N-acetyl-L-cysteine in the presence of Cu<Superscript>2+</Superscript> induces oxidative stress and death of granule neurons in dissociated cultures of rat cerebellum

Addition into the culture medium of the antioxidant N-acetylcysteine (NAC, 1 mM) in the presence of Cu²⁺ (0.0005-0.001 mM) induced intensive death of cultured rat cerebellar granule neurons, which was significantly decreased by the zinc ion chelator TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine). However, the combined action of NAC and Zn²⁺ did not induce destruction of the neurons. Measurement of the relative intracellular concentration of Zn²⁺ with the fluorescent probe FluoZin-3 AM or of free radical production using a CellROX Green showed that incubation of the culture for 4 h with Cu²⁺ and NAC induced an intensive increase in the fluorescence of CellROX Green but not of FluoZin-3. Probably, the protective effect of TPEN in this case could be mediated by its ability to chelate Cu²⁺. Incubation of cultures in a balanced salt solution in the presence of 0.01 mM Cu²⁺ caused neuronal death already after 1 h if the NAC concentration in the solution was within 0.005–0.05 mM. NAC at higher concentrations (0.1–1 mM) together with 0.01mM Cu²⁺ did not cause the death of neurons. These data imply that the antioxidant NAC can be potentially harmful to neurons even in the presence of nanomolar concentrations of variable valence metals.     

The cytoprotective properties of prostaglandin E2 against the toxic effects of actinomycin C on embryonic neural retina cells.

Prostaglandins (PGs) have been shown to cytoprotect various tissue types against the toxic effects of many chemicals. The mechanism of this protection is poorly understood, but the involvement of cAMP is often implied. Only one previous study examined nervous tissue and PG protection. The present study was designed to determine if PGE2 affords cytoprotection to a more specific nervous tissue (embryonic neural retina) from the toxicity of actinomycin C (AMC) using a trypan blue exclusion assay. The lowest concentration of PGE2 (2 x 10(-5)M) had no effect, but as the concentration increased (3 x 10(-5)M and 5 x 10(-5)M), PGE2 did afford protection against AMC in a dose dependent fashion. Theophylline treated cells were not protected, suggesting that cAMP may not be the primary mechanism of protection.     

Disruption of endothelial barrier function: relationship to fluidity of membrane extracellular lamella

• 1.1. Endothelial cells were cultured in tissue culture flasks or on microcarrier beads and labeled with a lipid specific spin-label.
• 2.2. Exposure of endothelial cells to benzyl alcohol caused a dose- and time-dependent increase in membrane fluidity using electron spin resonance (ESR). Maximum fluidity was reached after a 5-min exposure to 100 mM benzyl alcohol.
• 3.3. Albumin permeability across endothelial cells cultured on micropore filters was used as an indication of endothelial monolayer integrity.
• 4.4. A significant increase in permeability occurred with 50 mM benzyl alcohol. Maximal albumin permeability was reached after a 5-min exposure to 100 mM benzyl alcohol.

     

