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In addition to light, water and CO(2), plants require a number of mineral nutrients, in particular the macronutrients nitrogen, sulphur, phosphorus, magnesium, calcium and potassium. After uptake from the soil by the root system they are either immediately assimilated into organic compounds or distributed within the plant for usage in different tissues. A good understanding of how the transport of macronutrients into and between plant cells is adjusted to different environmental conditions is essential to achieve an increase of nutrient usage efficiency and nutritional value in crops. Here, we review the current state of knowledge regarding the regulation of macronutrient transport, taking both a physiological and a mechanistic approach. We first describe how nutrient transport is linked to environmental and internal cues such as nutrient, carbon and water availability via hormonal, metabolic and physical signals. We then present information on the molecular mechanisms for regulation of transport proteins, including voltage gating, auto-inhibition, interaction with other proteins, oligomerization and trafficking. Combining of evidence for different nutrients, signals and regulatory levels creates an opportunity for making new connections within a large body of data, and thus contributes to an integrative understanding of nutrient transport.

We carried out a "pathway" screen of 50,000 small molecules to identify novel modulators of cAMP signaling. One class of compounds, the 2-(acylamino)-3-thiophenecarboxylates, strongly suppressed cAMP and cGMP in multiple cell lines in response to different agonists acting on G-protein-coupled receptors, adenylyl cyclase, and guanylyl cyclase. The best compounds from structure-activity analysis of 124 analogs, including several synthesized chiral analogs, had and IC(50) of <5 microM for suppression of agonist-induced cAMP and cGMP elevation. Measurements of cAMP, cGMP, and downstream signaling in response to various activators/inhibitors suggested that the 2-(acylamino)-3-thiophenecarboxylates function as nonselective phosphodiesterase activators, although it was not determined whether their action on phosphodiesterases is direct or indirect. The 2-(acylamino)-3-thiophenecarboxylates suppressed CFTR-mediated Cl(-) current in T84 colonic cells in response to cholera and Escherichia coli (STa) toxins, and prevented intestinal fluid accumulation in a closed-loop mouse model of secretory diarrhea. They also prevented cyst growth in an in vitro renal epithelial cell model of polycystic kidney disease. The 2-(acylamino)-3-thiophenecarboxylates represent the first small-molecule cyclic nucleotide suppressors, whose potential therapeutic indications include secretory diarrheas, polycystic kidney disease, and growth inhibition of cAMP-dependent tumors.

Toxicity data from laboratory rodents are widely available and frequently used in human health assessments as animal model data. This study explores the possibility of using single rodent acute toxicity values to predict chemical toxicity to a diversity of wildlife species and estimate hazard levels from modeled species sensitivity distributions (SSD). Interspecies correlation estimation (ICE) models predict toxicity values for untested species using the sensitivity relationship between measured toxicity values of two species. Predicted toxicity values can subsequently populate SSD for application in ecological risk assessments. Laboratory mouse and rat toxicity values were used to estimate toxicity to wildlife and the predicted values then were used to derive SSD hazard dose levels. Toxicity values were predicted within fivefold of measured toxicity values for 78% of ICE models using laboratory rat or mouse toxicity as a surrogate value. Hazard dose levels (HD5) were within fivefold of measured estimates for 72% of SSD developed using laboratory rodent ICE models. Rodents were most often in the least sensitive quartile of species sensitivity distributions, and therefore toxicity values alone may not adequately represent the toxicity to many species of concern without appropriate safety or assessment factors. Laboratory rodent toxicity data offer an additional source of information that can be used to predict hazard levels for wildlife species, and thus offer a starting point for both health and ecological risk assessment.

Eukaryotic PsbO, the photosystem II (PSII) manganese-stabilizing protein, has two N-terminal sequences that are required for binding of two copies of the protein to PSII [Popelkova, H., et al. (2002) Biochemistry 41, 10038-10045; Popelkova, H., et al. (2003) Biochemistry 42, 6193-6200]. In the work reported here, a set of selected N-terminal truncation mutants of PsbO that affect subunit binding to PSII were used to determine the effects of PsbO stoichiometry on the Mn, Ca(2+), and Cl(-) cofactors and to characterize the roles of each of the PsbO subunits in PSII function. Results of the experiments with the PsbO-depleted PSII membranes reconstituted with the PsbO deletion mutants showed that the presence of PsbO does not affect Ca(2+) retention by PSII in steady-state assays of activity, nor is it required for Ca(2+) to protect the Mn cluster against reductive inhibition in darkness. In contrast to the results with Ca(2+), PsbO increases the affinity of Cl(-) for the active site of the O(2)-evolving complex (OEC) as expected. These results together with other data on activity retention suggest that PsbO can stabilize the Mn cluster by facilitating retention of Cl(-) in the OEC. The data presented here indicate that each of two copies of PsbO has a distinctive function in PSII. Binding of the first PsbO subunit fully stabilizes the Mn cluster and enhances Cl(-) retention, while binding of the second subunit optimizes Cl(-) retention, which in turn maximizes O(2) evolution activity. Nonspecific binding of some PsbO truncation mutants to PSII has no functional significance.

Multifunctional shell-core nano/microbeads with a hydrophobic biopolymer core and a designed protein coat for selective binding of an inorganic substance and antibodies were self-assembled inside engineered bacteria. Hybrid genes were constructed to produce tailormade bead-coating proteins in the bacterium Escherichia coli. These fusion proteins contained a binding peptide for an inorganic material, the antibody binding ZZ domain, and a self-assembly promoting as well as biopolymer synthesizing enzyme. Production of these multidomain fusion proteins inside E. coli resulted in self-assembly of beads comprising a biopolyester core and displaying covalently bound binding sites for specific and selective binding of an inorganic substance and any antibody belonging to the immunoglobulin G class. Engineered beads were isolated and purified from the respective E. coli cells by standard cell disruption procedures. Bead morphology and the binding functionalities displayed at the bead surface were assessed by the enzyme-linked immunosorbent assay, transmission electron microscopy, elemental analysis, backscattering electron density, analytical density ultracentrifugation, and atomic force microscopy. These analyses showed that bacteria can be engineered to produce fusion proteins mediating self-assembly of spherical biopolymer beads with binding affinity to gold and/or silica and antibodies. Spherical structures of this type could conceivably serve as nano/microdevices for bioimaging in medical approaches where an antibody mediated targeted delivery of an inorganic contrast agent would be desired.

