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Red blood cells (RBCs) make up the overwhelming majority of cells in the vascular system, spending most of their lives wandering the vast network of vessels that permeate every tissue of our bodies. Therefore, the delivery of any class of therapeutic agent that must stay in the circulatory system may benefit from being carried by RBCs. Toward this direction, we have re-engineered a synthetic liposome with the membranes of RBCs and incorporated a magnetic resonance imaging (MRI) contrast agent gadolinium along with the chemotherapeutic drug doxorubicin (DOX) to form a biomimetic liposome (BML). The BMLs proposed herein consist of biocompatible/biodegradable synthetic phospholipids, which include 1,2-distearoyl

Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC50 = 11.44 μM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings.

BackgroundOmicron a new variant of SARS COV2 was first detected in November 2021. This was believed to be highly transmissible and evade immunity as a result urgent need was felt to screen all positive, identify Omicron cases and isolate them to prevent spread of infection and study their clinico-epidemiological profile.MethodologyAll positive cases detected in state of Rajasthan during November to January beginning were selected for next generation sequencing. Processing was done as per protocol on Ion Torrent S5 system for 1210 samples and bioinformatics analysis was done.ResultsAmong the 1210 samples tested 762(62.9%) were Delta/Delta like and other lineages, 291(24%) were Omicron and 157(12.9%) were invalid or repeat samples. Within a month the proportion of Delta and other variants was reversed, from zero omicron became 81% and delta and other variants 19%, initially all omicron cases were international travelers and their contacts but soon community transmission was seen. Majority of omicron patients were asymptomatic (56.7%) or had mild disease (33%), 9.2% had moderate symptoms and 2(0.7%) had severe disease requiring hospitalization, of which one (0.3%) died and rest (99.7%) recovered. History of vaccination was seen in 81.1%, of previous infection in 43.2%. Among the Omicron cases BA.1 (62.8%) was the predominant lineage followed by BA.2(23.7%) and B.1.529 (13.4%), however rising trends were seen initially for BA.1 and later for BA.2 also.ConclusionIn very short time Omicron has spread in community and has taken over the preexisting Delta/Delta like and other lineages, it evades immunity, but the good part is most of the cases were asymptomatic or had mild disease and mortality rate was very low.

Purpose: Suddenly, many cases of fever with jaundice were reported from Sodala area at Jaipur. This outbreak of acute hepatitis at Jaipur Rajasthan was investigated for aetiology and subsequent phylogenetic analysis. Methods: Blood samples were collected from 106 symptomatic patients of acute hepatitis and 39 pregnant females (with or without symptoms of hepatitis) during an outbreak at Jaipur. The samples were tested for hepatitis A virus (HAV) and hepatitis E virus (HEV) by serological and molecular methods (polymerase chain reaction [PCR]). Sequencing of nested PCR product was done for phylogenetic analysis. Hepatitis B surface antigen (HBs antigen), anti-hepatitis C virus (HCV), anti-Leptospira and anti-scrub typhus IgM enzyme-linked immunosorbent assay (ELISA) was done for patients negative for HEV and HAV. Results: Among 106 symptomatic patients, HEV IgM was positive in 84/106 (79.2%) patients and HEV RNA in 72/106 (67.9%) patients. Among pregnant women, 6/39 (15.4%) were HEV IgM positive and 5/39 (12.8%) for HEV RNA. One (2.5%) pregnant woman died due to hepatitis. All the isolates belonged to genotype 1A of HEV. All HAV, HEV-negative samples were negative for HBs antigen, HCV antibody, Leptospira and scrub typhus IgM ELISA. Conclusion: The outbreak was due to HEV genotype 1A. The municipal water supply was contaminated and sanitary conditions and waste disposal were poor in the area. Boiling of drinking water, fixing the water supply pipes and frequent hand washing helped in controlling the outbreak.

