Effects of flow on the synthesis and release of fibronectin by endothelial cells

Summary Human umbilical vein endothelial cells at confluence were subjected to steady shear flow. The effect of flow on the synthesis of fibronectin, its release into the medium, and incorporation into the extracellular matrix were investigated. The total content of fibronectin in endothelial cells exposed to flow was found to be lower than that in static controls after periods of 12 to 48 h. In the presence of cycloheximide there was no difference in the fibronectin content of sheared and unsheared cells. Our results suggest that the synthesis of fibronectin is inhibited by the flow-induced perturbation of endothelial cells. This work was supported by grant EET 8708692 from the National Science Foundation, Washington, DC; grant HL-40696 from the National Institutes of Health, Bethesda, MD; and a Research Initiation Grant from the Pennsylvania State University.     

The role of isoprene in insect herbivory

Several hypotheses have previously been proposed to explain the function of isoprene in plants, including its ability to protect the leaf metabolic machinery from transient high temperature^1,2 and from oxidative stress.³ Isoprene may also serve as a metabolic overflow mechanism for carbon or photosynthetic energy^4–6 and may promote flowering in neighbouring plants.⁷ We have reported recently that isoprene can be detected by a herbivore, Manduca sexta, and that it directly deters them from feeding, with an isoprene emission threshold level of <6 nmol m⁻² s⁻¹.⁸ We demonstrated this using both in vivo experiments, using isoprene-emitting transgenic tobacco plants (Nicotiana tabacum cv. Samsun) and non-emitting azygous control plants, and in vitro experiments, using an artificial (isoprene-emitting and non-emitting control) diet. Here we discuss the potential role of isoprene in plant-herbivore interactions and the possibility that isoprene actually serves multiple purposes in plants.Key words: multiple functions, deterrence, signal, Manduca sextaIsoprene (C₅H₈; 2-methyl 1,3-butadiene) is a volatile organic compound that is produced in many, but not all, plant species.⁹ Because of its high volatility, once produced, isoprene is rapidly released from the leaf surface into the atmosphere, the chemical and physical properties of which can be altered due to the high chemical reactivity and large mass flux rate of isoprene.^10,11 While isoprene production in plants consumes considerable amounts of energy,¹² its role in plants has not yet been fully explained. Isoprene production and emission may confer significant benefits for plants that balance or outweigh its high production costs,¹³ but why only some plants produce the compound and why several beneficial effects have been demonstrated is not clear.Isoprene has recently been reported to directly deter M. sexta caterpillars from feeding. This is supported both by in vivo and in vitro studies and we propose that isoprene may confer competitive advantage to plant against herbivory.⁸ The reason why isoprene functions as a deterrent is not clear. The very obvious explanation is that isoprene is a harmful substance acting, for example as a digestibility reducing substance or toxin, and that it might operate together with other types of plant defences. However, another possibility is that isoprene might be a harmless deterrent as, for example, in the case of the alkaloid gramine in grasses. A grasshopper (Locusta migratoria) normally avoids this compound, but when force fed, habituation occurs and chronic intake does not affect herbivore fitness.¹⁴ This type of substance may be a chemical mimic or be associated with other more toxic compounds.¹⁵ It is also possible that the avoidance behaviour towards harmless deterrents may not be associated at all with toxicity but rather with the avoidance of non-host plants,¹⁶ or avoidance of unsuitable food sources to reduce the risk of feeding on a toxic plant.¹⁷Using nutritional indices, it is possible to investigate the effect of a compound on food consumption and utilisation.¹⁸ We therefore investigated the effect of isoprene on food consumption and utilization by M. sexta using nutritional indices. In this experiment, third instar caterpillars were force fed on isoprene-emitting or non-emitting artificial diets for 24 hour. There were no significant differences (p > 0.05, n = 27) in relative growth rate, consumption index, approximate digestibility, efficiency of conversion of ingested food to body mass and efficiency of conversion of digested food to body mass between the caterpillars that fed on the isoprene emitting-diet and those that fed on the non-emitting diet (Fig. 1). This indicates that isoprene does not directly influence food uptake and utilization by the caterpillars, and that the observed avoidance behaviour⁸ may be related to avoidance of isoprene behaving as a ‘non-host plant’ signal, or isoprene might act in association with other defence mechanism in plants.Open in a separate windowFigure 1Quantification of food consumption and utilization of third instar M. sexta larvae. Larvae fed on isoprene-emitting (55 nmol m⁻² s⁻¹) or non-emitting artificial diet for 24 hours. Larval weight gained, frass produced and leaf weight consumed were measured and analysed according to Waldbauer nutritional indices (1968). ANOVA was used to analyse the data at 95% confidence and no significant confidence was found (n = 27). RGR, relative growth rate; CI, consumption index; AD, approximate digestibility; ECI, efficiency of conversion of ingested food to body mass; ECD, efficiency of conversion of digested food to body mass.It should be noted that this experiment was only performed over 24 hours and it therefore does not probe the longer term effect of isoprene on caterpillar fitness. The function of isoprene in plant-herbivore interactions should therefore be investigated further by extending the experimental period and measurement of caterpillar development, both by using in vitro (isoprene-emitting and non-emitting artificial diet) and in vivo (isoprene-emitting and non-emitting transgenic plants) model systems. The recent availability of transgenic plants, including (a) one that does not normally emit isoprene but is induced to produce isoprene^19,20 and (b) one that normally produces isoprene but has its isoprene emission suppressed,² can facilitate this research and provide insights into the function of isoprene in planta.Various adaptation strategies for reducing the production costs of terpenes have been proposed, including sharing biosynthetic enzymes among multiple pathways, minimizing enzyme turnover rate, using a single enzyme to generate multiple products and using products for more than one function.²¹ The possibility of isoprene being a compound with multiple functions has been hypothesized,⁸ as isoprene has been shown, by inhibitor studies and genetic manipulation, to be able to defend plants from high temperature episodes^1,2 and against ozone³ and now also to protect against herbivory.⁸ It has also been shown to promote flowering in neighboring plants.⁷ Further investigations are, however, required to determine if isoprene emission can deter herbivore insects from a wide range of plant species, or whether this and other effects are specific to plant species. The multiple functions of isoprene may explain how the benefits of isoprene production by plants can outweigh the costs of its production, and why some, but not all, plants produce this reactive and volatile compound.     

