Enantiomeric Composition of the trans-Dihydrodiols Produced from Phenanthrene by Fungi

The trans-dihydrodiols produced during the metabolism of phenanthrene by Cunninghamella elegans, Syncephalastrum racemosum, and Phanerochaete chrysosporium were purified by high-performance liquid chromatography (HPLC). The enantiomeric compositions and optical purities of the trans-dihydrodiols were determined to compare interspecific differences in the regio- and stereoselectivity of the fungal enzymes. Circular dichroism spectra of the trans-dihydrodiols were obtained, and the enantiomeric composition of each preparation was analyzed by HPLC with a chiral stationary-phase column. The phenanthrene trans-1,2-dihydrodiol produced by C. elegans was a mixture of the 1R,2R and 1S,2S enantiomers in variable proportions. The phenanthrene trans-3,4-dihydrodiol produced by P. chrysosporium was the optically pure 3R,4R enantiomer, but that produced by S. racemosum was a 68:32 mixture of the 3R,4R and 3S,4S enantiomers. The phenanthrene trans-9,10-dihydrodiol produced by P. chrysosporium was predominantly the 9S,10S enantiomer, but those produced by C. elegans and S. racemosum were predominantly the 9R,10R enantiomer. The results indicate that although different fungi may exhibit similar regioselectivity, there still may be differences in stereoselectivity that depend on the species and the cultural conditions.     

Properties of Phosphoglycolate Phosphatase from Chlamydomonas reinhardtii and Anacystis nidulans

The levels of activity of 2-phosphoglycolate phosphatase in the green algae, Chlamydomonas reinhardtii and Chlorella vulgaris, were in the range of 37 to 60 micromoles per milligram chlorophyll per hour and in the blue-green algae, Anacystis nidulans and Anabaena variabilis were 204 to 310 micromoles per milligram chlorophyll per hour. The activity in each species was similar regardless of whether the algae were grown with air or 5% CO₂ in air. The enzyme purified 530-fold from Chlamydomonas was stable, had a broad pH optimum between 6 and 8.5, and was specific for the hydrolysis of P-glycolate with a K_m of 23 micromolar. The enzyme purified 18-fold from Anacystis was labile, had a sharp pH optimum at 6.3, and was also specific for P-glycolate with a K_m of 94 micromolar. The molecular weight of the enzyme from Chlamydomonas was estimated to be 92,000 by gel filtration.

The phosphatase from both sources required a divalent cation for activity. The Chlamydomonas enzyme was most effectively activated by Co²⁺, but was also activated by Mg²⁺ (K_a = 30 micromolar), Mn²⁺, and Zn²⁺. The Anacystis enzyme was most effectively activated by Mg²⁺ (K_a = 140 micromolar), and was also activated by Co²⁺ and Mn²⁺, but not by Zn²⁺. Anions were also required for maximum activity of the enzyme from both sources. The Chlamydomonas enzyme was activated about 2- to 3-fold by chloride (K_a = 140 micromolar), bromide, nitrate, bicarbonate (K_a = 600 micromolar) and formate. The Anacystis enzyme was activated over 10-fold by chloride (K_a = 870 micromolar), bromide, iodide, and nitrate, but was not activated by bicarbonate or formate.

The properties of the algal enzymes were similar to those previously reported for higher plants. The levels and kinetic properties of the enzyme seemed sufficient to account for the flux through the glycolate pathway that occurs in these algae. The phosphatase was not associated with the ribulose 1,5-bisphosphate carboxylase/oxygenase responsible for P-glycolate formation in the carboxysomes of Anacystis.

     

Molecular and Functional Characterization of an Invertase Secreted by Ashbya gossypii

The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-μm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii.     

