Presence of benzodiazepine binding sites (receptors) and amplification thereof by imprinting inTetrahymena

Summary LiveTetrahymena cells bound³H-diazepam specifically, as demonstrated by autoradiographic evidence of displacement of about 25% of labeled diazepam in the presence of a 1000-fold amount of cold diazepam. The³H-diazepam bound to membrane preparations isolated from untreated (control) cells was not displaced by cold diazepam, whereas cells involved in primary interaction (imprinting) with diazepam showed amplification and specificity of diazepam binding in both in vivo (cell suspension) and in vitro (pellicle) systems, as well as displacement of bound label in the presence of 1000-fold cold diazepam. It appears that diazepam induced imprinting and, consequently, also the formation of specific receptors inTetrahymena.     

Role of proline in the imprinting developed by dipeptides — in Tetrahymena Possible role in hormone evolution

The effects of proline and serine dipeptides containing phenylalanine, alanine and leucine on the behaviour of receptors of Tetrahymena were investigated. Only proline-containing dipeptides were able to develop positive imprinting, and the activity depended on which other amino acid was present in the dipeptide. In contrast to the positive imprinting effect of the dipeptides Pro-Phe and Pro-Ala, the dipeptide Pro-Pro and Pro-Leu caused negative imprinting. Serine dipeptides produced negative imprinting in all cases. The possible importance of proline in the evolution of hormone specificity is discussed.     

Maternal vitamin A restriction alters biochemical development of the brain in rats

Summary The biochemical development of the fetal brain in relation to maternal vitamin A restriction was studied in rats. The vitamin A status of pregnant rats was varied by supplying low, medium and adequate amounts (6, 40, and 100 g retinol/day/kg body weight, respectively) of vitamin A during pregnancy and suckling. The maternal vitamin A restriction caused an altered brain development in terms of tissue weight, DNA, RNA and protein levels, and biosynthesis of DNA and protein from [³H]-thymidine and [³H]-leucine, respectively. A dose-dependent effect of maternal vitamin A restriction on the metabolism of DNA, RNA and protein was noticed in the developing fetal brain of rats.     

Ube3a,the E3 ubiquitin ligase causing Angelman syndrome and linked to autism,regulates protein homeostasis through the proteasomal shuttle Rpn10

Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. In particular, Angelman syndrome and one of the most common genomic forms of autism, dup15q, are caused respectively by lack of or excess of UBE3A, a ubiquitin E3 ligase. Its Drosophila orthologue, Ube3a, is also active during brain development. We have now devised a protocol to screen for substrates of this particular ubiquitin ligase. In a neuronal cell system, we find direct ubiquitination by Ube3a of three proteasome-related proteins Rpn10, Uch-L5, and CG8209, as well as of the ribosomal protein Rps10b. Only one of these, Rpn10, is targeted for degradation upon ubiquitination by Ube3a, indicating that degradation might not be the only effect of Ube3a on its substrates. Furthermore, we report the genetic interaction in vivo between Ube3a and the C-terminal part of Rpn10. Overexpression of these proteins leads to an enhanced accumulation of ubiquitinated proteins, further supporting the biochemical evidence of interaction obtained in neuronal cells.     

Imprinting of the Peking duck (anas platyrhynchos) and dependence on exposure to light during ontogenesis

Summary Embryos of Peking ducks were either incubated in complete darkness up to hatching or were put into light one week before hatching. Control embryos were incubated under dim light conditions which corresponded broadly to the natural conditions. Under standardized imprinting conditions the controls and both groups of the light deprived ducklings showed the following response. Most of the dark-incubated embryos, however, did not distinguish between imprinting and test objects of different shapes. Since most of the embryos kept in darkness only for 21 days also failed to develop the capacity for shape discrimination, there is apparently a, critical period for light influences on the development of this capacity at some time during the early prenatal period.Acknowledgments. We are grateful to Dr E. Pröve for his advice on the carrying out of our imprinting tests, to K. Weigel for help with the drawings and to Dr. J. R. Wolff for helpful discussions.     

Investigations of receptor-mediated phagocytosis by hormone-induced (imprinted)Tetrahymena pyriformis

Receptor-mediated endocytosis byTetrahvmena pyriformis was studied using tetramethylrhodamine isothiocyanate-labeled concanavalin A (TRITC-Con A) with fluorescence and confocal microscopy. In the presence of insulin, or 24 h after insulin pretreatment (hormonal imprinting), the binding and uptake of TRITC-Con A increased when compared to controls, owing to the binding of TRITC-Con A to sugar oligomers of insulin recptors. Mannose inhibited the binding of Con A, thus demonstrating the specificity of binding. Histamine, a phagocytosis-promoting factor in mammals andTetrahymena, and galactose, did not influence the uptake of TRITC-Con A.     

