Did the ambient ozone affect stem increment of Scots Pines (Pinus sylvestris L.) on territories under regional pollution load? Step III of Lithuanian studies

This study aimed to explore if changes in stem increment of Scots pines (Pinus sylvestris L.) could be related to changes in ambient ozone concentration when the impact of tree dendrometric parameters (age, diameter) and crown defoliation are accounted for. More than 200 dominant and codominant trees from 12 pine stands, for which crown defoliation had been assessed since 1994, were chosen for increment boring and basal area increment computing. Stands are located in Lithuanian national parks, where since 1994-95 Integrated Monitoring Stations have been operating. Findings of the study provide statistical evidence that peak concentrations of ambient ozone (O3) can have a negative impact on pine tree stem growth under field conditions where O3 exposure is below phytotoxic levels.     

USER friendly DNA engineering and cloning method by uracil excision

Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the nicking and restriction sites are tailored to create ss extensions of custom sequence. The vector is then linearized by digestion with nicking and restriction endonucleases. To generate target molecules, a single deoxyuridine (dU) residue is placed 6–10nt away from the 5′-end of each PCR primer. 5′ of dU the primer sequence is compatible either with an ss extension on the vector or with the ss extension of the next-in-line PCR product. After amplification, the dU is excised from the PCR products with the USER enzyme leaving PCR products flanked by 3′ ss extensions. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary ss extensions. By varying the design of the PCR primers, the protocol is easily adapted to perform one or more simultaneous DNA manipulations such as directional cloning, site-specific mutagenesis, sequence insertion or deletion and sequence assembly.     

The impacts of heavy metals on oxidative stress and growth of spring barley

Oxidative stress is accepted to play a significant role in stress symptoms, caused by different stressors in a variety of organisms. In this study seedlings of spring barley (Hordeum vulgare L.) were exposed to a wide range of copper, zinc, chromium, nickel, lead and cadmium concentrations in order to determine the relationships between heavy metals-induced oxidative stress and plant growth inhibition. All investigated heavy metals induced an essential increase in lipid peroxidation and a reduction of dry biomass along with an increase in metal concentration in the nutrient solution. A very close and statistically significant exponential relationship between lipid peroxidation and growth inhibition was detected in this study. According to the results of analysis of variance (ANOVA), the intensity of nonspecific oxidative stress is identified as the main factor of barley growth inhibition, explaining 75% of total variance. Almost 10% of growth inhibition is attributed to the specific impact of heavy metals. The most pronounced increase of malondialdehyde content and growth inhibition was observed in Cu and Cd treatments, whereas the lowest changes in observed indicators were detected after exposure to Zn and Pb.     

Fast in vitro translation system immobilized on a surface via specific biotinylation of the ribosome

The ribosome is the macromolecular machine responsible for translating the genetic code into polypeptide chains. Despite impressive structural and kinetic studies of the translation process, a number of challenges remain with respect to understanding the dynamic properties of the translation apparatus. Single-molecule techniques hold the potential of characterizing the structural and mechanical properties of macromolecules during their functional cycles in real time. These techniques often necessitate the specific coupling of biologically active molecules to a surface. Here, we describe a procedure for such coupling of functionally active ribosomes that permits single-molecule studies of protein synthesis. Oxidation with NaIO4 at the 3' end of 23S rRNA and subsequent reaction with a biotin hydrazide produces biotinylated 70S ribosomes, which can be immobilized on a streptavidin-coated surface. The surface-attached ribosomes are fully active in poly(U) translation in vitro, synthesizing poly(Phe) at a rate of 3-6 peptide bonds/s per active ribosome at 37 degrees C. Specificity of binding of biotinylated ribosomes to a streptavidin-coated quartz surface was confirmed by observation of individual fluorescently labeled, biotinylated 70S ribosomes, using total internal reflection fluorescence microscopy. Functional interactions of the immobilized ribosomes with various components of the protein synthesis apparatus are shown by use of surface plasmon resonance.     