The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two β2 subunits ((α4)3(β2)2 nAChR) harbors two high-affinity "canonical" acetylcholine (ACh) binding sites located in the two α4:β2 intersubunit interfaces and a low-affinity "non-canonical" ACh binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:β2 interfaces), TC-2559 (which binds at the α4:β2 but not at the α4:α4 interface), and (3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:β2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(β2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod-Wyman-Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:β2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(β2)2 nAChR channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain.

Neuronal nicotinic acetylcholine receptors (nAChRs) are promising drug targets to manage several neurological disorders and nicotine addiction. Growing evidence indicates that positive allosteric modulators of nAChRs improve pharmacological specificity by binding to unique sites present only in a subpopulation of nAChRs. Furthermore, nAChR positive allosteric modulators such as NS9283 and CMPI have been shown to potentiate responses of (α4)3(β2)2 but not (α4)2(β2)3 nAChR isoforms. This selective potentiation underlines that the α4:α4 interface, which is present only in the (α4)3(β2)2 nAChR, is an important and promising drug target. In this report we used site-directed mutagenesis to substitute specific amino acid residues and computational analyses to elucidate CMPI's binding mode at the α4:α4 subunit extracellular interface and identified a unique set of amino acid residues that determined its affinity. We found that amino acid residues α4Gly-41, α4Lys-64, and α4Thr-66 were critical for (α4)3(β2)2 nAChR potentiation by CMPI, but not by NS9283, whereas amino acid substitution at α4His-116, a known determinant of NS9283 and of agonist binding at the α4:α4 subunit interface, did not reduce CMPI potentiation. In contrast, substitutions at α4Gln-124 and α4Thr-126 reduced potentiation by CMPI and NS9283, indicating that their binding sites partially overlap. These results delineate the role of amino acid residues contributing to the α4:α4 subunit extracellular interface in nAChR potentiation. These findings also provide structural information that will facilitate the structure-based design of novel therapeutics that target selectively the (α4)3(β2)2 nAChR.

Disposition of beta-amyloid peptide 1-42 (Aβ1-42) in the space around the synapses and formation of Aβ-containing aggregates known as neuritic or senile plaques are hallmark features of neurodegenerative pathologies associated with Alzheimer's disease (AD). While AD is a multifactorial disease that includes other proteinopathies (e.g., hyperphosphorylated tau aggregates) and neurotransmitter disturbances (e.g., loss of cortical cholinergic innervation), Aβ (soluble or in senile plaques) remains the major undisputed factor that contributes to the pathological and behavior presentation of AD. Overproduction of Aβ and mutations in Aβ precursor (amyloid precursor protein) or enzymes involved in Aβ1-42 production and removal (γ secretase/presenilins) have been shown in cases of early onset of AD and produced AD-like pathologies in animal models. In addition, the level of soluble Aβ1-42 has been shown to correlate with cognitive impairment in animal models before the presence of senile plaques or other histological features of AD. However, much still is unknown about the biochemical processes leading to amyloid formation and its relation to the pathogenesis, neuronal damage/dysfunction, and behavioral changes associated with AD. In this article, we review animal models that have been developed to study AD-like pathologies and then provide detailed methodology to develop an acute rat model of Aβ-induced cognitive impairment. We use this model to examine the cognitive-enhancing effect of novel pharmacological interventions targeting nicotinic acetylcholine receptors.