Profiling of the secreted proteins during 3T3-L1 adipocyte differentiation leads to the identification of novel adipokines

Adipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation was screened, and 41 proteins that responded to blocking of secretion by 20°C treatment and/or brefeldin A treatment were identified. Prohibitin, stress-70 protein, and adhesion-regulating molecule 1 are reported for the first time as secreted proteins. In addition, procollagen C-proteinase enhancer protein, galectin-1, cyclophilin A and C, and SF20/IL-25 are newly identified as adipocyte secreted factors. Secretion profiles indicated a dynamic environment including an actively remodeling extracellular matrix and several factors involved in growth regulation.Received 15 June 2004; received after revision 26 July 2004; accepted 2 August 2004     

Maternal vitamin A restriction alters biochemical development of the brain in rats.

The biochemical development of the fetal brain in relation to maternal vitamin A restriction was studied in rats. The vitamin A status of pregnant rats was varied by supplying low, medium and adequate amounts (6, 40, and 100 micrograms retinol/day/kg body weight, respectively) of vitamin A during pregnancy and suckling. The maternal vitamin A restriction caused an altered brain development in terms of tissue weight, DNA, RNA and protein levels, and biosynthesis of DNA and protein from [3H]-thymidine and [3H]-leucine, respectively. A dose-dependent effect of maternal vitamin A restriction on the metabolism of DNA, RNA and protein was noticed in the developing fetal brain of rats.     

Presence in and effects of pineal indoleamines at very low level of phylogeny

The unicellular organism Tetrahymena contains serotonin and is able to take up the hormone from its mileiu. The serotonin content of the cell changes as a function of the presence of foreign exogenous hormones. This indicates a possible role of serotonin as a chemical mediator. Exogenous serotonin stimulates the RNA synthesis of Tetrahymena, and it was the only one among the hormones studied which kept the RNA level durably high. Serotonin stimulates phagocytosis and growth of Tetrahymena, and its precursors also stimulate growth. Serotonin can imprint Tetrahymena, and as a consequence of this the effect of the hormone increases in the case of further encounters. Treatment with serotonin-related molecules soon after imprinting can reduce the effect of imprinting. Melatonin can contract the pigment cells of Planaria; however, its precursors serotonin and tryptamine can do this more intensely. Both melatonin and serotonin can influenc the regeneration of Planaria, with effects which differ when different phenomena are studied. Evolutionary theories are discussed.     

A 30-kDa fragment of insulin-like growth factor (IGF) binding protein-3 in human pregnancy serum with strongly reduced IGF-I binding

Proteolytic cleavage of insulin-like growth factor (IGF) binding protein (IGFBP)-3 during pregnancy is likely to have both IGF-dependent and -independent effects on maternal, placental and fetal growth and metabolism. A 30-kDa proteolytic IGFBP-3 fragment was isolated from third trimester pregnancy human serum and identified by N- and C-terminal amino acid sequence analysis and mass spectrometry to correspond to residues 1–212 of the parent protein. This fragment is the dominating IGFBP-3 immunoreactive species in pregnancy serum. The 30-kDa fragment was also detected in serum of non-pregnant women where it coexists with intact IGFBP-3. Using biosensor technology, (1–212)IGFBP-3 was found to have 11-fold lower affinity for IGF-I compared to intact IGFBP-3, while a 4-fold decrease in affinity was found for IGF-II. Tests with des(1–3)IGF-I suggest fast binding of IGF-I to the N-terminal region of IGFBP-3 and similar affinity to a slow binding site in the C-terminal region. Received 24 April 2007; received after revision 11 June 2007; accepted 13 June 2007     

Interferon-alpha and transforming growth factor-β co-induce growth inhibition of human tumor cells

A hallmark of resistance to type I interferons (IFNs) is the lack of antiproliferative responses. We show here that costimulation with IFN-alpha and transforming growth factor beta-1 (TGF-beta) potentiates antiproliferative activity in a sensitive (ME15) and resistant (D10) human melanoma cell line. A DNA microarray-based search for proliferation control genes involved that are cooperatively activated by IFN-alpha and TGF-beta, yielded 28 genes. Among these are the insulin-like growth factor-binding protein 3 (IGFBP3) and the calcium-binding protein S100A2; we demonstrate, that recombinant IGFBP3 protein is a potent growth inhibitor requiring TGF-beta activity. The antiproliferative activity of S100A2 is significantly enhanced by IFN-alpha in stably transfected ME15 or D10 cell lines. We show for the first time that IFN-alpha is a potent inducer of intracellular calcium release required for activation of S100A2. Our study provides a functional link between IFN-alpha and TGF-beta signaling and extends the function of IFN signaling to calcium-sensitive processes.     

