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Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS).

A widely applicable, positive cDNA selection method was developed to identify RNAs synthesized by Mycobacterium tuberculosis in response to phagocytosis by cultured human primary macrophages. cDNAs for sigE and sigH (alternative sigma factors), aceA (isocitrate lyase), ponA (class I penicillin-binding protein), pks2 (polyketide synthase), uvrA (UvrABC endonuclease), and ctpV (putative cation transporter) were obtained from macrophage-grown bacteria. cDNAs for ORFs Rv3070, Rv3483c, Rv0903c (encoding a putative bacterial two-component transcriptional activator), and Rv0170 of the mce1 virulence operon also were obtained from phagocytized bacilli. cDNAs for these genomic regions were not obtained from approximately 1, 000-fold more bacteria grown in laboratory broth. Methods described here, which have identified M. tuberculosis genes expressed in response to host interaction, will allow the study of gene expression in a variety of microorganisms, including expression resulting from interaction with human tissues in natural disease states.

Bacterial Proteins↗

Isotope studies to the sorption behavior of atmospheric sulfate in humus layers of scots pine ecosystems.

The sorption potential for SO4(2-) in humus layer samples from field sites along a deposition gradient was determined experimentally in batch experiments. The Freundlich equation was used to quantify the sorption of added SO4(2-) in humus layer samples and to determine site-dependent sorption parameters. SO4(2-) sorption in humus layers is a concentration-dependent process. The linearity of isotherms reveals that SO4(2-) is reversibly bound in the organic surface layer, as long as soil solution concentrations remain above 26 to 44 mg SO4(2-) L(-1). Natural isotope variations of sulfur in SO4(2-) were analysed to investigate the degree of sorption of dissolved atmospheric and added SO4(2-). Both sulfate species differed significantly in their isotope composition. The pattern of delta34S values for SO4(2-) in all equilibrium solutions confirm the findings from sorption isotherms, showing a close relationship between the sulfur isotope ratios of SO4(2-) in soil solutions and the amount of SO4(2-) sorbed at the humus layer matrix. Stored atmospheric SO4(2-) in humus layers is released at sites where sulfate concentration in throughfall drops below 26 mg SO4(2-) L(-1). Concentration of soluble Fe decreased with increasing sulfate sorption, thus supporting the assumption that active Fe for example is important. Iron probably stabilizes the reactive surface of humus complexes and therefore has a positive influence on the SO4(2-) sorption in humus layers.

Adsorption↗

Influence of gibberellic acid and stock plant irradiance on carbohydrate content and rooting in cuttings of Scots pine seedlings (Pinus sylvestris L.).

Seedlings of Pinus sylvestris L. were grown at three different irradiances (12, 30, or 60 W m(-2)) under a 17 h photoperiod. After six weeks, cuttings were excised and rooted at 30 W m(-2). Gibberellic acid (GA(3), 5 microl of 2 x 10(-4) or 2 x 10(-3) M) was applied to each cutting immediately after excision. Cuttings from stock plants grown at the low irradiance rooted most quickly, whereas the highest number of roots per cutting was obtained in cuttings from stock plants grown at 30 W m(-2). Gibberellic acid delayed rooting and reduced the number of roots in cuttings from stock plants grown at 12, 30, or 60 W m(-2). The initial carbohydrate content of cuttings increased with stock plant irradiance. For all carbohydrates, except myoinositol and an unknown inositol derivative, the content per cutting increased during the first 28 days of rooting. The GA(3) treatment significantly reduced the starch content of cuttings during the rooting period, but did not significantly change the content of ethanol soluble carbohydrates.

Journal Article↗

Fine root biomass of Scots pine stands differing in age and soil fertility in southern Finland.

The biomass of small and large fine roots (</= 2 mm and 2-5 mm in diameter, respectively) in mineral soils of southern Finland was compared with estimated foliage biomass in the same stands. Study material was collected from stands differing in site fertility and age. The humus layer was deeper at the more fertile sites than at the less fertile sites. Fine root density was greater in humus than in mineral soil, and the fine root density in mineral and humus layers was greater at the less fertile sites than at the more fertile sites. Although the amount of fine root biomass was not correlated with any commonly recorded stand characteristic, small fine root biomass was proportional to foliage biomass for both site types. The coefficient of proportionality was larger for the less fertile sites than for the more fertile sites, supporting the theory of functional balance. A similar relationship could not be established for the large fine roots. A trend of increasing fine root:needle mass ratio with stand age was observed for stands on the less fertile sites.

Journal Article↗

Regeneration patterns in boreal Scots pine glades linked to cold-induced photoinhibition.