Polar auxin transport controls suspensor fate

Polar auxin transport is critical for normal embryo development in angiosperms. It has been proposed that auxin accumulates dynamically at specific positions, which in early Arabidopsis embryos correlates with developmental decisions such as specification of the apical cell lineage, specification of the hypophysis, and differentiation of the two cotyledons. In conifers, pattern formation during embryo development is different, and includes a free nuclear stage, nondividing suspensor cells, presence of tube cells, lack of hypophysis and formation of a crown of cotyledons surrounding the shoot apical meristem. We have recently shown that polar auxin transport is important for normal embryo development also in conifers. Here we suggest a model where auxin is transported from the suspensor cells to the embryonal mass during early embryogeny in conifers. This transport is essential for the developmental decisions of the tube cells and the suspensor, and affects both the amount of programmed cell death and the embryo patterning.Key words: conifer, embryo development, 1-N-naphtylphthalamic acid (NPA), patterning, polar auxin transport, programmed cell death, somatic embryogenesis, suspensorIn the model plant Arabidopsis thaliana auxin is transported, already from the first cell division of the zygote, from the basal cell to the apical cell, where it is involved in establishing the identity of the apical cell lineage. At the 32-cell stage the polar auxin transport is reversed, leading to an auxin accumulation in the uppermost suspensor cell, which occurs concomitantly with the specification of the hypophysis. During the heart stage auxin is transported towards the cotyledonary primordia, giving positional information about the cotyledon outgrowth.¹ Formation of the apical-basal embryonic pattern during early embryogeny in conifers is quite different from that in Arabidopsis and proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass, the embryonal tube cells and terminally differentiated nondividing suspensor cells.²The somatic embryo system of Picea abies (Norway spruce) includes a stereotyped sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments.^3,4 We are using this as a model system for elucidating the regulation of embryo development in conifers.² Early somatic embryos differentiate from proembryonic masses (PEMs) after withdrawal of the plant growth regulators (PGRs) auxin and cytokinin (Fig. 1A and B). We have previously shown that the organisation of the apical-basal polarity in early embryos is dependent on a gradient of PCD from the embryonal tube cells committed to death, to the cell corpses at the basal end of the suspensor.^5–7 Dysregulation of the PCD leads to aberrant apical-basal patterning.Open in a separate windowFigure 1Model for polar auxin transport control of early embryo patterning in conifers. (A) Embryogenic cultures proliferate as proembryonic masses (PEMs) in the presence of the plant growth regulators (PGRs) auxin and cytokinin. (B) Early embryos start to differentiate from PEMs after withdrawal of PGRs. Endogenous auxin is transported to the newly formed embryonal mass. (C) Early embryos are formed within two weeks in PGR-free medium. Early embryos have a distinct embryonal mass, tube cells and a suspensor. IAA is transported from the suspensor and the tube cells to the embryonal mass. (D) Fully matured cotyledonary embryos are formed after 5–6 weeks on maturation medium. (E) Treatment with NPA blocks the polar auxin transport to the embryonal mass, leading to an IAA accumulation in the suspensor cells, tube cells and perhaps also in the cells of the embryonal mass most adjacent to the tube cells. (F) Embryos with supernumerary suspensor cells are formed if polar auxin transport is inhibited only during the earliest stages of suspensor differentiation. (G) Embryos with meristematic cells in the suspensor are formed if polar auxin transport is inhibited during both differentiation and elongation of the suspensor. We assume that these abnormalities abort further development and maturation of viable embryos. em, embryonal mass; s, suspensor; tc, tube cells. Green arrows indicate polar auxin transport, T indicates blocked polar auxin transport, green shadings indicate auxin accumulation.We recently showed that in embryogenic cultures of Norway spruce treated with the polar auxin transport inhibitor NPA, the number of cells undergoing PCD decreases. As a consequence the balance between the number of cells in the embryonal mass and the number of cells in the suspensor develop abnormally, and concomitantly the endogenous free IAA content increases almost two-fold.⁸In order to visualise the IAA accumulation within the embryos we used a -318 bp deletion of the auxin-responsive IAA4/5 promoter from Pisum sativum (pea), previously characterized by Oeller et al.,⁹ and Ballas et al.,¹⁰ fused to the GUS reporter gene.¹¹ In tobacco (Nicotiana tabacum) the promoter is expressed in rapidly elongating hypocotyls,¹² (our unpublished observations) and strong induction by auxin is clear in elongating zones of both roots and hypocotyls in transgenic pIAA4/5-GUS Arabidopsis plants.¹¹ However, to our knowledge, expression of IAA4/5 has not been reported in embryonal shoot apical meristems. Hence, the pIAA4/5-GUS may preferentially be used as a biosensor of auxin activity in non-meristematic cells during spruce embryo development. During normal somatic embryo development in spruce, pIAA4/5-GUS activity is detected in PEMs, tube cells and suspensor cells, but not in the embryonal mass. Early embryos of Norway spruce that are treated with NPA show increased pIAA4/5-GUS activity in tube cells and suspensor cells (unpublished), well in line with the increment of free IAA levels.Our results indicate that IAA under normal conditions is transported from the suspensor cells to the cells in the embryonal mass (Fig. 1B and C). NPA-treatment blocks this polar transport of endogenous IAA, which results in an accumulation of IAA and increased pIAA4/5-GUS activity in the suspensor cells, the tube cells, and perhaps also in the cells of the embryonal mass most adjacent to the tube cells (Fig. 1F and G). Blocked polar auxin transport during early differentiation of the suspensor stimulates abnormal cell divisions of the meristematic cells most adjacent to the tube cells or perhaps even of the tube cells themselves. Consequently, embryos with supernumerary tube and suspensor cells are formed (Fig. 1F). If the polar auxin transport is blocked for a longer time, i.e., during both differentiation and elongation of the suspensor, the auxin accumulation leads to maintenance of meristematic fate and a failure to undergo PCD (Fig. 1G).It has been proposed that the fate of the suspensor cells is regulated by signals from the embryo proper which impede developmental potential and initiate PCD.¹³ In accordance, we assume that the abnormal embryo morphologies formed after NPA-treatment may result from adverse inhibitory signals from the embryonal mass.     

