scholarly journals The Caenorhabditis elegans unc-13 gene product is a phospholipid-dependent high-affinity phorbol ester receptor

1992 ◽  
Vol 287 (3) ◽  
pp. 995-999 ◽  
Author(s):  
S Ahmed ◽  
I N Maruyama ◽  
R Kozma ◽  
J Lee ◽  
S Brenner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Phorbol Ester ◽  
Catalytic Domain ◽  
Amino Acid Residues ◽  
C Elegans ◽  
High Affinity ◽  
Cell Extracts ◽  
Neuronal Connections ◽  
Phorbol Ester Receptor

The Caenorhabditis elegans unc-13 mutant is a member of a class of mutants that have un-coordinated movement. Mutations of the unc-13 gene cause diverse defects in C. elegans, including abnormal neuronal connections and modified synaptic transmission in the nervous system. unc-13 cDNA encodes a protein (UNC-13) of 1734 amino acid residues with a predicted molecular mass of 198 kDa and sequence identity to the C1/C2 regions but not to the catalytic domain of the ubiquitously expressed protein kinase C family [Maruyama & Brenner (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5729-5733]. To characterize the phorbol ester binding site of the UNC-13 protein, cDNA encoding the C1/C2-like regions (amino acid residues 546-940) was expressed in Escherichia coli and the 43 kDa recombinant protein was purified. Phorbol ester binding to the 43 kDa protein was zinc- and phospholipid-dependent, stereospecific and of high affinity (Kd 67 nM). UNC-13 specific antisera detected a protein of approx. 190 kDa in wild-type (N2) but not in mutant (e1019) C. elegans cell extracts. We conclude that UNC-13 represents a novel class of phorbol ester receptor.

scholarly journals Identification of a Gene Involved in Assembly ofActinomyces naeslundii T14V Type 2 Fimbriae

1998 ◽  
Vol 66 (4) ◽  
pp. 1482-1491 ◽  
Author(s):  
Maria K. Yeung ◽  
Jacob A. Donkersloot ◽  
John O. Cisar ◽  
Pamela A. Ragsdale
Keyword(s):  
Amino Acid ◽  
Kanamycin Resistance ◽  
Amino Acid Residues ◽  
Subunit Protein ◽  
Actinomyces Naeslundii ◽  
Cell Extracts ◽  
Acid Protein ◽  
Structural Subunit ◽  
Antipeptide Antibody

ABSTRACT The nucleotide sequence of the Actinomyces naeslundiiT14V type 2 fimbrial structural subunit gene, fimA, and the 3′ flanking DNA region was determined. The fimA gene encoded a 535-amino-acid precursor subunit protein (FimA) which included both N-terminal leader and C-terminal cell wall sorting sequences. A second gene, designated orf365, that encoded a 365-amino-acid protein which contained a putative transmembrane segment was identified immediately 3′ to fimA. Mutants in which either fimA or orf365 was replaced with a kanamycin resistance gene did not participate in type 2 fimbriae-mediated coaggregation with Streptococcus oralis34. Type 2 fimbrial antigen was not detected in cell extracts of thefimA mutant by Western blotting with anti-A. naeslundii type 2 fimbrial antibody, but the subunit protein was detected in extracts of the orf365 mutant. The subunit protein detected in this mutant also was immunostained by an antibody raised against a synthetic peptide representing the C-terminal 20 amino acid residues of the predicted FimA. The antipeptide antibody reacted with FimA isolated from the recombinant Escherichia coliclone containing fimA but did not react with purified type 2 fimbriae in extracts of the wild-type strain. These results indicate that synthesis of type 2 fimbriae in A. naeslundii T14V may involve posttranslational cleavage of both the N-terminal and C-terminal peptides of the precursor subunit and also the expression oforf365.

scholarly journals Four Amino Acid Residues Are Critical for High Affinity Binding of Neuromedin B to the Neuromedin B Receptor

1998 ◽  
Vol 273 (26) ◽  
pp. 15927-15932 ◽  
Author(s):  
Eduardo Sainz ◽  
Mark Akeson ◽  
Samuel A. Mantey ◽  
Robert T. Jensen ◽  
James F. Battey
Keyword(s):  
Amino Acid ◽  
Affinity Binding ◽  
Amino Acid Residues ◽  
High Affinity Binding ◽  
High Affinity ◽  
Neuromedin B

scholarly journals Characterization of the Cysteine-rich Region of theCaenorhabditiselegansProtein Unc-13 as a High Affinity Phorbol Ester Receptor

1995 ◽  
Vol 270 (18) ◽  
pp. 10777-10783 ◽  
Author(s):  
Marcelo G. Kazanietz ◽  
Nancy E. Lewin ◽  
Jay D. Bruns ◽  
Peter M. Blumberg
Keyword(s):  
Phorbol Ester ◽  
Rich Region ◽  
High Affinity ◽  
Phorbol Ester Receptor

Reporter gene constructs suggest that the Caenorhabditis elegans avermectin receptor beta-subunit is expressed solely in the pharynx.