The cells of Saccharomyces cerevisiae accumulate inorganic polyphosphate (polyP) when reinoculated on a phosphate-containing medium after phosphorus starvation. Total polyP accumulation was similar at cultivation on both glucose and ethanol. Five separate fractions of polyP: acid-soluble fraction polyP1, salt-soluble fraction polyP2, weakly alkali-soluble fraction polyP3, alkali-soluble fraction polyP4, and polyP5, have been obtained from the cells grown on glucose and ethanol under phosphate overplus. The dynamics of polyP fractions depend on a carbon source. The accumulation rates for fractions polyP2 and polyP4 were independent of the carbon source. The accumulation rates of polyP1 and polyP3 were higher on glucose, while fraction polyP5 accumulated faster on ethanol. As to the maximal polyP levels, they were independent of the carbon source for fractions polyP2, polyP3, and polyP4. The maximal level of fraction polyP1 was higher on glucose than on ethanol, but the level of fraction polyP5 was higher on ethanol. It was assumed that accumulation of separate polyP fractions has a metabolic interrelation with different energy-providing pathways. The polyphosphate nature of fraction polyP5 was demonstrated for the first time by (31)P nuclear magnetic resonance spectroscopy, enzymatic assay, and electrophoresis.

The histochemical distribution of mercury in the kidneys and gut of frogs (Rana ridibunda) exposed to inorganic mercury was analyzed with autometallography (AMG). It was found that most mercury in the kidneys accumulated in the proximal convoluted tubules as Hg-S nanocrystal, while control animals were totally void of AMG grains. In the gut the highest concentration of mercury was observed in the large intestine. The AMG grains were primarily located in the apical part of the absorptive cells, although rather high concentrations of silver enhanced mercury quantum dots were also detected in a special cell type of gut epithelium and the glycocalyx. A certain amount of AMG grains were detected in the lumen of the gut. We hypothesize that this pool of quantum dots results from sloughed off epithelial cells and macrophages. Such still intact cells and red blood cells containing AMG grains were also found in the lumen of the gut.

The adsorption properties of reference minerals may be considerably modified by the presence of the inorganic and organic coatings that are ubiquitous in soils. It is therefore important to assess the effect of such coatings to evaluate the relevance of adsorption studies on pure minerals. The adsorption of trace amounts of (85)Sr and (137)Cs has been studied in dilute suspensions for various minerals that are common components of soils: quartz, calcium carbonate, kaolinite, montmorillonite and illite. We studied the effect of coatings with either Fe or Al oxide with varying additions of soil-extracted humic or fulvic acid. Both adsorption and desorption were measured and data presented as distribution coefficients, Kd. No adsorption was detected on quartz and it was not possible to coat this surface. Adsorption on calcium carbonate was small and not influenced by coatings. Adsorption of Sr on the three clay minerals was very similar, enhanced by the Al-coating, but not affected by Fe and organic coatings. The presence of organic coatings decreased Cs adsorption on illite. Similar but smaller effects were seen on montmorillonite and kaolinite. Aluminum coating enhanced Cs adsorption on illite, whereas both inorganic coatings caused decreases in adsorption on montmorillonite, and there was no effect on kaolinite. Effects were not additive with mixed, organic-inorganic coatings. Adsorption of both Cs and Sr on all minerals was strongly irreversible, with Kd (desorption) being up to four-times greater than adsorption Kd. The ratio of desorption and adsorption Cs Kd values (an assessment of irreversibility) was inversely related to adsorption Kd. This is consistent with a decreasing contribution of high-affinity adsorption as adsorption increases, but may also reflect the partial loss of organic coatings during desorption.

Submarine groundwater discharge to coastal waters can be a significant source of both contaminants and biologically limiting nutrients. Nitrogen cycling across steep gradients in salinity, oxygen and dissolved inorganic nitrogen in sandy 'subterranean estuaries' controls both the amount and form of nitrogen discharged to the coastal ocean. We determined the effect of these gradients on betaproteobacterial ammonia-oxidizing bacteria (beta-AOB) and ammonia-oxidizing archaea (AOA) in a subterranean estuary using the functional gene encoding ammonia monooxygenase subunit A (amoA). The abundance of beta-AOB was dramatically lower in the freshwater stations compared with saline stations, while AOA abundance remained nearly constant across the study site. This differing response to salinity altered the ratio of beta-AOB to AOA such that bacterial amoA was 30 times more abundant than crenarchaeal amoA at the oxic marine station, but nearly 10 times less abundant at the low-oxygen fresh and brackish stations. As the location of the brackish mixing zone within the aquifer shifted from landward in winter to oceanward in summer, the location of the transition from a beta-AOB-dominated to an AOA-dominated community also shifted, demonstrating the intimate link between microbial communities and coastal hydrology. Analysis of ammonia-oxidizing enrichment cultures at a range of salinities revealed that AOA persisted solely in the freshwater enrichments where they actively express amoA. Diversity (as measured by total richness) of crenarchaeal amoA was high at all stations and time points, in sharp contrast to betaproteobacterial amoA for which only two sequence types were found. These results offer new insights into the ecology of AOA and beta-AOB by elucidating conditions that may favour the numerical dominance of beta-AOB over AOA in coastal sediments.

We investigated the impacts of forest thinning, prescribed fire, and contour ripping on community level physiological profiles (CLPP) of the soil microbial population in postmining forest rehabilitation. We hypothesized that these management practices would affect CLPP via an influence on the quality and quantity of soil organic matter. The study site was an area of Jarrah (Eucalyptus marginata Donn ex Sm.) forest rehabilitation that had been mined for bauxite 12 years previously. Three replicate plots (20 x 20 m) were established in nontreated forest and in forest thinned from 3,000-8,000 stems ha(-1) to 600-800 stems ha(-1) in April (autumn) of 2003, followed either by a prescribed fire in September (spring) of 2003 or left nonburned. Soil samples were collected in August 2004 from two soil depths (0-5 cm and 5-10 cm) and from within mounds and furrows caused by postmining contour ripping. CLPP were not affected by prescribed fire, although the soil pH and organic carbon (C), total C and total nitrogen (N) contents were greater in burned compared with nonburned plots, and the coarse and fine litter mass lower. However, CLPP were affected by forest thinning, as were fine litter mass, soil C/N ratio, and soil pH, which were all higher in thinned than nonthinned plots. Furrow soil had greater coarse and fine litter mass, and inorganic phosphorous (P), organic P, organic C, total C, total N, ammonium, microbial biomass C contents, but lower soil pH and soil C/N ratio than mound soil. Soil pH, inorganic P, organic P, organic C, total C and N, ammonium, and microbial biomass C contents also decreased with depth, whereas soil C/N ratio increased. Differences in CLPP were largely (94%) associated with the relative utilization of gluconic, malic (greater in nonthinned than thinned soil and mound than furrow soil), L-tartaric, succinic, and uric acids (greater in thinned than nonthinned, mound than furrow, and 5-10 cm than 0-5 cm soil). The relative utilization of amino acids also tended to increase with increasing soil total C and organic C contents but decreased with increasing nitrate content, whereas the opposite was true for carboxylic acids. Only 45% of the variance in CLPP was explained using a multivariate multiple regression model, but soil C and N pools and litter mass were significant predictors of CLPP. Differences in soil textural components between treatments were also correlated with CLPP; likely causes of these differences are discussed. Our results suggest that 1 year after treatment, CLPP from this mined forest ecosystem are resilient to a spring prescribed fire but not forest thinning. We conclude that differences in CLPP are likely to result from complex interactions among soil properties that mediate substrate availability, microbial nutrient demand, and microbial community composition.

