Source and Magnitude of Ammonium Generation in Maize Roots1

Studies with 15N indicate that appreciable generation of NH4+ from endogenous sources accompanies the uptake and assimilation of exogenous NH4+ by roots. To identify the source of NH4+ generation, maize (Zea mays L.) seedlings were grown on 14NH4+ and then exposed for 3 d to highly labeled 15NH4+. More of the entering 15NH4+ was incorporated into the protein-N fraction of roots in darkness (approximately 25%) than in the light (approximately 14%). Although the 14NH4+ content of roots declined rapidly to less than 1 μmol per plant, efflux of 14NH4+ continued throughout the 3-d period at an average daily rate of 14 μmol per plant. As a consequence, cumulative 14NH4+ efflux during the 3-d period accounted for 25% of the total 14N initially present in the root. Although soluble organic 14N in roots declined during the 3-d period, insoluble 14N remained relatively constant. In shoots both soluble organic 14N and 14NH4+ declined, but a comparable increase in insoluble 14N was noted. Thus, total 14N in shoots remained constant, reflecting little or no net redistribution of 14N between shoots and roots. Collectively, these observations reveal that catabolism of soluble organic N, not protein N, is the primary source of endogenous NH4+ generation in maize roots.

Single-molecule spectroscopy of the β2 adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule studies of the conformations of the intact β2 adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the β2 adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

Multiple DNA and protein sequence alignment based on segment-to-segment comparison.

In this paper, a new way to think about, and to construct, pairwise as well as multiple alignments of DNA and protein sequences is proposed. Rather than forcing alignments to either align single residues or to introduce gaps by defining an alignment as a path running right from the source up to the sink in the associated dot-matrix diagram, we propose to consider alignments as consistent equivalence relations defined on the set of all positions occurring in all sequences under consideration. We also propose constructing alignments from whole segments exhibiting highly significant overall similarity rather than by aligning individual residues. Consequently, we present an alignment algorithm that (i) is based on segment-to-segment comparison instead of the commonly used residue-to-residue comparison and which (ii) avoids the well-known difficulties concerning the choice of appropriate gap penalties: gaps are not treated explicity, but remain as those parts of the sequences that do not belong to any of the aligned segments. Finally, we discuss the application of our algorithm to two test examples and compare it with commonly used alignment methods. As a first example, we aligned a set of 11 DNA sequences coding for functional helix-loop-helix proteins. Though the sequences show only low overall similarity, our program correctly aligned all of the 11 functional sites, which was a unique result among the methods tested. As a by-product, the reading frames of the sequences were identified. Next, we aligned a set of ribonuclease H proteins and compared our results with alignments produced by other programs as reported by McClure et al. [McClure, M. A., Vasi, T. K. & Fitch, W. M. (1994) Mol. Biol. Evol. 11, 571-592]. Our program was one of the best scoring programs. However, in contrast to other methods, our protein alignments are independent of user-defined parameters.

Induction of a Cryphonectria parasitica cellobiohydrolase I gene is suppressed by hypovirus infection and regulated by a GTP-binding-protein-linked signaling pathway involved in fungal pathogenesis.

Extracellular cellulase activity is readily induced when the chestnut blight fungus Cryphonectria parasitica is grown on cellulose substrate as the sole carbon source. However, an isogenic C. parasitica strain rendered hypovirulent due to hypovirus infection failed to secrete detectable cellulase activity when grown under parallel conditions. Efforts to identify C. parasitica cellulase-encoding genes resulted in the cloning of a cellobiohydrolase (exoglucanase, EC 3.2.1.91) gene designated chb-1. Northern blot analysis revealed an increase in cbh-1 transcript accumulation in a virus-free virulent C. parasitica strain concomitant with the induction of extracellular cellulase activity. In contrast, induction of cbh-1 transcript accumulation was suppressed in an isogenic hypovirus-infected strain. Significantly, virus-free C. parasitica strains rendered hypovirulent by transgenic cosuppression of a GTP-binding protein alpha subunit were also found to be deficient in the induction of cbh-1 transcript accumulation.

Apoptosis induced by a human milk protein.

