Reporter-linked monitoring of transgene expression in living cells using the ecdysone-inducible promoter system

Inducible promoter systems such as the ecdysone-inducible system or the tetracycline-regulated expression systems have proven to be powerful tools in studying gene function. In practice, such systems have met with the difficulty that either the vector expressing the transactivator gene or the vector carrying the response element are frequently silenced by flanking genomic sequences after stable integration. In order to identify those cells in a heterogeneous population in which a transgene is expressed from an ecdysone-inducible promoter, we have created the vector p2ER-EGFP/mcs that contains two ecdysone-inducible expression cassettes in tandem. Using two reporter genes, lacZ and green fluorescent protein (EGFP), we demonstrate that the expression of both genes can be co-induced from a very low baseline in CHO cells expressing the modified ecdysone receptor and the retinoid X receptor. The expression of EGFP and lacZ from vector p2ER-EGFP/lacZ follows the same Muristerone A concentration-dependence as that of EGFP from vector pER-EGFP, indicating that the juxtaposition of the two inducible promoters in vector p2ER-EGFP/mcs does not cause cross interference between them. We suggest that this modification of the ecdysone-inducible promoter system will allow for the visual control of the induced expression of other genes by Muristerone A.     

Application of randomly amplified polymorphic DNA (RAPD) analysis for typing avian Salmonella enterica subsp. enterica

Randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular genetic typing of 30 Salmonella enterica subsp. enterica strains isolated from chickens and ducks in Thailand. Six different primers were tested for their discriminatory ability. While some of the primers could only differentiate between the different serovars, the use of multiple primers showed that the RAPD method could also subdivide within a given serovar. The Ready-To-Go RAPD analysis beads used, resulted in reproducible and stable banding patterns. As the RAPD technique is simple, rapid and rather cheap, we suggest that it may be a valuable new tool for studying the molecular genetic epidemiology of S. enterica ssp. enterica, both inter- and intra-serovars.     

Telomere length as a biological marker in malignancy

Telomere maintenance is important for tumor cell growth and survival. Telomere length (TL) is determined by the balance between positive and negative factors impacting telomere homeostasis. In the last decade, TL has emerged as a promising clinical marker for risk and prognosis prediction in patients with malignant disorders. Tumor TL, as well as TL in healthy tissues such as peripheral blood, may carry valuable information for future treatment strategies. Here we discuss the present status of TL as a biological marker in cancer patients.     

Acoustic microscopy of cultured cells. Distribution of forces and cytoskeletal elements.

The scanning acoustic microscope (SAM) allows one to measure mechanical parameters of living cells with high lateral resolution. By analyzing single acoustic images' sound attenuation and sound velocity, the latter corresponding to stiffness (elasticity) of the cortical cytoplasm can be determined. In this study, measurements of stiffness distribution in XTH-2 cells were compared with the organization of F-actin and microtubules. Single XTH-2 cells exhibit relatively high stiffness at the free margins; toward the cell center this value decreases and reaches a sudden minimum where the slope of the surface topography enlargens at the margin of the dome-shaped cell center. The steepness of the increase in slope is linearly related to the decrease in sound velocity at this site. Thus, a significant determinant of cell shape is paralleled by an alteration of stiffness. In the most central parts, no interferences could be distinguished, therefore, this region had to be excluded from the calculations. Stiffness distribution roughly coincided with the distribution of F-actin, but no correlation to microtubule arrangement was found. Following the treatment of XTH-2 cells with ionomycin in the presence of calcium (in the culture medium), the cell cortex first contracted as indicated by shape changes and by a marked increase in stiffness (deduced from sound velocity). This contraction phase was followed by a phase of microtubule and F-actin disassembly. Concomittantly, sound velocity decreased considerably, indicating the loss of elasticity in the cell cortex. No structural equivalent to sound attenuation has been identified.     

Population structure and dynamics of wood mouse Apodemus sylvaticus in Iceland

Five-year-studies of two wood mouse populations (Apodemus sylvaticus L.) were carried out outside the village Vík. S. Iceland, and in a birch forest close to the lake Mývatn, N. Iceland. The population at Vík was studied on a 1.10 ha grid on a slope covered with Angelica archangelica L., the seeds of which constituted a major food resource for the mice. Altogether 948 individuals were caught, marked and followed during 1973–1978. No other free-living small mammals occur in Iceland, and predator pressure is low and considered non-existent on the Angelica slope. The population density was extremely high; highest autumn density recorded was 150 ind ha^?1. Like other Apodemus populations, the one at Vík showed a typical seasonal pattern in numbers: a winter decrease, relatively stable numbers in late summer, and an autumn peak. Breeding started in late April or early May and ended in late September; no winter breeding was observed. Three or four litters were produced, and the last litter(s) of the season greatly contributed to the autumn increase. The output of young per reproductive female throughout the year was negatively correlated with spring density, and autumn peak density with the number of females in August. Also overwinter success and weight changes appeared to be associated with population density (and weather). Survival rates did not vary much between years and seasons and were consistently rather high. Autumn survival of juveniles was positively correlated with mean minimum temperatures. Juvenile survival was, at least at times, negatively affected by the presence of adult males. Trapping outside the main grid did not indicate any marked dispersal, though some immigration to the grid must have occurred, especially in spring and early summer. Among the factors governing the population density and dynamics of wood mouse at Vík we emphasize the absence of interspecific competition and predation, the rich and dependable food resources (mainly Angelica seeds), the mild (though moist and windy) climate, the shelter of dense Angelica stands, the abundance of crevices (e.g. nest-sites), and the social interactions. The birch forest in northern Iceland was low productive and the wood mouse population differed from the Vík population by exhibiting low density, short breeding season, and high vagility; characteristics we consider to be representative for most Apodemus populations in Iceland.     

Application of cyanide-metabolizing enzymes to environmental control; enzyme thermistor assay of cyanide using immobilized rhodanese and injectase

The application of the enzyme thermistor in the analysis of cyanide in standard solutions as well as in blast furnace waste water is described. The heat signal is generated in the conversion of cyanide, catalyzed by the immobilized enzymes rhodanese (E.C. 2.8.1.1) and injectase (E.C. 4.4.19). Using the combination of cyanide-metabolizing enzymes and the enzyme thermistor unit, assays down to 20μM cyanide can be carried out. Linear relationships were obtained at 20–600μM cyanide for injectase and 20–1000μM for rhodanese. The stability at 27°C of the heat response was initially decreased, but soon stabilized at about 80% of the initial value and remained so for at least 200 hr. The technique was easily adapted to continuous analysis, applicable to environmental control (e.g., a “cyanide guard”) with a response time at present within 2–3 min after a sudden change in cyanide concentration has appeared.     

Influence of sexual intercourse on genital tract microbiota in infertile couples

Several studies have suggested the association of disturbed genital tract microbiota with infertility. Our aim was to clarify the influence of sexual intercourse on partner’s genital tract microbiota in infertile couples. Seventeen couples were studied, and in 5 men inflammatory prostatitis (IP) was diagnosed. Semen samples were collected during menstruation of the female counterpart, two self-collected vaginal samples were taken 3–5 days later – before intercourse and 8–12 h after intercourse. Ureaplasma parvum was found in 59% of women, its prevalence was higher in women whose partner had IP, as well as in half of their male partners. Sexual intercourse caused significant shifts in vaginal microbiota – increase of Nugent score and shifts in cultured microbiota (emergence and disappearance of several species). These changes were less expressed in the presence of normal vaginal microbiota but more prominent in the partners of IP men. These changes may interfere with fertilization.