Methods of <Emphasis Type="BoldItalic">Candida dubliniensis</Emphasis> identification and its occurrence in human clinical material

Candida dubliniensis was reported as a new species in 1995. This species is often misidentified as Candida albicans. The aims of this work were to determine the occurrence of C. dubliniensis in various clinical materials, to evaluate several ways to identify it and to examine the genetic variability of isolates. Among 7706 isolates originally identified as C. albicans, 237 were identified as C. dubliniensis (3.1%). Most of the C. dubliniensis isolates were obtained from the upper and lower respiratory tract (61.4 and 22.9%). Five phenotypic methods including latex agglutination were used (cultivation on CHROMagar Candida, on Staib agar, at 42 °C and in medium with 6.5% NaCl), but only cultivation on the medium with an increased concentration of NaCl and latex agglutination gave reliable results. Species-specific polymerase chain reaction was used as the confirmation method. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided less reliable results. In fact, 78.9% of C. dubliniensis isolates had scores above 1.7. However, the rest of them (21.1%) were also identified as C. dubliniensis even when the scores were lower than 1.7. Divergences among C. dubliniensis strains were evaluated by means of pulsed-field gel electrophoresis. Eighty-six selected C. dubliniensis isolates showed a 69.6% level of similarity. The results of this study expand the knowledge of the incidence, means of identification and genotypic divergence of C. dubliniensis isolates.     

Cultivation of mesenchymal tissue with reference to antibody formationin vitro

The optimal conditions for the cultivation of rabbit lymphocytes in suspension and antibody formation by these cellsin vitro were studied. The lymphocytes were cultivated in medium in which serum was replaced by serum albumin containing the alpha globulin fraction. Several media were used, with different amounts and proportions of amino acids, vitamins and glucose. The cells survived best in medium in which a component with a high molecular weight was present and in which the glutamine concentration was 0.5–1 mM and the amount of glucose was 0.4–0.5%. Among the other factors determined, the initial pH had a significant influence on survival of the tissue, the optimal limits being 6.9–7.2. Under these conditions 55–60% of the cells survived during 120 hours’ cultivation. The conditions and factors influencing these findings are discussed. Study of the formation of antibodies againstBrucella suis in vitro showed that antibody formation increased only up to 48 hours, after which it remained at the same level.     

<Emphasis Type="Italic">N</Emphasis>-Glycosylation of an IgG antibody secreted by <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> BY-2 cells can be modulated through co-expression of human β-1,4-galactosyltransferase

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.     

Antibody formation by cells isolated from donors of different ages after transfer to young rabbits and to X-irradiated adult rabbits

Antibody formation by spleen cells isolated from adult, non-immunized donors and mixed withBrucella suis antigenin vitro was compared after transfer to young rabbits and to adult recipients irradiated with a dose of 450 r. It was found that this dose did not produce complete inhibition and that the recipients still responded actively to the given antigen. Only young animals, therefore, are a suitable experimental model in which to study antibody formation by transferred isolated cells. Development of the antibody reaction in young rabbits was studied by transferring cells isolated from the spleen of animals aged 8–30 days and mixed with antigenin vitro. Antibodies were formed after the transfer of these cells to X-irradiated recipients, but by the active response of the latter to the antigen. Antibody formation by cells of donors of different ages can be determined after transfer to young animals. The youngest donors whose cells form antibodies after transfer are three-week-old rabbits, but demonstration of antibodies requires the transfer of quite a large number of cells. The results are discussed in relation to the experiments of other authors on the transfer of cells to young animals.     

A study of the nitrate and nitrite discharge from the mutant cells of <Emphasis Type="Italic">Neurospora crassa</Emphasis> lacking nitrate and nitrite reductase activities

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.     

