Choline Transport Research Articles

Protein kinaseC promotes choline transporter‑like protein1 function via improved cell surface expression in immortalized human hepatic cells.

Choline is used to synthesize phospholipids and a lack of choline induces a number of liver-related diseases, including non-alcoholic steatohepatitis. The current study characterized the choline uptake system, at molecular and functional levels, in the immortalized human hepatic cell line, Fa2N-4, to identify the specific choline transporter involved in choline uptake. The present study also assesed whether choline deficiency or the inhibited choline uptake affected cell viability and apoptosis. Reverse transcription-quantitative polymerase chain reaction (PCR) revealed choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein expression in Fa2N-4 cells. [Methyl-3H]choline studies revealed choline uptake was saturable and mediated by a single transport system that functioned in a Na+-independent but pH-dependent manner, which was similar to CTL1. Hemicholinium-3 (HC-3), which is a choline uptake inhibitor, and choline deficiency inhibited cell viability, increased caspase-3 and −7 activities, and increased fluorescein isothiocyanate-Annexin V immunofluorescent staining indicated apoptosis. Immunofluorescent staining also revealed CTL1 and CTL2 localized in plasma and mitochondrial membranes, respectively. [Methyl-3H]choline uptake was enhanced by a protein kinase C (PKC) activator, phorbol-12-myristate 13-acetate (PMA). Immunofluorescence staining and western blot analysis demonstrated increased CTL1 expression on the cell membrane following PMA treatment. The results of current study indicated that extracellular choline is primarily transported via CTL1, relying on a direct H+ gradient that functions as a driving force in Fa2N-4 cells. Furthermore, it was hypothesized that CTL1 and the choline uptake system are strongly associated with cell survival, and that the choline uptake system is modulated by PKC signaling via increased CTL1 expression on the cell surface. These findings provide further insights into the pathogenesis of liver disease involving choline metabolism.

Functional Expression of Choline Transporter-Like Protein 1 in LNCaP Prostate Cancer Cells: A Novel Molecular Target.

Prostate cancer is one of the most common cancers in men. Choline PET or PET/CT has been used to visualize prostate cancer, and high levels of choline accumulation have been observed in tumors. However, the uptake system for choline and the functional expression of choline transporters in prostate cancer are not completely understood. In this study, the molecular and functional aspects of choline uptake were investigated in the LNCaP prostate cancer cell line along with the correlations between choline uptake and cell viability in drug-treated cells. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA were highly expressed in LNCaP cells. CTL1 and CTL2 were located in the plasma membrane and mitochondria, respectively. [3H]Choline uptake was mediated by a single Na+-independent, intermediate-affinity transport system in the LNCaP cells. The anticancer drugs, flutamide and bicalutamide, inhibited cell viability and [3H]choline uptake in a concentration-dependent manner. The correlations between the effects of these drugs on cell viability and [3H]choline uptake were significant. Caspase-3/7 activity was significantly increased by both flutamide and bicalutamide. Furthermore, these drugs decreased CTL1 expression in the prostate cancer cell line. These results suggest that CTL1 is functionally expressed in prostate cancer cells and are also involved in abnormal proliferation. Identification of this CTL1-mediated choline transport system in prostate cancer cells provides a potential new therapeutic target for the treatment of this disease.

Acetylcholine Receptor Activation as a Modulator of Glioblastoma Invasion.