A model of isoprene emission based on energetic requirements for isoprene synthesis and leaf photosynthetic properties for Liquidambar and Quercus

We present a physiological model of isoprene (2-methyl-1,3-butadiene) emission which considers the cost for isoprene synthesis, and the production of reductive equivalents in reactions of photosynthetic electron transport for Liquidambar styraciflua L. and for North American and European deciduous temperate Quercus species. In the model, we differentiate between leaf morphology (leaf dry mass per area, M_A, g m ^{? 2}) altering the content of enzymes of isoprene synthesis pathway per unit leaf area, and biochemical potentials of average leaf cells determining their capacity for isoprene emission. Isoprene emission rate per unit leaf area ( μ mol m ^{? 2} s ^{? 1}) is calculated as the product of M_A, the fraction of total electron flow used for isoprene synthesis ( ? , mol mol ^{? 1}), the rate of photosynthetic electron transport (J) per unit leaf dry mass (J_m, μ mol g ^{? 1} s ^{? 1}), and the reciprocal of the electron cost of isoprene synthesis [mol isoprene (mol electrons ^{? 1})]. The initial estimate of electron cost of isoprene synthesis is calculated according to the 1-deoxy- D -xylulose-5-phosphate pathway recently discovered in the chloroplasts, and is further modified to account for extra electron requirements because of photorespiration. The rate of photosynthetic electron transport is calculated by a process-based leaf photosynthesis model. A satisfactory fit to the light-dependence of isoprene emission is obtained using the light response curve of J, and a single value of ? , that is dependent on the isoprene synthase activity in the leaves. Temperature dependence of isoprene emission is obtained by combining the temperature response curves of photosynthetic electron transport, the shape of which is related to long-term temperature during leaf growth and development, and the specific activity of isoprene synthase, which is considered as essentially constant for all plants. The results of simulations demonstrate that the variety of temperature responses of isoprene emission observed within and among the species in previous studies may be explained by different optimum temperatures of J and/or limited maximum fraction of electrons used for isoprene synthesis. The model provides good fits to diurnal courses of field measurements of isoprene emission, and is also able to describe the changes in isoprene emission under stress conditions, for example, the decline in isoprene emission in water-stressed leaves.     