Sorption of some ions by algae related to their trophic conditions

The unicellular algaeScenedesmus obliquus (125),Chlorella pyrenoidosa (82) andCoccomyxa solorinae saccatae (111) were studied with respect to the form of uptake of potassium, phosphate, calcium and zinc ions and to the energy sources involved: light under autotrophic conditions, glucose under mixotrophic or heterotrophic conditions (in light and in darkness or together with yeast extract as an auxotrophic substrate). We respected the trophic conditions of algae when preparing the experimental material (precultivation). The following conditions were reached:

1. (1)
   The three algae grow faster in a glucose medium under mixotrophic conditions and are capable of growing on it also heterotrophically:Ch. pyrenoidosa andSc. obliquus grow substantially better thanC. solorinae saccatae. The first two algae grow more intensively in a glucose medium containing yeast extract whileCoccomyxa does not. After cultivation under mixotrophic conditions the first two diminish endogenous respiration, the third raises it. Glucose stimulates respiration in the first two when grown autotrophically, while after mixotrophic cultivation the effect of glucose is suppressed inCh. pyrenoidosa and in the other two only after growth on glucose with yeast extract.     
   
   

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

A semi-continuous cultivation method for <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> from non-motile cells to motile cells

Non-motile cells of Haematococcus pluvialis grow slowly, whereas motile cells grow fast and divide frequently. Cultivation from non-motile cells to motile cells of H. pluvialis was implemented to promote semi-continuous production. When old cultures which consist of non-motile cells were inoculated in fresh medium with an inoculation amount less than 15%, zoospores were produced in the non-motile cells and developed into motile cells, as the concentration of astaxanthin inducer in the medium was below the threshold value. This process was accomplished within 3 days after inoculation. Furthermore, enhancing KNO₃ content to 1200 mg L^?1 or reducing light intensity to 20 μmol photons m^?2?s^?1 could increase growth during the late culturing period of H. pluvialis and postpone the next round of transformation from motile cells to non-motile cells. A semi-continuous cultivation method for H. pluvialis from non-motile cells to motile cells is proposed in order to regulate the life cycle and promote industrial production. This cultivation mode shortens the inoculum cultivation stage and simplifies the production process of H. pluvialis, showing considerable commercial potential.     

Improved Tolerance of <Emphasis Type="Italic">Escherichia coli</Emphasis> to Propionic Acid by Overexpression of Sigma Factor RpoS

Propionic acid (PA) is an economically important compound, but large-scale microbial production of PA confronts obstacle such as acid stress on microbial cells. Here, we show that overexpressing sigma factor RpoS improves the acid tolerance of Escherichia coli. Four genes including rpoS, fur, pgi and dnaK (encoding RNA polymerase sigma factor, ferric uptake regulator, phosphoglucoisomerase, and chaperone, respectively) were independently overexpressed in E. coli. The recombinant E. coli overexpressing rpoS showed the highest PA tolerance. This strain could grow in M9 medium at pH 4.62, whereas wild type E. coli survived only at pHs above 5.12. Moreover, in the shake-flask cultivation, the E. coli strain overexpressing rpoS grew faster than wild type. Notably, the minimum inhibitory concentration of PA for this recombinant strain was 7.81 mg/mL, which was 2-fold higher in comparison with wild type. Overall these results indicated that overexpression of sigma factor rpoS significantly enhanced E. coli tolerance to PA.     

Biotic factors in induced defence revisited: cell aggregate formation in the toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC 7806 is triggered by spent <Emphasis Type="Italic">Daphnia</Emphasis> medium and disrupted cells