Role of oxidative stress on growth responses of spring barley exposed to different environmental stressors

Aims Oxidative stress is one of the most important mechanisms in a plant's reaction to the effects of different stressors; however, its role in plants' resistance is still poorly understood. The objective of this study is to evaluate an influence of oxidative stress induced by stress factors of different origin—ozone, ultraviolet (UV)-B radiation, drought, cadmium (Cd) and copper (Cu), to growth of spring barley and to check the hypothesis, that intensification of oxidative stress is the main factor of growth depression induced by strong treatments of different stressors; meanwhile, mitigation of oxidative stress determines eustress-induced growth stimulation.Methods A pot experiment was carried out in phytotron chambers with a controlled environment. Spring barley (Hordeum vulgare L.) plants were exposed to different doses of investigated environmental stress factors (O 3, UV-B radiation, drought, Cd and Cu), and their effects on shoots growth, accumulation of superoxide (O 2 .?), intensification of lipid peroxidation and antioxidative protection (superoxide dismutase, glutathione reductase and catalase activities and concentration of carotenoids) were measured. Analysis of variance (ANOVA) with classical eta-squared (η 2) values was used to evaluate and to compare the contribution of non-specific oxidative stress and stressor-specific mechanisms on plants growth.Important findings Low doses of most stressors stimulated antioxidative protection and growth of barley shoots, reduced the concentration of O 2 .? and/or intensity of lipid peroxidation. Whereas an impairment of growth and intensification of oxidative stress as well as a reduction in concentration of carotenoids and further increase in activity of antioxidative enzymes were noticed when the intensity of the stressors was increased. In the cases of ozone and UV-B stress, the effects of oxidative stress on plant growth was mitigated by strong antioxidative protection—highly increased catalase (CAT) and superoxide dismutase (SOD) activities, respectively. In the cases of drought and Cu, relatively strong oxidative stress was the major cause of plant growth depression. Additionally, mitigation of oxidative stress due to increased SOD activity was likely to be one of the main causes of growth stimulation induced by low doses of UV-B, Cd and Cu stress. Possible reasons for O 3 -induced growth stimulation were increased CAT activity and concentration of carotenoids. Generalizing the effects of different stressors, the contribution of non-specific oxidative stress on plant growth was stronger compared with stressor-specific action mechanisms: oxidative stress determined 42% of the changes in plants' dry biomass, whereas the contribution of stressor-specific mechanisms accounted for 35% of variability in barley growth.     

Discovery and distribution of super-integrons among Pseudomonads

Until recently, integrons (systems for acquisition and expression of new genetic materials) have been associated generally with antibiotic resistance gene cassettes. The discovery of 'super-integrons' in Vibrionaceae suggests a greater impact of this gene acquisition mechanism on bacterial genome evolution than initially believed. Super-integrons may contain more than 100 gene cassettes and may encode other determinants, including biochemical functions or virulence factors. Here, we report the genetic organization of a super-integron from Pseudomonas alcaligenes ATCC 55044. This is the first evidence of a super-integron in a non-pathogenic bacterium, one which is widely distributed in a great number of ecological niches such as soil and aquatic habitats. Here, the sequence composition, open reading frame (ORF) content and organization of In55044 are described and found to have features intermediate between the multidrug-resistant integrons and the Vibrio cholerae super-integron. Similar structures are inferred to be present in several Pseudomonas species, based on polymerase chain reaction (PCR) experiments.     

Subsequent drought and re-watering events cause a shift in productivity and nutritive value of alfalfa and hybrid fescue plant

Global climate change leads to increasing frequency of droughts, threatening the productivity and quality of forage plants. Therefore, this glasshouse experiment was conducted to investigate the effects of reoccurring droughts on productivity and nutritive value of alfalfa and hybrid fescue plants. Starting from 63 days after sowing, seedlings of plants were exposed to double drought-recovery treatments, each separate period lasting for 1 week. At the end of each treatment, growth of above- and below-ground dry weight, lipid peroxidation, total antioxidative capacity, content of polyphenols, soluble and insoluble carbohydrates and proteins, as well as elemental composition of shoots were investigated. The findings confirmed our hypothesis, as growth of both plant species were mostly insignificantly affected by subsequent drought events. Whereas effect on leave redox homeostasis and plant nutritive value was much stronger, highly depending on both plant species and the order of stress event. We found significant drought-induced increase in the content of water-soluble carbohydrates (WSCs), starch, soluble and insoluble proteins, and polyphenols. Water shortage tended to decrease and increase the content of most macro- and microelements in alfalfa and fescue, respectively. These effects were reversed by re-watering with several exceptions, such as constant reduction of Ca content in alfalfa. To conclude, alfalfa was found to be more sensitive than fescue to the first drought, but able to recover after both stress events. The second drought-initiated prolonged accumulation of WSC implies a shift in plants metabolism towards increased resistance and herewith nutritive value with respect to soluble sugars.     

Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: Interactions with human β2 integrins and erythrocytes

Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α_Lβ₂ (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express β₂ integrin. Cells expressing α_Mβ₂ (CD11b/CD18) or α_Xβ₂ (CD11c/CD18) were killed as efficiently as cells expressing α_Lβ₂. Erythrocytes, which do not express β₂ integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β₂ chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α_Mβ₂ and α_Xβ₂ as novel receptors for LtxA.