Chronic nicotine exposure selectively activates a carrier-mediated release of endogenous glutamate and aspartate from rat hippocampal synaptosomes

The effect of chronic nicotine treatment on the release of endogenous glutamate (GLU), aspartate (ASP) and GABA evoked in vitro by KCl, 4-aminopyridine (4AP) and nicotinic agonists in synaptosomes of rat hippocampus was investigated. Rats were chronically administered with nicotine bitartrate or saline vehicle each for 14 days using osmotic mini-pumps. Hippocampal synaptosomes were stimulated with KCl, 4AP, nicotine or with choline (Ch) and 5-iodo-A-85380 dihydrochloride (5IA85380). The GLU and ASP overflow evoked by Ch, nicotine, KCl and 4AP were increased in treated animals while the nicotine-evoked GABA overflow was reduced and that evoked by Ch, KCl and 4AP was unaffected. The 5IA85380-evoked overflow of the three aminoacids (AAs) was always reduced. The increase of ASP and GLU overflow evoked by KCl, 4AP or Ch was blocked by dl-threo-β-benzyloxyaspartic acid (dl-TBOA), a carrier transporter inhibitor, and by inhibitors of the Na(+)/Ca(2+) exchangers 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-4-thiazolidinecarboxylic acid ethyl ester (SN-6) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate (KB-R7943). In conclusion long-term nicotine treatment may selectively increase GLU and ASP overflow elicited by KCl, 4AP and Ch through the activation of a carrier-mediated release mechanism and completely abolished the stimulatory effects of α4β2 nAChRs which modulate the release of all the three AA.     

Presynaptic nicotinic α7 and non-α7 receptors stimulate endogenous GABA release from rat hippocampal synaptosomes through two mechanisms of action

Background

Although converging evidence has suggested that nicotinic acetylcholine receptors (nAChR) play a role in the modulation of GABA release in rat hippocampus, the specific involvement of different nAChR subtypes at presynaptic level is still a matter of debate. In the present work we investigated, using selective α7 and α4β2 nAChR agonists, the presence of different nAChR subtypes on hippocampal GABA nerve endings to assess to what extent and through which mechanisms they stimulate endogenous GABA release.

Methodology/Findings

All agonists elicited GABA overflow. Choline (Ch)-evoked GABA overflow was dependent to external Ca²⁺, but unaltered in the presence of Cd²⁺, tetrodotoxin (TTX), dihydro-β-erythroidine (DHβE) and 1-(4,4-Diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride SKF 89976A. The effect of Ch was blocked by methyllycaconitine (MLA), α-bungarotoxin (α-BTX), dantrolene, thapsigargin and xestospongin C, suggesting that GABA release might be triggered by Ca²⁺ entry into synaptosomes through the α7 nAChR channel with the involvement of calcium from intracellular stores. Additionally, 5-Iodo-A-85380 dihydrochloride (5IA85380) elicited GABA overflow, which was Ca²⁺ dependent, blocked by Cd²⁺, and significantly inhibited by TTX and DHβE, but unaffected by MLA, SKF 89976A, thapsigargin and xestospongin C and dantrolene. These findings confirm the involvement of α4β2 nAChR in 5IA85380-induced GABA release that seems to occur following membrane depolarization and opening calcium channels.

Conclusions/Significance

Rat hippocampal synaptosomes possess both α7 and α4β2 nAChR subtypes, which can modulate GABA release via two distinct mechanisms of action. The finding that GABA release evoked by the mixture of sub-maximal concentration of 5IA85380 plus sub-threshold concentrations of Ch was significantly larger than that elicited by the sum of the effects of the two agonists is compatible with the possibility that they coexist on the same nerve terminals. These findings would provide the basis for possible selective pharmacological strategies to treat neuronal disorders that involve the dysfunction of hippocampal cholinergic system.     

The role of calpain in an in vivo model of oxidative stress-induced retinal ganglion cell damage

Purpose

In this study, we set out to establish an in vivo animal model of oxidative stress in the retinal ganglion cells (RGCs) and determine whether there is a link between oxidative stress in the RGCs and the activation of calpain, a major part of the apoptotic pathway.

