GeneJockeyII. Primer design.
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DNA Primers↗
Biomedical subjects
P Taylor
Publications and source records attributed to P Taylor.
Stereological estimation of blood vessel surface and volume densities in human normal and rhinitic nasal mucosa.
A technique is proposed for applying well-established stereological methods to study fixed nasal biopsy material to obtain an unbiased estimate of blood vessel surface and volume densities. Biopsies of the nasal mucosa from the anterior 10 mm of an inferior turbinate were obtained from 18 subjects (15 males, 3 females with a mean age of 28.5 years [range: 17-54 years]), ten of whom had perennial allergic rhinitis, and eight control subjects. The mucosal tissue volumes were estimated by water displacement. Zamboni's-fixed cryostat sections (10 microns thick), stained with haematoxylin and eosin, were examined histologically. Computerised images of randomly selected tissue sections were analysed with point-counting and intercept-counting techniques to determine large blood vessel volume and surface densities, respectively. There were no significant differences between the volumes of tissue analysed from the control and rhinitic subjects (p = 0.35). The average volume density of the vessels was similar in the control group (6.17 +/- 1.41%) and the rhinitic group (7.8 +/- 5.59%; p = 0.38), but with a greater variability in the rhinitic group. Surface density estimations were 3.14 +/- 0.74 mm-1 in the control group and 3.10 +/- 1.41 mm-1 in the rhinitic group. Therefore, on average, the volume and surface densities of the cavernous blood vessels in rhinitis were unaltered and there was no evidence of vascular remodelling.
Adolescent↗
Evidence-based approach to treating depression.
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Antidepressive Agents, Tricyclic↗
Anti-tumor necrosis factor alpha therapy of rheumatoid arthritis. Mechanism of action.
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Arthritis, Rheumatoid↗
Determining ligand orientation and transphosphonylation mechanisms on acetylcholinesterase by Rp, Sp enantiomer selectivity and site-specific mutagenesis.
Acetylcholinesterase, an enzyme of the serine hydrolase family, catalyzes the rapid hydrolysis of certain carboxyl esters. Other acyl esters efficiently transacylate the enzyme with a subsequent, slow deacylation step. Of these, the phosphoryl and phosphonyl esters are perhaps of greatest mechanistic interest since individual enantiomers of known absolute stereochemistry can be isolated and their interactions with the dissymmetric enzyme active site examined. We describe here studies of a series of enantiomeric Rp- and Sp-alkylphosphonates interacting with mouse acetylcholinesterase. Since the acetylcholinesterase is generated by recombinant DNA methods, mutant enzymes can be made with specific replacements of individual amino acid side chains. Individual amino acid replacements in the acyl pocket, the choline subsite and at the active center gorge entry have been generated, and the reaction kinetics of the mutant enzymes analyzed. These studies have shown that substitution of aliphatic amino acids for phenylalanines 295 and 297 in the acyl pocket diminishes, and in some cases, actually inverts chiral preferences. The combined structure-activity approach, where both ligand and enzyme are modified systematically, has enabled us to show that the restricted dimensions of the acyl pocket in the active center dictate enantiomeric selectivity. Moreover, the reactions of compounds of known absolute stereochemistry show three requirements for efficient transphosphonylation: (a) apposition of the phosphate with the gamma-oxygen on Ser 203 to form a pentavalent, presumed trigonal bipyramidal intermediate, (b) polarization of the phosphonyl oxygen bond by its positioning in the oxyanion hole, and (c) positioning the leaving group towards the gorge exit.
Acetylcholinesterase↗
Anionic residue in the alpha-subunit of the nicotinic acetylcholine receptor contributing to subunit assembly and ligand binding.
To ascertain the anionic sites on the nicotinic receptor to which acetylcholine and other quaternary ammonium ligands bind, we have examined the role of an aspartyl residue (Asp-152) in the alpha-subunit. Prior photolytic labeling with agonist analogues of the neighboring residues Trp-149 and Tyr-151 suggests that their side chains reside on the binding face (also termed the (+)- or counterclockwise face) of the alpha-subunit. Asp-152 presents an anionic charge in the vicinity of these aromatic residues. Modification of the aspartate to asparagine (D152N) creates a glycosylation signal (Asn-152-Gly-Ser), and we find, on the basis of altered electrophoretic migration, that glycosylation occurs at this position upon cotransfection of the mutant alpha-subunit with beta-, gamma-, and delta-subunits. Glycosylation results in a reduction in the capacity of the receptor to assemble; this reduction is manifest in the initial step of dimer formation between the alphagamma- and alphadelta-subunits. The alpha-subunit mutant receptor reaching the assembled pentamer exhibits an altered selectivity for certain ligands. Little reduction in alpha-bungarotoxin binding is observed, whereas affinities for agonists and competitive alkaloid antagonists are reduced substantially. Separation of the contributions of charge removal and glycosylation addition shows that both factors affect agonist affinity, with the charge influence being far more predominant. These findings raise the possibility that a component of the coulombic attraction stabilizing the binding of agonists comes from the aspartyl residue at position 152 in the alpha-subunit.
