Biological activities of the natural imidazole-containing peptidomimetics n-acetylcarnosine, carcinine and L-carnosine in ophthalmic and skin care products

Apart from genetically programmed cell aging, different external aggressors related to oxidative stress and lipid peroxidation (LPO) can accelerate the skin aging phenomenon. Oxidative stress associated with the formation of lipid peroxides is suggested to contribute to pathological processes in aging and systemic diseases known as the risk factors for cataract. Despite the fact that L-carnosine-related peptidomimetics N-acetylcarnosine (N-acetyl-beta-alanyl-L-histidine) (NAC) and carcinine (beta-alanylhistamine) are metabolically related to L-carnosine and have been demonstrated to occur in tissues of many vertebrates, including humans, these compounds were shown resistant toward enzymatic hydrolysis. A series of related biocompatible imidazole-containing peptidomimetics were synthesized in order to confer resistance to enzymatic hydrolysis and ex vivo improvement of protective antioxidative properties related to L-carnosine. The included findings revealed a greater role of N-acetylcarnosine (NAC) and carcinine ex vivo in the prolongation and potentiation of physiological responses to the therapeutical and cosmetics treatments with L-carnosine as antioxidant. 3-D molecular conformation studies proposed the antioxidant activity of peptidomimetics (carcinine, L-prolylhistamine, N-acetylcarnosine, L-carnosine) for metal ion binding, quenching of a number free radicals, and binding of hydroperoxide or aldehyde (including dialdehyde LPO products) in an imidazole-peroxide adducts. NAC can act as a time release (carrier) stable version of L-carnosine during application in ophthalmic pharmaceutical and cosmetics formulations which include lubricants. Carcinine, L-prolylhistamine show efficient deactivation of lipid hydroperoxides monitored by HPLC and protection of membrane phospholipids and water soluble proteins from the lipid peroxides-induced damages. This activity is superior over the lipophilic antioxidant vitamin E. The biologically significant applications of carnosine mimetics were patented by Dr. Babizhayev and the alliance Groups (WO 2004/028536 A1; WO 94/19325; WO 95/12581; WO 2004/064866 A1).     

cAMP-Dependent protein kinase inhibits the chloride conductance in apical membrane vesicles of hunman placenta

Summary The role of adenosine 3,5-monophosphate (cAMP) dependent protein kinase (PK-A) on the Cl^– conductance has been studied in the apical membrane vesicles purified from the chorionic villi of human placenta. In order to phosphorylate the cytosolic side of the membranes, vesicles have been hypotonically lysed, loaded with 100nm catalytic subunit of PK-A purified from human placenta and 1mm of the phosphatase resistant adenosine 5-thiotriphosphate (ATP-gamma-S) and resealed. Cl^– conductance has been measured by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) at 23°C with membrane potential clamped at 0 mV. The actual volume of the resealed vesicles was measured in each experiment by trapping an impermeable radioactive molecule ([¹⁴C]-sucrose) and included in each Cl^– flux calculation. In 19 independent experiments, the mean Cl^– conductance in placental membranes in the absence of phosphorylation was 3.67±3.18 whereas with the addition of PK-A and ATP-gamma-S it was 1.97±1.75 nmol·sec^–1·(mg protein)^–1 (mean±sd). PK-A dependent phosphorylation reduced the Cl^– conductance in 14/19 experiments. The same protocol applied to the apical membranes of bovine trachea, where PK-A is known to activate the Cl^– channels, confirmed that the PK-A dependent phosphorylation increased the Cl^– conductance in 11/13 experiments, from 1.01±0.61 to 1.85±0.99 nmol·sec^–1·(mg protein)^–1(mean±sd). These studies indicate that the PK-A dependent phosphorylation inhibits one or more Cl^– channel(s) of the apical membranes of human placenta.     

Opioid activity of synthetic deltorphin II analogues and their spin labeled derivatives

Deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH₂, Del II), an endogenous linear heptapeptide, is a highly selective agonist of the -opioid receptor. To study the effect of the position 4 residue (Glu) on the opioid activity of Del II, we designed and synthesized three analogues of Del II by solid-phase peptide synthesis. They were [Val⁴,Glu⁵]Del II, [Val⁴,Glu⁶]Del II and [Gly⁴,Glu⁷]Del II. To study the effect of spin labeling on peptide bioactivities, all the peptides were labeled using a free radical. The labeling material was a stable nitrogen–oxygen free radical which was linked to the N-terminal via an amide bond. We investigated the opioid bioactivities of these analogues both in vivo and in vitro, and concluded that the differences in opioid activity of Del II and its analogues were due to structural differences. When the Glu residue is at position 5 or 6, the internal hydrogen bonds in Del II are affected and there is a change in three-dimensional structure and opioid activity. The antinociceptive activity of all the peptides decreased after spin labeling. This indicates that the stable nitrogen–oxygen free radical is a dual-function spin-labeling molecule.     

