Peptide substrates for G protein-coupled receptor kinase 2

G protein-coupled receptor kinases (GRKs) control the signaling and activation of G protein-coupled receptors through phosphorylation. In this study, consensus substrate motifs for GRK2 were identified from the sequences of GRK2 protein substrates, and 17 candidate peptides were synthesized to identify peptide substrates with high affinity for GRK2. GRK2 appears to require an acidic amino acid at the −2, −3, or −4 positions and its consensus phosphorylation site motifs were identified as (D/E)X_1–3(S/T), (D/E)X_1–3(S/T)(D/E), or (D/E)X_0–2(D/E)(S/T). Among the 17 peptide substrates examined, a 13-amino-acid peptide fragment of β-tubulin (DEMEFTEAESNMN) showed the highest affinity for GRK2 (K_m, 33.9 μM; V_max, 0.35 pmol min⁻¹ mg⁻¹), but very low affinity for GRK5. This peptide may be a useful tool for investigating cellular signaling pathways regulated by GRK2.     

Mitochondrial DNA Sequence Divergence among Meloidogyne incognita,Romanomermis culicivorax,Ascaris suum,and Caenorhabditis elegans

Mitochondrial DNA sequences were obtained from the NADH dehydrogenase subunit 3 (ND3), large rRNA, and cytochrome b genes from Meloidogyne incognita and Romanomermis culicivorax. Both species show considerable genetic distance within these same genes when compared with Caenorhabditis elegans or Ascaris suum, two species previously analyzed. Caenorhabditis, Ascaris, and Meloidogyne were selected as representatives of three subclasses in the nematode class Secernentea: Rhabditia, Spiruria, and Diplogasteria, respectively. Romanomermis served as a representative out-group of the class Adenophorea. The divergence between the phytoparasitic lineage (represented by Meloidogyne) and the three other species is so great that virtually every variable position in these genes appears to have accumulated multiple mutations, obscuring the phylogenetic information obtainable from these comparisons. The 39 and 42% amino acid similarity between the M. incognita and C. elegans ND3 and cytochrome b coding sequences, respectively, are approximately the same as those of C. elegans-mouse comparisons for the same genes (26 and 44%). This discovery calls into question the feasibility of employing cloned C. elegans probes as reagents to isolate phytoparasitic nematode genes. The genetic distance between the phytoparasitic nematode lineage and C. elegans markedly contrasts with the 79% amino acid similarity between C. elegans and A. suum for the same sequences. The molecular data suggest that Caenorhabditis and Ascaris belong to the same subclass.     

Sann: solvent accessibility prediction of proteins by nearest neighbor method

We present a method to predict the solvent accessibility of proteins which is based on a nearest neighbor method applied to the sequence profiles. Using the method, continuous real-value prediction as well as two-state and three-state discrete predictions can be obtained. The method utilizes the z-score value of the distance measure in the feature vector space to estimate the relative contribution among the k-nearest neighbors for prediction of the discrete and continuous solvent accessibility. The Solvent accessibility database is constructed from 5717 proteins extracted from PISCES culling server with the cutoff of 25% sequence identities. Using optimal parameters, the prediction accuracies (for discrete predictions) of 78.38% (two-state prediction with the threshold of 25%), 65.1% (three-state prediction with the thresholds of 9 and 36%), and the Pearson correlation coefficient (between the predicted and true RSA's for continuous prediction) of 0.676 are achieved An independent benchmark test was performed with the CASP8 targets where we find that the proposed method outperforms existing methods. The prediction accuracies are 80.89% (for two state prediction with the threshold of 25%), 67.58% (three-state prediction), and the Pearson correlation coefficient of 0.727 (for continuous prediction) with mean absolute error of 0.148. We have also investigated the effect of increasing database sizes on the prediction accuracy, where additional improvement in the accuracy is observed as the database size increases. The SANN web server is available at http://lee.kias.re.kr/~newton/sann/.     

Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation

Beta adrenergic receptors (β-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of β-ARs in zebrafish are, however, largely unknown. We have identified zebrafish β-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these β-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.     

In silico characterization of a RNA binding protein of cattle filarial parasite Setaria digitata

Human lymphatic filariasis (HLF) is a neglected tropical disease which threatens nearly 1.4 billion people in 73 countries worldwide. Wuchereria bancrofti is the major causative agent of HLF and it closely resembles cattle filarial parasite Setaria digitata. Due to difficulties in procuring W. bancrofti parasite material, S. digitata cDNA library has been constructed to identify novel drug targets against HLF and many of the cDNA sequences are yet to be assigned structure and function. In this study, a 549 bp long cDNA (sdrbp) has been sequenced and characterized in silico. The shortest ORF of 249 bp from the isolated cDNA encodes a polypeptide of 82 amino acids and shows an amino acid identity of 54% with the RRM domain of human cleavage stimulation factor-64 kDa subunit (CstF-64). Structure of the protein (sdRBP) obtained by homology modelling using RRM of CstF-64 as template adopts classical RRM topology (β1α1β2β3α2β4). sdRBP model built was validated by superimposition tools and Ramachandran plot analysis. CstF-64 plays an important role in pre-mRNA polyadenylation by interacting with specific GU-rich downstream sequence element. Molecular docking studies of sdRBP with different RNA molecules revealed that sdRBP has greater binding affinity to GU-rich RNA and comparable results were obtained upon similar docking of RRM of CstF-64 with the same RNA molecules. Therefore, sdRBP is likely to perform homologous function in S. digitata. This study brings new dimensions to the functional analysis of RNA binding proteins of S. digitata and their evaluation as new drug targets against HLF.     

Detection of microsatellite instability during somatic embryogenesis of oak (Quercus robur L.)

Five microsatellite loci (QpZAG1/5, QpZAG9, QpZAG36, MSQ4, MSQ13) were used to test for genetic stability of three somatic embryogenic culture lines of Quercus robur L. and plantlets derived therefrom. DNA variation was detected among somatic embryos within all embryogenic lines, whereas no genetic instability was found among the regenerated plants. Two microsatellite loci revealed variation, and a locus-dependent instability was observed. The most polymorphic and useful microsatellite locus for detecting genetic variation was QpZAG9, with 28.5% of the investigated loci being variable.     

Quantitative Trait Loci Mapping of Leaf Morphological Traits and Chlorophyll Content in Cultivated Tetraploid Cotton

Genetic mapping provides a powerful tool for quantitative trait loci （QTL） analysis at the molecular level. A simple sequence repeat （SSR） genetic map containing 590 markers and a BCI population from two cultivated tetraploid cotton （Gossypium hirsutum L.） cultivars, namely TM-1 and Hai 7124 （G. barbadense L.）, were used to map and analyze QTL using the composite interval mapping （CIM） method. Thirty one QTLs, 10 for lobe length, 13 for lobe width, six for lobe angle, and two for leaf chlorophyll content, were detected on 15 chromosomes or linkage groups at logarithm of odds （LOD）≥2.0, of which 15 were found for leaf morphology at LOD≥3.0. The genetic effects of the QTL were estimated. These results are fundamental for marker-assisted selection （MAS） of these traits in tetraploid cotton breeding.