Effect of IAA on Growth and Soluble Cell Wall Polysaccharides Centrifuged from Pine Hypocotyl Sections

Auxin-induced elongation and cell wall polysaccharide metabolism were studied in excised hypocotyl sections of ponderosa pine (Pinus ponderosa) seedlings. Sections excised from hypocotyls of ponderosa pine elongate in response to the addition of auxin. The neutral sugar composition of the extracellular solution removed from hypocotyl sections by centrifugation was examined. In cell wall solution from freshly excised sections, glucose, galactose, xylose, and arabinose make up more than 90% of the neutral sugars, while rhamnose, fucose, and mannose are relatively minor components. The neutral sugar composition of the polysaccharides of the pine cell wall solution is both qualitatively and quantitatively similar to that of pea. Following auxin treatment of pine hypocotyls, the neutral sugar composition of the cell wall changes; glucose, xylose, rhamnose, and fucose increase by nearly 2-fold relative to controls in buffer without auxin. These changes in neutral sugars in response to auxin treatment are similar to those found in pea, with the exception that in pea, rhamnose levels decline in response to auxin treatment.     

Effects of NaCl on water relations and cell wall elasticity and composition of red-osier dogwood (Cornus stolonifera) seedlings

Red-osier dogwood ( Cornus stolonifera Michx, Syn. Cornus sericea ), a species relatively well adapted to moderately saline conditions compared with other boreal species, was used to test the effects of NaCl on plant water relations, cell wall elasticity, and cell wall composition of seedlings. Three month-old seedlings were treated hydroponically with 0, 25, and 50 m m NaCl for 21 days. The osmotic potential at full turgor, osmotic potential at turgor loss, pressure potential at full turgor, and relative water content at turgor loss of red-osier dogwood shoot tissue were not significantly affected by the NaCl treatments. Cell wall elasticity of the shoot tissues did not change following NaCl treatments, suggesting that elastic adjustment did not play a role in the adaptation mechanism. Hemicellulose content of the cell wall increased in salt treated seedlings. The primary sugar found in the cell wall hemicellulose fraction was xylose. In the pectin fraction arabinose and galacturonic acid were the main sugars. Sodium chloride stress did not alter the sugar composition of the hemicellulose fraction; however, NaCl did increase the amount of rhamnose in the pectin fraction. The results of this study suggest that at moderate salinity red-osier dogwood does not make any osmotic or elastic adjustments in the shoot tissue, but some changes in the cell wall composition do occur. These changes could contribute to the decrease in growth recorded in red-osier dogwood during NaCl stress.     

Response of Chlorella to Scandium and Potassium at Various pH

The response of Chlorella pyrenoidosa cells to moderate potassium deficiency and to the addition of various scandium amounts in the nutrient solution were studied at different pH. The effects on growth, chlorophyll content, oxygen consumption, and the ¹⁴CO₂ incorporation in photosynthesizing cells were measured. Considerable metabolic changes inside the cells were registered under the given nutrient conditions. The separated ¹⁴C-labelled compounds showed, at acid and neutral pH in complete nutrient solution, an increase in the relative content of sugar phosphates and a decrease in the relative content of sucrose, caused by increasing scandium concentrations. Moderate potassium deficiency caused a diminution of the relative content of sugar phosphates. The relative content of amino acids was increased by scandium added to complete nutrient solution, but it decreased under potassium deficiency.     

Changes in Cell Wall Composition during Ripening of Grape Berries

Cell walls were isolated from the mesocarp of grape (Vitis vinifera L.) berries at developmental stages from before veraison through to the final ripe berry. Fluorescence and light microscopy of intact berries revealed no measurable change in cell wall thickness as the mesocarp cells expanded in the ripening fruit. Isolated walls were analyzed for their protein contents and amino acid compositions, and for changes in the composition and solubility of constituent polysaccharides during development. Increases in protein content after veraison were accompanied by an approximate 3-fold increase in hydroxyproline content. The type I arabinogalactan content of the pectic polysaccharides decreased from approximately 20 mol % of total wall polysaccharides to about 4 mol % of wall polysaccharides during berry development. Galacturonan content increased from 26 to 41 mol % of wall polysaccharides, and the galacturonan appeared to become more soluble as ripening progressed. After an initial decrease in the degree of esterification of pectic polysaccharides, no further changes were observed nor were there large variations in cellulose (30–35 mol % of wall polysaccharides) or xyloglucan (approximately 10 mol % of wall polysaccharides) contents. Overall, the results indicate that no major changes in cell wall polysaccharide composition occurred during softening of ripening grape berries, but that significant modification of specific polysaccharide components were observed, together with large changes in protein composition.     