In vivo real-time measurement of superoxide anion radical with a novel electrochemical sensor

The dynamics of superoxide anion (O₂⁻) in vivo remain to be clarified because no appropriate method exists to directly and continuously monitor and evaluate O₂⁻ in vivo. Here, we establish an in vivo method using a novel electrochemical O₂⁻ sensor. O₂⁻ generated is measured as a current and evaluated as a quantified partial value of electricity (Q_part), which is calculated by integration of the difference between the baseline and the actual reacted current. The accuracy and efficacy of this method were confirmed by dose-dependent O₂⁻ generation in xanthine–xanthine oxidase in vitro in phosphate-buffered saline and human blood. It was then applied to endotoxemic rats in vivo. O₂⁻ current began to increase 1 h after lipopolysaccharide, and Q_part increased significantly for 6 h in endotoxemic rats, in comparison to sham-treated rats. These values were attenuated by superoxide dismutase. The generation and attenuation of O₂⁻ were indirectly confirmed by plasma lipid peroxidation with malondialdehyde, endothelial injury with soluble intercellular adhesion molecule-1, and microcirculatory dysfunction. This is a novel method for measuring O₂⁻ in vivo and could be used to monitor and treat the pathophysiology caused by excessive O₂⁻ generation in animals and humans.     

Electron microscopic studies on the polytene chromosomes of Chironomus thummi salivary glands

The method of ultrathin sections of unsquashed salivary gland polytene chromosomes of Ch. thummi was applied to their ultrastructural mapping. There was a good agreement between electron micrographs and Hägele's light microscopic map (1970) with respect to the pattern and number of bands. 94% of bands were identified in larval and prepupal chromosomes. In Ch. thummi, band thickness varied from 0.05–0.5 m. Most characteristic were 0.2–0.3 m bands. Morphologically, bands were classified as: continuous (frequently with holes and gaps), discrete, dotted and continuous-discrete, discrete-dotted.Band morphology is related to band size, such that smaller bands, as a rule, were also dotted. Bands beginning to puff likewise became dotted. Interbands in unsquashed chromosome sections were from 0.05–0.15 m. The smallest interbands contained only fibrils, in the larger interbands few granules could be observed. This makes interbands distinguishable from a typical puff with many such granules.     

Evaluation of antioxidant activity of epigallocatechin gallate in biphasic model systems in vitro

The antioxidant activity of epigallocatechin gallate (EGCG) was studied in different in vitro model systems, which enabled evaluation of both chemical and physical factors involved in assessing the role of EGCG in oxidative reactions. EGCG suppressed the initiation rate and prolonged the lag phase duration of peroxyl radical-induced oxidation in a phospholipid liposome model to a greater extent (p < 0.01) compared to both Trolox and -tocopherol. Effectiveness of these antioxidants to prolong the peroxyl radical-induced lag phase was inversely related to lipophilic character. EGCG also protected against both peroxyl radical and hydroxyl radical-induced supercoiled DNA nicking. The rate constant describing EGCG reaction against hydroxyl radical was 4.22 ± 0.07 × 10¹⁰ M^–1·sec^–1, which was comparable to those of Trolox and -tocopherol, respectively. EGCG exhibited a synergistic effect with -tocopherol in scavenging 1,1-diphenyl-2-picylhydrazyl (DPPH) radical, thus displaying a direct free radical scavenging capacity. In vitro Cu²⁺-induced-human LDL oxidation was accelerated in the presence of EGCG and attributed to the conversion of Cu²⁺ to Cu⁺. We conclude that the particularly effective antioxidant properties of EGCG noted in both chemical and biological biphasic systems were related to a unique hydrophilic and lipophilic balance which enabled effective free radical scavenging. The same chemical-physical properties of EGCG also enabled prooxidant activity, only when in contact with unbound transition metal ions in a multiphasic system.     

