas aeruginosa: Effect of growth conditions on the expression and construction of a mutant by gene disruption

Abstract The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.     

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.     

Hatching plasticity in a Southeast Asian tree frog mitigates submergence-induced mortality

Environmentally cued hatching has been well-documented in amphibians in response to a wide range of abiotic and biotic factors. The hatching of terrestrial amphibian eggs in response to flooding may be basal within the group, but amphibian lineages in tropical Asia and sub-Saharan Africa have not received as much attention as their Neotropical counterparts. We investigated submergence-induced hatching in Feihyla hansenae, a Rhacophorid tree frog with terrestrial eggs. We quantified natural rates of clutch submergence at our study site in Thailand. Using submergence experiments, we found that embryos are capable of hatching early to escape flooding, and that failure to hatch results in mortality. Among the embryos that were able to hatch early, only the earliest, youngest hatchlings experienced a trade-off in body size that persisted for 6 days, while later, older hatchlings were not significantly smaller than spontaneous hatchlings under control conditions. By incorporating our natural and experimental data into Monte Carlo methods to simulate and compare survival probabilities with and without hatching plasticity, we found an overall 3.1% increase in submergence survival due to hatching plasticity. Our findings support the idea that flooding-induced hatching is widespread across amphibians with terrestrial eggs and highlight the importance of researching understudied tropical regions. As climate change is projected to affect rainfall patterns, the ability of embryos to escape abiotic egg-stage threats may be an indicator of species' ability to flexibly navigate a changing environment.     

The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13.

Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.     

Locomotor Activity in the Freshwater Crab Potamon fluviatile: The Analysis of Temporal Patterns by Radio-telemetry

Locomotor activity in a field population of the freshwater crab, Potamon fluviatile, was studied during the breeding season by means of radio-telemetry and by direct counts of active animals along a transect of the stream. The basic pattern of crabs' locomotor activity can be described as a sequence of short distance movements around the shelters (foraging movements), followed by rarer long distance displacements (wandering movements). Whilst direct counting showed that the crabs exhibit a broadly nocturnal rhythm in foraging movements, no daily periodicity in wandering activity was revealed by telemetry. There is sexual difference in these latter excursions: females move farther along the stream and into the surrounding terrestrial habitat than do males. This behaviour is in contrast to observations made during the non-breeding season and is probably related to the stage of female reproduction.     

Mortality and sterility induced in Piophila casei by X-ray and neutron irradiation

Different doses of neutrons and X-rays were given to 5-day-old pupae of Piophila casei L. (Diptera, Piophilidae), just before their emergence. The mortality and sterility induced by the different types of radiation were measured. Neutrons are more effective than X-rays in provoking lethal lesions in somatic cells. Females are more resistant than males to the sterilizing action of neutrons, the relative biological efficiency of neutrons being 6 and 3.5, respectively.

---

Iduktion von mortalität und sterilität durch röntgenstrahlen und neutronen bei piophila casei

Zusammenfassung Puppen von Piophila casei im Alter von 5 Tagen wurden mit verschiedenen Dosierungen von Neutronen und Röntgenstrahlen bestrahlt. Dadurch was es möglich, Dosis-Effekt-Kurven für Mortalität und Sterilität zu bilden. Die Neutronen erwiesen sich als wirksamer als die Röntgenstrahlen für die Auslösung von Letalstörungen bei den Puppen. Die Relative Biologische Wirkung (RBE) beider Strahlenarten auf die Mortalität ist nicht im ganzen Mortalitätsbereich gleichartig. Die durch Bestrahlung verursachte Sterilität wurde für beide Geschlechter bestimmt und zwar anhand der Überlebensrate der Eier von Einzelpaaren. Neutronen sind wirksamer als Röntgenstrahlen, um letale dominante Mutationen in Spermatozoen zu verursachen (RBE: 6). Neutronen reduzieren die Fertilität von Weibchen, welche aus bestrahlten Puppen stammen, ebenfalls stärker (RBE: 3,5). Die Fekundität der Weibchen wird wesentlich vermindert bei Neutronenbestrahlung von über 2000 rad und bei Röntgenbestrahlung von 7500–10000 rad. Die strahlenbedingte Schädigung der Ovarien konnte auch histologisch nachgewiesen werden.

