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.     

Picornavirus internal ribosome entry site elements can stimulate translation of upstream genes

Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.     

Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release

The death of Medicago sativa L. cells cultivated in a batch culture was investigated by measuring both the appearance of intact dead cells determined on the basis of the trypan blue (TB) dye exclusion, and the release of the cytoplasmic esterase activity into the culture medium upon cell death. Taking into account the strong instability of this released esterase activity, the total dead cell and lysed cell densities have been estimated. A mechanism for cell death and lysis is proposed and the specific rates of cell growth, death and lysis estimated. The specific rate of appearance of TB dead cells was low and essentially constant (0.25 day(-1)) during the first 8 days of the batch culture, and then increased above 1.5 day(-1) after 2 weeks of cultivation. Whereas no lysis occurred during the first seven days, this phenomenon occurred during the second period and accounted for about 20% of the total cell death by the end of the process. Thus, the viability determined by the trypan blue exclusion method appeared to be invalid after 7 days of culture. When lysis of viable cells is taken into consideration, the specific growth rate was significantly increased and growth was shown to continue for a further 8 days. Increased sensitivity of the cells to shear stresses and consequent cell lysis could be the result of a 35% increase in the cell size Copyright 1999 John Wiley & Sons, Inc.     

Flexibility of the metabolism of Corynebacterium glutamicum 2262, a glutamic acid-producing bacterium,in response to temperature upshocks

In order to test the temperature sensitivity of glutamate production metabolism, several temperature shifts, from 33 to 37, 38, 39, 40 or 41°C, were applied to the temperature-sensitive strain, Corynebacterium glutamicum 2262, cultivated in a 24-h fed-batch process. Whereas glucose was entirely dedicated to biomass synthesis when cells were grown at 33°C, applying temperature upshocks, whatever their range, triggered a redistribution of the carbon utilisation between glutamate, biomass and lactate production. Although increasing the culture temperature from 33 to 37, 38, 39 or 40°C resulted in final glutamate titers superior to 80 g/l, temperatures resulting in the best chanelling of the carbon flow towards glutamic acid synthesis were 39 and 40°C. Moreover, this study showed that the higher the temperature, the slower the growth rate and the higher the lactate accumulation. Journal of Industrial Microbiology & Biotechnology (2002) 28, 333–337 DOI: 10.1038/sj/jim/7000251 Received 26 September 2001/ Accepted in revised form 23 February 2002     

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.     

Effect of temperature on tether extraction, surface protrusion, and cortical tension of human neutrophils

Neutrophil rolling on endothelial cells, the initial stage of its migrational journey to a site of inflammation, is facilitated by tether extraction and surface protrusion. Both phenomena have been studied extensively at room temperature, which is considerably lower than human body temperature. It is known that temperature greatly affects cellular mechanical properties such as viscosity. Therefore, we carried out tether extraction, surface protrusion, and cortical tension experiments at 37 degrees C with the micropipette aspiration technique. The experimental temperature was elevated using a custom-designed microscope chamber for the micropipette aspiration technique. To evaluate the constant temperature assumption in our experiments, the temperature distribution in the whole chamber was computed with finite element simulation. Our simulation results showed that temperature variation around the location where our experiments were performed was less than 0.2 degrees C. For tether extraction at 37 degrees C, the threshold force required to pull a tether (40 pN) was not statistically different from the value at room temperature (51 pN), whereas the effective viscosity (0.75 pN.s/microm) decreased significantly from the value at room temperature (1.5 pN.s/microm). Surface protrusion, which was modeled as a linear deformation, had a slightly smaller spring constant at 37 degrees C (40 pN/microm) than it did at room temperature (56 pN/microm). However, the cortical tension at 37 degrees C (5.7+/-2.2 pN/microm) was substantially smaller than that at room temperature (23+/-8 pN/microm). These data clearly suggest that neutrophils roll differently at body temperature than they do at room temperature by having distinct mechanical responses to shear stress of blood flow.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.