Bioassays with the toxic cyanobacterium Microcystis aeruginosa PCC 7806, its non-toxic mutant ΔmcyB, and Daphnia magna as grazer were used to evaluate biotic factors in induced defence, in particular cyanobacterial and grazer-released info-chemicals. Three main questions were addressed in this study: Does Daphnia grazing lead to a loss of cyanobaterial biomass? Is the survival time of Daphnia shorter in a culture of the toxic cyanobacterium? Does direct grazing or the presence of spent Daphnia medium or a high number of disrupted toxic Microcystis cells in the assays lead to an increase in the cellular microcystin content in the remaining intact cells? The biovolume (growth) as well as size and abundance of Microcystis aggregates were determined by particle analysis, while the survival time of Daphnia individuals was recorded by daily observation and counting, with the relative concentration of cell-bound microcystin-LR, was measured by HPLC analysis. Compared to some recent studies in the field of induced defence, in this study, evidence was found for a direct grazing effect, i.e. the loss of biovolume in the toxic culture. In addition, Daphnia magna ingested more non-toxic than toxic cells, and survived longer with non-toxic cells. In terms of increased cell-bound toxin concentration as a means of defence reported in some studies, a higher cell-bound microcystin-LR content was not measured in this study in any of the treatments (P > 0.05). Under low light conditions with impaired growth of Microcystis, and the presence of a high number of particles with less than 1-μm diameter (possibly heterotrophic bacteria), Daphnia medium was associated with a strong reduction in cell-bound toxin concentration (P < 0.05). This study showed no increased cell aggregation under direct grazing (P > 0.05), but increased aggregation with spent Daphnia medium under high light conditions (P < 0.05). Further, the addition of cell-free extract from disrupted toxic Microcystis cells strongly increased the aggregation of the intact cells under low light (P < 0.05). These findings are discussed with the possible role of microcystin and other infochemicals in the expression of proteins and morphology changes in Microcystis.     

The Effects of Supplementary Cr3 (Chromium(III) Propionate Complex) on the Mineral Status in Healthy Female Rats

Circulating concentration of the essential trace element selenium (Se) was significantly lower in inflammatory disorders. Although Se plays physiological roles mainly through the function of 25 selenoproteins, the response of the selenogenome in immune tissues during inflammatory reactions remains unclear. The objective of this study was to determine the Se retention and selenogenome expression in immune tissues during the lipopolysaccharide (LPS)-induced inflammatory response in porcine. A total of 12 male pigs were randomly divided into two groups and injected with LPS or saline. After 4 h postinjection, blood samples were collected and pigs were euthanized. Pigs challenged with LPS had 36.8 and 16.6 % lower (P < 0.05) Se concentrations in the serum and spleen, respectively, than those injected with saline. Moreover, the activities of GPX decreased (P < 0.05) by 23.4, 26.6, and 30.4 % in the serum, thymus, and lymph node, respectively, in the pigs injected with LPS. Furthermore, the LPS challenge altered (P < 0.05) the mRNA expression of 14, 16, 10, and 6 selenoprotein genes in the liver, spleen, thymus, and lymph node, respectively. Along with 10 previously reported selenoprotein genes, the response of Txnrd2, Txnrd3, Sep15, Selh, Seli, Seln, Selo, Selt, Selx, and Sephs2 to inflammatory reaction in immune tissues were newly illustrated in this study. In conclusion, the LPS-induced inflammatory response impaired Se metabolism and was associated with dysregulation of the selenogenome expression in immune tissues.     

Transport of acetylated sugars intoSaccharomyces cerevisiae RXII cells

The only acetylated sugar taken up bySaccharomyces cerevisiae RXII was 2,3,4,6-tetra-0-acetyl-β, D-glucopyranose and the only cells which took it up were those which grew under aerobic conditions and were harvested during the logarithmic phase of growth. The rate of the uptake of 2,3,4,6,-tetra-0-acetyl-β, D-glucopyranose was considerably slower than that of galactose and its “space” was approximately half the “space” of galactose. The results indicate that 2,3,4,6-tetra-0-acetyl-β, D-glucopyranose is transported into yeast cells by simple diffusion. The results of inhibition of growth ofSaccharomyces cerevisiae andEscherichia coli by 2,3,4,6-tetra-0-acetyl-β, D-glucopyranose are described. Inhibition differed in relation to the composition of the medium during cultivation.     

Serologic characteristics of certain root-nodule bacteria of legumes

Rhizobium trifolii organisms associated with eleven species ofTrifolium were isolated and studied by means of antigen-antibody agglutination and immune diffusion techniques. Ten serologic types were differentiated from the eleven species of nativeTrifolium. Certain serologic types of rhizobia were widely distributed among the native species ofTrifolium, whereas other rhizobial types were found only on one or two species. The distribution pattern appears to be independent of the proportion of each type present in the soil population. This may indicate specificity of selection between a host legume and serologic types of rhizobia.     