Regeneration patterns of Pinus sylvestris L. juveniles in central Siberian glades were studied in relation to cold-induced photoinhibition. Spatial distribution of seedlings in different height classes revealed higher seedling densities beneath the canopy than beyond the canopy, and significantly higher densities of seedlings < 50 cm tall on the north side of the trees. These patterns coincided with differences in light conditions. Compared with plants on the north side of canopy trees (north-exposed), photosynthetic photon flux (PPF) received by plants on the south side of canopy trees (south-exposed) was always higher, making south-exposed plants more susceptible to photoinhibition, especially on cool mornings. Chlorophyll fluorescence data revealed lower photochemical efficiency and increased non-photochemical quenching of small (20-50 cm in height), south-exposed seedlings from spring to early autumn, indicating increased excitation pressure on photosynthesis. Maximum rate of oxygen evolution was less in south-exposed plants than in north-exposed plants. Increased pools of xanthophyll cycle pigments and formation of the photoprotective zeaxanthin provided further evidence for the higher susceptibility to photoinhibition of south-exposed seedlings. A linear mixed model analysis explained many of the physiological differences observed in seedlings according to height class and aspect with early morning temperature and PPF as predictors. The link between photoinhibition and differential distribution of seedlings by height class suggests that photoinhibition, together with other environmental stresses, decreases the survival of small, south-exposed P. sylvestris seedlings, thereby significantly affecting the regeneration pattern of central Siberian pine glades.

Chlorophyll↗

Purification and characterization of NADP+-linked isocitrate dehydrogenase from scots pine . Evidence for different physiological roles of the enzyme in primary development

NADP+-isocitrate dehydrogenase (NADP+-IDH; EC 1.1.1.42) is involved in the supply of 2-oxoglutarate for ammonia assimilation and glutamate synthesis in higher plants through the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Only one NADP+-IDH form of cytosolic localization was detected in green cotyledons of pine (Pinus spp.) seedlings. The pine enzyme was purified and exhibited molecular and kinetic properties similar to those described for NADP+-IDH from angiosperm, with a higher catalytic efficiency (10(5) M-1 s-1) than the deduced efficiencies for GS and GOGAT in higher plants. A polyclonal antiserum was raised against pine NADP+-IDH and used to assess protein expression in the seedlings. Steady-state levels of NADP+-IDH were coordinated with GS during seed germination and were associated with GS/GOGAT enzymes during chloroplast biogenesis, suggesting that NADP+-IDH is involved in the provision of carbon skeletons for the synthesis of nitrogen-containing molecules. However, a noncoordinated pattern of NADP+-IDH and GS/GOGAT was observed in advanced stages of cotyledon development and in the hypocotyl. A detailed analysis in hypocotyl sections revealed that NADP+-IDH abundance was inversely correlated with the presence of GS, GOGAT, and ribulose-1,5-bisphosphate carboxylase/oxygenase but was associated with the differentiation of the organ. These results cannot be explained by the accepted role of the enzyme in nitrogen assimilation and strongly suggest that NADP+-IDH may have other, as-yet-unknown, biological functions.

Journal Article↗

Identification and Quantification of Indole-3-methanol in Etiolated Seedlings of Scots Pine (Pinus sylvestris L.).

Combined gas chromatography-mass spectrometry has been used to identify indole-3-methanol in a purified buffer extract from etiolated seedlings of Pinus sylvestris L. Quantitative estimates obtained by high performance liquid chromatography with fluorescence detection, corrected for losses occurring during purification, indicated that etiolated seedlings of P. sylvestris contained 19.7 +/- 1.4 nanograms (+/- standard deviation) indole-3-methanol per gram fresh weight. The stability of indole-3-methanol at different pH levels was investigated. The rate of conversion, to a less polar unidentified substance, was enhanced with increasing acidity.

Journal Article↗

Tracheid production in response to changes in the internal level of indole-3-acetic Acid in 1-year-old shoots of scots pine.

Different concentrations of indole-3-acetic acid (IAA) were applied in lanolin to 1-year-old shoots of Pinus sylvestris (L.) in a manner known to stimulate cambial activity. The internal concentration of free IAA was measured at a distance below the application point by combined gas chromatography-selected ion monitoring-mass spectrometry using [(13)C(6)]IAA as a quantitative internal standard, and related to the production of tracheids at the same site. The experiment was performed with: (a) debudded cuttings, where the major source of endogenous IAA, the apical buds, were replaced with exogenous IAA, and (b) intact, attached shoots, where endogenous IAA was supplemented by applying IAA around the circumference of the shoot. In both experimental systems, an increase in the internal IAA level was positively related to increased tracheid production. It was also demonstrated that the concentration of internal IAA measured at the sampling site was comparable with endogenous IAA levels found in intact control shoots, and that a wide range of applied IAA concentrations was associated with a relatively small range of internal IAA levels.