Membrane damage associated with inositol-less death in Saccharomyces cerevisiae.

The effect of inositol deficiency was studied on fermentation, respiration, and sugar and amino acid transport. It was found that the loss of fermetnation and respiration and sugar transport activity parallel the loss of cell viability. The loss of sugar transport activity is associated with the development of cell membrane damage. It is concluded that the ultimate cause of cell death is cell membrane leakiness.     

Sphingolipids of transplantable rat nephroma-RA

Contents of sphingolipids (ceramide, sphingomyelin, gangliosides) and the composition of their sphingoid bases were studied in the transplantable rat nephroma-RA and in rat kidneys. The content of sphingomyelin was about 1.3-fold decreased and the content of ceramide was about 1.4-fold increased in the nephroma compared to normal kidneys, and this correlated with a 1.4-fold increased activity of neutral sphingomyelinase; however, the activity of the acidic isoform of the enzyme was virtually unchanged. The content of gangliosides was also increased in the nephroma. Ceramide and sphingomyelin of the nephroma, in addition to sphingosine, contained a significant amount of sphinganine, although a considerable amount of the latter was also found in the renal ceramide. The ratio sphingosine/sphinganine in sphingomyelins changed from 65:1 in kidneys to 5:1 in the nephroma. Thus, the biosynthesis of sphingoid bases seems to be disturbed in the transplantable rat nephroma-RA compared to normal kidneys.     

Sugarcane Internode Composition During Crop Development

Sugarcane sugar and bagasse can be utilized for the production of ethanol or other biofuels. A better understanding of the changes in composition with development along the stalk and with crop development will maximize the usage of sugarcane for this purpose. Two experiments were designed to elucidate internode composition changes during the growing season. In experiment 1, an internode of stalks of 5 modern cultivars were marked at the start of elongation, and then sampled every 1 to 2?weeks from July until October. Sugars were extracted and assayed, and a sequential detergent method was used to estimate hemicellulose, cellulose, and lignin contents. In experiment 2, internodes 1, 3, 5, 7, 9, and 11 down the stalk were sampled in late July (grand growth) and late September (ripening). Internode length, fresh weight, dry weight, water content, and sugar contents were determined as well as cell wall composition. Both experiments were repeated in 2?years. As internodes elongated, total sugar increased, and hemicellulose decreased as a proportion of neutral detergent fiber, while cellulose and lignin increased. After elongation, sucrose and lignin increased, and cellulose content decreased with internode age. The variability in cell wall composition among the five cultivars suggests that selection for desirable composition may be possible. In Experiment 2, hemicellulose contents were lower, and lignin and ash contents were higher at ripening than during grand growth. Delaying sugarcane harvest to maximize sucrose content may decrease bagasse suitability for cellulosic ethanol production because of the increased lignin content.     

Sodium-calcium interactions in two wheat species differing in salinity tolerance

White spruce [ Picea glauca (Moench) Voss.] seedlings were used to study the changes in cell wall composition and elasticity in mature needles before and after the resumption of growth following winter dormancy. Dormant seedlings showed high cell wall elasticity that decreased after the resumption of shoot growth. Cell wall hemicellulose content increased 3 days after planting and decreased after the buds flushed. Non-cellulosic glucose and arabinose were the sugars showing the most pronounced changes related to shoot growth. Arabinose was the most abundant sugar residue in the pectin and hemicellulose fractions and it decreased until day 10 after planting. At the same time, the levels of glucose in pectin and hemicellulose increased. The results provide evidence for cell wall carbohydrate turnover in dormant and active seedlings before and after bud flushing.     

