Mechanisms of the tRNA wobble cytidine modification essential for AUA codon decoding in prokaryotes

Bacteria and archaea have 2-lysylcytidine (L or lysidine) and 2-agmatinylcytidine (agm²C or agmatidine), respectively, at the first (wobble) position of the anticodon of the AUA codon-specific tRNA^Ile. These lysine- or agmatine-conjugated cytidine derivatives are crucial for the precise decoding of the genetic code. L is synthesized by tRNA^Ile-lysidine synthetase (TilS), which uses l-lysine and ATP as substrates. Agm²C formation is catalyzed by tRNA^Ile-agm²C synthetase (TiaS), which uses agmatine and ATP for the reaction. Despite the fact that TilS and TiaS synthesize structurally similar cytidine derivatives, these enzymes belong to non-related protein families. Therefore, these enzymes modify the wobble cytidine by distinct catalytic mechanisms, in which TilS activates the C2 carbon of the wobble cytidine by adenylation, while TiaS activates it by phosphorylation. In contrast, TilS and TiaS share similar tRNA recognition mechanisms, in which the enzymes recognize the tRNA acceptor stem to discriminate tRNA^Ile and tRNA^Met.     

Enzymatic resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase

2-(Substituted phenoxy)-1-propanols, e.g. 2-(4-chlorophenoxy)-1-propanol, belonging to primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, as judged by the value of enantiomeric ratio E (up to 27), through the enantioselective acylation with vinyl butanoate mediated by the little-known lipase from Achromobacter sp. in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors.     

Cloning, sequencing, and expression of the genes encoding an isocyclomaltooligosaccharide glucanotransferase and an alpha-amylase from a Bacillus circulans strain

The gene for a novel glucanotransferase, isocyclomaltooligosaccharide glucanotransferase (IgtY), involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}] from starch, was cloned from the genome of B. circulans AM7. The IgtY gene, designated igtY, consisted of 2,985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The deduced amino-acid sequence showed similarities to 6-alpha-maltosyltransferase, alpha-amylase, and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8,325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme was purified. The enzyme acted on maltooligosaccharides that have a degree of polymerization (DP) of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme (IgtZ), which has a novel amino-acid sequence, should be assigned to alpha-amylase. It is notable that both IgtY and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to a family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.     

Purification and characterization of highly branched α-glucan-producing enzymes from Paenibacillus sp. PP710

Highly branched α-glucan molecules exhibit low digestibility for α-amylase and glucoamylase, and abundant in α-(1→3)-, α-(1→6)-glucosidic linkages and α-(1→6)-linked branch points where another glucosyl chain is initiated through an α-(1→3)-linkage. From a culture supernatant of Paenibacillus sp. PP710, we purified α-glucosidase (AGL) and α-amylase (AMY), which were involved in the production of highly branched α-glucan from maltodextrin. AGL catalyzed the transglucosylation reaction of a glucosyl residue to a nonreducing-end glucosyl residue by α-1,6-, α-1,4-, and α-1,3-linkages. AMY catalyzed the hydrolysis of the α-1,4-linkage and the intermolecular or intramolecular transfer of maltooligosaccharide like cyclodextrin glucanotransferase (CGTase). It also catalyzed the transfer of an α-1,4-glucosyl chain to a C3- or C4-hydroxyl group in the α-1,4- or α-1,6-linked nonreducing-end residue or the α-1,6-linked residue located in the other chains. Hence AMY was regarded as a novel enzyme. We think that the mechanism of formation of highly branched α-glucan from maltodextrin is as follows: α-1,6- and α-1,3-linked residues are generated by the transglucosylation of AGL at the nonreducing ends of glucosyl chains. Then AMY catalyzes the transfer of α-1,4-chains to C3- or C4-hydroxyl groups in the α-1,4- or α-1,6-linked residues generated by AGL. Thus the concerted reactions of both AGL and AMY are necessary to produce the highly branched α-glucan from maltodextrin.     

Antidiuretic activity of teleost urophysial extracts in the rat

Summary Extracts of the carp urophysis elicit a marked decrease in urine flow in the anaesthetized hydrated rat. Reproducible dose-dependent responses are obtained within the range of 2 to 16 g of acetonedried carp urophysis per 100 gBW of the rat. The carp urophysial antidiuretic substance is peptidic, and is different from the neurohypophysial peptides. The bulk of antidiuretic activity is located in the electrondense granules in the carp urophysis. The antidiuretic substance, probably urotensin I, is found generally in teleost urophyses. The activity per mg of acetonedried urophysis is higher in freshwater teleost species than in seawater species.Abbreviations AVP arginine vasopressin - AVT arginine vasotocin - LVP lysine vasopressin - US carp urophysial standard preparation     

