Characteristics and N-terminal amino acid sequence of manganese peroxidase from solid substrate cultures of Agaricus bisporus

Extracellular manganese peroxidase (MnP) was purified from the compost extract of Agaricus bisporus using anion exchange chromatography, gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two forms (MnP1 and MnP2) were separated by isoelectric focusing and their isoelectric points were determined to be 3.25 (MnP1) and 3.3 (MnP2). Both forms had a molecular mass of 40 kDa. The first 25 amino acids of the N-terminal end of MnP1 sequence was found to share 68% identity with a Pleurotus ostreatus and a P. eryngii MnP. Lignin peroxidase was not detected during any of the steps in the purification process. In liquid cultures with both soluble and insoluble carbon sources in defined medium (D-glucose, glycerol, Whatman CC-41 microcrystalline cellulose or Solka-floc cellulose) MnP protein was detected in culture fluid by Western blot, but no MnP activity could be detected. A. bisporus appears to be in the group of ligninolytic fungi which do not produce lignin peroxidase.     

Tumor necrosis factor-alpha, sphingomyelinase, and ceramide inhibit store-operated calcium entry in thyroid FRTL-5 cells

Tumor necrosis factor alpha (TNF-alpha) is a potent inhibitor of proliferation in several cell types, including thyroid FRTL-5 cells. As intracellular free calcium ([Ca2+]i) is a major signal in activating proliferation, we investigated the effect of TNF-alpha on calcium fluxes in FRTL-5 cells. TNF-alpha per se did not modulate resting [Ca2+]i. However, preincubation (10 min) of the cells with 1-100 ng/ml TNF-alpha decreased the thapsigargin (Tg)-evoked store-operated calcium entry in a concentration-dependent manner. TNF-alpha did not inhibit the mobilization of sequestered calcium. To investigate whether the effect of TNF-alpha on calcium entry was mediated via the sphingomyelinase pathway, the cells were pretreated with sphingomyelinase (SMase) prior to stimulation with Tg. SMase inhibited the Tg-evoked calcium entry in a concentration-dependent manner. Furthermore, an inhibition of calcium entry was obtained after preincubation of the cells with the membrane-permeable C2-ceramide and C6-ceramide analogues. The inactive ceramides dihydro-C2 and dihydro-C6 showed only marginal effects. Neither SMase, C2-ceramide, nor C6-ceramide affected the release of sequestered calcium. C2- and C6-ceramide also decreased the ATP-evoked calcium entry, without affecting the release of sequestered calcium. The effect of TNF-alpha and SMase was inhibited by the kinase inhibitor staurosporin and by the protein kinase C (PKC) inhibitor calphostin C but not by down-regulation of PKC. However, we were unable to measure a significant activation of PKC using TNF-alpha or C6-ceramide. The effect of TNF-alpha was not mediated via activation of either c-Jun N-terminal kinase or p38 kinase. We were unable to detect an increase in the ceramide (or sphingosine) content of the cells after stimulation with TNF-alpha for up to 30 min. Thus, one mechanism of action of TNF-alpha, SMase, and ceramide on thyroid FRTL-5 cells is to inhibit calcium entry.     

A stable bis-allyloxycarbonyl biotin aldehyde derivative for biotinylation via reductive alkylation: application to the synthesis of a biotinylated doxorubicin derivative

A novel, stable, biotin aldehyde derivative is reported in which the biotin moiety is N1,N3-protected by the allyloxycarbonyl group. The derivative is stable to sodium cyanoborohydride mediated reductive alkylation and is cleaved under mild Pd [0] catalysis. This novel biotin aldehyde should have wide application in avidin- and streptavidin-based detection systems and bioassays. The derivative is utilized in the synthesis of a biotinylated doxorubicin analogue that retains topoisomerase activity.     

