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.     

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.     

Expression profiles of <Emphasis Type="Italic">amh</Emphasis> and <Emphasis Type="Italic">foxl2</Emphasis> in <Emphasis Type="Italic">Schizothorax kozlovi</Emphasis>, and their response to temperature during the early developmental stage

To elucidate the role of amh and foxl2 in sex differentiation of the teleost fish Schizothorax kozlovi, the full-length cDNAs were cloned from the mature testis and ovary by rapid amplification of cDNA ends (RACE), and their relative mRNA expression levels were determined by quantitative real-time polymerase chain reaction among tissues and temperature groups. The complete amh and foxl2 cDNAs of S. kozlovi were 2060 bp and 1750 bp, which encoded 568 and 306 amino acids, respectively. The amh were expressed only in gonads, while foxl2 was expressed in the gills, brain and gonads, both exhibiting relatively high tissue specificity. The amh exhibited sex-specific expression pattern in the gonads. No sex differences in the foxl2 expression were observed in the brain and gonads, but significant sex differences were found in the gills. No significant differences were found in the foxl2 expression, from the larval to the juvenile stage, and also between different temperature groups. However, significant differences were found in the expression levels of amh from the larval (12–63 days posthatching (dph)) to the juvenile stage (190 dph), and also among the \(18{^{\circ }}\hbox {C}\) and \(10{^{\circ }}\hbox {C}\) groups at 31 dph. This result suggested that amh plays an important role in male sex differentiation of S. kozlovi during the early developmental stage, but no similar effect was observed in foxl2.     

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.     

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.     

<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.     

Mapping and characterization of wheat stem rust resistance genes <Emphasis Type="Italic">SrTm5</Emphasis> and <Emphasis Type="Italic">Sr60</Emphasis> from <Emphasis Type="Italic">Triticum monococcum</Emphasis>

Key message

The new stem rust resistance gene Sr60 was fine-mapped to the distal region of chromosome arm 5A^mS, and the TTKSK-effective gene SrTm5 could be a new allele of Sr22.

Abstract

The emergence and spread of new virulent races of the wheat stem rust pathogen (Puccinia graminis f. sp. tritici; Pgt), including the Ug99 race group, is a serious threat to global wheat production. In this study, we mapped and characterized two stem rust resistance genes from diploid wheat Triticum monococcum accession PI 306540. We mapped SrTm5, a previously postulated gene effective to Ug99, on chromosome arm 7A^mL, completely linked to Sr22. SrTm5 displayed a different race specificity compared to Sr22 indicating that they are distinct. Sequencing of the Sr22 homolog in PI 306540 revealed a novel haplotype. Characterization of the segregating populations with Pgt race QFCSC revealed an additional resistance gene on chromosome arm 5A^mS that was assigned the official name Sr60. This gene was also effective against races QTHJC and SCCSC but not against TTKSK (a Ug99 group race). Using two large mapping populations (4046 gametes), we mapped Sr60 within a 0.44 cM interval flanked by sequenced-based markers GH724575 and CJ942731. These two markers delimit a 54.6-kb region in Brachypodium distachyon chromosome 4 and a 430-kb region in the Chinese Spring reference genome. Both regions include a leucine-rich repeat protein kinase (LRRK123.1) that represents a potential candidate gene. Three CC–NBS–LRR genes were found in the colinear Brachypodium region but not in the wheat genome. We are currently developing a Bacterial Artificial Chromosome library of PI 306540 to determine which of these candidate genes are present in the T. monococcum genome and to complete the cloning of Sr60.

     

<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.     

<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.     

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.     

Fertility and precocity of <Emphasis Type="Italic">Osmunda</Emphasis> × <Emphasis Type="Italic">intermedia</Emphasis> offspring in culture

The feasibility of later-generation hybrid production in ferns has not been previously studied, although it is a significant factor in relation to reproductive isolation. Osmunda × intermedia, a hybrid between O. japonica and O. lancea, is semifertile and has moderate spore germination rates. Under the artificial conditions of this study, F2 and F3 offspring were formed. Some of the F2 offspring showed precocity, and some of the F3 offspring also showed precocity. This fertility suggests that introgressive hybridization might be ongoing in nature. This also indicates a currently unknown genetic control over the timing of fertile frond production in Osmunda.     

Geometric characters of the radius and tibia in <Emphasis Type="Italic">Macaca mulatta</Emphasis> and <Emphasis Type="Italic">Macaca fascicularis</Emphasis>

We performed comparative analyses of four cross-sections of the distal radius and tibia in two species of macaque to clarify the relationships between bone morphology and locomotor type. The lengths of bones and five bone geometric properties in each section were examined and compared separately in both female and male Macaca mulatta and Macaca fascicularis. In M. mulatta, there were no significant gender-specific differences in either the radius or the tibia. In contrast, the radius and tibia of male M. fascicularis had greater geometric parameters in the 20% and 40% positions relative to the 5% and 10% positions from the distal end than those of their female counterparts. The radius and tibia of M. mulatta were relatively longer than those of M. fascicularis, and the sectional parameters of the tibia of M. mulatta were relatively larger than those of M. fascicularis. Standardization of the log-transformed bone length between the species revealed larger radial cortical bone areas in M. fascicularis. In contrast, there were minimal differences in the tibial cortical bone areas between the two species. This study suggests that the observed distinctions in bone geometry in female and male M. fascicularis may be due to gender-specific differences in the muscle weights of the forearm and calf, which may underlie the divergence in the leaping abilities of females and males of this species. Taken together, these results of interspecies comparisons may be related to the fact that arboreal primates such as M. fascicularis undergo compressive mechanical stress due to the forelimb lead that occurs as the animal descends a sloping trunk or bridges a tree gap downward, while terrestrial primates such as M. mulatta move on nearly flat substrates. Differences in fore- and hind-limb bone properties between the two species are discussed with regard to functional morphology and locomotor type.     

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.     

Differential expression of <Emphasis Type="Italic">Gnrh2</Emphasis>, <Emphasis Type="Italic">Gthβ</Emphasis>, and <Emphasis Type="Italic">Gthr</Emphasis> genes in sterile triploids and fertile tetraploids

Gonadotropin-releasing hormone (GnRH), gonadotropin hormone (GTH), and gonadotropin hormone receptor (GTHR) are the pivotal signal molecules of the hypothalamic-pituitary-gonad (HPG) axis, which plays a crucial role in regulating gonadal development in vertebrate. In this study, we comparatively analyze the expression characteristics of Gnrh2, Gthβ, and Gthr in red crucian carp diploids, triploids, and allotetraploids. The expression patterns of these genes are similar in the three fish ploidy types: the Gnrh2 gene is expressed in midbrains, pituitaries, and gonads; the Gthβ gene is expressed in pituitaries; the Gthr gene is mainly expressed in gonads. These results indicate that the three genes participate in the regulation of gonadal development. By real-time polymerase chain reaction and in situ hybridization, we find that, among these three fish ploidy types, the expression level of Gthr in the gonads of triploids is lower than those of diploids and tetraploids; this weakens the combination of GTHR with GTH released from the pituitary and leads to the sterility of triploids, since the gonad cannot produce enough sex steroids. In addition, the low expression of Gthr in triploids may affect the down-regulation of Gthβ, which then affects the down-regulation of Gnrh2; hence, the expression levels of Gnrh2 and Gthβ genes in triploids are the highest after the breeding season. In conclusion, the differential expression of Gnrh2, Gthβ, and Gthr in triploids and tetraploids is related to their sterility and bisexual fertility, respectively.     

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.