Systemic and Mucosal T-Lymphocyte Activation Induced by Recombinant Adenovirus Vaccines in Rhesus Monkeys

The administration of vectors designed to elicited cell-mediated immune responses may have other consequences that are clinically significant. To explore this possibility, we evaluated T-cell activation during the first 2 months after recombinant adenovirus serotype 5 (rAd5) prime or boost immunizations in rhesus monkeys. We also evaluated the kinetics of T-lymphocyte activation in both the systemic and the mucosal compartments after rAd5 administration in monkeys with preexisting immunity to Ad5. The rAd5 immunization induced lower-frequency Gag epitope-specific CD8⁺ T cells in the colonic mucosa than in the peripheral blood. There was evidence of an expansion of the simian immunodeficiency virus Gag-specific CD8⁺ T-cell responses, but not the Ad5 hexon-specific T-cell responses, following a homologous rAd5 boost. A striking but transient T-lymphocyte activation in both the systemic and the mucosal compartments of rhesus monkeys was observed after rAd5 immunization. These findings indicate that the administration of a vaccine vector such as Ad5 can induce a global activation of T cells.Considerable effort has been invested in the development of vaccine strategies for eliciting cell-mediated immune responses to human immunodeficiency virus (HIV). Studies in simian immunodeficiency virus (SIV)/SHIV-infected nonhuman primates and HIV-infected humans demonstrated a central role for cell-mediated immune responses in the containment of HIV replication (1, 12). These findings led to the hypothesis that vaccine-elicited cell-mediated immunity might contribute to improved control of HIV in infected individuals. Studies in the SIV and SHIV/macaque models have supported this hypothesis, demonstrating a decrease in peak plasma virus RNA levels during primary infection, protection against memory CD4⁺ T-cell lymphocyte loss, and prolonged survival of monkeys that had vaccine-elicited cell-mediated immunity to the virus prior to challenge (8, 15, 16).Despite promising results in preclinical nonhuman primate studies, a prophylactic HIV vaccine trial of the Merck recombinant adenovirus serotype 5 (rAd5) vector expressing HIV gag, pol, and nef genes (STEP trial) was recently halted due to a 2.3-fold increase of HIV acquisition in vaccinees with preexisting neutralizing antibodies (NAbs) to Ad5 (2, 9, 10). This finding raised the possibility that T lymphocytes that are activated in response to vaccination might represent an increased pool of potential targets for HIV infection, and the persistence of such activated cells may increase the susceptibility of the vaccinated individual to acquiring an HIV infection (5, 11). HIV replicates most readily in activated, CCR5⁺CD4⁺ T lymphocytes. It has been suggested that vaccines that elicit potent cellular immune responses may also activate subpopulations of CD4⁺ T lymphocytes. In fact, in the aftermath of the failed STEP trial, it was proposed that the activation of Ad5-specific T cells in individuals with prior Ad5 immunity may have contributed to their increased acquisition of HIV after vaccination.The contribution of cellular activation in mucosal tissues to acquisition of HIV remains unexplored (2). HIV transmission occurs most often across mucosal barriers. There is increasing evidence that CD4⁺ T lymphocytes are among the first cells infected during the transmission event (4). Activation of mucosal populations of lymphocytes as a consequence of vaccination could contribute to increasing the incidence of HIV transmission at a mucosal site.To examine these issues, the present study was initiated to explore vaccine-induced activation of T-lymphocyte populations in rhesus monkeys. The character and kinetics of the activation of both circulating and mucosal T-lymphocyte populations were evaluated after immunization with a variety of immunogens. These experiments demonstrate a striking but transient T-lymphocyte activation induced by adenovirus-based vaccine vectors in both the systemic and mucosal compartments of rhesus monkeys.     

