<Emphasis Type="Italic">Spartina versicolor</Emphasis> Fabre: Another case of <Emphasis Type="Italic">Spartina</Emphasis> trans-Atlantic introduction?

Intercontinental introductions are widespread in the genus Spartina, with important ecological and evolutionary consequences. The native or introduced status of Spartina species is then critical with regard to biodiversity assessment, especially for vulnerable Mediterranean coastline ecosystems. Spartina versicolor was first recorded in southern France in 1849, then successively in various places on the European and North-African Mediterranean and Atlantic coasts. This species is considered to be either a European native or an invasive species introduced from North America which has a high morphological similarity to the Atlantic American species Spartina patens. We performed extensive sampling of S. versicolor in Europe and North Africa (from natural populations and herbarium collections) and compared these samples to other European and American Spartina species (including S. patens). Chromosome counts were reported for the first time and revealed that S. versicolor is tetraploid (2n = 4x = 40). Phylogenetic analyses based on chloroplast and nuclear ribosomal DNA sequences did not reveal any molecular variation within S. versicolor. In this species, a single haplotype, that is identical to one haplotype of S. patens, was found in the four chloroplast and the nuclear ribosomal ITS regions investigated. In addition, simple sequence repeat markers were used and revealed a low level of genetic diversity within S. versicolor, suggesting that the introduction of S. versicolor occurred from a narrow genetic pool of S. patens from North America.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

How promising is <Emphasis Type="Italic">Lasioseius floridensis</Emphasis> as a control agent of <Emphasis Type="Italic">Polyphagotarsonemus</Emphasis><Emphasis Type="Italic">latus</Emphasis>?

The blattisociid mite Lasioseius floridensis Berlese was found associated with the broad mite, Polyphagotarsonemus latus (Banks), on gerbera leaves in Mogi das Cruzes, State of Sao Paulo, Brazil. Blattisociid mites are not common on aerial plant parts, except under high air humidity levels. Some Lasioseius species have been mentioned as effective control agents of rice pest mites, but nothing is known about the biology of L. floridensis. The objective of this study was to evaluate whether the observed co-occurrence of L. floridensis and P. latus was just occasional or whether the latter could be important as food source for the former, assumed by laboratory evaluation of the ability of the predator to maintain itself, reproduce and develop on that prey. Biological parameters of L. floridensis were compared when exposed to P. latus and to other items as food. The study showed that mating is a pre-requisite for L. floridensis to oviposit and that oviposition rate was much higher on the soil nematode Rhabditella axei (Cobbold) (Rhabditidae) than on P. latus. Ovipositon on the acarid mite Tyrophagus putrescentiae (Schrank) was about the same as on P. latus, but it was nearly zero when the predator was fed the fungi Aspergillus flavus Link or Penicillium sp., or cattail (Typha sp.) pollen. Survivorship was higher in the presence of pollen and lower in the presence of A. flavus or Penicillium sp. than in the absence of those types of food. Life table parameters indicated that the predator performed much better on R. axei than on P. latus. To evaluate the potential effect of L. floridensis as predator of P. latus, complementary studies are warranted to determine the frequency of migration of L. floridensis to aerial plant parts, when predation on P. latus could occur.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic">trx</Emphasis>B <Emphasis Type="Italic">gor</Emphasis> Mutants

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Heterologous expression of a gene for thermostable xylanase from <Emphasis Type="Italic">Chaetomium</Emphasis> <Emphasis Type="Italic">thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia</Emphasis> <Emphasis Type="Italic">pastoris</Emphasis> GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml⁻¹ while that of recombinant without intron (xyn669) was 1.26 U ml⁻¹ after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.     

Whole Mitochondrial Genome of Blakiston’s Fish Owl <Emphasis Type="Italic">Bubo</Emphasis> (<Emphasis Type="Italic">Ketupa</Emphasis>) <Emphasis Type="Italic">blakistoni</Emphasis> Suggests Its Redescription in the Genus <Emphasis Type="Italic">Ketupa</Emphasis>

The paper reports the whole mitochondrial genome (approximately 13 kb) sequencing in three individual representatives of the continental population of Blakiston’s fish owl Bubo blakistoni (Seebohm 1884), the IUCN Red List species in the family Strigidae. The analysis revealed extremely low mtDNA genetic diversity, which may be indicative of the critical state of the studied population. The phylogenetic analysis performed on the basis of the whole mitochondrial genome sequencing data showed that Blakiston’s fish owl is more closely related to the Strix genus than to the Bubo genus with the genetic divergence between blakistoni and either of the two genera being statistically significant and close to intergeneric level (p-distance of 0.135 in the case of the Strix genus and p-distance of 0.151 in the case of the Bubo genus). The results obtained in this work do not match the published data on the mitochondrial cytochrome b gene and the nuclear RAG-1 gene, which laid the basis for the assignment of Blackiston’s fish owl to the Bubo genus in the recent taxonomic bulletins, but rather support the earlier taxonomic classification according to which all four Asian forms, blakistoni, flavipes, zeylonensis, and ketupu, constituted a separate Ketupa genus.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Synthesis and transformations of a pyrazole containing <Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">β</Emphasis>-didehydro-<Emphasis Type="Italic">α</Emphasis>-amino acid derivatives

Summary.  2H-Pyran-2-ones 1 were transformed with various hydrazines into (E)- or (Z)-α,β-didehydro-α-amino acid (DDAA) derivatives 4 (and 7) containing a highly substituted pyrazolyl moiety attached at the β-position. With heterocyclic hydrazines, the products 4 were accompanied also by decarboxylated enamines E-6. In order to separate (E/Z)-mixtures of acids, they were transformed to the corresponding methyl esters 9 and 10 by the application of diazomethane. Catalytic hydrogenation under high pressures with Pd/C as a catalyst resulted in the formation of racemic alanine derivatives 11. Received January 29, 2002 Accepted May 27, 2002 Published online December 18, 2002 RID="*" ID="*"  Dedicated with deep respect to Professor Waldemar Adam on the occasion of his 65^th birthday. Acknowledgements We thank the Ministry of Education, Science and Sport of the Republic of Slovenia for the financial support (P0-0503-103). Dr. B. Kralj and Dr. D. Žigon (Center for Mass Spectroscopy, “Jožef Stefan” Institute, Ljubljana, Slovenia) are gratefully acknowledged for the mass measurements. Authors' address: Prof. Marijan Kočevar, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia, E-mail: marijan.kocevar@uni-lj.si     

Population variation of invasive <Emphasis Type="Italic">Spartina</Emphasis> <Emphasis Type="Italic">alterniflora</Emphasis> can differentiate bacterial diversity in its rhizosphere

While several studies have documented that invasive plants can change the microbial communities, little is known about how soil microbial communities respond to population variation of invasive plants. Here, nine populations of Spartina alterniflora were selected from the east coast of China along latitudinal gradient to compare bacterial diversity of rhizospheres among these populations. The bacterial diversity in S. alterniflora rhizospheres was valued by denaturing gradient gel electrophoresis (DGGE) analysis. Shannon–Weaver diversity index (H′) and number of DGGE bands showed that rhizosphere bacterial diversity of S. alterniflora populations increased along a latitudinal gradient when all the populations were grown in a common garden. These findings suggest that population variation of S. alterniflora can differentiate the rhizosphere bacterial diversity, and the latitudinal gradient can shape the specific plant–bacterial diversity relationship. Our results adding to the recent literature suggest that invasive plant–soil biota interactions would have clinal variation with environmental gradients and improve our understanding of the mechanisms and processes of plant invasions.