Nucleic acid-based detection of Mycobacterium tuberculosis infections has the potential to improve the analysis of the tuberculosis epidemiology and patient care by increasing the specificity and sensitivity of diagnosis. One potential diagnostic sequence, the DR locus, is present in all isolates of M. tuberculosis complex bacteria. It encodes no known gene product but is useful for molecular typing of M. tuberculosis because of its fortuitous absence in non-tuberculosis strains of mycobacteria. The DR locus contains a variable number of short direct repeats interspersed with non-repetitive spacers and is commonly used as a target for the spoligotyping method, a technique based on the detection of the presence or absence of distinct spacers between the repeats. In this study, we attempted to combine the specificity of molecular inversion probe (MIP) technology with the sensitivity of modified pyrosequencing readout in order to detect a short conserved 18 bp sequence included in DR locus in 25 isolates of M. tuberculosis. Additional sensitivity was obtained by introducing modifications in pyrosequencing methodology, by these means we achieved to detect 500 fg of M. tuberculosis DNA.

The design, synthesis and properties of a new class of enzyme/DNA/inorganic nanobiomaterials are described here. DNA has been used to stabilize the enzymes intercalated in the galleries of the inorganic solid, alpha-Zr(iv) phosphate (alpha-Zr(HPO(4))(2).H(2)O, abbreviated as alpha-ZrP). Interestingly, the presence of DNA improved the activity and stability of the bound enzymes. Key studies leading to the current strategy are presented initially, and these are followed by more recent developments. Several enzymes and proteins, including horseradish peroxidase, lysozyme, glucose oxidase, chymotrypsin, bovine serum albumin, cytochrome c, met-hemoglobin and met-myoglobin are successfully intercalated in the galleries of alpha-ZrP, under benign ambient conditions (aqueous buffered solutions, at room temperature and neutral pH). These novel materials are characterized by XRD, SEM and TEM as well as by biochemical, calorimetric and spectroscopic methods. Spectroscopic studies (circular dichroism, CD), for example, indicated that co-intercalation of DNA improved the retention of bound enzyme structure. The activity was enhanced markedly (five-fold) when DNA is co-intercalated, when compared to the activity in the absence of DNA. Addition of DNA to the sample, after enzyme intercalation, did not make any improvements. Our hypothesis is that enzyme-DNA supramolecular complex binds to the solid and the unfavorable interactions between the enzyme and the solid are minimized. These novel nanobiocomposite materials provide a simple method for packaging DNA and aid in engineering more effective synthetic materials for gene/RNA-delivery and drug delivery applications.

The Black Sea is the largest anoxic water basin on Earth and its stratified water column comprises an upper oxic, middle suboxic and a lower permanently anoxic, sulfidic zone. The abundance of sulfate-reducing bacteria (SRB) in water samples was determined by quantifying the copy number of the dsrA gene coding for the alpha subunit of the dissimilatory (bi)sulfite reductase using real-time polymerase chain reaction. The dsrA gene was detected throughout the whole suboxic and anoxic zones. The maximum dsrA copy numbers were 5 x 10(2) and 6.3 x 10(2) copies ml(-1) at 95 m in the suboxic and at 150 m in the upper anoxic zone, respectively. The proportion of SRB to total Bacteria was 0.1% in the oxic, 0.8-1.9% in the suboxic and 1.2-4.7% in the anoxic zone. A phylogenetic analysis of 16S rDNA clones showed that most clones from the anoxic zone formed a coherent cluster within the Desulfonema-Desulfosarcina group. A similar depth profile as for dsrA copy numbers was obtained for the concentration of non-isoprenoidal dialkyl glycerol diethers (DGDs), which are most likely SRB-specific lipid biomarkers. Three different DGDs were found to be major components of the total lipid fractions from the anoxic zone. The DGDs were depleted in (13)C relative to the delta(13)C values of dissolved CO(2) (delta(13)C(CO2)) by 14-19 per thousand. Their delta(13)C values [delta(13)C(DGD(II-III))] co-varied with depth showing the least (13)C-depleted values in the top of the sulfidic, anoxic zone and the most (13)C-depleted values in the deep anoxic waters at 1500 m. This co-variation provides evidence for CO(2) incorporation by the DGD(II-III)-producing SRB, while the 1:2 relationship between delta(13)C(CO2) and delta(13)C(DGD(II-III)) indicates the use of an additional organic carbon source.

The growing commerce in micro- and nanotechnology is expected to increase human exposure to submicrometer and nanoscale particles, including certain forms of amorphous silica. When inhaled, these particles are likely to reach the alveoli, where alveolar type II epithelial cells that are distinguished by apical microvilli are found. These cells play critical roles in the function of the alveoli and participate in the immune response to amorphous silica and other particles by releasing chemokines. The cellular interactions of the particles, which drive the cellular responses, are still unclear. Adverse effects of nanoparticles have been attributed, in part, to the unique properties of materials at the nanoscale. However, little is known about the cellular interactions of individual or small nanoparticle aggregates, mostly because of their tendency to agglomerate under experimental conditions. Here we investigate the interaction and internalization pathway of individual precipitated amorphous silica particles with specific surface properties and size, by following one particle at a time. We find that both 100 and 500 nm particles can take advantage of the actin turnover machinery within filopodia and microvilli-like structures to advance their way into alveolar type II epithelial cells. This pathway is strictly dependent on the positive surface charge of the particle and on the integrity of the actin filaments, unraveling the coupling of the particle with the intracellular environment across the cell membrane. The retrograde pathway brings a new mechanism by which positive surface charge supports particle recruitment, and potential subsequent toxicity, by polarized epithelial cells bearing microvilli.

