The Effect of Nutritional Factors and Plant Growth Regulators on Micropropagation and Production of Phenolic Acids and Saponins from Plantlets and Adventitious Root Cultures of Eryngium maritimum L.

Eryngium maritimum L. is a valuable medicinal species, but since it is protected plant, collection from natural populations is forbidden. Therefore, establishing an efficient system for micropropagation of this species is desirable. To determine the optimal nutritional factors needed for shoot multiplication, root development and secondary metabolites accumulation, different media and plant growth regulators were tested. The highest plant regeneration efficiency (over 96 %), with 4.4 shoots per explant was induced on Murashige and Skoog (MS) medium supplemented with 1.0 mg L^?1 benzyladenine (BA) and 0.1 mg L^?1 indole-3-acetic acid (IAA). The in vitro-regenerated shoots were rooted (83.3–100 %) and transferred to an experimental plot with 62 % efficiency. Flow cytometric analysis revealed no variation in nuclear DNA content in field- and in vitro-delivered plant material. Ultra high performance liquid chromatography (UHPLC) indicated that multiple shoots and roots from in vitro-regenerated plantlets and adventitious root cultures maintained the production of rosmarinic (RA) and chlorogenic (CGA) acids and triterpenoid saponins found in the rosette leaves and roots of E. maritimum intact plants. UHPLC revealed a 12-fold increase of RA and CGA and 3.2-fold higher accumulation of triterpenoid saponins in roots of in vitro-derived plantlets in comparison to roots from field-grown plants. Adventitious root cultures allowed continuous growth of excised root in liquid media with or without exogenous auxins. The roots grown in liquid medium supplemented with 0.1 mg L^?1 IAA showed higher (227-fold) phenolic acids accumulation than those without auxin. Obtained results confirmed that micropropagation is a useful strategy in the protection of endangered species and a renewable source of a high quality plant material for secondary metabolites production.     

Development and use of an efficient system for random mariner transposon mutagenesis to identify novel genetic determinants of biofilm formation in the core Enterococcus faecalis genome

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, most of the genetic analysis of these organisms has focused on mobile genetic elements, and existing tools for manipulation and analysis of the core E. faecalis chromosome are limited. We are interested in a comprehensive analysis of the genetic determinants for biofilm formation encoded within the core E. faecalis genome. To identify such determinants, we developed a substantially improved system for transposon mutagenesis in E. faecalis based on a mini-mariner transposable element. Mutagenesis of wild-type E. faecalis with this element yielded predominantly mutants carrying a single copy of the transposable element, and insertions were distributed around the entire chromosome in an apparently random fashion. We constructed a library of E. faecalis transposon insertion mutants and screened this library to identify mutants exhibiting a defect in biofilm formation. Biofilm-defective mutants were found to carry transposon insertions both in genes that were previously known to play a role in biofilm formation and in new genes lacking any known function; for several genes identified in the screen, complementation analysis confirmed a direct role in biofilm formation. These results provide significant new information about the genetics of enterococcal biofilm formation and demonstrate the general utility of our transposon system for functional genomic analysis of E. faecalis.     

MxaJ structure reveals a periplasmic binding protein‐like architecture with unique secondary structural elements

MxaJ is a component of type II methanol dehydrogenase (MDH) that mediates electron transfer during methanol oxidation in methanotrophic bacteria. However, little is known about how MxaJ structurally cooperates with MDH and Cytochrome c_L. Here, we report for the first time the crystal structure of MxaJ. MxaJ consists of eight α‐helices and six β‐strands, and resembles the “bi‐lobate” folding architecture found in periplasmic binding proteins. Distinctive features of MxaJ include prominent loops and a β‐strand around the hinge region supporting the ligand‐binding cavity, which might provide a more favorable framework for interacting with proteins rather than small molecules. Proteins 2017; 85:1379–1386. © 2017 Wiley Periodicals, Inc.     

Host range factor 1 from Lymantria dispar Nucleopolyhedrovirus (NPV) is an essential viral factor required for productive infection of NPVs in IPLB-Ld652Y cells derived from L. dispar

Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells.     