Selection for novel,acid-tolerant <Emphasis Type="Italic">Desulfovibrio</Emphasis> spp. from a closed Transbaikal mine site in a temporal pH-gradient bioreactor

Almost all the known isolates of acidophilic or acid-tolerant sulphate-reducing bacteria (SRB) belong to the spore-forming genus Desulfosporosinus in the Firmicutes. The objective of this study was to isolate acidophilic/acid-tolerant members of the genus Desulfovibrio belonging to deltaproteobacterial SRB. The sample material originated from microbial mat biomass submerged in mine water and was enriched for sulphate reducers by cultivation in anaerobic medium with lactate as an electron donor. A stirred tank bioreactor with the same medium composition was inoculated with the sulphidogenic enrichment. The bioreactor was operated with a temporal pH gradient, changing daily, from an initial pH of 7.3 to a final pH of 3.7. Among the bacteria in the bioreactor culture, Desulfovibrio was the only SRB group retrieved from the bioreactor consortium as observed by 16S rRNA-targeted denaturing gradient gel electrophoresis. Moderately acidophilic/acid-tolerant isolates belonged to Desulfovibrio aerotolerans-Desulfovibrio carbinophilus-Desulfovibrio magneticus and Desulfovibrio idahonensis-Desulfovibrio mexicanus clades within the genus Desulfovibrio. A moderately acidophilic strain, Desulfovibrio sp. VK (pH optimum 5.7) and acid-tolerant Desulfovibrio sp. ED (pH optimum 6.6) dominated in the bioreactor consortium at different time points and were isolated in pure culture.     

Assessment of the diagnostic value of specific anti-<Emphasis Type="Italic">Toxocara</Emphasis> IgA in Slovakian patients suspected to have toxocarosis

Human toxocarosis is one of the most widespread and prevalent helminthic zoonosis in many countries, including Slovakia. The aim was to evaluate the usefulness of IgA anti-Toxocara antibody detection in the serodiagnosis of toxocarosis. The levels of specific IgA antibodies were determined by excretory-secretory (ES)-enzyme-linked immunosorbent assay (ELISA). The IgA seropositivity in IgG anti-Toxocara seropositive patients (n?=?52) was 32.7% and found to be highest in the oldest age groups (P?=?0.026). The presence of IgA in suspected patients for toxocarosis were evaluated in respect to some characteristics of examined persons. Substantially higher IgA seropositivity was detected in patients with increased total IgE (44.8%) than in subjects with a normal level of IgE (17.4%; P?=?0.036). No associations (P?>?0.05) were found between IgA seropositivity and sex, level of specific IgG antibodies, avidity of IgG, eosinophilia, domicile, geophagia, traveling abroad, dog/cat ownership, or clinical symptoms. The IgA-ELISA showed sensitivity of 57.1% and specificity of 100%. Mild correlations (r?=?0.302, r?=?0.305, r?=???0.409) were observed between the levels of anti-Toxocara IgA antibodies and age, the amounts of eosinophils and IgA antibody levels, the amounts of eosinophils, and the values of IgG avidity, respectively. The presence of anti-Toxocara IgA may facilitate the diagnosis of toxocarosis and may well be useful for the determination of acute Toxocara infection. Moreover, this test should be accompanied by other immunological markers of examined patients (e.g., increased total IgE, eosinophilia, and low-avidity IgG antibodies).     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> seroprevalence in breeding pigs in Estonia

Background

Toxoplasma gondii is a widespread occurring parasite infecting warm-blooded animals, including pigs and humans. The aims of this study were to estimate the prevalence of anti-T. gondii antibodies and to evaluate risk factors for T. gondii seropositivity in breeding pigs raised in Estonia. Sera from 382 pigs were tested with a commercial direct agglutination test, using a cut-off titer of 40 for seropositivity, for the presence of anti-T. gondii immunoglobulin G antibodies.

Results

Twenty-two (5.8%) of the 382 pigs tested seropositive for T. gondii, and 6 of the 14 herds had at least one seropositive pig. The proportion of seropositive pigs within the herds ranged between 0 and 43%. Gender appeared as a significant factor, with sows having 5.6 times higher odds to be seropositive to T. gondii than boars. Seroprevalence did not increase with age.