Changes in composition of harvested mushrooms (Agaricus bisporus)

Post-harvest changes in the biochemical composition of the mushroom were studied. Non-structural polysaccharide was found at levels greater than 10% dry wt in the fresh mushroom. After 4 days storage, the level had decreased to below 5% dry weight. The polysaccharide appeared to contain only glucose residues joined by α-1,4 and α-1,6 linkages. The chitin content of cell walls increased by ca 50% during 4 days storage, while cell wall glucan decreased. There was a large increase in urea content.     

Up-regulation of ceramide glucosyltransferase during the differentiation of U937 cells

The phorbol ester tetradecanoylphorbol acetate (TPA) induces promyelocytic leukaemia cells to differentiate to macrophage-like cells in vitro. During the course of this differentiation, the cells adhere to the bottom of the culture dish, a process that requires an increase in cell surface glycosphingolipids (GSLs). We examined the cellular content of glucosylceramide (GlcCer), the simplest of the GSLs, in a TPA-treated leukaemia cell line, U937. Following TPA treatment, we observed a 3.5-fold increase in GlcCer levels that was caused by enhanced activity of ceramide glucosyltransferase (GlcT-1), which catalyses ceramide glycosylation. Furthermore, in TPA-treated cell GlcT-1 amounts were increased at both the mRNA and protein levels. We also found decreased activity of lactosylceramide synthase in TPA-treated cells, which could also contribute to the increase in cellular GlcCer content.     

Stearoyl-CoA desaturase-1 deficiency reduces ceramide synthesis by downregulating serine palmitoyltransferase and increasing beta-oxidation in skeletal muscle

Stearoyl-CoA desaturase (SCD) has recently been shown to be a critical control point of lipid partitioning and body weight regulation. Lack of SCD1 function significantly increases insulin sensitivity in skeletal muscles and corrects the hypometabolic phenotype of leptin-deficient ob/ob mice, indicating the direct antilipotoxic action of SCD1 deficiency. The mechanism underlying the metabolic effects of SCD1 mutation is currently unknown. Here we show that SCD1 deficiency reduced the total ceramide content in oxidative skeletal muscles (soleus and red gastrocnemius) by approximately 40%. The mRNA levels and activity of serine palmitoyltransferase (SPT), a key enzyme in ceramide synthesis, as well as the incorporation of [14C]palmitate into ceramide were decreased by approximately 50% in red muscles of SCD1-/- mice. The content of fatty acyl-CoAs, which contribute to de novo ceramide synthesis, was also reduced. The activity and mRNA levels of carnitine palmitoyltransferase I (CPT I) and the rate of beta-oxidation were increased in oxidative muscles of SCD1-/- mice. Furthermore, SCD1 deficiency increased phosphorylation of AMP-activated protein kinase (AMPK), suggesting that AMPK activation may be partially responsible for the increased fatty acid oxidation and decreased ceramide synthesis in red muscles of SCD1-/- mice. SCD1 deficiency also reduced SPT activity and ceramide content and increased AMPK phosphorylation and CPT I activity in muscles of ob/ob mice. Taken together, these results indicate that SCD1 deficiency reduces ceramide synthesis by decreasing SPT expression and increasing the rate of beta-oxidation in oxidative muscles.     

Cell wall changes in white spruce (Picea glauca) needles subjected to repeated drought stress

White spruce [ Picea glauca (Moench) Voss] seedlings were preconditioned by subjecting them to 3 cycles of a mild drought stress. After 1 week of stress relief their water status, soluble carbohydrate content and cell wall composition in newly formed needles were examined and compared with those in control seedlings. Both preconditioned and control seedlings were subsequently subjected to a severe drought stress and again analyzed. Preconditioning treatment both before and during subsequent stress exposure lowered osmotic potentials at full hydration, and after the loss of turgor, decreased lignin content and increased hemicellulose content of the cell walls. Severe drought had similar but more drastic effects on seedling water relations, sugar accumulation and cell wall hemicellulose content; it also decreased cell wall pectin levels. The decrease in pectin levels was accompanied by a loss of galactose and glucose from pectic substances. Little change in cellulose content was observed as a result of preconditioning and severe drought.     

