<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.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

<Emphasis Type="Italic">Wax</Emphasis><Emphasis Type="Italic">crystal</Emphasis>-<Emphasis Type="Italic">sparse leaf 3</Emphasis> encoding a β-ketoacyl-CoA reductase is involved in cuticular wax biosynthesis in rice

Key message

WSL3 encodes β-ketoacyl-CoA reductase (KCR) in rice, in a similar way to YBR159w in yeast, and is essential for VLCFA biosynthesis and leaf wax accumulation.

Abstract

Cuticular waxes on plant surfaces limit non-stomatal water loss, protect plants against deposits of dust and impose a physical barrier to pathogen infection. We identified a wax-deficient mutant of rice, wax crystal-sparse leaf 3 (wsl3), which exhibits a pleiotropic phenotype that includes reduced epicuticular wax crystals on the leaf surface and altered wax composition. Map-based cloning demonstrated that defects in the mutant were caused by two adjacent single-nucleotide changes in a gene encoding β-ketoacyl-CoA reductase (KCR) that catalyzes the second step of the fatty acid elongation reaction. The identity of WSL3 was further confirmed by genetic complementation. Transient assays of fluorescent protein-tagged WSL3 in tobacco protoplasts showed that WSL3 localizes to the endoplasmic reticulum, the compartment of fatty acid elongation in cells. Quantitative PCR and histochemical staining indicated that WSL3 is universally expressed in tissues. RNA interference of WSL3 caused a phenotype that mimicked the wsl3 mutant. Very long-chain fatty acids (VLCFAs) 20:0 and 22:0, or 20:1Δ¹¹ and 22:1Δ¹³, were detected when WSL3 and Arabidopsis fatty acid elongation 1 (FAE1) were co-expressed in a yeast ybr159wΔ mutant strain. Our results indicated that WSL3 affects rice cuticular wax production by participating in VLCFA elongation.

     

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.     

Characterization of the DNA Adenine 5′-GATC-3′ Methylase <Emphasis Type="Italic">Hpy</Emphasis>IIIM from <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

The effect of inactivation of the 5-GATC-3 methylase HpyIIIM in Helicobacter pylori (H. pylori) on mismatch repair, adherence, and in vitro fitness was examined. Chromosomal DNA from 90 H. pylori strains was isolated, and restriction enzyme digestion indicated all strains examined possess HpyIIIM. Wild-type H. pylori and a strain with an inactive HpyIIIM were found to have rifampicin mutation frequencies of 2.93 × 10^–7 and 1.05 × 10^–7 (p > 0.05), respectively, indicating that HpyIIIM does not appear to be important in mismatch repair. Adherence of H. pylori in an in vitro model cell system was also unaffected by inactivation of HpyIIIM. Inactivation of HpyIIIM did not result in a decrease in fitness, as determined by liquid in vitro competition experiments.     

<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.     

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.     

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.     

In vitro growth response of <Emphasis Type="Italic">Phytophthora cactorum</Emphasis>, <Emphasis Type="Italic">P. nicotianae</Emphasis> and <Emphasis Type="Italic">P.</Emphasis> × <Emphasis Type="Italic">pelgrandis</Emphasis> to antibiotics and fungicides

The reactions of isolates of Phytophthora cactorum, P. nicotianae and P. × pelgrandis to metalaxyl, mancozeb, dimethomorph, streptomycin and chloramphenicol were tested to obtain information about the variability of resistance in these pathogens. Distinct genetic groups showed significant differences in resistance to all tested substances except streptomycin. In response to streptomycin, the growth inhibition rates of distinct groups did not differ significantly. The most remarkable differences were detected in the reactions to chloramphenicol and metalaxyl. Discriminant analysis evaluating the effect of all substances confirmed the differences among the groups, which are in agreement with the differences revealed by earlier DNA analyses.     

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.     

Isolation and characterization of a <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N′</Emphasis>-dicyclohexylcarbodiimide-resistant mutant of <Emphasis Type="Italic">Methanothermobacter thermautotrophicus</Emphasis> with alterations to the ATP synthesis machinery

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant toward the ATP-synthase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD) was isolated. DCCD normally inhibits methanogenic electron-transport-driven ATP synthesis, however, the DCCD-resistant strain exhibited methanogenesis in the presence of 300 μmol/L DCCD. Total ATP synthesis was shown to be higher in the mutant strain, both in the presence and absence of DCCD. These results suggested a modification in the ATP-synthesizing system of the mutant strain. Using Blue Native PAGE combined with MALDI TOF/TOF mass spectrometry, increased concentrations of both the A₁ and A_o subcomplexes of the A₁A_o-type synthase were identified in the mutant strain. However, no alterations were found in the structural genes (atp) for the A₁A_o ATP synthase. The results imply that DCCD resistance is a consequence of increased A₁A_o ATP synthase expression, and suggest that genes involved in regulating synthase expression are responsible for DCCD resistance.     

