AXY8 encodes an α-fucosidase, underscoring the importance of apoplastic metabolism on the fine structure of Arabidopsis cell wall polysaccharides

An Arabidopsis thaliana mutant with an altered structure of its hemicellulose xyloglucan (XyG; axy-8) identified by a forward genetic screen facilitating oligosaccharide mass profiling was characterized. axy8 exhibits increased XyG fucosylation and the occurrence of XyG fragments not present in the wild-type plant. AXY8 was identified to encode an α-fucosidase acting on XyG that was previously designated FUC95A. Green fluorescent protein fusion localization studies and analysis of nascent XyG in microsomal preparations demonstrated that this glycosylhydrolase acts mainly on XyG in the apoplast. Detailed structural analysis of XyG in axy8 gave unique insights into the role of the fucosidase in XyG metabolism in vivo. The genetic evidence indicates that the activity of glycosylhydrolases in the apoplast plays a major role in generating the heterogeneity of XyG side chains in the wall. Furthermore, without the dominant apoplastic glycosylhydrolases, the XyG structure in the wall is mainly composed of XXXG and XXFG subunits.     

An Insect Herbivore Microbiome with High Plant Biomass-Degrading Capacity

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome''s predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy.     

Historical ecology of the Raja Ampat Archipelago, Papua Province, Indonesia

This work presents a review of the status of marine resources of the Raja Ampat Archipelago, Papua Province, Indonesia, based on narratives of early European expeditions in various museums and libraries in Europe, Canada, and local archives in Papua. More than 500 pertinent documents on the study area were identified and located in various European museums and at the University of British Columbia library. About half of these were scanned (25,000 pages), which yielded the equivalent of 900 pages of text (or 4% of the total number of pages scanned) with observations on abundance and impact of the human population on the marine ecosystem within 2 degrees North and 2 degrees South between 127 degrees and 132 degrees East. In general, these observations, which spanned the period from 1810 to the present, suggest a decrease in the perceived occurrences of turtles, fish, and invertebrates; perceived abundance of turtles, fish, and algae; percieved subsistence exploitation of marine resources; and an increase in perceived commercial exploitation of marine resources. We conclude with a discussion of the problems and potential of contents analysis, and its use in the historical reconstruction of broad biodiversity trends.     

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.     

High-throughput functional assessment of polysaccharide-active enzymes using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry as exemplified on plant cell wall polysaccharides

Despite a wealth of sequence information on genes encoding carbohydrate-active enzymes (e.g., transferases, esterases, hydrolases), very few of these enzymes have been described in detail, particularly regarding substrate specificities. A facile and rapid method for the characterization of substrate specificities of polysaccharide-active enzymes that uses matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) has been developed. This method has been applied to characterize a xyloglucan fucosyltransferase and a pectin methyl-esterase. Reactions were performed in liquid phase, and aliquots of the reaction mixtures were spotted on a polyvinylidene fluoride (PVDF) membrane. Reaction products were precipitated onto the membrane and cleaned by treatment with an ethanol-water mixture. Subsequently, the reaction products were hydrolyzed by specific endoglycanases, and the resulting oligosaccharides were directly analyzed onto the PVDF membrane by MALDI-TOF MS. The new method is amenable to high-throughput analysis and, thus, constitutes an emerging avenue to rapidly fill the gap in our knowledge of the specificities of polysaccharide-active enzymes.     

The nucleus together with the cytosol generates patterns of specific cellular calcium signatures in tobacco suspension culture cells

Plant cell suspension cultures respond to osmotic changes by alterations in levels of free cellular calcium. Using the aequorin recombinant method, we have measured the spatial and temporal characteristics of calcium signatures in the nucleus and the cytosol of BY-2 tobacco suspension cells challenged with hypo- or hyper-osmotic shock. We show here that the nuclear compartment contributes together with the cytosol to produce calcium signal patterns that discriminate hypo- from hyper-osmotic treatments, i.e. turgor from tension. We also demonstrate that calcium responses in the nucleus and the cytosol are differentially modulated by the strength and the nature of hyper-osmotic treatments. We conclude that qualitative and quantitative changes in the parameters of an external stimulus such as osmotic changes are converted into calcium signatures, distinctive in their temporal and subcellular characteristics, involving both the nucleus and the cytosol. Our results illustrate the versatility of calcium signaling in plant cells. In addition to the physiological 'address' of the cell, the compartmentation of the calcium signal is probably an important parameter in encoding response specificity.     

Population dose from natural radionuclides in phosphate fertilizers

Summary The natural radionuclide content of mineral phosphate fertilizers has been determined gammaspectrometrically. The investigations comprised ca. 70% of the mineral phosphate fertilizers authorized and used in the Federal Republic of Germany (FRG). At maximum, we found specific activities of 62 nCi U_nat/kg, 23 nCi²²⁶Ra/kg, 1.6 nCi Th_nat/kg and 262 nCi⁴⁰K/kg. The mean values, weighted by the percentual agricultural consumption of the main phosphate fertilizer groups in 1973/74 and related to the phosphate content, amounted to 58, 40, 2, and 584 nCi/kg P₂O₅ for U_nat,²²⁶Ra, Th_nat, and⁴⁰K respectively. This resulted in an annual distribution due to phosphate fertilizing of about 3.9 µCi U_nat, 2.7 µCi²²⁶Ra, 0.1 µCi Th_nat, and 39.9 µCi⁴⁰K per ha of arable or pasture land in 1973/74 on the average. From these values the air dose rates over agricultural areas have been estimated under extreme conservative assumptions resulting in an additional external exposure of members of the population of 0.02 mrd/a on the average and 0.4 mrd/a in the region of highest phosphate fertilizing intensity. If it is assumed that radium contained in phosphate fertilizers were completely accumulated in the soils during the last 80 years, this value would be raised to 0.3 mrd/a on the average. The occupational external radiation exposure due to natural radionuclides contained in phosphate fertilizers was estimated to be 0.1 mrd/a on the average and 2.3 mrd/a at maximum for persons working in agriculture. These estimates show that natural radionuclides in phosphate fertilizers contribute but very little to the mean terrestrial radiation exposure of the population which is 50 to 55 mrd/a in Germany. Only for the small group of persons working in fertilizer production plants or storehouses a significant increase of the external radiation exposure has to be expected which could reach a doubling of the mean natural exposure value.Dedicated to Prof. Dr. H. Muth on the occasion on his 60th birthday     

Evaluation of growth rate in adhering cell cultures using a simple colorimetric method

In this paper we present a simple colorimetric method for evaluating cell growth in adhering cell cultures. This technique is based on the staining of basophilic cellular compounds (mainly nucleic acids) with methylene blue. To check its reliability, we have compared the quantity of dye fixed by the cells with the number of cells released from the culture substratum by trypsinization, and with the total cellular protein content determined by the Lowry's method. In these experiments we have used human skin fibroblasts and human epithelial cells derived from the epithelium of the full-term umbilical cord. Like other alternative methods, the methylene blue colorimetric technique can be used in 96-well microplates and allows the rapid collection of large amounts of data. As an illustration, we report on the selection of optimal composition of culture medium for human skin fibroblasts.