Lichens as biomonitors of air quality around a diamond mine, northwest territories, Canada.

Lichens are known to be bioaccumulators of atmospheric pollutants and are abundant in the Canadian arctic. Mining in this region may negatively impact the tundra communities and these impacts may be detected by increased accumulation of heavy metals, greenhouse gas constituents, and organic compounds in lichen tissue. The effect of sampling direction and distance from a diamond mine on bioaccumulation in three lichen species, Flavocetraria nivalis, Flavocetraria cucullata, and Cladina arbuscula, was investigated. Eight sample sites were located immediately adjacent to a diamond mine, one in each cardinal and ordinal direction, and six sample sites each were located 30 and 60 km from the mine (cardinal, NE, and SE). Thirty-three major and trace elements, sulfate (SO(4)), nitrate (NO(3)), ammonium (NH(4)), polycyclic aromatic hydrocarbons (PAH), and phthalates were analyzed in lichen tissue and soil. A significant interaction occurred between distance and direction from the mine. Highest concentrations of Al, Cr, Cu, Fe, Ni, Ti, and V in lichen were at the mine site regardless of direction. Highest concentrations for all other elements were at the mine in at least two directions. Although present in lichen tissue, there was no significant difference among sites for Hg, Mn, S, and three phthalates. PAHs were below detection limits in lichen tissue. The effect of direction was dependent on element and species, although concentrations of most elements were greatest east or southeast of the mine site. At distance from the mine, direction had less of an effect on concentrations. Elevated concentrations in tissue did not negatively impact lichen or plant cover or lichen richness. This research strongly suggests selection of sample sites and species can impact results and interpretation of data from air quality monitoring programs that use lichens as biomonitors.

Bermudagrass management in the southern Piedmont, USA: IX. Trace elements in soil with broiler litter application.

An understanding of the long-term cycling of trace elements in soil with broiler litter fertilization under various forage utilization strategies is needed to develop sustainable agricultural production systems. We evaluated differences in Cu, Mn, Zn, and six other trace elements in response to 5 yr of bermudagrass [Cynodon dactylon (L.) Pers.] management varying in fertilization and harvest strategies on a Typic Kanhapludult in Georgia. Chicken (Gallus gallus) broiler litter was a significant source of trace elements that led to 3.4 +/- 0.5 times higher Cu, 2.0 +/- 0.3 times higher Mn, and 2.1 +/- 0.2 times higher Zn in the surface 3 cm of soil than when forage was fertilized inorganically. There were variable effects of broiler litter fertilization on other trace elements, depending upon element, depth of sampling, and forage utilization strategy. Concentrations of all trace elements in soil were below levels considered toxic to plants. Soil at a depth of 0 to 3 cm under grazed paddocks had 33 +/- 5% greater Cd, 18 +/- 1% greater Cr, 53 +/- 24% greater Cu, and 24 +/- 7% greater Zn compared with unharvested and hayed management. Trace elements in soil were unaffected whether forage was unharvested or removed as hay. These results suggest that broiler litter is a significant source of several trace elements and that ruminant processing of forage and subsequent deposition of excreta on the paddock allow these trace elements to accumulate more at the soil surface where they might interact with the high concentration of organic matter.

Biochemical characterization of a trypanosome enzyme with glutathione-dependent peroxidase activity.

In most eukaryotes, glutathione-dependent peroxidases play a key role in the metabolism of peroxides. Numerous studies have reported that trypanosomatids lack this activity. Here we show that this is not the case, at least for the American trypanosome Trypanosoma cruzi. We have isolated a single-copy gene from T. cruzi with the potential to encode an 18 kDa enzyme, the sequence of which has highest similarity with glutathione peroxidases from plants. A recombinant form of the protein was purified following expression in Escherichia coli. The enzyme was shown to have peroxidase activity in the presence of glutathione/glutathione reductase but not in the presence of trypanothione/trypanothione reductase. It could metabolize a wide range of hydroperoxides (linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide>cumene hydroperoxide>t-butyl hydroperoxide), but no activity towards hydrogen peroxide was detected. Enzyme activity could be saturated by glutathione when both fatty acid and short-chain organic hydroperoxides were used as substrate. For linoleic acid hydroperoxide, the rate-limiting step of this reaction is the reduction of the peroxidase by glutathione. With lower-affinity substrates such as t-butyl hydroperoxide, the rate-limiting step is the reduction of the oxidant. The data presented here identify a new arm of the T. cruzi oxidative defence system.

Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia.

The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A-binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1. 4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild-type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post-exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth.

A new insertion sequence, ISCb1, from Clostridium beijernickii NCIMB 8052.

The NCIMB 8052 strain of Clostridium beijerinckii contains nine copies of a novel insertion sequence, ISCb1, belonging to the IS4 family. The 1764 bp element has 18 bp inverted repeats at its extremities, and generates 11 bp target repeats upon insertion. It contains a 1365 bp ORF whose predicted product (455 amino acids) resembles bacterial transposases. The highly conserved DD(35)E motif is present, as are signatures characteristic of the N3 and C1 domains of bacterial transposases. Codon usage of the ORF is somewhat different from that of other C. beijerinckii genes, suggesting that ISCb1 may have been acquired from another organism by horizontal gene transfer in the evolutionary past. One ISCb1 copy lies close to the site of insertion of Tn 1545 in a mutant strain, C10, which shows a reduced tendency to degenerate (i.e. loss of the potential to form solvents) compared with the wild type. In the C10 strain, the characteristic pattern of DNA fragments detected by an IS-specific probe was altered, but this was due to the Tn1545 insertion itself, rather than an ISCb1-mediated genome re-arrangement. There is currently no evidence that the element is involved in strain degeneration, since 12 independently isolated spontaneous mutants that had lost the ability to form solvents had the same ISCb1 profile as that of the wild type strain. The element is apparently restricted to a series of closely related solvent-forming clostridia.

Distinct mitochondrial and cytosolic enzymes mediate trypanothione-dependent peroxide metabolism in Trypanosoma cruzi.

The American trypanosome Trypanosoma cruzi is exposed to toxic oxygen metabolites that are generated by drug metabolism and immune responses in addition to those produced by endogenous processes. However, much remains to be resolved about the parasite oxidative defense system, including the mechanism(s) of peroxide reduction. Here we show that reduction of peroxides in T. cruzi is catalyzed by two distinct trypanothione-dependent enzymes. These were localized to the cytosol and mitochondrion. Both are members of the peroxiredoxin family of antioxidant proteins and are characterized by the presence of two conserved domains containing redox active cysteines. The role of these proteins in protecting T. cruzi from peroxide-mediated damage was demonstrated following overexpression of enzyme activity. The parasite-specific features of T. cruzi cytoplasmic peroxiredoxin and T. cruzi mitochondrial peroxiredoxin may be exploitable in terms of drug development.

Optimization of conditions for growth of wild-type and genetically transformed Trypanosoma cruzi on agarose plates.

Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0.65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28 degrees C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0.6% agarose overlay containing G418 as the selective drug, on top of a 0.8% agar base. Transformation efficiencies were as high as 10(-5) cells per microgram of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of 'shotgun transformation' to generate libraries of T. cruzi recombinants.

Overexpression of superoxide dismutase in Trypanosoma cruzi results in increased sensitivity to the trypanocidal agents gentian violet and benznidazole.

The parasitic protozoan Trypanosoma cruzi is exposed to toxic oxygen metabolites which arise from drug metabolism or immune mechanisms, in addition to those produced by endogenous processes. Identification and functional analysis of parasite enzymes which confer protection against oxidative stress is therefore of importance. To investigate the role of T. cruzi superoxide dismutase (SOD) we transfected epimastigotes with an expression vector containing a putative Fe-SOD gene homologue and achieved overexpression of enzyme activity (5-8 fold). Inhibition studies carried out on the partially purified enzyme revealed azide and H2O2 sensitivity and cyanide insensitivity, the profile expected of an Fe-isoform. Phenotypic analysis of transformed parasites showed that they were more susceptible than control cells to growth inhibition by the trypanocidal drug benznidazole and by gentian violet, an agent which can be used to decontaminate blood supplies in endemic areas. These results may reflect an imbalance in the antioxidant defences of the parasite produced as a result of overexpression of Fe-SOD.