Oxidative formation of glycolic acid in photosynthesizing cells of Chromatium

Upon exposure to atmospheric oxygen photosynthesizing cellsof Chromatium synthesize glycolic acid and excrete it extracellularly.Under anaerobic conditions, glycolic acid is not detectableas a photoassimilation product. We found that the lower thelevel of NaHCO₃ in the culture medium the greater the amountof glycolic acid produced oxidatively by the bacterial cells. ¹Structure and Function of Chloroplast Proteins, Part XXV. Thisinvestigation was supported in part by research grants fromthe Toray Science Foundation (Tokyo) and the Naito Science Foundation(Tokyo). (Received February 7, 1974; )     

Stimulation of the synthesis and release of lipids in tumor cells under attack by antibody and C

Antibody-sensitized line-1 or line-10 tumor cells treated with GPC (TAC) incorporated fatty acids into complex cellular lipids and released increased amounts of fatty acids within 5 to 10 min after the addition of GPC as compared to control cells. This effect was dependent on the concentration of GPC used; however, under conditions where the cells were not killed, the enhanced synthesis and release of lipids were not dependent on the antibody concentration used to sensitize the cells. Treatment of the cells with antibody alone, GPC alone, or antibody plus heat-inactivated GPC did not result in enhanced synthesis or release of lipids. No enhancement in DNA, RNA, or protein synthesis in TAC was noted. Line-1 cells, which can be killed by GPC when sensitized with excess anti-Forssman IgM antibody, demonstrated enhanced lipid synthesis within 1 to 3 min after the addition of GPC to the antibody-sensitized cells, before measurable killing of the cells had occurred. This effect persisted in the surviving cells when tested 5 and 10 min after the formation of TAC. Addition of GPC deficient in C4 to antibody-sensitized cells did not result in enhanced lipid synthesis or release. These data suggest that the synthesis of macromolecules of which lipids are a major component is of central importance for the ability of the cells to resist antibody-GPC mediated attack.     

Biological synthesis and anti-HeLa cells effect of glycosylated bafilomycins

Bafilomycin A1 (BAF A1) is a macrolide antibiotic that, in addition to its antibacterial activity, can induce apoptosis of cancer cells. Glycosylation plays an important role in the modification of antibiotics as it can improve their bioactivity. However, glycosylation of BAF A1 has not been previously reported. Bacillus licheniformis glycosyltransferase (GTs) Bl-YjiC and Bacillus subtilis GTs Bs-YjiC were expressed successfully in a heterologous manner. The glycosylation of BAF A1 with UDP-glucose, UDP-galactose or UDP-N-acetylglucosamine was catalyzed using the enzymes Bl-YjiC and Bs-YjiC. Our results demonstrated that Bl-YjiC can only utilize UDP-glucose as the donor, while Bs-YjiC can utilize all three glycosyl donors. The glycosylation site was demonstrated by MS/MS to be the hydroxyl group at the C21 position of BAF A1. The anti-proliferative effects of glucosyl BAF A1 (BAF-Glc) on HeLa cells indicate that this novel antibiotic is superior to BAF A1. The IC50 for BAF-Glc was determined to be 5.47 μM. Here, we report the production of glycosylated BAF A1 for the first time, and we show that the produced BAF-Glc exhibited better anticancer activity than BAF A1. This work provides theoretical and experimental support for the development of novel anticancer bafilomycins.     