Journal Article↗

Biochemical Plant Responses to Ozone : II. Induction of Stilbene Biosynthesis in Scots Pine (Pinus sylvestris L.) Seedlings.

Formation of the stilbenes pinosylvin and pinosylvin 3-methyl ether, as well as the activity of the biosynthetic enzyme stilbene synthase (pinosylvin-forming), were induced several hundred- to thousandfold in primary needles of 6-week-old pine (Pinus sylvestris L.) seedlings upon exposure to a single pulse of ozone of at least 0.15 microliters per liter. The seedlings required 4 hours of exposure as a minimum for the induction of stilbene biosynthesis when exposed to 0.2 microliters per liter ozone. Both stilbene synthase activity and stilbene accumulation increased with the duration of ozone treatment. The activity of phenylalanine ammonia-lyase and the activity of chalcone synthase, a key enzyme of the flavonoid pathway that uses the same substrates as stilbene synthase, were also stimulated about twofold by ozone. Stilbene biosynthesis appears to represent the first example of a dose-dependent biochemical response to ozone in a conifer species and may serve as a useful biomarker to study stress impacts on pine trees.

Journal Article↗

Effect of inoculation of a TOL plasmid containing mycorrhizosphere bacterium on development of Scots pine seedlings, their mycorrhizosphere and the microbial flora in m-toluate-amended soil.

The purpose of this study was to evaluate the influence of introduced bacteria containing a contaminant degrading plasmid on the growth and survival of pine seedlings and mycorrhizosphere microbial flora in contaminated soil. The Pseudomonas fluorescens strain OS81, originally isolated from fungal hyphae in contaminated soil, was supplied with the TOL plasmid pWW0::Km (to generate OS81(pWW0::Km)) and inoculated in humus-soil microcosms with and without pine seedlings mycorrhized with Suillus bovinus. After 3 months of regular treatment with m-toluate (mTA) solutions, the introduced catabolic plasmid was found to be disseminated in the indigenous bacterial population of both mycorrhizosphere and soil uncolonized by the fungus. Transconjugants were represented by bacteria of the genera Pseudomonas and Burkholderia and their number correlated positively with the concentration of mTA applied. Indigenous mTA degrading bacteria with low similarity to Burkholderia species were also enriched in microcosms. They were mostly associated with mycorrhizal soil or fungal structures and virtually absent in microcosms without pines. The total number of Tol(+) bacteria was higher in mycorrhizospheric soil compared with bulk soil. Inoculation with P. fluorescens OS81(pWW0::Km) had a positive effect on the development of roots and fungus in contaminated soil. Both inoculation with the P. fluorescens OS81(pWW0::Km) and mTA contamination as well as the presence of mycorrhized pine roots and fungal hyphae had an effect on the microbial community structure of soil as measured by carbon source oxidation patterns. However, the impact of mTA on the microbial community was more prominent. The study indicates that an effect on plant and fungal development can be obtained by manipulating the mycorrhizosphere. Both introduction of the bacterium carrying the degradative plasmid and the plasmid itself are likely to have a positive effect not only on the organisms involved, but also on bioremediation of contaminated soil, a factor that was not directly monitored here.

Journal Article↗

Prevalence and natural history of periodontal disease in prehistoric Scots (pre-900 AD).

The periodontal status of a Scottish prehistoric population was studied. No individual over the age of 10 yr had an entirely healthy periodontium. Gingivitis was the most widespread disease state in the adolescent and younger age groups. The progression towards periodontitis was at a constant rate and mirrored modern epidemiological studies. A small proportion of individuals proved to be either more susceptible or resistant to periodontal disease. There was no evidence of periodontal disease prevalence being higher than that of modern societies with access to dental treatment. Much can be learnt regarding the natural history of periodontal disease by the study of archaeological material.

Age Distribution↗

Bereavement reactions of female Scots and Swazis: a preliminary comparison.

Forty matched Scottish and Swazi girls and women reported the intensity of the bereavement reactions which they had experienced two weeks and one year after the death of a close relative. The Swazi respondents reported more intense grief reactions during the first two weeks of bereavement than the Scottish participants, but the only way in which the two nationalities differed one year after the death was that the Scottish participants had a greater tendency to report feelings of guilt than the Swazi respondents. It is suggested that ritualistic crying and saying farewell to a dying relative may intensify feelings of grief during the early days of bereavement but, in the context of a clearly defined period of mourning, may play a part in reducing later feelings of guilt. Nationality was not a good predictor of the overall bereavement reaction at either of the time periods. A better prediction was obtained from the number of exchanges with other people which were perceived as being unhelpful, and the number of interactions which were absent but longed for.

Adaptation, Psychological↗