Grade IV astrocytomas, or glioblastomas (GBMs), are the most common malignant primary brain tumor in adults. The median GBM patient survival of 12–15 months has remained stagnant, in spite of treatment strategies, making GBMs a tremendous challenge clinically. This is at least in part due to the complex interaction of GBM cells with the brain microenvironment and their tendency to aggressively infiltrate normal brain tissue. GBMs frequently invade supratentorial brain regions that are richly innervated by neurotransmitter projections, most notably acetylcholine (ACh). Here, we asked whether ACh signaling influences the biology of GBMs. We examined the expression and function of known ACh receptors (AChRs) in large GBM datasets, as well as, human GBM cell lines and patient-derived xenograft lines. Using RNA-Seq data from the “The Cancer Genome Atlas” (TCGA), we confirmed the expression of AChRs and demonstrated the functionality of these receptors in GBM cells with time-lapse calcium imaging. AChR activation did not alter cell proliferation or migration, however, it significantly increased cell invasion through complex extracellular matrices. This was due to the enhanced activity of matrix metalloproteinase-9 (MMP-9) from GBM cells, which we found to be dependent on an intracellular calcium-dependent mechanism. Consistent with these findings, AChRs were significantly upregulated in regions of GBM infiltration in situ (Ivy Glioblastoma Atlas Project) and elevated expression of muscarinic AChR M3 correlated with reduced patient survival (TCGA). Data from the Repository for Molecular Brain Neoplasia Data (REMBRANDT) dataset also showed the co-expression of choline transporters, choline acetyltransferase, and vesicular acetylcholine transporters, suggesting that GBMs express all the proteins required for ACh synthesis and release. These findings identify ACh as a modulator of GBM behavior and posit that GBMs may utilize ACh as an autocrine signaling molecule.

Functional Expression of Choline Transporters in the Blood-Brain Barrier.

Cholinergic neurons in the central nervous system play a vital role in higher brain functions, such as learning and memory. Choline is essential for the synthesis of the neurotransmitter acetylcholine by cholinergic neurons. The synthesis and metabolism of acetylcholine are important mechanisms for regulating neuronal activity. Choline is a positively charged quaternary ammonium compound that requires transporters to pass through the plasma membrane. Currently, there are three groups of choline transporters with different characteristics, such as affinity for choline, tissue distribution, and sodium dependence. They include (I) polyspecific organic cation transporters (OCT1-3: SLC22A1-3) with a low affinity for choline, (II) high-affinity choline transporter 1 (CHT1: SLC5A7), and (III) choline transporter-like proteins (CTL1-5: SLC44A1-5). Brain microvascular endothelial cells, which comprise part of the blood–brain barrier, take up extracellular choline via intermediate-affinity choline transporter-like protein 1 (CTL1) and low-affinity CTL2 transporters. CTL2 is responsible for excreting a high concentration of choline taken up by the brain microvascular endothelial cells on the brain side of the blood–brain barrier. CTL2 is also highly expressed in mitochondria and may be involved in the oxidative pathway of choline metabolism. Therefore, CTL1- and CTL2-mediated choline transport to the brain through the blood–brain barrier plays an essential role in various functions of the central nervous system by acting as the rate-limiting step of cholinergic neuronal activity.

Biomimetic Magnetic Nanocarriers Drive Choline Kinase Alpha Inhibitor inside Cancer Cells for Combined Chemo-Hyperthermia Therapy.

Choline kinase α1 (ChoKα1) has become an excellent antitumor target. Among all the inhibitors synthetized, the new compound Ff35 shows an excellent capacity to inhibit ChoKα1 activity. However, soluble Ff35 is also capable of inhibiting choline uptake, making the inhibitor not selective for ChoKα1. In this study, we designed a new protocol with the aim of disentangling whether the Ff35 biological action is due to the inhibition of the enzyme and/or to the choline uptake. Moreover, we offer an alternative to avoid the inhibition of choline uptake caused by Ff35, since the coupling of Ff35 to novel biomimetic magnetic nanoparticles (BMNPs) allows it to enter the cell through endocytosis without interacting with the choline transporter. This opens the possibility of a clinical use of Ff35. Our results indicate that Ff35-BMNPs nanoassemblies increase the selectivity of Ff35 and have an antiproliferative effect. Also, we demonstrate the effectiveness of the tandem Ff35-BMNPs and hyperthermia.

Repetitive mild concussion in subjects with a vulnerable cholinergic system: Lasting cholinergic-attentional impairments in CHT+/- mice.