1997 ◽  
Vol 200 (10) ◽  
pp. 1509-1514 ◽  
Author(s):  
D L Laughton ◽  
G G Lunt ◽  
A J Wolstenholme
Keyword(s):  
Caenorhabditis Elegans ◽  
Genomic Sequence ◽  
Gene Promoter ◽  
Pcr Amplification ◽  
Beta Subunit ◽  
Amino Acid Residues ◽  
C Elegans ◽  
Larval Stages ◽  
Major Mode ◽  
Flanking Sequences

Gene promoter/LacZ reporter constructs were made in order to analyse the expression of the beta-subunit of the Caenorhabditis elegans glutamate-gated Cl- channel (Glu-Cl) receptor. Southern blot analysis of the C. elegans cosmid C35E8 identified a 4kbp EcoRI fragment which contained the 5' portion of the Glu-Cl beta coding sequence together with 5' flanking sequences. This was subcloned and used as the template for polymerase chain reaction (PCR) amplification of a DNA fragment encoding the first 24 amino acid residues of Glu-Cl beta together with 1.4 kbp of 5' genomic sequence. The fragment was subcloned into the LacZ expression vector pPD22.11 to form a translational reporter fusion. After injection of the construct into worms, six stably transformed lines were established and assayed for beta-galactosidase activity. Stained nuclei were observed in the pharyngeal metacorpus in adults and in all larval stages, and stained nuclei were seen in many embryos undergoing morphogenesis. Additional stained nuclei towards the terminal bulb of the pharynx were observed in larval stages. These results provide further evidence that the Glu-Cl receptor mediates the glutamatergic inhibition of pharyngeal muscle via the M3 motor neurone and point to inhibition of pharyngeal pumping as a major mode of action for avermectins.

A water channel of the nematode C. elegans and its implications for channel selectivity of MIP proteins

1998 ◽  
Vol 275 (6) ◽  
pp. C1459-C1464 ◽  
Author(s):  
Michio Kuwahara ◽  
Kenichi Ishibashi ◽  
Yong Gu ◽  
Yoshio Terada ◽  
Yuji Kohara ◽  
...  
Keyword(s):  
Amino Acid ◽  
Water Channel ◽  
Genome Project ◽  
Amino Acid Residues ◽  
C Elegans ◽  
Extra Amino Acid ◽  
Urea Permeability ◽  
Glycerol Facilitator ◽  
A Genome ◽  
Channel Selectivity

A genome project focusing on the nematode Caenorhabditis elegans has demonstrated the presence of eight cDNAs belonging to the major intrinsic protein superfamily. We functionally characterized one of these cDNAs named C01G6.1. Injection of C01G6.1 cRNA increased the osmotic water permeability ( P f) of Xenopusoocytes 11-fold and the urea permeability 4.5-fold but failed to increase the glycerol permeability. It has been speculated that the MIP family may be separated into two large subfamilies based on the presence or absence of two segments of extra amino acid residues (∼15 amino acids) at the second and third extracellular loops. Because C01G6.1 (designated AQP-CE1), AQP3, and glycerol facilitator (GlpF) all have these two segments, we replaced the segments of AQP-CE1 with those of AQP3 and GlpF to identify their roles. The functional characteristics of these mutants were principally similar to that of wild-type AQP-CE1, although the values of P f and urea permeability were decreased by 39–74% and 28–65%, respectively. These results suggest that the two segments of extra amino acid residues may not contribute to channel selectivity or formation of the route for small solutes.

scholarly journals Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain

2003 ◽  
Vol 373 (2) ◽  
pp. 369-379 ◽  
Author(s):  
Maria-Dolores MONTIEL ◽  
Marie-Ange KRZEWINSKI-RECCHI ◽  
Philippe DELANNOY ◽  
Anne HARDUIN-LEPERS
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Human Gene ◽  
Short Form ◽  
Cytoplasmic Tail ◽  
Gene Organization ◽  
Sialic Acid Residue ◽  
Chromosome 17 ◽  
Soluble Form ◽  
Amino Acid Residues

The human Sda antigen is formed through the addition of an N-acetylgalactosamine residue via a β1,4-linkage to a sub-terminal galactose residue substituted with an α2,3-linked sialic acid residue. We have taken advantage of the previously cloned mouse cDNA sequence of the UDP-GalNAc:Neu5Acα2-3Galβ-R β1,4-N-acetylgalactosaminyltransferase (Sda β1,4GalNAc transferase) to screen the human EST and genomic databases and to identify the corresponding human gene. The sequence spans over 35 kb of genomic DNA on chromosome 17 and comprises at least 12 exons. As judged by reverse transcription PCR, the human gene is expressed widely since it is detected in various amounts in almost all cell types studied. Northern blot analysis indicated that five Sda β1,4GalNAc transferase transcripts of 8.8, 6.1, 4.7, 3.8 and 1.65 kb were highly expressed in colon and to a lesser extent in kidney, stomach, ileum and rectum. The complete coding nucleotide sequence was amplified from Caco-2 cells. Interestingly, the alternative use of two first exons, named E1S and E1L, leads to the production of two transcripts. These nucleotide sequences give rise potentially to two proteins of 506 and 566 amino acid residues, identical in their sequence with the exception of their cytoplasmic tail. The short form is highly similar (74% identity) to the mouse enzyme whereas the long form shows an unusual long cytoplasmic tail of 66 amino acid residues that is as yet not described for any other mammalian glycosyltransferase. Upon transient transfection in Cos-7 cells of the common catalytic domain, a soluble form of the protein was obtained, which catalysed the transfer of GalNAc residues to α2,3-sialylated acceptor substrates, to form the GalNAcβ1-4[Neu5Acα2-3]Galβ1-R trisaccharide common to both Sda and Cad antigens.