---

---

This work has been supported by grant nr. 74.01552.06 from the Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy.     

Production of amylase(s) by Schwanniomyces castellii and Endomycopsis fibuligera

Schwanniomyces castellii and Endomycopsis fibuligera Produced extracellular amylase(s) when grown on various carbon sources and at different pH values. Both yeast species showed significant amylase synthesis in the presence of either maltose or soluble starch. On the other substrates tested (glucose, cellobiose, sucrose, trehalose, melezitose, raffinose, ethanol, glycerol) differences were found regarding growth and amylase production. Free glucose in the culture medium apparently inhibited enzyme synthesis. The pH range allowing maximal growth and amylase production was 4.5–6.0 for E. fibuligera and 5.5–7.0 for S. castellii.     

MK-801 Prevents 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinsonism in Primates

In cynomologus monkeys, systemic administration of MK-801, a noncompetitive antagonist for the N-methyl-D-aspartate receptor, prevented the development of the parkinsonian syndrome induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MK-801 also attenuated dopamine depletion in the caudate and putamen and protected dopaminergic neurons in the substantia nigra from the degeneration induced by the neurotoxin. Nevertheless, 7 days after MPTP administration in the caudate and putamen of monkeys also receiving MK-801, the levels of toxic 1-methyl-4-phenylpyridinium were even higher than those measured in monkeys receiving MPTP alone. This indicates that the protective action of MK-801 is not related to MPTP metabolism and strongly suggests that, in primates, the excitatory amino acids could play a crucial role in the mechanism of the selective neuronal death induced by MPTP.     

A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers

Model membranes with unsaturated lipid chains containing various amounts of M13 coat protein in the -helical form were studied using time-resolved fluorescence and ESR spectroscopy. The lipid-to-protein (L/P) ratios used were > 12 to avoid protein-protein contacts and irreversible aggregation leading to -polymeric coat protein. In the ESR spectra of the 12-SASL probe in dioleoyl phosphatidylcholine (DOPC) bilayers no second protein induced component is observed upon incorporation of M13 coat protein. However, strong effects are detected on the ESR lineshapes upon changing the protein concentration. The ESR lineshapes are simulated by assuming a fixed ratio between the parallel (D) and perpendicular (D) diffusion coefficients of 4, and an order parameter equal to zero. It is found that increasing the protein concentration from L/P to L/P 15 results in a decrease of the rotational diffusion coefficient D from 3.4 × 10⁷ to 1.9 × 10⁷ s^–1. In the time-resolved fluorescence experiments with DPH-propionic acid as a probe, it is observed that increasing the M13 coat protein concentration causes an increase of the two fluorescent lifetimes, indicating an increase in bilayer order. Analysis of the time-resolved fluorescence anisotropy decay allows one to quantitatively determine the order parameters P₂ and P₄, and the rotational diffusion coefficient D of the fluorescent probe. The order parameters P₂ and P₄ increase from 0.34 to 0.55 and from 0.59 to 0.77, respectively, upon adding M13 coat protein to DOPC bilayers with an L/P ratio of 35. The rotational diffusion coefficient D of the DPH-propionic acid probe decreases on incorporating M13 coat protein, in accordance with the ESR results. It is concluded that M13 coat protein in the -monomeric state is not able to produce a long living lipid boundary shell and consequently an immobilization of the lipids. An overall effect on the lipids is induced, resulting in a reduction in the dynamics and an increase in average lipid order. The hydrophobic region of M13 coat protein is proposed to perfectly match the lipid bilayer, resulting in a relatively small distortion of the bilayer structure of the lipid system.     

Single-stranded molecules in DNA preparations from cultured mammalian cells at different moments of cell cycle

Long single-stranded DNA molecules have been observed at electron microscope in DNA preparations from synchronized Chinese hamster cells. The amount of single strandedness in parental DNA increases following a prolonged block of DNA synthesis by hydroxyurea as judged by the results obtained using an improved hydroxyapatite chromatography (Hanania et al., 1975). As far as newly replicated DNA is concerned, an increase of the single strand amount has been observed in DNA preparations from cells actively synthesizing DNA.