Previous research emphasized the impact of traumatic brain injury on cholinergic systems and associated cognitive functions. Here we addressed the converse question: Because of the available evidence indicating cognitive and neuronal vulnerabilities in humans expressing low-capacity cholinergic systems or with declining cholinergic systems, do injuries cause more severe cognitive decline in such subjects, and what cholinergic mechanisms contribute to such vulnerability? Using mice heterozygous for the choline transporter (CHT+/- mice) as a model for a limited cholinergic capacity, we investigated the cognitive and neuronal consequences of repeated, mild concussion injuries (rmCc). After five rmCc, and compared with wild type (WT) mice, CHT+/- mice exhibited severe and lasting impairments in sustained attention performance, consistent with effects of cholinergic losses on attention. However, rmCc did not affect the integrity of neuronal cell bodies and did not alter the density of cortical synapses. As a cellular mechanism potentially responsible for the attentional impairment in CHT+/- mice, we found that rmCc nearly completely attenuated performance-associated, CHT-mediated choline transport. These results predict that subjects with an already vulnerable cholinergic system will experience severe and lasting cognitive-cholinergic effects after even relatively mild injuries. If confirmed in humans, such subjects may be excluded from, or receive special protection against, activities involving injury risk. Moreover, the treatment and long-term outcome of traumatic brain injuries may benefit from determining the status of cholinergic systems and associated cognitive functions. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Abstract 2064: CHRM1-mediated acetylcholine signaling protects prostate cancer cells from the chemotherapy agent docetaxel

Abstract Background: Chemotherapy-resistant prostate cancer (PCa) remains a leading cause of cancer-related deaths in advanced PCa patients. The current study investigates the role of acetylcholine (ACh) signaling in the development and maintenance of chemotherapy resistance in PCa. Methods: Human PCa cell cultures were used to harvest total RNA and protein for qPCR and Western blot analyses, respectively. The chemotherapy drug docetaxel’s (DTX) ability to induce apoptosis was measured with crystal violet survival assays and Western blot analysis of pro-apoptotic proteins. Results: Treatment of the androgen-independent human PCa cell lines 22Rv1 and DU145 with a low concentration of DTX at 1 nM increases the expression of the key ACh production and secretion enzymes choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and the high-affinity choline transporter 1 (CHT1), which is accompanied by a 4-fold increase in ACh secretion. These genes also show increased expression in DTX-resistant 22Rv1 cells. The muscarinic receptor system further shows alterations in gene expression with low-concentration DTX treatment and acquired DTX resistance. Specifically, across all 5 muscarinic cholinergic receptors (CHRM1-5), the expression of CHRM1 is increased in both circumstances. Treatment of both DTX-sensitive and -resistant cell lines with a combination of DTX and the CHRM1 antagonist dicyclomine decreases cell survival, lowers DTX IC50 values (DU145: 62%; 22Rv1: 48%; DTX-resistant 22Rv1: 23%), and enhances the expression of the pro-apoptotic proteins cleaved caspase-3 and cleaved PARP1 compared to treatment with DTX alone. This is paralleled by the observations that treatment with the ACh mimetic carbachol (CCh) protects cells from DTX treatment. Mechanistically, ACh signaling protects PCa cells from DTX through activation of the extracellular signal-regulated kinase / cAMP response element binding protein 1 (ERK/CREB) pathway. Treatment of 22Rv1 cells with CCh increases levels of the active phosphorylated forms of both ERK1/2 and CREB within 30 min. This increase is inhibited by the pan muscarinic receptor antagonist atropine and is not affected by the pan nicotinic receptor antagonist tubocurarine. Treatment of 22Rv1 cells with a combination of DTX and either the ERK inhibitor UO-126 or the CREB inhibitor 666-15 decreases cell survival in a synergistic manner. Conclusion: DTX induces an enhancement of ACh signaling in PCa cells, which may contribute to the development and maintenance of DTX resistance through CHRM1 and the downstream ERK/CREB pathway. Antagonism of CHRM1 and its downstream effectors may be useful in preventing and reverting chemotherapy resistance in PCa. Citation Format: Tyler Bland, Karen Vo, Baron Bechtold, Boyang Wu. CHRM1-mediated acetylcholine signaling protects prostate cancer cells from the chemotherapy agent docetaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2064.

