Extracellular mitochondrial DNA and oxidatively damaged DNA in synovial fluid of patients with rheumatoid arthritis

We investigated whether plasma and synovial fluid (SF) samples from patients with rheumatoid arthritis (RA) contained extracellular mitochondrial DNA (mtDNA) or the oxidatively damaged DNA adduct 8-hydroxy-2'-deoxyguanosine (8-oxodG). Moreover, we correlated the laboratory findings of the patients with RA with their levels of mtDNA and 8-oxodG. SF and plasma samples from 54 patients with RA, SF from 30 non-arthritic control subjects, and plasma from 22 healthy volunteers were collected. The samples were subjected to polymerase chain reaction (PCR) using mitochondrial genomic primers, and the products were analyzed by SDS–polyacrylamide-gel electrophoresis. The intensities of the PCR-amplified bands were quantified and normalized to a reference sample. Furthermore, the SF samples were assayed by enzyme-linked immunosorbent assay for 8-oxodG. Extracellular PCR-amplifiable mtDNA was detected in the SF of 38 of 54 (70%) patients with RA, but not in any of the SF controls. PCR-amplifiable mtDNA was detected in the plasma of 30 of 54 (56%) of patients with RA and in 6 of 22 (27%) of the healthy volunteers. The levels of mtDNA in the plasma and SF samples of patients with RA were significantly higher (P < 0.0001) than in the respective control samples. The presence of both mtDNA and 8-oxodG in SF was significantly correlated with the presence of rheumatoid factor in the patients with RA. Extracellular mtDNA and oxidized DNA were detected in the SF of the great majority of patients with RA, but were absent or present at low levels in the control SF. These findings indicate that endogenous nucleic acid compounds might participate in joint inflammation by activating immune cells in the joints to produce proinflammatory cytokines.     

Electrical capacitance of lipid bilayer membranes of hydrogenated egg lecithin at the temperature phase transition

Electrical capacitance of the planar bilayer lipid membrane (BLM) formed from hydrogenated egg lecithin (HEL) has been studied during many passages through the phase transition temperature. In contrast to the BLM from individual synthetic phospholipids, membranes from HEL did not demonstrate any capacitance change at the phase transition temperature maximum, as measured by differential scanning calorimeter at 52 degrees C. Instead, two temperatures have been discerned by capacitance records: thickening at 42-43 degrees C and thinning at 57-59 degrees C. The first temperature region is close to the transition temperature of dipalmitoyllecithin, whereas the second is close to that of distearoyllecithin, two main components of the HEL. It was suggested that capacitance measurements were able to reveal a phase separation in the BLM from HEL which was not detected by differential scanning calorimetry. The phase transition of the BLM from the liquid crystal state to the gel state is followed by thickening of the bilayer structure, partly due to a gauche- trans transition of lipid molecules but mainly due to redistribution of the solvent n-decane.     

Photosynthetic UV-B Response of Beech (Fagus sylvatica L.) Saplings

Cloned saplings of beech (7-y-old) were exposed to enhanced UV-B irradiation (+25 %) continuously over three growing seasons (1999–2001). Analysis of CO₂ assimilation, variable chlorophyll (Chl) a fluorescence, and pigment composition was performed in late summer of the third growing season to evaluate the influence of long-term elevated UV-B irradiation. This influence was responsible for the stimulation of the net assimilation rate (P _N) over a range of irradiances. The increase in P _N was partially connected to increase of the area leaf mass, and thus to the increased leaf thickness. Even a higher degree of UV-B induced stimulation was observed at the level of photosystem 2 (PS2) photochemistry as judged from the irradiance response of electron transport rate and photochemical quenching of Chl a. The remarkably low irradiance-induced non-photochemical quenching of maximum Chl a fluorescence (NPQ) in the UV-B plants over the entire range of applied irradiances was attributed both to the reduced demand on non-radiative dissipation processes and to the considerably reduced contribution of the quenching localised in the inactivated PS2 reaction centres. Neither the content of Chls and total carotenoids expressed per leaf area nor the contents of lutein, neoxanthin, and the pool of xanthophyll cycle pigments (VAZ) were affected under the elevated UV-B. However, the contributions of antheraxanthin (A) and zeaxanthin (Z) to the entire VAZ pool in the dark-adapted UV-B treated plants were 1.61 and 2.14 times higher than in control leaves. Surprisingly, the retained A+Z in UV-B treated plants was not accompanied with long-term down-regulation of the PS2 photochemical efficiency, but it facilitated the non-radiative dissipation of excitation energy within light-harvesting complexes (LHC) of PS2. Thus, in the beech leaves the accumulation of A+Z, induced by other factors than excess irradiance itself, supports the resistance of PS2 against combined effects of high irradiance and elevated UV-B.     

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).