The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the -subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled -bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide 122–138. In addition, low-binding activities were obtained with peptides 34–49 and 194–210. It is concluded that the region within residues 122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

Molecular evolution of the nicotinic acetylcholine receptor: An example of multigene family in excitable cells

An extensive phylogenetic analysis of the nicotinic-acetylcholine-receptor subunit gene family has been performed by cladistic and phenetic methods. The conserved parts of amino acid sequences have been analyzed by CLUSTAL V and PHYLIP software. The structure of the genes was also taken in consideration. The results show that a first gene duplication may have occurred before the appearance of Bilateria. Three subfamilies then appeared: I-the neuronal -bungarotoxin binding-site subunits (7, 8); III-the neuronal nicotinic subunits (2–6, 2–4), which also contain the muscle acetylcholine-binding subunit (1); and IV—the muscle non- subunits (1, , ). The Insecta subunits (subfamily II) could be orthologous to family III and IV. Several tissular switches of expression from neuron to muscle and the converse can be inferred from the extant expression of subunits and the reconstructed trees. The diversification of the neuronal nicotinic subfamily begins in the stem lineage of chordates, the last duplications occurring shortly before the onset of the mammalian lineage. Such evolution parallels the increase in complexity of the cholinergic systems.Abbreviations -Bgt -bungarotoxin - ACh acetylcholine - MP maximum of parsimony - MYA million years ago - NJ neighbor-joining - nAChR nicotinic acetylcholine receptor Correspondence to: N. Le Novère     

Efficient membrane targeting of the chick nicotinic acetylcholine receptor α-subunit in stable insect cell lines

We have synthesised the -subunit of the chick nicotinic acetylcholine receptor (nAChR) in stable, continuous insect (Spodoptera frugiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovius immediate early gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1.nAChR, encoding the -subunit cDNA, and pIEK1.neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising the chick nAChR -subunit. Using fluorescence microscopy and ligand binding studies we were able to demonstrate efficient membrane targeting of the receptor subunit in the insect cell plasma membrane. Stable insect cell lines may thus have significant advantages over transient baculovirus vectors for the synthesis and characterisation of heterologous receptor proteins.Abbreviations AcNPV Autographa californica nuclear polyhedrosis virus - BTX -bungarotoxin - BSA bovine serum albumin - FITC Fluoroscein isothiocyanate - G418 geneticin-418 - hpi hours post-infection - ie-1 immediate early 1 gene - nAChR nicotinic acetylcholine receptor alpha subunit - Sf Spodoptera frugiperda - tPA tissue plasminogen activator     

The sialyl-α2,6-lactosaminyl-structure: Biosynthesis and functional role

Sialylation represents one of the most frequently occurring terminations of the oligosaccharide chains of glycoproteins and glycolipids. Sialic acid is commonly found ,3- or ,6-linked to galactose (Gal), ,6-linked to N-acetylgalactosamine (GalNAc) or ,8-linked to another sialic acid. The biosynthesis of the various linkages is mediated by the different members of the sialyltransferase family. The addition of sialic acid in ,6-linkage to the galactose residue of lactosamine (type 2 chains) is catalyzed by -galactoside ,6-sialyltransferase (ST6Gal.I). Although expressed by a single gene, this enzyme shows a complex pattern of regulation which allows its tissue- and stage-specific modulation. The cognate oligosaccharide structure, NeuAc,6Gal1,4GIcNAc, is widely distributed among tissues and is involved in biological processes such as the regulation of the immune response and the progression of colon cancer. This review summarizes the current knowledge on the biochemistry of ST6Gal.I and on the functional role of the sialyl-,6-lactosaminyl structure.     