Materials and methods

Oxidative stress was induced in the RGCs of C57BL/6 mice by the intravitreal administration of 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH, 30 mM, 2 μl). Control eyes were injected with 2 μl of vehicle. Surviving Fluorogold (FG)-labeled RGCs were then counted in retinal flat mounts. Double staining with CellROX and Annexin V was performed to investigate the co-localization of free radical generation and apoptosis. An immunoblot assay was used both to indirectly evaluate calpain activation in the AAPH-treated eyes by confirming α-fodrin cleavage, and also to evaluate the effect of SNJ-1945 (a specific calpain inhibitor: 4% w/v, 100 mg/kg, intraperitoneal administration) in these eyes.

Results

Intravitreal administration of AAPH led to a significant decrease in FG-labeled RGCs 7 days after treatment (control: 3806.7 ± 575.2 RGCs/mm², AAPH: 3156.1 ± 371.2 RGCs/mm², P < 0.01). CellROX and Annexin V signals were co-localized in the FG-labeled RGCs 24 h after AAPH injection. An immunoblot assay revealed a cleaved α-fodrin band that increased significantly 24 h after AAPH administration. Intraperitoneally administered SNJ-1945 prevented the cleavage of α-fodrin and had a neuroprotective effect against AAPH-induced RGC death (AAPH: 3354.0 ± 226.9 RGCs/mm², AAPH+SNJ-1945: 3717.1 ± 614.6 RGCs/mm², P < 0.01).

Conclusion

AAPH administration was an effective model of oxidative stress in the RGCs, showing that oxidative stress directly activated the calpain pathway and induced RGC death. Furthermore, inhibition of the calpain pathway protected the RGCs after AAPH administration.     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

Quantitative in vitro assay to evaluate the capability of yeast cell wall fractions from Trichosporon mycotoxinivorans to selectively bind gram negative pathogens

Yeast cell wall fractions have been proposed to bind enteropathogenic bacteria. The aim of this study was to develop a quantitative assay by measuring the optical density as growth parameter of adhering bacteria. The exponential growth phase of adhering bacteria was determined by optical density reading and compared with the colony count (CFU/mL). A linear regression was compiled and the bacterial number bound to the yeast cell wall product could be determined. Further focus was the investigation of a yeast cell wall from strain Trichosporon mycotoxinivorans (MTV) for its ability to bind gram negative Salmonella, E. coli and Campylobacter strains and gram positive probiotic bacteria of the genera lactobacilli and bifidobacteria as well as gram positive Clostridium perfringens quantitatively. The gram negative probiotic strain E. coli Nissle 1917 was also investigated. Seven out of 10 S. Typhimurium and S. Enteritidis strains adhered to the cell wall product with an amount between 10³ and 10⁴ CFU/10 μg. Four out of 7 E. coli strains showed an average binding capability (10² CFU/10 µg) whereas 4 × 10³E. coli F4 cells bound per 10 μg yeast cell wall. E. coli 0149 K91, E. coli 0147 K89, C. jejuni and C. perfringens as well the genera lactobacilli and bifidobacteria did not bind to the yeast cell wall. E. coli Nissle 1917 was bound with 2 × 10² CFU/10 μg. These results demonstrate that cell wall from MTV can be used to differentially bind E. coli spp. and Salmonella spp. up to 8 × 10⁴ CFU/10 μg. Thus certain yeast cell walls may prevent enteric infections caused by selective bacteria. This methodical approach would be an accurate tool in the feed industry for quality control of yeast cell wall products.     

