The occurrence and accumulation of <Emphasis Type="SmallCaps">d</Emphasis>-pinitol in fenugreek (<Emphasis Type="Italic">Trigonella foenum graecum</Emphasis> L.)

This article presents changes in concentrations of d-pinitol (and other cyclitols as well as low molecular weight carbohydrates) in vegetative and reproductive organs of fenugreek (Trigonella foenumgraecum L.) during an entire plant growing period. d-Pinitol was the major cyclitol in all tested organs, representing 43–94% of total cyclitols and 2–77% of total soluble carbohydrates. The highest concentration of d-pinitol was found in pods (14–23 mg g^?1 of dry weight, DW), lower in leaves and stems (5–20 and 9–10 mg g^?1 DW, respectively), and the lowest in maturing seeds (2–5 mg g^?1 DW). Although maturing seeds accumulate α-d-galactosides of d-pinitol (galactosyl pinitols, up to 6.6 mg g^?1 DW), the major storage sugars were raffinose family oligosaccharides (RFOs, 65.37 mg g^?1 DW). Both RFOs and galactosyl pinitols are hydrolyzed during seed germination, releasing sucrose and d-pinitol, respectively. Accumulation of free galactose was not detected. Owing to the high concentration of d-pinitol (up to 23.70 mg g^?1 DW) and low concentration of soluble sugars, developing pods seem to be the best source of d-pinitol.     

Changes in soluble carbohydrates in polar Caryophyllaceae and Poaceae plants in response to chilling

Four species of flowering plants comprising Arctic populations of Cerastium alpinum and Poa arctica var. vivipara and indigenous Antarctic species Colobanthus quitensis and Deschampsia antarctica were investigated. Plants derived from natural origins were grown in an experimental greenhouse in Poland (53°47′N and 20°30′E latitude). Plants for experiment were collected during spring of 2010. Soluble carbohydrates in the intact shoots of C. alpinum and C. quitensis, polar plants of the family Caryophyllaceae, and D. antarctica and P. arctica var. vivipara, representatives of the family Poaceae, were analyzed by gas chromatography, and their involvement in the plants’ response to chilling stress was examined. Plant tissues of the examined families growing in a greenhouse conditions (18–20 °C, short day 10/14 h light/darkness) differed in the content and composition of soluble carbohydrates. In addition to common monosaccharides, myo-inositol and sucrose, Caryophyllaceae plants contained raffinose family oligosaccharides (RFOs), d-pinitol and mono-galactosyl pinitols. RFOs and d-pinitol were not detected in plants of the family Poaceae which contain 1-kestose, a specific tri-saccharide. The accumulation of significant quantities of sucrose in all investigated plants, RFOs in Caryophyllaceae plants and 1-kestose in Poaceae plants in response to chilling stress (4 °C for 48 h with a long day photoperiod, 20/4 h) indicates that those compounds participate in the stress response. The common sugar accumulating in cold stress response and probably most important for chilling tolerance of four investigated plants species seems to be sucrose. On the other hand, the accumulation of above-mentioned carbohydrates during chilling stress can be a return to sugars metabolism, occurring in natural environmental conditions. No changes in d-pinitol concentrations were observed in the tissues of C. alpinum and C. quitensis plants subjected to both low and elevated temperatures, which probably rules out the protective effects of d-pinitol in response to cold stress.     

Ectopic expression of sorbitol-6-phosphate 2-dehydrogenase gene from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis> enhances salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

d-Sorbitol-6-phosphate 2-dehydrogenase (S6PDH, E.C. 1.1.1.140) catalyzes the NADH-dependent conversion of d-fructose 6-phosphate (F6P) to d-sorbitol 6-phosphate (S6P). In this work, recombination and characterization of Haloarcula marismortui d-sorbitol-6-phosphate 2-dehydrogenase are reported. Haloarcula marismortui d-sorbitol-6-phosphate 2-dehydrogenase was expressed in P. pastoris and Arabidopsis thaliana. Enzyme assay indicated that HmS6PDH catalyzes the reduction of d-fructose 6-phosphate to d-sorbitol 6-phosphate and HmS6PDH activity was enhanced by NaCl. Furthermore, transgenic A. thaliana ectopic expressing HmS6PDH accumulate more sorbitol under salt stress. These results suggest that the ectopic expression of HmS6PDH in plants can facilitate future studies regarding the engineering and breeding of salt-tolerant crops.     

