Molecular cloning and characterization of a murine hemicholinium-3-sensitive choline transporter

2001 ◽  
Vol 29 (6) ◽  
pp. 711-716 ◽  
Author(s):  
S. Apparsundaram ◽  
S. M. Ferguson ◽  
R. D. Blakely
Keyword(s):  
Spinal Cord ◽  
Amino Acid ◽  
Genomic Dna ◽  
Cholinergic Neurons ◽  
Membrane Vesicles ◽  
Predicted Amino Acid Sequence ◽  
Sequence Information ◽  
Choline Uptake ◽  
Choline Transporter ◽  
Mouse Tissues

In cholinergic neurons, a specific requirement for precursor choline in the biosynthesis of acetylcholine (ACh) is thought to be sustained by a presynaptic, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). This transporter exhibits micromolar affinity for choline and transport activity is Na+- and Cl−-dependent. Based on the sequence information available with the recent cloning of rat and human CHTs [Okuda, Haga, Kanai, Endou, Ishihara and Katsura (2000) Nat. Neurosci. 3, 120–125; Apparsundaram, Ferguson, George Jr and Blakely (2000) Biochem. Biophys. Res. Commun. 276, 862–867; Okuda and Haga (2000) FEBS Lett. 484, 92–97], we have identified a murine CHT orthologue (mCHT) by reverse transcriptase-PCR of spinal cord mRNA and confirmed this sequence using assembled mouse genomic DNA. Inferred splice junctions for mCHT exons are conserved with those of hCHT. The mCHT cDNA encodes a protein of 580 amino acids with 93 % and 98 % amino acid identity to human CHT and rat CHT1, respectively. Hydropathy analysis of the predicted amino acid sequence of mCHT indicates a protein containing 13 transmembrane domains (TMDs), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analysis of mouse tissues reveals the expression of mCHT as a single transcript of ~ 5 kb with highest expression in regions that are rich in cholinergic cell bodies, e.g. the spinal cord, brainstem, mid-brain and striatum, whereas hybridization signals are absent in regions lacking cholinergic soma, e.g. the cerebellum and kidney. Expression of mCHT in COS-7 cells results in high-affinity [3H]HC-3-binding sites (Kd = 5 nM), and Na+- and Cl−-dependent HC-3-sensitive choline uptake (Km = 2 μM), assessed in resealed membrane vesicles. The availability of cloned, functional mCHT and its cognate genomic DNA should prove useful for studies of mCHT regulation and should open possibilities for evaluation of CHT dysfunction in murine models.

scholarly journals Distinct distribution of vimentin and cytokeratin in Xenopus oocytes and early embryos

1992 ◽  
Vol 101 (1) ◽  
pp. 151-160 ◽  
Author(s):  
N.P. Torpey ◽  
J. Heasman ◽  
C.C. Wylie
Keyword(s):  
Spinal Cord ◽  
Amino Acid ◽  
Xenopus Oocytes ◽  
Germ Plasm ◽  
Predicted Amino Acid Sequence ◽  
Larval Stages ◽  
Functional Differences ◽  
Early Embryos ◽  
Strong Staining ◽  
Distinct Distribution

We report the identity of a major component of Triton-insoluble extracts from Xenopus oocytes and early embryos. In a previous paper we showed that an antibody, Z9, cross-reacts with two polypeptides from such extracts (Mr 56,000 and 57,000) as well as Xenopus vimentin. Direct microsequencing of the Mr 57,000 protein shows near identity of three tryptic fragments with regions of the predicted amino acid sequence of XCK1(8), a basic cytokeratin whose mRNA is known to be expressed in Xenopus oocytes. We have raised an antibody, CK7, against a fusion protein generated from this cDNA. The specificity of this antibody has been tested using 1- and 2-dimensional immunoblotting, which show that it is specific for the Mr 56,000 and 57,000 proteins, suggesting that these two proteins may be the products of two non-allelic XCK1(8) genes. The antibody does not cross-react with vimentin. We have used CK7 to follow the distribution of XCK1(8) throughout development by immunoblotting and immunocytochemistry. In larval stages, strong staining is seen in the notocord, the apical epithelia of the gut, the mesentery, and a few cells in the spinal cord. In oocytes and early embryos, two distinct intermediate filament (IF) networks can be distinguished: a cortical cytokeratin network, and a deeper vimentin one. In addition, the oocyte germ plasm stains with Z9 but not CK7. We propose that such distinct distributions of each IF protein reflect functional differences during early development.