Innate immunity to <Emphasis Type="Italic">Legionella</Emphasis> and toll-like receptors — <Emphasis Type="Italic">review</Emphasis>

Legionella is a parasite of eukaryotic cells, able to survive and replicate in a wide range of protozoan hosts. It can also infect humans as an opportunistic pathogen, primarily by interaction with alveolar macrophages. These bacteria can cause life-threatening pneumonia, especially in immunocompromised individuals. However, most infections triggered by Legionella are cleared by an efficient host immune system. The protective immune responses against Legionella are complex and multifaceted, involving many components of the immune system. Recognition of such components as LPS, flagellum, and peptidoglycan of L. pneumophila by the TLRs, which orchestrates the innate immune responses to Legionella, lays an important role in activation of monocytes and alveolar macrophages and, thus, in inhibition of intracellular proliferation of bacteria. MyD88-dependent signaling pathways are important for host protection against Legionella.     

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     

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.     

Butterfly diversity in Mediterranean islands and in Pentadaktylos <Emphasis Type="Italic">Pinus</Emphasis><Emphasis Type="BoldItalic"> </Emphasis><Emphasis Type="Italic">brutia</Emphasis> forests of Cyprus

We analysed the influence of contemporary geography on butterfly diversity for islands in the Mediterranean Basin. We found that island size and distance from the mainland has a significant effect on the number of species. We also used butterflies as an indicator group to identify the importance of forest habitats for biodiversity conservation in the island of Cyprus. To understand the relative importance of local vegetation characteristics of butterflies in the Pentadaktylos mountains transect counts were used to assess the abundance and butterfly diversity in two different forest types. A total of 1,602 butterflies and 23 species were recorded during this research. We observed highly significant effects of forest type on abundance and species richness of butterflies. For example, number of butterflies was significantly higher in old forest than young pine forest. Also, the abundance of endemic butterflies was highest in old forest habitats. Therefore, the survival of the majority of endemic butterflies in Cyprus may depend on conservation of old forests and their understorey plants.     

QTL mapping of grain quality traits from the interspecific cross <Emphasis Type="Italic">Oryza sativa</Emphasis> × <Emphasis Type="Italic">O. glaberrima</Emphasis>

International rice export markets are increasing demands for rapid improvements in grain quality characteristics. The African rice Oryza glaberrima is a new potential source of genes that will enhance the eating, cooking, and milling properties of the rice grain. The objective of this research was to identify and characterize quantitative trait loci (QTLs) among 312 doubled haploid lines derived from the BC₃F₁ of an interspecific cross of O. sativa × O. glaberrima. Genetic material was planted in replicated plots and evaluated for ten grain quality traits in 2001 in Colombia. A linkage map was constructed with 100 polymorphic microsatellite markers using the mapdisto software program to adjust for segregation distortion. Transgressive segregation was observed for all traits. Interval and composite interval analyses identified 27 QTLs for nine characters located on 11/12 chromosomes. The chromosomal positions of QTLs for percentage amylose, alkali-spreading score, and percentage protein were in agreement with data reported by others, whereas QTL markers for percentage head rice, percentage milled rice, percentage protein, and percentage brown rice were different in our mapping population. Five major QTLs were found to be associated with improved percentage rice bran, percentage amylose, and alkali-spreading score. Seven QTLs for improved percentage rice bran, percentage milled rice, alkali-spreading score, percentage protein, and grain length/width ratio were derived from the O. glaberrima accession. Three new QTLs for percentage rice bran are reported here for the first time. Results from this study suggest that the African rice might be a valuable new source for introgression and improvement of several traits that affect quality traits demanded by the different rice export markets.Approved for publication by the Director of the Louisiana Agricultural Experiment Station as paper no. 04-14-0054.     

AFLP and SSR polymorphism in a <Emphasis Type="Italic">Coffea</Emphasis> interspecific backcross progeny [(<Emphasis Type="Italic">C. heterocalyx</Emphasis> × <Emphasis Type="Italic">C. canephora</Emphasis>) × <Emphasis Type="Italic">C. canephora</Emphasis>]

An interspecific cross (BC 1) involving a species with one of the largest genomes in the Coffea genus [Coffea heterocalyx (HET), qDNA = 1.74 pg] and a species with a medium-sized genome [Coffea canephora (CAN), qDNA = 1.43 pg] was studied using two types of molecular markers, AFLP and SSR. One hundred and eighty eight AFLP bands and 34 SSR primer pairs were suitable for mapping. The total map length was 1,360 cM with 190 loci distributed in 15 linkage groups. The results were compared to those obtained previously on an interspecific BC 1 progeny involving a species with a medium-sized genome (Coffea liberica var dewevrei, DEW) and a species with one of the smallest genomes (Coffea pseudozanguebariae, PSE). They are discussed relative to three main points: (1) the relevance of the different marker types, (2) the genomic distribution of AFLP and SSR markers, and (3) the relation between AFLP polymorphism and genome size.Communicated by H.F. Linskens