Developmental changes of sugar contents in the gall on the leaf of elm (Zelkowa serrata Makino) formed byParacolopha morrisoni Baker (homopetra)

To understand cell wall polysaccharide synthesis and the role of gall in interaction with aphids, the changes of sugar contents in the galls during their growth and development were determined from May 2 to June 8, 1996. The sugar content in the symplastic (MeOH and hot water) fractions decreased as the developmental stages progressed. In the cell wall fraction, the amount of pectic substances (2-3 mg per gram fresh weight) did not change. The hemicellulosic substance increased by 40% from May 14 to May 31. Among the neutral sugar components of hemicellulosic polysaccharides, xylose and arabinose contents increased during development of the gall, suggesting that xylans with arabinose residues were massively synthesized. On the other hand, glucose content decreased during development of the gall. The cellulose substance consistently increased 5 folds from May 2 to 31. The relationship between the aphid and the changes in sugar contents of cell walls during the development of aphid and the gall formation was discussed.     

Effects of NaCl-salinity on amino acid and carbohydrate contents of Phragmites australis

The effects of NaCl-salinity on growth, free amino acid and sugar content and composition were assayed in roots, rhizomes and leaves of Phragmites australis (Cav.) Trin. ex Steud. Juvenile plants produced from freshwater clones, were cultured under greenhouse hydroponic conditions for 21 days. Relative growth rates were highest at a salinity level of 0 and 1.5‰, respectively, but decreased significantly at 10‰. All plants cultured at 35‰ salinity died. The osmolality in rhizomes and leaves increased with salinity. The total contents of free amino acids were highest in rhizomes>leaves>roots. In rhizomes, the amino acid content increased significantly up to four-fold from 0 to 10‰ salinity. This increase was caused by up to 200-fold increase of proline and 11-fold increase of glutamine at 10‰, whilst the share of asparagine and glutamate decreased. Leaves showed a similar response to salinity with increasing amino acid contents, and shares of proline and glutamine whereas roots did not react significantly. The contents of sucrose, glucose and fructose were highest in leaves>rhizomes>roots. In rhizomes of all three clones, the sugar contents increased up to 3.5-fold from 1.5 to 10‰ salinity level, but were lower at 1.5‰ versus the control (0‰). Sugar contents were lowest (roots) and highest (leaves) at 1.5‰ salinity. The sugar composition did not vary significantly except for leaves where the fraction of sucrose decreased with increasing salinity level at all three clones from 89.1 to 61.7% of total dissolved sugar (pooled data). The importance of free amino acids and sugars as osmolytes was similar in rhizomes and leaves (13–15% of total osmolality at 10‰). In rhizomes, free amino acids were more important as osmolyte than sugars, while the opposite was true for leaves. Proline contributed up to 2.7% to total osmolality. It is hypothesised that a strong proline accumulation indicates the exceeding of a critical salinity level.     

Phospholipid composition of oligodendroglial cells during normal development and in 18 day old hyperthyroid and malnourished rats.

The phospholipid composition of isolated oligodendroglial cell perikarya was studied in normal rats during development and in 18 day old malnourished and hyperthyroid rats. Phosphatidyl choline and phosphatidyl ethanolamine were found to be the major phospholipid constituents of oligodendroglial cells. Phospholipid content increased during development, mainly due to an increase of the above mentioned phospholipids. The major changes were observed in sphingomyelin, phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine between 18 and 30 days of age. The phospholipid and protein content per cell was significantly decreased in the oligodendroglial cells isolated from malnourished rats as compared to controls. When data were expressed as a function of total proteins, the composition was similar to that of normal animals. In the hyperthyroid rats on the other hand, there were no changes in the amount of phospholipids per cell, while phospholipids per milligram of total oligodendroglial cell protein were markedly decreased. The changes in myelin composition produced by hyperthyroidism that we have previously described, do not follow closely those produced by this experimental condition in oligodendroglial cells, suggesting that the metabolism of myelin might be to a certain extent, independent of that in the parent cell.     

Surviving High-Intensity Field Pulses: Strategies for Improving Robustness and Performance of Electrotransfection and Electrofusion

Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10–20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.     