A possible role for cyclic AMP in the initiation of DNA synthesis by isoproterenol-activated parotid gland cells

An intraperitoneal injection of the β-adrenergic agonist dl-isoproterenol hydrochloride (100 mg/Kg body weight) into a rat caused an early, very large (400-fold) cyclic AMP surge (peaking at 10 minutes) in the parotid gland which was followed by a second, much smaller (two-fold) surge 12 to 16 hours later. DNA synthesis began about 16 to 20 hours after the isoproterenol injection and peaked between 24 and 28 hours. The maximum level of DNA-synthetic activity at 24 hours was correlated positively to the magnitude of the small cyclic AMP surge at 12 hours, but not to the size of the much larger cyclic AMP surge at 10 minutes. An intraperitoneal injection of dl-propranolol hydrochloride (59 mg/Kg body weight) at 8 hours after isoproterenol injection abolished the second cyclic AMP surge at 12 hours and markedly (65-75%) reduced the incorporation of [³H]-thymidine into DNA. Injection of dibutyryl cyclic AMP (6.3 mg/Kg body weight) and theophylline (25 mg/Kg body weight) at 8 hours prevented propranolol from inhibiting DNA synthesis. Propranolol appeared specifically to affect the cyclic AMP-dependent pre-DNA-synthetic step because it did not reduce [³H]-thymidine incorporation when injected after the second cyclic AMP surge had passed and DNA synthesis had just begun. Thus, the initial, large cyclic AMP surge following β-adrenergic stimulation may not be necessary for the initiation of prereplicative development, while the much smaller second surge may be needed for the initiation of DNA synthesis.     

Plastid-nuclear complexes in the photosynthesizing cells from their mitosis up to programmed death

Permanent plastid-nuclear complexes (PNCs) exist in tobacco cells from their mitosis up to programmed cell death (PCD). PNCs in senescing cells of tobacco leaves were typical by enclosure of peroxisomes and mitochondria among chloroplasts which were in contact with nucleus. Such a complex position provides simultaneous interaction of these organelles and direct regulation of metabolism and PCD avoiding the cytosol.     

Modification of isoprene synthesis to enable production of curcurbitadienol synthesis in Saccharomyces cerevisiae

Journal of Industrial Microbiology & Biotechnology - Cucurbitane-type triterpenoids such as mogrosides and cucurbitacins that are present in the plants of Cucurbitaceae are widely used in Asian...     

Natural abundance carbon isotope composition of isoprene reflects incomplete coupling between isoprene synthesis and photosynthetic carbon flow

Isoprene emission from leaves is dynamically coupled to photosynthesis through the use of primary and recent photosynthate in the chloroplast. However, natural abundance carbon isotope composition (delta(13)C) measurements in myrtle (Myrtus communis), buckthorn (Rhamnus alaternus), and velvet bean (Mucuna pruriens) showed that only 72% to 91% of the variations in the delta(13)C values of fixed carbon were reflected in the delta(13)C values of concurrently emitted isoprene. The results indicated that 9% to 28% carbon was contributed from alternative, slow turnover, carbon source(s). This contribution increased when photosynthesis was inhibited by CO(2)-free air. The observed variations in the delta(13)C of isoprene under ambient and CO(2)-free air were consistent with contributions to isoprene synthesis in the chloroplast from pyruvate associated with cytosolic Glc metabolism. Irrespective of alternative carbon source(s), isoprene was depleted in (13)C relative to mean photosynthetically fixed carbon by 4 per thousand to 11 per thousand. Variable (13)C discrimination, its increase by partially inhibiting isoprene synthesis with fosmidomicin, and the associated accumulation of pyruvate suggested that the main isotopic discrimination step was the deoxyxylulose-5-phosphate synthase reaction.     

DNA-damaging ability of isoprene and isoprene mono-epoxide (EPOX I) in human cells evaluated with the comet assay

Isoprene is produced in combustion processes and is widely used as an industrial chemical. It is a natural product emitted by plants and endogenously produced by humans and other mammals. Therefore, exposure to isoprene from both endogenous and exogenous sources is unavoidable and occurs during the entire human life. Based on evaluations of the International Agency for Research on Cancer (IARC), isoprene has been classified in Group 2B (possibly carcinogenic to humans). In the present work, we have demonstrated, by use of the single-cell gel electrophoresis assay (SCGE or comet assay), that isoprene is able to induce DNA damage in peripheral blood mononuclear cells (PBMCs) in the presence of metabolic activation. In addition, treatment of cells with the main isoprene mono-epoxide (EPOX I) induced time- and dose- dependent DNA damage in both PBMCs and human leukaemia cells (HL60). The metabolic activation system, represented by rat liver post-mitochondrial fractions (S9), was obtained from rats that had been treated - or not - with inducing agents such as phenobarbital and ethanol. The inclusion of S9 fractions (4mg protein/mL) from non-induced or phenobarbital-induced rats resulted in a statistically significant enhancement of isoprene genotoxicity. A different pattern was obtained by the addition of ethanol-induced S9, which appeared highly genotoxic by itself even in the absence of isoprene. Reducing the concentration of ethanol-induced S9 to 0.25mg protein/mL resulted in a considerable enhancement of isoprene genotoxicity. In the absence of clear epidemiological evidence of the carcinogenicity of isoprene in humans, the results of this study seem to be particularly important since they add new findings to support the classification of this chemical as possibly carcinogenic to humans.     