Positive allosteric modulators (PAMs) of nicotinic acetylcholine (ACh) receptors (nAChRs) have potential clinical applications in the treatment of nicotine dependence and many neuropsychiatric conditions associated with decreased brain cholinergic activity, and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI) has been identified as a PAM selective for neuronal nAChRs containing the α4 subunit. In this report, we compare CMPI interactions with low-sensitivity (α4)3(β2)2 and high-sensitivity (α4)2(β2)3 nAChRs, and with muscle-type nAChRs. In addition, we use the intrinsic reactivity of [3H]CMPI upon photolysis at 312 nm to identify its binding sites in Torpedo nAChRs. Recording from Xenopus oocytes, we found that CMPI potentiated maximally the responses of (α4)3(β2)2 nAChR to 10 μM ACh (EC10) by 400% and with an EC50 of ∼1 µM. CMPI produced a left shift of the ACh concentration-response curve without altering ACh efficacy. In contrast, CMPI inhibited (∼35% at 10 µM) ACh responses of (α4)2(β2)3 nAChRs and fully inhibited human muscle and Torpedo nAChRs with IC50 values of ∼0.5 µM. Upon irradiation at 312 nm, [3H]CMPI photoincorporated into each Torpedo [(α1)2β1γδ] nAChR subunit. Sequencing of peptide fragments isolated from [3H]CMPI-photolabeled nAChR subunits established photolabeling of amino acids contributing to the ACh binding sites (αTyr190, αTyr198, γTrp55, γTyr111, γTyr117, δTrp57) that was fully inhibitable by agonist and lower-efficiency, state-dependent [3H]CMPI photolabeling within the ion channel. Our results establish that CMPI is a potent potentiator of nAChRs containing an α4:α4 subunit interface, and that its intrinsic photoreactivy makes it of potential use to identify its binding sites in the (α4)3(β2)2 nAChR.

In this article, an attempt has been made to study the behavior of non-Newtonian viscoelastic model of a Williamson fluid flow problem containing nanoparticles and is assumed to be flowing over a stretching sheet stretched along its surface in both directions with the same constant surface stretching velocity. The boundary layer governing equations of the conservation of mass, conservation of linear momentum and the energy are first modeled into a set of nonlinear coupled partial differential equations along with the appropriate boundary conditions. A numerical study of impact of nanofluid flow over a stretching sheet is tabulated. The basic equations of Williamson fluid are modeled with the help of Navier-Stokes equations for momentum and heat transfer. With the assistance of appropriate similarity transformations these pair of PEDs is transformed into up-to-date system of coupled nonlinear ODEs. These transformed systems of equations are evaluated numerically with the assistance of shooting method using forth order. The dominant physical properties for system of equations of the model that is wall shear stress, and the coefficient of skin friction are acquired. The behavior of nondimensional velocity and thermal flow profiles are discussed for the important involved dimensionless parameters like the Williamson fluid parameter and the nanoparticles volume fraction through tables and graphs.

Receptors containing α4 and β2 subunits are a major neuronal nicotinic acetylcholine receptor (nAChR) subtype in the brain. This receptor plays a critical role in nicotine addiction, with potential smoking cessation therapeutics producing modulation of α4β2 nAChR. In addition, compounds that act as agonists at α4β2 nAChR may be useful for the treatment of pathological pain. Further, as the α4β2 nAChR has been implicated in cognition, therapeutics that act as α4β2 nAChR agonists are also being examined as treatments for cognitive disorders and neurological diseases that impact cognitive function, such as Alzheimer's disease and schizophrenia. This review will cover the molecular in vitro evidence that allosteric modulators of the α4β2 neuronal nAChR provide several advantages over traditional α4β2 nAChR orthosteric ligands. Specifically, we explore the concept that nAChR allosteric modulators allow for greater pharmacological selectivity, while minimizing potential deleterious off-target effects. Further, here we discuss the development and preclinical in vivo behavioral assessment of allosteric modulators at the α4β2 neuronal nAChR as therapeutics for smoking cessation, pathological pain, as well as cognitive disorders and neurological diseases that impact cognitive function. This article is part of the special issue on 'Contemporary Advances in Nicotine Neuropharmacology'.