Urinary excretion of lignans after administration of isolated plant lignans to rats: the effect of single dose and ten-day exposures

The difference in urinary excretion of mammalian and plant lignans in rats was determined after oral administration of equivalent doses (25 mg/kg of body weight) of 7-hydroxymatairesinol (HMR), lariciresinol (LAR), matairesinol (MR), and secoisolariciresinol (SECO). Twenty-four hours-urine samples were collected after a single dose and after administration of one dose/day for 10 days. Eight lignans were analysed in urine extracts using a high-performance liquid chromatography-tandem mass spectrometry method showing good sensitivity and repeatability. After a single dose of HMR, LAR, MR, and SECO, the main metabolites were 7-hydroxyenterolactone (HENL), cyclolariciresinol (CLAR), enterolactone (ENL), and enterodiol (END), respectively, but after 10-day exposure ENL was the main metabolite of all the tested lignans, showing a considerably higher excretion than after a single dose. Metabolic transformations of plant lignans into each other could also be observed.     

Allelopathic Effects of Toxic Haptophyte <Emphasis Type="Italic">Prymnesium parvum</Emphasis> Lead to Release of Dissolved Organic Carbon and Increase in Bacterial Biomass

The haptophyte Prymnesium parvum has lytic properties, and it affects coexisting phytoplankton species through allelopathy. We studied the effect of P. parvum allelochemicals on the lysis of the nontoxic and nonaxenic cryptomonad Rhodomonas salina and the consequent release of dissolved organic carbon (DOC). Changes in production, cell density, and biomass of associated bacteria were measured over 12 h. Six different combinations of P. parvum and R. salina cultures, their cell- and bacteria-free filtrates, and growth media as controls were used in the experiments. When P. parvum and R. salina cells were mixed, a significant increase in DOC concentration was measured within 30 min. Bacterial biomass increased significantly during the next 6 to 12 h when R. salina was mixed either with the P. parvum culture or the cell-free P. parvum filtrates (allelochemicals only). In contrast, bacterial biomass did not change in the treatments without the allelopathic action (without R. salina cells). Blooms of P. parvum alter the functioning of the planktonic food web by increasing carbon transfer through the microbial loop. In addition, P. parvum may indirectly benefit from the release of DOC as a result of its ability to ingest bacteria, by which it can acquire nutrients during limiting conditions.     

Assessment of microalgae species,biomass, and distribution from spectral images using a convolution neural network

Journal of Applied Phycology - Effective monitoring of microalgae growth is crucial for environmental observation, while the applications of this monitoring could also be expanded to commercial and...     

Forehead Patch Size Predicts the Outcome of Male–Male Competition in the Pied Flycatcher

Males of many animal species express ornaments that affect their reproduction opportunities through male–male competition or female mate choice. Such ornaments can, for example, inform conspecifics about the fighting ability, condition or territory ownership of the bearer. Pied flycatcher (Ficedula hypoleuca) males have a conspicuous white forehead patch that varies greatly in size. We examined whether the white forehead patch is an intrasexually selected trait in a Finnish population. We artificially manipulated forehead patch size to represent two naturally occurring extremes and competed males against each other in the presence of a female. Males with a large forehead patch were more aggressive than males with a small patch, whereas the original patch size of the male had no influence on aggression. Neither manipulated nor original patch size influenced resource dominance (over female or nest box). These results indicate that forehead patch signals fighting ability of the bearer in the pied flycatcher. The next step is to find out what kind of costs may maintain the honesty of this signal.     

Is the Pathogenic Ergot Fungus a Conditional Defensive Mutualist for Its Host Grass?

