NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo

NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo.     

PCR- and ligation-mediated synthesis of split-marker cassettes with long flanking homology regions for gene disruption in Candida albicans

Because Candida albicans is a diploid organism, two consecutive steps of gene disruption are required to generate a gene knock-out. The same marker (URA3) is often used for disruption of both copies of the gene. This is possible because, after the first round of disruption, homologous recombination between direct repeats flanking the URA3 marker and the subsequent counterselection allow for the efficient recovery of Ura- revertants. Unfortunately, the URA-blaster disruption cassette cannot be used in a PCR-based disruption approach. The hisG repeats flanking the URA3 gene in the disruption cassette anneal to one another during PCR and thereby prevent amplification of the complete cassette. We explored the use of transformation based on split-marker recombination to circumvent this problem. To avoid any cloning steps and to retain the advantage of long flanking regions for disruption, we combined this with a PCR- and ligation-mediated approach for generating marker cassettes. We used this approach to disrupt the C. albicans FAL1 (ATP-dependent RNA helicase) gene. Long 5' and 3' FAL1-specific regions were amplified by PCR and individually ligated to a URA-blaster cassette. The resulting ligation reactions were used separately as templates to generate two FAL1 disruption cassettes with overlapping URA3 marker regions. Simultaneous transformation with both overlapping disruption cassettes yielded efficient disruption of one FAL1 allele.     

The northern Baltic Sea phytoplankton communities in 1903–1911 and 1993–2005: a comparison of historical and modern species data

Despite over 100 years of phytoplankton research in the Baltic Sea, little is known about how the species composition has changed during this period, characterised by severe anthropogenic eutrophication. We investigated the phytoplankton communities in the northern Baltic Sea using data from 1903 to 1911 and 1993 to 2005; to minimise effects of methodological differences we focused on occurrence frequencies. We found that the historical and modern communities differed regarding both species composition and the relative importance of taxonomical groups. The most obvious differences were the increase of dinoflagellates and decrease in the diatom to dinoflagellate ratio in all seasons. Contrary to the widely held view that cyanophytes have gained significance, our results indicate that in terms of occurrence the group has not increased in summer, and has lost ground in both spring and autumn. Several shifts in the occurrence frequency and seasonality of individual taxa transpired. Examining our results in relation to environmental conditions we found that some changes may be symptomatic of climate change related pressures. However, statistical analyses revealed that an undefined ‘period effect’ was the most important factor separating the historical and modern phytoplankton communities. We interpret this ‘period effect’ as evidence for the direct and/or indirect influence of eutrophication.     

Targeting endothelial cells overexpressing VEGFR-2: selective toxicity of Shiga-like toxin-VEGF fusion proteins.

Growing endothelial cells at the sites of angiogenesis express high numbers of VEGF receptors and therefore may be particularly sensitive to VEGF-mediated drug delivery. To test this hypothesis we have constructed a protein containing the catalytic A-subunit of Shiga-like toxin I fused to VEGF121 (SLT-VEGF/L). Wild-type A-subunit is a site-specific N-glycosidase of 28S rRNA that inhibits protein synthesis after being delivered into cells by separate cell-binding B-subunits. SLT-VEGF/L retains functional activities of both SLT and VEGF121 moieties, since it inhibits protein synthesis in a cell-free translation system and induces VEGFR-2 tyrosine autophosphorylation. SLT-VEGF/L selectively inhibits growth of porcine endothelial cells expressing 2.5 x 10(5) VEGFR-2/cell with an IC50 of 0.2 nM and rapidly induces apoptosis at concentrations >1 nM. We found that sensitivity of VEGFR-2 transfected PAE cells to SLT-VEGF/L declined as the cellular VEGFR-2 density decreased; PAE cells expressing 25000 VEGFR-2/cell were as sensitive as parental cells lacking the receptor. Growth inhibition and induction of apoptosis by SLT-VEGF/L require intrinsic N-glycosidase activity of the SLT moiety, but take place without significant inhibition of protein synthesis. Selective cytotoxicity of SLT-VEGF/L against growing endothelial cells overexpressing VEGFR-2 suggests that it may be useful in targeting similar cells at the sites of angiogenesis.     

Microflora of the penile skin-lined neovagina of transsexual women

Background  

The microflora of the penile skin-lined neovagina in male-to-female transsexuals is a recently created microbial niche which thus far has been characterized only to a very limited extent. Yet the knowledge of this microflora can be considered as essential to the follow-up of transsexual women. The primary objective of this study was to map the neo-vaginal microflora in a group of 50 transsexual women for whom a neovagina was constructed by means of the inverted penile skin flap technique. Secondary objectives were to describe possible correlations of this microflora with multiple patients' characteristics, such as sexual orientation, the incidence of vaginal irritation and malodorous vaginal discharge.     

