Polymerase chain reaction in diagnosis of toxoplasmosis of the central nervous system: effect of methods for isolating DNA from samples of cerebrospinal fluid on results of the reaction

We examined influence of the method of isolation of DNA from cerebrospinal fluid samples on results of PCR in the diagnosis of toxoplasmosis of the central nervous system. Three different protocols of DNA isolation were used for DNA extraction from 360 samples made of cerebrospinal fluid spiked with tachyzoites of Toxoplasma gondii: thermic, enzymatic and enzymatic-filtering. Purified DNA samples were tested by PCR with primers T15 and T16 designed for the B1 gene of the parasite. Enzymatic method of DNA isolation appeared most effective allowing detection of T. gondii DNA in 50% of samples containing single parasite cell.     

Cytological and morphological differences between two species of the genus Tettigonia (Orthoptera,Tettigoniidae) from Korea

Tettigonia ussuriana and T. dolichopoda maritima differ in the length of tegmina, details in venation, and in females in details of the subgenital plate. The two species of the genus Tettigonia have the same number and morphology of autosomes but a different morphology of the X chromosome: in T. ussuriana it is metacentric, whereas in T. dolichopoda maritima acrocentric. In both species, euchromatic zones and breaks of one or to chromatids during meiosis and mitosis in the X chromosome were observed. Additionally, B chromosomes were noted in most individuals of both species.     

Chromosome C-banding patterns in isolated population of the grasshopper Podisma pedesis (L.) (Orthoptera,Acrididae) from the Altai Mts

The C-banding patterns in the embryo chromosomes of the grasshopper Podisma pedestris (L.) from the Altai Mts are reported. The additional second C-heterochromatic arms in at least five pairs of autosomes and in the X-chromosome were revealed. The paracentromeric, interstitial, and telomeric C-bands were observed. The studied population of P. pedestris shows some differences in the distribution and amount of the heterochromatin in comparison with European populations.     

The effect of pre-eclamptic umbilical cord serum on fibroblast division in culture

Edema, proteinuria, hypertension (EPH-gestosis), most commonly termed as pre-eclampsia, is the most common pregnancy-associated pathological syndrome. It is accompanied by a thorough remodelling of extracellular matrix in the umbilical cord tissues. It is commonly known that the presence of serum in culture medium strongly stimulates many functions of cells cultured in vitro. It was decided to check how the pre-eclamptic serum affects the fibroblast division in culture. Ki-67 is a protein present in proliferating cells and can be detected during all phases of the cell cycle (G1, S, G2/M) but not in resting (G0) cells. PCNA (proliferating cell nuclear antigen) is an intranuclear polypeptide whose synthesis rate is at its maximum during the S-phase of the cell cycle. The expression of Ki-67 and PCNA was measured by immunocytochemical methods and biosynthesis of DNA was evaluated by [14C]-thymidine incorporation. The activity of pre-eclamptic umbilical cord serum (UC-serum) was found to be distinctly lower in comparison to control one. The expression of Ki and PCNA in fibroblast cultures treated with pre-eclamptic serum was also distinctly lower. Also the incorporation of [14C]-thymidine to DNA was lower than in the cultures treated with control UC-serum. It may by concluded that pre-eclampsia reduces the mitogenic activity of the umbilical cord serum.     

The influence of various modified nucleotides placed as 3'-dangling end on thermal stability of RNA duplexes

The ribonucleic acids (RNA) form highly folded structures, which behind the helical fragments contain several secondary and tertiary structural motives. All of them have an influence on thermodynamic stability of the RNA. The 5'- and 3'-dangling ends are one of those structural motives, which effect stability of the adjacent helixes. In this paper, we described the influence of 14 different modified nucleotides, placed as 3'-dangling ends, on thermal stability of the RNA duplexes. Collected data demonstrate that: (i) 5-substituents of the uridine have an impact on the 3'-dangling end effect and the largest changes were observed for 5-chloro, bromo and methyl substituents; (ii) position of the methyl group within the uracil residue affect the thermal stability of the duplex; (iii) increasing a size of the heterocycle base placed as the 3'-terminal unpaired nucleotide enhances stabilization of duplexes.     