To the breast-fed infant, human milk is more than a source of nutrients; it furnishes a wide array of molecules that restrict microbes, such as antibodies, bactericidins, and inhibitors of bacterial adherence. However, it has rarely been considered that human milk may also contain substances bioactive toward host cells. While investigating the effect of human milk on bacterial adherence to a human lung cancer cell line, we were surprised to discover that the milk killed the cells. Analysis of this effect revealed that a component of milk in a particular physical state--multimeric alpha-lact-albumin--is a potent Ca(2+)-elevating and apoptosis-inducing agent with broad, yet selective, cytotoxic activity. Multimeric alpha-lactalbumin killed all transformed, embryonic, and lymphoid cells tested but spared mature epithelial elements. These findings raise the possibility that milk contributes to mucosal immunity not only by furnishing antimicrobial molecules but also by policing the function of lymphocytes and epithelium. Finally, analysis of the mechanism by which multimeric alpha-lactalbumin induces apoptosis in transformed epithelial cells could lead to the design of antitumor agents.

Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein ELI3.

Mannitol is the most abundant sugar alcohol in nature, occurring in bacteria, fungi, lichens, and many species of vascular plants. Celery (Apium graveolens L.), a plant that forms mannitol photosynthetically, has high photosynthetic rates thought to results from intrinsic differences in the biosynthesis of hexitols vs. sugars. Celery also exhibits high salt tolerance due to the function of mannitol as an osmoprotectant. A mannitol catabolic enzyme that oxidizes mannitol to mannose (mannitol dehydrogenase, MTD) has been identified. In celery plants, MTD activity and tissue mannitol concentration are inversely related. MTD provides the initial step by which translocated mannitol is committed to central metabolism and, by regulating mannitol pool size, is important in regulating salt tolerance at the cellular level. We have now isolated, sequenced, and characterized a Mtd cDNA from celery. Analyses showed that Mtd RNA was more abundant in cells grown on mannitol and less abundant in salt-stressed cells. A protein database search revealed that the previously described ELI3 pathogenesis-related proteins from parsley and Arabidopsis are MTDs. Treatment of celery cells with salicylic acid resulted in increased MTD activity and RNA. Increased MTD activity results in an increased ability to utilize mannitol. Among other effects, this may provide an additional source of carbon and energy for response to pathogen attack. These responses of the primary enzyme controlling mannitol pool size reflect the importance of mannitol metabolism in plant responses to divergent types of environmental stress.

Bronchiolar nonciliated secretory (Clara) cells: source of guanylin in the mammalian lung.

The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia.

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.

Enrichment, isolation and characterisation of ruminal bacteria that degrade non-protein amino acids from the tropical legume Acacia angustissima

Acacia angustissima has been proposed as a protein supplement in countries where low quality forages predominate. A number of non-protein amino acids have been identified in the leaves of A. angustissima and these have been linked to toxicity in ruminants. The non-protein amino acid 4-n-acetyl-2,4-diaminobutyric acid (ADAB) has been shown to be the major amino acid in the leaves of A. angustissima. The current study aimed to identify micro-organisms from the rumen environment capable of degrading ADAB by using a defined rumen-simulating media with an amino acid extract from A. angustissima. A mixed enrichment culture was obtained that exhibited substantial ADAB-degrading ability. Attempts to isolate an ADAB-degrading micro-organism were carried out, however no isolates were able to degrade ADAB in pure culture. This enrichment culture was also able to degrade the non-protein amino acids diaminobutyric acid (DABA) and diaminopropionic acid (DAPA) which have structural similarities to ADAB. Two isolates were obtained which could degrade DAPA. One isolate is a novel Grain-positive rod (strain LPLR3) which belongs to the Firmicutes and is not closely related to any previously isolated bacterium. The other isolate is strain LPSR1 which belongs to the Gammaproteobacteria and is closely related (99.93% similar) to Klebsiella pneumoniae subsp. ozaenae. The studies demonstrate that the rumen is a potential rich source of undiscovered micro-organisms which have novel capacities to degrade plant secondary compounds. (c) 2005 Elsevier B.V. All rights reserved.