The lipid composition,fluidity, and Mg2+-ATPase activity of rice (Oryza sativa L. cv. Bahia) shoot plasma membranes: effects of ABA and GA3

Six-day-old rice plants (Oryza sativa L., cv Bahia) were grown for 5 days more in nutrient solution culture containing 10^–5 M abscisic acid (ABA) or gibberellic acid (GA₃) (treated plants). Plasma membrane (PM) vesicles were isolated from the shoots of treated or control plants, and ATPase hydrolytic and proton-pumping activity, fluidity, and free sterol and phospholipid composition were determined. Both treatments resulted in modified plant growth and increases in PM fluidity. The ATPase hydrolytic activity was decreased by 25% of control values with ABA treatment and by 35% with GA₃. Both treatments reduced proton-pumping by 23%. GA₃ treatment reduced the relative amount (%) of stigmasterol in the PM from 38–34%, and increased ⁵-avenasterol from 4–7%. ABA and GA₃ did affect the percentage composition of the fatty acyl chains of phosphatidylinositol (PI). GA₃ treatment also resulted in a reduction of the total amount of PI from 73–37 mg mg^–1 protein, as well as a decrease in the overall PM C₁₆/C₁₈ ratio and an increase in the degree of unsaturation. Our results show that there is no common link between a specific change in lipid composition and fluidity in these membranes. On the other hand, the reduction in Mg²⁺-ATPase activity found with both hormones suggests that there is no relationship between membrane-bound enzyme activity and membrane fluidity.     

Micropropagation of Limonium cavanillesii Erben, a threatened statice, from inflorescence stems

Micropropagation of Limonium cavanillesii Erben, a threatened and endemic statice species from Valencia Community (Eastern Spain), was successfully achieved using inflorescence stem pieces as initial explants. Segments 20 mm long from basal parts of immature inflorescences and with axillary buds were cut, sterilised and established in vitro. Shoots obtained from indifferentiated buds were sectioned and then transferred to Murashige and Skoog (MS) medium with 2 mg l^–1 kinetin to provide a plant stock.Shoot multiplication was achieved on MS medium with different cytokinins. The best results for shoot formation were obtained with 2–5 mg l^–1kinetin, 5 mg l^–1 6---dimethylallylaminopurine or 0.1 mg l^–1 6-benzylaminopurine, without significant differences between them. High shoot rooting (80–85%) was obtained within four weeks with indolebutyric acid or indoleacetic acid (0.1 or 0.5 mg l^–1), and also on medium without plant growth regulators. Plant survival to hardened greenhouse conditions was 90% four weeks after plantlet removal from in vitro conditions.This protocol for micropropagation of Limonium cavanillesii is very useful for conservation purposes of endangered statice species, because by using inflorescence stem as initial material it is easier to establish aseptic cultures while preserving the mother plant.     

Effect of β-alanyl-L-histidinato zinc on differentiation of osteoblastic MC3T3-El cells: Increases in alkaline phosphatase activity and protein concentration

The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10^–7–10^–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10^–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10^–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10^–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells.     

The Antioxidant Vitamin E Modulates Amyloid β-Peptide-Induced Creatine Kinase Activity Inhibition and Increased Protein Oxidation: Implications for the Free Radical Hypothesis of Alzheimer's Disease

Amyloid -peptide (A), the main constituent of senile plaques in Alzheimer's disease (AD) brain, is hypothesized to be a key factor in the neurodegeneration seen in AD. Recently it has been shown by us and others that the neurotoxicity of A occurs in conjunction with free radical oxidative stress associated with the peptide. A(1–40) and several other fragments of the A sequence are associated with free radicals in solution that are detectable using electron paramagnetic resonance spectroscopy. These free radicals were shown to attack brain cell membranes, initiate lipid peroxidation, increase Ca²⁺ influx and damage membrane and cytosolic proteins. In AD brain obtained under rapid autopsy protocol, the activity of the oxidatively-sensitive enzyme creatine kinase was shown to be significantly reduced. We reasoned that A-associated free radical-induced modification of creatine kinase activity and other markers of cellular damage might be modulated by free radical scavengers. Accordingly, this study demonstrates that vitamin E can modulate A(25–35)-induced oxidative damage to creatine kinase and cellular proteins in cultured embryonic hippocampal neurons. These results, consistent with the hypothesis of free radical-mediated A toxicity in AD, are discussed with deference to potential free radical scavengers as therapeutic agents for slowing the progression of AD.     

