Enrichment and isolation of non-specific aromatic degraders from unique uncontaminated (plant and faecal material) sources and contaminated soils

Microbial analysis of contaminated soil and uncontaminated plant and faecal material resulted in the enrichment of a number of microbial communities capable of utilizing a range of environmental pollutants. Growth was observed on polycyclic aromatic hydrocarbons, polychlorinated biphenyls, heterocyclic aromatic compounds and organochlorine pesticides. However, none of the communities could grow on pentachlorophenol. Pure cultures were isolated from microbial communities using phenanthrene and pyrene as the sole carbon and energy source. Isolates were also obtained using DDT, DOH, DBH and PCPA when peptone was supplemented to the medium. Strain AJR39,504, isolated using DDT and peptone, could not be positively identified on the basis of substrate utilization tests. However, it most closely resembled Stenotrophomonas maltophilia (0.424 similarity) using the Microlog 3 database software. Isolate AJR39, 504 could also grow on polycyclic aromatic hydrocarbons, chlorinated- and nitro-aromatic compounds. In addition, the degradation of DDT (100 mg l(-1)) by isolate AJR39,504 resulted in a 35% decrease in DDT concentration after 28 days with a concomitant increase in DDD concentration.     

Combination of gastrin-releasing peptide antagonist with cytotoxic agents produces synergistic inhibition of growth of human experimental colon cancers

We investigated the efficacy of a powerful antagonist of bombesin/gastrin-releasing peptide (BN/GRP) RC-3940-II administered as a single agent or in combination with cytotoxic agents on the growth of HT-29, HCT-116 and HCT-15 human colon cancer in vitro and in vivo. GRP-receptor mRNA and protein were found in all three cell lines tested. Exposure of HT-29 cells to 10 μM RC-3940-II led to an increase in the number of cells blocked in S phase and G₂/M and cells with lower G₀/G₁ DNA content. Similar changes on the cell cycle traverse of HT-29 cells could also be seen at lower concentrations of RC-3940-II (1 μM) after pretreatment with 100 nM GRP (14–27), indicating a dose-dependent mechanism of action based on the blockage of BN/GRP induced proliferation of tumor cells at lower concentrations. Daily in vivo treatment with BN/GRP antagonist RC-3940-II decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice by 25 to 67% (p < 0.005). Combined treatment with RC-3940-II and chemotherapeutic agents 5-FU and irinotecan resulted in a synergistic tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts by 43% to 78%. In HT-29 and HCT-116 xenografts the inhibition for the combinations of RC-3940-II and irinotecan vs. single substances (p < 0.05) was significantly greater. These findings support the use of RC-3940-II as an anticancer agent and may help to design clinical trials using RC-3940-II in combinations with cytotoxic agents.     

The clinical importance of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in the 5-fluoropyrimidine-based therapy of metastatic colorectal tumours

The authors investigated the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in 101 metastatic colorectal cancer patients treated with 5-fluoropyrimidine-based therapy and in 196 healthy individuals by PCR-RFLP method. There was no significant difference in genotype distribution of patients and healthy controls, and between subgroups investigated according to clinical parameters (age, gender, tumor location, grade and treatment type). However, after a 3-30 (median 18.5) months follow-up the survival of patients with T allele proved to be better than that of patients with wild type (CC) genotype (p=0.036). In case of CT and TT genotypes the survival of patients receiving only first line therapy was significantly shorter than that of patients receiving more lines of treatment (p=0.015). Determination of MTHFR C677T polymorphism has prognostic value in case of patients with metastatic colorectal cancer receiving 5-fluoropyrimidine-based therapy, and may help in designing the individual (group) tailored therapy.     