Disposition of beta-amyloid peptide 1-42 (Aβ1-42) in the space around the synapses and formation of Aβ-containing aggregates known as neuritic or senile plaques are hallmark features of neurodegenerative pathologies associated with Alzheimer's disease (AD). While AD is a multifactorial disease that includes other proteinopathies (e.g., hyperphosphorylated tau aggregates) and neurotransmitter disturbances (e.g., loss of cortical cholinergic innervation), Aβ (soluble or in senile plaques) remains the major undisputed factor that contributes to the pathological and behavior presentation of AD. Overproduction of Aβ and mutations in Aβ precursor (amyloid precursor protein) or enzymes involved in Aβ1-42 production and removal (γ secretase/presenilins) have been shown in cases of early onset of AD and produced AD-like pathologies in animal models. In addition, the level of soluble Aβ1-42 has been shown to correlate with cognitive impairment in animal models before the presence of senile plaques or other histological features of AD. However, much still is unknown about the biochemical processes leading to amyloid formation and its relation to the pathogenesis, neuronal damage/dysfunction, and behavioral changes associated with AD. In this article, we review animal models that have been developed to study AD-like pathologies and then provide detailed methodology to develop an acute rat model of Aβ-induced cognitive impairment. We use this model to examine the cognitive-enhancing effect of novel pharmacological interventions targeting nicotinic acetylcholine receptors.

Summary Total phenolic content, antioxidant and antibacterial activities of Ficus microcarpa aerial roots were determined on methanol extract and its sub-fractions (hexane, ethyl acetate, n-butanol and water). The ethyl acetate fraction possessed the highest amount of phenolic compounds evaluated by Folin-Ciocalteu reagent method. It also showed the highest antioxidant activity when tested the following methods: DPPH, ABTS+ radical-scavenging, superoxide radical scavenging, reducing power and β-carotene linoleic acid bleaching. On the other hand, high phenolics content extracts (ethyl acetate, methanol and n-butanol) showed significantly higher inhibitory activity than low phenolics content extracts (water and hexane) against five tested Gram positive and Gram negative bacteria. Protocatechuic acid, catechol, p-vinylguaiacol, syringol, p-propylphenol, vanillin, p-propylguaiacol, isovanillic acid, 4-n-Propylresorcinol, syringaldehyde and oleanolic acid contained in ethyl acetate fraction were identified by GC-MS and HPLC based on their cochromatography with standard compounds. Protocatechuic acid, catechol and syringol exhibited nearly same antioxidant activity as did by positive control Trolox using the mentioned assays. Results here showed that the ethyl acetate fraction has strong antioxidant and antibacterial activities, which are correlated with its high level of phenolic compounds.

The present study describes the chemical composition, antibacterial and antifungal activities of essential oils from Bidens pilosa, a traditional medicinal plant widely distributed in the subtropics and tropics. The essential oils from the fresh leaves and flowers of B. pilosa were analyzed by GC–MS. Forty-four components were identified, of which β-caryophyllene (10.9% and 5.1%) and τ-cadinene (7.82% and 6.13%) were the main compounds in leaves and flowers, respectively. The oils and aqueous extracts of leaves and flowers were subjected to screening for their possible antioxidant activities by using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene bleaching methods. In the former case, the essential oils from leaves and flowers were found to be superior to all aqueous extracts tested with an IC50 value of 47.5 and 49.7 μg/ml, respectively, whereas all extracts and essential oils seemed to inhibit the oxidation of linoleic acid in the latter case. The oils from B. pilosa exerted significant antibacterial and antifungal activities against six bacteria and three fungal strains. The inhibitory activity of the flower essential oils in Gram-negative bacteria was significantly higher than in Gram-positive. Our findings demonstrate that the essential oils and aqueous extracts of B. pilosa possess antioxidant and antimicrobial activities that might be a natural potential source of preservative used in food and other allied industries.

In Okinawa, leucaena (Leucaena leucocephala de Wit) has great potential as animal feed for its high protein content, but its use has been limited owing to the presence of mimosine (β-[N-(3-hydroxy-4-pyridone)]-α-aminopropionic acid) which, when ingested, causes diseases in animals. A detoxifying enzyme, mimosinase, was purified from leucaena leaves and cDNA was cloned based on the amino acid sequence. During essential oil production and fiber isolation from alpinia (Alpinia zerumbet), large volumes of solid and squeezed wastes are produced and subsequently discarded. An extraction protocol to obtain essential oils, dihydro-5,6-dehydrokawain (DDK) and enriched antioxidant phenolic extracts from fresh leaves or rhizomes of alpinia and its waste was developed.