Modulation of caffeine contractures in mammalian skeletal muscles by variation of extracellular potassium

Caffeine contractures were induced after K⁺ -conditioning of skeletal muscles from pigs and mice. K⁺ -conditioning is defined as the partial depolarization caused by increasing external potassium (K) with [K⁺]×[Cl^?] constant. Conditioning depolarizations that rendered muscles refractory to brief electrical stimulation still enhanced the contracture tension elicited by subsequent direct caffeine stimulation of sarcoplasmic reticulum (SR) calcium release. The effects of K⁺ -conditioning on caffeine-induced contractures of intact cell bundles reached a maximum at 15–30 mM K and then progressively declined at higher [K⁺]₀. Conditioning with 30 mM K⁺ for 5 min, which inactivates excitation-contraction (EC) coupling in response to action potentials, both increased the magnitude of caffeine contractures 2–10-fold and shifted the contracture threshold toward lower caffeine concentrations. Enhanced sensitivity to caffeine was inhibited by dantrolene (20 μM) and its watersoluble analogue azumolene (150 μM). These drugs decreased caffeine-induced contractures following depolarization with 4–15 mM K⁺ to 25–50% of control tension. The inorganic anion perchlorate (CIO), which like caffeine potentiates twitches, increased caffeine-induced contractures ～? twofold after K⁺ -conditioning (>4 mM). The results suggest that CIO and dantrolene, in addition to caffeine, also influence SR calcium release either directly or by mechanism(s) subsequent to depolarization of the sarcolemma. Moreover, since CIO is known to shift the voltage-dependence of intramembrane charge movement, CIO may exert effects on the transverse-tubule voltage sensors as well as the SR. © 1995 Wiley-Liss, Inc.     

The modulation of various physiochemical changes in <Emphasis Type="Italic">Bruguiera cylindrica</Emphasis> (L.) Blume affected by high concentrations of NaCl

Bruguiera cylindrica is a major mangrove species in the tropical mangrove ecosystems and it grows in a wide range of salinities without any special features for the excretion of excess salt. Therefore, the adaptation of this mangrove to salinity could be at the physiological and biochemical level. The 3-month-old healthy plantlets of B. cylindrica, raised from propagules were treated with 0 mM, 400 mM, 500 mM and 600 mM NaCl for 20 days under hydroponic culture conditions provided with full strength Hoagland medium. The modulation of various physiochemical changes in B. cylindrica, such as chlorophyll a fluorescence, total chlorophyll content, dry weight, fresh weight and water content, Na⁺ accumulation, oxidation and antioxidation (enzymatic and non-enzymatic) features were studied. Total chlorophyll content showed very minute decrease at 500 mM and 600 mM NaCl treatment for 20 days and the water content percentage was decreased both in leaf and root tissues with increasing concentration. A significant increase of Na⁺ content of plants from 84.505 mM/plant dry weight in the absence of NaCl to 543.38 mM/plant dry weight in plants treated with 600 mM NaCl was recorded. The malondialdehyde and the metabolites content associated with stress tolerance (amino acid, total phenols and proline) showed an increasing pattern with increasing NaCl concentration as compared to the control in both leaf and root tissues but the increase recorded in plantlets subjected to 500 mM was much less, indicating the tolerance potential of this species towards 500 mM NaCl. The significant decrease of sugar content was found only in 600 mM NaCl on 20 days of treatment, showing that the process of sugar synthesis was negatively affected but the same process remains less affected at 500 mM NaCl. A slight reduction in ascorbate and glutathione content and very less increase in carotenoid content were observed at 500 mM and 600 mM NaCl stress. Antioxidant enzymes (APX, GPX, SOD and CAT) showed an enhanced activity in all the treatments and the increased activity was more significant in 600 mM treated plants. The result establishes that B. cylindrica tolerates high NaCl concentration, to the extent of 500 mM NaCl without any major inhibition on photosynthesis and metabolite accumulation. Understanding the modulation of various physiological and biochemical changes of B. cylindrica at high levels of NaCl will help us to know the physiochemical basis of tolerance strategy of this species towards high NaCl.     

