Neuroprotection of Lubeluzole Is Mediated Through the Signal Transduction Pathways of Nitric Oxide

Abstract: Neuronal survival after ischemic injury is determined through the induction of several biological pathways. We examined whether lubeluzole, an agent efficacious in both clinical and experimental models of cerebral ischemia, modulated the signal transduction mechanisms of nitric oxide (NO), a downstream mediator of anoxic neurodegeneration. Both pretreatment [NO survival = 23 ± 3%, NO/lubeluzole (750 n M ) survival = 63 ± 2%, p < 0.001] and coadministration [NO survival = 25 ± 3%, NO/lubeluzole (750 n M ) survival = 59 ± 3%, p < 0.001] of lubeluzole with NO generators equally protected cultured hippocampal neurons in a dose-dependent manner against the toxic effects of NO, suggesting that the agent protects by acutely modifying toxic cellular pathways rather than preconditioning the neuron before injury. The protection observed with lubeluzole was stereospecific but was not limited to pre- or coadministration. Lubeluzole also was found to significantly protect against the toxicity of NO for a period of 4–6 h after NO exposure [NO survival = 31 ± 2%, NO/lubeluzole (750 n M ) survival at 6 h = 56 ± 3%, p < 0.001]. We conclude that the neuroprotective ability of lubeluzole is unique and involves the direct modulation of the NO pathway. In addition, the mechanisms of NO toxicity are dynamic and reversible processes that, if left unaltered, will lead to neuronal injury. Further investigation of the downstream signal transduction mechanisms below the level of NO generation may elucidate the specific cellular events responsible for neurodegeneration.     

Cerebral Taurine Release Mechanisms In Vivo: Pharmacological Investigations in Rats Using Microdialysis for Proof of Principle

Cerebral taurine acts as neurotransmitter, as neuromodulator, or as osmoregulator. To investigate its release mechanisms in vivo, we combined the microdialysis technique with a variety of experimental paradigms. Taurine release was stimulated by either NMDA, NO or a hypotonic solution locally with or without the addition of the NMDA antagonists APV or Ketamine, or the NO synthase inhibitor L-NAME. Alternatively, the neuroprotective drug lubeluzole was applied i.v. NMDA, NO or the hypotonic solution stimulated the release of taurine. NMDA-mediated taurine release was inhibited by either APV, Ketamine or the NO synthase inhibitor L-NAME. Lubeluzole had no effect. Under the hypotonic conditions only lubeluzole was effective. These data confirm in vivo that the NMDA-induced taurine release is mediated via the NO cascade. By contrast, the release after a hypotonic stimulus is not related to the NO cascade. Instead, Na⁺- and/or Ca²⁺- mediated events might have been attenuated by lubeluzole.     

An alternative propagation method ofAnanas through nodule culture

A micropropagation scheme forAnanas comosus Merr. was developed using nodule culture. Nodules were induced from leaf-base or chopped shoot-base explants on modified half-strength MS medium supplemented with 2.69-5.37 M NAA and 4.44 M BA and could be maintained long-term as nodules. The nodules proliferated into more nodules when chopped into pieces of 1–3 mm and placed onto the same medium. They regenerated shoots when transferred to medium supplemented with 0.54–10.74 M NAA and 0.44–8.88 M BA. The regeneration capacity of nodules is higher than that of direct regeneration or callus. Maximum regeneration was obtained from culture medium containing 0.54 M NAA and 0.44 M BA, where shoots could be observed as early as within 2 weeks. Many shoots formed roots in the same medium in which they were regenerated after 10 subcultures, but the best rooting occurred in medium containing 0.54 M NAA and 0.44 M BA. Rooted plantlets ofA. comosus Merr. could be routinely produced at 6-week intervals.Abbreviations NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

Nitric Oxide Modulates Muscarinic Acetylcholine Receptor Binding in the Cerebral Cortex of Gerbils