Host age determines parasite load of Laboulbeniales fungi infecting ants: Implications for host-parasite relationship and fungal life history

Arthropod-parasitic fungi of the order Laboulbeniales are known to exhibit specialization to individual host taxa in most cases. Some species exhibit ecological specificity to multiple, often unrelated hosts in certain microhabitats; and often position specificity to different host body parts. The myrmecophilous Rickia wasmannii (Ascomycota: Laboulbeniales) infects Myrmica species (Hymenoptera: Formicidae) (host specificity), and occasionally other arthropod inquilines inside the ant nest (ecological specificity). An effect of the position of infection on the thallus densities has also been reported. Another determinative factor that may also exist in the Rickia-Myrmica host-parasite system, the chronological age of ant worker hosts, has also been linked to parasite load. Comprehensive studies on the age-related infection intensity, however, are still lacking. Here we investigated whether the level of infection correlates with the age of the M. scabrinodis host consistently. We found that older hosts exhibited higher parasite load, even though the infection level of the different colonies varied widely. The results highlight that the level of R. wasmannii infections are strongly influenced by host individual and host colony factors.     

Hemin and bile pigments are the secondary structure regulators of intrinsically disordered antimicrobial peptides

The interaction of protoporphyrin compounds of human origin with the major bee venom component melittin (26 a.a., Z +6) and its hybrid derivative (CM15, 15 a.a., Z +6) were studied by a combination of various spectroscopic methods. Throughout a two‐state, concentration‐dependent process, hemin and its metabolites (biliverdin, bilirubin, bilirubin ditaurate) increase the parallel β‐sheet content of the natively unfolded melittin, suggesting the oligomerization of the peptide chains. In contrast, α‐helix promoting effect was observed with the also disordered but more cationic CM15. According to fluorescence quenching experiments, the sole Trp residue of melittin is the key player during the binding, in the vicinity of which the first pigment molecule is accommodated presumably making indole‐porphyrin π‐π stacking interaction. As circular dichroism titration data suggest, cooperative association of additional ligands subsequently occurs, resulting in multimeric complexes with an apparent dissociation constant ranged from 20 to 65 μM. Spectroscopic measurements conducted with the bilirubin catabolite urobilin and stercobilin refer to the requirement of intact dipyrrinone moieties for inducing secondary structure transformations. The binding topography of porphyrin rings on a model parallel β‐sheet motif was evaluated by absorption spectroscopy and computational modeling showing a slipped‐cofacial binding mode responsible for the red shift and hypochromism of the Soret band. Our results may aid to recognize porphyrin‐responsive binding motifs of biologically relevant, intrinsically disordered peptides and proteins, where transient conformations play a vital role in their functions.     

Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO₂ uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.Energy security and climate change as global problems urge increased efforts to use plants as renewable energy sources both for power generation and transportation fuel production. Selected wood species, such as willows (Salix spp.), can be cultivated as short-rotation coppice for the rapid accumulation of biomass and reduction of CO₂ emission. Coppicing reinvigorates shoot growth, resulting in a special woody plant life cycle that differs from natural tree development, which takes decades. In this cultivation system, small stem cuttings are planted at high densities (15,000–25,000 ha⁻¹). In the soil, these dormant wood cuttings first produce roots and shoots that emerge from reactivated buds. During the first year, the growing shoots mature to woody stems. In the winter, these stems are cut back, and in the following spring, the cut stumps develop multiple shoots. The short-rotation coppice plantations are characterized by a very short, 2- to 3-year rotation, and the most productive varieties can produce up to 15 tons of oven-dried wood per hectare per year (Cunniff and Cerasuolo, 2011). The high-density willow plantations can also be efficiently used for heavy metal or organic phytoremediation, as reviewed by Marmiroli et al. (2011).The biomass productivity of shrub willows is largely dependent on coppicing capability, early vigorous growth, shoot growth rate and final stem height, root system size, photosynthetic efficiency, formation and composition of woody stems, water and nutrient use, as well as abiotic and biotic stress tolerance. Genetic improvement of all these traits can be based on broad natural genetic resources represented by more than 400 species in the genus Salix. More than 200 species have hybrid origins, and ploidy levels vary from diploid up to dodecaploid (Suda and Argus, 1968; Newsholme, 1992). In addition to molecular marker-assisted clone selection, intraspecific and interspecific crosses have been shown to further extend genetic variability in breeding programs for biomass yield (Karp et al., 2011).During natural diversification and artificial crossings of Salix spp., the willow genomes frequently undergo polyploidization, resulting in triploid or tetraploid allopolyploids. In triploid hybrids, both heterosis and ploidy can contribute to the improved biomass yield (Serapiglia et al., 2014). While the alloploid triploids have attracted considerable attention in willow improvement, the potentials of autotetraploid willow genotypes have not been exploited so far. As shown for other short-rotation wood species (poplar [Populus spp.], black locust [Robinia pseudoacacia], Paulownia spp., and birch [Betula spp.]), doubling the chromosome set by colchicine treatment can cause significant changes in organ morphology or growth parameters (Tang et al., 2010; Cai and Kang, 2011; Harbard et al., 2012; Mu et al., 2012; Wang et al., 2013a, 2013b). In several polyploidization protocols, the in vitro cultured tissues are exposed to different doses of colchicine or other inhibitors of mitotic microtubule function, and plantlets are differentiated from polyploid somatic cells (Tang et al., 2010; Cai and Kang, 2011). Alternatively, seeds or apical meristems of germinating seedlings can be treated with a colchicine solution (Harbard et al., 2012). Allotetraploids of poplar were produced by zygotic chromosome doubling that was induced by colchicine and high-temperature treatment (Wang et al., 2013a).Since tetraploid willow plants with 2n = 4x = 76 chromosomes are expected to represent novel genetic variability, especially for organ development and physiological parameters, a polyploidization project was initiated that was based on a highly productive diploid energy willow (S. viminalis var. Energo). Colchicine treatment of reactivated axillary buds of the in vitro-grown energy willow plantlets resulted in autotetraploid shoots and, subsequently, plants. For comparison of diploid and tetraploid variants of willow plants, digital imaging of green organs and roots was used for phenotyping. Among the tetraploid lines, genotypes were identified with improved biomass production, better photosynthetic parameters, and altered organ structure and hormone composition. The new tetraploid willow variants produced can serve as a unique experimental material to uncover key factors in biomass production in this short-rotation energy plant. In the future, these plants can also serve as crossing partners of diploid lines for the production of novel triploid energy willow genotypes.     

Characteristics of attention-related body sensations. Temporal stability and associations with measures of body focus,affect, sustained attention,and heart rate variability

Aim: This study investigated the temporal stability and correlates of attention-related body sensations that emerge without external stimulation during rest and due to focused attention on a body part.

Materials: To assess attention-related body sensations, participants were asked to focus on a freely chosen body area with closed eyes, and had to report whether the sensation of that area had changed. Self-report questionnaires were used to assess various aspects of body focus (body awareness, body responsiveness, somatosensory amplification, subjective somatic symptoms), and positive and negative affectivity. Previous experiences in body–mind therapies were also measured. PEBL Continuous Performance Test was used to assess sustained attention. Heart rate variability scores were based on a 3-minute long resting heart rate measurement.

Methods: Fifty-eight university students (22.3?±?3.95 years; 34 females) participated in the study. The stability of attention-related body sensations was measured 8?weeks later on a randomly chosen sub-group (n?=?28).

Results: Attention-related body sensations showed a mediocre temporal stability (r_ρ?=?0.47, p?=?0.012). People reporting attention-related body sensations showed significantly higher body awareness, somatosensory amplification, and resting heart rate; and marginally higher somatic symptoms. No relation was found with body–mind practice, body responsiveness, positive and negative affect, the vagal component of heart rate variability, and performance in the sustained attention task.

Conclusion: Attention-related sensations are relatively stable over time. They are connected to some, but not to all of the aspects of body focus. Further studies are needed to elaborate the influencing stable and situational factors.     

