Screening of porous and cellular materials for covalent immobilisation of Agaricus bisporus tyrosinase

We conducted a systematic study of covalent immobilisation of Agaricus bisporus tyrosinase onto typical enzyme carriers. Acrylic beads, two commercial silica gels with different pore structures and mesoporous silica foam (MCFs) beads functionalised using different organosilanes showed that only aminated MCFs offer active preparations with immobilisation efficiencies greater than 100% and a similar ratio of diphenolase (L-DOPA) to monophenolase (L-tyrosine) activities as the free enzyme. The native enzyme was entirely inactivated during incubation at 55°C for 30 min, whereas the enzyme immobilised on acrylic carrier or MCF retained 46 and 35%, respectively, of the initial activity after similar treatment. Susceptibility of native and immobilised tyrosinase to suicide inactivation in the presence of L-tyrosine and L-DOPA was tested in repeated batch tests. However, none of the preparations obtained in the L-DOPA solution was operationally stable enough to be used for practical applications.     

Exposure to (1?→?3)-β-D-glucans in swine farms

The aim of this study was to assess the exposure to organic dust and (1 → 3)-β-D-glucans in the buildings where an intensive breeding of swine is going on and evaluation of the impact of the breeding technical conditions on the observed levels of bioaerosols. The study was carried out in 30 swine farms differentiated by the size of the herd and technical conditions of breeding. In 35 randomly selected buildings, air samples were collected by stationary measurements to determine the concentrations of organic dust and (1 → 3)-β-D-glucans in inhalable and respirable fractions. Furthermore, each of the investigated buildings was precisely characterized by means of a questionnaire for technical conditions and type of breeding. In each of the points, the microclimate parameters were measured, i.e., temperature, relative humidity, CO₂ concentration and air velocity. The analyzed levels of organic dust and (1 → 3)-β-D-glucans were characterized by a wide range of concentrations. For inhalable fraction, they reached respectively: organic dust (0.43–11.8 mg/m³), (1 → 3)-β-D-glucans (14–3,594 ng/m³). For respirable fraction, the results were as follows: organic dust (0.01–4.69 mg/m³), (1 → 3)-β-D-glucans (1–703 ng/m³). The concentrations of (1 → 3)-β-D-glucans were positively correlated with organic dust (r = 0.68; p < 0.001). The most significant factor increasing the concentrations of organic dust and (1 → 3)-β-D-glucans was the use of bedding in the form of cut straw. Additionally, the levels of (1 → 3)-β-D-glucans were affected by manual forage feeding, mechanical manure disposal and the lack of the liquid manure container in breeding buildings. In view of the hazardous effects of biological agents on the health of swine-breeding workers, the swine management systems without beddings should be used, along with automated dosing techniques.     

Probing the Monophyly of the Sphaeropleales (Chlorophyceae) Using Data From Five Genes

Molecular phylogenetic analyses have had a major impact on the classification of the green algal class Chlorophyceae, corroborating some previous evolutionary hypotheses, but primarily promoting new interpretations of morphological evolution. One set of morphological traits that feature prominently in green algal systematics is the absolute orientation of the flagellar apparatus in motile cells, which correlates strongly with taxonomic classes and orders. The order Sphaeropleales includes diverse green algae sharing the directly opposite (DO) flagellar apparatus orientation of their biflagellate motile cells. However, algae across sphaeroplealean families differ in specific components of the DO flagellar apparatus, and molecular phylogenetic studies often have failed to provide strong support for the monophyly of the order. To test the monophyly of Sphaeropleales and of taxa with the DO flagellar apparatus, we conducted a molecular phylogenetic study of 16 accessions representing all known families and diverse affiliated lineages within the order, with data from four plastid genes (psaA, psaB, psbC, rbcL) and one nuclear ribosomal gene (18S). Although single‐gene analyses varied in topology and support values, analysis of combined data strongly supported a monophyletic Sphaeropleales. Our results also corroborated previous phylogenetic hypotheses that were based on chloroplast genome data from relatively few taxa. Specifically, our data resolved Volvocales, algae possessing predominantly biflagellate motile cells with clockwise (CW) flagellar orientation, as the monophyletic sister lineage to Sphaeropleales, and an alliance of Chaetopeltidales, Chaetophorales, and Oedogoniales, orders having multiflagellate motile cells with distinct flagellar orientations involving the DO and CW forms.     