To determine whether nitric oxide (NO) acts as a modulator of muscarinic acetylcholine receptor (mACh-R) function, we performed a radioligand receptor assay using [³H]quinuclidinyl benzylate ([³H]QNB), the NO radical (NO·) donor 3-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC7) and a gerbil brain cortical membrane preparation. NOC7 (at 10 M, 100 M or 1 mM concentrations) significantly reduced the [³H]QNB binding K_d values (from 0.196 ± 0.009 nM in the control, to 0.151 ± 0.013, 0.144 ± 0.012 and 0.153 ± 0.007 nM respectively). NOC7 did not alter the displacement curves of atropine or carbachol. Reduction of SH groups with dithiothreitol, in the presence of the NO donor, significantly increased [³H]QNB binding affinity whereas alkylation by N-ethylmaleimide markedly decreased it. The observed enhancing effect on mACh-R binding affinity for [³H]QNB, may reflect conformational changes in the receptors mediated by the NO generated, and these changes might be explained by NO reactions with such groups through conditions supporting redox reactions intrinsic to the NO molecule, similar to those occurring in redox regulatory sites reported for other neurotransmitter pathways in the CNS.     

Activity of dihydrofolate reductase in the mollicutesAcholeplasma laidlawii andMycoplasma gallisepticum and their susceptibility to antifolates

Mycoplasma gallisepticum andAcholeplasma laidlawii were found to possess dihydrofolate reductases exhibiting similar specific activities and kinetics, with values in the range of those reported for other microorganisms. The apparent Km values for dihydrofolate in enzymes ofM. gallisepticum andA. laidlawii are 7.95±0.13 and 7.50±0.11 M, and for NADPNH 8.46±0.25 and 9.32±0.18 M, respectively.M. gallisepticum is 3300-fold more resistant to methotrexate than isA. laidlawii; concentrations causing 50% inhibition were 200.00 and 0.06 M, respectively. This is in contrast to almost the same sensitivity to that drug exhibited by the dihydrofolate reductases of both microorganisms.M. gallisepticum is also 3600-fold more resistant to trimethoprim than isA. laidlawii, and the concentrations for 50% inhibition of growth were 1800.0 and 0.5 M, respectively. The high resistance was found to be due partially to a 130-fold lower affinity of the target enzyme for this antifolate, but another mechanism, presumably impaired transport, must also be involved. This is the first report of dihydrofolate reductase activity in Mollicutes.     

Effect of Metabolic Inhibitors on Membrane Potential and Ion Conductance of Rat Astrocytes

1. The aim of this study was to elucidate the effect of metabolic inhibition on the membrane potential and ion conductance of rat astrocytes. The metabolic inhibitors investigated were dinitrophenol (DNP), carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), cyanide, and oligomycin.2. Primary cultures of astroglial cells from newborn rat cerebral cortex were cultivated for 13–20 days on chamber slides. The effect of metabolic inhibitors on the cellular ATP concentration was estimated from the decrease in peak chemiluminescence from the luciferin/luciferase reaction. The membrane potential and ion conductances were measured from whole-cell recordings with the patch-clamp technique.3. After 2.0 min of incubation ATP decreased from the control level to 43%with cyanide (2 mM), 58% with DNP (1 mM), 47% with FCCP (1 M), and 69% with oligomycin (10 M).4. Under normal conditions V was –74.4±1.0 mV. DNP and FCCP both caused a rapid and reversible depolarization equivalent to a shift in the I/V curve of 8.2±1.3 and 19.7±3.8 mV, respectively. DNP decreased the slope conductance (g) by 22.1% but FCCP had no significant effect on g. In contrast, neither oligomycin nor cyanide had any significant effect on the I/V curve.5. Tetraethylammonium (TEA; 10 mM) depolarized the cells by 7.1±2.0 mV but had no significant effect on g. In the presence of TEA, DNP caused a depolarization of 52.8±3.5 mV and increased g by 45.5±9.6%. The action of FCCP was not affected by the presence of TEA.6. Perfusion of the astrocytes with a Cl^– free solution inhibited the action of DNP and FCCP. Thus the depolarization was only 4.2±1.5mV in DNP and 3.7±0.3 mV in FCCP, which were significantly smaller effects than in the presence of a high intracellular [Cl^–].7. Block of tentative K_ATP channels with tolbutamide (1 mM) or Cl^– channels with Zn²⁺ (1 mM) did not inhibit the depolarization caused by DNP or FCCP.8. In conclusion, DNP and FCCP have specific effects on the plasmalemma in rat astrocytes which may be due to opening of Cl^– channels. This effect was not seen with cyanide or oligomycin and should be considered as a possible complication when DNP and FCCP are used for metabolic inhibition.     