Aims: To further improve the soy-based medium, devoid of animal and dairy products, for a production of tetanus toxin by nutritional studies and to scale-up the Clostridium tetani process into small fermentors., Methods and Results: Optimum production of tetanus toxin did not require addition of pantothenic acid, thiamine, riboflavin, pyridoxine, biotin and uracil, growth factors used by previous investigators. Furthermore, l-tyrosine and l-cysteine could be eliminated from our soy-based medium without effect. Seven carbon sources were compared with glucose in the soy-based medium, but none was found to be superior to glucose. The process was successfully scaled-up into 250-ml bottles, 1-l bottles and 1-l fermentors., Conclusions: Quite remarkably, when comparing the tetanus production process in our soy-based medium with the traditional animal/dairy-containing media, our medium does not require addition of expensive vitamins, uracil or carbon sources other than glucose. Furthermore, the l-tyrosine and l-cysteine components could be eliminated, making the medium (Hy-Soy, glucose, powdered iron and inorganic salts) much more simple and economical. The successful scale-up from test tubes into 1-l fermentors allows us to predict that further scale-up into large fermentors will be successful., Significance and Impact of the Study: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (BSE; mad cow's disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. Our vegetable-based process avoids such unfortunate possibilities. The medium, having been made simpler and less expensive, and shown to be scaleable from test tubes into small fermentors, should be excellent for large scale production of tetanus toxin.

Developing effective regulations on mercury (Hg) emissions requires a better understanding of how atmospheric Hg deposition affects methylmercury (MeHg) levels in aquatic biota. This study tested the hypothesis that MeHg accumulation in aquatic food webs is related to atmospheric Hg deposition. We simulated a range of inorganic Hg deposition rates by adding isotopically enriched Hg(II) (90.9% 202Hg) to 10-m diameter mesocosms in a boreal lake. Concentrations of experimentally added ("spike") Hg were monitored in zooplankton, benthic invertebrates, and fish. Some Hg(II) added to the mesocosms was methylated and incorporated into the food web within weeks, demonstrating that Hg(II) deposited directly to aquatic ecosystems can become quickly available to biota. Relationships between Hg(II) loading rates and spike MeHg concentrations in zooplankton, benthic invertebrates, and fish were linear and significant. Furthermore, spike MeHg concentrations in the food web were directly proportional to Hg(II) loading rates (i.e., a percent change in Hg(II) loading rate resulted in, statistically, the same percent change in MeHg concentration). This is the first experimental determination of the relationship between Hg(II) loading and MeHg bioaccumulation in aquatic biota. We conclude that changes in atmospheric Hg deposition caused by increases or decreases in Hg emissions will ultimately affect MeHg levels in aquatic food webs.

Here, we evaluated how the arbuscular mycorrhizal (AM) symbiosis regulates root hydraulic properties and root plasma membrane aquaporins (PIP) under different stresses sharing a common osmotic component. Phaseolus vulgaris plants were inoculated or not with the AM fungus Glomus intraradices, and subjected to drought, cold or salinity. Stress effects on root hydraulic conductance (L), PIP gene expression and protein abundance were evaluated. Under control conditions, L in AM plants was about half that in nonAM plants. However, L was decreased as a result of the three stresses in nonAM plants, while it was almost unchanged in AM plants. At the same time, PIP2 protein abundance and phosphorylation state presented the same trend as L. Finally, the expression of each PIP gene responded differently to each stress and was dependent on the AM fungal presence. Differential expression of the PIP genes studied under each stress depending on the AM fungal presence may indicate a specific function and regulation by the AM symbiosis of each gene under the specific conditions of each stress tested.

Inorganic carbon (IC), such as bicarbonate or carbon dioxide, stimulates the growth of Lactobacillus plantarum. At low IC levels, one-third of natural isolated L. plantarum strains are nutritionally dependent on exogenous arginine and pyrimidine, a phenotype previously defined as high-CO2-requiring (HCR) prototrophy. IC enrichment significantly decreased the amounts of the enzymes in the pyrimidine biosynthetic pathway encoded by the pyrR1BCAa1Ab1DFE operon, as demonstrated by proteomic analysis. Northern blot and reverse transcription-PCR experiments demonstrated that IC levels regulated pyr genes mainly at the level of transcription or RNA stability. Two putative PyrR regulators with 62% amino acid identity are present in the L. plantarum genome. PyrR1 is an RNA-binding protein that regulates the pyr genes in response to pyrimidine availability by a mechanism of transcriptional attenuation. In this work, the role of PyrR2 was investigated by allelic gene replacement. Unlike the pyrR1 mutant, the DeltapyrR2 strain acquired a demand for both pyrimidines and arginine unless bicarbonate or CO2 was present at high concentrations, which is known as an HCR phenotype. Analysis of the IC- and pyrimidine-mediated regulation in pyrR1 and pyrR2 mutants suggested that only PyrR2 positively regulates the expression levels of the pyr genes in response to IC levels but had no effect on pyrimidine-mediated repression. A model is proposed for the respective roles of PyrR1 and PyrR2 in the pyr regulon expression.

The 150th anniversary of the birth of Sergei N. Winogradsky is a special date for general microbiology. Apart from the discovery of chemosynthesis, the development of the elective method, the application of the direct method, and formulation of microbial ecology problems, Winogradsky, in his pioneering studies, addressed fundamental aspects of the naturalistic Weltanschauung from the position of a microbiologist who studies the role of microorganisms in nature. These problems are still important today. Expanding knowledge throws light on them from different angles of view and highlights them with varying degrees of completeness, but the problems remain beyond time. It may be useful for contemporary microbiologists to realize the extent to which they would recognize their own work in the system of questions and answers stated a hundred years ago.

The concept of scopes is applied to analyse large metabolic networks. Scopes are defined as sets of metabolites that can be synthesised by a metabolic network when it is provided with given seeds (Sets of initial metabolic compounds). Thus, scopes represent synthesising capacities of the seeds in the network. A hierarchy is discussed in the sense that compounds, which are part of the scope of another compound, possess scopes themselves that are subsets of the former scope. This hierarchy is analysed by means of a directed acyclic graph. Using a simple chemical model, it is found that this hierarchy contains specific structures that can, to a large extent, be explained by the chemical composition of the participating compounds. In this way, it represents a new kind of map of metabolic networks, arranging the metabolic compounds according to their chemical capacity.