Chemoenzymatic galactosialylation with integrated cofactor regeneration

As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac (2-8)Gal(1-4)GlcNAc(1-O)-pent-4-ene was synthesized starting from GlcNAc(1-O)-pent-4-ene, UDP-glucose andN-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and (2-6)sialyl-transferase in a complete cofactor regeneration system.Abbreviations Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5-monophosphosialate - CMP cytidine 5-monophosphate - CDP cytidine 5-diphosphate - CTP cytidine 5-triphosphate - Gal galactose - GlcNAc N-acetylglucosamine - UDP uridine 5-diphosphate - UDP-Glc uridine-5-diphosphoglucose - UDP-Gal uridine-5-diphosphogalactose - PEP phosphoenolpyruvate     

Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata,identification and specificity of a branching β1→6 galactosyltransferase

Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-³H]-uridine 5diphosphogalactose the incorporation of labelled d-galactose into the polysaccharide can be traeed in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltrasferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient -(16) galactosyltransferase was detected when methyl 3-O-(-d-galactopyranosyl)--d-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by ¹H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked 13. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer d-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(-d-galactopyranosyl)-d-galactopyranose. but without any for methyl-3-O-(-d-galactopyranosyl)--d-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan.Abbreviation Gal galactose - glc gas-liquid chromatography - Gro glycerol - tlc thin layer chromatography     

Modification of myosin subfragment 1 by carbodiimide in the presence of a nucleophile. Effect on adenosinetriphosphatase activities

The modification of myosin subfragment 1 by N-cyclohexyl-N'-[2-(4-morpholinyl)ethyl]carbodiimide methyl p-toluenesulfonate in the presence of the nucleophile nitrotyrosine ethyl ester was investigated. For elimination of interference of the thiol groups, the two most reactive thiols were protected by cyanylation with 2-nitro-5-(thiocyanato)benzoic acid. The ATPase activity of the cyanylated myosin subfragment 1 was not lost, but had changed. At pH 5.9, carbodiimide in the presence of the nucleophile rapidly inactivated the cyanylated enzyme. The inactivation followed first-order kinetics. The K+(EDTA)--, Ca2+--, and Mg2+--ATPase activities decreased at the same rate. Inactivation and incorporation of nucleophile occurred simultaneously. A full loss of activity resulted from the incorporation of 1 mol of nitrotyrosine per mol of myosin subfragment 1. Pyrophosphate, ITP, ADP, and ATP protected against inactivation, and the efficiency of the protection was parallel to the ligand binding strength. These results suggested that one carboxyl group was essential for the active conformation of myosin.     

Enzyme-catalysed synthesis of galactosylated 1D- and 1L-chiro-inositol, 1D-pinitol, myo-inositol and selected derivatives using the beta-galactosidase from the thermophile Thermoanaerobacter sp. strain TP6-B1

The products from the enzymatic beta-D-galactopyranosylation of 1D-chiro-inositol, 1D-pinitol, 1D-3-O-allyl-4-O-methyl-chiro-inositol, 1D-3,4-di-O-methyl-chiro-inositol, 1L-chiro-inositol and myo-inositol in combined yields ranging from 46% to 64% have been obtained using the beta-galactosidase isolated from an anaerobic extreme thermophile, Thermoanaerobacter sp. strain TP6-B1 and p-nitrophenyl beta-D-galactopyranoside as the donor. Analysis of the products from these reactions reveals information about the acceptor preferences of the enzyme.     

Emergence and serovar transition of Vibrio parahaemolyticus pandemic strains isolated during a diarrhea outbreak in Vietnam between 1997 and 1999

We characterized 523 Vibrio parahaemolyticus strains isolated during a survey of diarrhea patients in Khanh Hoa province, Vietnam between 1997 and 1999. Forty-nine percent of the strains were judged to belong to the pandemic strains that emerged around 1996 and spread to many countries. These strains were positive in the GS-PCR assay and carried the tdh gene. The ORF8 of the f237 phage genome, a possible marker of the pandemic clone, was absent in 10% of these strains. Eleven O: K serovars were detected among the pandemic strains and the strains representing all 11 serovars of pandemic strains were shown to be closely related regardless of the ORF8 genotype using arbitrarily primed PCR and pulsed field gel electrophoresis analyses. It was clear that a transition of major serovars occurred among the pandemic strains represented by the emergence of O3: K6 in 1997, O4: K68 in 1998, and O1: K25 in 1998 and 1999.     

Cryopreservation of invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure

Invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott.) were successfully cryopreserved by vitrification. Excised shoot tips precultured on solidified MS supplemented with 0.3M sucrose and maintained under a 16 h phtoperiod at 25°C for 16 h were loaded with a mixture of 2M glycerol plus 0.4M sucrose for 20 min at 25°C. The shoot tips were then sufficiently dehydrated with a highly concentrated vitrification solution (PVS2) for 20 min at 25°C prior to immersion into liquid nitrogen. Successfully vitrified and warmed shoot tips resumed growth within 7 days and developed shoots directly without intermediate callus formation. The average rate of shoot recovery amounted to around 80%, and the vitrification protocol appeared to be very promising for the cryopreservation of taro germplasm.Abbreviations DMSO Dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS Murashige & Skoog medium (1962) - TDZ thidiazuron