Effect of Ammonia and Methionine Sulfoximine on myo-Inositol Transport in Cultured Astrocytes

Ammonia causes astrocyte swelling which is abrogated by methionine sulfoximine (MSO). Since myo-inositol is an important osmolyte, we investigated the effects of ammonia and MSO on myo-inositol flux in cultured astrocytes for periods up to 72 hours. Uptake of myo-inositol was significantly decreased by 26.7 (P < 0.05) and 39.3 (P lt; 0.006) percent after 48 hours of exposure to 5 or 10 mM ammonia, respectively. The maximum rate of uptake was 14.0 ± 0.5 nmol/hour/mg protein which was reduced to 7.45 ± 0.27 and 7.02 ± 0.57 nmoles/hour/mg protein by 5 or 10 mM ammonia, respectively. The Kms by Michaelis-Menten equation for the control, and in the presence of 5, or 10 mM ammonia were 32.5 ± 4.52, 44.4 ± 5.82, and 39.3 ± 7.0 M, respectively. Kms by Hanes-Woolf plot for the control, 5, or 10 mM ammonia were 25, 45, and 40 M, respectively. Treatment of astrocytes with either 5 or 10 mM NH₄Cl for 6 hours caused a decrease in myo-inositol content by 66% and 58%, respectively. MSO (3 mM) partially diminished the ammonia-induced inhibition of myo-inositol uptake and decreased myo-inositol content by 31% after 24 hours. Additionally, ammonia increased myo-inositol efflux briefly through the fast efflux component but had little effect on myo-inositol efflux through the slow efflux component of astrocytes exposed to ammonia for up to 72 hours. Predominantly decreased myo-inositol influx coupled with brief efflux through the fast component may represent an adaptive response to diminish the extent of ammonia-induced astrocyte swelling.     

Early-flowering Scots pines through tissue culture for accelerating tree breeding

Scots pine plantlets were produced via tissue culture using cotyledons excised from germinated embryos as explants. The optimum tissue culture conditions were: 1/2GDbasal medium gelled with agar-Gelrite during shoot formation and with agar during rooting, inclusion of 5.0M benzylaminopurine (BAP) and 0.05 M naphthaleneacetic acid (NAA) for 2 weeks for shoot induction, and repeated 2.7 M NAA pulses of 1 week for rooting. Micropropagation success was genotype-dependent. Average multiplication rates varied among experiments from 3 to 15 shoots per embryo. The maximum shoot production from a single embryo was 35. Rooting was the most difficult phase in the propagation process. Most of the plantlets had a plagiotrophic and highly branched growth habit when growing in the greenhouse. Some individuals produced megasporangiate strobili at the age of 3 years and microsporangiate strobili with viable pollen at the age of 4 years. Early-flowering clones and the ability to conserve seedlings from which cotyledons have been cultured give new possibilities for accelerated tree breeding.     

In vitro somatic embryogenesis ofDalbergia sissoo Roxb. —a multipurpose timber-yielding tree

Somatic embryogenesis was achieved in callus cultures dervied from 40-day-old semimature zygotic embryos ofDalbergia sissoo on semi-solid Murashige and Skoog (MS) salts and vitamins supplemented with 0.46–1.16 M kinetin, 6.78–9.04 M 2,4-dichlorophenoxy acetic acid (2,4-D) and 30 g/1 sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to half-strength basal MS medium supplemented with 0.46-1.16 M kinetin and 6.78–9.04 M 2,4-D with 2% (w/v) sucrose. The light-green somatic embryos germinated on half-strength MS salts and vitamins supplemented with 0.5 mg/1 abscisic acid and 2% (w/v) sucrose. The developmental stages of somatic embryogenesis were studied by light and scanning electron microscopy.Abbreviations ABA Abscisic acid - BA 6-benzyladenine - Kn kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) basal medium     

Adaptation of inducible defense in Euplotes daidaleos (Ciliophora) to predation risks by various predators