Central cholinergic neuronal degeneration promotes the development of postoperative cognitive dysfunction

Postoperative cognitive dysfunction (POCD) is consistently associated with increased morbidity and mortality. However, its mechanism remains poorly understood. We hypothesized that central cholinergic neuronal degeneration facilitates the development of POCD. The impact of anesthesia/surgery (appendectomy) on learning and memory and the levels of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), vesicular acetylcholine transporter (VAChT), and choline transporter (CHT) in adult and aged mice were measured. Separate cohorts were analyzed after pretreatment with donepezil, an AChE inhibitor, in aged mice or with murine-p75-saporin (mu-p75-sap), a cholinergic-specific immunotoxin, in adult mice. Morris Water Maze was used to measure the learning and memory changes after anesthesia/surgery. Western blot was used to measure the changes in the protein levels of the biomarkers of the central cholinergic system. We found that anesthesia/surgery-induced memory decline and attenuation of central cholinergic biomarkers (ChAT and VAChT) in aged mice but not in adult mice. Donepezil pretreatment reduced central cholinergic impairment in the aged mice and prevented learning and memory declines after anesthesia/surgery. In contrast, when central cholinergic neurons were pre-injured with mu-p75-sap, cognitive dysfunction developed in the adult mice after anesthesia/surgery. These data suggest that central cholinergic neuronal degeneration facilitates the development of POCD.

Hepatocellular Carcinomas With Mutational Activation of Beta-Catenin Require Choline and Can Be Detected by Positron Emission Tomography

In one-third of hepatocellular carcinomas (HCCs), cancer cells have mutations that activate β-catenin pathway. These cells have alterations in glutamine, bile, and lipid metabolism. We investigated whether positron emission tomography (PET) imaging allows identification of altered metabolic pathways that might be targeted therapeutically. We studied mice with activation of β-catenin in liver (Apcko-liv mice) and male C57Bl/6 mice given injections of diethylnitrosamine, which each develop HCCs. Mice were fed a conventional or a methionine- and choline-deficient diet or a choline-deficient (CD) diet. Choline uptake and metabolism in HCCs were analyzed by micro-PET imaging of mice; livers were collected and analyzed by histologic, metabolomic, messenger RNA quantification, and RNA-sequencing analyses. Fifty-two patients with HCC underwent PET imaging with 18F-fluorodeoxyglucose, followed by 18F-fluorocholine tracer metabolites. Human HCC specimens were analyzed by immunohistochemistry, quantitative polymerase chain reaction, and DNA sequencing. We used hepatocytes and mouse tumor explants for studies of incorporation of radiolabeled choline into phospholipids and its contribution to DNA methylation. We analyzed HCC progression in mice fed a CD diet. Livers and tumors from Apcko-liv mice had increased uptake of dietary choline, which contributes to phospholipid formation and DNA methylation in hepatocytes. In patients and in mice, HCCs with activated β-catenin were positive in 18F-fluorocholine PET, but not 18F-fluorodeoxyglucose PET, and they overexpressed the choline transporter organic cation transporter 3. The HCC cells from Apcko-liv mice incorporated radiolabeled methyl groups of choline into phospholipids and DNA. In Apcko-livmice, the methionine- and choline-deficient diet reduced proliferation and DNA hypermethylation of hepatocytes and HCC cells, and the CD diet reduced long-term progression of tumors. In mice and humans, HCCs with mutations that activate β-catenin are characterized by increased uptake of a fluorocholine tracer, but not 18F-fluorodeoxyglucose, revealed by PET. The increased uptake of choline by HCCs promotes phospholipid formation, DNA hypermethylation, andhepatocyte proliferation. In mice, the CD diet reverses theseeffects and promotes regression of HCCs that overexpress β-catenin.

Choline uptake and metabolism modulate macrophage immunity

Abstract Choline is a vitamin-like nutrient that is taken up via specific transporters and metabolized by choline kinase (ChoKα) which converts it to phosphocholine needed for de novo synthesis of phosphatidylcholine (PC), the main phospholipid of cellular membranes. We found that Toll-like receptor (TLR) activation enhances choline uptake by macrophages through induction of the choline transporter CTL1. Choline is rapidly phosphorylated and mobilized to the Kennedy pathway leading to increased cellular phosphatidylcholine production. Inhibition of choline uptake or phosphorylation attenuated NLRP3 inflammasome activation and IL-1β and IL-18 production in stimulated macrophages. Mechanistically, reduced choline uptake altered mitochondrial lipid profile, attenuated mitochondrial ATP synthesis and activated the energy sensor AMP-activated protein kinase (AMPK). By potentiating mitochondrial recruitment of DRP1, AMPK stimulates mitophagy, which contributes to termination of NLRP3 inflammasome activation. Correspondingly, pharmacological ChoKα inhibition ameliorated acute and chronic macrophage-dependent inflammation in vivo.

