On-chip non-invasive and label-free cell discrimination by impedance spectroscopy.

OBJECTIVES: Many flow-cytometric cell characterization methods require costly markers and colour reagents. We present here a novel device for cell discrimination based on impedance measurement of electrical cell properties in a microfluidic chip, without the need of extensive sample preparation steps and the requirement of labelling dyes. MATERIALS AND METHODS, RESULTS: We demonstrate that in-flow single cell measurements in our microchip allow for discrimination of various cell line types, such as undifferentiated mouse fibroblasts 3T3-L1 and adipocytes on the one hand, or human monocytes and in vitro differentiated dendritic cells and macrophages on the other hand. In addition, viability and apoptosis analyses were carried out successfully for Jurkat cell models. Studies on several species, including bacteria or fungi, demonstrate not only the capability to enumerate these cells, but also show that even other microbiological life cycle phases can be visualized. CONCLUSIONS: These results underline the potential of impedance spectroscopy flow cytometry as a valuable complement to other known cytometers and cell detection systems.

The phytoestrogen genistein enhances osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells.

In the present study, we investigated the role of the phytoestrogen genistein and 17beta-estradiol in human bone marrow stromal cells, undergoing induced osteogenic or adipogenic differentiation. Profiling of estrogen receptors (ERs)-alpha, -beta1, -beta2, -beta3, -beta4, -beta5, and aromatase mRNAs revealed lineage-dependent expression patterns. During osteogenic differentiation, the osteoblast-determining core binding factor-alpha1 showed a progressive increase, whereas the adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARgamma) was sequentially decreased. This temporal regulation of lineage-determining marker genes was strongly enhanced by genistein during the early osteogenic phase. Moreover, genistein increased alkaline phosphatase mRNA levels and activity, the osteoprotegerin:receptor activator of nuclear factor-kappaB ligand gene expression ratio, and the expression of TGFbeta1. During adipogenic differentiation, down-regulation in the mRNA levels of PPARgamma and CCAAT/enhancer-binding protein-alpha at d 3 and decreased lipoprotein lipase and adipsin mRNA levels at d 21 were observed after genistein treatment. This led to a lower number of adipocytes and a reduction in the size of their lipid droplets. At d 3 of adipogenesis, TGFbeta1 was strongly up-regulated by genistein in an ER-dependent manner. Blocking the TGFbeta1 pathway abolished the effects of genistein on PPARgamma protein levels and led to a reduction in the proliferation rate of precursor cells. Overall, genistein enhanced the commitment and differentiation of bone marrow stromal cells to the osteoblast lineage but did not influence the late osteogenic maturation markers. Adipogenic differentiation and maturation, on the other hand, were reduced by genistein (and 17beta-estradiol) via an ER-dependent mechanism involving autocrine or paracrine TGFbeta1 signaling.

A spin-column procedure for estrogen receptor equilibrium and competition binding analysis.

Estrogen receptors, members of the nuclear hormone receptor family, are not only able to bind their endogenous hormone, 17beta-estradiol, but can also accommodate other naturally-occuring, non-steroidal molecules. Here, we describe a spin-column procedure to determine accurately equilibrium dissociation constants (Kds) and IC50 concentrations for estrogenic compounds. The human wild-type ERalpha was used to validate the protocol. We expressed the full-length ERalpha protein in an eukaryotic system to ensure all possible post-transcriptional modifications. The gel filtration-based assay revealed a temperature-dependent Kd shift for ERalpha. At physiological conditions (150 mM salt, 37 degrees C) we determined the 17beta-estradiol Kd for ERalpha to be 281 +/- 13 pmol/L. Positive cooperativity was only apparent at low temperatures and diminished to zero at 37 degrees C. In homologous competition binding experiments using 17beta-estradiol, we observed fifty fold higher IC50 values than the respective Kd. This paper presents a reliable and sensitive protocol to generate saturation binding curves and heterologous competition curves to test estrogenic compounds.