Sialidase of swine influenza A viruses: variation of the recognition specificities for sialyl linkages and for the molecular species of sialic acid with the year of isolation

The sialidase of swine influenza A viruses of N1 and N2 subtypes, isolated from 1930 to 1992, was studied for substrate specificity with ganglio-series, lacto-series type II and GM3 gangliosides containing Neu5Ac2-3Gal, Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. All viral sialidases tested showed that the activity for hydrolysing substrates with Neu5Ac2-3Gal was higher than the activities with Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. When GM1b, GM3 and sialylparagloboside were used as substrates, the earliest strain (A/Wisconsin/15/30 H1N1, isolated in 1930) showed the activity ratio of Neu5Ac2-6Gal to Neu5Ac2-3Gal to be 0.13:0.2, and the ratio Neu5Gc2-3Gal/Neu5Ac2-3Gal to be 0.19:0.37, while those strains isolated from 1978 to 1992 exhibited ratios of 0.29:0.58 for Neu5Ac2-6Gal/Neu5Ac2-3Gal and 0.51:0.76 for Neu5Gc2-3Gal/Neu5Ac2-3Gal. The above results indicate that the substrate specificities of sialidases from swine influenza A viruses towards sialyl linkages and the molecular species of sialic acid are related to the year of isolation, i.e. strains isolated after 1978 exhibited higher activity towards Neu5Ac2-6Gal and Neu5Gc2-3Gal linkages when compared with strains isolated in an earlier year, 1930.Abbreviation Neu5Ac 5-N-acetylneuraminic acid - Neu5Gc 5-N-glycolyneuraminic acid - Gal d-galactose - Glc d-glucose - Cer Ceramide - II³(Neu5Ac)Lac Neu5Ac2-3Gal1-4Glc - GM3(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-4Glc1-Cer - GM3(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-4Glc1-Cer - GM1b(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-3GalNac1-4Gal1-4Glc1-Cer - GMlb(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-3GalNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Ac)nLc4Cer Neu5Ac2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Gc)nLc4Cer Neu5Gc2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV⁶(Neu5Ac)nLc4Cer Neu5Ac2-6Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - TDC taurodeoxycholate.     

Simultaneous recognition of two chiral centers in α,β-diastereomeric amino acids by an N-carbamoyl-L-α-amino acid amidohydrolase from Alcaligenes xylosoxidans

Cells of Alcaligenes xylosoxidans containing N-carbamoyl-L--amino acid amidohydrolase strictly distinguished the configuration of not only the -carbon but also the -carbon of N-carbamoyl--methylphenylalanine, and produced threo-l--methylphenylalanine specifically from a mixture of the four stereoisomers.     

Confirmation of assignment of the human α1-crystallin gene (CRYA1) to chromosome 21 with regional localization to q22.3

Summary The crystallins are highly conserved structural proteins universally found in the eye lens of all vertebrate species. In mammals, three immunologically distinct classes are present, -, -, and -crystallins, and each class represents a multigene family. The -crystallin gene family consists of 1-crystallin (CRYA1) and 2-crystallin (CRYA2) genes (previously designated A-and B-crystallin, respectively), which show extensive sequence homology. We constructed a synthetic oligonucleotide probe of 25 bases corresponding to a specific region of the human 1-crystallin gene sequence. This 25-mer probe bears little sequence homology to human 2-crystallin gene and does not cross-hybridize to 2-crystallin sequences in Southern blot analysis. Using this unique synthetic probe, we have demonstrated the identity of the 1-crystallin gene in human genomic DNA. In addition, we have also confirmed its chromosomal location on human chromosome 21. Finally, we have regionally localized the gene to q22.3 by using both Southern blot analysis of a panel of cell hybrids containing different parts of human chromosome 21, and in situ hybridization to metaphase chromosomes. The use of synthetic oligonucleotide probes specific for individual genes should be useful in identifying and mapping members of multigene families.     

High frequency of triplicated α-globin loci and absence or low frequency of α thalassemia in Polynesian Samoans

Summary Most of the population in certain areas of Melanesia have one -globin gene deletion ( thal₂). It is thought that the high frequencies of thal₂ in this population is due to a selective advantage given by malaria infection to carriers of thal₂. We are interested in neighboring Polynesia which, although adjacent to Melanesia, has always been free of malaria due to the absence of the vector anopheles. We studied 60 Polynesian Samoans and 150 Malaysians by restriction endonuclease gene mapping using Eco RI, Bam HI, and Bgl II and hybridization to ³²P-labeled -globin gene probe. Seven among the 60 (11.7%) Samoans had triplicated -globin loci type 1, while none had thal₂. On digestion with Bgl II the third -globin gene was found in an additional 3.7kb fragment in all seven Samoans with triplicated -globin loci, while digestion with Bam HI produced an abnormal elongated 18.2 kb fragment carrying -globin genes in addition to the normal 14.5 kb fragment. None of the Polynesian Samoans had thal₂ or thal₁. Only two of the Malaysians had triplicated -globin loci.     

Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.     

Identification of amino acids involved in the binding of hMIP-1α to CC-CKR1, a MIP-lα receptor found on neutrophils

Human macrophage inflammatory protein-1 (hMIP-1) and human macrophage inflammatory protein-1 (hMIP-1) are chemokines involved in a diverse range of immunological effects. Both hMIP-1 and hMIP-1 are involved in the activation of monocytes and THP-1 cells probably through a common receptor(s). However, only hMIP-1 can bind to neutrophils with high affinity, presumably through CC-CKR1 (CKR1). Since the structure of these two proteins is highly conserved, non-conserved amino acids must define the disparate binding patterns that these two proteins exhibit. Measurements of binding, chemotaxis and calcium influx conducted with hMIP-1 and hMIP-1 chimeric proteins and mutants show that two amino acids (37K and 43L) are important in the binding and signaling of hMIP-1 through CKR1. Furthermore, we also show that mutations of the three charged amino acids at the C-terminus of hMIP-1 and hMIP-1 (amino acids 61, 65 and 67), do not adversely affect the binding to THP-1 cells.     

Changes in Cortical Activity in Altered States of Consciousness: The Study of Meditation by High-Resolution EEG

The specific features of the topology of spectral powers and coherent interregional interrelationships in the narrow, individually determined -, -, ₁-, ₂-, and ₃-frequency bands were studied by means of high-resolution EEG (62 channels) in novice and experienced meditators (NMs and EMs) at rest and under the conditions of generation of an altered state of consciousness characterized by inactivation of cognitive activity and the occurrence of a positive emotional experience of happiness. EMs in the meditation-free state were found to be characterized by a shift in the values of the individual frequency to a lower-frequency region of the spectrum, along with higher, compared to NMs, -, ₁-, ₂-, and ₃-band power values, which probably reflects the cumulative character of the influence of long-term meditative practice. The effective achievement of altered states of consciousness in EMs was associated with an increase in the local - and ₁ powers in the anterior cortical areas, as well as long-distance coherence between the prefrontal and posterior associative cortex with the formation of a center of gravity in the left prefrontal region (lead AF ₃). According to the data of the correlation analysis of the EEG power values and the data of subjective scaling of the meditation state, the -power values were positively associated with positive emotional experiences and negatively associated with the level of mental activity. The results of this study are consistent with current concepts that the and activities in narrow frequency bands reflect the activity of multifunctional neuronal networks selectively associated with processes of cognitive and affective activity.     

Looking back on the discovery of α-bungarotoxin

This review is a personal narration by a retiring pharmacologist from Taiwan who looks back at his discovery of -bungarotoxin from the historical perspective of Taiwan during the last 50 years, with accounts of his experiences and his efforts to overcome hardship. How the -toxin was isolated and characterized as an irreversible specific nicotinic acetylcholine (ACh) receptor antagonist,and how it subsequently became a useful experimental probe are presented here. The dilemma of differentiating the actions of tubocurarine and -bungarotoxin is analyzed. The author also outlines findings based on work done in his laboratory using -bungarotoxin as a tool on particular aspects of synaptic transmission. These include presynaptic receptor for positive feedback of transmitter release, explosive release of ACh, up- and down-regulation of ACh receptors after chronic drug treatment, autodesensitization of junctional ACh receptors, differences in action between natural transmitter and exogenous agonists and that between junctional and extra-junctional ACh receptors. Some experimental pitfalls, in which biomedical scientists are frequently trapped, are raised. Finally, some anecdotes are appended from which the reader may further understand scientific life in the 20th century, including its joys and regrets.     