Aims: Burkholderia cepacia complex (Bcc) isolates causing pulmonary infection in cystic fibrosis (CF) patients grow within an acidic environment in the lung. As exposure to acid pH has been shown to increase intracellular inorganic polyphosphate (polyP) formation in some bacteria, we investigated the inter-relationship between acidic pH and polyP accumulation in Bcc isolates., Methods and Results: The formation of polyP by one Burkholderia cenocepacia clinical isolate was initially examined at a range of pH values by measuring total intracellular polyP accumulation and phosphate uptake. The pattern of polyP accumulation corresponded with the pattern of phosphate uptake with the maximum for both occurring at pH 5.5. Phosphate uptake and formation of polyP by this isolate was further determined over 48 h at pH 5.5, 6.5 and 7.5; formation of polyP was maximal at pH 5.5 at all time points studied. Sixteen of 17 additional clinical and environmental Bcc isolates examined also exhibited maximum phosphate uptake at pH 5.5., Conclusions: Both clinical and environmental Bcc isolates, of five genomovars, show enhanced formation of polyP in an acidic environment. Given both the speculated role of polyP in pathogenesis, cell signalling and biofilm formation and the acidic nature of the CF lung, this may be of considerable clinical importance., Significance and Impact of the Study: Growth of Bcc in an acidic environment, such as that found in the lungs of CF patients may be influenced in part by polyP accumulation.

Separation of microbial cells by flotation recovery is usually carried out in industrial reactors or wastewater treatment systems, which contain a complex mixture of microbial nutrients and excretion products. In the present study, the separation of yeast cells by flotation recovery was carried out using a simple flotation recovery systems containing washed yeast cells resuspended in water in order to elucidate the effects of additives (defined amounts of organic and inorganic acids, ethanol, surfactants and sodium chloride) on the cellular interactions at interfaces (cell/aqueous phase and cell/air bubble). When sodium chloride, organic acids (notably propionic, succinic and acetic acids) and organic surfactants (sodium dodecyl sulphate (SDS), cetyltrimethylammonium bromide (CTAB) and Nonidet P40) were added to the flotation recovery system, significant increases in the cell recovery of yeast hydrophobic cells (Saccharomyces cerevisiae, strain FLT-01) were observed. The association of ethanol to acetic acid solution (a minor by-product of alcoholic fermentation) in the flotation recovery system, containing washed cells of strain FLT-01 resuspended in water, leading to an increased flotation recovery at pH 5.5. Thus, the association among products of the cellular metabolism (e.g., ethanol and acetic acid) can improve yeast cell recovery by flotation recovery.

The results showed that when Thellungiella halophila was treated with NaCl, the fresh and dry weight, the water content, the succulency of whole plant and the root/shoot ratio were decreased (Figs. 2-4, 7); the organic matter content in roots was increased and the inorganic matter content in roots was decreased, while those in shoots changed in the opposite direction (Fig. 6); osmotic adjustment ability, the Na+ content, the root activity were increased (Figs. 5, 7, 8); the nitrate reductase activity increased significantly; the O(-)(2*) content decreased at about NaCl 50 mmol/L but increased at about NaCl 100-400 mmol/L (Fig. 10). The micrographs of T. halophila leaf surface by scanning electron microscope (SEM) indicate that there is no salt gland or bladder on the surface of T. halophila (Fig. 1), so it is not a salt-secreting halophyte. The determination of growth parameters, the Na(+) content and Na(+) X-ray (Table 1) microanalysis of T. halophila indicate that T. halophila is not a salt-exclusing halophyte but it probability is a salt-dilution halophyte.

The community composition and ecophysiological features of microbial autotrophic biofilms were studied in Fuente Podrida, a cold sulfur spring located in East Spain. We demonstrated how different ecophysiological strategies, such as resistance and/or utilization of sulfide and oxygen, light adaptation, or resistance to high water flow, allow each of the microorganisms described to efficiently colonize several areas within the environmental gradient. In the zone of the spring constantly influenced by sulfide-rich waters, biofilms were formed by purple bacteria, cyanobacteria, and filamentous colorless sulfur bacteria. Purple bacteria showed higher photosynthetic efficiency per pigment unit than cyanobacteria, although they were dominant only in anoxic areas. Two filamentous cyanobacteria, strain UVFP1 and strain UVFP2, were also abundant in the sulfide-rich area. Whereas the cyanobacterial strain UVFP2 shows a strategy based on the resistance to sulfide of oxygenic photosynthesis, strain UVFP1, additionally, has the capacity for sulfide-driven anoxygenic photosynthesis. Molecular phylogenetic analyses cluster the benthic strain UVFP1 with genus Planktothrix, but with no particular species, whereas UVFP2 does not closely cluster with any known cyanobacterial species. The colorless sulfur bacterium Thiothrix sp. extended throughout the zone in which both sulfide and oxygen were present, exhibiting its capacity for chemolithoautotrophic dark carbon fixation. Downstream from the source, where springwater mixes with well-oxygenated stream water and sulfide disappears, autotrophic biofilms were dominated by diatoms showing higher photosynthetic rates than cyanobacteria and, by a lesser extent, by a sulfide-sensitive cyanobacterium (strain UVFP3) well adapted to low light availability, although in the areas of higher water velocity far from the river shore, the dominance shifted to crust-forming cyanobacteria. Both types of microorganisms were highly sensitive to sulfide impeding them from occupying sulfide-rich areas of the spring. Sulfide, oxygen, light availability, and water velocity appear as the main factors structuring the autotrophic community of Fuente Podrida spring.

The review presents the recent data demonstrating the important role high-molecular inorganic polyphosphates in regulatory processes in a yeast cell. It has been shown that polyphosphates are localized in different cell compartments, where they are metabolized by a special set of enzymes. The review presents the evidence in favor of the concept of multiple functions of these biopolymers in a cell, as well as the data on the pleiotropic effects of mutations in the genes encoding the enzymes of polyphosphate metabolism.

