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.     

<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.     

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.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

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.     

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.     

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.     

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.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> inhibition of α-amylases of stored-product mite <Emphasis Type="Italic">Acarus siro</Emphasis>

The stored-product mites are the most abundant and frequent group of pests living on the stored food products in Europe. They endanger public health since they produce allergens and transmit mycotoxin-producing fungi. Novel acaricidal compounds with inhibitory effects on the digestive enzymes of arthropods are a safe alternative to the traditional neurotoxic pesticides used for control of the stored-product pests. In this work, we explored the properties of acarbose, the low molecular weight inhibitor of -amylases (AI), as a novel acaricide candidate for protection of the stored products from infestation by Acarus siro (Acari: Acaridae). In vitro analysis revealed that AI blocked efficiently the enzymatic activity of digestive amylases of A. siro, and decreased the physiological capacity of mites gut in utilizing a starch component of grain flour. In vivo experiments showed that AI suppressed the population growth of A. siro. The mites were kept for three weeks on experimental diet enriched by AI in concentration range of 0.005 to 0.25%. Population growth of A. siro was negatively correlated with the content of AI in the treated diet with a half-population dose of 0.125%. The suppressive effect of AIs on stored-product mites is discussed in the context of their potential application in GMO crops     

New family of pectinase genes <Emphasis Type="Italic">PGU1</Emphasis>b–<Emphasis Type="Italic">PGU3b</Emphasis> of the pectinolytic yeast <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> var. <Emphasis Type="Italic">uvarum</Emphasis>

Using yeast genome databases and literature data, we have conducted a phylogenetic analysis of pectinase PGU genes from Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and hybrid taxon S. pastorianus (syn. S. carlsbergensis). Single PGU genes were observed in all Saccharomyces species, except S. bayanus. The superfamily of divergent PGU genes has been documented in S. bayanus var. uvarum for the first time. Chromosomal localization of new PGU1b, PGU2b, and PGU3b genes in the yeast S. bayanus var. uvarum has been determined by molecular karyotyping and Southern hybridization.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Somatic hybridization between the diploids of <Emphasis Type="Italic">S.</Emphasis> × <Emphasis Type="Italic">michoacanum</Emphasis> and <Emphasis Type="Italic">S. tuberosum</Emphasis>

Interspecific somatic hybrids between a diploid potato clone DG 81-68 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuber-bearing species Solanum × michoacanum were generated and analyzed. About 30 regenerants displaying an intermediate morphology were obtained as a result of three separate PEG-mediated fusion experiments. The RAPD analysis confirmed the hybridity of all the regenerants. About 50% of the hybrid plants exhibited vigorous growth and were stable in culture, while the rest of them rooted poorly and grew slowly in vitro. Most of the hybrid clones were at the tetraploid level (70%), while 30% of the clones examined were at the hexaploid level. The S. × michoacanum (+) DG 81-68 hybrids with growth anomalies were aneuploid. The variation in late blight resistance of the hybrid clones was found in detached leaflet tests, with enhanced resistance characteristic for three tetraploid hybrids.     

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     

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.     

<Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>. The most important bacterial warfare agents — <Emphasis Type="Italic">review</Emphasis>

There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.     

Gill cell toxicity of northern boreal scyphomedusae <Emphasis Type="Italic">Cyanea</Emphasis> <Emphasis Type="Italic">capillata</Emphasis> and <Emphasis Type="Italic">Aurelia</Emphasis> <Emphasis Type="Italic">aurita</Emphasis> measured by an in vitro cell assay

Scyphozoan medusae are very successful foragers which occasionally occur in high abundances in boreal waters and may impact many different groups in the marine ecosystem by means of a variety of toxins. A rainbow trout gill cell line, RTgill-W1, was tested for its suitability as quantitative indicator of the cytotoxicity of Cyanea capillata and Aurelia aurita; the major scyphozoan species in the North and Baltic seas. Cultures of rainbow trout gill cells were exposed to whole venoms extracted from fishing tentacles and oral arms at increasing protein concentrations. The venom caused detachment, clumping and lysis of cells, as well as a drop in vitality, in a dose-dependent manner. Morphological changes in the cells were evident within 1 h after venom addition. The damage to gill cells was quantified by measuring the metabolic activity of the cells by means of the fluorescence of resorufin derived from the nonfluorescent substrate, resazurin. In general, a decrease in the metabolic activity of the cells was detected at a venom (protein) concentration above 2.0 μg ml⁻¹ (corresponding to 0.2 μg 10⁴ cells⁻¹), and a total loss of activity was observed above 40.0 μg ml⁻¹ (corresponding to 4.0 μg 10⁴ cells⁻¹). C. capillata venoms had increased cytotoxic activity as compared to A. aurita venoms at the same concentration. Cnidocyst extracts from oral arms of A. aurita induced an 85% loss of gill cell viability at concentrations of 0.2 μg 10⁴ cells⁻¹, whereas crude venoms from fishing tentacles reduced cell viability by 18% at the same concentration. Gel electrophoresis of the venoms indicated that these consist of a large number of proteins in a fairly wide size range, from 6 to 200 kDa, including some that are the same size as those found in cubomedusae. It also appears that larger (i.e., older) medusae have more complex venoms and, in some cases, more potent venoms than smaller animals.