Genetic polymorphisms and protein structures in growth hormone,growth hormone receptor,ghrelin, insulin-like growth factor 1 and leptin in Mehraban sheep

The somatotropic axis, the control system for growth hormone (GH) secretion and its endogenous factors involved in the regulation of metabolism and energy partitioning, has promising potentials for producing economically valuable traits in farm animals. Here we investigated single nucleotide polymorphisms (SNPs) of the genes of factors involved in the somatotropic axis for growth hormone (GH1), growth hormone receptor (GHR), ghrelin (GHRL), insulin-like growth factor 1 (IGF-I) and leptin (LEP), using polymerase chain reaction–single-strand conformation polymorphism (PCR–SSCP) and DNA sequencing methods in 452 individual Mehraban sheep. A nonradioactive method to allow SSCP detection was used for genomic DNA and PCR amplification of six fragments: exons 4 and 5 of GH1; exon 10 of GH receptor (GHR); exon 1 of ghrelin (GHRL); exon 1 of insulin-like growth factor-I (IGF-I), and exon 3 of leptin (LEP). Polymorphisms were detected in five of the six PCR products. Two electrophoretic patterns were detected for GH1 exon 4. Five conformational patterns were detected for GH1 exon 5 and LEP exon 3, and three for IGF-I exon 1. Only GHR and GHRL were monomorphic. Changes in protein structures due to variable SNPs were also analyzed. The results suggest that Mehraban sheep, a major breed that is important for the animal industry in Middle East countries, has high genetic variability, opening interesting prospects for future selection programs and preservation strategies.     

The N’s and O’s of Drosophila glycoprotein glycobiology

The past 25 years have seen significant advances in understanding the diversity and functions of glycoprotein glycans in Drosophila melanogaster. Genetic screens have captured mutations that reveal important biological activities modulated by glycans, including protein folding and trafficking, as well as cell signaling, tissue morphogenesis, fertility, and viability. Many of these glycan functions have parallels in vertebrate development and disease, providing increasing opportunities to dissect pathologic mechanisms using Drosophila genetics. Advances in the sensitivity of structural analytic techniques have allowed the glycan profiles of wild-type and mutant tissues to be assessed, revealing novel glycan structures that may be functionally analogous to vertebrate glycans. This review describes a selected set of recent advances in understanding the functions of N-linked and O-linked (non-glycosaminoglycan) glycoprotein glycans in Drosophila with emphasis on their relatedness to vertebrate organisms.     

Substrate-mediated proton relay mechanism for the religation reaction in topoisomerase II

The DNA religation reaction of yeast type II topoisomerase (topo II) was investigated to elucidate its metal-dependent general acid/base catalysis. Quantum mechanical/molecular mechanical calculations were performed for the topo II religation reaction, and the proton transfer pathway was examined. We found a substrate-mediated proton transfer of the topo II religation reaction, which involves the 3′ OH nucleophile, the reactive phosphate, water, Arg781, and Tyr782. Metal A stabilizes the transition states, which is consistent with a two-metal mechanism in topo II. This pathway may be required for the cleavage/religation reaction of topo IA and II and will provide a general explanation for the catalytic mechanism in the topo IA and II.     

Japanese Encephalitis Virus Core Protein Inhibits Stress Granule Formation through an Interaction with Caprin-1 and Facilitates Viral Propagation

Stress granules (SGs) are cytoplasmic foci composed of stalled translation preinitiation complexes induced by environmental stress stimuli, including viral infection. Since viral propagation completely depends on the host translational machinery, many viruses have evolved to circumvent the induction of SGs or co-opt SG components. In this study, we found that expression of Japanese encephalitis virus (JEV) core protein inhibits SG formation. Caprin-1 was identified as a binding partner of the core protein by an affinity capture mass spectrometry analysis. Alanine scanning mutagenesis revealed that Lys⁹⁷ and Arg⁹⁸ in the α-helix of the JEV core protein play a crucial role in the interaction with Caprin-1. In cells infected with a mutant JEV in which Lys⁹⁷ and Arg⁹⁸ were replaced with alanines in the core protein, the inhibition of SG formation was abrogated, and viral propagation was impaired. Furthermore, the mutant JEV exhibited attenuated virulence in mice. These results suggest that the JEV core protein circumvents translational shutoff by inhibiting SG formation through an interaction with Caprin-1 and facilitates viral propagation in vitro and in vivo.     

Changes in 1-Aminocyclopropane-1-carboxylic Acid Content and Ethylene Production in Hiproly Barley Callus during Differentiation

During differentiation after auxin withdrawal, the change in the ethylene production of Hiproly barley callus paralleled the change in 1-aminocyclopropane-1-carboxylic acid (ACC) content. The levels of ACC and ethylene production decreased rapidly, and then increased in Hiproly barley callus.

Aminooxyacetic acid (AOA) prevented the ACC and ethylene production of the callus. Moreover, aminoisobutyric acid (AIB) also inhibited the ethylene production, but did not prevent the ACC synthesis of the callus. On the other hand, methylglyoxal-bis(guanylhydrazone) (MGBG) greatly enhanced the ACC and ethylene production. Formation of adventitious roots in Hiproly barley callus was enhanced by the cultivation in the medium containing AIB or AOA. However, differentiation of the callus was strongly inhibited by MGBG.

