New Facts about CdCl2 Action on the Photosynthetic Apparatus of Spinach Chloroplasts and Its Comparison with HgCl2 Action

Using EPR spectroscopy it was found that CdCl₂ and HgCl₂ interact (1) with the intermediates , i.e. with the tyrosine radicals on the donor side of photosystem (PS) 2 situated in the 161^st position in D₁ and D₂ proteins; (2) with the primary donor of PS1 (P700) whereby the oxidation of chlorophyll (Chl) a dimer in the reaction centre of PS1 occurs yet in the dark; (3) with the manganese cluster which is situated in the oxygen evolving complex. Due to these interactions of investigated metal chlorides with the photosynthetic apparatus, the interruption of the photosynthetic electron transport through photosynthetic centres occurs. Monitoring of time dependence of EPR signal I of chloroplasts treated with CdCl₂ or HgCl₂ after switching off the light suggests that all mechanisms, i.e. direct, cyclic, and non-cyclic reductions of P700⁺ are damaged. The formation of complexes between mercury or cadmium ions and amino acid residues constituting photosynthetic peptides was suggested as possible mechanism of their inhibitory action. The higher HgCl₂ efficiency in comparison with that of CdCl₂ was explained by higher ability of mercury ions to form complexes with amino acids, what was demonstrated by their apparent binding constants: K = 10 200 M^–1 for Hg²⁺ ions, and K = 3 700 M^–1 for Cd²⁺ ions.     

Glutamate decarboxylase activity in Trichoderma viride conidia and developing mycelia

Glutamic acid decarboxylase (GAD) activity was measured in homogenates of conidia and both submerged and aerial mycelia of Trichoderma viride. The GAD activity in conidia had a temperature optimum at 30 degrees C and a pH optimum at pH 4. GAD was stimulated by EDTA (2 mM) and was insensitive to treatment with calmodulin antagonists calmidazolium (10 microM) or phenothiazine neuroleptics (60 microM). Cyclosporin A (up to 300 microM) partially inhibited GAD in the homogenate, but not in the supernatant obtained after centrifuging the homogenate. Attempts to release GAD activity from the homogenate using high ionic strength, detergents, or urea failed. Freezing-thawing led to the partial increase of activity in the conidial homogenate. These results indicate that GAD is a membrane-bound enzyme. The highest specific activity of GAD was present in the mitochondrial/vacuolar organellar fraction. Germination of conidia in the submerged culture led to a temporary decrease in GAD activity. After prolonged cultivation, the activity displayed quasi-oscillatory changes. The stationary state was characterized by a high GAD activity. The presence of gamma-aminobutyric acid in the submerged mycelia was demonstrated. In surface culture in the dark, GAD activity increased in a monophasic manner until conidia formation. The illumination of dark-cultivated mycelia by a white-light pulse caused a dramatic increase in GAD activity. Light-induced changes were not observed in mutants with delayed onset of conidiation. In the dark or upon illumination by light pulse, the increase of GAD activity preceded the appearance of conidia. Thus, GAD activity in T. viride is closely associated with its developmental status and may represent a link between differentiation events and energy metabolism.     

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.     

Changes in the yeast metabolism at very high-gravity wort fermentation

The rate of ethanol production increased with increasing wort gravity up to the initial wort concentration of 24%, reaching the maximum ethanol concentration of 6.2%, but its attenuation reached only 49%. The intracellular trehalose accumulation was proportional to the inital wort gravity, at 24 or 30% wort fermentation increased 3 or 4.5 times, respectively, compared to 12% wort fermentation. Trehalose accumulation began after exhaustion of glucose, ceased after uptake of approximately 65% reducing saccharides, despite of increasing ethanol or remaining saccharide concentration in the environment.     

