Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells

DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.     

Genetic variability within Borrelia burgdorferi sensu lato genospecies established by PCR-single-strand conformation polymorphism analysis of the rrfA-rrlB intergenic spacer in ixodes ricinus ticks from the Czech Republic

In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a approximately 230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.     

Comparison of hCMV immediate early and CaMV 35S promoters in both plant and human cells

Cauliflower mosaic virus 35S promoter, widely used in transgenic crop plants, is known to be recognized in widely differing kinds of cells. Its activity in human cells may have impact on the risk assessment for the environmental release of genetically modified plants. In this study, transient expression of several constructs containing beta-glucuronidase (GUS) gene driven by cauliflower mosaic virus 35S promoter or by immediate early promoter of human cytomegalovirus (pCMV) was tested in both potato leaf protoplasts and cultured human cells. The results showed very low but measurable activity of 35S promoter in human 293T-cells (0.01% of that revealed when using pCMV) and in 293 cells that do not produce SV40 T antigen this activity was even lower. On the other hand, in potato protoplasts, pCMV displayed nearly 1% activity seen with p35S.     

Engineering tolerance and accumulation of lead and cadmium in transgenic plants

We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination. We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast. DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium. Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals. These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium.     

The content of four immunomodulatory steroids and major androgens in human semen

Seminal fluid fulfils a dual role: it provides optimal conditions for fertilization and protects male germ cells from infections. Besides both major sexual hormones and cortisol it contains a considerable amounts of dehydroepiandrosterone (DHEA), known to counteract the excessive actions of glucocorticoids. From this point of view of importance may be our recent finding of both 7-hydroxy-dehydroepiandrosterone epimers (7-OH-DHEA) in semen, believed to be in some instances the locally active immunoprotective agents. The concentrations of these steroids were of the same range or even higher than in blood. Here further data on 7-OH-DHEA in semen, along with other relevant steroid hormones, are given in 79 samples, either from healthy males or from patients with various sexual disorders. A method has been developed enabling us a simultaneous determination of DHEA, 7-OH-DHEA epimers, testosterone, dihydrotestosterone and cortisol in seminal fluid. It was based on ether extraction, solvent partition and HPLC separation, followed by specific radioimmunoassays in the respective fractions. In addition, the steroids were measured in serum and the concentrations in both fluids were compared. The concentrations of 7-OH-DHEA in seminal fluid varied from 1.8 to 15.7 nmol/l, while those of DHEA were about five times higher.     

2,6,8,9-tetrasubstituted purines as new CDK1 inhibitors

Purine inhibitors of cyclin-dependent kinases attract attention as potential anticancer drugs because their first representative roscovitine recently entered clinical trials. Although well described in terms of structure-activity relationships, we still present here a novel modification of the purine scaffold influencing their inhibitory properties. The introduced C-8 substituents, however, lowered the CDK inhibitory activity of roscovitine, whereas the antiproliferative potential of several derivatives remained high.     

On the relationship between the non-photochemical quenching of the chlorophyll fluorescence and the Photosystem II light harvesting efficiency. A repetitive flash fluorescence induction study

Plants respond to excess light by a photoprotective reduction of the light harvesting efficiency. The notion that the non-photochemical quenching of chlorophyll fluorescence can be reliably used as an indicator of the photoprotection is put to a test here. The technique of the repetitive flash fluorescence induction is employed to measure in parallel the non-photochemical quenching of the maximum fluorescence and the functional cross-section (sigma(PS II)) which is a product of the photosystem II optical cross-section a(PS II) and of its photochemical yield Phi(PS II) (sigma (PS II) = a(PS II) Phi(PS II)). The quenching is measured for both, the maximum fluorescence found in a single-turnover flash (F(M) (ST)) and in a multiple turnover light pulse (F(M) (MT)). The experiment with the diatom Phaeodactylum tricornutum confirmed that, in line with the prevalent model, the PS II functional cross-section sigma (PS II) is reduced in high light and restored in the dark with kinetics and amplitude that are closely matching the changes of the F(M) (ST) and F(M) (MT) quenching. In contrast, a poor correlation between the light-induced changes in the PS II functional cross-section sigma (PS II) and the quenching of the multiple-turnover F(M) (MT) fluorescence was found in the green alga Scenedesmus quadricauda. The non-photochemical quenching in Scenedesmus quadricauda was further investigated using series of single-turnover flashes given with different frequencies. Several mechanisms that modulate the fluorescence emission in parallel to the Q(A) redox state and to the membrane energization were resolved and classified in relation to the light harvesting capacity of Photosystem II.     

Accelerated HER-2 degradation enhanced ovarian tumor recognition by CTL. Implications for tumor immunogenicity

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369–377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.     

Melatonin concentrations in patients with large goiter before and after surgery

OBJECTIVES: Surgical removal of a very large goiter may traumatize adjacent anatomical structures. The manipulations that involve superior cervical ganglia may alter melatonin secretion. To test this hypothesis we decided to study diurnal serum melatonin profiles in patients with a very large goiter before and after the surgery. MATERIAL AND METHODS:The study was performed on 10 women (mean age-46.5+/-1.6 years; mean+/-SEM; range 39-54 years) with very large non-toxic nodular goiter (mean thyroid volume-125.8+/-25.9 cm (3); mean+/-SEM; range 82.6-326.7 cm(3)). Diurnal serum melatonin profiles were estimated two days before the operation and 10 days after the surgery. Blood samples were collected at 08:00, 12:00, 16:00, 20:00, 22:00, 24:00, 02:00, 04:00, 06:00 and 08:00 h. Melatonin concentration was measured using RIA kit. RESULTS: Nocturnal serum melatonin concentrations (at 24, 02, and 04 hours) were significantly higher after the surgery than before the operation. CONCLUSIONS: Very large goiter may compress the superior cervical ganglia altering indirectly the melatonin synthesis. It cannot be excluded, however, that the presence of the large goiter in some other way affects melatonin secretion.     

News & Notes: Detection of Microorganisms with Overall Cellulolytic Activity

A modification is described of the plate method for the detection of microorganisms with overall cellulolytic activity, including those like Cytophaga, in which the activity is cell bound. Within a few days of incubation colonies of cellulose-degrading bacteria formed holes in discs of lens paper placed on freshly inoculated agar plates. Received: 14 June 1999 / Accepted: 20 July 1999