An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications

We proposed a modification the procedure of genotyping based in labeled universal primer and tailed primer. In the standard protocol, three primers are used in the same PCR reaction, a forward primer with tail added at the 5′ end of the identical sequence to labeled universal primer with dye-fluorescent and a reverse primer. Unfortunately, the choice of a labeled primer characterized by a large number of complementary sequences in target genomes (which is more probable in larger genomes) result in unspecific amplifications (false positive) can cause absence or decrease amplification of the locus of interest and also false interpretation of the analysis. However, identification of possible homologies between the primer chosen for labelling and the genome is rarely possible from the available DNA data bases. In our approach, cycling is interrupted for the addition of the labeled primer only during the final cycles, thus minimizing unspecific amplification and competition between primers, resulting in the more fidelity amplification of the target regions.     

Pectolytic Bacteria Associated with Soft Rot of Calla Lily (Zantedeschia spp.) Tubers

Soft rot is the most important disease on calla lily in Poland. The isolation of the presumptive pathogen from symptomatic tubers on nutrient agar yielded bacteria with different colony morphology. Of 41 isolates collected, 10 showed pectolytic activity on crystal violet pectate medium and caused soft rot on potato slices. All pectolytic bacteria appeared to be Gram‐negative rods producing typical soft rot on inoculated leaf petioles of calla lily. Bacteria with colonies which morphologically resembled those used for inoculation were re‐isolated from diseased petioles. Their identification was based on phenotypic characters and sequence of the gene fragment coding 16S rRNA. It was found that, in addition to Pectobacterium carotovorum subsp. carotovorum, soft rot of calla lily can be caused by Pectobacterium carotovorum subsp. atrosepticum, Pseudomonas marginalis, Pseudomonas veronii and Chryseobacterium indologenes. The latter two are described for the first time as plant pathogens. The pectolytic activity of all identified bacteria, except that of P. carotovorum subsp. atrosepticum, was lower than that of P. carotovorum subsp. carotovorum, but strains of P. veronii showed a higher activity than P. marginalisand C. indologenes species.     

Biodegradation of epoxyconazole and piraclostrobin fungicides by Klebsiella sp. from soil

Three bacterial strains have been isolated from soil in which soybean had been continuously cropped and treated with Opera^®, a fungicide containing epoxyconazole and pyraclostrobin. The three strains (1,805, 2,801 and 3,803), obtained from soil at 80–100 cm depth, were selected on medium containing 0.03% Opera^®. Morphological examination revealed that the strains were Gram-negative, and two of them (1,805 and 2,801) exhibited polymorphism. The growth profiles demonstrated that 1,805 and 3,803 were more efficient growing in the presence of Opera^® than 2,801. Maximum growth was reached between 24 and 48 h, however, 2,801 was not able to survive after this period. The total protein content produced by 1,805, 2,801 and 3,803 in liquid selective medium containing Opera^® were 111.0 ± 0.02, 80.0 ± 0.05 and 130.5 ± 0.07 μg/ml, respectively. According to its biochemical and molecular features, strain 1,805 was identified as Klebsiella sp. On the basis of the characteristics presented (facultative anaerobic nature, polymorphic character and capacity of growing in the presence of Opera^®) strain 1,805 seems to be able to degrade the epoxyconazole and pyraclostrobin.     

In Vivo Formation and Binding of SeHg Complexes to the Erythrocyte Surface

The in vivo dynamics of selenium (Se) and mercury (Hg) interaction was studied in mouse tissues using direct visualization of individual Se, Hg, and SeHg particles on the surface of circulating erythrocytes. This high-resolution detection of Se and Hg was obtained by scanning electron microscopy coupled to X-ray microanalysis. BALB/c mice were injected in the peritoneal cavity with Se and Hg salts, and the animals were sacrificed 3 min after the Hg injection. Only a minority (9%) of the metal dots seen on mouse liver erythrocytes were SeHg complexes when Se and Hg salts were mixed together before injection. In contrast, the majority (73%) of metal dots on liver erythrocytes were SeHg complexes if Se was injected at least 5 min before Hg injection. All metal dots on liver erythrocytes were of SeHg complexes if Se was injected 9 or 12 min before the Hg injection. We conclude that the formation of stable in vivo SeHg complexes requires preliminary interaction of Se with a putative serum factor before complexes between Se and Hg are formed and are bound to the erythrocyte cell surface.     