Retroviral-mediated gene therapy for the treatment of citrullinemia. Transfer and expression of argininosuccinate synthetase in human hematopoietic cells

Citrullinemia is a recessive genetic disease caused by a deficiency in argininosuccinate synthetase (AS). Retroviruses were used to transduce the human AS gene into cultured human cells. Using amphotropic viruses with high titer (>10⁶ cfu/ml), we were able to correct the defect in cultured fibroblasts from citrullinemic patients. Retroviral transduction of the human AS gene into human bone marrow cells was also studied. Co-cultivation was used to infect the cells and up to 80% of progenitor cells were found to be carrying and expressing the AS retrovirus after infection. When the infected cells were kept in culture, integration and expression of the retrovirus was observed. Retroviral sequences were present and expressed in the cultured bone marrow-derived cells for up to 10 weeks.     

Inhibition of protein deacetylation by trichostatin A impairs microtubule-kinetochore attachment

Inhibition of protein deacetylation arrests cells in mitosis, but the mechanism is unknown. To understand why inhibiting protein deacetylation causes cell cycle arrest, we treated HeLa cells beyond G1/S transition with trichostatin A (TSA), a potent protein deacetylase inhibitor, and found that the cells arrested at prometaphase with ectopic spindles and unaligned chromosomes. The hyper-acetylated cells encountered a serious microtubule (MT)-kinetochore attachment problem, although the kinetochores are intact at ultrastructural level. By immunofluorescence staining of kinetochore proteins, we found that the pericentromeric H3K9Me3-HP1 pathway was disrupted and that the CENP-A-dependent outer plate protein dynamics of kinetochores was greatly diminished by the drug treatment. The treatment also caused the loss of chromosome passenger complex (CPC), the proposed error checking system, from centromere and impaired the microtubule dynamics of the cells. Overall, we propose that deacetylation inhibition impairs MT-kinetochore attachment through disrupting the centromere function and altering the kinetochore composition and MT dynamics. Received 30 April 2008; received after revision 28 July 2008; accepted 14 August 2008     

Genome-wide association study to identify SNPs conferring risk of myocardial infarction and their functional analyses

Myocardial infarction might result from the interactions of multiple genetic and environmental factors, none of which can cause disease solely by each of themselves. Although molecular biological studies revealed that a number of proteins are possibly involved in its pathogenesis, little, if any genetic findings have been reported so far. To reveal genetic backgrounds of myocardial infarction, we performed a large-scale, case-control association study using 92,788 gene-based single-nucleotide polymorphism (SNP) markers. We have identified functional SNPs within the lymphotoxin-α gene (LTA) located on chromosome 6p21 that conferred susceptibility to myocardial infarction. Furthermore, we could identify galectin-2 protein as a binding partner of LTA protein. The association study further revealed that a functional SNP in LGALS2 encoding galectin-2, which led to altered secretion of LTA, also indicated a risk of myocardial infarction. A combined strategy of genetic and molecularcellular biological approaches may be useful in clarifying pathogenesis of common diseases.Received 7 March 2005; received after revision 22 April 2005; accepted 25 April 2005     

Regulation of cell adhesion by PP2A and SV40 small tumor antigen: An important link to cell transformation

The serine/threonine protein phosphatase 2A (PP2A) represents a large family of highly conserved heterotrimeric enzymes. Their critical importance in cell homeostasis is underlined by the fact that they are targets of natural toxins like the tumor promoter okadaic acid, and of simian virus 40 small tumor antigen (SV40 small t), a viral protein known to promote cell transformation. Furthermore, mutated or lower expression levels of PP2A subunits have been found in certain cancers. One major known event in PP2A-dependent cell transformation is the alteration of key signaling pathways that control cell growth and survival. In this review, we focus on how PP2A enzymes also affect cell adhesion and cytoskeletal dynamics, the disruption of which is linked to loss of cell polarity, increased cell motility and invasiveness. We also examine how those various pathways participate in the transforming activity of SV40 small t. Received 29 June 2006; received after revision 3 August 2006; accepted 20 September 2006     

Presence of benzodiazepine binding sites (receptors) and amplification thereof by imprinting in Tetrahymena

Live Tetrahymena cells bound 3H-diazepam specifically, as demonstrated by autoradiographic evidence of displacement of about 25% of labeled diazepam in the presence of a 1000-fold amount of cold diazepam. The 3H-diazepam bound to membrane preparations isolated from untreated (control) cells was not displaced by cold diazepam, whereas cells involved in primary interaction (imprinting) with diazepam showed amplification and specificity of diazepam binding in both in vivo (cell suspension) and in vitro (pellicle) systems, as well as displacement of bound label in the presence of 1000-fold cold diazepam. It appears that diazepam induced imprinting and, consequently, also the formation of specific receptors in Tetrahymena.