The effects of rumen fluid on the in vitro aflatoxin binding capacity of different sequestering agents and in vivo release of the sequestered toxin

In an in vitro experiment, aluminosilicates (Atox^® and Novasil™ Plus) and a yeast cell wall derivate (Mycosorb^®) were used as sequestering agents (SAs) to verify their capacity for binding aflatoxin B1 (AFB1) in vitro. SAs were individually mixed at three different ratios with AFB1 (1:5000, 1:50,000 and 1:500,000, w/w) in water (CTR), rumen fluid from a lactating cow with a low rumen pH (LRS) or rumen fluid from a dry cow with a high rumen pH (HRS), and then used in a 3 × 3 × 3 factorial arrangement of a completely randomized design. At the 1:500,000 AF:SA ratio Atox^® and Novasil™ Plus sequestered over 0.87 and 0.98 of the AFB1 in the CTR and rumen solutions (LRS and HRS), respectively. This efficacy decreased when the amount of clays was reduced, with higher values (P<0.001) for Atox^® compared with Novasil™ Plus (0.50 vs. 0.28 in CTR; 0.58 vs. 0.16 in LRS and 0.44 vs. 0.27 in HRS). Mycosorb^® had a lower sequestering efficacy (P<0.001) in all of tested experimental conditions, with 0.34 being the maximum value obtained in the CTR solution.     

Rare sesquiterpenes from South African Pteronia species

The genus Pteronia consists of approximately 80 species which are widely distributed in southern Africa. The essential oils isolated from the aerial parts of eleven species, analyzed by GC-MS varied both qualitatively and quantitatively. In Pteronia pallens, Pteronia empetrifolia and Pteronia flexicaulis uncommon sesquiterpenes such as presilphiperfolol-7-ene, 7-α-(H)-silphiperfol-5-ene, 7-β-(H)-silphiperfol-5-ene, α-campholene aldehyde, silphiperfol-5-ene, cameroonan-7-α-ol, silphiperfol-7-β-ol, presilphiperfolan-9-α-ol and presilphiperfolan-8-ol (a major compound in P. pallens) were identified. Cluster analysis based of the chemical composition of the oils revealed that individual plants of Pteronia camphorata collected in the same population had similar oil profiles with a high correlation coefficient (S_corr ≈ 0.98). Similarly, the essential oil composition of P. pallens collected from two distinct localities also showed high levels of congruency (S_corr ≈ 0.99).     

Developing an in vitro method for Eimeria tenella attachment to its preferred and non-preferred intestinal sites

A frozen section method utilising chicken intestinal tissue was developed to study the Eimeria tenella attachment ex vivo. In order to examine Eimeria-epithelial cell attachment, 10⁵E. tenella sporozoites were incubated with each caecal frozen section (6, 10 and 14 μm) for 1 h in 5% CO₂ incubator at 41 °C. E. tenella sporozoites attached successfully to enterocytes in 14 μm thick of caecal sections. Sporozoite attachment to caecal sections was shown to be dependent on the number of parasites added. To evaluate the method, E. tenella sporozoites were incubated to its preferred (caecum) and non-preferred (duodenum and jejunum) intestinal sites. The number of sporozoites attached to the caecal enterocytes was significantly greater (P < 0.0001) in comparison with the limited number of sporozoites attached to enterocytes of non-preferred intestinal sites. This method was shown to be able to reveal differences in binding capability and allows for comparison of intestinal site attachment.     

Factors influencing in vitro shoot regeneration from leaf segments of Chrysanthemum

The objective of this research was to develop an efficient protocol for shoot regeneration from leaf segments of the Chrysanthemum cv. Vivid Scarlet by examining the effects of plant growth regulators, dark incubation period, gelling agents, and silver nitrate. The highest number of shoots per explant (12.3) was regenerated from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with a combination of 1 mg L⁻¹ of 6-benzyladenine (BA) and 2 mg L⁻¹ of α-naphthaleneacetic acid (NAA) under light conditions without any initial dark period. Gelrite was the most effective gelling agent for shoot regeneration among those tested, whereas the presence of silver nitrate distinctly inhibited shoot regeneration. Superior plant growth and rooting was observed on a hormone-free MS medium solidified with Gelrite. Flow cytometry analysis revealed no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions. The established protocol was applicable to shoot regeneration for four out of six cultivars tested. This research will facilitate the genetic transformation and micropropagation of Chrysanthemum cultivars.     

The metabolism of anatabine to α,β-dipyridyl in Nicotiana species

α,β-Dipyridyl isolated from Nicotiana tabacum plants which had been fed anatabine-[2′-¹⁴C, ¹³C], and then allowed to dry in air for 20 days was radioactive (82% specific incorporation)An examination of its ¹³C NMR spectra established that it was enriched only at C-2, indicative of its direct formation from anatabineThe labelled anatabine was also fed to Nglauca and Nglutinosa plants, which were extracted immediately after harvestingIn these experiments no radioactive α,β-dipyridyl was detected, suggesting that α,β-dipyridyl is an artifact produced by the oxidation of anatabine in the drying leaves of tobaccoAnabasine isolated from the Nicotiana species which had been fed anatabine-[2′- ¹⁴C, ¹³C] was unlabelled, indicating that none of this alkaloid is formed by the reduction of anatabine.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Endogenous isoflavone methylation correlates with the in vitro rooting phases of Spartium junceum L. (Leguminosae)

Spartium junceum L. (Leguminosae) is a perennial shrub, native to the Mediterranean region in southern Europe, widespread in all the Italian regions and, as a leguminous species, it has a high isoflavone content. An in vitro culture protocol was developed for this species starting from stem nodal sections of in vivo plants, and isoflavone components of the in vitro cultured tissues were studied by means of High Performance Liquid Chromatography (HPLC) analytical techniques. Two main isoflavones were detected in the S. junceum tissues during the in vitro propagation phases: Genistein (4′,5,7-Trihydroxyisoflavone), already reported in this species, and its methylated form 4′,5,7-Trimethoxyisoflavone, detected for the first time in this plant species (0.750 ± 0.02 mg g⁻¹ dry tissue). The presence of both of these compounds in S. junceum tissues was consistently detected during the in vitro multiplication phase. The absence of the methylated form within plant tissues in the early phases of the in vitro adventitious root formation was correlated with its negative effect displayed on root induction and initiation phases, while its presence in the final “root manifestation” phase influenced positively the rooting process. The unmethylated form, although detectable in tissues in the precocious rooting phases, was no longer present in the final rooting phase. Its effect on rooting, however, proved always to be beneficial.     

Pro-fibrotic activity of lysophosphatidic acid in adipose tissue: In vivo and in vitro evidence

Lysophosphatidic acid (LPA) is a pro-fibrotic mediator acting via specific receptors (LPARs) and is synthesized by autotaxin, that increases with obesity. We tested whether LPA could play a role in adipose tissue (AT)-fibrosis associated with obesity. Fibrosis [type I, III, and IV collagens (COL), fibronectin (FN), TGFβ, CTGF and αSMA] and inflammation (MCP1 and F4/80) markers were quantified: (i) in vivo in inguinal (IAT) and perigonadic (PGAT) AT from obese-diabetic db/db mice treated with the LPAR antagonist Ki16425 (5 mg/kg/day ip for 7 weeks); and (ii) in vitro in human AT explants in primary culture for 72 h in the presence of oleoyl-LPA (10 μM) and/or Ki16425 (10 μM) and/or the HIF-1α inhibitor YC-1 (100 μM). Treatment of db/db mice with Ki16425 reduced Col I and IV mRNAs in IAT and PGAT while Col III mRNAs were only reduced in IAT. This was associated with reduction of COL protein staining in both IAT and PGAT. AT explants showed a spontaneous and time-dependent increase in ATX expression and production of LPA in the culture medium, along with increased levels of Col I and III, TGFβ and αSMA mRNAs and of COL protein staining. In vitro fibrosis was blocked by Ki16425 and was further amplified by oleoyl-LPA. LPA-dependent in vitro fibrosis was blocked by co-treatment with YC1. Our results show that endogenous and exogenous LPA exert a pro-fibrotic activity in AT in vivo and in vitro. This activity could be mediated by an LPA1R-dependent pathway and could involve HIF-1α.     