异戊二烯合成酶(IspS)在大肠杆菌中的表达及其产异戊二烯的研究

将来源于银白杨的异戊二烯合成酶基因按照大肠杆菌密码子偏爱性进行优化,克隆到表达载体pACYCDu-et-1上,在大肠杆菌BL21(DE3)中异源表达,采用镍柱亲和层析纯化重组蛋白并测定其异戊二烯合成酶活性,通过摇瓶发酵实验对重组菌产异戊二烯进行进一步研究。结果显示:银白杨异戊二烯合成酶在大肠杆菌中能够高效表达,经过镍柱纯化后,电泳检测到特异性表达条带;该重组异戊二烯合成酶能够催化异戊二烯的合成,重组菌的异戊二烯产量可达到60μg/L。     

The cytoplasmic pH in photosynthesizing cells of the green alga Chlorella fusca,measured by P-31 NMR spectroscopy

P-31 NMR investigations were performed with the green alga Chlorella fusca under anaerobic conditions in the dark and in the light.In spectra of cells in the dark the signal of intracellular, nonvacuolar P_i indicates a pH in its chemical environment of 7.0–7.2. Upon illumination this signal looses intensity and shifts to lower field, corresponding to a pH of 7.7. Further downfield no other signal that could be attributed to a P_i-pool in more alkaline environment was detected. By the use of 2-deoxyglucose-6-phosphate as an indicator of cytoplasmic pH, this P_i-signal was assigned to the cytoplasm. The pH increase in the cytoplasm upon transfer of cells from the dark to the light is the same as that previously observed upon transfer of cells from anaerobic to aerobic conditions.In cells performing only cyclic photophosphorylation the cytoplasmic pH is lower than in photosynthesizing cells but still 0.2 pH units higher than in the cells in the dark. The reasons for the missing of a signal of stromal P_i and for the difference in cytoplasmic pH in photosynthesizing cells and those capable only of cyclic photophosphorylation are discussed.Non-standard abbreviations 2dG 2-Deoxyglucose - dG-6-P 2-deoxyglucose-6-phosphate - DCMU 3,4-dichlorophenyl-dimethylurea - MOPSO 3-(N-morpholino)-2-hydroxypropane sulfonic acid - P-31 NMR P-31 nuclear magnetic resonance     

Rapid regulation of the methylerythritol 4-phosphate pathway during isoprene synthesis

More volatile organic carbon is lost from plants as isoprene than any other molecule. This flux of carbon to the atmosphere affects atmospheric chemistry and can serve as a substrate for ozone production in polluted air. Isoprene synthesis may help leaves cope with heatflecks and active oxygen species. Isoprene synthase, an enzyme related to monoterpene synthases, converts dimethylallyl diphosphate derived from the methylerythritol 4-phosphate pathway to isoprene. We used dideuterated deoxyxylulose (DOX-d(2)) to study the regulation of the isoprene biosynthetic pathway. Exogenous DOX-d(2) displaced endogenous sources of carbon for isoprene synthesis without increasing the overall rate of isoprene synthesis. However, at higher concentrations, DOX-d(2) completely suppressed isoprene synthesis from endogenous sources and increased the overall rate of isoprene synthesis. We interpret these results to indicate strong feedback control of deoxyxylulose-5-phosphate synthase. We related the emission of labeled isoprene to the concentration of labeled dimethylallyl diphosphate in order to estimate the in situ K(m) of isoprene synthase. The results confirm that isoprene synthase has a K(m) 10- to 100-fold higher for its allylic diphosphate substrate than related monoterpene synthases for geranyl diphosphate.