Ceramide mediates nanovesicle shedding and cell death in response to phosphatidylinositol ether lipid analogs and perifosine

Anticancer phospholipids that inhibit Akt such as the alkylphospholipid perifosine (Per) and phosphatidylinositol ether lipid analogs (PIAs) promote cellular detachment and apoptosis and have a similar cytotoxicity profile against cancer cell lines in the NCI60 panel. While investigating the mechanism of Akt inhibition, we found that short-term incubation with these compounds induced rapid shedding of cellular nanovesicles containing EGFR, IGFR and p-Akt that occurred in vitro and in vivo, while prolonged incubation led to cell detachment and death that depended on sphingomyelinase-mediated generation of ceramide. Pretreatment with sphingomyelinase inhibitors blocked ceramide generation, decreases in phospho-Akt, nanovesicle release and cell detachment in response to alkylphospholipids and PIAs in non-small cell lung cancer cell lines. Similarly, exogenous ceramide also decreased active Akt and induced nanovesicle release. Knockdown of neutral sphingomyelinase decreased, whereas overexpression of neutral or acid sphingomyelinase increased cell detachment and death in response to the compounds. When transferred in vitro, PIA or Per-induced nanovesicles increased ceramide levels and death in recipient cells. These results indicate ceramide generation underlies the Akt inhibition and cytotoxicity of this group of agents, and suggests nanovesicle shedding and uptake might potentially propagate their cytotoxicity in vivo.     

Effects of inositol starvation on phospholipid and glycan syntheses in Saccharomyces cerevisiae.

The early biochemical consequences of inositol starvation in an inositol auxotroph of Saccharomyces cerevisiae were examined as a means of determining the cellular role of inositol. Upon withdrawal of inositol, the rate of incorporation of 32P-labeled inorganic phosphate into phosphatidylinositol and into the phosphoinositol-containing sphingolipids immediately dropped by 80 and 50%, respectively; however, synthesis of the other major phospholipids continued for 2 to 3 h at control rates. The incorporation of [U-14C]glucose into cell wall glycans began to decline immediately poststarvation and decreased to 50% of the initial rate by 80 min for mannan and by 140 min for alkali- and acid-insoluble glucan. These changes in the rates of synthesis of cell wall glycan and phosphatidylinositol were the earliest effects of inositol starvation, preceding inhibition of the synthesis of protein and ribonucleic acid as measured by incorporation of radioactive precursors into trichloroacetic acid-insoluble cell material. These results suggest that phosphatidylinositol may play a direct role in the synthesis or secretion of yeast glycans.     

Ceramide enhances cholesterol efflux to apolipoprotein A-I by increasing the cell surface presence of ATP-binding cassette transporter A1

It is widely accepted that functional ATP-binding cassette transporter A1 (ABCA1) is critical for the formation of nascent high density lipoprotein particles. However, the cholesterol pool(s) and the cellular signaling processes utilized by the ABCA1-mediated pathway remain unclear. Sphingomyelin maintains a preferential interaction with cholesterol in membranes, and its catabolites, especially ceramide, are potent signaling molecules that could play a role in ABCA1 regulation or function. To study the potential role of ceramide in this process, we treated a variety of cell lines with 20 microM C2-ceramide and examined apolipoprotein-mediated cholesterol efflux to lipid-free apoA-I. We found that cell lines expressing ABCA1 displayed 2-3-fold increases in cholesterol efflux to apoA-I. Cell lines not expressing ABCA1 were unaffected by ceramide. We further characterized the cholesterol efflux effect in Chinese hamster ovary cells. Ceramide treatment did not cause significant cytotoxicity or apoptosis and did not affect cholesterol efflux to non-apolipoprotein acceptors. Raising endogenous ceramide levels increased cholesterol efflux to apoA-I. Using a cell surface biotinylation method, we found that the total cellular ABCA1 and that at the plasma membrane were increased with ceramide treatment. Also ceramide enhanced the binding of fluorescently labeled apoA-I to Chinese hamster ovary cells. These data suggest that ceramide may increase the plasma membrane content of ABCA1, leading to increased apoA-I binding and cholesterol efflux.     

Major changes in the cell wall during silique development in Arabidopsis thaliana

Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.