Characterization of TRPM8-like channels activated by the cooling agent icilin in the macrophage cell line RAW 264.7

Icilin is recognized as a chemical agonist of nociceptors and can activate TRPM8 channels. However, whether this agent has any effects on immune cells remains unknown. In this study, the effects of icilin on ion currents were investigated in RAW 264.7 murine macrophage-like cells. Icilin (1–100 μM) increased the amplitude of nonselective (NS) cation current (I _NS) in a concentration-dependent manner with an EC₅₀ value of 8.6 μM. LaCl₃ (100 μM) or capsazepine (30 μM) reversed icilin-induced I _NS; however, neither apamin (200 nM) nor iberiotoxin (200 nM) had any effects on it. In cell-attached configuration, when the electrode was filled with icilin (30 μM), a unique population of NS cation channels were activated with single-channel conductance of 158 pS. With the use of a long-lasting ramp pulse protocol, increasing icilin concentration produced a left shift in the activation curve of NS channels, with no change in the slope factor of the curve. The probability of channel opening enhanced by icilin was increased by either elevated extracellular Ca²⁺ or application of ionomycin (10 μM), while it was reduced by BAPTA-AM (10 μM). Icilin-stimulated activity is associated with an increase in mean open time and a reduction in mean closed time. Under current-clamp conditions, icilin caused membrane depolarization. Therefore, icilin interacts with the TRPM8-like channel to increase I _NS and depolarizes the membrane in these cells.     

Liposomal-Glutathione Provides Maintenance of Intracellular Glutathione and Neuroprotection in Mesencephalic Neuronal Cells

A liposomal preparation of glutathione (GSH) was investigated for its ability to replenish intracellular GSH and provide neuroprotection in an in vitro model of Parkinson’s disease using paraquat plus maneb (PQMB) in rat mesencephalic cultures. In mixed neuronal/glial cultures depleted of intracellular GSH, repletion to control levels occurred over 4 h with liposomal-GSH or non-liposomal-GSH however, liposomal-GSH was 100-fold more potent; EC_50s 4.75 μM and 533 μM for liposomal and non-liposomal-GSH, respectively. Liposomal-GSH utilization was also observed in neuronal cultures, but with a higher EC₅₀ (76.5 μM), suggesting that glia facilitate utilization. Blocking γ-glutamylcysteine synthetase with buthionine sulfoxamine prevented replenishment with liposomal-GSH demonstrating the requirement for catabolism and resynthesis. Repletion was significantly attenuated with endosomal inhibition implicating the endosomal system in utilization. Liposomal-GSH provided dose-dependent protection against PQMB with an EC₅₀ similar to that found for repletion. PQMB depleted intracellular GSH by 50%. Liposomal-GSH spared endogenous GSH during PQMB exposure, but did not require GSH biosynthesis for protection. No toxicity was observed with the liposomal preparation at 200-fold the EC₅₀ for repletion. These findings indicate that glutathione supplied in a liposomal formulation holds promise as a potential therapeutic for neuronal maintenance.     

Effects of the antidepressant pirlindole and its dehydro-derivative on the activity of monoamine oxidase-a and on GABAA receptors

The effects of pirlindole and dehydro-pirlindole on GABA_A receptors and MAO-A activity were investigated in vitro. Pirlindole was inactive as a GABA antagonist. Dehydro-pirlindole exhibited partial and selective blockade of a subset of GABA_A receptors with an EC₅₀ of 12μM and maximum reversal (ΔB_opt) of 42%. Inhibition of rat brain and human placenta MAO-A by both compounds was much more potent (with IC₅₀ range 0.3–0.005 μM). Their effects on MAO-A activity were partially reversible in vitro. In contrast to pirlindole, dehydro-pirlindole may act not only as MAO-A inhibitor but also as a clozapine-like selective GABA_A receptor blocker, preferentially blocking a subset of GABA_A receptors that are not sensitive to DMCM or Ro 5-4864. Pirlindole is the generic name of the drug pyrazidol.     