A MADS domain gene involved in the transition to flowering in Arabidopsis.

Flowering time in many plants is triggered by environmental factors that lead to uniform flowering in plant populations, ensuring higher reproductive success. So far, several genes have been identified that are involved in flowering time control. AGL20 (AGAMOUS LIKE 20) is a MADS domain gene from Arabidopsis that is activated in shoot apical meristems during the transition to flowering. By transposon tagging we have identified late flowering agl20 mutants, showing that AGL20 is involved in flowering time control. In previously described late flowering mutants of the long-day and constitutive pathways of floral induction the expression of AGL20 is down-regulated, demonstrating that AGL20 acts downstream to the mutated genes. Moreover, we can show that AGL20 is also regulated by the gibberellin (GA) pathway, indicating that AGL20 integrates signals of different pathways of floral induction and might be a central component for the induction of flowering. In addition, the constitutive expression of AGL20 in Arabidopsis is sufficient for photoperiod independent flowering and the over-expression of the orthologous gene from mustard, MADSA, in the classical short-day tobacco Maryland Mammoth bypasses the strict photoperiodic control of flowering.

[Imaging methods in the diagnosis of carotid stenosis: is angiography obsolete?]. / Bildgebende Verfahren bei der Diagnostik der Karotisstenose: Ist die Angiographie veraltet?

The aim of diagnosis is exact grading of carotid stenosis. The existence of different methods of measuring stenosis causes problems. Also, measurement of stenosis using Doppler ultrasound is based on a wholly different principle. Investigative methods for measurement of stenosis are selective angiography, CT angiography, MR angiography and duplex Doppler ultrasound. On the basis of the literature on the advantages and drawbacks, risks and accuracy of the various methods in symptomatic patients, ultrasound is recommended as the primary diagnostic tool. If Doppler ultrasound shows a stenosis of > 70%, MR angiography or CT angiography is recommended. If the results correspond, no further investigation is needed before surgery. If they do not correspond a selective carotid angiogram is required. Sonographic diagnosis of carotid occlusion needs confirmation by MR angiogram or CT angiogram.

FPF1 modulates the competence to flowering in Arabidopsis.

During the transition to flowing the FPF1 gene is expressed in the peripheral zone of apical meristems and in floral meristems of Arabidopsis. Constitutive expression of FPF1 causes early flowering in Arabidopsis under both long-day and short-day conditions and leads to a shortened juvenile phase as measured by the trichome distribution on the abaxial leaf surface. In the classical late flowering mutants, overexpression of FPF1 compensates partially for the late flowering phenotype, indicating that FPF1 acts downstream or in a parallel pathway to the mutated genes. The co-overexpression of 35S::AP1 with 35S::FPF1 leads to a synergistic effect on the shortening of the time to flowering under short-day conditions. The co-overexpression of 35S::FPF1 and 35S::LFY, however, shows only an additive reduction of flowering time and the conversion of nearly every shoot meristem, except the inflorescence meristem, to a floral meristem under the same light conditions. In addition, the constitutive expression of FPF1 attenuates the severe lfy-1 phenotype under short days and phenocopies to a great extent the lfy-1 mutant grown under long-day conditions. Thus, we assume that FPF1 modulates the competence to flowering of apical meristems.

Interplay between global regulators of Escherichia coli: effect of RpoS, Lrp and H-NS on transcription of the gene osmC.