Choline Uptake and Metabolism Modulate Macrophage IL-1β and IL-18 Production

Choline is a vitamin-like nutrient that is taken up via specific transporters and metabolized by choline kinase, which converts it to phosphocholine needed for de novo synthesis of phosphatidylcholine (PC), the main phospholipid of cellular membranes. We found that Toll-like receptor (TLR) activation enhances choline uptake by macrophages and microglia through induction of the choline transporter CTL1. Inhibition of CTL1 expression or choline phosphorylation attenuated NLRP3 inflammasome activation and IL-1β and IL-18 production in stimulated macrophages. Mechanistically, reduced choline uptake altered mitochondrial lipid profile, attenuated mitochondrial ATP synthesis, and activated the energy sensor AMP-activated protein kinase (AMPK). By potentiating mitochondrial recruitment of DRP1, AMPK stimulates mitophagy, which contributes to termination of NLRP3 inflammasome activation. Correspondingly, choline kinase inhibitors ameliorated acute and chronic models of IL-1β-dependent inflammation.

Silica nanoparticle-exposure during neuronal differentiation modulates dopaminergic and cholinergic phenotypes in SH-SY5Y cells

BackgroundSilica-ε-polycaprolactone-nanoparticles (SiPCL-NPs) represent a promising tool for laser-tissue soldering in the brain. After release of the SiPCL-NPs in the brain, neuronal differentiation might be modulated. The present study was performed to determine effects of SiPCL-NP-exposure at different stages of neuronal differentiation in neuron-like SH-SY5Y cells. The resulting phenotypes were analyzed quantitatively and signaling pathways involved in neuronal differentiation and degeneration were studied. SH-SY5Y cells were differentiated with all-trans retinoic acid or staurosporine to obtain predominantly cholinergic or dopaminergic neurons. The resulting phenotype was analyzed at the end of differentiation with and without the SiPCL-NPs given at various times during differentiation.ResultsExposure to SiPCL-NPs before and during differentiation led to a decreased cell viability of SH-SY5Y cells depending on the differentiation protocol used. SiPCL-NPs co-localized with the neuronal marker β-3-tubulin but did not alter the morphology of these cells. A significant decrease in the number of tyrosine hydroxylase (TH) immunoreactive neurons was found in staurosporine-differentiated cells when SiPCL-NPs were added at the end of the differentiation. TH-protein expression was also significantly downregulated when SiPCL-NPs were applied in the middle of differentiation. Protein expression of the marker for the dopamine active transporter (DAT) was not affected by SiPCL-NPs. SiPCL-NP-exposure predominantly decreased the expression of the high-affinity choline transporter 1 (CHT1) when the NPs were given before the differentiation. Pathways involved in neuronal differentiation, namely Akt, MAP-K, MAP-2 and the neurodegeneration-related markers β-catenin and GSK-3β were not altered by NP-exposure.ConclusionsThe decrease in the number of dopaminergic and cholinergic cells may implicate neuronal dysfunction, but the data do not provide evidence that pathways relevant for differentiation and related to neurodegeneration are impaired.

A Bioinspired Platform for Effective Delivery of Protein Therapeutics to the Central Nervous System.

Central nervous system (CNS) diseases are the leading cause of morbidity and mortality; their treatment, however, remains constrained by the blood-brain barrier (BBB) that impedes the access of most therapeutics to the brain. A CNS delivery platform for protein therapeutics, which is achieved by encapsulating the proteins within nanocapsules that contain choline and acetylcholine analogues, is reported herein. Mediated by nicotinic acetylcholine receptors and choline transporters, such nanocapsules can effectively penetrate the BBB and deliver the therapeutics to the CNS, as demonstrated in mice and non-human primates. This universal platform, in general, enables the delivery of any protein therapeutics of interest to the brain, opening a new avenue for the treatment of CNS diseases.

Choline transport for phospholipid synthesis: An emerging role of choline transporter-like protein 1.