Derivatives of N-amidinoproline and their use in conventional and solid phase peptide synthesis

N-Amidinoproline, a hybrid structure modeling key features of the Arg-Pro sequence, was synthesized. The activation of carboxyl group of free N-amidinoproline was found to result in the formation of a cyclic side product, whose structure was confirmed by ESI MS, ¹H NMR, and ¹³C NMR spectra. The preparation of N-(mesitylenesulfonylamidino)-L-proline using the mesitylenesulfonyl derivative of 2-methylisothiourea was demonstrated to be accompanied by partial racemization. The target product was synthesized by modification of N-amidinoproline by mesitylenesulfonyl chloride. The possibility of using N-amidinoproline in the N-terminal modification of a peptide chain was shown by the example of synthesis of an analogue of the 95–98 fragment of fibrinogen α chain.     

Suppression of the Biocontrol Agent Trichoderma harzianum by Mycelium of the Arbuscular Mycorrhizal Fungus Glomus intraradices in Root-Free Soil

Trichoderma harzianum is an effective biocontrol agent against several fungal soilborne plant pathogens. However, possible adverse effects of this fungus on arbuscular mycorrhizal fungi might be a drawback in its use in plant protection. The objective of the present work was to examine the interaction between Glomus intraradices and T. harzianum in soil. The use of a compartmented growth system with root-free soil compartments enabled us to study fungal interactions without the interfering effects of roots. Growth of the fungi was monitored by measuring hyphal length and population densities, while specific fatty acid signatures were used as indicators of living fungal biomass. Hyphal ³³P transport and β-glucuronidase (GUS) activity were used to monitor activity of G. intraradices and a GUS-transformed strain of T. harzianum, respectively. As growth and metabolism of T. harzianum are requirements for antagonism, the impact of wheat bran, added as an organic nutrient source for T. harzianum, was investigated. The presence of T. harzianum in root-free soil reduced root colonization by G. intraradices. The external hyphal length density of G. intraradices was reduced by the presence of T. harzianum in combination with wheat bran, but the living hyphal biomass, measured as the content of a membrane fatty acid, was not reduced. Hyphal ³³P transport by G. intraradices also was not affected by T. harzianum. This suggests that T. harzianum exploited the dead mycelium but not the living biomass of G. intraradices. The presence of external mycelium of G. intraradices suppressed T. harzianum population development and GUS activity. Stimulation of the hyphal biomass of G. intraradices by organic amendment suggests that nutrient competition is a likely means of interaction. In conclusion, it seemed that growth of and phosphorus uptake by the external mycelium of G. intraradices were not affected by the antagonistic fungus T. harzianum; in contrast, T. harzianum was adversely affected by G. intraradices.     

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein (hsp) 23.7 promoter, with a neomycin-resistance gene (neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.     

The Common Nodulation Genes of Astragalus sinicus Rhizobia Are Conserved despite Chromosomal Diversity

The nodulation genes of Mesorhizobium sp. (Astragalus sinicus) strain 7653R were cloned by functional complementation of Sinorhizobium meliloti nod mutants. The common nod genes, nodD, nodA, and nodBC, were identified by heterologous hybridization and sequence analysis. The nodA gene was found to be separated from nodBC by approximately 22 kb and was divergently transcribed. The 2.0-kb nodDBC region was amplified by PCR from 24 rhizobial strains nodulating A. sinicus, which represented different chromosomal genotypes and geographic origins. No polymorphism was found in the size of PCR products, suggesting that the separation of nodA from nodBC is a common feature of A. sinicus rhizobia. Sequence analysis of the PCR-amplified nodA gene indicated that seven strains representing different 16S and 23S ribosomal DNA genotypes had identical nodA sequences. These data indicate that, whereas microsymbionts of A. sinicus exhibit chromosomal diversity, their nodulation genes are conserved, supporting the hypothesis of horizontal transfer of nod genes among diverse recipient bacteria.     

Pathogen-inducible CaUGT1 is involved in resistance response against TMV infection by controlling salicylic acid accumulation

Capsicum annuum L. Bugang exhibits a hypersensitive response against Tobacco mosaic virus (TMV) P₀ infection. The C. annuumUDP-glucosyltransferase 1 (CaUGT1) gene was upregulated during resistance response to TMV and by salicylic acid, ethephon, methyl viologen, and sodium nitroprusside treatment. When the gene was downregulated by virus-induced gene silencing, a delayed HR was observed. In addition, free and total SA concentrations in the CaUGT1-downregulated hot pepper were decreased by 52% and 48% compared to that of the control plants, respectively. This suggested that the CaUGT1 gene was involved in resistance response against TMV infection by controlling the accumulation of SA.     