Effect of down-regulating 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose-5-phosphate reductoisomerase by RNAi on growth and artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), an important enzyme in the 2-c-methyl-d-erythritol-4-phosphate (MEP) pathway in plant plastids, provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the roles of the MEP pathway in regulating growth, development and artemisinin biosynthesis of Artemisia annua L., we used RNA interference technology to generate transgenic plants with suppressed expression of DXR in A. annua (AaDXR). Suppression of AaDXR resulted in shorter stems, decreased branch numbers and leaf area, lower density of leaf trichomes. Although AaDXR-RNAi plants had no significant changes on the stomatal conductance, the net photosynthesis rate was decreased by 20.0–31.4% due to the marked decline in the contents of chlorophyll. Decreased levels of endogenous gibberellic acid (GA₃) and abscisic acid were also detected in the transgenic lines. The artemisinin contents in leaves of all tested transgenic lines declined by 41.8–73.4% at the vegetative stage and 61.5–63.6% at the stages of flowering. The enhancement of artemisinin contents by methyl jasmonate at 300 µM has been abolished at seedling and vegetative stages in AaDXR-RNAi plants. These results demonstrate that AaDXR play import roles in the control of plan vegetative growth and artemisinin biosynthesis in A. annua.     

Synthesis of a tetra- and a trisaccharide related to an anti-tumor saponin “Julibroside J<Subscript>28</Subscript>” from <Emphasis Type="Italic">Albizia julibrissin</Emphasis>

Simple and convergent synthesis of a tetra- and a trisaccharide portions of an antitumor compound Julibroside J₂₈, isolated from Albizia julibrissin, that showed significant in vitro antitumor activity against HeLa, Bel-7402 and PC-3M-1E8 cancer cell lines is reported. The tetrasaccharide has been synthesized as its p-methoxyphenyl glycoside starting from commercially available d-glucose, l-rhamnose and l-arabinose. The trisaccharide part has been synthesized from commercially available N-acetyl d-glucosamine, d-fucose and d-xylose using simple protecting group manipulations. Sulfuric acid immobilized on silica has been used successfully as a Brönsted acid catalyst for the crucial glycosylation steps.     

General assay for enzymes in the heptose biosynthesis pathways using electrospray ionization mass spectrometry

The ADP-l-glycero-β-d-manno-heptose and the GDP-6-deoxy-α-d-manno-heptose biosynthesis pathways play important roles in constructing lipopolysaccharide of Gram-negative bacteria. Blocking the pathways is lethal or increases antibiotic susceptibility to pathogens. Therefore, the enzymes involved in the pathways are novel antibiotic drug targets. Here, we designed an efficient method to assay the whole enzymes in the pathways using mass spectrometry and screened 148 compounds. One promising lead is (?)-nyasol targeting d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC) included in the GDP-6-deoxy-α-d-manno-heptose biosynthesis pathway from Burkholderia pseudomallei. The inhibitory activity of the lead compound against HddC has been confirmed by blocking the system transferring the guanosine monophosphate (GMP) moiety to α-d-glucose-1-phosphate. (?)-Nyasol exhibits the half maximal inhibitory concentration (IC50) value of 17.6 μM. A further study is going on using (?)-nyasol derivatives to find better leads with high affinity.     

