Activation of the estrogen receptor from N-nitrosomethylurea-induced rat mammary tumors

The activation of the estrogen receptor (ER) from N-nitrosomethylurea (NMU)-induced rat mammary tumors was studied in vitro. The activation of the receptor induced by heating of the cytosol containing occupied ER was measured by a 3-4-fold increase of receptor binding to nuclei in comparison with the nuclear binding of the nonactivated ER. The activation of the ER was further shown by alteration of the elution profile from DEAE-cellulose. A shift of the receptor peak from 234 mM (Peak II, nonactivated ER) to 70 mM (Peak I, activated ER) phosphate buffer could be obtained. The overall recoveries of activated ER following chromatography on DEAE-cellulose were significantly lower than the recoveries of the nonactivated ER, 71 and 85%, respectively. Binding of the activated ER to nuclei and chromatography of the supernatant which is not able to bind to nuclei on DEAE-cellulose resulted in a decrease of Peak I and in an increase of the overall recovery. These findings suggest that the nuclear bound ER consists of two parts. One is represented partially by Peak I of the elution profile and the other one by that part of the receptor which can not be eluted from the column under the conditions used. Furthermore, the dissociation of tritiated estradiol (E3H) from the nonactivated ER followed a two component exponential function whereas after activation a monophasic dissociation curve could be observed. The mean half times for the dissociation of E3H from the activated and nonactivated ER were 101 and 7.2 min, respectively. Finally, the nonactivated molybdate stabilized ER sedimented in 5-20% sucrose density gradients as two peaks, one at 9.5 S and the other at 4 S. After activation of the ER only the smaller 4 S peak was evident. Molybdate inhibited the activation of the ER measured by nuclear binding assays, sucrose density gradient analysis, dissociation kinetics or ion exchange chromatography but not completely in every case.     

Evaluation and routine application of the novel restricted-access precolumn packing material Alkyl-Diol Silica: coupled-column high-performance liquid chromatographic analysis of the photoreactive drug 8-methoxypsoralen in plasma

A fully automated coupled-column HPLC method for on-line sample processing and determination of the photoreactive drug 8-methoxypsoralen (8-MOP) in plasma has been developed. The method is based on the novel internal-surface reversed-phase precolumn packing materials Alkyl-Diol Silica (ADS). This new family of restricted-access materials has a hydrophilic, electroneutral outer particle surface and a hydrophobic internal pore surface. The supports tolerate the direct and repetitive injection of proteinaceous fluids such as plasma and allow a classical C₁₈-, C₈- or C₄-reversed-phase partitioning at the internal (pore) surface. The total protein load, i.e. the lifetime of the precolumn used in this study (C₈-Alkyl-Diol Silica, 25 μm, 25 × 4 mm I.D.), exceeds more than 100 ml of plasma. 8-MOP was detected by its native fluorescence (excitation 312 nm, emission 540 nm). Validation of the method revealed a quantitative and matrix-independent recovery (99.5–101.3% measured at five concentrations between 21.3 and 625.2 ng of 8-MOP per milliliter of plasma), linearity over a wide range of 8-MOP concentrations (1.2–3070 ng of 8-MOP/ml, r = 0.999), low limits of detection (0.39 ng of 8-MOP/ml) and quantitation (0.79 ng of 8-MOP/ml) and a high between-run (C.V. 1.47%, n = 10) and within-run (C.V. 1.33%, n = 10) reproductivity. This paper introduces coupled-column HPLC as a suitable method for on-site analysis of drug plasma profiles (bedside-monitoring).     

LaPSvS1, a (1-->3)-beta-galactan sulfate and its effect on angiogenesis in vivo and in vitro

LaPSvS1, a highly sulfated branched (1-->3)-beta-galactan was prepared from the arabino-galactan from Larix decidua Miller by partial hydrolysis and subsequent sulfation with SO(3)-pyridine in DMF. The molecular weight was analyzed by GPC and the sulfate content was determined by ion chromatography. LaPSvS1 exhibited good antiangiogenic and antiinflammatory effects in two different modifications of the known CAM-assay. In vitro results obtained in the FGF-2-trypsin-assay and in fluorospectrometric experiments revealed that LaPSvS1 interacts with the fibroblast growth factor 2 system. This interaction is correlated with the in vivo effect of LaPSvS1 on FGF-2 induced angiogenesis.     

A high-throughput Arabidopsis reverse genetics system

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.     

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.     

Connections of the olfactory bulb in the piranha (Serrasalmus nattereri)

Summary The connections of the olfactory bulb were studied in the piranha using the Nauta and horseradish-peroxidase methods. Three olfactory tracts project to seven terminal fields in the telencephalon and one in the diencephalon, all of them bilaterally. The contralateral olfactory bulb also receives a small input. All contralateral projections decussate in the anterior commissure and are relatively weak compared to the ipsilateral projections. HRP-containing cells were found in all of the ipsilateral telencephalic aggregates receiving an olfactory tract projection; the contralateral side was free of labeled cell bodies. Although only about one fourth of the entire telencephalon receives a direct olfactory input, the high degree of differentiation of the olfactory system suggests that the piranha depends substantially on the sense of olfaction and that this species may be a good model for further studies on olfactory mechanisms.     

Mesurement of longitudinal ion profiles in single roots of Hordeum and Atriplex by use of flameless atomic absorption spectroscopy

Summary A method is described by which the Na⁺ and K⁺ content in 0.5 mm sections of single roots of Hordeum distichon L. and Atriplex hortensis L. can be determined by use of flameless atomic absorption spectroscopy. By this method the longitudinal profiles of K⁺ and Na⁺ along low salt roots and roots which had been equilibrated with or grown in K⁺-free 1 mM Na⁺-solution were determined. The profiles reveal that high K⁺/Na⁺ ratios in the cytoplasm are maintained also in K⁺-free solutions. In solutions containing 1 mM Na⁺ a high K⁺/Na⁺ selectivity was found to be dependent on sufficient aeration. From the ion profiles the cytoplasmic (110 mM) and vacuolar (20 mM) K⁺ concentration in low salt barley roots—values which are unobtainable by compartmental analysis—could be estimated.     

Role of the 3' splice site in U12-dependent intron splicing

U12-dependent introns containing alterations of the 3' splice site AC dinucleotide or alterations in the spacing between the branch site and the 3' splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5' splice site AU dinucleotide, any nucleotide could serve as the 3'-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3' splice site function. A branch site-to-3' splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3' splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3' splice site but that formation of the prespliceosomal complex A requires an active 3' splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3' splice site.     

Mom1 Is a Semi-Dominant Modifier of Intestinal Adenoma Size and Multiplicity in Min/+ Mice

The intestinal tumor multiplicity in mice heterozygous for Apc(Min) is strongly modulated by genetic background. On the sensitive C57BL/6J (B6) background, mice develop large numbers of intestinal adenomas. The AKR/J (AKR) strain carries alleles that correlate with a strong reduction in tumor multiplicity. To study the effect of one of these modifiers, Mom1, we have generated a mouse line in which the AKR allele of Mom1 is carried on the sensitive B6 genetic background. This strain was produced by using a marker-assisted selection method to eliminate unlinked AKR alleles more rapidly. The application and efficiency of this method are discussed. We used this strain to determine that Mom1 affects both tumor multiplicity and tumor size in a semi-dominant fashion.