Piggery pond sludge as a nitrogen source for crops - 2. Assay of wet and stockpiled piggery pond sludge by successive cereal crops or direct measurement of soil available N

The appropriate use of wastes is a significant issue for the pig industry due to increasing pressure from regulatory authorities to protect the environment from pollution. Nitrogen contained in piggery pond sludge ( PPS) is a potential source of supplementary nutrient for crop production. Nitrogen contribution following the application of PPS to soil was obtained from 2 field experiments on the Darling Downs in southern Queensland on contrasting soil types, a cracking clay ( Vertosol) and a hardsetting sandy loam (Sodosol), and related to potentially mineralisable N from laboratory incubations conducted under controlled conditions and NO3- accumulation in the field. Piggery pond sludge was applied as-collected ( wet PPS) and following stockpiling to dry ( stockpiled PPS). Soil NO3- levels increased with increased application rates of wet and stockpiled PPS. Supplementary N supply from PPS estimated by fertiliser equivalence was generally unsatisfactory due to poor precision with this method, and also due to a high level of NO3- in the clay soil before the first assay crop. Also low recoveries of N by subsequent sorghum ( Sorghum bicolor) and wheat ( Triticum aestivum) assay crops at the 2 sites due to low in-crop rainfall in 1999 resulted in low apparent N availability. Over all, 29% ( range 12 - 47%) of total N from the wet PPS and 19% ( range 0 - 50%) from the stockpiled PPS were estimated to be plant-available N during the assay period. The high concentration of NO3- for the wet PPS application on sandy soil after the first assay crop ( 1998 barley, Hordeum vulgare) suggests that leaching of NO3- could be of concern when high rates of wet PPS are applied before infrequent periods of high precipitation, due primarily to the mineral N contained in wet PPS. Low yields, grain protein concentrations, and crop N uptake of the sorghum crop following the barley crop grown on the clay soil demonstrated a low residual value of N applied in PPS. NO3- in the sandy soil before sowing accounted for 79% of the variation in plant N uptake and was a better index than anaerobically mineralisable N ( 19% of variation explained). In clay soil, better prediction of crop N uptake was obtained when both anaerobically mineralisable N (39% of variation explained) and soil pro. le NO3- were used in combination (R-2 = 0.49).

CyBase: a database of cyclic protein sequence and structure

CyBase is a curated database and information source for backbone-cyclized proteins. The database incorporates naturally occurring cyclic proteins as well as synthetic derivatives, grafted analogues and acyclic permutants. The database provides a centralized repository of information on all aspects of cyclic protein biology and addresses issues pertaining to the management and searching of topologically circular sequences. The database is freely available at http://research.imb.uq.edu.au/cybase.

Effects of nitrogen source and ectomycorrhizal association on growth and delta N-15 of two subtropical Eucalyptus species from contrasting ecosystems

Ectomycorrhizal (EM) associations facilitate plant nitrogen (N) acquisition, but the contribution of EM associations to tree N nutrition is difficult to ascertain in ecosystems. We studied the abilities of subtropical EM fungi and nutritionally contrasting Eucalyptus species, Eucalyptus grandis W. Hill ex Maiden and Eucalyptus racemosa Cav, to use N sources in axenic and soil cultures, and determined the effect of EM fungi on plant N use and plant N-15 natural abundance (delta N-15). As measured by seedling growth, both species showed little dependence on EM when growing in the N-rich minerotrophic soil from E. grandis rainforest habitat or in axenic culture with inorganic N sources. Both species were heavily dependent on EM associations when growing in the N-poor, organotrophic soil from the E. racemosa wallum habitat or in axenic culture with organic N sources. In axenic culture, EM associations enabled both species to use organic N when supplied with amide-, peptide- or protein-N. Grown axenically with glutamine- or protein-N, delta N-15 of almost all seedlings was lower than source N. The delta N-15 of all studied organisms was higher than the N source when grown on glutathione. This unexpected N-15 enrichment was perhaps due to preferential uptake of an N moiety more N-15-enriched than the bulk molecular average. Grown with ammonium-N, the delta N-15 of non-EM seedlings was mostly higher than that of source N. In contrast, the delta N-15 of EM seedlings was mostly lower than that of source N, except at the lowest ammonium concentration. Discrimination against N-15 was strongest when external ammonium concentration was high. We suggest that ammonium assimilation via EM fungi may be the cause of the often observed distinct foliar delta N-15 of EM and non-EM species, rather than use of different N sources by species with different root specialisations. In support of this notion, delta N-15 of soil and leaves in the rainforest were similar for E. grandis and co-occurring non-mycorrhizal Proteaceae. In contrast, in wallum forest, E. racemosa leaves and roots were strongly N-15-depleted relative to wallum soil and Proteaceae leaves. We conclude that foliar delta N-15 may be used in conjunction with other ecosystem information as a rapid indicator of plant dependency on EM associations for N acquisition.