Conclusions

Anti-T. gondii antibodies were present in a substantial proportion of breeding pig herds in Estonia. On the other hand, the presence of herds without seropositive pigs illustrates that porcine T. gondii infections can be avoided even in a country where the parasite is endemic and common in several other host species.

     

In vitro synthesis of primary specific anti-breast cancer antibodies by normal human peripheral blood mononuclear cells

In this study, we developed a unique in vitro model to mimic the endogenous tumor microenvironment to understand the effect of immunotherapy with activated T-cells (ATC) armed with anti-CD3 × anti-Her2 bispecific antibody (aATC) on antibody response by naive immune cells. This model contained a co-culture of naïve peripheral blood mononuclear cells (PBMC), breast cancer cells (SK-BR-3), ATC or aATC and CpG ODNs. Culture supernatants were tested at various time points for anti-SK-BR-3 antibodies by ELISA, Western blot and flow cytometry. PBMC cocultured with non-irradiated aATC or irradiated (*) aATC showed significant increases in anti-tumor antibody production at day 14 (P < 0.0001) in the presence of CpG-ODN compared to unstimulated PBMC cultures (n = 9). Antibody specificity was confirmed by ELISA, Western blot and flow cytometry. Co-cultures containing *aATC and CpG showed significantly enhanced levels of IgG₂ (P < 0.001) and cytokines that promote IgG₂ synthesis including IL-13 (P < 0.02), IFNγ (P < 0.01) and GM-CSF (P < 0.05) compared to unstimulated PBMC control (n = 3). We show that aATC targeting and lysis of tumor cells induces an anti-tumor antibody response in our in vitro model. This model provides a unique opportunity to evaluate the interactions of T-cells, B-cells, and antigen-presenting cells leading to specific anti-tumor antibody responses.     

Cloning and Functional Analysis of <Emphasis Type="Italic">SaCLCc1</Emphasis>, a Gene Belonging to the Chloride Channel Family (<Emphasis Type="Italic">CLC</Emphasis>), from the Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis> (L.) Pall.

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe²⁺-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn²⁺. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl^– ions.     

Interaction of rabbit lymph node cells with bacteria (Salmonella paratyphi B)

In five rabbits immunized withSalmonella paratyphi B antigen in all four paws, incubation of the regional lymph node cells with the same bacteria was followed by a significant shift of some of the bacteria from the supernatant of the culture medium to the sediment (as compared with the controls). In six rabbits, bactericidal activity was demonstrated in extracts prepared from the regional lymph nodes only 30 hours after immunization. No antibodies were present in extracts from control rabbits up to the fifth day after immunization.     

Cloning,antibody production,expression and cellular localization of universal stress protein gene (<Emphasis Type="Italic">USP1</Emphasis>-GFP) in transgenic cotton

This study was aimed to clone the universal stress protein (GUSP1) gene isolated from Gossypium arboreum in E. coli expression vector pET30(a) and to raise the specific antibody in rabbit to devise a system that could be used for localization and expression of this gene under drought stress. The amplification of GUSP1 transgene revealed a fragment of 500 bp via PCR in genomic DNA of transgenic cotton plants and expression was confirmed through ELISA and Western blot by using the GUSP1 specific polyclonal antibodies. ELISA showed the expression of GUSP1 protein in roots, stem and leaves of transgenic plants at seedling, vegetative and mature plant developmental stages. Total protein isolated from drought stressed transgenic plants revealed a fragment of 47 kDa (GUSP1-GFP fusion protein) in Western blot which confirmed the expression of transgene. Confocal microscopy detected the GFP fluorescence as localization of GUSP1 in the midrib, guard cells of stomata, trichome and globular trichome of intact leaf of transgenic plants. The co-localization was observed within cytoplasm, palisade, spongy mesophyll, guard cells of stomata, vascular bundle, trichome and globular trichome of transgenic plants by using the GUSP1 specific primary antibodies and Alexa fluor conjugated secondary antibodies. This study of GUSP1 gene will advance the mechanism of abiotic stress tolerance in plants.