Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi

Selected strains of three species of white rot fungi, Pleurotus ostreatus, Phanerochaete chrysosporium and Trametes versicolor, were grown in sterilized soil from straw inocula. The respective colonization rates and mycelium density values decreased in the above mentioned order. Three- and four-ringed PAHs at 50 ppm inhibited growth of fungi in soil to some extent. The activities of fungal MnP and laccase (units per g dry weight of straw or soil), extracted with 50 mM succinate-lactate buffer (pH 4.5), were 5 to 20-fold higher in straw compared to soil. The enzyme activities per g dry soil in P. ostreatus and T. versicolor were similar, in contrast to P. chrysosporium, where they were extremely low. Compared to the aerated controls, P. ostreatus strains reduced the levels of anthracene, pyrene and phenanthrene by 81–87%, 84–93% and 41–64% within 2 months, respectively. During degradation of anthracene, all P. ostreatus strains accumulated anthraquinone. PAH removal rates in P. chrysosporium and T. versicolor soil cultures were much lower.     

The Effect of Cauda Equina Constriction on Nitric Oxide Synthase Activity

Nitric oxide synthase (NOS) activity was studied in the gray and white matter regions of the spinal cord 2 and 5 days after multiple cauda equina constrictions of the central processes of L7-Co5 dorsal root ganglia neurons. The results show considerable differences in enzyme activity in the thoracic, upper lumbar, lower lumbar, and sacral segments. Increased NOS activity was observed at 2 days after multiple cauda equina constrictions in the dorsal, lateral, and ventral columns of the lower lumbar segments and in the ventral column of the upper lumbar segments. The values returned to control levels within 5 postconstriction days. In the lateral columns of thoracic segments taken 2 and 5 days after surgery, NOS activity was enhanced by 54% and 55% and in the upper lumbar segments by 130% and 163%, respectively. Multiple cauda equina constrictions performed surgically for 2 and 5 days caused a significant increase in NOS activity predominantly in the gray matter regions of thoracic segments. A quite different response was found 5 days postconstriction in the upper lumbar segments, where the enzyme activity was significantly decreased in the dorsal horn, intermediate zone, and ventral horn. No such extreme differences could be seen in the lower lumbar segments, where NOS activity was significantly enhanced only in the ventral horn. The data correspond with a higher number of NOS immunoreactive somata, quantitatively evaluated in the ventral horn of the lower lumbar segments at 5 days after multiple cauda equina constrictions. While the great region-dependent heterogeneity in NOS activity seen 2 and 5 days after multiple cauda equina constrictions is quite apparent and suggestive of an active role played by nitric oxide in neuroprotective or neurotoxic processes occurring in the gray and white matter of the spinal cord, the extent of damage or the degree of neuroprotection caused by nitric oxide in compartmentalized gray and white matter in this experimental paradigm would be possible only using longer postconstriction periods.     

Molecular characterization of binding of calcium and carbohydrates by an early activation antigen of lymphocytes CD69

CD69 is the earliest leukocyte activation antigen playing a pivotal role in cellular signaling. Here, we show that a globular C-terminal domain of CD69 belonging to C-type lectins binds calcium through Asp 171, Glu 185, and Glu 187 with K(d) approximately 54 microM. Closure of the calcium-binding site results in a conformational shift of Thr 107 and Lys 172. Interestingly, structural changes in all of these amino acids lead to the formation of high-affinity binding sites for N-acetyl-D-glucosamine. Similarly, a structural change in Glu 185 and Glu 187 contributes to a high-affinity site for N-acetyl-D-galactosamine. Site-directed mutagenesis and molecular modeling allowed us to describe the structural details of binding sites for both carbohydrates. These studies explain the importance of calcium for recognition of carbohydrates by CD69 and provide an important paradigm for the role of weak interactions in the immune system.     

PCR identification of Salmonella cells in food and stool samples after immunomagnetic separation

The purpose of the present study was to investigate the application of various sample preparation methods (cell washing before lysis, purification of DNA using phenol extraction method, immunomagnetic separation-IMS) for the final PCR identification of Salmonellacells. The presence of PCR inhibitors in processed food products (milk powder and dried eggs) can be the cause of false-negative results in PCR without IMS of target cells. It was also demonstrated that IMS-PCR was successfully used for identification and quick confirmation of untypical Salmonella strains isolated from human stool samples and rabbit meat. However, IMS cannot eliminate intracellular PCR inhibitors present in immunoseparated Salmonella cells. These inhibitors must be taken into consideration in evaluation of PCR procedure.     