Industrialised Building System (IBS) has been identified as a potential method to improve overall construction performance in term of quality, cost effectiveness, safety and health, waste reduction and productivity. There are several issues in managing IBS construction projects which lead to delays, poor qualities and cost overrun. This paper aims to analyze the issues in managing the construction phases of IBS projects. Based on this review, it shows that less effective use of IBS in Malaysia construction industry is due to unwillingness of the players to change from conventional system because they are not convinced with the IBS system. Therefore, appropriate bodies should help to increase the contractor’s understanding and prepareness in using IBS system in the construction industry.

Momilactones A (MA) and B (MB) are phytoalexins derived from rice plant (Oryza sativa) and were considered to be a part of the mechanism of rice self-defense system. The present study was to evaluate the comparative efficacies in vitro of antibacterial, fungicidal, antioxidant, and herbicidal activities of MA and MB. In general, MB shows higher antifungal, antibacterial, and herbicidal action than MA, although its antioxidant property was less than MA. In herbicidal trial, the IC50 values of MB against germination, shoot and root elongation of barnyardgrass and monochoria were 40.9, 45.5, and 27.5, and 27.1, 17.3, and 0.9 µg, respectively. For MA, these values were 40.3, 35.6, and 55.1, and 43.9, 24.3, and 0.5 µg, respectively. For antifungal activity, momilactone B (IC50: 1.2, 123.9, and 53.4 µg) exerted significantly greater inhibition than MA (IC50: 78.1, 198.1, and 95.3 µg) against Botrytis cinerea, Fusarium solani, Colletrotrichum gloeosporioides, respectively, except for Fusarium oxysporum ...

This study was carried out to examine the possibility of utilizing Bidens pilosa, a weed widely distributed in the subtropics and the tropics, for weed and plant fungus control. The extract of B. pilosa shows strong phytotoxic action against the growth of Raphanus sativus and Echinochloa crus-galli and antifungal activity against phytopathogens in bioassays. The extract of the roots exhibited plant growth inhibitory activity more than those of the leaves and stems. Fifteen phenolic compounds, including pyrocatechin, salicylic acid, p-vinylguaiacol, dimethoxyphenol, eugenol, 4-ethyl-1,2-benzenediol, iso-vanillin, 2-hydroxy-6-methylbenzaldehyde, vanillin, vanillic acid, p-hydroxybenzoic acid, protocatechuic acid, p-coumaric acid, ferulic acid, and caffeic acid were identified by gas chromatography‐mass spectrometry. Caffeic acid was in the highest amount among the detected substances. Dimethoxyphenol and vanillic acid were found only in the roots of B. pilosa. Ferulic acid and p-hydroxybenzoic acid were higher in the roots than in the leaves and stems. These identified constituents might be responsible for the phytotoxic and fungitoxic activities of B. pilosa.