Synaptonemal complex damage in relation to meiotic chromosome aberrations after exposure of male mice to cyclophosphamide

The genetic implications of induced synaptonemal complex (SC) damage are not known. However, on theoretical grounds, such aberrations could be involved in mechanisms leading to potentially heritable defects. Cyclophosphamide (CP), a chemical reported to cause structural and numerical chromosomal aberrations in the mouse, was used to determine if SC damage observed in meiotic prophase is related to subsequent metaphase chromosomal aberrations. Male mice were injected i.p. with CP. In some instances, mice were also injected simultaneously with tritiated thymidine to label DNA so that cells could be tracked autoradiographically through spermatogenesis. Prophase, primary metaphase (M1), and secondary metaphase (M2) samples were sequentially harvested at appropriate times from the same individual, and nuclei were examined for aberrations. Correlation coefficients between SC and metaphase chromosome aberrations were calculated. The inclusion of tritium labeling increased the number and significance of positive correlations. Positive correlations were found between (1) dose-dependent total SC damage and damage to M1, and to a lesser extent, M2 chromosomes; (2) SC breaks/fragments and M1 chains/rings as well as isochromatid breaks/fragments; (3) SC asynapsis and M1 chromatid breaks/fragments; (4) SC multi-axial configurations and M1 chains/rings as well as isochromatid and chromatid breaks/fragments; and (5) SC multi-axial configurations and M2 hyperploidy. These correlations do not define mechanistic or causal relationships between SC and chromosomal damage. However, taken together with the observation that induced SC damage is many times greater than ensuing metaphase chromosome damage, they substantiate SC analysis as a highly sensitive indicator of potentially heritable effects of this (and presumably other) genotoxic agents.     

Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells.

Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called pp185, were originally found in anti-phosphotyrosine antibody (alpha PY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to alpha PY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X. J., et al. (1991) Nature 352, 73-77]. IRS-1 and pp185 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of pp185. The pp185 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp185 (HMW-pp185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-pp185, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-pp185. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-pp185, which may play unique roles in insulin signal transmission.     

Simultaneous quantitation of ephedrines in urine by gas chromatography–nitrogen–phosphorus detection for doping control purposes

A gas chromatographic method for the simultaneous quantitation of ephedrine, pseudoephedrine, norephedrine (phenylpropanolamine), norpseudoephedrine (cathine) and methylephedrine in urine is described. The method consists of a liquid–liquid extraction with tert.-butyl methyl ether at pH 14. The extracts are analysed on a GC system equipped with an Rtx-5 Amine column and a nitrogen–phosphorus detector. Method validation shows excellent separation, linearity, specificity, accuracy, precision, intra-laboratory repeatability and reproducibility, making the method especially suitable for quantitation of ephedrines in urine samples for doping control purposes. A statistical analysis on the abuse of the different ephedrines in urine from athletes controlled in the Flemish doping control laboratory during the period 1993–2000 is included.     

In vitro characterization of fetal hematopoietic stem cells: range and kinetics of cell production from individual stem cells

We have developed methods for detailed characterization of the proliferation kinetics and lineage potential of single human hematopoietic progenitor cells in an in vitro culture system. Fetal bone marrow CD34(hi)/lin(-) cells were cultured at one cell per well in the presence of c-kit ligand (KL), interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) on a murine stroma cell monolayer. Individual wells were scored for growth between 1 and 10 weeks of culture and analyzed by flow cytometry for lineage composition. A wide variation in time (1 to 8 weeks) was observed before initial cell division, even in the presence of cytokines promoting cell division in primitive progenitors. Eleven percent of the plated cells eventually produced a confluent culture well of approximately 20,000 progeny. Confluent wells were harvested and individually analyzed by flow cytometry for cell surface phenotype. Forty-eight percent of confluent wells contained primitive progenitors (CD34(+)lin(-)), 16% contained B-lymphoid cells (CD19(+) or CD10(+)), and 100% contained cells committed to the myelo-erythroid lineage (CD33(+)). CD34(+)/lin(-) cells from confluent wells were replated at one cell per well in secondary culture and the analysis repeated. One of 216 original single cells plated produced populations of B-lymphoid cells, myeloid cells, and primitive progenitors (CD34(+)/lin(-)) which persisted through two expansion cycles. We estimate that more than 36 million cells can be produced from a single cell under these culture conditions. A very small percentage of the CD34(hi)/lin(-) population (about 1%) was responsible for the majority of subsequent cell production. Our estimate of stem cell content in fetal bone marrow, defined by self-renewal as well as both B-lymphoid and myeloid differentiation from one cell, is approximately 1/13,000. This assay system provides direct in vitro measurements of the expected characteristics of hematopoietic stem cells (high proliferation potential, multilineage potential, self-renewal, and quiescence), and is therefore well suited to assessment of stem cell activity within various cell populations. (c) 1996 John Wiley & Sons, Inc.     

Monoclonal antibodies against the native or denatured forms of muscarinic acetylcholine receptors.

BALB/c mice were immunized with affinity-purified muscarinic acetylcholine receptors from calf brain and their splenocytes fused with NS1 myeloma cells. Hybrid cultures were grown and selected for production of antibodies on the basis of enzyme immunoassays on calf and rat forebrain membrane preparations. Thirty-four clones were retained and six of them further subcloned. Two of these subclones produced antibodies that selectively recognized muscarinic acetylcholine receptor-bearing membranes. The M-35b antibodies interacted only with native digitonin-solubilized receptors, and not with denatured receptors. The M-23c antibodies did not react with active digitonin-solubilized receptors but recognized the denatured form. The M-23c antibodies should thus be useful in the purification of the receptor and its precursor translation products, while the M-35b antibodies could be used for the immunocytochemical localization of the receptor in cells and tissues of different species.