It is well recognized, that outcomes of mutualistic plant-microorganism interactions are often context dependent and can range from mutualistic to antagonistic depending on conditions. Instead, seemingly pathogenic associations are generally considered only harmful to plants. The ergot fungus (Claviceps purpurea) is a common seed pathogen of grasses and cereals. Ergot sclerotia contain alkaloids which can cause severe toxicity in mammals when ingested, and thus the fungal infection might provide protection for the host plant against mammalian herbivores. Theoretically, the net effect of ergot infection would positively affect host seed set if the cost is not too high and the defensive effect is strong enough. According to our empirical data, this situation is plausible. First, we found no statistically significant seed loss in wild red fescue (Festuca rubra) inflorescences due to ergot infection, but the seed succession decreased along increasing number of sclerotia. Second, in a food choice experiment, sheep showed avoidance against forage containing ergot. Third, the frequency of ergot-infected inflorescences was higher in sheep pastures than surrounding ungrazed areas, indicating a protective effect against mammalian grazing. We conclude that, although ergot can primarily be categorized as a plant pathogen, ergot infection may sometimes represent indirect beneficial effects for the host plant. Ergot may thus serve as a conditional defensive mutualist for its host grass, and the pathogenic interaction may range from antagonistic to mutualistic depending on the situation.     

Low Dose Ultraviolet B Irradiation Increases Hyaluronan Synthesis in Epidermal Keratinocytes via Sequential Induction of Hyaluronan Synthases Has1–3 Mediated by p38 and Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Signaling

Hyaluronan, a major epidermal extracellular matrix component, responds strongly to different kinds of injuries. This also occurs by UV radiation, but the mechanisms involved are poorly understood. The effects of a single ultraviolet B (UVB) exposure on hyaluronan content and molecular mass, and expression of genes involved in hyaluronan metabolism were defined in monolayer and differentiated, organotypic three-dimensional cultures of rat epidermal keratinocytes. The signals regulating the response were characterized using specific inhibitors and Western blotting. In monolayer cultures, UVB increased hyaluronan synthase Has1 mRNA already 4 h postexposure, with a return to control level by 24 h. In contrast, Has2 and Has3 were persistently elevated from 8 h onward. Silencing of Has2 and especially Has3 decreased the UVB-induced accumulation of hyaluronan. p38 and Ca²⁺/calmodulin-dependent protein kinase II pathways were found to be involved in the UVB-induced up-regulation of Has2 and Has3 expression, respectively, and their inhibition reduced hyaluronan deposition. However, the expressions of the hyaluronan-degrading enzymes Hyal1 and Hyal2 and the hyaluronan receptor Cd44 were also up-regulated by UVB. In organotypic cultures, UVB treatment also resulted in increased expression of both Has and Hyal genes and shifted hyaluronan toward a smaller size range. Histochemical stainings indicated localized losses of hyaluronan in the epidermis. The data show that exposure of keratinocytes to acute, low dose UVB increases hyaluronan synthesis via up-regulation of Has2 and Has3. The simultaneously enhanced catabolism of hyaluronan demonstrates the complexity of the UVB-induced changes. Nevertheless, enhanced hyaluronan metabolism is an important part of the adaptation of keratinocytes to radiation injury.     

Traditional microscopy instruction versus process-oriented virtual microscopy instruction: a naturalistic experiment with control group

Background

Virtual microscopy is being introduced in medical education as an approach for learning how to interpret information in microscopic specimens. It is, however, far from evident how to incorporate its use into existing teaching practice. The aim of the study was to explore the consequences of introducing virtual microscopy tasks into an undergraduate pathology course in an attempt to render the instruction more process-oriented. The research questions were: 1) How is virtual microscopy perceived by students? 2) Does work on virtual microscopy tasks contribute to improvement in performance in microscopic pathology in comparison with attending assistant-led demonstrations only?

Method

During a one-week period, an experimental group completed three sets of virtual microscopy homework assignments in addition to attending demonstrations. A control group attended the demonstrations only. Performance in microscopic pathology was measured by a pre-test and a post-test. Student perceptions of regular instruction and virtual microscopy were collected one month later by administering the Inventory of Intrinsic Motivation and open-ended questions.

Results

The students voiced an appreciation for virtual microscopy for the purposes of the course and for self-study. As for learning gains, the results indicated that learning was speeded up in a subgroup of students consisting of conscientious high achievers.

Conclusions

The enriched instruction model may be suited as such for elective courses following the basic course. However, the instructional model needs further development to be suited for basic courses.