Investigation of the Cytotoxicity of Antimicrobial Peptide P40 on Eukaryotic Cells

The in vitro cytotoxicity of the antimicrobial peptide P40 was investigated. The food grade bacteriocin nisin was also analyzed for comparison. VERO cells were treated with different concentrations (0.02–2.5 μg ml⁻¹) of nisin and P40, and cell viability and plasma membrane integrity were checked by MTT, neutral red uptake (NRU), and lactate dehydrogenase (LDH) assays. In MTT and NRU assays the EC₅₀ to the purified peptide P40 were 0.30 and 0.51 μg ml⁻¹, while values found to nisin were 0.35 and 0.79 μg ml⁻¹, respectively. In the LDH assay, the EC₅₀ was 0.57 and 0.62 μg ml⁻¹ for P40 and nisin, respectively. The peptide P40 revealed higher hemolytical activity (19%) when compared to nisin (4.9%) at the highest concentration tested (2.5 μg ml⁻¹). Relatively few studies about the cytotoxicity of antimicrobial peptides are available. The determination of the cytotoxicity of antimicrobial peptides is an essential step to warrant their safe use.     

Inhibition of oxygen consumption in skeletal muscle-derived mitochondria by pinacidil,diazoxide, and glibenclamide,but not by 5-hydroxydecanoate

Cell intermediary metabolism and energy production succeeds by means of mitochondria, whose activity is in relation to transmembrane potential and/or free radical production. Adenosine triphosphate (ATP)-dependent potassium channels (K_ATP) in several cell types have shown to couple cell metabolism to membrane potential and ATP production. In this study, we explore whether oxygen consumption in isolated skeletal-muscle mitochondria differs in the presence of distinct respiration substrates and whether these changes are affected by K_ATP-channel inhibitors such as glibenclamide, 5-Hydroxydecanoate (5-HD), and K_ATP channel activators (pinacidil and diazoxide). Results demonstrate a concentration-dependent diminution of respiration rate by glibenclamide (0.5–20 μM), pinacidil (1–50 μM), and diazoxide (50–200 μM), but no significant differences were found when the selective mitochondrial K_ATP-channel inhibitor (5-HD, 10–500 μM) was used. These results suggest that these K_ATP-channel agonists and antagonists exert an effect on mitochondrial respiration and that they could be acting on mito-K_ATP or other respiratory-chain components.     

Recent improvements in the development of A2B adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis. This article has previously been published in issue 4/4, under doi:.     

Peptide Pheromone Plantaricin A Produced by Lactobacillus plantarum Permeabilizes Liver and Kidney Cells

Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH₄ cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10–100 μM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca²⁺]_i, and diffusional loss of fluorophore from the cytosol. At a concentration of 5 μM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 μM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.     

Design, synthesis and biological evaluation of non-peptide PAR1 thrombin receptor antagonists based on small bifunctional templates: arginine and phenylalanine side chain groups are keys for receptor activity

In the present study, we report the synthesis and biological evaluation of a series of new non-peptide PAR₁ mimetic receptor antagonists, based on conformational analysis of the S₄₂FLLR₄₆ tethered ligand (TL) sequence of PAR₁. These compounds incorporate the key pharmacophore groups in the TL sequence, guanidyl, amino and phenyl, which are essential for triggering receptor activity. Compounds 5 and 15 (50–100 μM) inhibited both TFLLR-amide (10 μM) and thrombin-mediated (0.5 and 1 U/ml; 5 and 10 μM) calcium signaling in a cultured human HEK cell assay.     