scholarly journals Syntheses and In-vitro Evaluation of Tetrahydroaminoacridine (THA) Based Analogues as High Affinity Choline Transporter (HAChT) Imaging Probe

2016 ◽  
Vol 17 (2) ◽  
pp. 97-102
Author(s):  
Mohammad Anwar Ul Azim ◽  
Takashi Kozaka ◽  
Izumi Uno ◽  
Daisuke Miwa ◽  
Yoji Kitamura ◽  
...  
Keyword(s):  
Binding Assay ◽  
Research Work ◽  
Cholinergic Neurons ◽  
Choline Uptake ◽  
Choline Transporter ◽  
High Affinity ◽  
20 Nm ◽  
High Affinity Choline Transporter

Introduction: In cholinergic neurons, high affinity choline uptake (HACU) by the high affinity choline transporter (HAChT) is a rate-limiting and regulatory step for the synthesis of Acetylcholine (Ach).Thus, HAChT appear to be a relatively specific presynaptic marker for cholinergic neurons in Alzheimer’s disease.Objectives: The principle objective of the study is to check the affinity of tetrahydroaminoacridine (THA) derivatives for HAChT. Another objective of the research work is to clarify whether the hemicholinium-3 (ChT inhibitor) and HACU enhancer molecules share the same binding sites or not.Materials and Methods: The inhibition activities of tacrine, the 2,3-dimethylfuran derivative of tacrine (DMTA) and their corresponding 2-oxo-1-pyrrolidineacetyl derivatives, namely PTAA and MKC-231 were measured by displacement of a typical HAChT antagonist [3H]HC-3 in rat cerebral membrane. The percentage of inhibition against the binding of [3H]HC-3 to HAChT were calculated using GraphPad Prism v4 software.Results: Hemicholinium-3 showed affinity for HAChT (IC50 = 20 nM) in the in vitro binding assay. A very insignificant inhibition activity (IC50 = 1000 nM) of Tacrine was revealed. The newly synthesized tacrine derivatives, DMTA and PTAA did not show any affinity for HAChT. Although MKC-231 was reported to enhance cholinergic activity at synaptic terminals, it did not show any affinity for the HAChT in [3H]HC-3 binding assay.Conclusion: In vitro [3H]HC-3 binding assay revealed no affinity of MKC-231, tacrine and its corresponding2-oxo-1-pyrrolidineacetate derivative towards HAChT. So, it is worthy to develop radiolabeled HC-3 derivatives with high affinity for HAChT, which can diffuse the BBB, to enable the in vivo investigation of HACU system.Bangladesh J. Nuclear Med. 17(2): 97-102, July 2014

scholarly journals Noncatalytic Docking Domains of Cellulosomes of Anaerobic Fungi

2001 ◽  
Vol 183 (18) ◽  
pp. 5325-5333 ◽  
Author(s):  
Peter J. M. Steenbakkers ◽  
Xin-Liang Li ◽  
Eduardo A. Ximenes ◽  
Jorik G. Arts ◽  
Huizhong Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cdna Library ◽  
Genomic Dna ◽  
Catalytic Domain ◽  
Sequence Information ◽  
Anaerobic Fungi ◽  
Degenerate Primers ◽  
New Genes ◽  
Genes Encoding ◽  
Pcr Products

ABSTRACT A method is presented for the specific isolation of genes encoding cellulosome components from anaerobic fungi. The catalytic components of the cellulosome of anaerobic fungi typically contain, besides the catalytic domain, mostly two copies of a 40-amino-acid cysteine-rich, noncatalytic docking domain (NCDD) interspaced by short linkers. Degenerate primers were designed to anneal to the highly conserved region within the NCDDs of the monocentric fungusPiromyces sp. strain E2 and the polycentric fungusOrpinomyces sp. strain PC-2. Through PCR using cDNA fromOrpinomyces sp. and genomic DNA fromPiromyces sp. as templates, respectively, 9 and 19 PCR products were isolated encoding novel NCDD linker sequences. Screening of an Orpinomyces sp. cDNA library with four of these PCR products resulted in the isolation of new genes encoding cellulosome components. An alignment of the partial NCDD sequence information obtained and an alignment of database-accessible NCDD sequences, focusing on the number and position of cysteine residues, indicated the presence of three structural subfamilies within fungal NCDDs. Furthermore, evidence is presented that the NCDDs in CelC from the polycentric fungus Orpinomyces sp. strain PC-2 specifically recognize four proteins in a cellulosome preparation, indicating the presence of multiple scaffoldins.