Microbial activities in soils of a healthy and a declining reed stand

Microbial processes were investigated in the soil of a declining, more eutrophic (Romberk West) and a healthy looking, less eutrophic (Romberk East) freshwater reed stand. Soil was sampled monthly from June to September 1997. Glucose induced carbon dioxide (CO₂) production in oxic and anoxic conditions, methane (CH₄) production, nitrification and denitrification activities were measured in laboratory conditions in suspensions prepared from homogenised soil samples. Within a stand the proportion of anaerobic (as opposed to aerobic) microbial activity was greatest in June. Potential methanogenesis was highest in June and decreased later in both stands. Methane production was approximately the same in June at both stands but it was higher at Romberk East than at Romberk West stand in later months. Denitrifying activity was higher in August than July at both stands. Nitrifying activity was undetectable at both stands over the entire study period. Generally Romberk West was more anaerobic than Romberk East, with lower redox potential, higher amounts of oxygen-consuming organic matter and a lower ratio of CO₂ production in oxic conditions to CO₂ production in anoxic conditions. Microbial activity was apparently restricted at Romberk West stand in comparison to Romberk East. The shift from aerobic to anaerobic microbial metabolism and a coinciding restriction of metabolic activities at Romberk West are thought to be indicative of a strengthened oxygen stress in the soil, associated with accumulation of metabolites toxic to both the microorganisms and the reed. Possible links between eutrophication, microbial characteristics and reed performance are discussed.     

Photosynthetic Traits in Wheat Grown under Decreased and Increased CO2 Concentration, and after Transfer to Natural CO2 concentration

Wheat plants were grown from sowing to day 18 in 26-dm³ chambers at three different CO₂ concentrations: 150 (-CO₂), 350 (C, control), 800 (+CO₂) mol mol^-1. Afterwards, plants of the three variants were grown at the same natural CO₂ concentration. Plant characteristics were measured just before the transfer (0 days after CO₂ treatment, DAT), and at 5 – 8 DAT on the 1^st leaf, and at 12 – 22 DAT on the 4^th leaf. Decreased or increased CO₂ concentrations caused acclimations which persisted after transplantation to natural CO₂ concentration. At 5 – 8 DAT, stomatal density, stomatal conductance (g_s), CO₂ saturated net photosynthetic rate (P_Nsat0), radiation saturated net photosynthetic rate (P_Nsat1), and carboxylation efficiency () were higher in -CO₂ plants and lower in +CO₂ plants than in C plants. As compared with C plants, the photochemical efficiency () was lower in -CO₂ and higher in -CO₂ plants, however, chlorophyll (Chl) a, Chl b, Chl a–b and carotenoid contents were lower in both -CO₂ and +CO₂ plants. On the 4^th leaf, which emerged on plant after finishing CO₂ treatments, at 12 – 22 DAT, no differences in stomatal density and g, between treatments were observed. In -CO₂ plants, pigment content and P_Nsat0 were higher, was lower, and P_Nsat1 and were not different from C plants. In contrast, in +CO₂ plants, pigment content, P_Nsat1 and were lower, and P_Nsat0 and were unchanged. Leaf area, dry mass, and tiller development increased in +CO₂ plants and decreased in -CO₂ plants. In the interval between 8 and 22 DAT, lower net assimilation rate in +CO₂ than in -CO₂ plants was observed.     

Accumulation of pathogenesis-related proteins in barley leaf intercellular spaces during leaf senescence

Accumulation of the pathogenesis-related (PR) proteins localised in intercellular spaces of barley primary leaves, chlorophyll content, structure of chloroplasts, and photosynthesis were examined during natural and in vitro induced leaf senescence (cultivation of whole plants in the dark or detached leaves under nutrient deficiency). Some of PR proteins accumulated during natural senescence, but their accumulation pattern was different from those of pathogen-induced as well as during in vitro-induced senescence, which indicate different molecular bases of these processes. Photosynthetic rate and chlorophyll content indicate that natural senescence of barley primary leaves began from 15th day after sowing. In 35-d-old first leaves, the chloroplasts showed typical characteristics of senescence as significant decrease of size, greater grana, and prominent plastoglobuli. The chloroplasts contained more grana under in vitro induced senescence and they had reduced length in the dark. Correspondingly, accumulation of PR proteins was detectable on about the 15th day but the content of some PR proteins increased in later stages of senescence. This revised version was published online in July 2006 with corrections to the Cover Date.