The extent of induced morphological defense in Euplotes daidaleos correlates to this ciliate's predation risk from the defense-inducing predator species. Euplotes daidaleos responded by morphological transformation only to organisms that are able to feed on typically formed Euplotes cells (63 ± 5 m cell width in E. daidaleos). Three of those potential predator species caused defensive changes to various degrees (Student's t-test, P < 0.1 to P < 0.0001): Lembadion bullinum (Ciliata) induced 82 ± 6 m cell width in E. daidaleos; Chaetogaster diastrophus (Oligochaeta) induced 85 = 6 m width; and Stenostomum sphagnetorum (Turbellaria) induced 89 ± 8 m width (at a density of 10 predators per milliliter, respectively). At higher predator densities (50 or 100 organisms per milliliter), Euplotes developed a correspondingly larger width (to a maximum of 103 ± 10 m in the presence of S. sphagnetorum). Euplotes did not respond to organisms (e.g., Blepharisma japonicum, Colpidium campylum, Didinium nasutum, Paramecium caudatum, Spirostomum ambiguum, Stentor coeruleus) that cannot feed on this ciliate species. Daphnia longispina and Bursaria truncatella predators, which can feed on large prey of 125, or 200 m in diameter, respectively, also had no effect on the morphology of Euplotes. The extent of defense in Euplotes that was induced by 10 predators per milliliter during 24 h decreased the predation risk from those predators to 67% in the presence of S. sphagnetorum, to 50% with L. bullinum, and to 15% with C. diastrophus, compared to the typical form of Euplotes. In a natural population, the defensive form of E. daidaleos was found with average cell widths of 88 ± 8 m. The results indicate that predator-induced defense in natural Euplotes populations is beneficial to this prey and that it is adapted to the predation abilities of Euplotes predators, whereby energetical costs related to defensive changes may be saved.     

The Tyrosine Phosphatase SHP2 Modulates MAP Kinase p38 and Caspase 1 and 3 to Foster Neuronal Survival

1. The Src homology protein tyrosine phosphatase SHP2 is associated with cytoskeletal maintenance, cell division, and cell differentiation, but the role of SHP2 during central nervous system injury requires further definition. We therefore characterized the role of SHP2 during nitric oxide (NO)-induced programmed cell death (PCD).2. Employing primary hippocampal neurons from mice with a dominant negative SHP2 mutant to render the phosphatase site of the SHP2 protein biologically inactive, but functionally capable of binding substrate, neuronal injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidyl serine (PS) exposure, mitogen-activated protein (MAP) kinase phosphorylation, and cysteine protease activity. NO was administered through the NO generators SIN-1 (300 M) or NOC-9 (300 M).3. Following NO exposure, neuronal survival decreased from 89 ± 3% in untreated controls to 37 ± 2% in wild-type neurons and to 21 ± 4% in SHP2 mutant neurons. In sister cultures following NO exposure, this increased susceptibility to neuronal injury paralleled enhanced genomic DNA degradation and membrane PS exposure with PCD induction increasing in SHP2 mutant neurons by approximately 42% during specified time periods when compared to wild-type neurons. Interestingly, modulation of the MAP kinase p38 appears to represent an initial level of neuronal protection employed by SHP2. In addition, both the rate and degree of caspase 1- and caspase 3-like activities in SHP2 mutant neurons were significantly increased over a 24-h course when compared to wild-type neurons. Inhibition of caspase 1- and caspase 3-like activities reversed the progression of neuronal PCD, suggesting that inhibition of cysteine protease activity is a downstream mechanism for SHP2 to afford neuronal protection.4. Our work supports the premise that the tyrosine phosphatase SHP2 plays a dominant role during NO-induced PCD and may offer a potential molecular checkpoint against neurodegenerative disease.     

Excitation-calcium release uncoupling in aged single human skeletal muscle fibers

The biological mechanisms underlying decline in muscle power and fatigue with age are not completely understood. The contribution of alterations in the excitation-calcium release coupling in single muscle fibers was explored in this work. Single muscle fibers were voltage-clamped using the double Vaseline gap technique. The samples were obtained by needle biopsy of the vastus lateralis (quadriceps) from 9 young (25–35 years; 25.9 ± 9.1; 5 female and 4 male) and 11 old subjects (65–75 years; 70.5 ± 2.3; 6 f, 5 m). Data were obtained from 36 and 39 fibers from young and old subjects, respectively. Subjects included in this study had similar physical activity. Denervated and slow-twitch muscle fibers were excluded from this study. A significant reduction of maximum charge movement (Q_max) and DHP-sensitive Ca current were recorded in muscle fibers from the 65–75 group. Q_max values were 7.6 ± 0.9 and 3.2 ± 0.3 nC/F for young and old muscle fibers, respectively (P < 0.01). No evidences of charge inactivation or interconversion (charge 1 to charge 2) were found. The peak Ca current was (–)4.7 ± 0.08 and (–)2.15 ± 0.11 A/F for young and old fibers, respectively (P < 0.01). The peak calcium transient studied with mag-fura-2 (400 m) was 6.3 ± 0.4 m and 4.2 ± 0.3 m for young and old muscle fibers, respectively. Caffeine (0.5 mm) induced potentiation of the peak calcium transient in both groups. The decrease in the voltage-/ Ca-dependent Ca release ratio in old fibers (0.18 ± 0.02) compared to young fibers (0.47 ± 0.03) (P < 0.01), was recorded in the absence of sarcoplasmic reticulum calcium depletion. These data support a significant reduction of the amount of Ca available for triggering mechanical responses in aged skeletal muscle and, the reduction of Ca release is due to DHPR-ryanodine receptor uncoupling in fast-twitch fibers. These alterations can account, at least partially for the skeletal muscle function impairment associated with aging.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association, and National Institutes of Health (2-P60AG18484-06)     

