Purification,cDNA cloning,and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense)

Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.     

Water-Soluble Glycoconjugates of Vegetables I. Isolation and Properties of Arabinogalactan-Proteins from Cabbage

We present a new method for isolating and purifying water-soluble arabinogalactan-proteins from cabbage and give their chemical properties. The water-soluble nondialyzable material from fresh cabbage was separated into three fractions (A-I, II, and III) by gel filtration on Sepharose CL-4B. A-I and A-II can be purified by HPLC. Borate is necessary to avoid formation of insoluble aggregates during isolation and purification. The molecular weights of A-I, II, and III were estimated to be 4.0×10⁵, 1.0×10⁵, and 1.0~4.0×10⁴, respectively. A-I and A-II are arabinogalactan-proteins with different carbohydrate/protein ratios: 5.5/1 for A-I and 11.4/1 for A-II. The carbohydrate moieties of A-I and A-II were both arabino-3,6-galactans having d-galactose/l-arabinose ratios of 1.9/1 and 1.5/1, respectively. The amino acid composition indicates an abundance of hydroxyproline, serine, threonine and alanine, the sum of which amounted to about 50% of the total amino acids. A-I contained 1.5 times more hydroxyproline (20%) than A-II (14%), while A-II contained higher proportions of serine, glycine, and alanine. A-III was not a glycoprotein but was a mixture of carbohydrate and polypeptides.     

Purification,Crystallization and Properties of Tyrosine Phenol Lyase from Erwinia herbicola

Crystalline tyrosine phenol lyase was prepared from the cell extract of Erwinia herbicola grown in a medium supplemented with l-tyrosine. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 259,000. The crystalline enzyme catalyzed the conversion of l-tyrosine into phenol, pyruvate and ammonia, in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from d-tyrosine, S-methyl-l-cysteine, 3, 4-dihydroxyphenyl-l-alanine, l- and d-serine, and l- and d-cysteine, but at lower rates than from l-tyrosine. l-Phenyl-alanine, l-alanine, phenol and pyrocatechol inhibited pyruvate formation from l-tyrosine.

Crystalline tyrosine phenol lyase from Erwinia herbicola is inactive in the absence of added pyridoxal phosphate. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 425 mμ. The amount of pyridoxal phosphate bound to the apoenzyme was determined by equilibrium dialysis to be 2 moles per mole of enzyme. Addition of the substrate, l-tyrosine, or the competitive inhibitors, l-alanine and l-phenyl-alanine, to the holoenzyme causes appearance of a new absorption peak near 500 mμ which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor.     

Associations among Erythroferrone and Biomarkers of Erythropoiesis and Iron Metabolism,and Treatment with Long-Term Erythropoiesis-Stimulating Agents in Patients on Hemodialysis

Background

We aimed to identify associations between erythroferrone (ERFE), a regulator of hepcidin 25, and biomarkers of erythropoiesis and iron metabolism. We also aimed to determine the effects of erythropoiesis-stimulating agents (ESA), continuous erythropoietin receptor activator (CERA) and darbepoetin-α (DA) on ERFE production in patients on hemodialysis (HD).

Methods

Blood samples were obtained from 59 patients before HD sessions on day 0 (baseline). Twenty patients who were injected with either CERA (N = 10) or DA (N = 10) at the end of the dialysis week (day 0), who had ferritin ≥ 100 ng/mL and/or transferrin saturation ≥ 20%, and hemoglobin > 9 g/dL were selected from among the 59 patients. Blood was sampled serially before HD sessions on days 3, 5, 7 from patients on DA and on the same days plus day 14 from those on CERA.

Results

Levels of ERFE correlated inversely with those of hepcidin 25 and ferritin, and positively with those of soluble transferrin receptor. The hepcidin 25: ERFE ratio and hepcidin 25 levels positively correlated with ferritin levels. Levels of ERFE significantly increased from day 3 of treatment with DA and CERA and decreased by days 7 and 14, respectively. Erythropoiesis-stimulating agents concomitantly decreased levels of hepcidin 25 as those of ERFE increased.

Conclusion

We identified a novel association between ESA and ERFE in patients on HD. Both DA and CERA increased levels of ERFE that regulated hepcidin 25 and led to iron mobilization from body stores during erythropoiesis.     

Incidence of AIDS-Defining Opportunistic Infections and Mortality during Antiretroviral Therapy in a Cohort of Adult HIV-Infected Individuals in Hanoi, 2007-2014

Background

Although the prognosis for HIV-infected individuals has improved after antiretroviral therapy (ART) scale-up, limited data exist on the incidence of AIDS-defining opportunistic infections (ADIs) and mortality during ART in resource-limited settings.

Methods

HIV-infected adults in two large hospitals in urban Hanoi were enrolled to the prospective cohort, from October 2007 through December 2013. Those who started ART less than one year before enrollment were assigned to the survival analysis. Data on ART history and ADIs were collected retrospectively at enrollment and followed-up prospectively until April 2014.