BchJ and BchM interact in a 1 : 1 ratio with the magnesium chelatase BchH subunit of Rhodobacter capsulatus

Substrate channeling between the enzymatic steps in the (bacterio)chlorophyll biosynthetic pathway catalyzed by magnesium chelatase (BchI/ChlI, BchD/ChlD and BchH/ChlH subunits) and S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase (BchM/ChlM) has been suggested. This involves delivery of magnesium-protoporphyrin IX from the BchH/ChlH subunit of magnesium chelatase to BchM/ChlM. Stimulation of BchM/ChlM activity by BchH/ChlH has previously been shown, and physical interaction of the two proteins has been demonstrated. In plants and cyanobacteria, there is an added layer of complexity, as Gun4 serves as a porphyrin (protoporphyrin IX and magnesium-protoporphyrin IX) carrier, but this protein does not exist in anoxygenic photosynthetic bacteria. BchJ may play a similar role to Gun4 in Rhodobacter, as it has no currently assigned function in the established pathway. Purified recombinant Rhodobacter capsulatus BchJ and BchM were found to cause a shift in the equilibrium amount of Mg-protoporphyrin IX formed in a magnesium chelatase assay. Analysis of this shift revealed that it was always in a 1 : 1 ratio with either of these proteins and the BchH subunit of the magnesium chelatase. The establishment of the new equilibrium was faster with BchM than with BchJ in a coupled magnesium chelatase assay. BchJ bound magnesium-protoporphyrin IX or formed a ternary complex with BchH and magnesium-protoporphyrin IX. These results suggest that BchJ may play a role as a general magnesium porphyrin carrier, similar to one of the roles of GUN4 in oxygenic organisms.     

Antimicrobial activity of wax and hexane extracts from Citrus spp. peels

Antibacterial and antifungal properties of wax and hexane extracts of Citrus spp. peels were tested using bioautographic and microdilution techniques against three plant pathogenic fungi (Penicillium digitatum, Curvularia sp., and Colletotrichum sp.), two human pathogens (Trichophyton mentagrophytes and Microsporum canis), and two opportunistic bacteria (Escherichia coli and Staphylococcus aureus). Two polymethoxylated flavonoids and a coumarin derivative, were isolated and identified from peel extracts, which presented antimicrobial activity especially against M. canis and T. mentagrophytes: 4',5,6,7,8-pentamethoxyflavone (tangeritin) and 3',4',5,6,7,8-hexamethoxyflavone (nobiletin) from C. reticulata; and 6,7-dimethoxycoumarin (also known as escoparone, scoparone or scoparin) from C. limon.     

Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 2⁴ full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL⁻¹ of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor.     

Long-term stability of marker gene expression in Prunus subhirtella: a model fruit tree species

Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. Since trees are particularly suited for long-term evaluations of the impact of the technology, Prunus subhirtella autumno rosa (PAR) was chosen as model fruit tree species and transformed with a reporter gene (uidA) under the control of the 35S promoter. Using Southern and GUS fluorometric techniques, we compared transgene copy numbers and observed stability of transgene expression levels in 34 different transgenic plants, grown under in vitro, greenhouse and screenhouse conditions, over a period of 9 years. An influence of grafting on gene expression was not observed. No silenced transgenic plant was detected. Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs, confirming the value of PAR as model species to study season-dependent regulation in mature stone fruit tissues. While the Agrobacterium-derived Prunus transformants contained one to two copies of the transgenes, 91% of the transgenic events also contained various lengths of the bacterial plasmid backbone, indicating that the Agrobacterium-mediated transformation is not as precise as previously perceived. The implications for public acceptance and future applications are discussed.     

In vitro induction of mucosa-type dendritic cells by all-trans retinoic acid

Efficient induction of mucosal immunity usually employs nasal or oral vaccination while parenteral immunization generally is ineffective at generating mucosal immune responses. This relates to the unique ability of resident mucosal dendritic cells (DC) to induce IgA switching and to imprint mucosa-specific homing receptors on lymphocytes. Based on the well-established plasticity of the DC system, this study sought to investigate whether peripheral DC could be modulated toward "mucosa-type" DC by treatment with immunomodulatory, and therefore potentially adjuvant-like, factors. In this study, we show that monocyte-derived DCs pretreated with the vitamin A derivative all-trans retinoic acid (RA) indeed acquired several attributes characteristic of mucosal DC: secretion of TGF-beta and IL-6 and the capacity to augment mucosal homing receptor expression and IgA responses in cocultured lymphocytes. Addition of a TGF-beta-neutralizing Ab to cocultures significantly inhibited alpha4beta7 integrin, but not CCR9 mRNA expression by the lymphocytes. Both alpha4beta7 integrin and CCR9 mRNA expression, but not IgA production, were suppressed in the presence of a RA receptor antagonist. None of the observed effects on the lymphocytes were influenced by citral, a retinal dehydrogenase inhibitor, arguing against a role for de novo-synthesized RA. Collectively, our findings identified a novel role for RA as a mucosal immune modulator targeting DC. Our results further demonstrate that DC can act as efficient carriers of RA at least in vitro. Consequently, RA targeting of DC shows potential for promoting vaccine-induced mucosal immune responses via a parenteral route of immunization.