Mechanisms of Na and Cl absorption across the distal colon epithelium of the pig

Summary Porcine distal colon epithelium was mounted in Ussing chambers and bathed in plasma-like Ringer solution. Tissue conductances ranged from 10 to 15 mS and the short-circuit current (I_sc) ranged from-15 to 220 A·cm^-2. Variations in basal I_sc resulted from differences in the amount of amiloride (10M mucosal addition)-sensitive Na⁺ absorption. Ion substitution and transepithelial flux experiments showed that 10 M amiloride produced a decrease in the mucosal-to-serosal (M-S) and net Na flux, and that this effect on I_sc was independent of Cl^- and HCO ₃ ^- replacement. When the concentration of mucosal amiloride was increased from 10 to 100 M, little change in I_sc was observed. However, increasing the concentration to 1 mM produced a further inhibition, which often reversed the polarity of the I_sc. The decrease in I_sc due to 1 mM amiloride was dependent on both Cl^- and HCO ₃ ^- , and was attributed to reductions in the M-S and net Na⁺ fluxes as well as the M-S unidirectional Cl^- flux. Ion replacement experiments demonstrated that Cl^- substitution reduced the M-S and net Na fluxes, while replacement of HCO ₃ ^- with HEPES abolished net Cl^- absorption by reducing the M-S unidirectional Cl^- flux. From these data it can be concluded that: (1) Na⁺ absorption is mediated by two distinct amiloride-sensitive transport pathways, and (2) Cl^- absorption is completely HCO ₃ ^- -dependent (presumably mediated by Cl^-/HCO ₃ ^- exchange) and occurs independently of Na⁺ absorption.Abbreviations G_t tissue conductance - HEPES tris (hydroxymethyl) aminomethane - (tris) N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - I_sc short-circuit current - J_r residual flux - M-S mucosal-to-scrosal - S-M serosal-to-mucosal - TTX tetrodotoxin     

Killing ofLegionella pneumophila by human serum and iron-binding agents

The inhibitory effect of serum on the growth and survival ofLegionella pneumophila Bloomington 2 was investigated. When incubated in the presence of 20%–50% normal human serum for 10 h, viability was decreased by >99%. Heat-inactivated or <40% normal serum supplemented with 50 M iron was not inhibitory. The addition of guinea pig complement to heat-inactivated serum resulted in killing of approximately 98% of the cells. Growth in buffered yeast extract broth was inhibited by the addition of ferric iron-binding compounds. Minimum bactericidal concentrations at 37°C were 10 M apotransferrin, 35 M 1,10-phenanthroline, and 50 M deferoxamine. Addition of iron chelators to normal serum did not accelerate killing. Egg yolk-passaged virulent strains and agar-grown avirulent strains exhibited similar serum sensitivity. Results of this study indicate that complement and serum transferrin are antagonistic to the growth ofLegionella in serum.     

The cyclic-2,3-diphosphoglycerate fromMethanobacterium thermoautotrophicum is thed enantiomer

The cyclic pyrophosphate obtained fromMethanobacterium thermoautotrophicum was converted tosn-glycerol 3-phosphate by a stereospecific route. This conversion establishes the structure as cyclic-2,3-diphospho-d-glycerate. The same method was used to determine the cellular content of this metabolite under two conditions: batch culture in a medium containing 2 mM inorganic phosphate and continuous culture with 0.1 mM phosphate in the inflowing medium. The values found were 194±13 and 27.5±1.1 mol/g dry weight, respectively. Computer modeling indicated that the pyrophosphate group cannot adopt a staggered conformation.     

