Transient expression of the CD2 cell surface antigen as a sortable marker to monitor high frequency transfection of human primary B cells.

We have used the T cell surface molecule CD2 gene, expressed from the human cytomegalovirus promoter as a reporter to optimize a transfection system for human primary B cells. The CD2-encoding DNA was transfected into cells by electroporation and transient expression was monitored by flow cytometric analysis. By using our optimal electroporation conditions on activated primary B cells, more than 30% of the resulting viable cells expressed CD2 on the cell surface. Moreover, unactivated primary B cells could also be transfected using this system but subsequent expression of CD2 required cellular activation. Magnetic beads or plastic culture bottles coated with anti-CD2 antibodies have been used to selectively purify transfected cells. The high transfection efficiency combined with the ability to specifically purify transfected cells may allow future studies on specific genes transiently expressed in human primary B cells.     

Expression in Escherichia coli and purification of a translocation-competent precursor of the chloroplast protein ferredoxin

The precursor of the chloroplast protein ferredoxin from Silene pratensis was expressed in Escherichia coli. When a low copy number plasmid was used, the preferredoxin level was low, and the protein was soluble. The expression level was increased by using a high copy number plasmid. In protease-deficient cells transformed with the latter plasmid, the preferredoxin accumulated up to 1% of total protein, and it was found in insoluble aggregates. These aggregates were dissolved in 4 M urea, and the protein was purified to homogeneity. Amino-terminal sequencing confirmed the amino acid sequence as deduced from the copy DNA. However, the first methionine residue of the expected sequence was absent in E. coli. The purified precursor was readily imported by isolated chloroplasts and processed to the mature size.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

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.     

Post-Glacial Climate–Fire Interactions Control Tree Composition of Mesic Temperate Forests in Eastern North America

Stand-scale gap-phase dynamics is generally viewed as the main driver of development in mesic deciduous forests of the temperate biome. Soil charcoal of temperate forests in eastern North America are unnoticed in most surveys, thus explaining why fire is undervalued as a driver of forest succession. The extent to which gap-phase, fire, or other processes are responsible for the regeneration and maintenance of mesic deciduous forests is unknown because paleoecological evidence is lacking. We tested the fire-driven succession hypothesis on the development of this major forest type. Based on charcoal ¹⁴C dates of two sites, 44 and 55 fires occurred since early Holocene, with a mean interval of 170 to 215 years. The vegetation of both sites followed comparable post-glacial trajectories consisting of three distinct periods. Conifers dominated the two first periods during 5200–6000 years and were replaced by hardwoods–conifers over the last 3500 years. The first period was represented by boreal conifers, whereas the second period, dominated by white pine (Pinus strobus) forests, persisted during 3000–4300 years. The third period marked the development of hardwood (sugar maple, Acer saccharum) forests. Fires occurred continuously on the sites since early Holocene likely under dry conditions during the conifer periods and cooler and moister conditions during the hardwood–conifer period. Recurrent fires appear with climate as key drivers of the long-term dynamics of several temperate forests in eastern North America. Similar studies on other temperate forests should be pursued to test the hypothesis of climate–fire interactions influencing tree composition change.

     

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.     

Grassland root demography responses to multiple climate change drivers depend on root morphology

Aims

We examine how root system demography and morphology are affected by air warming and multiple, simultaneous climate change drivers.

Methods

Using minirhizotrons, we studied root growth, morphology, median longevity, risk of mortality and standing root pool in the upper soil horizon of a temperate grassland ecosystem for 3 years. Grassland monoliths were subjected to four climate treatments in a replicated additive design: control (C); elevated temperature (T); combined T and summer precipitation reduction (TD); combined TD and elevated atmospheric CO₂ (TDCO₂).

Results

Air warming (C vs T) and the combined climate change treatment (C vs TDCO₂) had a positive effect on root growth rate and standing root pool. However, root responses to climate treatment varied depending on diameter size class. For fine roots (≤ 0.1 mm), new root length and mortality increased under warming but decreased in response to elevated CO₂ (TD vs TDCO₂); for coarse roots (> 0.2 mm), length and mortality increased under both elevated CO₂ and combined climate change drivers.

Conclusions

Our data suggest that the standing roots pool in our grassland system may increase under future climatic conditions. Contrasted behaviour of fine and coarse roots may correspond to differential root activity of these extreme diameter classes in future climate.