Preferential expression of cellular retinoic acid binding protein in a subpopulation of neural cells in the developing mouse embryo

The cellular retinoic acid binding protein is thought to be involved in the retinoic-acid-mediated signal transduction pathway. We have isolated the mouse cellular retinoic acid binding protein cDNA from an embryonal-carcinoma-derived cell line by using differential cDNA cloning strategies. In situ hybridization on sections of mouse embryos of various developmental stages indicated that the cellular retinoic acid binding protein gene, which we localized on mouse chromosome 9, is preferentially expressed in a subpopulation of neurectodermal cells. This restricted expression pattern suggests an important role for cellular retinoic acid binding protein in murine neurogenesis.     

Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.     

Transforming growth factor-β in the early mouse embryo: Implications for the regulation of muscle formation and implantation

In a search for functions of transforming growth factor-β during early embryonic development we used two different experimental approaches. In the first we made use of embryonic stem (ES) cells. ES cells in culture differentiate to derivatives of all three germ layers and mimic some aspects of organogenesis when grown as aggregates in suspension to form embryoid bodies. Differentiation procedes further when the embryold bodies attach to suitable substrates. Muscle and neuronal cells are among the most readily identified cell types then formed. We examined the effect of all-trans retinoic acid (RA) and members of the transforming growth factor-β family(TGF-βl, TGF-β2) under these conditions in an assay where single aggregates formed in hanging microdrops in medium supplemented with serum depleted of lipophilic substances which would include retinoids. Endoderm-like cells formed under all conditions tested. RA at concentrations of 10⁸ M and 10⁷ M induced the formation of neurons but in the absence of RA or at concentrations up to 10?⁹ M, neurons were not observed. Instead, beating muscle formed in about one-third of the plated aggregates; this was greatly reduced when RA concentrations increased above 10?⁹ M. Immunofluorescent staining for muscle specific myosin showed that two muscle cell types could be distinguished: elongated, non-contractile myoblasts and mononucleate flat cells. The mononucleate flat cells appeared to correspond with rhythmically contracting muscle. The number of non-contractile myoblasts increased 3-fold over controls in the presence of 10?⁹ M RA. TGF-βs increased the number of contractile and non-contractile muscle cells by a factor 3 to 7 over controls, depending on the TGF-β isoform added and the muscle cell type formed. TGF-β2 also invariably increased the rate at which contracting muscle cells were first observed in replated aggregates. The stimulatory effect of TGF-βs on the formation of mononucleate flat cells was completely abrogated by RA at 10?⁹ M while the number of myoblasts under similar conditions was unchanged. These data suggest that a complex interplay between retinoids and TGF-β isoforms may be involved in regulation of differentiation in early myogenesis. In the second approach, neutralizing polyclonal rabbit antibodies specific for TGF-β2 were injected into the cavity of mouse blastocysts 3.5 days post coitum (pc). After 1 day in culture, embryos were transferred to pseudopregnant females. The number of decidua, embryos and resorptions were counted at day 8.5–9.5 pc. Control antibody injected embryos implanted with high efficiency (87%) compared with anti-TGF-β2 injected embryos which implanted with an efficiency of only 43%. If empty decidua (resorptions) were included, the overall recovery was 71% and 32% for control and experimental embryos, respectively. Embryos that were recovered showed no overt macroscopic abnormalities. These results together impiy functions for TGF-βs in implantation as well as in later development of the embryo. © 1993Wiley-Liss, Inc.     

The effect of monochromatic radiation in the range 350 to 750 nm on the carotenogenesis in Verticillium agaricinum

An action spectrum for carotenogenesis in V. agaricinum has maxima at 395, 433, 660 and 737 nm. In a previous study it had been shown that a light-minus-dark difference spectrum of a crude extract from V. agaricinum had maxima at 390 and 420 nm, and furthermore a red, far-red interaction suggesting phytochrome involvement has been proposed. All these data suggest that there may be at least two photoreceptor systems operating in the photoinduction process here; one for the near-ultraviolet (UV-A)-mediated carotenogenesis, presumably a novel pigment, and the other for the red, far-red region, most likely phytochrome.     

The carotenoids of a black variety of the larva of the moth, Manduca sexta

The black mutant larva of Manduca sexta contains approximately the same quantity of carotenoids as the normal green type, but there is possibly a slight qualitative difference between them, the former storing more β-carotene and less violaxanthin.     

Regulation of stem cell therapies under attack in Europe: for whom the bell tolls

At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non‐medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context—social, financial, medical, legal—in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of ‘translational medicine’. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients.     

Polycistronic lentivirus induced pluripotent stem cells from skin biopsies after long term storage,blood outgrowth endothelial cells and cells from milk teeth

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative.     

The Characterisation of Residual Strain in Ensis siliqua Shells

1 Introduction Molluscan species exhibit a range of morphologies associated with their particular function and habitat. The razor clam (Ensis siliqua) is a bivalve mollusc, usually found in fine sands, that burrows up to depths of 20 fathoms (～36 m)[1]. The species is found in several coastal regions of the British Isles, the Norwegian Sea, the Baltic south, the Iberian Peninsula, the Mediterranean and along the Atlantic coast of Morrocco[2]. The protec- tive outer shell, which is often …     

Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice

The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.