Biosynthesis of silver nanoparticles with the participation of extracellular Mn-dependent peroxidase from <Emphasis Type="Italic">Azospirillum</Emphasis>

The accumulation of nanoparticles of colloidal silver with spherical shape in culture liquid of Azospirillum brasilense has been shown by transmission electron microscopy. Bacterial extracellular Mn-peroxidases were found to participate in silver reduction from silver nitrate with the formation of nanoparticles. A mechanism of extracellular biosynthesis of silver nanoparticles by A. brasilense bacteria was proposed.     

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (³⁵S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.     

Response of plant species richness and primary productivity in shrublands along a north–south gradient in Europe to seven years of experimental warming and drought: reductions in primary productivity in the heat and drought year of 2003

We used a nonintrusive field experiment carried out at six sites – Wales (UK), Denmark (DK), the Netherlands (NL), Hungary (HU), Sardinia (Italy – IT), and Catalonia (Spain – SP) – along a climatic and latitudinal gradient to examine the response of plant species richness and primary productivity to warming and drought in shrubland ecosystems. The warming treatment raised the plot daily temperature by ca. 1 °C, while the drought treatment led to a reduction in soil moisture at the peak of the growing season that ranged from 26% at the SP site to 82% in the NL site. During the 7 years the experiment lasted (1999–2005), we used the pin‐point method to measure the species composition of plant communities and plant biomass, litterfall, and shoot growth of the dominant plant species at each site. A significantly lower increase in the number of species pin‐pointed per transect was found in the drought plots at the SP site, where the plant community was still in a process of recovering from a forest fire in 1994. No changes in species richness were found at the other sites, which were at a more mature and stable state of succession and, thus less liable to recruitment of new species. The relationship between annual biomass accumulation and temperature of the growing season was positive at the coldest site and negative at the warmest site. The warming treatment tended to increase the aboveground net primary productivity (ANPP) at the northern sites. The relationship between annual biomass accumulation and soil moisture during the growing season was not significant at the wettest sites, but was positive at the driest sites. The drought treatment tended to reduce the ANPP in the NL, HU, IT, and SP sites. The responses to warming were very strongly related to the Gaussen aridity index (stronger responses the lower the aridity), whereas the responses to drought were not. Changes in the annual aboveground biomass accumulation, litterfall, and, thus, the ANPP, mirrored the interannual variation in climate conditions: the most outstanding change was a decrease in biomass accumulation and an increase in litterfall at most sites during the abnormally hot year of 2003. Species richness also tended to decrease in 2003 at all sites except the cold and wet UK site. Species‐specific responses to warming were found in shoot growth: at the SP site, Globularia alypum was not affected, while the other dominant species, Erica multiflora, grew 30% more; at the UK site, Calluna vulgaris tended to grow more in the warming plots, while Empetrum nigrum tended to grow less. Drought treatment decreased plant growth in several studied species, although there were some species such as Pinus halepensis at the SP site or C. vulgaris at the UK site that were not affected. The magnitude of responses to warming and drought thus depended greatly on the differences between sites, years, and species and these multiple plant responses may be expected to have consequences at ecosystem and community level. Decreases in biodiversity and the increase in E. multiflora growth at the SP site as a response to warming challenge the assumption that sensitivity to warming may be less well developed at more southerly latitudes; likewise, the fact that one of the studied shrublands presented negative ANPP as a response to the 2003 heat wave also challenges the hypothesis that future climate warming will lead to an enhancement of plant growth and carbon sequestration in temperate ecosystems. Extreme events may thus change the general trend of increased productivity in response to warming in the colder sites.     

The in vitro motility assay to study the calcium-mechanical relationship in skeletal and cardiac muscles

In a series of experiments on regulated contractile systems (i.e., in vitro mobile systems with reconstructed thin filaments), the velocities of the movement of a thin filament on the surface covered by either rabbit skeletal or rat cardiac myosin at various concentrations of calcium ions in solution (in the pCa range from 4 to 8) were assessed. The corresponding "pCa-velocity" relationships were plotted, which proved to be of the sigmoid form. It was found that, at a saturating calcium concentration (pCa 4), the velocity of regulated thin filaments was 65% higher than for unregulated ones in the case of skeletal myosin and 87% higher than for unregulated thin filaments in the case of cardiac myosin. It was also found that the Hill coefficient was 1.95 and 2.5 for skeletal and cardiac myosins, respectively. The difference in the Hill coefficients for skeletal and cardiac myosins is discussed in terms of the difference in contribution of cooperativity mechanisms of contractile and regulatory proteins in the regulation of contraction in these types of muscles.     

