Cloning ofCandida boidinii DNA fragments promoting autonomous replication of plasmids inSaccharomyces cerevisiae

Fragments of Candida boidinii chromosomal DNA were inserted into the integrative vector YIp-kanr and examined for the presence of sequences promoting autonomous replication of plasmids in Saccharomyces cerevisiae. Restriction maps of two plasmids, designated S6/4 and S6/5, originating from the same S. cerevisiae transformant, were constructed. Southern hybridization data confirmed that the plasmids carry sequences from the C. boidinii chromosome. Both plasmids transform S. cerevisiae strains at 4-5-fold higher frequency than cloning vectors based on the replication origin of the 2 microns plasmid. Mitotic stability of the constructed plasmids is similar to that of the 2 mu-based vector pNF2 in S. cerevisiae.     

Expression of the threonine operon fromEscherichia coli inBrevibacterium flavum andCorynebacterium glutamicum

Summary The threonine operon fromEscherichia coli was cloned in plasmid pBR322, subcloned into the shuttle vector pCEM300 and the resulting recombinant plasmid was transferred intoBrevibacterium flavum andCorynebacterium glutamicum. The expression ofE. coli threonine genes in these coryneform bacteria was demonstrated by complementing thethrA andthrB mutations and by assaying homoserine dehydrogenase activity.     

Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.     

Vibrational circular dichroism spectroscopy study of paroxetine and femoxetine precursors

The solution structures of (3R,4S)- and (3S,4R)- 4-(4-fluorophenyl)-3-hydroxylmethyl- 1-methylpiperidine, which are intermediates in the synthesis of the two pharmaceuticals paroxetine and femoxetine, were studied by vibrational circular dichroism (VCD) spectroscopy. In addition, six derivatives with different substituents attached to the C3 atom were prepared and their VCD and absorption spectra discussed with the aid of ab initio simulations. The VCD spectra were found to be sensitive to the geometry changes. In addition, a subtle variation caused by intermolecular aggregation was apparent in the spectra. The VCD technique can be applied for structural analysis of chiral pharmaceuticals in solutions.     

Usefulness of PCR-HRMA in identification of non-fermentative Gram-negative rods recovered from patients suffering from cystic fibrosis or chronic obstructive pulmonary disease

Adequate treatment of microbial infections requires rapid and accurate identification of the etiological agent. In routine diagnostics, identification of bacteria conventionally relies on phenotypic testing, which can be hindered by phenotypic variations. Therefore, genotyping techniques should perform faster and more accurately. Recently, the technique of high-resolution melting analysis (HRMA) of PCR amplicons promises to provide a convenient and economic tool of genotypic identification. In our study, we performed prospective routine testing of a PCR-HRMA system that was recently published in a proof-of-the-principle study. The system was evaluated by analysing 275 clinical isolates of bacteria acquired from 65 patients suffering from cystic fibrosis or chronic obstructive pulmonary disease. Our results show that its routine use may result in partial worsening of its discriminatory power; however, it still outmatched conventional phenotyping in the group of non-fermentative Gram-negative rods. Moreover, when supplemented by rapid, simple and economic oxidase test, it can be even simplified for more economic performance.     

Experiences of the Czech toxicological information centre with ethylene glycol poisoning

The objective was to evaluate the severity of ethylene glycol (EG) intoxications in a 3-year retrospective study of the calls to the Toxicological Information Centre (TIC). Data about clinical course of patients with EG poisoning reported to the TIC in the years 2000-2002 were analysed. They were completed by the data from discharge records from the hospitals and by toxicological analyses. The chi-square test, Student's t-test, Fisher's test and the calculation of linear correlation coefficient were used for statistical analysis. The significance level was set at 0.05. TIC received total 188 calls concerning EG, from which 33 discharge reports were gained. There were 30 males (age 5-74 years) and 3 females (age 10-54 years). The patients ingested 252 ml on average (30-1000 ml); lethal dose (100 ml) was exceeded in 14 patients. Mean time interval from ingestion to admission was 3 hours (3-24 hours), mean length of hospitalisation 6 days (1-76 days). Fourteen patients developed metabolic acidosis, nine unconsciousness, thirteen signs of nephrotoxicity and nine signs of hepatotoxicity. Three patients died. Antidote ethanol was given in 30 patients. Other treatment included haemodialysis (20 cases) and B vitamins (23 cases). Ingested dose and the time interval between ingestion and admission correlated with severity of kidney damage. These data confirm that EG poisoning could seriously threaten the life. Renal parameters were abnormal in 30 % of patients who were discharged from the hospital. Those patients will be followed to evaluate the reversibility of EG toxic kidney damage.     