Uncoupling of mitochondrial oxidative phosphorylation alters lipid peroxidation-derived free radical production but not recovery of postischemic rat hearts and post-hypoxic endothelial cells

The contribution of mitochondrial free radical production towards the initiation of lipid peroxidation (LPO) and functional injury in the post-ischemic heart is unclear. Using the isolated rat heart model, the effects of the uncoupler of mitochondrial oxidative phosphorylation dinitrophenol (DNP, 50 M final) on post-ischemic lipid peroxidation-derived free radical production and functional recovery were assessed. Hearts were subjected to 30 min total global ischemia followed by 15 min of reperfusion in the presence of DNP. As expected, DNP enhanced oxygen consumption before (11.3 ± 0.9 mol/min, p < 0.001) and during reperfusion (at 10 min: 7.9 ± 0.7 umol/min), compared to the heart with control treatment (8.2 ± 0.5 and 6.7 ± 0.3, respectively). This effect was only associated with a higher incidence of ventricular tachycardia during reperfusion (80 vs. 50% for control treatment, p < 0.05). Electron spin resonance spectroscopy (ESR) and spin trapping with u.-phenyl-tert-butylnitrone (PBN, 3 mM final) were used to monitor free radical generation during reperfusion. The vascular concentration of PBN-radical adducts (untreated: 6.4 ±1.0 nM, at 10 min) decreased in the presence of DNP (1.7 ± 0.4 nM, p < 0.01). The radical concentration inversely correlated with myocardial oxygen consumption. Total liberation of free radical adducts during the initial 10 min of reperfusion was reduced by DNP (0.59 ± 0.09 nmol, p < 0.01) compared to the respective control treatment (1.26 ± 0.16 nmol). Similar effects, prevention of PBN adduct formation and unchanged viability in the presence of DNP, were obtained with endothelial cells during post-hypoxic reoxygenation. Since inhibition of mitochondrial phosphorylation can inhibit the formation of LPO-derived free radicals after an ischemic/hypoxic interval, mitochondria may represent an important source of free radicals capable of initiating lipid peroxidative injury during reperfusion/reoxygenation. (Mol Cell Biochem 160/161: 167–177, 1996)     

Zingerone [4-(4-hydroxy-3-methoxyphenyl)-2-butanone] Prevents 6-Hydroxydopamine-induced Dopamine Depression in Mouse Striatum and Increases Superoxide Scavenging Activity in Serum

As superoxide (·O₂^–) and hydroxyl radical (·OH) have been implicated in pathogenesis of Parkinsons disease, free radical scavenging, antioxidant, and neuroprotective agents have attracted attention as ways to prevent progression. We examined effects of zingerone, an alkaloid extracted from ginger root, on 6-hydroxydopamine (6-OHDA)-induced dopamine (DA) reduction in mouse striatum. Zingerone administration 1 h before and for 6 more days following one intracerebroventricular 6-OHDA injection prevented reductions of striatal DA and its metabolites, and increased serum ·O₂^– scavenging activity. Zingerone did not change activities of catalase or glutathione peroxidase in striatum or serum, or ·O₂^– scavenging activity in striatum. Treatment with diethyldithiocarbamate, SOD inhibitor, abolished the protective effect of zingerone against 6-OHDA-induced DA reduction. In vitro, zingerone scavenged ·O₂^– and ·OH and suppressed lipid peroxidation only weakly. Thus, direct antioxidant effects may be a minor component of its putative neuroprotective effect; instead, zingerone acted mainly by increasing systemic superoxide dismutase activity. Effects of zingerone treatment in this model suggest possible value in treatment of Parkinsons disease.     

Subcellular distribution of glycoside hydrolase and polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum

The distribution of 12 acid hydrolase and two polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum, isolated from the ovine rumen 2 h after feeding, was examined by differential and density-gradient centrifugation. Approximately 60%–70% of the recovered activity was sedimentable in fractions prepared by centrifugation at 10³ g for 10 min (F1) and 10⁴ g for 10 min (F2) with 25%–35% of the acid hydrolases and 15%–20% of acid phosphatase and the polysaccharidases remaining nonsedimentable (in fraction F5) after centrifugation at 10⁵ g for 60 min. Approximately 60% of the sedimentable activity was located in fraction F1. Latency of the hydrolase activity was demonstrated. After isopycnic centrifugation in sucrose density gradients, the hydrolytic enzymes cosedimented in acid phosphatase-containing, membrane-bound, pleomorphic lysosomelike vesicles 0.1–1.0 m in size, with a mean equilibrium density of 1.17 (1.15–1.19) g/ml.     