Influence of glutathione on zinc-mediated cellular toxicity

The effect of zinc on various pulmonary cell lines has been studied by measuring the depletion of total cellular glutathione after exposure to zinc(II) chloride at different concentrations. Total cellular glutathione (cGS) was measured at 31 ± 3 nmol/mg, 3.8 ± 0.6 nmol/mg, and 3.7 ±1.2 nmol/mg protein in A549, L2, and 11Lu cells, respectively. After treatment with buthionine sulfoximine (BSO), the cGS levels decreased by 20% in A549 cells and below 0.2 nmol/mg in L2 and 11Lu cells. Exposure of A549 cells to 25–200 μM ZnCl₂ for 4 h alone decreased the cGS content to 60–80%. There was little additional effect in BSO-pretreated cells. In L2 and 11Lu cells, the decrease of cGS was 70–85% following exposure to 15–150 μM ZnCl₂ for 2 h. If BSO was also used, the decrease in cGS was 85–95% in L2 cells and 75–85% in 11Lu cells. Exposure to 25–250 μM ZnCl₂ for 2 h diminished protein synthesis as determined by radiolabeled methionine incorporation, with half-maximum inhibition (EC₅₀) from 40–160 μM ZnCl₂. To attain similar EC₅₀ values in BSO-pretreated cells, only about half the zinc concentrations were required as compared to cells without pretreatment. The decrease of cGS was accompanied by an increased ratio of oxidized : reduced glutathione that was more pronounced in cells with low glutathione content.     

A fast algal bioassay for assessment of copper toxicity in water using <Emphasis Type="Italic">Euglena gracilis</Emphasis>

A rapid, sensitive algal bioassay was investigated to monitor copper toxicity using photosynthetic activity and motility parameters as end points in the photosynthetic flagellate Euglena gracilis. Effects on motility parameters were determined using the automatic bioassay ECOTOX. Photosynthetic efficiency was measured using chlorophyll fluorescence by means of a fast, noninvasive pulse amplitude modulated fluorometer. These parameters were assessed regarding their effectiveness as end points for short- (0–24 h) and long-term (1–5 days) toxicity tests. The model organism showed significant responses to the tested concentrations at both motility and photosynthesis examined levels. EC₅₀ values for movement parameters immediately after the cells were mixed with copper are 19.09 (300.4 μM), 20.4 (312 μM) and 23.09 (363.35 μM) mg L⁻¹ for motility, r-value and velocity, respectively. The current study proofs that Euglena is a convenient biotest organism where motility parameters show rapid sensitive responses to copper (direct exposure to copper). Also, the photosynthetic parameters appear to be appropriate for acute and chronic toxicity tests for higher and lower copper concentrations, respectively.     

Somatic embryogenesis and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Establishment, maintenance, regeneration, and transformation of somatic embryos by both direct and indirect means (callus-mediated) was achieved for Bixa orellana, a tropical plant whose seeds produce commercially edible ‘annatto pigment,’ which mainly constitutes an apocarotenoid called bixin. Callus-mediated methodology was found to be efficient in producing a greater number of embryos in a short time. The maximum of 28 somatic embryos were produced in 16–18 weeks when immature zygotic embryonic stalks were inoculated onto Murashige and Skoog (MS) medium containing B5 vitamins supplemented with 0.44 μM benzyladenine (BA), 0.054 μM α-naphthaleneacetic acid (NAA), 2.89 μM gibberellic acid (GA₃), 0.02 μM triiodobenzoic acid (TIBA), and 0.011 μM triacontanol (TRIA). Callus initiation from hypocotyl explants was obtained on MS medium supplemented with 1.07–2.14 μM NAA and 10.2 μM BA. In 3 months, somatic embryos were produced when callus was inoculated onto MS medium supplemented with 4.44 μM BA, 40 μM AgNO₃, and 0.011 μM TRIA. Somatic embryos were efficiently regenerated on MS basal solid and liquid media supplemented with 0.44–4.4 μM BA, 0.54–2.69 μM NAA, 4.92 μM 2iP, 2.1 μM calcium d-pantothenate, 0.21 μM biotin, 227.7 μM cysteine HCl monohydrate, and 108.6 μM adenine sulfate. Agrobacterium tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector-mediated stable transformation of somatic embryos exhibited a transformation frequency of 2.56%. As somatic embryogenesis in any perennial system is useful in terms of both commercial and scientific nature, this somatic embryo-based transformation protocol for the commercially important dye-yielding tropical plant B. orellana is useful for its improvement through genetic engineering.     