Negative second virial coefficients as predictors of protein crystal growth: Evidence from sedimentation equilibrium studies that refutes the designation of those light scattering parameters as osmotic virial coefficients

The effects of ammonium sulphate concentration on the osmotic second virial coefficient (B-AA/M-A) for equine serum albumin (pH 5.6, 20 degrees C) have been examined by sedimentation equilibrium. After an initial steep decrease with increasing ammonium sulphate concentration, B-AA/M-A assumes an essentially concentration-independent magnitude of 8-9 ml/g. Such behaviour conforms with the statistical-mechanical prediction that a sufficient increase in ionic strength should effectively eliminate the contributions of charge interactions to B-AA/M-A but have no effect on the covolume contribution (8.4 ml/g for serum albumin). A similar situation is shown to apply to published sedimentation equilibrium data for lysozyme (pH 4.5). Although termed osmotic second virial coefficients and designated as such (B-22), the negative values obtained in published light scattering studies of both systems have been described incorrectly because of the concomitant inclusion of the protein-salt contribution to thermodynamic nonideality of the protein. Those negative values are still valid predictors of conditions conducive to crystal growth inasmuch as they do reflect situations in which there is net attraction between protein molecules. However, the source of attraction responsible for the negative virial coefficient stems from the protein-salt rather than the protein-protein contribution, which is necessarily positive. (c) 2005 Elsevier B.V. All rights reserved.

Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet A source

Investigations were undertaken to study the role of the protein cross-linking enzyme tissue transglutaminase in changes associated with the extracellular matrix and in the cell death of human dermal fibroblasts following exposure to a solarium ultraviolet A source consisting of 98.8% ultraviolet A and 1.2% ultraviolet B. Exposure to nonlethal ultraviolet doses of 60 to 120 kJ per m2 resulted in increased tissue transglutaminase activity when measured either in cell homogenates, "in situ" by incorporation of fluorescein-cadaverine into the extracellular matrix or by changes in the epsilon(gamma-glutamyl) lysine cross-link. This increase in enzyme activity did not require de novo protein synthesis. Incorporation of fluorescein-cadaverine into matrix proteins was accompanied by the cross-linking of fibronectin and tissue transglutaminase into nonreducible high molecular weight polymers. Addition of exogenous tissue transglutaminase to cultured cells mimicking extensive cell leakage of the enzyme resulted in increased extracellular matrix deposition and a decreased rate of matrix turnover. Exposure of cells to 180 kJ per m2 resulted in 40% to 50% cell death with dying cells showing extensive tissue transglutaminase cross-linking of intracellular proteins and increased cross-linking of the surrounding extracellular matrix, the latter probably occurring as a result of cell leakage of tissue transglutaminase. These cells demonstrated negligible caspase activation and DNA fragmentation but maintained their cell morphology. In contrast, exposure of cells to 240 kJ per m2 resulted in increased cell death with caspase activation and some DNA fragmentation. These cells could be partially rescued from death by addition of caspase inhibitors. These data suggest that changes in cross-linking both in the intracellular and extracellular compartments elicited by tissue transglutaminase following exposure to ultraviolet provides a rapid tissue stabilization process following damage, but as such may be a contributory factor to the scarring process that results.

Understanding the yeast host cell response to recombinant membrane protein production

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.