<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Glucose oxidase-polypyrrole electrodes synthesized in <Emphasis Type="Italic">p</Emphasis>-toluenesulfonic acid and sodium <Emphasis Type="Italic">p</Emphasis>-toluenesulfonate

Amperometric glucose biosensors have been developed based on entrapment on platinum (Pt) electrode using cyclic voltammetry technique in glucose oxidase (GOD) and pyrrole containing p-toluenesulfonic acid (pTSA) or sodium p-toluenesulfonate (NapTS) as supporting electrolyte solutions. Both of electrolyte solutions were suitable media for the formation and deposition of polypyrrole-GOD (PPy-GOD) layers on Pt substrate. Pt/PPy-GOD electrodes brought about in different morphological properties as well as different electrochemical and biochemical response. The highest responses obtained in pTSA and NapTS electrolytes were observed at pH of 4.5 and 7.0 for Pt/PPy-GOD electrodes, respectively. While linearity was observed between 0.0–1.0 mM glucose substrate for both electrodes, I _max value of Pt/PPy-GOD_NapTS electrode was approximately twice as high as that of Pt/PPy-GOD_pTSA electrode as 25.4 and 14.2 μA, respectively. Five commercial drinks were tested with enzyme electrodes and compared with results obtained spectropho-tometrically using glucose kit. Results revealed that Pt/PPy-GOD_NapTS electrode exhibited better biosensor response.     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.     

Biotransformation of polychlorinated dibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin by <Emphasis Type="Italic">Coprinellus</Emphasis> species

A degradation experiment on dibenzo-p-dioxin (DD) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) was carried out using basidiomycetous fungi belonging to the genera Coprinus, Coprinellus, and Coprinopsis. Some species showed a high rate of decrease in DD for the 2-week test period. Among them, Coprinellus disseminatus showed the highest ability to decrease the DD level, close to 100% by the end of 2 weeks. Further examination showed that Coprinellus disseminatus and Coprinellus micaceus, belonging to the genus Coprinellus, were able to metabolize 2,7-DCDD to a monohydroxylated compound, probably mediated by the P450 system. The metabolism of chlorinated DD by fungi capable of living in soil conditions is reported here for the first time.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

A new gene coding for <Emphasis Type="Italic">p</Emphasis>-coumarate 3-hydroxylase from <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

p-Coumarate 3-hydroxylase (C3H) is a rate-limiting enzyme involved in monolignol biosynthesis. The full-length cDNA from Ginkgo biloba and genomic DNA sequence encoding C3H (designated as GbC3H) were cloned and characterized for the first time by rapid amplification of cDNA ends technique. The full-length cDNA of GbC3H was of 1860 bp containing a 1527 bp open reading frame encoding a cytochrome P450 protein of 508 amino acids with a calculated mol wt of 57.46 kD and an isoelectric point of 7.09. Two introns were present in the GbC3H gene. Comparative and bioinformatic analyses revealed that GbC3H had close similarity with C3Hs from other species and contained a conserved cytochrome P450 cysteine heme-iron ligand signature. Phylogenetic analysis indicated that GbC3H shared a common evolutionary origin based on sequence and had the closest relationship to C3H from gymnosperm species. Southern blot analysis indicated that GbC3H belonged to a small-gene family. Tissue expression pattern analysis revealed the highest expression of GbC3H in roots followed by leaves, and no expression was detected in stems. Only a few proteins of this class have been found, so the cloning and characterization of GbC3H will be useful in understanding the role of C3Hs in the lignin biosynthesis at the molecular level. This text was submitted by the authors in English.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Removal of <Emphasis Type="Italic">p</Emphasis>-chlorophenol by the marine microalga <Emphasis Type="Italic">Tetraselmis marina</Emphasis>

The ability of Tetraselmis marina, a green coastal microalga, to remove chlorophenols under photoautotrophic conditions was investigated. T.marina was able to grow in the presence of 20 mg L⁻¹ of the phenolic compounds tested. The EC₅₀ (growth rate) value of p-chlorophenol (p-CP) to T.marina was found to be 25.5 mg L⁻¹. The microalga was able to remove chlorophenols, showing higher efficiency for p-CP. The effect of photoregime and NaHCO₃ concentration on p-CP removal was investigated. Under continuous illumination with 1 g L⁻¹ NaHCO₃ initial concentration T.marina removed 65% of 20 mg L⁻¹ in a 10-day cultivation period.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

Characterization of <Emphasis Type="Italic">p</Emphasis>-hydroxybenzaldehyde dehydrogenase,the final enzyme of <Emphasis Type="Italic">p</Emphasis>-hydroxybenzoic acid biosynthesis in hairy roots of <Emphasis Type="Italic">Daucus carota</Emphasis>