Rate-limiting steps of 2-deoxyglucose uptake in rat adipocytes

2-Deoxy[1-¹⁴C]glucose uptake in rat adipocytes was measured as a function of time in the absence and presence of unlabelled glucose or 2-deoxyglucose. Uptake of tracer alone was linear from 2 s to 6 min. At 37°C the rate of uptake in insulin-stimulated cells decreased markedly after a few seconds in the presence of glucose (0.5–10 mM) and after 0.5–2 min in the presence of deoxyglucose (2–10 mM). Similar data were obtained at 22°C. With 10 mM glucose (37°C, 30 s) approx. 80% of the intracellular radioactivity was non-phosphorylated deoxyglucose and with 10 mM deoxyglucose approx. 40% was non-phosphorylated. The results show that deoxy[¹⁴C]glucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose and at least partially in the presence of deoxyglucose. The data suggest caution in using deoxyglucose uptake as a measure of transport, especially in complex kinetic studies.In addition, the initial velocity of tracer ¹$\text{3-O-}\text{methylglucose}$ was found to be approx. 2-fold higher than that of tracer deoxyglucose even though both sugars inhibited the initial velocity of labelled methylglucose half-maximally at a concentration of 5 mM. These data suggest a fundamental difference between deoxyglucose and methylglucose transport.     

Phytochromabhängiger Ionentransport in Erbsensämlingen

Summary Five to 6 day old dark-adapted dwarf and tall pea seedlings grown in water culture were illuminated for ten minutes with red light and/or ten minutes with far-red light, and 90 to 170 minutes later their roots were immersed in a 0.2 mM K⁺ solution containing labeled ⁸⁶Rb⁺. After two hours uptake the fresh-weights and radioactivities of the shoot organs were determined. It was found that red light inhibits K⁺uptake into internodes and promotes uptake into the plumula. The red-light effect on K⁺transport precedes the red-light induced growth inhibition of internodes and growth promotion of leaves and is abolished by far-red light given immediately after red. The red-light effect on K⁺transport is independent of the concentration of K⁺ given to the roots in the range between 0.2 to 125 mM.     

The cytoprotective properties of prostaglandin E2 against the toxic effects of actinomycin C on embryonic neural retina cells

Prostaglandins (PGs) have been shown to cytoprotect various tissue types against the toxic effects of many chemicals. The mechanism of this protection is poorly understood, but the involvement of cAMP is often implied. Only one previous study examined nervous tissue and PG protection. The present study was designed to determine if PGE₂ affords cytoprotection to a more specific nervous tissue (embryonic neural retina) from the toxicity of actinomycin C (AMC) using a trypan blue exclusion assay. The lowest concentration of PGE₂ (2 × 10⁻⁵M) had no effect, but as the concentration increased (3 × 10⁻⁵M and 5 × 10⁻⁵M), PGE₂ did afford protection against AMC in a dose dependent fashion. Theophylline treated cells were not protected, suggesting that cAMP may not be the primary mechanism of protection.     

