Extracellular matrix proteins influence phenotype and cytokine expression in human breast cancer cell lines

Tumor growth and invasion are not only the result of malignant transformation but are also dependent on environmental influences from surrounding stroma, extracellular matrix (ECM), local cytokines and systemic hormones. We have investigated the influence of ECM components on three human breast cancer cell lines of different malignant potential: MCF-7, T47D and MDA-MB-231 were cultured on collagen I, collagen IV, laminin, fibronectin or poly-D-lysine, and we analyzed the proliferation rate and cytokine expression pattern. Among the three cell lines investigated we observed a distinct response to each ECM component. We hypothesize that ECM may have a significant modulatory effect on malignant behavior in vivo which might depend on individual responses and on the differentiation state of tumor cells. This study also shows that the surface on which cells are cultured greatly influences cell kinetics and the cytokine expression pattern.     

BiP binding keeps ATF6 at bay

The erythropoietin receptor transduces signals leading to the growth, differentiation, and survival of red blood cell precursors via interaction with Janus kinase 2 (JAK2). This interaction was thought to occur only at the plasma membrane. Recent evidence, however, shows that JAK2 assembles with newly synthesized erythropoietin receptors in the endoplasmic reticulum, and that this assembly is essential for efficient expression of the receptors at the cell surface.     

Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3" exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3" pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5" phosphate of the 3" fragment, which is not required for U removal. The ability of the 3" U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3" exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (R_p)α-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.     

Separation of carbamazepine and five metabolites,and analysis in human plasma by micellar electrokinetic capillary chromatography

A rapid and feasible method was developed for the analysis of carbamazepine and its five metabolites (10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 10,11-dihydro-10-hydroxycarbamazepine, 2-hydroxycarbamazepine and 3-hydroxycarbamazepine) in human plasma. Separation of the analytes is based on micellar electrokinetic chromatography, in untreated fused-silica capillary (48.5/40.0 cm length, 50 microm I.D.) with phosphate buffer (30 mM, pH 8.00) as background electrolyte, containing 50 mM sodium dodecylsulfate, and methanol (15%, v/v) as organic modifier. Clean up of human plasma samples was carried out by means of a solid-phase extraction procedure, which gave a high extraction yield for all six carbamazepines (>88%). The overall precision of the method gives a mean RSD of about 1.8%. The limit of quantitation for all analytes is < or = 0.30 microg ml(-1), the limit of detection < or = 0.12 microg ml(-1).     

Structure and dynamics of human interphase chromosome territories in vivo

A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5–10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture. Received: 10 November 1997 / Accepted: 24 November 1997     

Evidence for a Conjugation-Like Mechanism of DNA Transfer in Helicobacter pylori

Many strains of Helicobacter pylori are naturally competent for transformation in vitro. Since there is a high degree of genetic variation among H. pylori strains, we sought to determine whether mechanisms of DNA exchange other than transformation exist in these organisms. Studies were done with H. pylori cells that each were resistant to two different antibiotics; the procedure used involved mating of cells on plates or in broth, in the absence or presence of DNase. In each experiment, such matings produced progeny with the markers of both parents. Examination of the full resistance profile and random arbitrarily primed DNA PCR (RAPD-PCR) profiles of the progeny indicated that DNA transfer was bidirectional. DNase treatment reduced but did not eliminate transfer; only the presence of both DNase and a membrane separating the cells did so. For progeny derived from matings in the presence of DNase, antibiotic resistance and RAPD profiles indicated that transfer was unidirectional. DNase-treated cell-free supernatants also did not transform, ruling out transduction. These experiments indicate that both a DNase-sensitive mechanism (transformation) and a DNase-resistant conjugation-like mechanism involving cell-to-cell contact may contribute to DNA transfer between H. pylori cells.     

Distribution of cadmium in leaves of cadmium tolerant and sensitive ecotypes of Silene vulgaris

It has been postulated that vacuolar compartmentation might play an important role in naturally selected cadmium tolerance in Silene vulgaris (Moench.) Garcke (Bladder campion). Additionally, a tendency of heavy metals to accumulate in the epidermis has been reported. Since these factors would affect the distribution of cadmium in leaves, we determined the distribution of cadmium in leaves of cadmium tolerant and sensitive ecotypes of Silene vulgaris at different levels of exposure and at different time intervals. Cadmium concentrations were higher in leaves of sensitive plants than in those of cadmium tolerant ones after identical exposure to cadmium for a period of 8 days. The highest cadmium concentrations were found in the lower epidermis of plants of both ecotypes. The amount of cadmium located at the lower epidermis was highest for sensitive plants, although the stomatal density was lower in the sensitive ecotype than in the tolerant one. A possible explanation for this phenomenon is the weak relationship between transpiration (water flow) and element allocation. Our results support the hypothesis that vacuolar storage of cadmium plays an important role in the mechanism of cadmium tolerance in Silene vulgaris .     

Water uptake by roots of maize and sunflower affects the radial transport of abscisic acid and its concentration in the xylem

The radial movement of cis-abscisic acid (ABA) has been investigated in young excised roots of Zea mays L. and Helianthus annuus L. which were grown hydroponically. In addition to the symplastic path, ABA was largely translocated across the root apoplast by solvent drag with the water in the transpiration stream. On the apoplastic path ABA may even cross the endodermis. Depending on the ABA concentration of the medium (range: 5–500 nM) and in the root apoplast, the solvent-drag component of the flow of ABA counteracted the dilution of ABA in the xylem caused by transpirational water flow. Acidification of the rhizosphere and of the root apoplast increased the apoplastic transport component. In sunflower, the apoplastic flow of ABA was significantly weaker than in maize roots. This was also indicated by the larger apparent reflection coefficient (σ_ABA) of sunflower roots for ABA (sunflower: σ_ABA = 0.97 ± 0.02, n = 6 roots; maize: σ_ABA = 0.68 ± 0.06, n = 6 roots; ±SD). For both species, σ_ABA was smaller than unity. Root reflection coefficients were affected by factors such as pH, ABA concentration of the medium, and by the suction force applied to excised root systems. Due to the complex composite structure of the permeation barrier in the root, the reflection coefficient estimated from solvent drag is also complex. Since unstirred layers affected the absolute value of the reflection coefficient, σ_ABA has been termed `apparent'. It is concluded that the pH and ABA concentration of the soil solution as well as the transpiration rate (suction force) modify the intensity of the root-to-shoot signal which is influenced by an apoplastic bypass flow of ABA. The latter may be substantially affected by the existence of Casparian bands in the exodermis, which were lacking in the roots studied in this paper. Received: 25 February 1998 / Accepted: 16 July 1998     

Chromosomal location of genes for resistance to powdery mildew in common wheat (Triticum aestivum L. em Thell.). 5. Alleles at the Pm1 locus

 The chromosomal location and genetic characterization of powdery mildew resistance genes were determined in the common wheat lines MocZlatka, Weihenstephan Stamm M1N and in a resistant line of Triticum aestivum ssp. spelta var. duhamelianum. Monosomic analyses revealed that one major dominant gene is located on chromosome 7A in each of the lines tested. Allelism tests with Pm1 in the backcross-derived line Axminster/8^*Cc on 7A indicated that the resistance genes are alleles at the Pm1 locus. These alleles are now designated Pm1a in line Axminster/8^*Cc, Pm1b in MocZlatka, Pm1c in Weihenstephan Stamm M1N, and Pm1d in T. spelta var. duhamelianum, respectively. Received: 10 November 1997 / Accepted: 29 January 1998