Amino Acid Sequence↗
Involvement of the chaperone protein calnexin and the acetylcholine receptor beta-subunit in the assembly and cell surface expression of the receptor.
The nicotinic acetylcholine receptor at the neuromuscular junction is a ligand-gated ion channel assembled in the endoplasmic reticulum from four distinct glycoprotein subunits into the pentameric configuration of alpha2betagammadelta. The individual homologous subunits form specific contacts at interfaces with neighboring subunits to achieve the appropriate orientation and order of each subunit in surrounding the ion channel. Assembly is thought to proceed through the formation of intermediates composed of dimers of the alphadelta and alphagamma subunits which are eventually joined by the beta-subunit to achieve a circular structure enclosing the gated ion channel. In this study, we transfect cDNAs encoding receptor subunits in various combinations into HEK-293 cells to identify intracellular factors that influence the assembly and cell surface expression of the receptor. Our data derived from brefeldin A-treated cells indicate that intracellular association of the receptor subunits with the beta-subunit increases the pool of fully assembled receptors available for transport to the cell surface, presumably by protection from degradation. In addition, we determined that the chaperone protein calnexin is associated with the isolated alpha-, beta-, and delta-subunits of the receptor, but calnexin is not detected in association with assembled alphadelta subunit dimers. Calnexin is also detected in association with maturely folded, unassembled alpha-subunits, as observed by the recognition of this complex by the monoclonal antibody mAb 35, believed to be specific for correctly folded alpha-subunits. Thus, calnexin appears to associate with the individual nascent subunits, thereby facilitating their assembly into the mature pentameric receptor.
Animals↗
Aspartate 74 as a primary determinant in acetylcholinesterase governing specificity to cationic organophosphonates.
Through site-specific mutagenesis, we examined the determinants on acetylcholinesterase which govern the specificity and reactivity of three classes of substrates: enantiomeric alkyl phosphonates, trifluoromethyl acetophenones, and carboxyl esters. By employing cationic and uncharged pairs of enantiomeric alkyl methylphosphonyl thioates of known absolute stereochemistry, we find that an aspartate residue near the gorge entrance (D74) is responsible for the enhanced reactivity of the cationic organophosphonates. Removal of the charge with the mutation D74N causes a near equal reduction in the reaction rate constants for the Rp and Sp enantiomers and exerts a greater influence on the cationic organophosphonates than on the charged trimethylammonio trifluoromethyl acetophenone and acetylthiocholine. This pattern of reactivity suggests that the orientation of the leaving group for both enantiomers is directed toward the gorge exit and in apposition to Asp 74. Replacement of tryptophan 86 with alanine in the choline subsite also diminishes the reaction rates for cationic organophosphonates, although to a lesser extent than with the D74N mutation, while not affecting the reactions with the uncharged compounds. Hence, reaction with cationic OPs depends to a lesser degree on Trp 86 than on Asp 74. Docking of Sp and Rp cycloheptyl methylphosphonyl thiocholines and thioethylates in AChE as models of the reversible complex and transition state using molecular dynamics affords structural insight into the spatial arrangement of the substituents surrounding phosphorus prior to and during reaction. The leaving group of the Rp and Sp enantiomers, regardless of charge, is directed to the gorge exit and toward Asp 74, an orientation unique to tetrahedral ligands.
Acetylcholinesterase↗
Pulse oximetry for assessing oxygen status of neonatal calves.
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Animals↗
Regulation of acetylcholinesterase expression during neuronal differentiation.
We have examined the developmental expression of acetylcholinesterase (AChE) during the process of neuronal differentiation from a pluripotent stem cell. P19 embryonic carcinoma cells form embryoid bodies, which, when cultured with retinoic acid, are induced to differentiate into neurons and glia. No AChE activity is present in the undifferentiated stem cells, and mRNA protection analyses do not detect AChE mRNA. Commitment to a neuronal differentiation pathway results in increased levels of AChE mRNA, production of a tetrameric form of the enzyme, and secretion of AChE into the culture medium. Concomitant with subsequent morphological differentiation into neurons, enzyme secretion diminishes and AChE becomes largely tethered to the neuronal cell membranes. The enzyme is attached to the cell surface as a globular tetramer. Its hydrodynamic properties are consistent with association through a noncatalytic hydrophobic subunit rather than anchorage by a glycophospholipid tail. No change in the rate of transcription of the Ache gene was detected during the course of differentiation, suggesting that the gene is actively transcribed at very early stages of development. Results suggest that stabilization of a labile mRNA governs the increase in AChE mRNA and gene product. The studies presented indicate that an early event in neuronal differentiation is the stabilization of the mRNA leading to expression of a secreted form of AChE. A subsequent step associated with neurite outgrowth results in a transition from secretion of the tetrameric enzyme to its localization on the cell membrane.
Acetylcholinesterase↗
Conformational polymorphism in peptidic and nonpeptidic drug molecules.