scholarly journals Expression of rat liver ketohexokinase in yeast results in fructose intolerance

1993 ◽  
Vol 291 (1) ◽  
pp. 179-186 ◽  
Author(s):  
I A Donaldson ◽  
T C Doyle ◽  
N Matas
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Molecular Mass ◽  
Rat Liver ◽  
Amino Acid Sequences ◽  
Growth Media ◽  
Predicted Amino Acid Sequence ◽  
Sequence Information ◽  
Coding Region ◽  
Pcr Product

Rat liver ketohexokinase (ATP:D-fructose 1-phosphotransferase; EC 2.7.1.3) was purified to homogeneity and the molecular mass of the protein was found by mass spectrometry to be 32,800 Da. The enzyme was cleaved and the amino acid sequences of seven peptides, comprising 24% of the total sequence, were determined. This sequence information was used to design oligonucleotide primers for a PCR using rat liver single-stranded cDNA as a template. The 224 bp PCR product was used as a probe to screen a rat liver cDNA library. A cDNA sequence of 1342 bp was obtained from three positive clones. This contained the entire coding region for ketohexokinase, and all seven peptides were identified in the predicted amino acid sequence. When ketohexokinase was expressed in Saccharomyces cerevisiae using the yeast expression vector pMA91, the cells became intolerant of the presence of fructose in their growth media. The growth of an exponential-phase culture was completely arrested within 90 min by the addition of fructose to a final concentration as low as 0.1% (w/v). This response is associated with an accumulation of fructose 1-phosphate. The cDNA for ketohexokinase encodes a protein composed of 299 amino acids with a combined molecular mass of 32,728 Da. This is in close agreement with the value for the isolated protein determined by mass spectrometry. The primary structure does not show any significant homology with those of other eukaryotic hexokinases, but it contains a highly conserved region that is present in three prokaryotic phosphotransferases that have furanose substrates.

Age- and gender-dependent D-amino acid oxidase activity in mouse brain and peripheral tissues: implication for aging and neurodegeneration

2019 ◽  
Vol 166 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Soo Hyeon Kim ◽  
Yuji Shishido ◽  
Hirofumi Sogabe ◽  
Wanitcha Rachadech ◽  
Kazuko Yorita ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hydrogen Peroxide ◽  
Spinal Cord ◽  
Amino Acid ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Age And Gender ◽  
Peripheral Tissues ◽  
Mouse Tissues ◽  
And Gender

Abstract D-amino acid oxidase (DAO) is a flavoenzyme, catalysing oxidative deamination of D-amino acids to produce corresponding α-keto acids, ammonia and hydrogen peroxide. In our search for DAO activity among various tissues, we developed a sensitive assay based on hydrogen peroxide production involving enzyme-coupled colorimetric assay with peroxidase. We first optimized buffer components to extract DAO protein from mouse tissues. Here we show that DAO activity was detected in kidney, cerebellum, medulla oblongata, midbrain and spinal cord, but not in liver. In addition, we observed that DAO activity and expression were decreased in thoracic and lumbar regions of spinal cord in aged mice when compared with young mice, indicating that decreased DAO is involved in motoneuron degeneration during senescence. We also found gender difference in DAO activity in the kidney, suggesting that DAO activity is influenced by sexual dimorphism. We newly detected DAO activity in the epididymis, although undetected in testis. Furthermore, DAO activity was significantly higher in the caput region than corpus and cauda regions of epididymis, indicating that D-amino acids present in the testis are eliminated in epididymis. Taken together, age- and gender-dependent DAO activity in each organ may underlie the human pathophysiology regulated by D-amino acid metabolism.

Paris I Dysfibrinogenemia: A Point Mutation in Intron 8 Results in Insertion of a 15 Amino Acid Sequence in the Fibrinogen γ-Chain

1993 ◽  
Vol 69 (03) ◽  
pp. 217-220 ◽  
Author(s):  
Jonathan B Rosenberg ◽  
Peter J Newman ◽  
Michael W Mosesson ◽  
Marie-Claude Guillin ◽  
David L Amrani
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Point Mutation ◽  
Genomic Dna ◽  
Comparative Sequence Analysis ◽  
Chain Gene ◽  
Molecular Defect ◽  
Nucleotide Position ◽  
Normal Individuals ◽  
Polymerase Chain