Hairy root cultures of Daucus carota respond to methyl-jasmonate treatment with enhanced accumulation of p-hydroxybenzoic acid. The final C₁-side chain of this compound is shaped by p-hydroxybenzaldehyde dehydrogenase (HBD) that catalyzes the formation of p-hydroxybenzoic acid from p-hydroxybenzaldehyde in the presence of NAD⁺. HBD was biochemically characterized from cell-free hairy root extracts of D. carota. The preferred substrate for HBD was p-hydroxybenzaldehyde. The apparent K _m values were 54.8 and 74.4 μM for p-hydroxybenzaldehyde and NAD⁺, respectively. Divalent metal cations did not significantly affect enzyme activity.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

<Emphasis Type="Italic">egl</Emphasis>-<Emphasis Type="Italic">4</Emphasis> modulates electroconvulsive seizure duration in <Emphasis Type="Italic">C. elegans</Emphasis>

Increased neuronal excitability causes seizures with debilitating symptoms. Effective and noninvasive treatments are limited for easing symptoms, partially due to the complexity of the disorder and lack of knowledge of specific molecular faults. An unexplored, novel target for seizure therapeutics is the cGMP/protein kinase G (PKG) pathway, which targets downstream K⁺ channels, a mechanism similar to Retigabine, a recently FDA-approved antiepileptic drug. Our results demonstrate that increased PKG activity decreased seizure duration in C. elegans utilizing a recently developed electroconvulsive seizure assay. While the fly is a well-established seizure model, C. elegans are an ideal yet unexploited model which easily uptakes drugs and can be utilized for high-throughput screens. In this study, we show that treating the worms with either a potassium channel opener, Retigabine or published pharmaceuticals that increase PKG activity, significantly reduces seizure recovery times. Our results suggest that PKG signaling modulates downstream K⁺ channel conductance to control seizure recovery time in C. elegans. Hence, we provide powerful evidence, suggesting that pharmacological manipulation of the PKG signaling cascade may control seizure duration across phyla.     

Facile synthesis of optically pure (<Emphasis Type="Italic">S</Emphasis>)-3-<Emphasis Type="Italic">p</Emphasis>-hydroxyphenyllactic acid derivatives

Summary. Optically pure (S)-3-p-hydroxyphenyllactic acid derivatives are important intermediates of peroxisome proliferator-activated receptor α/γ dual agonists and heteropeptides. Many efforts have been made for synthesis of those intermediates, but there exist some flaws yet. We observed that dielectric constants of organic solvents drastically affected diazotization of O-benzyl-L-tyrosine. Optically pure (S)-3-p-benzyloxyphenyllactic acid was obtained by simple recrystallization when DMF or DMSO of higher dielectric constant was used as a co-solvent in diazotization of O-benzyl-L-tyrosine. It was easily turned into various optically pure (S)-3-p-hydroxyphenyllactic acid derivatives.     

Harvest-inducibility of the promoter of alfalfa<Emphasis Type="Italic"> S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine:<Emphasis Type="Italic"> trans</Emphasis>-caffeoyl-CoA3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase gene

A major limitation on the expression of some foreign proteins in transgenic plants is the toxic effect of such proteins on the host plant resulting in inhibition of normal growth and development. A solution to this problem is to control the expression of genes for such proteins by means of inducible promoters, as is frequently done in microbial systems. A cDNA clone was obtained from subtractive hybridization of non-harvested and harvested alfalfa leaf tissue, named hi12. The hi12 cDNA was identified as part of the S-adenosyl-l-methionine: trans-caffeoyl-CoA3-O-methyltransferase gene of alfalfa, a gene encoding an essential key enzyme in lignin synthesis. The hi12 gene was strongly induced by harvesting and wounding but not by heat shock. The promoter of the hi12 gene, isolated by genomic walking, contained several stress response cis-elements. Transgenic plants of tobacco and Medicago truncatula containing the GUS gene driven by the promoter showed GUS expression following harvesting, demonstrating the activity of these regulatory regions in other plant species.     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

Phenotypic Switching of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> and <Emphasis Type="Italic">Cryptococcus</Emphasis><Emphasis Type="Italic">gattii</Emphasis>

Microorganisms that live in fluctuating environments must constantly adapt their behavior to survive. The host constitutes an important microenvironment in opportunistic and primary fungal pathogens like Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii). In clonal populations, adaptation may be achieved through the generation of diversity. For fungi phenotype switching constitutes a mechanism that allows them to change rapidly. Both C. neoformans and C. gattii undergo phenotypic switching, which allows them to be successful pathogens and cause persistent disease. Similar to other encapsulated microbes that exhibit phenotypic variation, phenotypic switching in Cryptococcus changes the polysaccharide capsule. Most importantly, in animal models phenotypic switching affects virulence and can change the outcome of infection. Virulence changes because C. neoformans and C. gattii switch variants elicit different inflammatory responses in the host. This altered host response can also affect the response to antifungal therapy and in some cases may even promote the selection of switch variants. This review highlights the similarity and differences between phenotypic switching in C. neoformans and C. gattii, the two dominant species that cause cryptococcosis in humans.