Differences in metallothionein gene expression in primary cultures of rainbow trout hepatocytes and the RTH-149 cell line

Primary cultures of rainbow trout, Salmo gairdneri, hepatocytes were used to study the expression of metallothionein (MT) genes in response to steroid hormone treatment. The expression pattern was compared to that of an immortal cell line (RTH-149). MT mRNA accumulated in both cell cultures after exposure to zinc while 17 beta-oestradiol had no effect in either system. Treatment with cortisol and corticosterone resulted in a 2-fold increase of metallothionein mRNA levels in the primary cultures but had no effect in the RTH-149 cell culture. Primary cultures that were exposed to zinc or cortisol showed a high temporal correlation (r = 0.974) between MT mRNA and MT protein levels. The basal level expression was 3-4-fold higher in primary cultures than in RTH-149 cells. The present study demonstrates the inducibility of rainbow trout MT genes in response to glucocorticoids. It further indicates that primary cultures are to be preferred to immortal cell lines when investigating the inducibility of MT mRNA.     

Analysis of rpsD mutations in Escherichia coli

Summary A certain proportion of protein S7 exists in an altered form in E. coli rpsD (S4) mutants. Depending on the type of S4 mutation involved, two different forms of the altered S7 can be distinguished. The unusual form is longer than normal S7 by about 500 daltons due to extra material at the carboxyl end of the protein. It is suggested that a mutationally altered S4 might lower the efficiency of termination during translation of the messenger for S7. This results in an increased frequency of translational read-through, which gives the observed longer forms of S7. Data are interpreted to mean that one class of S4 mutants might suppress UGA and UAG whereas another class only suppresses UGA.     

Purification and properties of four species of lysyl oxidase from bovine aorta

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49-60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000-33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of alpha-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu(2+), and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.     

Susceptibility to antifungal agents of Candida sp. and biofilm formation

In recent years the increase in frequency of fungal infections with Candida sp. was noticed. These infections are connected with ability of Candida sp. to form biofilm on surfaces of biomaterials used in medicine. Furthermore fungal infections make serious therapeutic problems because ofbiofilm resistance to antifungal agents actually. The aim of the study was to evaluate the susceptibility to antifungal agents of Candida sp. and their ability to form biofilm on different biomaterials. 50 strains of Candida sp. isolated from patients of University Hospital No. 1 of dr A. Jurasz in Bydgoszcz were examined. API Candida (bioMérieux) tests were used to identify Candida sp. strains. The susceptibility of the yeast strains to antifungal agents was evaluated by ATB FUNGUS 2 INT (bioMérieux) tests. The susceptibility of examined strains to voriconazole, posaconazole, caspofungin and anidulafungin was assessed by means ofEtests (AB BIODISK) method employing drug concentrations from 0,002 to 32 microg/ml. All analysed strains were susceptible to amphotericin B and caspofungin. Biofilm formation on different biomaterials (silicon, latex, polychloride vinyl, polypropylene, nylon) was measured after 72 hour incubation at 37 degrees C. All examined yeasts formed biofilm on all analysed biomaterials. The highest number of strains formed biofilm on surface of polychloride vinyl: 23 (92,0%) by C. albicans strains and 24 (96,0%) Candida non-albicans strains. The lowest number of the strains formed biofilm on the surface of nylon: 12 (48,0%) of C. albicans strains and 9 (36,0%) of Candida non-albicans strains. The studied strains resistant to azoles and anidulafungin display stronger ability to form biofilm on surfaces of all analysed biomaterials.     

Seasonal carbon allocation to arbuscular mycorrhizal fungi assessed by microscopic examination, stable isotope probing and fatty acid analysis

Background and Aim

Climate change models are limited by lack of baseline data, in particular carbon (C) allocation to – and dynamics within – soil microbial communities. We quantified seasonal C-assimilation and allocation by plants, and assessed how well this corresponds with intraradical arbuscular mycorrhizal fungal (AMF) storage and structural lipids (16:1ω5 NLFA and PLFA, respectively), as well as microscopic assessments of AMF root colonization.

Methods

Coastal Hypochoeris radicata plants were labeled with ¹³CO₂ in February, July and October, and ¹³C-allocation to fine roots and NLFA 16:1ω5, as well as overall lipid contents and AM colonization were quantified.

Results

C-allocation to fine roots and AMF storage lipids differed seasonally and mirrored plant C-assimilation, whereas AMF structural lipids and AM colonization showed no seasonal variation, and root colonization exceeded 80 % throughout the year. Molecular analyzes of the large subunit rDNA gene indicated no seasonal AMF community shifts.

Conclusions

Plants allocated C to AMF even at temperatures close to freezing, and fungal structures persisted in roots during times of low C-allocation. The lack of seasonal differences in PLFA and AM colonization indicates that NLFA analyses should be used to estimate fungal C-status. The implication of our findings for AM function is discussed.     

The incorporation of deoxyuridine monophosphate into DNA increases the sister-chromatid exchange yield

The effect of a treatment with 5-fluoro-2'-deoxyuridine (FdUrd) in combination with 2'-deoxyuridine (dUrd) on cell proliferation, incorporation of DNA precursors into DNA and sister-chromatid exchanges (SCEs) has been analyzed in Allium cepa meristem cells. FdUrd in the range 10(-9)-5 X 10(-7) M produced a dose- and time-dependent decrease in the amount of cells in mitosis. This inhibitory effect could be reversed by 70-80% in short-term (6 h) experiments, by exogenously supplied dUrd at a concentration of 10(-4) M. However, at the highest FdUrd dose tested (10(-7) M), 10(-4) M dUrd could not reverse the FdUrd effect in long-term experiments (20 h, about one cell cycle interval), as shown by analyzing the kinetics of synchronous cell populations. DNA extracted from cells pulsed with [6-3H]dUrd in the presence of FdUrd and 6-amino-uracil (6-AU), an inhibitor of uracil-DNA glycosylase, contained a small amount of label (at least 3% of the total radioactivity incorporated into DNA) in the form of [6-3H]dUMP. Thus, we conclude that, under our experimental conditions, exogenously supplied dUrd may be metabolized intracellularly to 2'-deoxyuridine triphosphate (dUTP) and that this deoxynucleotide may eventually be mis-incorporated into DNA. As far as the formation of SCEs is concerned, analysis of second division chromosomes showed that 2'-deoxyuridine monophosphate (dUMP) residues present in newly-synthesized DNA strands are probably not relevant to SCE formation. However, by analyzing SCE levels in third division chromosomes of cells treated with FdUrd and dUrd during their second cycle, we have scored a 6-fold increase in the reciprocal SCE level which demonstrates that the replication of a dUMP-containing DNA template leads to a higher SCE yield.