Macrolide ligands that bind FK506 binding proteins and cyclosporins that a bind cyclophilins are chemically dissimilar but can share a number of structural and biological properties. Both families of ligands have very different conformations in the free state compared to those adopted when complexed with their binding protein. These transformations involve twisting from cis to trans about specific amide bonds, which result in significant changes in the hydrogen-bonding capabilities of the molecular surfaces. The three-dimensional structure of a new cyclosporin-like ligand (SDZ214 - 103) is described in the free crystalline state and bound to cyclophilin, and is shown to have a very different conformation from cyclosporin A in the free crystal, but a very similar conformation when bound to cyclophilin.
Amino Acid Isomerases↗
Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.
A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.
Acetylcholinesterase↗
Humoral and cellular responses raised against the human HER2 oncoprotein are cross-reactive with the homologous product of the new proto-oncogene, but do not protect rats against B104 tumors expressing mutated neu.
The neu proto-oncogene encodes a plasma membrane protein belonging to the epidermal growth factor receptor family. The cell line B104, derived from BDIX rat neuroblastoma, carries a point mutation in neu, and forms a tumor when injected into these rats. The human homologue of the neu oncogene (here called HER2) is overexpressed in certain types of cancer. Rats were immunized with HER2 protein (HER2) to investigate a possible cross-reaction between the homologous proteins which could protect them against subsequent inoculation with B104. Specific antibody in the serum was measured by cell-based enzyme-linked immunoabsorbent assay and fluorescence immunocytochemistry, and delayed-type hypersensitivity by an ear assay. Sera from animals immunized with the HER2 extracellular domain (HER2-ECD) reacted with both HER2- and neu-expressing cells. In the ear assay, a significant cellular response to both HER-ECD (P < 0.05) and neu protein (P < 0.001) was observed in HER2-ECD-immunized rats. However, the growth of B104 tumors in rats was not affected by preimmunization with HER2-ECD. The results indicate that an autoreactive immune response to neu was induced by immunization with HER2-ECD, but was too weak to affect the growth of the neu-bearing tumor.
3T3 Cells↗
Acetylcholinesterase and nicotinic acetylcholine receptor expression diverge in muscular dysgenic mice lacking the L-type calcium channel.
L-type Ca2+ channels play critical roles in achieving stabilization of acetylcholinesterase (AChe) mRNA during myogenesis in C2-C12 skeletal muscle cells. To ascertain the importance of this signaling pathway in AChE expression during skeletal muscle development in the animal, we examined AChE mRNA levels in skeletal muscle and heart from control (+/+) and muscular dysgenic (mdg/mdg) mice that lack the skeletal, but not the cardiac, muscle L-type Ca2+ channels. RNase protection analysis showed 40-60% reductions in content of AChE mRNA in leg muscle, but not heart, from newborn and day 18 embryonic dysgenic mice. AChE activity was also reduced uniquely in skeletal muscle. In contrast to AChE transcripts, mRNA levels of the alpha-subunit of the nicotinic acetylcholine receptors (nAChRs) were increased in dysgenic skeletal muscle. Similar alterations in activity and mRNA levels of AChE were also observed form skeletal muscle cell lines derived from mdg mice. Because run-on transcription revealed no corresponding decrease in transcription rate, the decrease in mRNA content is likely a consequence of the inability of the dysgenic muscle cells to stabilize AChE mRNA during differentiation. These findings indicate that L-type Ca2+ channels play an important role in regulation of AChE expression during skeletal muscle development in vivo. The differential influence of muscle dysgenesis on mRNA levels of AChE and nAChRs provides additional evidence for distinct mechanisms of regulation of these two proteins.
Acetylcholinesterase↗
Lack of efficacy of intra-articular opioids for analgesia after day-case arthroscopy.
A randomised double-blind placebo-controlled trial was undertaken to assess the analgesic efficacy of intra-articular opioids following arthroscopy of the knee. At the completion of surgery, patients received an intra-articular injection of: morphine 1 mg, buprenorphine 30 micrograms or 0.9% saline. There were no differences in pain scores among groups for the first 24 h postoperatively. We have found no clinical evidence for a peripherally-mediated opioid analgesic effect.
Adult↗
Intra-articular morphine and saline injections induce release of large molecular weight proteoglycans into equine synovial fluid.
Both morphine and physiologic saline injected intra-articularly into healthy equine tarsocrural joints induced a release of large molecular size proteoglycan (PG) subunits into the synovial fluid (SF) analysed 24 h postinjection. High-performance liquid chromatography (HPLC) with a size-exclusion column was used to assess the high molecular weight proteoglycans in equine synovial fluid (SF). The PG peaks of SF samples eluated separately from SF hyaluronate and other molecular components of the SF in the HPLC chromatographies indicating no interaction between hyaluronate and PG in the SF. Individual elution profiles varied between joints and horses. The amount of PG release was measured by relative area index from the HPLC chromatograms. The synovial fluid PG content was significantly increased (P < 0.05) after morphine but not in saline injected joints compared with pretreatment but there were no significant differences between the two groups. It was concluded that intra-articular injections of both morphine and physiologic saline are able to elicit a marked PG release into the SF from articular cartilage within 24 h of injection.
Animals↗