Hepatitis E Virus in Cambodia: Prevalence among the General Population and Complete Genome Sequence of Genotype 4

Hepatitis E virus (HEV) is a growing public health problem in many countries. In this study, we investigated HEV seroprevalence among the general population in the Siem Reap province, Cambodia, and performed HEV genetic analysis with the aim to develop an HEV prevention strategy. This seroepidemiological cross-sectional study conducted from 2010 to 2014 included 868 participants from four different locations in Siem Reap province, Cambodia. They answered questionnaires and provided blood samples for the analysis of hepatitis virus infections. Among the participants (360 men and 508 women; age range, 7–90 years), the prevalence of anti-HEV IgG was 18.4% (95% confidence interval: 15.9–21.0); HEV RNA was detected in two participants (0.23%) and was classified as genotype 3 and 4. Full-length genome of the genotype 4 isolate, CVS-Sie10, was sequenced; it contained 7,222 nucleotides and three ORFs and demonstrated high sequence identity with the swine China isolates swGX40 (95.57%), SS19 (94.37%), and swDQ (91.94%). Multivariate logistic regression analysis revealed that men, elderly people, and house workers were risk groups significantly associated with the positivity for anti-HEV IgG. This is the first report on the detection of HEV genotype 4 in humans in Cambodia and on the complete genome sequence of HEV genotype 4 from this country. Our study demonstrates that new HEV infection cases occur frequently among the general population in Cambodia, and effective preventive measures are required.     

Fine-mapping of qRL6.1, a major QTL for root length of rice seedlings grown under a wide range of NH4 + concentrations in hydroponic conditions

Root system development is an important target for improving yield in cereal crops. Active root systems that can take up nutrients more efficiently are essential for enhancing grain yield. In this study, we attempted to identify quantitative trait loci (QTL) involved in root system development by measuring root length of rice seedlings grown in hydroponic culture. Reliable growth conditions for estimating the root length were first established to renew nutrient solutions daily and supply NH₄ ⁺ as a single nitrogen source. Thirty-eight chromosome segment substitution lines derived from a cross between ‘Koshihikari’, a japonica variety, and ‘Kasalath’, an indica variety, were used to detect QTL for seminal root length of seedlings grown in 5 or 500 μM NH₄ ⁺. Eight chromosomal regions were found to be involved in root elongation. Among them, the most effective QTL was detected on a ‘Kasalath’ segment of SL-218, which was localized to the long-arm of chromosome 6. The ‘Kasalath’ allele at this QTL, qRL6.1, greatly promoted root elongation under all NH₄ ⁺ concentrations tested. The genetic effect of this QTL was confirmed by analysis of the near-isogenic line (NIL) qRL6.1. The seminal root length of the NIL was 13.5–21.1% longer than that of ‘Koshihikari’ under different NH₄ ⁺ concentrations. Toward our goal of applying qRL6.1 in a molecular breeding program to enhance rice yield, a candidate genomic region of qRL6.1 was delimited within a 337 kb region in the ‘Nipponbare’ genome by means of progeny testing of F₂ plants/F₃ lines derived from a cross between SL-218 and ‘Koshihikari’.     

Ethacrynic Acid Exhibits Selective Toxicity to Chronic Lymphocytic Leukemia Cells by Inhibition of the Wnt/β-Catenin Pathway

Background

Aberrant activation of Wnt/β-catenin signaling promotes the development of several cancers. It has been demonstrated that the Wnt signaling pathway is activated in chronic lymphocytic leukemia (CLL) cells, and that uncontrolled Wnt/β-catenin signaling may contribute to the defect in apoptosis that characterizes this malignancy. Thus, the Wnt signaling pathway is an attractive candidate for developing targeted therapies for CLL.

Methodology/Principal Findings

The diuretic agent ethacrynic acid (EA) was identified as a Wnt inhibitor using a cell-based Wnt reporter assay. In vitro assays further confirmed the inhibitory effect of EA on Wnt/β-catenin signaling. Cell viability assays showed that EA selectively induced cell death in primary CLL cells. Exposure of CLL cells to EA decreased the expression of Wnt/β-catenin target genes, including LEF-1, cyclin D1 and fibronectin. Immune co-precipitation experiments demonstrated that EA could directly bind to LEF-1 protein and destabilize the LEF-1/β-catenin complex. N-acetyl-L-cysteine (NAC), which can react with the α, β-unsaturated ketone in EA, but not other anti-oxidants, prevented the drug''s inhibition of Wnt/β-catenin activation and its ability to induce apoptosis in CLL cells.

Conclusions/Significance

Our studies indicate that EA selectively suppresses CLL survival due to inhibition of Wnt/β-catenin signaling. Antagonizing Wnt signaling in CLL with EA or related drugs may represent an effective treatment of this disease.     

Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche

Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.