Studies in the taxonomy of the Polypores II

The following genera are redefined:Albatrellus S. F. Gray,Heterobasidion Bref.,Haploporus Bond. et Sing. ex Sing.,Fomitopsis P. Karst. andRigidoporus Murrill two new subgenera are described:Polyporus subgen.Dendropolyporus Pouz. (type:Polyporus umbellatus) andRigidoporus subgen.Neooxyporus Pouz. (type:Polyporus latemarginatus); the genusOxyporus (Bourd. etGalz.)Donk is classified as a subgenus of the genusRigidoporus,Murrill and the generaBjerkandera P. Karst. andLeptoporus quél. are classified as subgenera of the genusTyromyces P. Karst. The new subfamilyAlbatrelloideae Pouz. (genera:Albatrellus andGrifola) is described and 14 new specific combinations are made. The new genusIrpicodon Pouz. (type:Irpex pendulus) is proposed.     

Heterologous expression of a β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidase in <Emphasis Type="Italic">Caldicellulosiruptor bescii</Emphasis> has a surprisingly modest effect on the activity of the exoproteome and growth on crystalline cellulose

Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, β-d-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. In spite of its ability to grow efficiently on crystalline cellulose, no extracellular β-d-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted β-d-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable β-d-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that β-d-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.     

Enantiomer-Selective Photo-Induced Reaction of Protonated Tryptophan with Disaccharides in the Gas Phase

In order to investigate chemical evolution in interstellar molecular clouds, enantiomer-selective photo-induced chemical reactions between an amino acid and disaccharides in the gas phase were examined using a tandem mass spectrometer containing an electrospray ionization source and a cold ion trap. Ultraviolet photodissociation mass spectra of cold gas-phase noncovalent complexes of protonated tryptophan (Trp) enantiomers with disaccharides consisting of two d-glucose units, such as d-maltose or d-cellobiose, were obtained by photoexcitation of the indole ring of Trp. NH₂CHCOOH loss via cleavage of the C_α–C_β bond in Trp induced by hydrogen atom transfer from the NH₃ ⁺ group of a protonated Trp was observed in a noncovalent heterochiral H⁺(l-Trp)(d-maltose) complex. In contrast, a photo-induced chemical reaction forming the product ion with m/z 282 occurs in homochiral H⁺(d-Trp)(d-maltose). For d-cellobiose, both NH₂CHCOOH elimination and the m/z 282 product ion were observed, and no enantiomer-selective phenomena occurred. The m/z 282 product ion indicates that the photo-induced C-glycosylation, which links d-glucose residues to the indole moiety of Trp via a C–C bond, can occur in cold gas-phase noncovalent complexes, and its enantiomer-selectivity depends on the structure of the disaccharide.     

A new recombinant <Emphasis Type="Italic">endo-</Emphasis>1,3-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucanase from the marine bacterium <Emphasis Type="Italic">Formosa algae</Emphasis> KMM 3553: enzyme characteristics and transglycosylation products analysis

A specific endo-1,3-β-d-glucanase (GFA) gene was found in genome of marine bacterium Formosa algae KMM 3553. For today this is the only characterized endo-1,3-β-d-glucanase (EC 3.2.1.39) in Formosa genus and the only bacterial EC 3.2.1.39 GH16 endo-1,3-β-d-glucanase with described transglycosylation activity. It was expressed in E. coli and isolated in homogeneous state. Investigating the products of polysaccharides digestion with GFA allowed to establish it’s substrate specificity and classify this enzyme as glucan endo-1,3-β-d-glucosidase (EC 3.2.1.39). The amino-acid sequence of GFA consists of 556 residues and shows sequence similarity of 45–85% to β-1,3-glucanases of bacteria belonging to the CAZy 16th structural family of glycoside hydrolases GH16. Enzyme has molecular weight 61 kDa, exhibits maximum of catalytic activity at 45?°C, pH 5.5. Half-life period at 45 °С is 20 min, complete inactivation happens at 55?°C within 10 min. K_m for hydrolysis of laminarin is 0.388 mM. GFA glucanase from marine bacteria F. algae is one of rare enzymes capable to catalyze reactions of transglycosylation. It catalyzed transfer of glyconic part of substrate molecule on methyl-β-d-xylopyranoside, glycerol and methyl-α-d-glucopyranoside. The enzyme can be used in structure determination of β-1,3-glucans (or mixed 1,3;1,4- and 1,3;1,6-β-d-glucans) and enzymatic synthesis of new carbohydrate-containing compounds.     