Liver protein phosphatases: studies of the presumptive native forms of phosphorylase phosphatase activity in liver extracts and their dissociation to a catalytic subunit of Mr 35,000.

Several aspects of the properties of phosphorylase phosphatase in crude rat liver extracts were investigated. Treatment of tissue extracts with either trypsin, ethanol, or urea was found to dissociate phosphorylase phosphatase activity to a form of M_r 35,000. The M_r 35,000 enzyme form was derived from three native enzyme forms. The major cytosolic form of phosphorylase phosphatase had a molecular weight of 260,000 as determined by gel filtration and was dissociated to a M_r 35,000 form by treatment with either ethanol or urea. Treatment of the M_r 260,000 form with trypsin led to its conversion to M_r 225,000 and a M_r 35,000 form. A minor cytosolic form of M_r 200,000 was also present. This minor activity was latent until activated by trypsin treatment and was converted to a M_r 35,000 form by such treatment. The third form was found to chromatograph as a form of molecular weight greater than 500,000 on gel filtration and, like the M_r 200,000 form, was only detected after activation with trypsin. Subsequent to this treatment, it too behaved as a M_r 35,000 enzyme. Although a single major enzyme form was present in the cytosol, multiple molecular weight forms could be generated in crude extracts simply by the use of vigorous mechanical homogenization procedures. This suggested that artifactual forms of enzyme may readily be produced, possibly by proteolytic cleavage of the native enzyme.     

Phosphatidyl inositol: myo-Inositol exchange enzyme from rat liver: Partial purification and characterization

The enzyme which catalyzes CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol was solubilized from rat liver microsomes by sodium cholate and was partially purified by ammonium sulfate fractionation and sucrose density gradient centrifugation. Addition of phospholipids during purification and assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation contained about 3.7% of the protein and 35% of the original activity of the microsomal fraction. The activity of the enzyme preparation was strongly enhanced by addition of phosphatidyl inositol. The enzyme required Mn²⁺ for activity. The K_m for myo-inositol was 4 × 10^?5m. The pH optimum was 7.4. The activity was inhibited by thiol-reactive reagents and also to some extent by inosose-2 but not by scyllitol. Phosphorus-containing acidic substances such as acidic phospholipids and nucleotides were generally inhibitory. It was found that the preparation catalyzed liberation of inositol moiety from phosphatidyl inositol in a manner dependent on the concentration of free myo-inositol and also on Mn². The K_m of this reaction for free myo-inositol was estimated to be 7 × 10^?5m. This result indicates that CDP-diglyceride-independent incorporation, which has been assumed to show inositol exchange reaction, actually represents an exchange reaction between the myo-inositol moiety of phosphatidyl inositol and free myo-inositol. Phosphatidyl choline and phosphatidyl ethanolamine did not play a role as acceptor of the exchange reaction.     

The Effects of Fluctuations in the Nutrient Supply on the Expression of Five Members of the AGL17 Clade of MADS-Box Genes in Rice

The ANR1 MADS-box gene in Arabidopsis is a key gene involved in regulating lateral root development in response to the external nitrate supply. There are five ANR1-like genes in Oryza sativa, OsMADS23, OsMADS25, OsMADS27, OsMADS57 and OsMADS61, all of which belong to the AGL17 clade. Here we have investigated the responsiveness of these genes to fluctuations in nitrogen (N), phosphorus (P) and sulfur (S) mineral nutrient supply. The MADS-box genes have been shown to have a range of responses to the nutrient supply. The expression of OsMADS61 was transiently induced by N deprivation but was not affected by re-supply with various N sources. The expression of OsMADS25 and OsMADS27 was induced by re-supplying with NO₃ ⁻ and NH₄NO₃, but downregulated by NH₄ ⁺. The expression of OsMADS57 was significantly downregulated by N starvation and upregulated by 3 h NO₃ ⁻ re-supply. OsMADS23 was the only gene that showed no response to either N starvation nor NO₃ ⁻ re-supply. OsMADS57 was the only gene not regulated by P fluctuation whereas the expression of OsMADS23, OsMADS25 and OsMADS27 was downregulated by P starvation and P re-supply. In contrast, all five ANR1-related genes were significantly upregulated by S starvation. Our results also indicated that there were interactions among nitrate, sulphate and phosphate transporters in rice.     