This review will provide a summary of recent advances in choline transport research and highlight important novel areas of focus in the field.

Acetylcholine receptors in the equatorial region of intrafusal muscle fibres modulate mouse muscle spindle sensitivity.

Acetylcholine receptors are aggregated in the central regions of intrafusal muscle fibres. Single unit muscle spindle afferent responses from isolated mouse extensor digitorum longus muscle were recorded in the absence of fusimotor input to ramp and hold stretches as well as to sinusoidal vibrations in the presence and absence of the acetylcholine receptor blockers d-tubocurarine andα-bungarotoxin. Proprioceptive afferent responses to both types of stretch were enhanced in the presence of either blocker. Blocking acetylcholine uptake and vesicular acetylcholine release by hemicholinium-3 also enhanced stretch-evoked responses. These results represent the first evidence that acetylcholine receptors negatively modulate muscle spindle responses to stretch. The data support the hypothesis that the sensory nerve terminal is able to release vesicles to fine-tune proprioceptive afferent sensitivity. Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibres, called intrafusal fibres, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. Previously it was shown that acetylcholine receptors (AChR) are concentrated in the equatorial region at the contact site between the sensory neuron and the intrafusal muscle fibre. To address the function of these AChRs, single unit sensory afferents were recorded from an isolated mouse extensor digitorum longus muscle in the absence of γ-motoneuron activity. Specifically, we investigated the responses of individual sensory neurons to ramp-and-hold stretches and sinusoidal vibrations before and after the addition of the competitive and non-competitive AChR blockers d-tubocurarine and α-bungarotoxin, respectively. The presence of either drug did not affect the resting action potential discharge frequency. However, the action potential frequencies in response to stretch were increased. In particular, frequencies of the dynamic peak and dynamic index to ramp-and-hold stretches were significantly higher in the presence of either drug. Treatment of muscle spindle afferents with the high-affinity choline transporter antagonist hemicholinium-3 similarly increased muscle spindle afferent firing frequencies during stretch. Moreover, the firing rate during sinusoidal vibration stimuli at low amplitudes was higher in the presence of α-bungarotoxin compared to control spindles also indicating an increased sensitivity to stretch. Collectively these data suggest a modulation of the muscle spindle afferent response to stretch by AChRs in the central region of intrafusal fibres possibly fine-tuning muscle spindle sensitivity.

Elucidating the pathogenesis of esophageal adenocarcinoma through meta-analysis of public data.

71 Background: Esophageal adenocarcinoma (EAC) is the sixth most common cause of cancer-related deaths worldwide. It commonly arises in the setting of reflux disease and Barett’s esophagus. It remains incurable and holds a poor prognosis. Dissecting the genetic signature of EAC can pave new therapeutic avenues. Methods: We employed our Search Tag Analyze Resource (STARGEO) platform to conduct meta-analysis using the National Center for Biotechnology’s (NCBI) Gene Expression Omnibus (GEO). We tagged 151 tumor samples from EAC patients and 62 normal esophageal samples as a control. We then analyzed the signature in Ingenuity Pathway Analysis, restricting genes that showed statistical significance (p < 0.05) and an absolute experimental log ratio greater than 0.15 between tumor and control. Results: Our analysis revealed granulocyte adhesion and diapedesis and FXR/RXR signaling as top canonical pathways. TGFB1, TNF, and beta-estradiol were top upstream regulators with predicted activation. TGFB1 and TNF expression have been correlated with poor prognosis in EAC. Also, beta-estradiol has tumorigenic activity in several cancers but has not been investigated in EAC. Among the top upregulated genes were oncogenic genes such as tetraspanin 8, the antiapoptotic factor OLFM4, and the protease cathepsin E (CTSE). SPINK1, a trypsin inhibitor with recently suggested role in cancer, and the choline transporter SLC44A4, a drug target for pancreatic cancer, were also upregulated. The most downregulated genes included alcohol dehydrogenase 7, associated with EAC in alcohol-drinkers, and the pro-apoptotic gene CRCT1. We also found downregulation of the serine peptidase inhibitor SPINK7. SPINK7 is involved in maintaining epithelial-barrier integrity and is implicated in eosinophilic esophagitis pathogenesis. Lastly, there was downregulation of the candidate tumor suppressor gene transglutaminase 3. Conclusions: Despite screening efforts, EAC incidence and mortality continues to increase as does the need for better treatment. This meta-analysis defines the significant gene expression changes within causal disease processes to provide markers for detection, prognostic insight, and therapeutic value for EAC.