The variability of critical species ofLotus L. in Iran and in the neighbouring countries I. the circle ofL. corniculatus L. andL. gebelia Vent.

The region of Iran, Iraq, Afghanistan and the neighbouring countries is important for some groups of the speciesLotus L., especially those of the circle ofL. corniculatus L. andL. gebelia Vent. The first group is represented by the speciesL. corniculatus L. with 4 subspecies (3 of which are important for this region), andL. tenuis Waldst. etKit. which here attains the eastern boundary of the continuous area of distribution, and by the eastern speciesL. krylovii Schischk. etSerg. andL. rechingeri Chrtková-?ertová. The second group is represented by the speciesL. gebelia Vent.,L. michauxianus Ser. in DC. andL. libanoticus Boiss. their areas of distribution covering mostly those regions. Most of the species show considerable variability within the species.     

Change of the <Emphasis Type="Italic">N</Emphasis>-terminal codon bias combined with tRNA supplementation outperforms the selected fusion tags for production of human <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidase as active inclusion bodies

Objectives

To optimize the production of active inclusion bodies (IBs) containing human d-amino acid oxidase (hDAAO) in Escherichia coli.

Results

The optimized initial codon region combined with the coexpressed rare tRNAs, fusion of each of the N-terminal partners including cellulose-binding module, thioredoxin, glutathione S-transferase and expressivity tag, deletion of the incorporated linker, and improvement of tRNA abundance affected the production and activity for oxidizing d-alanine of the hDAAO in IBs. Compared with the optimized fusion constructs and expression host, IBs yields and activity were increased to 2.6- and 2.8-fold respectively by changing the N-terminal codon bias of the hDAAO. The insoluble hDAAO codon variant displayed the same substrate specificity as the soluble one for oxidizing d-alanine, d-serine and d-aspartic acid. The freshly prepared hDAAO codon variant was used for analyzing the l-serine racemization activity of the bacterially expressed maize serine racemase.

Conclusions

Optimization of the N-terminal codon bias combined with the coexpression of rare tRNAs is a novel and efficient approach to produce active IBs of the hDAAO.

     

Differentiation of species in Gonioloboceras

The type specimen ofGonioloboceras goniolobum (Meek), rediscovered by Spath in the British Museum, is the foundation for a more accurate comparative study of this and other species ofGonioloboceras.Gonioloboceras described asG. goniolobum byElias in 1938 is differentiated asGonioloboceras schmidti, new species. Suture sets (new term) for several growth stages inG. goniolobum (Meek),G. welleriSmith,G. schmidtiElias, G.eliasiMiller &Owen, andG. asiaticumLibrovitch are assembled and used for differentiation of the species.The Kazakhstan goniatite faunule containingG. asiaticum is considered of very late Pennsylvanian age.     

Structural and functional characterization of aspartate racemase from the acidothermophilic archaeon <Emphasis Type="Italic">Picrophilus torridus</Emphasis>

Functional and structural characterizations of pyridoxal 5′-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60 °C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5′-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of d-amino acids. Unexpectedly, the proportion of d-aspartate to total aspartate was not very high. In contrast, both d-proline and d-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus.     