Guanidino Acids Act as ρ1 GABA<Subscript>C</Subscript> Receptor Antagonists

GABA_C receptors play a role in myopia, memory-related disorders and circadian rhythms signifying a need to develop potent and selective agents for this class of receptors. Guanidino analogs related to glycine, β-alanine and taurine were evaluated at human ρ₁GABA_C receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods. Of the 12 analogs tested, 8 analogs were active as antagonists and the remaining were inactive. (S)-2-Guanidinopropionic acid (IC₅₀ = 2.2 μM) and guanidinoacetic acid (IC₅₀ = 5.4 μM; K _B = 7.75 μM [pK _B = 5.11 ± 0.06]) were the most potent being competitive antagonists at this receptor. In contrast, the β-alanine and GABA guanidino analogs showed reduced activity, indicating the distance between the carboxyl carbon and terminal nitrogen of the guanidino group is critical for activity. Substituting the C2-position of guanidinoacetic acid with various alkyl groups reduced activity indicating that steric effects may impact on activity. The results of this study contribute to the structure–activity-relationship profile required in developing novel therapeutic agents.     

Pharmacological characterization of a nicotinic autoreceptor in rat hippocampal synaptosomes

The modulation of [³H]ACh release by nicotinic compounds was studied in superfused rat hippocampal synaptosomes loaded with [³H]choline. (−)-Nicotine (0.1–10 μM) evoked a dose-dependent increase in [³H]ACh release; higher concentrations were less effective. Nicotine-evoked release was Ca²⁺-dependent, and blocked by the nicotinic antagonists dihydro-β-erythroidine, mecamylamine, and pempidine. The α7-selective antagonist methyllycaconitine did not inhibit nicotine-evoked release when tested at 1 μM, although at 10 μM some attenuation of the response was observed. Six agonists tested were equally efficacious in stimulating [³H]ACh release, as judged by the maximum responses, and gave the following EC₅₀ values: (±)-epibatidine 0.12 μM; (+)-anatoxin-a 0.14 μM; (−)-nicotine 0.99 μM; (−)-cytisine 1.06 μM; ABT-418 2.6 μM; isoarecolone 43 μM. Each agonist generated a “bell-shaped” dose response curve, suggesting desensitisation at higher concentrations. This is supported by analysis of repetitive stimulation with (−)-nicotine and (−)-cytisine: S2/S1 ratios declined sharply with increasing concentration, whereas subsequent KCl-evoked release remained constant. These results are discussed in terms of possible nicotinic receptor subtypes that might be present on hippocampal nerve terminals. Special issue dedicated to Dr. Herman Bachelard.     

Optimization of growth regulators and silver nitrate for micropropagation of <Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L. with the aid of a response surface experimental design

This paper reports on the optimum concentrations of naphthalene acetic acid (NAA) and 6-benzyladenine (BA) to stimulate callus growth and NAA; kinetin and silver nitrate (AgNO₃) for callus redifferentiation in Dianthus caryophyllus L. Meristems were excised and placed in MS medium with 30 g l⁻¹ sucrose and 9.0 μM 2,4-d. Callus clusters were transferred to MS medium containing NAA (0, 1.7, 3.3, and 5.0 μM) and BA (0, 1.7, 3.3, and 5.0 μM) for proliferation and to MS medium with 30 g l⁻¹ sucrose, 2.5 g l⁻¹ phytagel, kinetin (0, 33, and 66 μM); NAA (0, 7.95, and 15.9 μM) and AgNO₃ (0, 23.54 and 47.08 μM) for shoot and root induction. Treatments were applied according to a Box–Behnken design. After callus growth and redifferentiation, plants were incubated in the greenhouse at 18 ± 2°C for 4 wk and at 20–26°C for 4 wk. Finally, plants were changed to near-commercial greenhouse conditions with different day (30–35°C) and night (16–24°C) temperatures. Results showed better callus growth at higher NAA concentrations. A maximum callus weight was found with 5.0 μM NAA but without BA. A maximum of 78% calluses with shoots was obtained with 15.9 μM NAA, 47.08 μM AgNO₃, and 0.74 μM kinetin and 58% with roots with 15.7 μM NAA and 47.08 μM AgNO₃, but without kinetin. The shoots obtained showed little hyperhydricity. Vigorous plants were obtained after gradual acclimatization with an 80% survival rate under nursery conditions.     