Diversity of Fusarium Species Isolated from Weeds and Plant Debris in Croatia

Weeds are alternative hosts of plant pathogens and when colonized may not exhibit disease symptoms. In 2008 and 2009, samples of weeds and plant debris were collected from 12 locations in eastern Croatia, and 300 Fusarium isolates colonizing them were identified. Strains were grouped and identified based on morphology and amplified fragment length polymorphism (AFLP) patterns. Portions of the β‐tubulin and translocation elongation factor 1‐α genes were sequenced from representative strains of each group to confirm the identifications. Fourteen Fusarium species were identified with F. graminearum (20%), F. verticillioides (18%), F. oxysporum (16%), F. subglutinans (13%) and F. proliferatum (11%) all present as more than 10% of the population. Fusarium acuminatum, F. avenaceum, F. concolor, F. crookwellense (F. cerealis), F. equiseti, F. semitectum, F. solani, F. sporotrichioides and F. venenatum, were all present at frequencies < 8%. Our results indicate that economically important Fusarium spp. may be isolated from numerous alternative hosts during the off season and that weeds and plant debris can serve as a reservoir of genetically diverse inoculum.     

The effect of adipokinetic hormones on the activity of digestive enzymes

The role of the adipokinetic hormone (AKH) in the control of protease, amylase and lipase activities is examined using the cockroach Periplaneta americana and the fruit fly Drosophila melanogaster as model species. The effects of Peram‐CAH‐I and ‐II on the activity of cockroach digestive enzymes in the gastric caeca and midgut are measured both in vivo and in vitro. The results show the activity of proteases, amylases and lipases in both parts of the gut: amylase activity is higher in the gastric caeca than in the midgut; lipase activity presents the opposite trend; and protease activity is similar in both organs. The applied hormones stimulate the activity of all digestive enzymes, although this stimulation is not uniform; AKHs affect enzymes selectively, and in some cases unequally, in the gastric caeca and midgut. No substantial differences between Peram‐CAH‐I and ‐II stimulation are recorded. The in vitro results demonstrate that AKH stimulates digestive enzyme activity directly. In agreement with the cockroach results, enzymatic activity in D. melanogaster larvae producing nonfunctional AKH is lower than that in the larvae with ectopically expressed Akh gene, where enzyme activity reaches or even exceeds that of the controls. Overall, the results demonstrate the active role of AKHs in the stimulation of digestive enzyme activity in insects.     

Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of ‘poisons’, namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.     

Ditopic tris(2-mercaptoimidazol-1-yl)borate ligands and their coordination behavior toward [Ru(p-cymene)]

The ditopic tris(2-mercaptoimidazol-1-yl)borate ligand K₂[(mt^Et)₃B-B(mt^Et)₃] cannot be prepared from B₂(NMe₂)₄/4 Hmt^Et/2 Kmt^Et, because the stable intramolecular diadduct (mt^Et)B(μ-mt^Et)₂B(mt^Et) is generated instead (Hmt^Et = 2-mercapto-1-ethylimidazole). Introduction of a meta- or para-phenylene spacer between the two boron atoms precludes the 2-mercaptoimidazol-1-yl groups from adopting a bridging position so that the potassium salts K₂[(mt^Et)₃B-(m-C₆H₄)-B(mt^Et)₃] and K₂[(mt^Et)₃B-(p-C₆H₄)-B(mt^Et)₃] become readily accessible. These ligands react with [(p-cym)RuCl₂]₂ to give the dinuclear Ru^II complexes [(p-cym)Ru(mt^Et)₃B-(m-C₆H₄)-B(mt^Et)₃Ru(p-cym)]Cl₂ and [(p-cym)Ru(mt^Et)₃B-(p-C₆H₄)-B(mt^Et)₃Ru(p-cym)]Cl₂ (p-cym = p-cymene). After the exchange of the Cl⁻ counterions for [PF₆]⁻, both complexes have been crystallized and structurally characterized by X-ray diffraction.