SummaryParis I dysfibrinogenemia results in the production of a fibrinogen molecule containing a functionally abnormal γ-chain. We determined the basis of the molecular defect using polymerase chain reaction (PCR) to amplify the γ-chain region of the Paris I subject’s genomic DNA. Comparative sequence analysis of cloned PCR segments of normal and Paris I genomic DNA revealed only an A→G point mutation occurring at nucleotide position 6588 within intron 8 of the Paris I γ-chain gene. We examined six normal individuals and found only normal sequence in this region, indicating that this change is not likely to represent a normal polymorphism. This nucleotide change leads to a 45 bp fragment being inserted between exons 8 and 9 in the mature γparis I chain mRNA, and encodes a 15 amino acid insert after γ350 [M-C-G-E-A-L-P-M-L-K-D-P-C-Y]. Alternative splicing of this region from intron 8 into the mature Paris I γ-chain mRNA also results after translation into a substitution of S for G at position γ351. Biochemical studies of 14C-iodoacetamide incorporation into disulfide-reduced Paris I and normal fibrinogen corroborated the molecular biologic predictions that two additional cysteine residues exist within the γpariS I chain. We conclude that the insertion of this amino acid sequence leads to a conformationallyaltered, and dysfunctional γ-chain in Paris I fibrinogen.

scholarly journals Commissural Interneurons in Rhythm Generation and Intersegmental Coupling in the Lamprey Spinal Cord

1999 ◽  
Vol 81 (5) ◽  
pp. 2037-2045 ◽  
Author(s):  
James T. Buchanan
Keyword(s):  
Spinal Cord ◽  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Rhythmic Activity ◽  
Ventral Root ◽  
Rhythm Generation ◽  
Spinal Segment ◽  
Spinal Segments ◽  
Autocorrelation Method

Commissural interneurons in rhythm generation and intersegmental coupling in the lamprey spinal cord. To test the necessity of spinal commissural interneurons in the generation of the swim rhythm in lamprey, longitudinal midline cuts of the isolated spinal cord preparation were made. Fictive swimming was then induced by bath perfusion with an excitatory amino acid while recording ventral root activity. When the spinal cord preparation was cut completely along the midline into two lateral hemicords, the rhythmic activity of fictive swimming was lost, usually replaced with continuous ventral root spiking. The loss of the fictive swim rhythm was not due to nonspecific damage produced by the cut because rhythmic activity was present in split regions of spinal cord when the split region was still attached to intact cord. The quality of this persistent rhythmic activity, quantified with an autocorrelation method, declined with the distance of the split spinal segment from the remaining intact spinal cord. The deterioration of the rhythm was characterized by a lengthening of burst durations and a shortening of the interburst silent phases. This pattern of deterioration suggests a loss of rhythmic inhibitory inputs. The same pattern of rhythm deterioration was seen in preparations with the rostral end of the spinal cord cut compared with those with the caudal end cut. The results of this study indicate that commissural interneurons are necessary for the generation of the swimming rhythm in the lamprey spinal cord, and the characteristic loss of the silent interburst phases of the swimming rhythm is consistent with a loss of inhibitory commissural interneurons. The results also suggest that both descending and ascending commissural interneurons are important in the generation of the swimming rhythm. The swim rhythm that persists in the split cord while still attached to an intact portion of spinal cord is thus imposed by interneurons projecting from the intact region of cord into the split region. These projections are functionally short because rhythmic activity was lost within approximately five spinal segments from the intact region of spinal cord.

scholarly journals B0AT1 amino acid transporter complexed with SARS-CoV-2 receptor ACE2 forms a heterodimer functional unit: in situ conformation using radiation inactivation analysis

Function ◽  
2021 ◽  
Author(s):  
Bruce R Stevens ◽  
J Clive Ellory ◽  
Robert L Preston
Keyword(s):  
Amino Acid ◽  
Membrane Trafficking ◽  
Functional Unit ◽  
Membrane Vesicles ◽  
High Energy ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Transport Activity ◽  
Acid Transporter

Abstract The SARS-CoV-2 receptor, Angiotensin Converting Enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared to all other human tissues. Enterocyte ACE2 is co-expressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2: B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2: B0AT1] heterodimer pair constitutes a physiological “functional unit.” This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles that were exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on established target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size mw = 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2: B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2: B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance the understanding of the physiology of B0AT1 interaction with ACE2 in the gut, and thereby potentially contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul Post-Acute Sequelae of SARS-CoV-2 (PASC).

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