Molecular genetics and the initiation of solventogenesis in Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052.

A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. The 14 rrn operons together with about 40 genes have been assigned positions on the map. Genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. Experiments using the available genetic tools have shown that spo0A plays a cardinal role in controlling several aspects of the transition from exponential growth to stationary phase in C. beijerinckii. These include initiation of sporulation, accumulation of the storage polysaccharide, granulose, and production of acetone and butanol. Several C. beijerinckii and C. acetobutylicum genes concerned with fermentative metabolism, whose expression is modulated at the onset of solventogenesis, contain sequence motifs resembling 0A boxes in their 5' regulatory regions. This invites the speculation that they are under the direct control of Spo0A, and additional data are now required to test this prediction.

Physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome.

A combined physical and genetic map of the single, circular, 6.7-Mbp chromosome of the NCIMB 8052 strain of Clostridium beijerinckii (formerly Clostridium acetobutylicum) has been constructed by using a combination of cloned DNA fragments as hybridization probes and a bank of strains harboring insertions of the conjugative transposon Tn1545. The positions of 81 restriction endonuclease cleavage sites and 32 genes have been determined. Eight genes concerned with solventogenic fermentation are found at three different locations. The chromosome contains at least 13 rrn operons, 11 of which have been located on the map. Their transcriptional orientation diverges from the presumed location of the replication origin.

Characterization of spo0A homologues in diverse Bacillus and Clostridium species identifies a probable DNA-binding domain.

Spo0A is a phosphorylation-activated transcription factor of Bacillus subtilis. It is a member of the response regulator superfamily of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of sporulation. To identify the domains of Spo0A most critical for determining its structural and functional features, presumptive homologues of the spo0A gene were characterized in a collection of eight Bacillus species and six Clostridium species representing phylogenetically diverse members of these genera. An alignment of the partial or complete DNA sequences of these homologues revealed three regions of especially high conservation in the effector domain. We speculate that the most highly conserved of these corresponds to the recognition helix of a putative helix-turn-helix motif, and, therefore, represents the actual DNA-containing surface of the protein. In the case of homologues identified in Bacillus anthracis and Clostridium acetobutylicum and retrieved by polymerase chain reaction amplification, we confirmed by gene-disruption analysis that the homologue actually is required for initiation of sporulation. Apparent homologues of the B. subtilis spoIVB gene were also discovered immediately upstream from the spo0A homologues in all Bacillus and Clostridium species examined. The discovery of homologues of B. subtilis sporulation genes in these diverse species implies that the gene products required for specifying pathways of sporulation-specific gene activation and for determining key morphogenetic changes may be highly conserved and suggests that an approach similar to that undertaken here might be used as a general strategy to retrieve and compare their gene sequences. Exhaustive efforts to detect a spo0A-like gene in non-endospore formers, including close relatives of Bacillus such as Listeria and Staphylococcus, were uniformly unsuccessful, suggesting that regulation of gene activity during the transition into stationary phase mediated by Spo0A-like proteins may be exclusive to the endospore-forming bacteria.

[Child psychiatry at a general hospital. The aims of preventive "liaison child psychiatry"]. / Barnepsykiatri i et somatisk sykehus. Mål for forebyggende "liaison barnepsykiatri".

The department of child psychiatry, Ullevål Hospital, has started a liaison child psychiatry service for persons under 18 years old admitted to the hospital. The different tasks involved are analysed with a view to meeting the requirements of the different wards. This facilitates purposeful liaison, establishment of jointly agreed priorities and evaluation of the service.