Plant Regeneration from Decapitated Mature Embryo Axis and Agrobacterium Mediated Genetic Transformation of Pigeonpea

A reliable method of plant regeneration has been achieved from decapitated mature embryo axes (DCMEA) explants. Shoots appear directly from explants of genotype T-15-15 when cultured on Maheswaran and Williams (EC₆) basal medium supplemented with N⁶-benzylaminopurine (BAP) and indole-3-acetic acid (IAA) at various combinations. The shoots elongated on half strength Murashige and Skoog (MS) medium fortified with 3 μM gibberellic acid. Elongated shoots were rooted with 80 – 85 % efficiency on half strength MS medium with 0.5 μM indole-3-butyric acid. Survival of plants in the pots was 75 – 80 %. This protocol was used in Agrobacterium mediated transformation. The DCMEA explants were treated independently with two A. tumefaciens (LBA 4404) strains harbouring a binary vector carrying the green fluorescent protein (GFP) and β-glucuronidase (GUS) reporter genes, respectively. Both the strains contained neomycin phosphotransferase selectable marker gene. After co-cultivation, the explants were cultured on EC₆ basal medium supplemented with 5 μM BAP and 1 μM IAA. The selection of putative transformants was on a medium containing 50 mg dm⁻³ kanamycin. Expression of GUS and GFP gene was confirmed by histochemical assay and fluorescence microscopy, respectively. The elongated shoots expressing GFP reporter gene were rooted and transferred to pots for hardening. The integration of GFP gene into the genome of putative transformants was confirmed by Southern blotting. This revised version was published online in July 2006 with corrections to the Cover Date.     

Domoic acid neurotoxicity in hippocampal slice cultures

Summary.  The neurotoxicity of domoic acid was studied in 2–3 week old rat hippocampal slice cultures, derived from 7 day old rat pups. Domoic acid 0.1–100 μM was added to the culture medium for 48 hrs, alone or together with the glutamate receptor antagonists NS-102 (5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3-oxime), NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) or MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), followed by transfer of the cultures to normal medium for additional 48 hrs. Neuronal degeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was monitored and EC₅₀ values estimated by densitometric measurements of the cellular uptake of propidium iodide (PI). The CA1 region was most sensitive to domoic acid, with an EC₅₀ value of 6 μM domoic acid, estimated from the PI-uptake at 72 hrs. Protective effects of 10 μM NBQX against 3 and 10 μM domoic acid were observed for both dentate granule cells and CA1 and CA3c pyramidal cells. NS102 and MK 801 only displayed protective effects when combined with NBQX. MK801 significantly increased the combined neuroprotective effect of NBQX and NS102 against 10 μM domoic acid in both CA1 and FD, but not in CA3. We conclude, that domoic acid neurotoxicity in CA3 and in hippocampal slice cultures in general primarily involves AMPA/kainate receptors. At high concentrations (10 μM domic acid) NMDA receptors are, however, also involved in the toxicity in CA1 and FD. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Role of Prostanoid Production and Receptors in the Regulation of Retinal Endogenous Amino Acid Neurotransmitters by 8-Isoprostaglandin E<Subscript>2</Subscript>, Ex Vivo