AT-1 Receptor and Phospholipase C Are Involved in Angiotensin III Modulation of Hypothalamic Noradrenergic Transmission

SUMMARY 1. We previously reported that angiotensin III modulates noradrenergic neurotransmission in the hypothalamus of the rat. In the present work we studied the effects of angiotensin III on norepinephrine release and tyrosine hydroxylase activity. We also investigated the receptors and intracellular pathways involved in angiotensin III modulation of noradrenergic transmission.2. In rat hypothalamic tissue labeled with [³H]norepinephrine 1, 10, and 100 nM and 1 M losartan (AT1 receptor antagonist) had no effect on basal neuronal norepinephrine release, whereas 10 and 100 nM and 1 M losartan partially diminished norepinephrine secretion evoked by 25 mM KCl. The AT2 receptor antagonist PD 123319 showed no effect either on basal or evoked norepinephrine release. The increase in both basal and evoked norepinephrine output induced by 1 M angiotensin III was blocked by 1 M losartan, but not by 1 M PD 123319.3. The phospholipase C inhibitor 5 M neomicin inhibited the increase in basal and evoked norepinephrine release produced by 1 M angiotensin III.4. Tyrosine hydroxylase activity was increased by 1 M angiotensin III and this effect was blocked by 1 M LST and 5 M neomicin, but not by PD 123319. On the other hand, 1 M angiotensin III enhanced phosphatidyl inositol hydrolysis that was blocked by 1 M losartan and 5 M neomicin. PD 123319 (1 M) did not affect ANG III-induced phosphatidyl inositol hydrolysis enhancement.5. Our results confirm that angiotensin III acts as a modulator of noradrenergic transmission at the hypothalamic level through the AT1-phospholipase C pathway. This enhancement of hypothalamic noradrenergic activity suggests that angiotensin III may act as a central modulator of several biological processes regulated at this level by catecholamines, such as cardiovascular, endocrine, and autonomic functions as well as water and saline homeostasis.     

A convenient synthesis of lubeluzole and its enantiomer: Evaluation as chemosensitizing agents on human ovarian adenocarcinoma and lung carcinoma cells

Lubeluzole, a neuroprotective anti-ischemic drug, and its enantiomer were prepared following a convenient procedure based on hydrolytic kinetic resolution. The ee values were >99% and 96%, respectively, as assessed by HPLC analysis. The chemosensitizing effects of both enantiomers were evaluated in combination with either doxorubicin (human ovarian adenocarcinoma A2780 cells) or paclitaxel (human lung carcinoma A549 cells) by the MTT assay. At the lowest concentrations used, lubeluzole showed an overall and remarkable tendency to synergize with both anticancer drugs. In ovarian cancer cells a clear prevalence of antagonistic effect was observed for the R-enantiomer. The synergistic effects of lubeluzole for both drugs were observed over a wide concentration window (0.005–5 μM), the lowest limit being at least 40 times lower than human plasma concentrations previously reported as causing serious side effects.     

The BCL-xL and ACR-1 Genes Promote Differentiation and Reduce Apoptosis in Muscle Fibers of mdx Mice

The effects of the human BCL-xL and ACR-1genes on dystrophin expression in cross-striated muscle fibers (CSMF) and on CSMF viability were studied in mdx mice after ballistic cotransfection with the human dystrophin minigene. In control mice, the proportion of dystrophin-positive (D(+)) and dying CSMF were 2.1 ± 0.1 and 2.1 ± 0.3%, respectively. Introduction of the dystrophin minigene (20 g of the pSG5dys plasmid) increased the proportions of D(+) and dying CSMF to 5.6 ± 1.4% and 4.5 ± 0.9%, respectively. When pSG5dys was introduced along with the pSFFV-Neo plasmid carrying the BCL-xL gene (10 g of each plasmid per shot), the death of CSMF decreased to 3.7 ± 1% and the proportion of D(+) CSMF significantly (P < 0.05) increased to 12.2 ± 2.2%. Cotransfection with the dystrophin minigene and the BCL-xL gene at 20 g of each plasmid per shot did not stimulate generation of D(+) CSMF, but did reduce the CSMF death to 1.5 ± 0.3%. Introduction of pSG5dys along with the pRc-CMV-10.1 plasmid containing the ACR-1 gene (10 g of each plasmid per shot) reduced the proportion of D(+) CSMF to 1.1 ± 0.5% and significantly reduced the proportion of dying CSMF to 0.9 ± 0.3% as compared with the proportions observed in intact mice or in mice subjected to transfection with pSG5dys. Introduction of the pSG5dys plasmid substantially reduced the proportion of CSMF with peripheral nuclei, suggesting disturbed CSMF differentiation. After cotransfection with the human dystrophin minigene, the BCL-xL and ACR-1 genes did not affect the extent of CSMF differentiation as compared with that observed in the case of the dystrophin minigene alone. Thus, ballistic transfection of mdx mice with the human dystrophin gene used along with the BCL-xLor ACR-1 gene was shown to suppress the death of muscle fibers and to expedite dystrophin synthesis and cell differentiation.     