Effect of ventilation on the production of acetaldehyde by Zymomonas mobilis

The production of acetaldehyde, a flavoring agent in food, by Zymomonas mobilis was carried out in batch culture. The volatilization rate constant (k_v) of acetaldehyde and the initial volumetric oxygen transfer coefficient (k_La⁰) in an Erlenmeyer flask with a cotton-plug (cotton-flask) and an aerated-flask with a forced-air system (aerated-flask) were measured. The culture environment in the aerated-flask was found to be very different from that in the cotton-flask. Cell growth in a cotton-flask was strongly inhibited, making practical acetaldehyde production in cotton-flask very difficult. On the other hand, acetaldehyde production in the aerated-flask increased while the fermentation time decreased with increases in the air flow rate. The k_v value of acetaldehyde in a jar fermentor was affected mainly by air flow rate. By considering both the oxygen transfer rate and the ventilation effect on the culture, it was possible to scale-up from the aerated-flask to a jar fermentor. In the jar fermentor, production of acetaldehyde and growth inhibition by acetaldehyde were affected mainly by the k_La⁰ and k_v, values, respectively. The overall production of acetaldehyde in the jar fermentor under the optimum k_La⁰ and k_v conditions was 6.64 g/l (Y_p/s: 0.27), which was about 1.5 times higher than the maximum concentration obtained in the aerated-flask.     

Laser spectroscopy for measuring the parameters of a plasma containing helium and argon

Laser spectroscopy diagnostics used in experiments on the PNX-U facility are described. The working gas was argon with an additive of helium. The 2³ P → 3³ D transition was excited by means of optical pumping, and helium fluorescence at wavelengths of 388 and 706.5 nm was observed. The Doppler temperature of helium atoms was determined by scanning the profile of the absorption line with the help of a tunable laser. The sum of the signals so obtained provides information on the local density of helium atoms. It was proposed to determine the local value of the electron density N _e (R) from the ratio between the fluorescence intensities at wavelengths of 388 and 706.5 nm. The ratio of these intensities as a function of N _e for He I was calculated in the collisional-radiative model, and relevant measurements of N _e in the PNX-U facility were performed. When diagnosing the argon component, the main attention was paid to measurements of the ion temperature T _i (R, t). In the course these measurements, anomalous heating of Ar II ions was revealed. The concentration of singly charged argon ions was estimated.     

Expression of multi-functional cellulase gene mfc in Coprinus cinereus under control of different basidiomycete promoters

Multi-functional cellulase gene mfc was expressed in Coprinus cinereus under naturally non-inductive conditions using three heterologous promoters. Endo-β-1,4-glucanase expression was achieved in solid and liquid media with promoter sequences from the Lentinula edodesgpd gene, the Flammulina velutipes gpd gene and the Volvariella volvaceagpd gene. As measured by enzyme activity in liquid cultures, a 613-bp gpd promoter fragment from L. edodes was most efficient, followed by a 752-bp gpd fragment from F. velutipes. The V. volvacea gpd promoter sequence was less active, in comparison. Irrespective of the promoter used, enzymatic activities increase 34-fold for highly active transformants and 29-fold for less active one by using cellulase-inducing medium. The highest activities of endo-β-1,4-glucanase (34.234 U/ml) and endo-β-1,4-xylanase (263.695 U/ml) were reached by using the L. edodesgpd promoter.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.