Prostaglandin E2 receptor type 2-selective agonist prevents the degeneration of articular cartilage in rabbit knees with traumatic instability

Introduction

Osteoarthritis (OA) is a common cause of disability in older adults. We have previously reported that an agonist for subtypes EP2 of the prostaglandin E2 receptor (an EP2 agonist) promotes the regeneration of chondral and osteochondral defects. The purpose of the current study is to analyze the effect of this agonist on articular cartilage in a model of traumatic degeneration.

Methods

The model of traumatic degeneration was established through transection of the anterior cruciate ligament and partial resection of the medial meniscus of the rabbits. Rabbits were divided into 5 groups; G-S (sham operation), G-C (no further treatment), G-0, G-80, and G-400 (single intra-articular administration of gelatin hydrogel containing 0, 80, and 400 μg of the specific EP2 agonist, ONO-8815Ly, respectively). Degeneration of the articular cartilage was evaluated at 2 or 12 weeks after the operation.

Results

ONO-8815Ly prevented cartilage degeneration at 2 weeks, which was associated with the inhibition of matrix metalloproteinase-13 (MMP-13) expression. The effect of ONO-8815Ly failed to last, and no effects were observed at 12 weeks after the operation.

Conclusions

Stimulation of prostaglandin E2 (PGE2) via EP2 prevents degeneration of the articular cartilage during the early stages. With a system to deliver it long term, the EP2 agonist could be a new therapeutic tool for OA.     

Salinity-dependence of the properties of gill (Na++K+-ATPase in rainbow trout Oncorhynchus mykiss

• 1.1. The expected higher gill (Na⁺+K⁺)-ATPase activity in rainbow trout adapted to brackish water (BW) with respect to fresh water (FW) is accompanied by some changes in the enzyme kinetics while the enzyme sensitivity to ouabain is unaffected
• 2.2. Maximal activation is attained under the optimal conditions of 4 mM ATP, 7.5 mM Mg²⁺, 50 mM Na⁺, 2.5 mM K⁺, pH 7.0 in FW, and 3 mM ATP, 10 mM Mg²⁺, 100 mM Na⁺, 10 mM K⁺, pH 7.5 in BW.
• 3.3. The change of the enzyme activation kinetics by Mg²⁺, ATP, Na⁺ and K⁺ from simple saturation in FW to cooperativity in BW and other habitat-dependent variations including the pH alkaline shift in BW are hypothetically related to an adaptive significance to the different environmental salinity.
• 4.4. Gill total lipids and phospholipids are 30% lower in BW than in FW while their ratio is constant; some differences in gill total lipid fatty acid composition between FW and BW do not significantly affect the unsaturation parameters.

     

Culture medium optimization for improved puerarin production by cell suspension cultures of Pueraria tuberosa (Roxb. ex Willd.) DC.

The effects of carbon, nitrogen, phosphate, and copper on cell growth and production of the isoflavone puerarin by suspension cultures of Pueraria tuberosa (Roxb. ex. Willd.) DC were investigated. Among the various sugars evaluated (glucose, galactose, fructose, maltose, and sucrose), use of sucrose in the medium led to the maximum accumulation of puerarin. A sucrose-feeding strategy in which additional sucrose was added to the flasks 15?d into the culture cycle stimulated both cell biomass and puerarin production. The maximum production of puerarin was obtained when a concentration balance of 20:60?mM NH ₄ ⁺ /NO ₃ ^? was used as the nitrogen source. Alteration in the concentration balance of nitrogen components (NH ₄ ⁺ /NO ₃ ^? 60:20?mM) or the use of either NH ₄ ⁺ or NO ₃ ^? alone decreased biomass production and puerarin accumulation compared with the control culture (NH ₄ ⁺ /NO ₃ ^? 20:20?mM). High amounts of phosphate (2.5 and 5?mM) in the medium inhibited puerarin production whereas 0.625?mM phosphate promoted puerarin production (68.3???g/g DW on day?25). An increase in Cu²⁺ concentration from 0.025 to 0.05?mg/l in the P. tuberosa cell culture medium resulted in a 2.2-fold increase in puerarin production (up to 141???g/g DW on day?25) but reduced cell culture biomass.