Cloning of a two-component regulatory system probably involved in the regulation of chitinase inStreptomyces cœlicolor A3(2)

Using the method for the identification of promoters recognized by the sporulation specific σ factor (σ^F), we identified a positive 950 pbSau3Al DNA fragment inStreptomyces cœlicolor A3(2). High-resolution S1-nuclease mapping identified a potential promoter, P_F35, in theE. coli two-plasmid system similar to the consensus sequence ofBacillus subtilis promoters recognized by the general stress-response σ factor (σ^B). However, the putativesigF-dependent promoter, P_F35, was inactive inS. cœlicolor in the course of diffenentiation and it was located divergently in the promoter region directing expression of thechiC gene encoding chitinase. Sequence analysis of the region potentially governed by P_F35 revealed two translationally coupled genes encoding proteins similar to bacterial two-component regulatory systems, and with the highest similarity to the two-component systemchiS, chiR, regulating chitinase activity inStreptomyces thermoviolaceus. However, the genes had a divergent orientation with respect to the P_F35 promoter. Disruption of theS. cœlicolor chiR gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin. Moreover, thechiR disruption did not affect the overall chitinase activity.     

Prevalence ofCryptococcus neoformans in clinical specimens

Cryptococcus neoformans isolated from various clinical materials in 14 cases, was identified by (1) cultivation on Sabouraud glucose agar and CHROMagar Candida, (2) microscopic examination of Indian-ink-stained preparations and (3) determination of biochemical properties (assimilation and fermentation of saccharides, assimilation of KNO3, production of urease and phenol monooxygenase). C. neoformans was determined in five specimens from paediatric patients in the intensive care unit and in nine specimens from adult patients, most frequently from liquor at meningitis (n = 3).     

Determination of thirty-two elements in human autopsy tissue

Inductively coupled plasma-mass spectrometry (ICP-MS) was used to determine the concentrations of 32 elements in the human liver and kidney and 20 elements in the bone, obtained from 70 autopsied dead individuals (54 males, 16 females) between 18 and 76 yr of age from the North Bohemia territory of the Czech Republic. Geometric means, median, minimal-maximal range, as well as distribution and correlation analysis were calculated. Some significant differences among tissue concentrations of trace elements of the women and men were found. In the liver, medians of the concentrations of some elements were higher for men than that for women (Al: 770 vs 610 microg/kg; As: 42 vs 27 microg/kg; Cd: 1800 vs 1390 microg/kg; Rb: 3955 vs 3210 microg/kg; V: 160 vs 105 microg/kg). On the contrary, the content of other elements for men was lower (Bi: 0.8 vs 3.2 microg/kg; Cr: 57 vs 72 microg/kg; Hg: 228 vs 325 microg/kg; Zn: 57.1 vs 68.5 mg/kg). In the kidney of men, there were higher contents of Al (360 vs 245 microg/kg) and Hg (135 vs 75 microg/kg) and lower contents of Zn (47.7 vs 59.7 mg/kg) and I (135 vs 220 microg/kg) than those of women. In the case of bone, the concentrations of Cu and Rb were higher for men (1410 microg Cu/kg and 405 microg Rb/kg, respectively) than for women (655 microg Cu/kg and 285 microg Rb/kg, respectively). On the contrary, the content of Mn was considerably lower for men (110 microg Mn/kg) than for women (215 microg Mn/kg).     

Screening of the occurrence of copper amine oxidases in Fabaceae plants

Aim of this work was to find the best source for obtaining high amount of copper amine oxidase (EC 1.4.3.6) that can be further used for analytical or industrial applications. The study focused on plant enzymes, because they occur in much higher content in the starting material than the enzymes from other sources, have higher specific activity and are also more thermostable. Presence of the amine oxidase was tested in extracts from 4 to 7-d-old seedlings of thirty-four various Fabaceae plants. Amine oxidases from nine selected plants were purified by general method involving ammonium sulfate fractionation, controlled heat denaturation, and three chromatographic steps. Kinetic properties of the amine oxidases purified were tested with a wide range of substrates and inhibitors and were found to be very similar. Best purification yield, and total and specific activities were obtained for the enzyme from grass pea (Lathyrus sativus) throughout all purification steps. Hence, the grass pea extract was chosen as a suitable candidate for massive production of the amine oxidase. This revised version was published online in July 2006 with corrections to the Cover Date.