The transcription of the osmC gene of Escherichia coli is regulated as a function of the phase of growth. It is induced during the decelerating phase, before entry into stationary phase. osmC expression is directed by two overlapping promoters, osmCp1 and osmCp2. osmCp2 is mainly transcribed by E-sigma(s), the RNA polymerase using the sigma(s) (RpoS) sigma factor, and is responsible for the growth phase regulation. Transcription from osmCp1 is independent of sigma(s). The leucine-responsive protein (Lrp) has been shown to bind the osmC promoter region in band shift experiments. In vivo analysis using osmC-lacZ transcriptional fusions demonstrated that Lrp affects the expression of both promoters. It represses the transcription of osmCp1 and activates the transcription of osmCp2 by E-sigma(s). An absence of Lrp results in an increase in the amount of RpoS during exponential growth in minimal medium. The nucleoid-associated protein H-NS also represses osmC transcription from both promoters. However, this happens through different mechanisms. The effect on osmCp2 is probably mediated by the increase in sigma(s) concentration in the cytoplasm of hns- mutants, while the effect on osmCp1 is independent of sigma(s). No binding of H-NS to the promoter region DNA could be detected, indicating that the effect on osmCp1 could also be indirect.

AIDS anxieties of adolescents: determinants of "state" and "trait" anxiety dimensions in a linear structural model.

This study presents the effects of general psychologic characteristics on acquired immunodeficiency syndrome (AIDS) anxieties and sexual behaviour of adolescents. To this end, data were collected in a complex interview and subsequently subjected to a linear structural model analysis. The questioned adolescents were divided into one representative group (n = 256) and a second group who had participated in a voluntary human immunodeficiency virus (HIV) antibody test (n = 45). AIDS anxieties have to be divided into two independent dimensions: first, a relatively stable feeling of AIDS anxiety (trait anxiety) and second, a manifest personal anxiety toward AIDS experienced in a concrete situation (state anxiety). A principal component analysis of the primary data brought forth four variables described as depression/general anxiety, extent of phobic anxieties, compulsion, and tendency to self-consciousness. The present study reveals that the AIDS trait anxiety is more pronounced among those subjects who are not well informed about AIDS, who tend to phobic anxieties, and who observe themselves in a particularly intensive manner. The AIDS state anxiety however, is stronger among subjects who are well informed about AIDS, have sexual experience, and observe themselves intensively. Among the participants who took part in the HIV test, there were more individuals with a higher manifest AIDS anxiety and stronger tendency to depression. The percentage of adolescents who were indeed exposed to a possible risk of getting infected was relatively low. Generally speaking, those young people who are depressed, anxious, and sexually active agreed more easily to take the test than young people with a pronounced phobia toward the risk of infection and less sexual experience. As a conclusion, we can state that those adolescents with less sexual experience tend to externalize their general sexual anxieties in the form of concrete AIDS anxieties.

[Quantitative flow determinations in the superior femoral artery and the popliteal artery using angiodynography (color-coded duplex sonography) before and after percutaneous transluminal laser angioplasty]. / Quantitative Flussbestimmungen in der A. fem. sup. und A. poplitea mit der Angiodynographie (Farbkodierte Duplexsonographie) vor und nach perkutaner transluminaler Laserangioplastie.

The primary angiographic PTLA-success correlates with the angiodynographically measured blood flow. 24 hours after the treatment the measured flow values in the AFS and AP allow prognoses: 1. Flow volumes in the AP less than 15 ml/min do not allow successful PLTA-therapy. 2. Slight clinical improvement can be expected for flow values greater than 50 ml/min in the AFS and between 20 and 30 ml/min in the AP. 3. AP-values greater than 30 ml/min correlate in this pilot investigations with an excellent clinical result 4 months after intervention.

Investigations of the binding of 239Pu to liver cell membranes.

The binding of Pu to liver cell membranes was studied and compared with that of iron with which plutonium shares some physiological properties. The binding of both metals is sensitive to pH changes and they can be dissociated from their binding sites by chelating agents and transferrin. The metal-binding proteins can be extracted with detergents. Both metals have at least two binding sites, the molecular weights of which lie between 150 and 400 kDa; the isoelectric points for iron are 5.5 and 6.5, and for plutonium 6.0 and 6.5. The significance of these results for plutonium uptake into liver cells is discussed.