Ether lipid transfer across the blood-brain and placental barriers does not improve by inactivation of the most abundant ABC transporters.

Phospholipid transport from the periphery to the brain is an understudied topic. When certain lipid species are deficient due to impaired synthesis, though, transfer across the blood-brain barrier is essential for replenishing lipids in the brain. For example, the deficiency in plasmalogens, the most abundant ether lipids in mammals, has detrimental effects on the brain, which is a major issue in inherited peroxisomal disorders but also contributes to more common disorders like Alzheimer's disease. Oral administration of alkylglycerols like batyl alcohol, which carry a pre-formed ether bond, enables replenishment of ether lipids in various peripheral tissues. However, plasmalogen deficiency in the brain cannot be overcome by this approach. Here, we tried to increase cerebral plasmalogen uptake by modulating the efflux transport across the blood-brain barrier. We hypothesized, based on previous literature, that at least some ether lipid species readily enter endothelial cells of the barrier through the transporter MFSD2A but are re-exported by ATP-binding cassette (ABC) transporters. By crossbreeding Mdr1a-/-/Mdr1b-/-/Bcrp-/- and ether lipid-deficient Gnpat-/- mice as well as pharmacological inhibition with MK-571 to inactivate the major ABC transporters at the blood-brain barrier, we evaluated the potential of combined ABC transporter inhibition and oral batyl alcohol administration for the treatment of plasmalogen deficiency. We found that even in the absence of the most abundant ABC transporters, batyl alcohol supplementation did not restore plasmalogen levels in the brain, despite the presence of a wide spectrum of ether lipid subspecies in the plasma as demonstrated by lipidomic analysis. Surprisingly, batyl alcohol treatment of pregnant Gnpat+/- dams had beneficial effects on the plasmalogen levels of Gnpat-/- offspring with defective ether lipid biosynthesis, independently of ABC transporter status at the placental barrier. Our results underline the autonomy of brain lipid homeostasis and indicate that peripheral supplementation of ether lipids is not sufficient to supply the brain with larger amounts of plasmalogens. Yet, the findings suggest that alkylglycerol treatment during pregnancy may pose a viable option to ameliorate some of the severe developmental defects of inborn ether lipid deficiency.

Millifolide A, a dimeric ether of degraded sesquiterpene lactones, inhibited the proliferation of human lung cancer cell line A549.

The inhibitory effect of three degraded sesquiterpene lactones, iso-seco-tanapartholide, arteludooicinolide A and millifolide A isolated from Achillea millefolium L., on anti-human lung cancer cells was examined using MTT and reporter gene assays. Millifolide A has significant inhibitory effects on the proliferation of human lung cancer cells probably through inducing cell apoptosis.

Antiglycoxidative Properties of Extracts and Fractions from Reynoutria Rhizomes.

Hyperglycemia, when sustained over a long time in diabetes mellitus (DM), leads to biochemical and cellular abnormalities, primarily through the formation of advanced glycation end-products (AGEs). In the treatment of diabetes, beside blood-sugar-lowering medications, a consumption of herbal products that can inhibit the AGEs' formation is recommended. This study investigated the in vitro antiglycoxidative potential of extracts and fractions from the rhizomes of Japanese, Giant, and Bohemian knotweeds (Reynoutria japonica (Houtt.), R. sachalinensis (F. Schmidt) Nakai, and R.× bohemica Chrtek et Chrtkova). Their effects on glycooxidation of bovine and human serum albumin were evaluated by incubation of the proteins with a mixture of glucose and fructose (0.5 M) and 150 µg/mL of extract for 28 days at 37 °C, followed by measuring early and late glycation products, albumin oxidation (carbonyl and free thiol groups), and amyloid-ß aggregation (thioflavin T and Congo red assays). The highest antiglycoxidative activity, comparable or stronger than the reference drug (aminoguanidine), was observed for ethyl acetate and diethyl ether fractions, enriched in polyphenols (stilbenes, phenylpropanoid disaccharide esters, and free and oligomeric flavan-3-ols). In conclusion, the antiglycoxidative compounds from these three species should be further studied for potential use in the prevention and complementary treatment of DM.

Synthesis and Antiviral Bioassay of New Diphenyl Ether-based Compounds.

A set of diphenyl ether derivatives bearing different heterocycles were synthesized from 4-phenoxybenzohydrazide 1 in good yield. Synthesized compounds were screened against a broad panel of viruses in different cell cultures and some of the synthesized compounds showed promising antiviral properties.

Daucus carota Pentane/Diethyl Ether Fraction Inhibits Motility and Reduces Invasion of Cancer Cells.

Daucus carota (DC) is a herb used in folklore medicine in Lebanon to treat numerous diseases including cancer. Recent studies in our laboratory on DC oil and its fractions revealed potent anticancer activities in vitro and in vivo. The present study aims to investigate the effect of the most potent DC fraction, pentane/diethyl ether (50:50), on lung, skin, breast and glioblastoma cancer cell motility and invasion. Upon treatment, a pronounced decrease in cancer cell motility was observed in the 4 cell lines. The treatment also led to a decrease in cancer cell invasion and an increased cell adhesion. Additionally, the DC fraction caused a decrease in the activation of the ρ-GTPases Rac and CDC42, a finding that may partially explain the treatment-induced decrease in cell motility. The current study demonstrates a crucial effect of the DC pentane/diethyl ether fraction on cancer cell motility and metastasis, making it a potential candidate for cancer therapy specifically targeting cancer motility and metastasis.

Involvement of GABAergic non-benzodiazepine sites in the anxiolytic-like and sedative effects of the flavonoid baicalein in mice.

Baicalein (BA), one of the main flavonoids obtained from the Chinese medicinal herb Scutellaria baicalensis, usually exerts several pharmacological effects. In the central nervous system (CNS), BA exerts a protective effect on neurons against several neuronal insults among other effects, but it is not clear if this effect is due to its metabolite, baicalin. The purpose of the present study was to assess the anxiolytic-like and related properties of BA following its central administration (i.c.v.) in mice. BA (0.02, 0.2pmol) exerted an anxiolytic-like effect at low doses, increasing the time spent in open arms and the head-dipping whereas reducing the stretched-attend postures in the elevated plus-maze. BA also increased the duration of ether-induced sleep without affecting the pentylenetetrazol (PTZ)-induced convulsions. In addition, pretreatment with flumazenil (FMZ), PTZ, dehydroepiandrosterone sulfate (DHEAS), and dl-p-chlorophenilalanine ethyl ester (PCPA) were conducted in order to investigate its mechanism of action. PTZ and DHEAS, but not FMZ or PCPA, antagonized the BA's anxiolytic-like effect. Taken together our results showed that BA, when directly injected into the CNS, promotes anxiolytic-like and sedative effects, pharmacological activities dependent on GABAergic non-benzodiazepine sites but not on the 5-HT system.

Central nervous system activity of the proanthocyanidin-rich fraction obtained from Croton celtidifolius in rats.

OBJECTIVES: The aim of the present study was to evaluate the possible neurobehavioural effects in rats of the proanthocyanidin-rich fraction (PRF) isolated from the bark of Croton celtidifolius (Euphorbiaceae). METHODS: Adult Wistar rats were treated with the PRF (0.3-30 mg/kg) and evaluated in different behavioural paradigms classically used for the screening of drugs with psychoactive effects. KEY FINDINGS: Acute intraperitoneal (i.p.) administration of PRF decreased spontaneous locomotor activity (open field arena and activity cage), enhanced the duration of ethyl ether-induced hypnosis, increased the latency to the first convulsion induced by pentylenetetrazole (60 mg/kg, i.p.) and attenuated apomorphine-induced (0.5 mg/kg, i.p.) stereotyped behaviour. In lower doses, PRF (0.3 or 3 mg/kg, i.p.) increased the frequency of open arm entries in the elevated plus-maze test. CONCLUSIONS: The present findings suggest that the systemic administration of PRF induces a wide spectrum of behavioural alterations in rats, consistent with the putative existence of hypnosedative, anticonvulsant and anxiolytic compounds.

Techniques of celloidin removal from temporal bone sections.

OBJECTIVES: We sought to determine whether the technique of celloidin removal influences the results of immunostaining in celloidin-embedded cochleae. METHODS: We compared four protocols of celloidin removal, including those using clove oil, acetone, ether-alcohol, and methanol saturated with sodium hydroxide. By optimally fixing our tissue (perfused mice), and keeping constant the fixative type (formalin plus acetic acid), fixation time (25 hours), and decalcification time (ethylenediaminetetraacetic acid for 7 days), we determined whether the technique of celloidin removal influenced the immunostaining results. Six antibodies were used with each removal method: prostaglandin D synthase, sodium, potassium adenosine triphosphatase (Na+,K(+)-ATPase), aquaporin 1, connective tissue growth factor, tubulin, and 200 kd neurofilament. RESULTS: Clove oil, acetone, and ether-alcohol resulted in incomplete removal of the celloidin, thereby negatively affecting the results of immunostaining. The methanol-sodium hydroxide method was effective in completely removing the celloidin; it produced the cleanest and most reproducible immunostaining for all six antibodies. CONCLUSIONS: Freshly prepared methanol saturated with sodium hydroxide and diluted 1:2 with methanol was the best solvent for removing celloidin from mouse temporal bone sections, resulting in consistent and reproducible immunostaining with the six antibodies tested.

Extracts of Valeriana officinalis L. s.l. show anxiolytic and antidepressant effects but neither sedative nor myorelaxant properties.

Extracts of Valeriana officinalis L. s.l. are used for treating mild sleep disorders and nervous tension. Despite intensive research efforts, the pharmacological actions accounting for the clinical efficacy of valerian remain unclear. Thus, it was the aim of this study to evaluate CNS-related effects of different valerian extracts using behavioral paradigms (mice and rats). Following oral administration two commercially available preparations (extraction solvents: 45% methanol m/m and 70% ethanol v/v), a 35% ethanolic v/v extract and a refined extract derived from it (patented special extract phytofin Valerian 368) were tested for sedative (locomotor activity, ether-induced anaesthesia) and anxiolytic (elevated plus maze) activity. Using the forced swimming and the horizontal wire test the latter two extracts were additionally tested for antidepressant and myorelaxant properties. Up to maximum dosages of 500 or 1000 mg/kg bw none of the valerian extracts displayed sedative effects. Neither spontaneous activity was reduced nor the duration of ether-induced narcosis was prolonged. In contrast, results obtained in the elevated plus maze test revealed a pronounced anxiolytic effect of the 45% methanolic and 35% ethanolic extract as well as of phyotofin Valerian 368 in a dose range of 100-500 mg/kg bw. Additionally and different from its primary extract (35% ethanolic extract) phytofin Valerian 368 showed antidepressant activity in the forced swimming test after subacute treatment. Myorelaxant effects were not observed in dosages up to 1000 mg/kg bw. Due to these findings it is proposed that not sedative but anxiolytic and antidepressant activity, which was elaborated particularly in the special extract phytofin Valerian 368, considerably contribute to the sleep-enhancing properties of valerian.

Neuroendocrine profiling in inherited stress-induced arterial hypertension rat strain with stress-sensitive arterial hypertension.

The functions of the hypothalamic adrenal cortical and sympathetic adrenal medullary systems were studied in rats with inherited stress-induced arterial hypertension (ISIAH strain). A characteristic feature of the ISIAH strain is an increase in arterial blood pressure measured both under basal conditions and after restraint stress in particular. In the control ISIAH rats, the basal plasma ACTH concentration was slightly lower than that in the normotensive Wistar albino Glaxo (WAG) rats, and no differences were found in plasma corticosterone. However, the 0.5-h restraint stress produced higher activation of the adrenal cortex in the ISIAH rats. Gluco- and mineralocorticoid responses to the blood volume reduction stresses and ACTH and corticosterone responses to social stress were stronger in the ISIAH than in the control WAG rats. An increase in epinephrine content in adrenals in the basal state and enhanced response of the sympathetic adrenal medullary system to handling stress were observed in the ISIAH rats. Restraint stress produced significantly higher expression of genes encoding corticotropin-releasing hormone-mRNA in hypothalamus and proopiomelanocortin-mRNA in pituitary in the ISIAH than in the WAG rats. Restraint stress produced a decrease in glucocorticoid receptor (GR) gene expression (GR-mRNA) in hippocampus in the ISIAH, but not in the WAG rats. A persistent increase in tyrosine hydroxylase-mRNA in adrenals of the ISIAH rats was found. It is concluded that the ISIAH rat strain is an appropriate model of stress-sensitive hypertension with the predominant involvement of the hypothalamic adrenal cortical and sympathetic adrenal medullary systems in its pathogenesis.

Suppression of adrenocorticotrophic hormone secretion by simultaneous antagonism of vasopressin 1b and CRH-1 receptors on three different stress models.

BACKGROUND/AIMS: Corticotrophin-releasing hormone (CRH), adrenocorticotrophic hormone (ACTH) and corticosterone are secreted during stress. These mediators may be involved in anxiety, depression and post-traumatic stress disorder, therefore antagonists have been developed to treat such conditions. METHODS: The non-peptide CRH receptor type 1 antagonist CP154,526 and the vasopressin receptor type 1b antagonist SSR149415 were used to suppress the secretion of ACTH induced by ether exposure, forced swimming and restraint in adult male Wistar rats. Doses ranged from 3 to 60 mg/kg s.c. (controls with vehicle) alone or in combination, in varying time schedules to assess the duration and effectiveness of treatments. RESULTS: Stressors increased plasma ACTH by 2.5- to 5-fold in control rats. SSR149415 at doses of 30 mg/kg was more effective at suppressing ACTH secretion after ether exposure and restraint but was ineffective against forced swimming. CP154,526 mildly affected ACTH rise after restraint at doses of 30 mg/kg. The combination of both antagonists at doses of 30 mg/kg effectively blocked the rise in plasma ACTH in all three stresses. The drug effects lasted less than 6 h. CONCLUSION: We demonstrated for the first time that simultaneous blockade of both vasopressin 1b and CRH-R1 receptors effectively abolish the ACTH response to physical and psychological stress modalities.

Adjuvant effect of melatonin on anesthesia induced by thiopental sodium, ketamine, and ether in rats.

This study evaluated the anesthetic effects of thiopental sodium, ketamine, and ether with concurrent administration of melatonin. The loss of righting reflex was taken to assess the onset of anesthesia. Melatonin (20 mg/kg, p.o.) potentiated the anesthetic effects of thiopental sodium (20 mg/kg, i.v.) and ketamine (50 mg/kg, i.p.). Melatonin pretreatment caused rapid onset of anesthesia after ketamine and thiopental sodium administration while the duration of action of these agents was prolonged. Melatonin failed to alter anesthetic effects of ether (2 mg/kg by open method) in rats. This study suggests that melatonin modulate mechanisms involved in induction of thiopental sodium and ketamine anesthesia.

ADAM family protein Mde10 is essential for development of spore envelopes in the fission yeast Schizosaccharomyces pombe.

We report the identification of Schizosaccharomyces pombe mde10+ as a gene possessing a FLEX element, which forms a binding site for the meiosis-specific transcription factor Mei4. In fact, mde10+ is transcribed only in diploid cells that are induced to meiosis in a Mei4-dependent manner. Western blot analysis indicated that the epitope-tagged Mde10 protein accumulates transiently during meiosis and then rapidly decreases. Mde10 is a multidomain protein containing a metalloprotease catalytic domain, a disintegrin domain, a cysteine-rich domain, and membrane-spanning regions, all of which are shared by members of the mammalian ADAM family. A fusion protein of Mde10 and green fluorescent protein localized to the endoplasmic reticulum during meiosis and was located at the peripheral region of spores at the end of meiosis. An mde10Delta deletion mutant showed no apparent defects in meiosis, sporulation, or spore germination. However, the mutant spores exhibited an aberrant surface appearance, in which the ragged outer spore wall was lost to a large extent. Furthermore, mde10Delta spores were found to be less tolerant to ethanol and diethyl ether than were wild-type spores. The mutagenic replacement of the conserved glutamic acid in the putative protease active site with an alanine residue did not affect the surface morphology or the resistance of spores to environmental stress. Our observations indicate that Mde10 is important in the development of the spore envelope, although this function of Mde10 seems to be independent of its metalloprotease activity.

Cell wall reactive proteins in the coat and wall of maize pollen: potential role in pollen tube growth on the stigma and through the style.

The surface of a pollen grain consists of an outermost coat and an underlying wall. In maize (Zea mays L.), the pollen coat contains two major proteins derived from the adjacent tapetum cells in the anthers. One of the proteins is a 35-kDa endoxylanase (Wu, S. S. H., Suen, D. F., Chang, H. C., and Huang, A. H. C. (2002) J. Biol. Chem. 277, 49055-49064). The other protein of 70 kDa was purified to homogeneity and shown to be a beta-glucanase. Its gene sequence and the developmental pattern of its mRNA differ from those of the known beta-glucanases that hydrolyze the callose wall of the microspore tetrad. Mature pollen placed in a liquid medium released about nine major proteins. These proteins were partially sequenced and identified via GenBank trade mark data bases, and some had not been previously reported to be in pollen. They appear to have wall-loosening, structural, and enzymatic functions. A novel pollen wall-bound protein of 17 kDa has a unique pattern of cysteine distribution in its sequence (six tandem repeats of CX3CX10-15) that could chelate cations and form signal-receiving finger motifs. These pollen-released proteins were synthesized in the pollen interior, and their mRNA increased during pollen maturation and germination. They were localized mainly in the pollen tube wall. The pollen shell was isolated and found to contain no detectable proteins. We suggest that the pollen-coat beta-glucanase and xylanase hydrolyze the stigma wall for pollen tube entry and that the pollen secrete proteins to loosen or become new wall constituents of the tube and to break the wall of the transmitting track for tube advance.

Desflurane, enflurane, isoflurane and ether produce ethanol-like discriminative stimulus effects in mice.

In the present studies, drug discrimination procedures were used to compare the discriminative stimulus effects of ethanol (ETOH) and several volatile anesthetics. Male albino mice were trained to discriminate between IP injections of ETOH (1.25 g/kg) and saline in a two-lever operant task in which responding was under the control of a fixed-ratio 20 (FR20) schedule of food presentation. Stimulus generalization was examined after 20-min inhalation exposures to desflurane (4,000-32,000 ppm), enflurane (3,000-12,000 ppm), isoflurane (1,000-8,000 ppm) and ether (4,000-32,000 ppm). Concentration-related increases in ETOH-lever responding were observed for all four volatile anesthetics. For enflurane and ether, maximal levels of > 85% ETOH-lever responding were obtained at one or more concentrations. For desflurane and isoflurane, the maximal mean percentages of ETOH-lever responding were somewhat lower, but 6 out of 7 mice showed full substitution with desflurane and 5 out of 7 for isoflurane. The shared discriminative properties of these compounds with ETOH suggest that these anesthetics may share some of ETOH's pharmacological properties. These results are similar to previous research results showing ETOH-like discriminative stimulus effects in mice with other anesthetics and abused volatile inhalants (i.e. halothane, toluene and 1.1,1-trichloroethane) and may reflect the CNS-depressant drug-like effects of inhaled anesthetics and abused solvents.

Stress- and yohimbine-induced release of cholecystokinin in the frontal cortex of the freely moving rat: prevention by diazepam but not ondansetron.

The in vivo release of cholecystokinin (CCK)-like material (CCKLM) was measured in the frontal cortex of freely moving rats using the microdialysis technique combined with a sensitive radioimmunoassay. Local perfusion of K+ (100 mM)-enriched artificial CSF resulted in a 10-fold increase in CCKLM outflow, as compared with that occurring under basal resting (K+ = 3.0 mM) conditions, and this effect could be completely prevented by removal of Ca2+ in the perfusing fluid. Chromatographic analyses demonstrated that CCK-8S contributed to 70% of CCKLM. Stressful stimuli such as a 2-min exposure to diethyl ether and a 30-min restraint produced a marked but transient increase in cortical CCKLM release. In addition, anxiety-like behavior induced by the systemic administration of yohimbine (5 mg/kg i.p.) was associated with a long-lasting enhancement in the peptide outflow. Pretreatment with the potent anxiolytic drug diazepam (5 mg/kg i.p., 5 min before each condition), which exerted no effect on its own, completely prevented CCKLM overflow due to diethyl ether, restraint, or yohimbine administration. In contrast, neither the systemic injection (0.1 mg/kg i.p.) nor the local application (100 microM through the microdialysis probe) of the serotonin 5-HT3 antagonist ondansetron affected the increased release of CCKLM in rats restrained for 30 min or treated with yohimbine. These results indicate that cortical CCKergic neurotransmission is increased during stress or anxiety-like behavior in rats. Prevention of this effect by diazepam suggests that an inhibitory influence of benzodiazepines on cortical CCKergic neurons might participate in the anxiolytic action of these drugs.

Effect of development of habituation to restraint stress on hypothalamic noradrenaline release and adrenocorticotropin secretion.

The effect of repeated stress has been studied on noradrenaline release in the hypothalamic paraventricular nucleus and on adrenocorticotropin levels. Rats were stressed by 20-min immobilization once a day for 5 days. On day 6 they were exposed to the same stress or to a different one (ether vapors for 2 min). Immobilization and ether stress increased noradrenaline release in naive rats (271 +/- 43 and 197 +/- 9%, respectively) and raised adrenocorticotropin levels, showing activation of the hypothalamus-pituitary axis. Repeated daily restraint did not modify basal noradrenaline or adrenocorticotropin levels. The further immobilization session on day 6 did not change noradrenaline levels at any observation time (20-120 min). The adrenocorticotropin response was still present, although significantly reduced. In repeatedly restrained rats, exposure to ether vapors induced a maximal increase in noradrenaline level similar to that observed in naive rats, although prolonged. In these rats the adrenocorticotropin response did not differ from that in acutely stressed rats. These results suggest that habituation may develop to a stressful stimulus leading to suppression of the hypothalamic noradrenergic response and that this phenomenon is stress specific. Moreover, modifications of noradrenaline release in the paraventricular nucleus are not solely responsible for the adrenocorticotropin response during stress, suggesting that other pathways and/or neurotransmitters are involved too.

Effect of a high linoleate and a high alpha-linolenate diet on general behavior and drug sensitivity in mice.

Semi-purified diets supplemented with either a high linoleate (n-6) (safflower) oil or a high alpha-linolenate (n-3) (perilla) oil were fed to mouse mothers and their offspring through 6 weeks of age. The proportions of n-3 and n-6 highly unsaturated fatty acids in brain phospholipids reflected the n-3/n-6 balance of the diets while no difference was found in phospholipid compositions or cholesterol/phospholipid ratios. In the elevated plus maze task, the total number of entries into the open- and enclosed-arms was smaller and the time spent in the dark enclosed arms tended to be longer in the perilla group than the safflower group. The time required to reach a safe platform in Morris's water maze test was less in the perilla group, but no significant difference was observed in the entries into the arms darkened with a movable cover in Y-maze dark-preference task. The safflower group was more sensitive to pentobarbital; the anesthesia onset time was less and the anesthetic time was longer than in the perilla group. Increased locomotion induced by scopolamine injection was less in the safflower group as compared with the perilla group. These results indicate that in mice the dietary alpha-linolenate/linoleate balance affects the n-3/n-6 ratio of brain phospholipid acyl chains and that this is accompanied by general behavioral changes as well as changes in sensitivities to drugs known to affect behavior.

[Protoscolicidal effect of some chemical agents and drugs against Echinococcus granulosus].

The present paper reports the protoscolicidal action of hydrastine, ether-acetic acid-ethanol admixture, H2O2, pyquiton and albendazole through in vitro or in vivo exposure, for 15 minutes and transplantation studies. The mortality of protoscolices in vitro and in vivo were 70.2% and 68.9% for 0.3% hydrastine, 56.8% and 56.2% for 10% ether-acetic acid-ethanol admixture, 6.0% and 8.8% for 0.3% H2O2; 6.1% for 0.004% pyquiton in vitro and 5.0% were 10% and 25% for 0.3% hydrastine, 30% and for 0.004% albendazole in vitro. The survival rates after transplantation of protoscolices 37.5% for 10% ether-acetic acid-ethanol admixture, 100% and 95% for 0.3% H2O2 respectively. Disruption of external plasma membrane, hook detachment, sucker deformity of protoscolices exposed to hydrastine were demonstrated by scanning electron microscopy. It is suggested that hydrastine exerts a profound intracellular effect on the protoscolex of E. granulosus of sheep and man, and might be a promising protoscolicide as adjuvant to hydatid surgery.