コリントランスポーターを標的とした抗腫瘍活性を有する植物由来天然有機化合物の探索

コリントランスポーターを標的とした抗腫瘍活性を有する植物由来天然有機化合物の探索

Drosophila Choline transporter non-canonically regulates pupal eclosion and NMJ integrity through a neuronal subset of mushroom body

Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. Till date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. In this study, we report a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. We demonstrate that knockdown of ChT in a sub-type of MB neurons called α/β core (α/βc) and ϒ neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in α/βc and ϒ neurons nor by knockdown of ChT in cholinergic neurons. Thus, our study provides insights into non-canonical roles of ChT in MB.

Tetanic Facilitation of Neuromuscular Transmission by Adenosine A2A and Muscarinic M1 Receptors is Dependent on the Uptake of Choline via High-Affinity Transporters

Background/Aims: In this study, we evaluated the functional impact of facilitatory presynaptic adenosine A_2A and muscarinic M₁ receptors in the recovery of neuromuscular tetanic depression caused by the blockage of high-affinity choline transporter (HChT) by hemicholinium-3 (HC-3), a condition that mimics a myasthenia-like condition. Methods: Rat diaphragm preparations were indirectly stimulated via the phrenic nerve trunk with 50-Hz frequency trains, each consisting of 500–750 supramaximal intensity pulses. The tension at the beginning (A) and at the end (B) of the tetanus was recorded and the ratio (R) B/A calculated. Results: Activation of A_2A and M₁ receptors with CGS21680 (CGS; 2 nmol/L) and McN-A-343c (McN; 3 μmol/L) increased R values. Similar facilitatory effects were obtained with forskolin (FSK; 3 μmol/L) and phorbol 12-myristate 13-acetate (PMA; 10 μmol/L), which activate adenylate cyclase and protein kinase C respectively. HC-3 (4 μmol/L) decreased transmitter exocytosis measured by real-time videomicroscopy with the FM4-64 fluorescent dye and prevented the facilitation of neuromuscular transmission caused by CGS, McN, and FSK, with a minor effect on PMA. The acetylcholinesterase inhibitor, neostigmine (NEO; 0.5 μmol/L), also decreased transmitter exocytosis. The paradoxical neuromuscular tetanic fade caused by NEO (0.5 μmol/L) was also prevented by HC-3 (4 μmol/L) and might result from the rundown of the positive feedback mechanism operated by neuronal nicotinic receptors (blocked by hexamethonium, 120 μmol/L). Conclusion: Data suggest that the recovery of tetanic neuromuscular facilitation by adenosine A_2A and M₁ receptors is highly dependent on HChT activity and may be weakened in myasthenic patients when HChT is inoperative.

Identification and characterization of a carnitine transporter in Acinetobacter baumannii.

The opportunistic pathogen Acinetobacter baumannii is able to grow on carnitine. The genes encoding the pathway for carnitine degradation to the intermediate malic acid are known but the transporter mediating carnitine uptake remained to be identified. The open reading frame HMPREF0010_01347 (aci01347) of Acinetobacter baumannii is annotated as a gene encoding a potential transporter of the betaine/choline/carnitine transporter (BCCT) family. To study the physiological function of Aci01347, the gene was deleted from A. baumannii ATCC 19606. The mutant was no longer able to grow on carnitine as sole carbon and energy source demonstrating the importance of this transporter for carnitine metabolism. Aci01347 was produced in Escherichia coli MKH13, a strain devoid of any compatible solute transporter, and the recombinant E. coli MKH13 strain was found to take up carnitine in an energy‐dependent fashion. Aci01347 also transported choline, a compound known to be accumulated under osmotic stress. Choline transport was osmolarity‐independent which is consistent with the absence of an extended C‐terminus found in osmo‐activated BCCT. We propose that the Aci01347 is the carnitine transporter mediating the first step in the growth of A. baumannii on carnitine.