Efficient determination of angles subtended by Cα-Hα and N-HN vectors in proteins via dipole-dipole cross-correlation§

The angle CH,NHN subtended by the internuclear vectors 13C-H and 15N-HN in doubly-labeled proteins can be determined by observing the effect of cross-correlation between the dipolar interactions on zero- and double-quantum coherences involving 13C and 15N. Two complementary 2D experiments with the appearance of 15N-HN correlation spectra yield signal intensities that depend on the rate of interconversion through cross-correlated relaxation of in-phase and doubly antiphase zero- and double-quantum coherences. The ratio of the signal intensities in the two experiments bears a simple relationship to the cross-correlation rate, and hence to the angle CH,NHN. Assuming planarity of the peptide bond, the dihedral angle (between C and C) can be determined from the knowledge of CH,NHN. The experiments are very time-effective and provide good sensitivity and excellent spectral resolution.     

Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> oocytes

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken 7, chicken 42 and the Drosophila melanogaster/chicken hybrid receptors SAD/2 and ALS/2. No agonist action of NTX (0.1–100 M) was observed on chicken 7, chicken 42 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (7) or heteromeric (42) nicotinic AChRs. Block by NTX of the chicken 7, chicken 42 and the SAD/2 and ALS/2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.     

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

Cobra (<Emphasis Type="Italic">Naja</Emphasis> spp. ) Nicotinic Acetylcholine Receptor Exhibits Resistance to Erabu Sea Snake (<Emphasis Type="Italic">Laticauda semifasciata</Emphasis>) Short-Chain α-Neurotoxin

Snake -neutotoxins of Elapidae venoms are grouped into two structural classes, short-chain and long-chain -neutotoxins. While these two classes share many chemical and biological characteristics, there are also distinct dissimilarities between them, including their binding site on the nicotinic acetylcholine receptor (nAChR), specificity among species of Chordata, and the associated pharmacological effects. In the present study we test the hypothesis that structural motifs that evolved to confer natural resistance against conspecific long-chain -neurotoxins in Elapidae snakes also interfere with the biological action of short-chain -neurotoxins. We expressed functional nAChRs that contains segments or single residues of the Elapidae nAChR ligand binding domain and tested the effect of short-chain -neurotoxin erabutoxin-a (ETX-a) from the Erabu sea snake Laticauda semifasciata on the acetylcholine-induced currents as measured by two-microelectrode voltage clamp. Our results show that the Elapidae nAChR subunit segment T¹⁵⁴–L²⁰⁸ ligand binding domain has an inhibitory effect on the pharmacological action of ETX-a. This effect is primarily attributed to the presence of glycosylation at position N¹⁸⁹. If the glycosylation is removed from the T¹⁵⁴–L²⁰⁸ segment, the nAChR will be inhibited, however, to a lesser extent than seen in the mouse. This effect correlates with the variations in -neurotoxin sensitivity of different species and, importantly, reflects the evolutionary conservation of the binding site on the nAChR polypeptide backbone per se. Phylogenetic analysis of -neurotoxin resistance suggests that -neurotoxin-resistant nAChR evolved first, which permitted the evolution of snake venom -neurotoxins. A model describing -neurotoxin resistance in Elapidae snakes is presented. Present address: Schering-Plough Research Institute, CNS-CV Research, K-15 C205/2600, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA     

Effects of mutations in the N terminal region of the yeast G protein α-subunit Gpa1p on signaling by pheromone receptors

The sites and modes of interaction between G protein-coupled receptors and their cognate heterotrimeric G proteins remain poorly defined. The C-terminus of the G subunit is the best established site of contact of G proteins with receptors, but structural analyses and crosslinking studies suggest the possibility of interactions at the N-terminus of G as well. We screened for mutations in the N-terminal region of the G subunit encoded by the yeast GPA1 gene that specifically affect the ability of the G protein to be activated by the yeast -mating factor receptor. The screen led to identification of substitutions of glutamine or proline for Leu18 of Gpa1p that reduce the response to the pheromones -factor and a-factor without affecting cellular levels of the subunit or its ability to interact with and subunits. The mutations do not appear to affect the intrinsic ability of the G protein to be converted to the activated state. The low yield of different mutations with this phenotype indicates either that the N-terminal segment of the yeast G subunit does not undergo extensive interactions with the -factor receptor, or that this region can not be altered without detrimental effects upon the formation of G protein trimers.Communicated by D. Y. Thomas