Activity of <Emphasis Type="Italic">Lentinus edodes</Emphasis> intracellular lectins at various developmental stages of the fungus

We studied changes of the hemagglutinating activity of intracellular lectins of the basidiomycete Lentinus edodes (shiitake) at various stages of its morphogenetic development depending on erythrocyte type, growth medium, and lectin purification degree. Under certain experimental conditions, the specific lectin activity at the brown mycelium film stage exceeded the corresponding value for nonpigmented mycelium. The sensitivity of the lectins towards trypsin-treated rabbit erythrocytes was no less than a hundredfold higher than towards any other erythrocyte type studied. The general regularities of specific activity change did not depend on nutrient medium composition. With purification of intracellular shiitake lectins, their sensitivity to human erythrocytes decreased seventyfold or more, whereas their sensitivity to rabbit erythrocytes increased by the same factor.     

Lectin activity of Lentinus edodes

The hemagglutinating activity of submerged mycelium and culture liquid for four strains of Lentinus edodes (Berk.) Sing [L. edodes (Berk.) Pegler] was studied in the search for lectins. The hemagglutinating activity of culture liquid was substantially higher, compared with mycelium. The carbohydrate-binding capacity of the agglutinins was established, and the lectin activity of extracts from mycelia grown on several agar media was elucidated in relation to fruiting. The lectin activity of L. edodes was examined at different morphogenetic steps: mycelium, brown mycelial film, primordium, and fruiting body. Hemagglutination titers at the brown film step were higher than in the mycelium, whereas activity at the primordial and fruiting bodies steps decreased. Lectins seem to be involved in the formation of hyphal aggregates of brown mycelial film.     

Distribution and Physiological State of Microorganisms in Petrochemical Oily Sludge

The occurrence, vertical distribution, and physiological state of microorganisms in a petrochemical oily sludge deposit were studied. The total number and the number of viable microbial cells at depths of 0.2 and 3 m were about 10¹⁰ and 10⁸ cells/g dry wt sludge. Most microbial cells taken from the middle (1 m deep) and the bottom (3 m deep) sludge horizons showed a delayed colony-forming ability, which suggested that the cells occurred in a hypometabolic state. The relative number of microaerobic denitrifying microorganisms steeply increased with depth. The amount of microorganisms tolerant to 3, 5, and 10% NaCl and capable of growing at 7 and 40°C varied from 10² to 10⁸ CFU/g dry wt sludge. Petrochemical oily sludge was found to maintain the growth of heterotrophs, among which the degraders of oily sludge and ten different individual polycyclic aromatic hydrocarbons were detected. The occurrence of highly adaptable microorganisms with an adequate metabolic potential in the petrochemical oily sludge deposit implies that its bioremediation is possible without introducing special microorganisms.     

Isolation and characterization of lectin from the surface of Grifola frondosa (FR.) S.F. Gray mycelium

For the first time, from the surface of the dikaryotic mycelium of the xylotrophic basidiomycete Grifola frondosa 0917 a lectin has been isolated with a molecular mass of 68 +/- 1 kDa, consisting of two subunits of 33-34 kDa each. The lectin is a hydrophilic glycoprotein with the protein : glycan ratio of 3 : 1. It exhibits high affinity to native rabbit erythrocytes and to human erythrocytes of the 0 blood group, but not to trypsin-treated ones. The hemagglutination (HA) caused by lectin was not blocked by any of the 25 tested mono-, di-, and amino sugars; it was also not blocked by some of glyco derivatives. Only 13.9 microg/ml of the homogeneous preparation of a polysaccharide, a linear D-rhamnan with the structure of the repetitive component --> 2)-beta-D-Rhap-(1 --> 3)-alpha-D-Rhap-(1 --> 3)-alpha-D-Rhap-(1 --> 2)-alpha-D-Rhap-(1 --> 2)-alpha-D-Rhap-(1 --> blocked hemagglutination completely. The analysis of the amino acid composition of the lectin showed the greatest percentage of amino acids with positively charged R groups, arginine, lysine, and histidine, as well as the complete absence of sulfur-containing amino acids, cysteine, and methionine. D-glucose and D-glucosamine were detected in the carbohydrate part.     

Effect of lectins from <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> to peroxidase and oxalate oxidase activity regulation in wheat roots

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability of lectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect of lectin in under 10 min in a concentration of 10 μg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.