LY2087101 ([2-[(4-Fluorophenyl)amino]-4-methyl-5-thiazolyl]-3-thienylmethanone) was identified as a positive allosteric modulator (PAM) of nicotinic acetylcholine receptors (nAChR) in a high-throughput screen at Eli Lilly and Company. LY2087101 potentiated α7 and α4β2 nAChRs but not α3β2 nAChR (Broad et al. 2008, J. Pharmacol. Exp. Ther., 318: 1108). Mutational analyses and computer docking simulation with α7 nAChR predicted that PNU120596 and LY2087101 bind to a common site within the transmembrane domain (Young et al. 2008, PNAS 105:14686). In this study, we use mutational analyses and two-electrode voltage-clamp recording from Xenopus oocytes to begin pharmacological characterization of LY208101 interaction with α4β2 nAChR and to identify the molecular determinants of LY2087101 selectivity for α4β2 vs. α3β4 nAChRs. LY2078101 potentiated ACh-induced currents of low ACh sensitivity (α4)3(β2)2 nAChR and high ACh sensitivity (α4)2(β2)3 nAChR with similar potency. However, the maximum potentiation at 10 μM LY2087101 was higher in (α4)3(β2)2 than (α4)2(β2)3 nAChR. Mutational analyses of amino acids contributing to extracellular subunits interface (e.g. α4His114, α4Gln122, and α4Thr124, known selectivity determinants for the (α4)3(β2)2 nAChR selective PAM NS9283; Olsen et al 2013, JBC 288: 35977) had no effect on LY2087101 potentiation. Within the transmembrane domain, mutation at α4Ser285 but not α4Thr261 within the M1 helix significantly reduced LY2087101 potentiation of α4β2 nAChR α4Ser285 and α4Thr261 are equivalent to α7Ser222 and α7Ala225, respectively; two important positions for potentiation of α7 nAChR by LY2087101 and PNU120596. α4Thr261 projects to intra-subunit pocket within the α4 transmembrane helix bundle whereas α4Ser285 is exposed to intra-subunit pocket and inter-subunit space. Ongoing mutational analyses within the M1 transmembrane domain will define the contribution of intra-subunit vs. inter-subunit binding sites on LY2087101 potentiation of α4β2 nAChR.

The α4β2 nicotinic acetylcholine receptor (nAChR) is a potential drug target for treating neuropathological and tobacco use disorders. Positive allosteric modulators (PAMs), which bind o site(s) distinct from the ACh (agonist) binding sites, may provide the required specificity by binding to unique site(s) present only in the α4β2 nAChR. However, the molecular pharmacology of this class of compounds is unclear, and the diversity of PAM binding site(s) have not been determined. In previous work, two radiolabeled α4β2 nAChR PAMs, desformylflustrabromine; ([3H]dFBr, 76 Ci/mmol) and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H- pyrrazol-4-yl) isoxazole ([3H]CMPI, 16 Ci/mmol) have been prepared and their merits as photoprobes established using Torpedo nAChR as a model (Hamouda et al. 2015, Mol. Pharmacol. 88:1; Hamouda et al. 2016, Mol. Pharmacol. 89: 575). Here we expand these studies to α4β2 nAChR. Using membrane fractions isolated from a HEK-293 cell line stably expressing α4β2 nAChR (HEK-α4β2 nAChR), we are examining the effects of orthosteric ligands, ion channel blockers and other PAMs on the equilibrium binding of [3H]dFBr and [3H]CMPI. In addition, we purified α4β2 nAChRs from HEK-α4β2 nAChR membranes using a bromo-acetylcholine bromide affinity column and performed initial [3H]dFBr and [3H]CMPI analytical photolabelings. When ∼100 μg of affinity-purified α4β2 nAChR and 20 μCi of [3H]dFBr (76 Ci/mmol) or [3H]CMPI (16 Ci/mmol) were used, photolysis at 312 nm for 5 min ([3H]dFBr) or 3 min ([3H]CMPI) resulted in photolabeling efficiency of (pmol of 3H incorporated/pmol of α4β2 nAChR) of ∼0.05% and 0.7%, respectively. Additional analytical [3H]dFBr and [3H]CMPI photolabeling in the presence of other nAChR ligands and preparative photolabeling experiments are in progress to identify amino acids contributing to dFBr and CMPI binding sites.