The creation of a mathematical simulation model of photosynthetic microbial mats is important to our understanding of key biogeochemical cycles that may have altered the atmospheres and lithospheres of early Earth. A model is presented here as a tool to integrate empirical results from research on hypersaline mats from Baja California Sur (BCS), Mexico into a computational system that can be used to simulate biospheric inputs of trace gases to the atmosphere. The first version of our model, presented here, calculates fluxes and cycling of O(2), sulfide, and dissolved inorganic carbon (DIC) via abiotic components and via four major microbial guilds: cyanobacteria (CYA), sulfate reducing bacteria (SRB), purple sulfur bacteria (PSB) and colorless sulfur bacteria (CSB). We used generalized Monod-type equations that incorporate substrate and energy limits upon maximum rates of metabolic processes such as photosynthesis and sulfate reduction. We ran a simulation using temperature and irradiance inputs from data collected from a microbial mat in Guerrero Negro in BCS (Mexico). Model O(2), sulfide, and DIC concentration profiles and fluxes compared well with data collected in the field mats. There were some model-predicted features of biogeochemical cycling not observed in our actual measurements. For instance, large influxes and effluxes of DIC across the MBGC mat boundary may reveal previously unrecognized, but real, in situ limits on rates of biogeochemical processes. Some of the short-term variation in field-collected mat O(2) was not predicted by MBGC. This suggests a need both for more model sensitivity to small environmental fluctuations for the incorporation of a photorespiration function into the model.

Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted.

Cast immobilization is associated with decreases in muscle contractile area, specific force, and functional ability. The pathophysiological processes underlying the loss of specific force production as well as the role of metabolic alterations are not well understood. The aim of this study was to quantify changes in the resting energy-rich phosphate content and specific force production after immobilization. (31)P-magnetic resonance spectroscopy, three-dimensional magnetic resonance imaging, and isometric strength testing were performed in healthy subjects and patients with an ankle fracture after 7 wk of immobilization and during rehabilitation. Muscle biopsies were obtained in a subset of patients. After immobilization, there was a significant decrease in the specific plantar flexor torque and a significant increase in the inorganic phosphate (P(i)) concentration (P < 0.001) and the P(i)-to-phosphocreatine (PCr) ratio (P < 0.001). No significant change in the PCr content or basal pH was noted. During rehabilitation, both the P(i) content and the P(i)-to-PCr ratio decreased and specific torque increased, approaching control values after 10 wk of rehabilitation. Regression analysis showed an inverse relationship between the in vivo P(i) concentration and specific torque (r = 0.65, P < 0.01). In vitro force mechanics performed on skinned human muscle fibers demonstrated that varying the P(i) levels within the ranges observed across individuals in vivo (4-10 mM) changed force production by approximately 16%. In summary, our findings clearly depict a change in the resting energy-rich phosphate content of skeletal muscle with immobilization, which may negatively impact its force generation.

A submerged membrane bioreactor (SMBR) and a conventional activated sludge system (CAS) were compared in parallel over a period of more than 260 days on treating synthetic ammonia-bearing inorganic wastewater without sludge purge under decreased hydraulic retention times (HRTs). Conversion of NH4(+)-N to NO3(-)-N was achieved with an efficiency of over 98% at an HRT > or = 10 h in the SMBR, while similar performance was obtained at an HRT > or = 20 h in the CAS. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rDNA was used to monitor variations of community structures in the two systems. With the prolongation of operation, the number of DGGE bands in the SMBR gradually increased from the initial 11 bands to the final 22 bands, whereas that in the CAS varied in a range between 13 and 183 Sequence analysis indicates that Nitrosomonas sp. and Nitrospira sp. were the dominating nitrification species responsible for ammonia and nitrite oxidation, respectively. Heterotrophic bacteria like Pseudomonas sp. and Flavobacteria sp. existed in both of the systems although only inorganic wastewater was fed. Substantive accumulation of extracellular polymeric substances (EPS) in the SMBR was confirmed by scanning electron microscopy and EPS analysis.

Aims: The study aimed at increasing Cry3Aa delta-endotoxin production by a local isolate of Bacillus thuringiensis (B.t. strain Mm2). To this end, different nutritional conditions were tested for their effects on Cry3Aa yields., Methods and Results: Bacillus thuringiensis Mm2 was grown by shaking at 30 degrees C in different media. Samples were taken from the cultures at intervals and used for protein extraction. SDS-PAGE was performed for toxin analysis. Inclusion of inorganic phosphate (Pi) into the Difco's sporulation medium at an increased level of 200 mmol l-1 caused a fivefold increase (from 3 to 15.6 microg ml-1) in toxin production. Omission of FeSO4 from the medium decreased this yield by half. Resuspension experiments suggested catabolite repression of toxin biosynthesis by glucose. The inclusion of high Pi invariably increased toxin synthesis, even in the absence of sugars., Conclusions: Inorganic phosphate had the most striking effect on toxin biosynthesis. Iron effect was found to be unique to our isolate whereas Pi effect seemed to be common to the biosynthesis of Cry3Aa-type toxins. Stimulation of toxin synthesis by Pi did not seem to represent a relief from glucose repression., Significance and Impact of the Study: Bacillus thuringiensis is the most versatile biopesticide for use in pest management. Regarding cost-effectiveness of related fermentations, high Pi supplement drastically increases Coleoptera-specific toxin synthesis.

A novel extremely thermophilic, hydrogen- and sulfur-oxidizing bacterium, designated strain IBSK3(T), was isolated from a coastal hot spring in Ibusuki, Kagoshima Prefecture, Japan. The cells were motile, straight to slightly curved rods (1.2-3.0 microm long and 0.3-0.4 microm wide). Strain IBSK3(T) was an obligate chemolithoautotroph growing by respiratory nitrate reduction with H(2), forming N(2)O as an end product. Low concentrations of O(2) (0.4-7.7 %, v/v; optimum 2.0 %, v/v) could serve as an alternative electron acceptor to growth. In addition, strain IBSK3(T) was able to utilize elemental sulfur as a sole electron donor with either nitrate or low concentrations of O(2) as an electron acceptor. Growth was observed between 55 and 77.5 degrees C (optimum 75 degrees C; 2 h doubling time), pH 5.5 and 8.3 (optimum pH 6.5-7.0), and in the presence of 0.5 and 4.0 % NaCl (optimum 2.0 %). The G+C content of the genomic DNA was 49.2 mol%. On the basis of 16S rRNA gene sequence analysis, strain IBSK3(T) belonged to the family Aquificaceae, but it only demonstrated a distant phylogenetic relationship with any recognized species within the family (sequence similarity was less than 92 %). On the basis of the physiological and molecular characteristics of the novel isolate, a new genus and novel species are proposed: the type strain of Hydrogenivirga caldilitoris gen. nov., sp. nov. is IBSK3(T) (=JCM 12173(T)=ATCC BAA-821(T)).

The paper assesses health of the tropical urban Robertson Lake, Jabalpur which receives domestic sewage from neighboring human inhabitation and is infested with water hyacinth. Peak density of this macrophyte was 12.5 t dw ha(-1). The water-column was anaerobic (0.6 to 1.9 mg O2 L(-1)), neutral in pH, and enriched with inorganic carbon (23.5 to 37.1 mg L(-1)), NH4-N (0.48 to 2.96 mg L(-1)), and organic nitrogen and phosphorus. Density of heterotrophic bacteria was high (6.8 to 15x10(5) cfu ml(-1)) along with that of total coliforms and fecal bacteria. Species diversity of phytoplankton and submerged macrophytes was very low. Growing stands of water hyacinth could store up to 613 g C m(-2), 23.5 g N m(-2) and 5.5 g P m(-2) and released them during decomposition. The release of nutrients was 3-4 times faster than the uptake. Water hyacinth stabilized water quality and provided substantial support to bacterial density, which in turn contributed significantly to its growth and nutrient dynamics. Turnover of water hyacinth was only 70-80%, adding approximately 175 t humus in the lake. The results denote poor health of the lake, characterized by low species diversity, fast shallowing, dominance of detritus food--webs, and the water unsuitable for human consumption.

Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium Pseudomonas sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors.

A submerged membrane bioreactor (SMBR) and a conventional activated sludge system (CAS) were compared in parallel over a period of 210 days on treating synthetic ammonia-bearing inorganic wastewater under similar conditions. Except for a short period of pH control failure, almost complete conversion of NH(4)(+)?N to NO(3)(-)?N was constantly achieved over an NH(4)(+)?N concentration range from 180 to 1300mgl(-1) at a hydraulic retention time (HRT) of 24h in the SMBR, compared to an average conversion ratio of 95.0% in the CAS. Scanning electron micrographs (SEMs) demonstrated the accumulation of extracellular polymeric substances (EPSs) in the SMBR. Ubiquinone-8 (UQ-8), followed by UQ-10, UQ-7 and UQ-9, was the dominant ubiquinone in both the systems. The dominant menaquinone in the SMBR was menaquinone-6 (MK-6), while that in the CAS was MK-7, indicating that some differences existed between the two systems in terms of microbial community structure. Soluble microbial products (SMPs) tended to accumulate, and then biodegrade in SMBR.

A fixed-film (biofilm) reactor was designed and its performance was determined at various retention times. The goal was to find the optimal retention time for recycling plant nutrients in an advanced life support system, to minimize the size, mass, and volume (hold-up) of a production model. The prototype reactor was tested with aqueous leachate from wheat crop residue at 24, 12, 6, and 3 h hydraulic retention times (HRTs). Biochemical oxygen demand (BOD), nitrates and other plant nutrients, carbohydrates, total phenolics, and microbial counts were monitored to characterize reactor performance. BOD removal decreased significantly from 92% at the 24 h HRT to 73% at 3 h. Removal of phenolics was 62% at the 24 h retention time, but 37% at 3 h. Dissolved oxygen concentrations, nitric acid consumption, and calcium and magnesium removals were also affected by HRT. Carbohydrate removals, carbon dioxide (CO2) productions, denitrification, potassium concentrations, and microbial counts were not affected by different retention times. A 6 h HRT will be used in future studies to determine the suitability of the bioreactor effluent for hydroponic plant production.

Stable C and N isotopes have long been used to examine properties of various C and N cycling processes in soils. Unfortunately, relatively large sample sizes are needed for accurate gas phase isotope ratio mass spectrometric analysis. This limitation has prevented researchers from addressing C and N cycling issues on microbially meaningful scales. Here we explored the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) to detect 13C and 15N assimilation by individual bacterial cells and to quantify N isotope ratios in bacterial samples and individual fungal hyphae. This was accomplished by measuring the relative abundances of mass 26 (12C14N-) and mass 27 (13C14N- and 12C15N-) ions sputtered with a Ga+ probe from cells adhered to an Si contact slide. TOF-SIMS was successfully used to locate and quantify the relative 15N contents of individual hyphae that grew onto Si contact slides in intimate contact with a model organomineral porous matrix composed of kaolin, straw fragments, and freshly deposited manure that was supplemented with 15NO3-. We observed that the 15N content of fungal hyphae grown on the slides was significantly lower in regions where the hyphae were influenced by N-rich manure than in regions influenced by N-deficient straw. This effect occurred over distances of tens to hundreds of microns. Our data illustrate that TOF-SIMS has the potential to locate N-assimilating microorganisms in soil and to quantify the 15N content of cells that have assimilated 15N-labeled mineral N and shows promise as a tool with which to explore the factors controlling microsite heterogeneities in soil.

Silica-based hybrid organic-inorganic materials prepared by sol-gel chemistry exhibit chemical and physical properties revealing their anisotropic organisation. Besides the opportunities that this phenomenon opens for the preparation of new materials, it also provides arguments to the chemist looking for a better comprehension and control of the organisation of solids.

To illustrate the functions of the aromatic residue Phe35 of cytochrome b(5) and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b(5), we studied electron transfer reactions of cytochrome b(5) and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)](-), [Fe(CDTA)](-) and [Ru(NH(3))(6)](3+). The changes at Phe35 of cytochrome b(5) and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.