scholarly journals Molecular Interaction of Droperidol with Human Ether-a-go-go -related Gene Channels

2007 ◽  
Vol 106 (5) ◽  
pp. 967-976 ◽  
Author(s):  
Alexander P. Schwoerer ◽  
Carmen Blütner ◽  
Sven Brandt ◽  
Stephan Binder ◽  
Cornelia C. Siebrands ◽  
...  
Keyword(s):  
Amino Acid ◽  
Patch Clamp ◽  
Cardiac Myocytes ◽  
Action Potentials ◽  
Amino Acid Residues ◽  
Related Gene ◽  
Wild Type ◽  
Early Afterdepolarizations ◽  
High Affinity ◽  
Herg Channels

Background The cardiac safety of droperidol given at antiemetic doses is a matter of debate. Although droperidol potently inhibits human ether-a-go-go-related gene (HERG) channels, the molecular mode of this interaction is unknown. The role of amino acid residues typically mediating high-affinity block of HERG channels is unclear. It is furthermore unresolved whether droperidol at antiemetic concentrations induces action potential prolongation and arrhythmogenic early afterdepolarizations in cardiac myocytes. Methods Molecular mechanisms of HERG current inhibition by droperidol were established using two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing wild-type and mutant channels. The mutants T623A, S624A, V625A, Y652A, and F656A were generated by site-directed mutagenesis. The effect of droperidol on action potentials was investigated in cardiac myocytes isolated from guinea pig hearts using the patch clamp technique. Results Droperidol inhibited currents through HERG wild-type channels with a concentration of half-maximal inhibition of 0.6-0.9 microM. Droperidol shifted the channel activation and the steady state inactivation toward negative potentials while channel deactivation was not affected. Current inhibition increased with membrane potential and with increasing duration of current activation. Inhibition of HERG channels was similarly reduced by all mutations. Droperidol at concentrations between 5 and 100 nM prolonged whereas concentrations greater than 300 nm shortened action potentials. Early afterdepolarizations were not observed. Conclusions Droperidol is a high-affinity blocker of HERG channels. Amino acid residues typically involved in high-affinity block mediate droperidol effects. Patch clamp results and computational modeling allow the hypothesis that interaction with calcium currents may explain why droperidol at antiemetic concentrations prolongs the action potential without inducing early afterdepolarizations.

scholarly journals Bioinformatics Insights Into Microbial Xylanase Protein Sequences

2018 ◽  
Vol 15 (2) ◽  
pp. 275-294
Author(s):  
Deepsikha Anand ◽  
Jeya Nasim ◽  
Sangeeta Yadav ◽  
Dinesh Yadav
Keyword(s):  
Amino Acid ◽  
Phylogenetic Tree ◽  
Catalytic Domain ◽  
Genetic Manipulation ◽  
Protein Sequences ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Molecular Weights ◽  
Aliphatic Index ◽  
Chemical Attributes

Microbial xylanases represents an industrially important group of enzymes associated with hydrolysis of xylan, a major hemicellulosic component of plant cell walls. A total of 122 protein sequences comprising of 58 fungal, 25 bacterial, 19actinomycetes and 20 yeasts xylanaseswere retrieved from NCBI, GenBank databases. These sequences were in-silico characterized for homology,sequence alignment, phylogenetic tree construction, motif assessment and physio-chemical attributes. The amino acid residues ranged from 188 to 362, molecular weights were in the range of 20.3 to 39.7 kDa and pI ranged from 3.93 to 9.69. The aliphatic index revealed comparatively less thermostability and negative GRAVY indicated that xylanasesarehydrophilicirrespective of the source organisms.Several conserved amino acid residues associated with catalytic domain of the enzyme were observed while different microbial sources also revealed few conserved amino acid residues. The comprehensive phylogenetic tree indicatedsevenorganismsspecific,distinct major clusters,designated as A, B, C, D, E, F and G. The MEME based analysis of 10 motifs indicated predominance of motifs specific to GH11 family and one of the motif designated as motif 3 with sequence GTVTSDGGTYDIYTTTRTNAP was found to be present in most of the xylanases irrespective of the sources.Sequence analysis of microbial xylanases provides an opportunity to develop strategies for molecular cloning and expression of xylanase genes and also foridentifying sites for genetic manipulation for developing novel xylanases with desired features as per industrial needs.

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