Thus, prevention of ethylene production may be significant for differentiation of Hiproly barley callus.     

Bifunctional properties and characterization of a novel sialidase with esterase activity from Bifidobacterium bifidum

ABSTRACT

Sialidases catalyze the removal of terminal sialic acid from various complex carbohydrates. In the gastrointestinal tract, sialic acid is commonly found in the sugar chain of mucin, and many enteric commensals use mucin as a nutrient source. We previously identified two different sialidase genes in Bifidobacterium bifidum, and one was cloned and expressed as an extracellular protein designated as exo-α-sialidase SiaBb2. The other exo-α-sialidase gene (siabb1) from the same bifidobacterium encodes an extracellular protein (SiaBb1) consisting of 1795 amino acids with a molecular mass of 189 kDa. SiaBb1 possesses a catalytic domain that classifies this enzyme as a glycoside hydrolase family 33 member. SiaBb1 preferentially hydrolyzes α2,3-linked sialic acid over α2,6-linked sialic acid from sialoglycan, which is the same as SiaBb2. However, SiaBb1 has an SGNH hydrolase domain with sialate-O-acetylesterase activity and an N-terminal signal sequence and C-terminal transmembrane region. SiaBb1 is the first bifunctional sialidase identified with esterase activity.

Abbreviations: GalNAc: N-acetyl-D-galactosamine; Fuc: L-fucose; Gal: D-galactose     

Prognostic Significance of Promoter DNA Hypermethylation of cysteine dioxygenase 1 (CDO1) Gene in Primary Breast Cancer

Using pharmacological unmasking microarray, we identified promoter DNA methylation of cysteine dioxygenase 1 (CDO1) gene in human cancer. In this study, we assessed the clinicopathological significance of CDO1 methylation in primary breast cancer (BC) with no prior chemotherapy. The CDO1 DNA methylation was quantified by TaqMan methylation specific PCR (Q-MSP) in 7 BC cell lines and 172 primary BC patients with no prior chemotherapy. Promoter DNA of the CDO1 gene was hypermethylated in 6 BC cell lines except SK-BR3, and CDO1 gene expression was all silenced at mRNA level in the 7 BC cell lines. Quantification of CDO1 methylation was developed using Q-MSP, and assessed in primary BC. Among the clinicopathologic factors, CDO1 methylation level was not statistically significantly associated with any prognostic factors. The log-rank plot analysis elucidated that the higher methylation the tumors harbored, the poorer prognosis the patients exhibited. Using the median value of 58.0 as a cut-off one, disease specific survival in BC patients with CDO1 hypermethylation showed significantly poorer prognosis than those with hypomethylation (p = 0.004). Multivariate Cox proportional hazards model identified that CDO1 hypermethylation was prognostic factor as well as Ki-67 and hormone receptor status. The most intriguingly, CDO1 hypermethylation was of robust prognostic relevance in triple negative BC (p = 0.007). Promoter DNA methylation of CDO1 gene was robust prognostic indicator in primary BC patients with no prior chemotherapy. Prognostic relevance of the CDO1 promoter DNA methylation is worthy of being paid attention in triple negative BC cancer.     

Germline recombination in a novel Cre transgenic line,Prl3b1‐Cre mouse

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1‐cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL‐2) protein in placenta along with increased expression toward the end of pregnancy. PL‐2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1‐cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1‐cre;R26GRR mice revealed that tdsRed‐positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1‐cre;R26GRR testes suggested that Cre‐mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1‐cre mice line provides a unique resource to understand testicular germ‐cell development. genesis 54:389–397, 2016. © 2016 Wiley Periodicals, Inc.     

Non-genomic effects of aldosterone on intracellular ion regulation and cell volume in rat ventricular myocytes

Aldosterone has non-genomic effects that express within minutes and modulate intracellular ion milieu and cellular function. However, it is still undefined whether aldosterone actually alters intracellular ion concentrations or cellular contractility. To clarify the non-genomic effects of aldosterone, we measured [Na+]i, Ca2+ transient (CaT), and cell volume in dye-loaded rat ventricular myocytes, and we also evaluated myocardial contractility. We found the following: (i) aldosterone increased [Na+]i at the concentrations of 100 nmol/L to 10 micromol/L; (ii) aldosterone (up to 10 micromol/L) did not alter CaT and cell shortening in isolated myocytes, developed tension in papillary muscles, or left ventricular developed pressure in Langendorff-perfused hearts; (iii) aldosterone (100 nmol/L) increased the cell volume from 47.5 +/- 3.6 pL to 49.8 +/- 3.7 pL (n=8, p<0.05); (iv) both the increases in [Na+]i and cell volume were blocked by a Na+-K+-2Cl- co-transporter (NKCCl) inhibitor, bumetanide, or by a Na+/H+ exchange (NHE) inhibitor, 5-(N-ethyl-N-isopropyl) amiloride; and (v) spironolactone by itself increased in [Na+]i and cell volume. In conclusion, aldosterone rapidly increased [Na+]i and cell volume via NKCC1 and NHE, whereas there were no changes in CaT or myocardial contractility. Hence the non-genomic effects of aldosterone may be related to cell swelling rather than the increase in contractility.