Nitrogen nutrition can influence cluster root formation in many wild species, but the effect of N form on cluster root formation and root exudation by white lupin is not known. In a solution culture study, we examined the effect of N nutrition (ammonium, nitrate, both or N2 fixation) on cluster root formation and H+ extrusion by white lupin plants under deficient and adequate P supply. The number of cluster roots increased greatly when plants were supplied with I microM P compared with 50 microM P, the increase being 7.8-fold for plants treated with (NH4)2SO4, 3-fold for plants treated with KNO3 and NH4NO3, and 2-4-fold for N2-fixing plants. Under P deficiency. NH4+-N supply resulted in production of a greater number and biomass of cluster roots than other N sources. Dry weight of cluster roots was 30 % higher than that of non-cluster roots in P-deficient plants treated with (NH4)2SO4 and NH4NO3. In plants treated with sufficient P (50 microM), the weight of non-cluster roots was approx. 90 % greater than that of cluster roots. Both total (micromol per plant h(-1)) and specific (micromol g(-1) root d. wt h(-1)) H+ extrusions were greatest from roots of plants supplied with (NH4)2SO4, followed by those supplied with NH4NO3 and N2 fixation, whereas plants receiving KNO3 had negative net H+ extrusion between the third and fifth week of growth (indicating uptake of protons or release of OH- ions). The rate of proton extrusion by NH4+-N-fed plants was similar under P-deficient and P-sufficient conditions. In contrast, proton exudation by N2-fixing plants and KNO3-treated plants was ten-fold greater under P deficiency than under P sufficiency. In comparison with P deficiency, plants treated with 50 microM P had a significantly higher concentration of P in roots, shoots and youngest expanded leaves (YEL). Compared with the N2 fixation and KNO3 treatments, total N concentration was highest in roots, shoots and YEL of plants supplied with (NH4)2SO4 and NH4NO3, regardless of P supply. Under P deficiency, K concentrations in roots decreased at all N supplies, especially in plants treated with (NH4)2SO4 and NH4NO3, which coincided with the greatest H+ extrusion at these P and N supplies. In conclusion, NH4-N nutrition stimulated cluster root formation and H+ extrusion by roots of P-deficient white lupin.

The effects of various nutrient-limiting conditions on expression of the sty operon in Pseudomonas putida CA-3 were investigated. It was observed that limiting concentrations of the carbon source phenylacetic acid, resulted in high levels of phenylacetyl coenzyme A (CoA) ligase activity, this was accompanied also by upper pathway styrene monooxygenase enzyme activity. The introduction of inorganic nutrient limitations, (nitrate, sulfate and phosphate), caused a dramatic reduction in detectable levels of phenylacetyl CoA ligase activity, particularly in the presence of the primary carbon source, succinate. Under these conditions it was no longer possible to detect styrene monooxygenase activity. Reverse transcription PCR analysis of total RNA, isolated under each of the continuous culture conditions examined, revealed that variations in the levels of enzyme activity coincided with altered patterns of corresponding paaK (phenylacetyl CoA ligase) and styA (styrene monooxygenase) gene expression. Transcription of the upper pathway regulatory sensor kinase gene styS was also observed to be growth condition-dependent. These observations suggest that induction/repression of the sty operon in P. putida CA-3, during growth on phenylacetic acid under continuous culture conditions, involves regulatory mechanisms coordinately affecting both the upper and lower pathways and acting at the level of gene transcription.

In this article we show why inorganic metal elements from the environment were an essential part of the origin of living aqueous systems of chemicals in flow. Unavoidably such systems have many closely fixed parameters, related to thermodynamic binding constants, for the interaction of the essential exchangeable inorganic metal elements with both inorganic and organic non-metal materials. The binding constants give rise to fixed free metal ion concentration profiles for different metal ions and ligands in the cytoplasm of all cells closely related to the Irving-Williams series. The amounts of bound elements depend on the organic molecules present as well as these free ion concentrations. This system must have predated coding which is probably only essential for reproductive life. Later evolution in changing chemical environments became based on the development of extra cytoplasmic compartments containing quite different energised free (and bound) element contents but in feed-back communication with the central primitive cytoplasm which changed little. Hence species multiplied late in evolution in large part due to the coupling with the altered inorganic environment.

The structure and function of Shisham (Dalbergia sissoo Roxb.) forests were investigated in relation to nutrient dynamics in 5- to 15-year-old stands growing in central Himalaya. Nutrient concentrations and storage in different layers of vegetation were in the order: tree > shrub > herb. Forest soil, litter and vegetation accounted for 80.1-91.9, 1.0-1.5 and 7.0-18.4%, respectively, of the total nutrients in the system. There were considerable reductions (trees 32.8-43.1; shrubs 26.2-32.4; and herbs 18-8-22-2%) in nutrient concentrations of leaves during senescence. Nutrient uptake by the vegetation as a whole and also by the different components, with and without adjustment for internal recycling, was investigated. Annual transfer of litter nutrients to the soil from vegetation was 74.8-108.4 kg ha(-1) year(-1) N, 56.8-4 kg ha(-1) year(-1) P and 38.7-46.9 kg ha(-1) year(-1) K. Turnover rate and time for different nutrients ranged between 56 and 66 % year(-1) and 1.5 and 1.8 years, respectively. The turnover rate of litter indicates that over 50% of nutrients in litter on the forest floor are released, which ultimately enhances the productivity of the forest stand. The nutrient use efficiency in Shisham forests ranged from 136 to 143 kg ha(-1) year(-1) for N, 1,441 to 1,570 kg ha(-1) year(-1) for P and 305 to 311 kg ha(-1) year(-1) for K. Compared with natural oak forest (265 kg ha(-1) year(-1) and an exotic eucalypt plantation (18 kg ha(-1) year(-1), a higher proportion of nutrients was retranslocated in Shisham forests, largely because of higher leaf tissue nutrient concentrations. This indicates a lower nutrient use efficiency of Shisham compared with eucalypt and oak. Compartment models for nutrient dynamics have been developed to represent the distribution of nutrients pools and net annual fluxes within the system.

A quantitative study of the influence of inlet sulfur dioxide concentration (600-3000 ppm), relative humidity (20-60%), reactor temperature (56-86 degrees C) and different amounts (0-30 wt.%) of inorganic additives (NaCl, CaCl(2) and NaOH) on gas desulfurization has been carried out in a continuous downflow fixed-bed reactor containing calcium hydroxide diluted with silica sand. Results show that the reaction rate does not depend on sulfur dioxide partial pressure (zero-order kinetics) and that the temperature and the relative humidity have a positive influence on reaction rate. An apparent activation energy of 32 kJ/mol Ca(OH)(2) has been estimated for the reaction. An empirical reaction rate equation at 71.5 degrees C and 36.7% relative humidity that includes the type and amount of additive is proposed. It has been found that calcium chloride is the best additive studied because it allows for a higher degree of sulfur dioxide removal.

Peroxynitrite, a potent oxidising, nitrating and hydroxylating agent, results from the reaction of nitric oxide with superoxide. We show that peroxynitrite can be produced by the action of a single enzyme, xanthine oxidoreductase (XOR), in the presence of inorganic nitrite, molecular oxygen and a reducing agent, such as pterin. The effects of oxygen concentration on peroxynitrite production have been examined. The physiologically predominant dehydrogenase form of the enzyme is more effective than the oxidase form under aerobic conditions. It is proposed that XOR-derived peroxynitrite fulfils a bactericidal role in milk and in the digestive tract.