Protein and lipid content of the rotifer Brachionus plicatilis during variable growth and feeding condition

Rotifers (Brachionus plicatilis) grown atdifferent growth rate ( = 0.05–0.39 d^–1)were analyzed for protein, lipid, fatty acids, aminoacids and free amino acids, and values are expressedin terms of individuals and dry weight. Increase ingrowth rate is equivalent with increased food rationof the individual rotifer, which responded by higheregg ratio. The protein content per individualrotifer increased by 60–80% when the growth rate increased, whereas the protein content per dryweight showed a slight, although insignificant,increase (p > 0.05). The lipid content perindividual was constant, whereas lipid per dryweight decreased when the growth rate increased. Theratio DHA/EPA decreased when the growth ratesincreased. The amino acids profile in percent oftotal amino acids showed low variation betweencultures maintained at different growth rates,whereas the values expressed in terms of amino acidper individual showed higher variation. The range ofvariation for free amino acids was more pronouncedthan for total amino acids.Short-term food enrichment of poorly fed rotifers( = 0.05 d^–1) with balanced protein richdiet resulted in increased protein and lipid contentper rotifer. The protein content per dry weightshowed only minor changes whereas lipid per dryweight increased. Contrary, short term enrichmentwith a lipid rich diet resulted in increased lipidcontent per individual rotifer and per dry weight,whereas the protein content per individual remainedconstant and the protein content per dry weightshowed a slight decrease.Our experiments show that the amount of protein, wasquite variable in rotifers, and that feeding andgrowth condition were decisive factors affecting it.The range of variation was large enough to be animportant factor during first feeding of marinelarvae, and should therefore be considered infeeding larvae.     

Regulation of Free Radical Processes by Delta-Sleep Inducing Peptide in Rat Tissues under Cold Stress

An intraperitoneal injection of an exogenous delta-sleep inducing peptide (DSIP) at a dose of 12 g/100 g body weight shifted the prooxidant–antioxidant balance of free radical process (FRP) in tissues and erythrocytes of rats: the activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) and the concentrations of antioxidants (reduced glutathione in particular) increased. The DSIP stimulated the myeloperoxidase activity in blood neutrophils and had no effect on the activity of xanthine oxidase, a prooxidant enzyme, in the brain and liver. Cold stress displaced the prooxidant–antioxidant balance by increasing the xanthine oxidase activity in tissues and decreasing the myeloperoxidase activity in blood neutrophils; it also inhibited the enzyme antioxidant activities in tissues and erythrocytes that was neutralized by an increased ceruloplasmin activity in blood plasma and by an elevated level of antioxidants in rat blood and tissues. Preliminary administration of DSIP to animals exposed to cold stress restored the prooxidant–antioxidant balance: it normalized the myeloperoxidase activity in blood neutrophils, decreased the xanthine oxidase activity, and increased the activity of antioxidant enzymes in tissues and erythrocytes restoring the antioxidant level. The molecular regulation mechanism of free radical processes by DSIP in tissues under stressful conditions is discussed.     

Family 19 chitinase from rice (Oryza sativa L.): substrate-binding subsites demonstrated by kinetic and molecular modeling studies

A family 19 chitinase (OsChia1c, class I) from rice, Oryza sativa L., and its chitin-binding domain-truncated mutant (OsChia1cCBD, class II) were produced by the Pichia expression system, and the hydrolytic mechanism toward N-acetylglucosamine hexasaccharide [(GlcNAc)₆] was investigated by HPLC analysis of the reaction products. The profile of the time-course of (GlcNAc)₆ degradation obtained by OsChia1c was identical to that obtained by OsChia1cCBD, indicating that the chitin-binding domain does not significantly participate in oligosaccharide hydrolysis. From the theoretical analysis of the reaction time-course of OsChia1cCBD, the free energy changes of sugar residue binding were estimated to be –0.4, –4.7, +3.4, –0.5, –2.3, and –1.0 kcal/mol for the individual subsites of (–3), (–2), (–1), (+1), (+2), and (+3), respectively. The hexasaccharide substrate appears to bind to the enzyme through interactions at the high-affinity sites, (–2) and (+2), and the sugar residues at both ends more loosely bind to the corresponding subsites, (–3) and (+3). The docking study of (GlcNAc)₆ with the modeled structure of OsChia1cCBD supported the subsite structure estimated from the experimental time-course of hexasaccharide degradation. Since the class II chitinase from barley seeds was reported to possess a similar subsite structure from (–3) to (+3) and a similar free energy distribution, substrate-binding mode of plant chitinases of this class would be similar to each other.     

Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice,Spitzbergen

The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4°C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses and of Proteobacteria, the Bacillus–Clostridium group, the order Actinomycetales, and the Cytophaga–Flexibacter–Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25°C. Only a few strains were psychrophilic, with an optimal growth at 10–15°C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4°C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, -amylase and -galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10°C, and almost no production was detected at higher temperatures (20–30°C). Catalytic activity was detected even below the freezing point of water (at –5°C), demonstrating the unique properties of these enzymes.     

Transformation of haploid, microspore-derived cell suspension protoplasts of rice (Oryza sativa L.)

We compared the transient activity of three cereal gene-derived promoter-gus fusions and the efficiency of selection mediated by three different selectable genes in a polyethylene glycol transformation system with haploid cell suspension protoplasts of rice. The maize ubiquitin promoter was found to be the most active in transformed protoplasts, and selection on ammonium glufosinate mediated by the bar gene was the most efficient for producing resistant calluses. Cotransformation of protoplasts with two separate plasmids carrying the gus and the bar genes, at either a 21 or 11 ratio, led to 0.8 × 10^–5 and 1.6 × 10^–5 resistant callus recovery frequencies and 59.7 and 37.9 cotransformation efficiencies respectively. No escapes were detected in dot blot analyses of 100 resistant calluses with a probe consisting of the bar coding region. Cotransformation efficiency, based on resistance to basta and -glucuronidase staining of the leaf tissue of 115 regenerated plants, was 47%. Resistance tests and Southern analysis of seed progenies of three diploid transgenic plants demonstrated homozygous integration of multiple copies of the transgene at one locus at least in the first plant, heterozygous integration at one locus in the second plant and heterozygous integration at two loci in the third plant.Abbreviations PEG polyethylene glycol - T₀ regenerated transgenic plant - GUS -glucuronidase - CaMV cauliflower mosaic virus - ARE anaerobic responsive element - OCS octopine synthase - T₁ first generation progeny of transgenic plants     

The influence of the cation of quaternized chitosans on antioxidant activity

Various quaternized chitosans (QCSs) were synthesized according to previous method. Their reducing power and antioxidant potency against hydroxyl radicals (OH) and hydrogen peroxide (H₂O₂) were explored by the established systems in vitro. The QCSs exhibited markedly antioxidant activity, especially TCEDMCS, whose IC₅₀ on hydroxyl radicals was 0.235 mg/mL. They showed 65–80% scavenging effect on hydrogen peroxide at a dose of 0.5 mg/mL. Generally, the antioxidant activity decreased in the order TCEDMCS > TBEDMCS > EDMCS > PDMCS > IBDMCS > Chitosan. Furthermore, the order of their OH and H₂O₂ scavenging activity was consistent with the electronegativity of different substituted groups in the QCSs. The QCSs showed much stronger antioxidant activity than that of chitosan may be due to the positive charge density of the nitrogen atoms in QCSs strengthened by the substituted groups.     

Impermeant potential-sensitive oxonol dyes: III. The dependence of the absorption signal on membrane potential

Summary We have measured potential-dependent changes in the absorption of light by oxidized cholesterol bilayer lipid membranes in the presence of impermeant oxonol dyes. The magnitude of the absorption signal increased linearly with the size of potential steps over a range of 500 mV. The signal also increased when the offset voltage of the pulse train was increased from –150 to +150 mV. The data are consistent with the on-off mechanism proposed by E. B. George et al. (J. Membrane Biol.103:245–253, 1988) in which the probe undergoes potential-dependent movement between a binding site in the membrane and an aqueous region just off the surface of the membrane. An equilibrium thermodynamic analysis of the experimental data indicates that the negatively charged oxonol chromophore senses only 5–10% of the total membrane potential difference across the membrane when it is driven into a nonpolar binding site on the membrane.ASW, Carnegie-Mellon University