Trait plasticity alters the range of possible coexistence conditions in a competition–colonisation trade‐off

Most of the classical theory on species coexistence has been based on species‐level competitive trade‐offs. However, it is becoming apparent that plant species display high levels of trait plasticity. The implications of this plasticity are almost completely unknown for most coexistence theory. Here, we model a competition–colonisation trade‐off and incorporate trait plasticity to evaluate its effects on coexistence. Our simulations show that the classic competition–colonisation trade‐off is highly sensitive to environmental circumstances, and coexistence only occurs in narrow ranges of conditions. The inclusion of plasticity, which allows shifts in competitive hierarchies across the landscape, leads to coexistence across a much broader range of competitive and environmental conditions including disturbance levels, the magnitude of competitive differences between species, and landscape spatial patterning. Plasticity also increases the number of species that persist in simulations of multispecies assemblages. Plasticity may generally increase the robustness of coexistence mechanisms and be an important component of scaling coexistence theory to higher diversity communities.     

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

Barley aleurone layer cell protoplasts as a transient expression system

Protoplasts were prepared from barley aleurone layers using Onozuka cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of -amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley -amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes.     

Characterization of a 46 kda insect chitinase from transgenic tobacco

A 46 kDa Manduca sexta (tobacco hornworm) chitinase was isolated from leaves of transgenic tobacco plants containing a recombinant insect chitinase cDNA, characterized, and tested for insecticidal activity. The enzyme was purified by ammonium sulfate fractionation, Q-Sepharose anion-exchange chromatography and mono-S cation-exchange chromatography. Although the gene for the chitinase encoded the 85 kDa full-length chitinase as previously reported by Kramer et al. [Insect Biochem. Molec. Biol. 23, 691–701 (1993)], the enzyme is produced in tobacco as a 46 kDa protein that is approximately four-fold less active than the 85 kDa chitinase. The N-terminal amino acid sequence of the 46 kDa chitinase is identical to that of the 85 kDa chitinase. The former enzyme is not glycosylated, whereas the latter contains approximately 25% carbohydrate. The pH and temperature optima of the 46 kDa chitinaseare similar to those of the 85 kDa chitinase. The former enzyme is more basic than the latter. The 46 kDa chitinase likely consists of the N-terminal catalytic domain of the 85 kDa chitinase and lacks the C-terminal domain that contains several potential sites for glycosylation. The 46 kDa chitinase is expressed in a number of plant organs, including leaves, flowers, stems and roots. Enzyme levels are higher in leaves and flowers than in stems and roots, and leaves from the middle portion of the plant have more chitinase than leaves from the top and bottom portions. Little or no enzyme is secreted outside of the plant cells because it remains in the intracellular space, even though its transit sequence is processed. When fed at a 2% dietary level, the 46 kDa chitinase caused 100% larval mortality of the merchant grain beetle, Oryzaephilis mercator. The results of this study support the hypothesis that insect chitinase is a biopesticidal protein for insect pests feeding on insect chitinase gene-containing transgenic plants.     

Basic fibroblast growth factor is efficiently released from a cytolsolic storage site through plasma membrane disruptions of endothelial cells.

Cells of gut and skin frequently suffer mechanically-induced plasma membrane disruptions in vivo, and bioactive molecules, including basic fibroblast growth factor (bFGF), could enter and leave cytoplasm through these disruptions. We here provide three lines of evidence that bFGF is released with surprising efficiency through plasma membrane disruptions, resembling those known to occur in vivo, produced by scraping endothelial cells from their culturing substratum. First, 41% of the total of bFGF extractable in 1 M NaCl by freeze-thaw and sonication was released simply by scraping the endothelial cells. Second, relative to release of lactate dehydrogenase, cells wounded by scraping under conditions promoting greater than 60% cell survival released a significantly larger amount (up to twofold more) of growth promoting activity than did cells uniformly killed and irreversibly permeabilized by scraping in the cold or by freezing and thawing. Last, cells that survived membrane disruptions released, and contained, less bFGF on each subsequent wounding, consistent with release of bFGF through transient (i.e., survivable) membrane disruptions. A polyclonal antibody against bFGF completely neutralized the growth promoting activity released by scraping, confirming that bFGF is released through endothelial cell plasma membrane disruptions. Cell fractionation and immunolocalization, including a novel permeabilization technique for electron microscope immunolocalization, demonstrated a cytosolic location of bFGF. We conclude that many characteristics of bFGF--its broad spectrum of producing and target cell types, cytosolic location, efficient release through biologically and pathologically relevant plasma membrane wounds, and its release from cells that survive membrane wounds--make it a strong candidate as a "wound hormone" for rapidly initiating the cell growth required for routine maintenance of tissue integrity and/or repair after injury.     

Properties of Barley Seed Chitinases and Release of Embryo-Associated Isoforms during Early Stages of Imbibition

Barley (Hordeum vulgare L.) seeds contain at least five proteins with chitinase (CH) activity. Two of these (CH1 and CH2) are found primarily in the aleurone and endosperm tissues, and the other three (CH3, CH4, and CH5) are enriched in the embryo. From the bran fraction, three of these CHs (CH1, CH2, and CH3) were purified to apparent homogeneity. These three CHs have apparent molecular masses of 27, 34, and 35 kilodaltons and isoelectric points of 9.3, 9.2, and 8.7, respectively. CH2 and CH3 have amino terminal sequences resembling a portion of the chitin-binding domain of lectins and other plant defense proteins. CH1 lacks this domain. All three CHs exhibit antifungal activity and inhibit the mycelial growth of some species of trichoderma and Fusarium in vitro. During the early period of imbibition by seeds, two of the embryo-associated CHs are selectively released into the surrounding aqueous medium.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.