Mechanism of sugar retention by roots of intact maize and field bean plants

The re-uptake of sugars driven by the proton gradient was studied in sugar net-release and net-uptake experiments using roots of intact maize (Zea mays cv. Blizzard) and field bean (Vicia faba L. cv. Alfred) plants. The net release of sugars into the root medium (0.1 mM CaSO₄) was stimulated by: the protonophore CCCP (10 M); the sulfhydryl reagent NEM (300 M); the specific inhibitor of plasmalemma ATPase vanadate (0.5 mM); and the inhibitor of the glucose carrier phlorizin (2 mM). Net uptake of glucose, fructose and arabinose from 10 M external concentrations was also inhibited by these substances. Surprisingly fusicoccin, a stimulator of net proton release did not effect net sugar uptake. Medium pH values only influenced sugar net uptake if the pH was above 7. It is concluded that a degradation of the proton gradient across the plasmalemma stimulates net sugar release because of disturbed re-uptake of sugars (in particular glucose) via a proton/sugar cotransport system. Thus, the retention of sugars by root cells not only depends on the plasmalemma permeability but also on the electro-chemical proton gradient. If an electro-chemical proton gradient is established by plasmalemma ATPase activity the re-uptake of sugars by proton/sugar cotransport minimizes the release of sugars into the rhizosphere.     

Effects of growth period,plant age and changes in solution aluminium concentrations on aluminium toxicity in wheat

The effects of growth period (time between transplanting and harvesting), plant age at which aluminium (Al) was added to solution, changes in Al concentration, and solution culture techniques (monitoring and adjusting solution Al concentrations thrice weekly or weekly replacement of the solutions) were investigated using a low ionic strength (2.7×10^–3 M) solution culture technique. The wheat (Triticum aestivum L.) cultivars Waalt (Al-tolerant) and Warigal (Al-sensitive), or the near isogenic lines bred from these cultivars (RR for the Al-tolerant line and SS for the Al-sensitive line) were grown. In all experiments and treatments, Al additions were required to maintain the nominal concentration. The decline in solution Al concentrations was partially attributed to formation of an Al-hydroxy-phosphate precipitate with an Al:P molar ratio of 2.8 to 4.0. Increasing the growth period from 14 to 28 days increased Al sensitivity in Warigal but not in Waalt. When plants were exposed to Al for the same time, increasing the age of the plants that Al was added to solution decreased sensitivity to Al. Differential Al tolerance between the two lines was evident when solutions were monitored thrice weekly or replaced weekly. However, the Al concentration required to reduce relative yield by a given amount when the solutions were replaced weekly was about twice that when the solutions were monitored. With a constant growth period of 28 days, increasing solution Al concentrations for 3 or more days resulted in decreased yields at harvest. The exact effect depended on the cultivar, plant part (tops or roots), when solution Al concentrations were increased and the duration of the increase. For example, increasing Al concentrations from 5 M to 20 M for 10 days reduced yield in the RR line by approximately 50% in the tops and 30% in the roots beyond the effect of 5 M but had no effect in the SS line due to yields already being low at 5 M. Adding 10 M Al to solution for 6 days at the beginning of the experiment reduced yield by 25% in the RR line and 50% in the SS line. In contrast, adding 10 M Al for 6 days in the middle of the growth cycle had no effect on the RR line but reduced yield by approximately 25% in the SS line. These results show that growth period, the age of the plants at which Al is added and the technique used (monitored or weekly replacement) all need to be considered when comparing results from different experiments. These results also show that the Al concentrations in solution need to be regularly monitored in long term experiments.