Isozymes in Aegilops kotschyi and Ae. biuncialis x Secale cereale hybrids and Ae. kotschyi x S. cereale amphiploids in relation to their parents

Seven enzymatic systems in F1 Aegilops kotschyi and Ae. biuncialis x Secale cereale hybrids, Aegilops kotschyi x S. cereale amphiploids and their parental species (Ae. kotschyi, Ae. biuncialis and S. cereale) were analysed by starch and polyacrylamide gel electrophoresis. Five of them (phosphoglucose isomerase, glutamic oxalacetic transaminase, esterase, acid phosphatase, and diaphorase) were polymorphic and two (malic dehydrogenase and superoxide dismutase) were monomorphic. Several isophorms of phosphoclucose isomerase, esterase, acid phosphatase, and diaphorase were detected in some hybrids and amphiploids, but absent in the parents. The role of regulators, translocations and recombination is discussed in relation to the origin of these new isophorms. Some parental isozymes were absent both in hybrids and amphiploids, probably as a result of the suppression of structural genes in new combinations of the three genomes.     

Seed-expressed fluorescent proteins as versatile tools for easy (co)transformation and high-throughput functional genomics in Arabidopsis

We demonstrate that fluorescent proteins can be used as visual selection markers for the transformation of Arabidopsis thaliana by the floral dip method. Seed-specific expression of green fluorescent protein (GFP) variants, as well as DsRed, permits the identification of mature transformed seeds in a large background of untransformed seeds by fluorescence microscopy. In planta visualization of transformed seeds in siliques shows that susceptibility to floral dip transformation is limited to a small, defined window in flower development. In the competent stage, the random transformation of up to 25% of the seeds within a single silique may occur. The use of fluorescent proteins with different spectral characteristics allows a rapid identification and genetic analysis of seeds that have received multiple genes-of-interest in co-transformation experiments. The data reveal that co-transformation does not occur at random, since the co-transformed genes are integrated at a single genetic locus in approximately 70% of the cases. This genetic linkage of the co-transformed genes greatly simplifies metabolic pathway engineering by reverse genetics in Arabidopsis. Additional advantages of using visual selection instead of antibiotic resistance include a rapid identification of the effect of the T-DNA insertion or the transgene on seed development and/or germination. This technology, of tagging and identifying transformed seeds by fluorescence provides a novel high-throughput screening system with many potential applications in plant biotechnology.     

Stereocontrolled synthesis of diene and enyne sugar-modified nucleosides and their interaction with S-adenosyl-L-homocysteine hydrolase

Conjugated diene 5-7 and enyne 8 analogs derived from adenosine and uridine were synthesized employing Pd-catalyzed cross-coupling reactions.     

Biochemical and immunohistochemical study on physiological activity and distribution of hepatic cathepsin D

Cathepsin D (EC 3.4.23.5) is a lysosomal endopeptidase physiologically present at very low concentration in different tissues. The aim of the study was to estimate the physiological activity and distribution of cathepsin D in the liver. Four groups of ten-week-old male Wistar rats were raised without xenobiotics and sacrificed on day 4, 42, 47 and 84 of the experiment, and their livers were taken for immunohistochemical and biochemical investigation. Immunostaining for cathepsin D was evaluated by light microscope. Activity of the free and bound fractions of hepatic cathepsin D was measured spectrophotometrically. Immunohistochemical staining for cathepsin D was positive in Browicz-Kupffer cells in some but not in all rat liver specimens of each experimental group. The staining pattern was cytoplasmic and granular. Occasionally the positive stained endothelial cells were also found. No activity of cathepsin D in hepatocytes was detected. The positive immunostaining was found in livers with high enzyme activity in the biochemical investigation. No significant differences in activity of the free and bound fractions of cathepsin D among the different age groups were noted. However, the higher, age-dependent activity (p>0.05) of the free fraction was observed in the youngest and the two-middle groups of rats that were sacrificed on day 42 and 47 than in the oldest one. The bound fraction did not reveal such changes. It could be concluded that there were no differences in the activity of hepatic free and bound fractions of cathepsin D in male Wistar rats of various reproductive age. The rat Browicz-Kupffer cells revealed the highest activity of cathepsin D.