Results

Of 2,070 cohort participants, 1,197 were eligible for analysis and provided 3,446 person-years (PYs) of being on ART. Overall, 161 ADIs episodes were noted at a median of 3.20 months after ART initiation (range 0.03–75.8) with an incidence 46.7/1,000 PYs (95% confidence interval [CI] 39.8–54.5). The most common ADI was tuberculosis with an incidence of 29.9/1,000 PYs. Mortality after ART initiation was 8.68/1,000 PYs and 45% (19/45) died of AIDS-related illnesses. Age over 50 years at ART initiation was significantly associated with shorter survival after controlling for baseline CD4 count, but neither having injection drug use (IDU) history nor previous ADIs were associated with poor survival. Semi-competing risks analysis in 951 patients without ADIs history prior to ART showed those who developed ADIs after starting ART were at higher risk of death in the first six months than after six months.

Conclusion

ADIs were not rare in spite of being on effective ART. Age over 50 years, but not IDU history, was associated with shorter survival in the cohort. This study provides in-depth data on the prognosis of patients on ART in Vietnam during the first decade of ART scale-up.     

Characterization of the LP28 strain-specific exopolysaccharide biosynthetic gene cluster found in the whole circular genome of Pediococcus pentosaceus

We have previously isolated a lactic acid bacterium (LAB), Pediococcus pentosaceus LP28, from the longan fruit Euphoria longana. Since the plant-derived LAB strain produces an extracellular polysaccharide (EPS), in this study, we analyzed the chemical structure and the biosynthesizing genes for the EPS.The EPS, which was purified from the LP28 culture broth, was classified into acidic and neutral EPSs with a molecular mass of about 50 kDa and 40 kDa, respectively. The acidic EPS consisted of glucose, galactose, mannose, and N-acetylglucosamine moieties. Interestingly, since pyruvate residue was detected in the hydrolyzed acidic EPS, one of the four sugars may be modified with pyruvate. On the other hand, the neutral EPS consisted of glucose, mannose, and N-acetylglucosamine; pyruvate was scarcely detected in the polysaccharide molecule.As a first step to deduce the probiotic function of the EPS together with the biosynthesis, we determined the whole genome sequence of the LP28 strain, demonstrating that the genome is a circular DNA, which is composed of 1,774,865 bp (1683 ORFs) with a GC content of 37.1%. We also found that the LP28 strain harbors a plasmid carrying 6 ORFs composed of 5366 bp with a GC content of 36.5%. By comparing all of the genome sequences among the LP28 strain and four strains of P. pentosaceus reported previously, we found that 53 proteins in the LP28 strain display a similarity of less than 50% with those in the four P. pentosaceus strains. Significantly, 4 of the 53 proteins, which may be enzymes necessary for the EPS production on the LP28 strain, were absent in the other four P. pentosaceus strains and displayed less than 50% similarity with other LAB species. The EPS-biosynthetic gene cluster detected only in the LP28 genome consisted of 12 ORFs containing a priming enzyme, five glycosyltransferases, and a putative polysaccharide pyruvyltransferase.     

Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro

Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro.     

Finlay–Wilkinson's regression coefficient as a pre‐screening criterion for yield responsiveness to elevated atmospheric CO2 concentration in crops

The rising atmospheric CO₂ concentration ([CO₂]) can increase crop productivity, but there are likely to be intraspecific variations in the response. To meet future world food demand, screening for genotypes with high [CO₂] responsiveness will be a useful option, but there is no criterion for high [CO₂] responsiveness. We hypothesized that the Finlay–Wilkinson regression coefficient (RC) (for the relationship between a genotype's yield versus the mean yield of all genotypes in a specific environment) could serve as a pre‐screening criterion for identifying genotypes that respond strongly to elevated [CO₂]. We collected datasets on the yield of 6 rice and 10 soybean genotypes along environmental gradients and compared their responsiveness to elevated [CO₂] based on the regression coefficients (i.e. the increases of yield per 100 µmol mol^?1 [CO₂]) identified in previous reports. We found significant positive correlations between the RCs and the responsiveness of yield to elevated [CO₂] in both rice and soybean. This result raises the possibility that the coefficient of the Finlay–Wilkinson relationship could be used as a pre‐screening criterion for [CO₂] responsiveness.     

Ant-Repelling Pollinators of the Myrmecophytic <Emphasis Type="Italic">Macaranga winkleri</Emphasis> (Euphorbiaceae)

Many plants have mutualistic relationships with ants, whereby plants provide food and/or nesting sites for the symbiotic ants, and in turn the ants protect the host plants by excluding herbivores. While the ants are useful as guards, they may negatively affect host reproduction by excluding pollinators. Here we studied this potential conflict in the myrmecophytic Macaranga winkleri pollinated by the thrips Dolichothrips fialae. Behavioural responses of ant guards to pollinator thrips and their chemicals, and related chemical analyses, provide evidence that thrips deter ant-guards by secreting droplets containing ant-repelling n-decanoic acid from their anuses. This is the first report of insect pollinators repelling their host’s symbiotic guard ants to perform pollination. This is a novel strategy by which a plant host avoids interference with pollination by ant-guards in an ant–plant mutualism. The acquisition of a pollination system that is resistant to ant attacks may have facilitated the evolution of myrmecophytes in the genus Macaranga.