Interleukin-6 N-terminal peptides modulate the expression of junB protooncogene and the production of fibrinogen in HepG2 cells

Interleukin-6 (IL-6) is a helical cytokine exerting pleiotropic activities including the regulation of hematopoiesis, B cell activation and acute-phase reaction. The structure-function relationship of the molecule is the subject of intensive investigation using point and deletion mutants. Our objective was to analyse the role of the N-terminal 18-46 region in IL-6-mediated expression of junB protooncogene and fibrinogen production, reflecting the acute phase response, with synthetic overlapping peptides. mRNA expression of junB was monitored by competitive RT-PCR, while sandwich ELISA was used for the detection of fibrinogen in the supernatant of HepG2 human hepatoma cells. We found that even short synthetic octapeptides can be stimulatory (in the absence of IL-6) or inhibitory (in the presence of IL-6) in both assays. To establish the molecular mechanism by which synthetic peptides exert their biological effects electromobility shift assay was carried out using HepG2 nuclear extracts. Peptides inducing junB expression initiate gel shifts of STAT3/DNA complexes, which may indicate the involvement of this signal transduction pathway. Circular dicroism spectroscopy data suggest that 8-11-mer peptides representing different parts of the 18-46 region have a marked tendency to adopt ordered conformations in a water/trifluoroethanol (1:1 v/v) mixture. Competition studies with rhIL-6 and selected fluorophore-labelled peptides indicate the presence of more than one binding site on soluble IL-6 receptor. Considering the possible multiple etiologic role of IL-6 in the pathogenesis of various diseases, these peptides could be useful for dissection of IL-6 related biological effects.     

Evaluation of a creosote-based medium for the growth and preparation of a PAH-degrading bacterial community for bioaugmentation

Creosote was evaluated as an inexpensive carbon source for growing inocula of a polycyclic aromatic hydrocarbon (PAH)-degrading bacterial community (community five). Creosote was a poor growth substrate when provided as sole carbon source in a basal salts solution (BSM). Alternatively, peptone, yeast extract or glucose in BSM supported high growth rates, but community five could not subsequently degrade pyrene. A combination of creosote and yeast extract in BSM (CYEM) supported growth and maintained the pyrene-degrading capacity of community five. Optimum pyrene-degrading activity occurred when the inocula were grown in creosote and yeast extract concentrations of 2 ml L⁻¹ and 1 g L⁻¹ respectively: concentrations outside these values resulted in either low biomass yields or loss of PAH-degrading activity. CYEM-grown community five inocula degraded 250 mg L⁻¹ of pyrene in BSM at a rate comparable to cultures inoculated with community five grown in BSM-pyrene. However, the CYEM-grown community showed a 40% lower rate of PAH degradation in a synthetic PAH mixture compared with pyrene-grown cells and there was an increase in the lag period before the onset of PAH degradation. This appears to reflect a weaker induction of PAH catabolism by CYEM compared to BSM-pyrene. Journal of Industrial Microbiology & Biotechnology (2000) 24, 277–284. Received 24 August 1999/ Accepted in revised form 20 January 2000     

Treatment of cardiac myocytes with 8-(4-chlorophenylthio)-adenosine 3'',5''-cyclic monophosphate, forskolin or cholera toxin does not stimulate cellular or heparin-releasable lipoprotein lipase activities.

Incubation of isolated cardiac myocytes with 500 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) or 100 microM-forskolin for 2 1/2 h did not increase the heparin-induced release of lipoprotein lipase (LPL) into the medium. When LPL activity in cardiac myocytes was depleted by treatment of rats with cycloheximide (2 mg/kg; 2.5 h) and inclusion of the protein-synthesis inhibitor in the isolation solutions, incubation with CPT-cAMP or forskolin did not influence the rate of repletion of LPL activity in cells or the recovery of heparin-releasable LPL activity. Although the administration of cholera toxin (0.5 mg/kg; 16-17 h) to rats increased LPL activity in a low-speed supernatant fraction from heparin-perfused hearts, LPL activity was not increased in cardiac myocytes from cholera-toxin-treated rat hearts, and the heparin-induced release of LPL was unchanged. Incubation of cultured ventricular myocytes with 1 microgram of cholera toxin/ml or 500 microM-CPT-cAMP for 24 h did not increase cellular LPL activity or LPL released into the culture medium after a 40 min incubation with heparin. Therefore interventions that stimulate adenylate cyclase activity (forskolin, cholera toxin) or incubation with CPT-cAMP do not increase cellular LPL activity or promote the translocation of LPL to a heparin-releasable fraction in cardiac myocytes.