Gluconobacter in biosensors: applications of whole cells and enzymes isolated from gluconobacter and acetobacter to biosensor construction

Bacteria belonging to the genus Acetobacter and Gluconobacter, and enzymes isolated from them, have been extensively used for biosensor construction in the last decade. Bacteria used as a biocatalyst are easy to prepare and use in amperometric biosensors. They contain multiple enzyme activities otherwise not available commercially. The range of compounds analyzable by Gluconobacter biosensors includes: mono- and poly-alcohols, multiple aldoses and ketoses, several disaccharides, triacylglycerols, and complex parameters like utilizable saccharides or biological O₂ demand. Here, the recent trends in Gluconobacter biosensors and current practical applications are summarized. An erratum to this article can be found at     

Biotechnological methods of <Emphasis Type="Italic">in vitro</Emphasis> propagation in willows (<Emphasis Type="Italic">Salix</Emphasis> spp.)

Many populations of high-mountainous relic dioecious willows in Central Europe only consist of female individuals and are thus limited in their reproductive potential. We completed micropropagation experiments with shoot apexes and nodal segments of common and endangered willow (Salix) species, which can help to reintroduce autochthonous genotypes to their natural sites. Until recently, cultivation of green young shoot apexes of S. alba and S. lapponum showed the highest percentage of regeneration. We successfully applied the two-times-sterilisation due to high contamination of natural explants. The OK medium was the most efficient culture medium. In vitro propagation of willows with unisexual catkins, anther and ovule cultures were tested and optimised. Isolated anthers were cultivated on selected media and then microcallus and calluses of S. caprea and calluses of S. viminalis were formed on the A medium. Among various tested and optimised media for the ovule culture, the CP medium was the most efficient one. In this case, only the microcalluses of S. viminalis were observed. We developed biotechnological procedures that can be useful in conserving fragmented populations of high-mountainous willows.     

Epidermal UV-shielding and photosystem II adjustment in wild type and chlorina f2 mutant of barley during exposure to increased PAR and UV radiation

The wild-type barley (WT; Hordeum vulgare L.) and its chlorophyll (Chl) b-less mutant chlorina f2 (clo f2) grown under shaded conditions in a greenhouse were transferred to outdoor conditions in early June with predominantly bright sunny days. During 6 days following transfer of plants we monitored the content of photosynthetic pigments, functional state of photosystem II (PSII) by means of Chl fluorescence induction kinetics and epidermal UV-shielding efficiency using Chl fluorescence imaging technique. Clo f2 mutant was more sensitive to exposure to an enhanced natural solar irradiance than WT barley. Nevertheless, clo f2 as well as WT were able to cope with stressful outdoor conditions, as was documented by the recovery of Chl a content and the maximal photochemical efficiency of PSII (F_V/F_M) after an initial decline. This was due to the immediate carotenoid-mediated photoprotection, reflected by strongly increased total carotenoids content and thermal energy dissipation localized within light-harvesting complexes of PSII (assessed by non-photochemical quenching of minimal fluorescence level). The positive acclimation response was further documented by an enhanced light-saturated electron transport rate through PSII (ETR). Based on the ratios of blue- to UV-excited Chl fluorescence we found that for both WT and clo f2 epidermal UV-shielding increased clearly after transfer to outdoor conditions and reached a saturation level after 3 days. In comparison with WT, clo f2 exhibited lower ability to induce UV-shielding. The kinetics of UV-shielding development during the outdoor treatment was different for the particular leaf regions. We suggest that this is related to the different age and developmental stage of the tissue along the leaf blade. The complementarity of carotenoid-mediated photoprotection and UV-shielding in acclimation of the assimilatory apparatus to increased visible and UV radiation is discussed.