Structural diversity of xylans in the cell walls of monocots

Main conclusion

Xylans in the cell walls of monocots are structurally diverse. Arabinofuranose-containing glucuronoxylans are characteristic of commelinids. However, other structural features are not correlated with the major transitions in monocot evolution. Most studies of xylan structure in monocot cell walls have emphasized members of the Poaceae (grasses). Thus, there is a paucity of information regarding xylan structure in other commelinid and in non-commelinid monocot walls. Here, we describe the major structural features of the xylans produced by plants selected from ten of the twelve monocot orders. Glucuronoxylans comparable to eudicot secondary wall glucuronoxylans are abundant in non-commelinid walls. However, the α-d-glucuronic acid/4-O-methyl-α-d-glucuronic acid is often substituted at O-2 by an α-l-arabinopyranose residue in Alismatales and Asparagales glucuronoxylans. Glucuronoarabinoxylans were the only xylans detected in the cell walls of five different members of the Poaceae family (grasses). By contrast, both glucuronoxylan and glucuronoarabinoxylan are formed by the Zingiberales and Commelinales (commelinids). At least one species of each monocot order, including the Poales, forms xylan with the reducing end sequence -4)-β-d-Xylp-(1,3)-α-l-Rhap-(1,2)-α-d-GalpA-(1,4)-d-Xyl first identified in eudicot and gymnosperm glucuronoxylans. This sequence was not discernible in the arabinopyranose-containing glucuronoxylans of the Alismatales and Asparagales or the glucuronoarabinoxylans of the Poaceae. Rather, our data provide additional evidence that in Poaceae glucuronoarabinoxylan, the reducing end xylose residue is often substituted at O-2 with 4-O-methyl glucuronic acid or at O-3 with arabinofuranose. The variations in xylan structure and their implications for the evolution and biosynthesis of monocot cell walls are discussed.

     

Flechtensystematische Studien IV. Die GattungGyalidea Lett

Im Artikel wird die ursprüngliche Gattungsdiagnose derGyalidea Lett. um die Merkmale der Pilzkomponente ergänzt. Die angenommenen verwandtschaftlichen Beziehungen derGyalidea zu der FamilieAsterothyriaceae werden diskutiert. Alle bisher festgestellten Arten mit Übersichten der revidierten Proben werden kurz beschrieben. Neue Taxa und Kombinationen:Gyalidea dodgei spec. n.,G. epiphylla spec. n.,G. mayaguezensis spec. n.,G. portoricensis spec. n.,G. fritzei (Stein) comb. n.,G. fritzei var.rivularis (Eitn.) comb. n.,G. hyalinescens (Nyl.) comb. n.,G. lecideopsis var.convarians (Nyl.) comb. n.,G. lecideopsis var.stigmatoides (Nyl.) comb. n.,G. mexicana (B. de Lesd.) comb. n. undG. subscutellaris (Vězda) comb. n.     

Some chromosome numbers of the CzechoslovakLeguminosae

Chromosome numbers of the Czechoslovak species of the genusLotus, from various localities have been determined. The paper includes the speciesLotus uliginosus Schkuhr,L.tenuis Waldst. etKit. andL. borbásii Ujhelyi.     

Beiträge zur Sedimentation und fossilführung des hunsrückschiefers 14.Viriatellina fuchsi (Kutscher, 1931) im Hunsrückschiefer und im Tentaculitenknollenkalk thüringens

The new genusViriatellina Bou?ek, 1964 unites the two older species namedNovakia gemündina Runzheimer, 1932 andTentaculites fuchsi Kutscher, 1931. ThusViriatellina gemuendina (Runzheimer) is cancelled, on the other handViriatellina fuchsi (Kutscher) remains valid. RecentlyViriatellina fuchsi (Kutscher) has been found in the tentaculitiferous limestone of Thuringia.     

Engineering an ATP-dependent <Emphasis Type="SmallCaps">d</Emphasis>-Ala:<Emphasis Type="SmallCaps">d</Emphasis>-Ala ligase for synthesizing amino acid amides from amino acids

We successfully engineered a new enzyme that catalyzes the formation of d-Ala amide (d-AlaNH₂) from d-Ala by modifying ATP-dependent d-Ala:d-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of d-Ala-d-Ala from two molecules of d-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second d-Ala of d-Ala-d-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for d-AlaNH₂ production. The S293E variant, which was selected as the best enzyme for d-AlaNH₂ production, exhibited an optimal activity at pH 9.0 and 40 °C for d-AlaNH₂ production. The apparent K _m values of this variant for d-Ala and NH₃ were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of d-AlaNH₂ from 10 and 50 mM d-Ala and 3 M NH₄Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.