Background Anoctamin 1 (ANO1 or TMEM16A) Ca2+-gated Cl− channels of nociceptor neurons are emerging as important molecular components of peripheral pain transduction. At physiological intracellular Cl− concentrations ([Cl−]i) sensory neuronal Cl− channels are excitatory. The ability of sensory neuronal ANO1 to trigger action potentials and subsequent nocifensive (pain) responses were examined by direct activation with an N-aroylaminothiazole. ANO1 channels are also activated by intracellular Ca2+ ([Ca2+]i) from sensory neuronal TRPV1 (transient-receptor-potential vallinoid 1) ion channels and other noxicant receptors. Thus, sensory neuronal ANO1 can facilitate TRPV1 triggering of action potentials, resulting in enhanced nociception. This was investigated by reducing ANO1 facilitation of TRPV1 effects with: (1) T16A[inh]-A01 ANO1-inhibitor reagent at physiological [Cl−]i and (2) by lowering sensory neuronal [Cl−]i to switch ANO1 to be inhibitory. Results ANO1 effects on action potential firing of mouse dorsal root ganglia (DRG) neurons in vitro and mouse nocifensive behaviors in vivo were examined with an N-aroylaminothiazole ANO1-activator (E-act), a TRPV1-activator (capsaicin) and an ANO1-inhibitor (T16A[inh]-A01). At physiological [Cl−]i (40 mM), E-act (10 µM) increased current sizes (in voltage-clamp) and action potential firing (in current-clamp) recorded in DRG neurons using whole-cell electrophysiology. To not disrupt TRPV1 carried-Ca2+ activation of ANO1 in DRG neurons, ANO1 modulation of capsaicin-induced action potentials was measured by perforated-patch (Amphotericin–B) current-clamp technique. Subsequently, at physiological [Cl−]i, capsaicin (15 µM)-induced action potential firing was diminished by co-application with T16A[inh]-A01 (20 µM). Under conditions of low [Cl−]i (10 mM), ANO1 actions were reversed. Specifically, E-act did not trigger action potentials; however, capsaicin-induced action potential firing was inhibited by co-application of E-act, but was unaffected by co-application of T16A[inh]-A01. Nocifensive responses of mice hind paws were dramatically induced by subcutaneous injections of E-act (5 mM) or capsaicin (50 µM). The nocifensive responses were attenuated by co-injection with T16A[inh]-A01 (1.3 mM). Conclusions An ANO1-activator (E-act) induced [Cl−]i-dependent sensory neuronal action potentials and mouse nocifensive behaviors; thus, direct ANO1 activation can induce pain perception. ANO1-inhibition attenuated capsaicin-triggering of action potentials and capsaicin-induced nocifensive behaviors. These results indicate ANO1 channels are involved with TRPV1 actions in sensory neurons and inhibition of ANO1 could be a novel means of inducing analgesia. Electronic supplementary material The online version of this article (doi:10.1186/s12990-015-0061-y) contains supplementary material, which is available to authorized users.

Mimosine [a-amino-β-(3-hydroxy-4-oxo-l,4-dihydropyridin-1-yl)-propanoic acid] is a major constituent of Leucaena (Leucaena leucocephala de Wit) and responsible for the strong allelopathic potential of the legume tree. Mimosine showed strong herbicidal activities on six plants in a bioassay. All plant parts of Leucaena contain mimosine. The quantity of mimosine in the young leaves and mature seeds was the greatest, 2.66 and 2.38% of dry weight, respectively, while the quantity in the root xylems and xylems was the lowest: 0.18 and 0.11% of dry weight, respectively. Younger plant parts of Leucaena contained greater amounts of mimosine than the mature parts. Mimosine released from young parts and the seeds of Leucaena was proportional to the quantity of soaking water. Compared with the mature seeds, other mature parts of the plant did not give any mimosine through exuding. In soil to which mimosine was added, about 60% of the mimosine was adsorbed in 1-5 days, and only a minor volume of mimosine was decomposed: 5.30 and 0.16% after 1 and 5 days, respectively. Using a mixture of FeCl 3 and mimosine in a 4:6 ratio may be the most effective and economical method of lowering the toxicity of mimosine in Leucaena leaves, giving a safer cattle feed and decreasing the soil toxicity caused by mimosine in areas where Leucaena is grown. On the other hand, the incorporation of mimosine leaves into cultivated soils may be helpful for the biological control of weeds. The findings of this research reveal that mimosine may be useful for the development of bio-herbicides.

連合農学研究科博士論文(農学) ; 学位取得日: 平成20年3月14日 鹿児島大学