Increased antitumor efficacy by the combined administration of swainsonine and cisplatin in vivo

Swainsonine is a natural α-mannosidase inhibitor found in numerous poisonous plants, such as Astragalus lentiginosus. Its mechanism of action is through the inhibition of Golgi α-mannosidase II activity in the N-glycan biosynthesis pathway. As a result, swainsonine inhibits the production of complex β1,6-branched N-linked glycans, which are related to the malignant phenotype of tumor cells. In this study, we investigated whether treatment with swainsonine affects the sensitivity of Ehrlich ascites carcinoma (EAC) cells to cisplatin. To this end, male C57BL/6 mice were treated with swainsonine (SW - 0.5 mg/kg, i.p., twice-daily for ten days) and/or cisplatin (Cis - 0.25 mg/kg, i.p., every other day for a total of five applications) two days after transplantation with EAC cells. The results showed a greater reduction in the ascites volume in mice from the CisSW group (63.5%) than in mice from the Cis group (45.7%), an elevated induction of apoptosis by CisSW treatment when compared to Cis alone, as demonstrated by higher percentage of cells in the subG1 phase in that group (p < 0.0001 Kruskal-Wallis, p < 0.0001 control vs. CisSW, p < 0.001 Co vs. Cis post-test Dunn), and an increase in the median survival from 12.5 days observed in the control group to 27 days in the CisSW group, which corresponds to a 116% survival increase (p = 0.0022 Co vs. CisSW Log-rank test). In addition, the mice from the Cis group had a median survival of only 15 days, an increase of just 20% compared to controls. Our results indicate that swainsonine increases the sensitivity of EAC cells to cisplatin.     

Low molecular weight chitosans—Preparation with the aid of pronase,characterization and their bactericidal activity towards Bacillus cereus and Escherichia coli

The homogeneous low molecular weight chitosans (LMWC) of molecular weight 9.5–8.5 kDa, obtained by pronase catalyzed non-specific depolymerization (at pH 3.5, 37 °C) of chitosan showed lyses of Bacillus cereus and Escherichia coli more efficiently (100%) than native chitosan (< 50%). IR and ¹H-NMR data showed decrease in the degree of acetylation (14–19%) in LMWC compared to native chitosan (∼ 26%). Minimum inhibitory concentration of LMWC towards 10⁶ CFU ml^− 1 of B. cereus was 0.01% (w/v) compared to 0.03% for 10⁴ CFU ml^− 1 of E. coli. SEM revealed pore formation as well as permeabilization of the bacterial cells, as also evidenced by increased carbohydrate and protein contents as well as the cytoplasmic enzymes in the cell-free supernatants. N-terminal sequence analyses of the released proteins revealed them to be cytoplasmic/membrane proteins. Upon GLC, the supernatant showed characteristic fatty acid profiles in E. coli, thus subscribing to detachment of lipopolysaccharides into the medium, whereas that of B. cereus indicated release of surface lipids. The mechanism for the observed bactericidal activity of LMWC towards both Gram-positive and Gram-negative bacteria has been discussed.     

Molecular cloning and expression in vitro of a carboxylesterase gene from the Glanville fritillary butterfly (Melitaea cinxia)

Carboxylesterase (EC 3.1.1.1) is a member of the carboxyl/cholinesterase (CCE) superfamily, which is widely distributed in animals, plants and microorganisms. This enzyme has been known to be associated with insecticide resistance and detoxification. Although CCEs have been extensively studied in insects, including lepidopterans, the research on butterflies, a major subgroup in Lepidoptera, is still poor. In the present study, we cloned a CCE gene (McCCE1) from the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The full-length cDNA encoding McCCE1 was 1786 bp, containing a 1641 bp open reading frame encoding 546 amino acids, a 38 bp 5′-untranslated region (5′-UTR), and a 107 bp 3′-UTR with a poly(A) tail. The functionally conserved amino acids in McCCE1 shared the 55% identity with the cytoplasmic esterase CCE017a in Helicoverpa armigera (Lepidoptera: Noctuidae), which has been associated with detoxification. Assays in vitro showed that the recombinant McCCE1 could hydrolyze α- and β-naphthyl acetate. Thus, the present study adds to the body of knowledge concerning the detoxification of pesticides by lepidopterans.     

Solanopubamine,a steroidal alkaloid from Solanum pubescens

Aerial parts of Solanum pubescens yielded a new steroidal alkaloid, solanopubamine, the structure of which was elucidated as 3β-amino-5α,22αH,25βH-solanidan-23β-ol by ¹³C NMR, ¹H NMR, IR, mass spectral analysis and chemical degradation methods.     

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α ∼ 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.