The role of enzymes and receptors of the prostanoid pathway in the inhibitory effect of 8-isoprostaglandin E₂ (8-isoPGE₂) on endogenous amino acid neurotransmitter levels was examined, ex vivo. Freshly isolated bovine eyeballs were injected intravitreally with IsoPs, incubated in Krebs buffer for 30 min and retina prepared for HPLC-ECD detection of amino acids. 8-isoPGE₂ attenuated retinal glutamate and its metabolite, glutamine and glycine in a concentration-dependent manner. The non-selective cyclooxygenase (COX)-inhibitor, flurbiprofen, COX-2 selective inhibitor, NS-398 and thromboxane (Tx) synthase inhibitor, furegrelate had no effect on both basal amino acid levels and the inhibitory effects of 8-isoPGE₂ (1–100 μM) on the retinal amino acids. Whereas the TP-receptor antagonist SQ-29548(10 μM) exhibited no effect, SC-19220(EP₁; 30 μM), AH-6809(EP_1–3; 30 μM) and AH-23848(EP₄; 30 μM) reversed the inhibitory effects of 8-isoPGE₂ (0.01–100 μM) on glutamate, glutamine and glycine levels. We conclude that prostanoid EP-receptors regulate the inhibitory effect of 8-isoPGE₂ on basal levels of endogenous amino acids in bovine retina, ex vivo.     

Distribution and morphology of the ampullary organs of the estuarine long-tailed catfish, <Emphasis Type="Italic">Euristhmus lepturus</Emphasis> (Plotosidae,Siluriformes)

Ampullary organs of Euristhmus lepturus occur in high densities along the head and in four parallel pathways along the trunk of the body. Large ampullary pores (125–130 μm) are easily distinguishable from other sensory epithelial pores due to the differences in size and the presence of a collar-like structure. Simple, singular ampullary organs of the head region consist of an ampullary pore connected to a long canal with a diameter of 115–175 μm before terminating as a simple ampulla with an external diameter of 390–480 μm. The ampullary canal is composed of 1–2 layers of flattened squamous epithelial cells, the basement membrane and an interlocking collagen sheath. The innermost cells lining the canal wall are adjoined via tight junctions and numerous desmosomes, as are those of the receptor and supportive cells. Canal wall tissue gives rise to a sensory epithelium containing between 242 and 285 total receptor cells, with an average diameter of 11.7 ± 5.3 μm, intermixed with medially nucleated supportive cells. Each receptor cell (21.38 ± 4.41 μm, height) has an apically positioned nucleus and a luminal surface covered with numerous microvilli. Neural terminals abut the basal region of receptor cells opposite multiple presynaptic bodies and dense mitochondria. Supportive cells extend from the ampullary lumen to the basement membrane, which is adjacent to the complex system of collagen fibres.     

Haemolytic and cytotoxic action of latarcin Ltc2a

Activity and action mechanisms of latarcin 2a (Ltc2a), an antimicrobial peptide from the venom of the spider Lachesana tarabaevi (Zodariidae), were studied in vitro on human cells. Cytotoxicity of Ltc2a for erythrocytes (EC₅₀ = 3.4 μM), leukocytes (EC₅₀ = 19.5 μM) and erythroleukemia K562 cells (EC₅₀ = 3.3 μM) has been found to be primary related to plasma membrane destabilization. Using fluorescently labeled Ltc2a, three common features are found for erythrocytes and K562 cells: pronounced inhomogeneity of cellular response to Ltc2a; complex multistage character of Ltc2a-cell interactions; a positive feedback between Ltc2a binding to plasma membrane and development of toxic effects. Discocyte - echinocyte - spherocyte - ghost is a sequence of Ltc2a-induced transformations of erythrocytes that are accompanied by multistage enhancement of Ltc2a membrane binding, formation of small (ca. 2.0 nm) membrane pores, osmotic imbalance development and reorganization of the pores into large (ca. 13 nm) membrane openings that are preserved in ghosts. Ltc2a induces membrane blebbing and swelling of K562 cells followed by cell death. Cytotoxic action occurs through formation of membrane pores (ca. 3.7 nm) which show greater permeability for anionic than cationic molecules. The pore formation is accompanied with self-assisted Ltc2a internalization and accumulation in mitochondria, mitochondrion inactivation and apoptosis-independent phosphatidylserine externalization.     

Recent improvements in the development of A<Subscript>2B</Subscript> adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis.